University of Groningen Mechanisms of oxidative stress ......Mechanisms of oxidative stress-induced cell death in hepatocytes: Targets for protective intervention Laura Conde de la

University of Groningen

Mechanisms of oxidative stress-induced cell death in hepatocytesConde de la Rosa, Laura

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Mechanisms of oxidative stress-induced cell death in

hepatocytes: Targets for protective intervention

Laura Conde de la Rosa

PhD thesis, Groningen University, The Netherlands

14

Copyright © 2006 Laura Conde de la Rosa All rights reserved. No part of this book may be reproduced or transmitted in any form or by any means without written permission of the author and the publisher holding the copyright of the published articles. Cover picture: Mosque of Córdoba, world heritage. Mezquita de Córdoba, patrimonio de la humanidad. Cover design: Ángel Conde García Printed by Gildeprint Drukkerijen B.V., Enschede, The Netherlands.

RIJKSUNIVERSITEIT GRONINGEN

Mechanisms of oxidative stress-induced cell death in

hepatocytes: Targets for protective intervention

Proefschrift

ter verkrijging van het doctoraat in de Medische Wetenschappen

aan de Rijksuniversiteit Groningen op gezag van de

Rector Magnificus, dr. F. Zwarts, in het openbaar te verdedigen op

maandag 16 oktober 2006 om 16.15 uur

door

Laura Conde de la Rosa

geboren op 23 februari 1976 te Córdoba (Spanje)

14

Promotores

Prof. dr. H. Moshage Prof. dr. P.L.M. Jansen

Beoordelingscommissie

Prof. dr. F. Kuipers Prof. dr. K. Poelstra Prof. dr. J.C. Fernández Checa ISBN paper edition: 90-367-2727-8

ISBN electronic edition: 90-367-2728-6

A mis padres Ángel y Paqui a mi hermana Susana

y a Raúl

14

Paranimfen Lisette Bok Thierry Claudel Research The studies described in this thesis were conducted within the Groningen University Institute for Drugs Exploration (GUIDE), Center for Liver, Digestive and Metabolic Diseases, Department of Gastroenterology and Hepatology, University Medical Center Groningen, The Netherlands. Funding The printing of this thesis was financially supported by the following organizations: Their contribution is gratefully acknowledged! Rijksuniversiteit Groningen, Groningen, The Netherlands. J.E. Jurriaanse Stichting, Rotterdam, The Netherlands. Groningen University Institute for Drug Exploration (GUIDE), Groningen, The Netherlands. Diabetes Fonds, Amersfoort, The Netherlands. AstraZeneca, Zoetermeer, The Netherlands Nederlandse Vereniging voor Hepatologie (NVH), Haarlem, The Netherlands. Dr. Ir. Van de Laar Stichting, Heerlen, The Netherlands. Harlan Nederland, Horst, The Netherlands. Tebu-Bio B.V., Heerhugowaard, The Netherlands

TABLE OF CONTENT Chapter 1 Scope of the thesis 9

Chapter 2 General introduction 11

Chapter 3 Superoxide anions and hydrogen peroxide induce hepatocyte cell death 27

by different mechanisms: involvement of JNK and ERK MAP kinases.

Journal of Hepatology 44 (2006) 918-929

Chapter 4 Heme oxygenase-derived carbon monoxide prevents apoptosis 47

in primary hepatocytes via inhibition of JNK activation

Chapter 5 Metformin protects primary rat hepatocytes against oxidative 65

stress-induced apoptosis

Chapter 6 TUDCA protects rat hepatocytes from bile acid-induced apoptosis via 77

activation of survival pathways

Hepatology 2004;39:1563-1573

Chapter 7 Summary, general discussion and perspectives 97

Chapter 8 Nederlandse Samenvatting (in Dutch) 109

Chapter 9 Sumario en Español 115

Acknowledgements/ Agradecimientos 123

Curriculum Vitae 126

List of publications 127

CHAPTER 1

Scope of the thesis

Chapter 1

10

Most chronic liver diseases, such as non-alcoholic steatohepatitis (NASH), chronic cholestasis and alcoholic and chronic viral hepatitis, are almost invariably accompanied by exposure to reactive oxygen species (ROS). In addition to oxidative stress, liver cells are also exposed to cytokines and bile acids during chronic liver injury. Under these circumstances, cell damage followed by cellular death occurs, which may lead to liver injury and loss of liver function. There are no effective treatments available for chronic liver disorders yet. Hepatocytes, the parenchymal cells of the liver, are well equipped with protective mechanisms to prevent cell death. Once these protective pathways are activated, the balance will be in favour of cell survival. However, the balance between cell survival and cell death is delicate and easily disrupted during liver injury, leading to massive cell death and loss of liver function. Hence, knowledge about the cellular mechanisms leading to cell death is of relevance as it may enable the identification of novel therapeutic tools to treat liver diseases. The aim of this thesis is to elucidate the mechanisms of oxidative stress-induced hepatocyte cell death, in order to develop strategies to protect hepatocytes and prevent liver injury. In chapter 2, the current knowledge on oxidative stress induced cell death, with special emphasis on liver cells, is reviewed. In chapter 3, the mechanisms of cell death induced by different ROS are elucidated. Furthermore, the role of different signal transduction pathways (MAPK and NF-κB) and ROS detoxification mechanisms in ROS-induced cell death are investigated. Heme oxygenase-1 (HO-1) is an important stress-responsive cytoprotective protein. In chapter 4, we describe the regulation of HO-1 in an in vivo model of chronic cholestasis (bile duct ligation model) and in acute inflammation. Furthermore, the protective role of HO-1 against oxidative stress-induced apoptosis was investigated in primary hepatocytes. In addition, we study the contribution of carbon monoxide, a product of heme oxygenase-1, to the protective role of HO-1 against oxidative stress-induced apoptosis and we examine the mechanism involved in this process. Chapter 5 describes the protective effect of metformin, an insulin sensitizing drug frequently used in the treatment of type 2 diabetes, against oxidative stress-induced apoptosis in primary rat hepatocytes. We also examine the mechanisms involved in the process. Exposure to oxidative stress is also a hallmark of cholestatic liver diseases. Patients with cholestatic liver disease are often treated with ursodeoxycholic acid (UDCA), but its protective mechanism of action has not been elucidated yet. Chapter 6 of this thesis reports the possible mechanisms behind the anti-apoptotic action of taurine-conjugated UDCA. In conclusion, this thesis describes various modes of cell death induced by ROS and provides tools to selectively interfere with this process. This knowledge may contribute to the development of novel therapies for liver disorders.

CHAPTER 2

General introduction

Chapter 2

12

Oxidative stress is the inappropriate exposure to reactive oxygen species (ROS) and results from the imbalance between prooxidants and antioxidants leading to cell damage (damage of lipids, proteins, carbohydrates and nucleic acids) and tissue injury. Oxidative stress is associated to many diseases such as diabetes, atherosclerosis, arthritis, neurodegenerative disorders, cancer and most liver diseases, including chronic viral hepatitis (1), alcoholic hepatitis, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH) (2) and chronic cholestasis. In liver diseases, excess of ROS can induce hepatocyte cell death by either apoptosis or necrosis, leading to liver injury and loss of liver function. Sources of Reactive Oxygen Species Reactive Oxygen Species (ROS) represents a variety of diverse species including superoxide anions (O2

.-), hydrogen peroxide (H2O2) and hydroxyl radicals (HO.). Some of these species (HO., O2

.-) are known as radicals (molecules containing unpaired electrons) and are extremely unstable, while others like hydrogen peroxide are freely diffusible, relatively stable and long-lived. ROS are produced during normal intracellular metabolism (endogenously) and from exogenous substances. Endogenous sources include mitochondria, xanthine oxidase, cytochrome P450 metabolism, peroxisomes and inflammatory cell activation (macrophages, neutrophils, eosinophils). Mitochondria The mitochondrial respiratory chain, located in the inner membrane, is a powerful source of ROS. Molecular oxygen can accept four electrons and the corresponding number of protons to generate two molecules of water. Mitochondria are the main consumers of oxygen in the cell. Most of the oxygen consumed during oxidative phosphorylation is converted to water. However a minor percentage (1-2%) generates superoxide anions which may damage the mitochondria and form ROS (3). ROS are produced in two major respiratory chain regions, complex I (NADH deshydrogenase) and complex III (ubiquinone-cytochrome c reductase) (4-6). Superoxide anions are formed on both sides of mitochondrial membranes and are efficiently detoxified to hydrogen peroxide by Cu/Zn-SOD (intermembrane space) and Mn-SOD (matrix). Hydrogen peroxide in turn is converted into water by catalase or glutathione peroxidase (7) (fig. 1). Xanthine oxidase (XO) Xanthine oxidase is a molybdenum and iron containing hydroxylating enzyme involved in the degradation of purine like nucleotides. This enzyme catalyzes the reaction of hypoxanthine to xanthine, forming superoxide anions, and of xanthine to uric acid, forming hydrogen peroxide (8) (fig.1). Cytochrome P450 metabolism Cytochrome P450 enzymes metabolize a variety of substrates such as fatty acids, cholesterol or bile acids and exogenous compounds like drugs (e.g. acetaminophen) and alcohol. The


13

underlying biochemical reactions consume oxygen whereby small amounts of ROS are generated. One type of cytochrome molecule that is especially active in producing ROS is known as CYP2E1. This is also the enzyme that coverts alcohol to acetaldehyde Peroxisomes under physiologic condition produce hydrogen peroxide and not superoxide anions. Peroxisomal oxidation of fatty acids has recently been recognised as a potentially important source of hydrogen peroxide production during prolonged starvation. Macrophages and neutrophils contain a group of enzymes called the NADPH oxidase complex. Activated macrophages initiate an increase in oxygen uptake that gives rise to a variety of ROS, including superoxide anions, nitric oxide and hydrogen peroxide (9) (fig.1). ROS can also be produced by exogenous substances, including environmental toxins, xenobiotics, radiation, ultraviolet light, metal ions and barbiturates. The antioxidant systems Antioxidant defences can be classified into enzymatic and non-enzymatic systems. Enzymatic antioxidant defences Superoxide dismutase (SOD) There are three isoforms of SOD with different subcellular localizations. Those containing copper and zinc (Cu/Zn-SOD; SOD1) are located in the cytosol and in the mitochondrial intermembrane space, the manganese containing SOD (SOD2) in the mitochondria and the extracellular form usually is located on the outside of plasma membrane interacting with matrix components. This enzyme catalyses the dismutation of superoxide to hydrogen peroxide and oxygen

2 O2

.- + 2 H+ H2O2 + O2SOD

2 O2.- + 2 H+ H2O2 + O2

SOD

Superoxide anions rapidly react with nitric oxide to form the potent oxidant peroxynitrite (10, 11).

O2.- + NO . → ONOO -

Peroxynitrite reacts with a number of important biological molecules including thiols (GSH), lipids, proteins and nucleic acids (12). In mitochondria, peroxynitrite inactivates MnSOD, thus increasing the amount of superoxide anions that are available to react with nitric oxide to form more peroxynitrite (13), which damages the mitochondria and disrupts ATP synthesis, leading to cellular death. Exposure to large amounts of peroxynitrite quickly leads to necrotic cell death, due to disruption of cellular metabolism and membrane integrity (14). Hydrogen peroxide is a weak oxidant and, unlike superoxide, can rapidly diffuse across cell membranes. In the presence of metal ions it can be converted into toxic hydroxyl radicals via Fenton chemistry:

Fe+2 + H2O2 → Fe+3 + OH + OH -

Chapter 2

14

Catalase This iron-containing enzyme is primary found in peroxisomes. It is predominantly concentrated in liver and it detoxifies hydrogen peroxide by catalyzing a reaction between two hydrogen peroxide molecules, resulting in the production of water and oxygen (fig. 1).

2 H2O2 2 H2O + O2CAT2 H2O2 2 H2O + O2CAT

Glutathione peroxidase system This system consists of several components including the enzymes glutathione peroxidase and glutathione reductase and the cofactors glutathione and NADPH. All components together efficiently remove hydrogen peroxide (fig. 1).

2 GSH + H2O2 GSSH + 2 H2O GPx

2 GSH + H2O2 GSSH + 2 H2O GPx

Nonenzymatic defences Glutathione (GSH) This tripeptide is present in high amounts in all cells, including hepatocytes (15, 16). It is synthesized in the cytosol, in a two-step energy consuming process, and distributed in different organelles, such as endoplasmic reticulum and mitochondria. In the mitochondria, GSH is mainly found in the reduced form. It detoxifies ROS produced in the mitochondrial electron transport chain, thus protecting the organelle. Mitochondrial GSH depletion may compromise mitochondrial function and sensitizes cells to diverse oxidant-induced toxicity, leading to cell death (17). Metal-binding proteins These proteins ensure that metals (iron and copper) are maintained in a nonreactive state and avoid formation of hydroxyl radicals. Transferrin and lactoferrin bind iron while albumin binds copper. Vitamins Vitamin C (ascorbate), E (α-tocopherol) and carotenoids (vitamin A precursor) exert their antioxidant action as free-radical scavengers. Tocopherols and flavonoids inhibit peroxidation by acting as chain-breaking peroxyl-radical scavengers (fig. 1). Furthermore, other substances such as bilirubin, melatonin and uric acid have been proposed to act as antioxidant systems.


15

O2.-

H2O2

.OH

SOD

Lipid peroxidation

Mitochondria

2 H2O + O2CAT GPx+ GSH GSSG + 2 H2O

Fe+2

XO Hypoxanthine + O2

DNA damage Protein damage

Transferrin

Vitamin E

O2 Kuppfer CellsNeutrophils

O2.-

H2O2

.OH

SOD

Lipid peroxidation

Mitochondria

2 H2O + O2CAT GPx+ GSH GSSG + 2 H2O

Fe+2

XO Hypoxanthine + O2

DNA damage Protein damage

Transferrin

Vitamin E

O2 Kuppfer CellsNeutrophils

Figure 1. Schematic summary of different sources of reactive oxygen species and ROS detoxification mechanisms.

Cell death and liver disease During acute and chronic liver diseases, hepatocytes are exposed to increased levels of oxidative stress, cytokines (e.g. Tumor necrosis factor-alpha (TNF-α), Interleukin-1-beta (IL-1β) and Interferon-gamma (IFN-γ)) and bile acids. Hepatocytes have highly efficient detoxification mechanisms and an enormous capacity to defend themselves against these agents, however, over-exposure to high levels of ROS perturbs the normal redox balance and shift cells into a state of oxidative stress, resulting in cell death. Under oxidative stress conditions, the mode of cell death is primarily dependent on the variety of ROS, the cell type and the specific condition of ATP content in the cell. Therefore, it is important to know the mechanisms leading to hepatocyte cell death, because a more complete understanding of the cellular response to oxidative stress could be of significant relevance to comprehend and treat liver diseases. Cell death can occur via two different pathways, necrosis and apoptosis. However, features characteristic of both necrotic and apoptotic cell death often occur in the same tissue and even the same cell simultaneously (18). Necrosis is passive and associated with ATP depletion, rupture of the plasma membrane and spilling of the cellular content eliciting inflammation (19). In contrast, apoptosis, or programmed cell death (19-21), is an active process characterized by cell shrinkage, chromatin condensation, formation of apoptotic bodies (small membrane-surrounded cell fragments) and activation of caspases (22-24). Apoptosis represents a regulated form of cell death and it is important in processes such as cell selection during development, immunologic responses and homeostasis. Deregulated apoptosis contributes to pathologic states. The strict regulation of apoptotic cell death allows therapeutic intervention strategies. Therefore, the aim of this thesis focuses on the mechanisms of cell death induced by oxidative stress in normal primary hepatocytes, and in addition, we describe different ways to interfere with this process.

Chapter 2

16

Death receptor-mediated apoptosis Activation of death receptors on the cell membrane, that belong to the tumor necrosis/nerve growth factor (TNF/NGF) receptor super family, can initiate apoptotic cell death. Hepatocytes express Fas (CD95), but not Fas Ligand, preventing them from killing their neighbours. The expression of Fas is markedly increased in the livers of patients with non-alcoholic steatohepatitis (NASH) (25) or in fat-laden mouse hepatocytes (26). Furthermore, several other conditions leading to increased ROS production (drug/alcohol abuse/Wilson’s disease) cause Fas Ligand expression by hepatocytes, leading to apoptosis (27). Hepatocytes also express Tumour necrosis factor Related Apoptosis Inducing Ligand-receptor (TRAIL-R1), TRAIL receptor-2 (TRAIL-R2) and tumour necrosis factor-receptor type-1 (TNF-R1). Death receptors are type-1 transmembrane proteins with an extracellular ligand-binding N-terminal region, a membrane spanning region and a C-terminal intracellular tail. The extracellular region contains cysteine-rich domains, whereas the intracellular region contains the death domain essential for signalling apoptosis. Unlike Fas or TRAIL-R1 and TRAIL-R2, TNF-R1-mediated intracellular signalling is more complex as it activates both apoptotic and survival signals. ROS causes an increased synthesis of TNF-α. It has been described that patients with NASH have both high hepatic TNF-α mRNA levels and high TNF-R1 expression (28). In the liver, inflammatory cells, cholangiocytes and Kupffer cells are the main sources of TNF-α. Upon activation by TNF-α, trimerization of TNF-R1 is followed by recruitment of the adaptor protein TRADD (TNF-Receptor Associated protein with Death Domain). TRADD recruits signalling proteins like FADD (Fas Associated Death Domain) and it is also capable of activating survival pathways like Nuclear Factor kappa B (NF-κB) and Mitogen-activated protein kinases (MAPKs)(fig. 2). FADD contains a death effector domain, which, through death-inducing signalling complex (DISC), mediates the recruitment of cysteine aspartyl-specific proteases (caspases), such as caspase-8 and caspase-10 that activates the death signalling cascade. Active caspase is a heterotetrameric enzyme and consists of two large and two small subunits with two active sites per molecule. Caspases cleave their substrates at aspartic acid (Asp) residues in the context of tetrapeptide motifs. Active caspase-8 is involved in the cleavage and activation of effector caspase-3, which is considered one of the central executioner molecules and is responsible for cleaving various proteins thereby disabling important cellular structural and repair processes. Mitochondria play a crucial role in controlling apoptotic cell death, particularly in hepatocytes. Hepatocytes are type II cells in which only a small amount of active caspase-8 is formed at the DISC. Therefore, a mitochondrial amplification loop is essential to induce apoptotic cell death in hepatocytes. Knowledge of mitochondria-controlled apoptosis is needed to find suitable intervention targets for liver diseases.


17

FADDCaspase-8

Caspase-3

APOPTOSIS

Death Receptor activation (TNF-R, Fas-R)

Caspase-9ROS

Plasma membrane

Oxidative stressGrowth Factors

Survival genes

nucleusNF-κB

NF-κB

IκB

p38

MAPK

ERK

PI3-K

AktJNK

Cyt c

BID

tBID

FADDCaspase-8

Caspase-3

APOPTOSIS

Death Receptor activation (TNF-R, Fas-R)

Caspase-9ROS

Plasma membrane

Oxidative stressGrowth Factors

Survival genes

nucleusNF-κB

NF-κB

IκB

p38

MAPK

ERK

PI3-K

AktJNKJNK

Cyt c

BID

tBID

Figure 2. Schematic summary of Death Receptor-mediated and Mitochondria-mediated signal transduction pathways. TNF-R1-mediated intracellular signalling activates both apoptotic and survival signals.

Mitochondria-mediated apoptosis The activation of a death receptor, e.g. by ROS, activates caspase-8, but this is not sufficient in type II cells and an amplification of the apoptotic signal is needed to trigger apoptosis. In this type of cells, active caspase-8 cleaves the protein BH3 interacting domain death agonist (Bid) into a truncated form. Truncated Bid (tBid) can enter the outer mitochondrial membrane to disrupt its permeability. This phenomenon is known as the mitochondria permeability transition (MPT). However, the main effect of tBid is to induce a conformational change in Bcl-2-associated X protein (Bax). Bax associates with Bak and the complex translocates to the outer mitochondrial membrane to form channels in this membrane (29). The increased outer membrane permeability allows the release of proteins, such as cytochrome c from the intermembrane space into the cytoplasm. This event abolishes the flow of electrons in the respiratory chain, blocks ATP production and increases mitochondrial ROS production (30). ROS can then act on the inner membrane of the same or other mitochondria to open a pore, called the mitochondrial permeability transition pore (MPTP). Pore opening induces matrix expansion and outer membrane rupture (31), which causes the release of cytochrome c and other proapoptotic proteins from the intermembrane space to the cytosol. Once in the cytosol, cytochrome c complexes with apoptosis protein-activating factor 1 (Apaf-1), dATP and cytosolic pro-caspase-9 to form a caspase-activating complex known as the apoptosome (32-36). Cytochrome c and dATP induce refolding of Apaf-1, which allows interaction with pro-caspase-9. In this way, caspase-9 is activated, which subsequently cleaves and activates caspase-3 (31). Depending on cell type and stimulus, caspase-3 can be involved in the activation of procaspase-8, procaspase-6, procaspase-9 and Bid, that results in a feedback amplification of the apoptotic signal (37, 38). Other mitochondrial apoptogenic factors that are released from the intermembrane space are Smac/DIABLO and Omi/HtrA2. Proteolytic processing in the mitochondria is required by these proteins to become active (39, 40). The release of Smac/DIABLO requires active caspases, occurs downstream of cytochrome c translocation and can be controlled by Bax (41, 42). In addition, Smac/DIABLO sequesters members of the Inhibitor of Apoptosis Family (IAP family) such as XIAP, cIAP1 and cIAP2, which has been shown to bind the activated

Chapter 2

18

forms of caspase-3 and -9, blocking apoptosome-mediated caspase activation (43). This results in the release of active caspases and the propagation of caspase cascades (44, 45). Omi/HtrA2 exerts a similar function although it also contributes to caspase-independent apoptosis due to its N-terminal serine protease catalytic domain (46-48). Anti-apoptotic members like Bcl-2, Bcl-XL and A1/Bfl-1, and pro-apoptotic Bak are integral membrane proteins that are predominantly present in the outer mitochondrial membrane, but may also be present in other organellar membranes. In contrast, pro-apoptotic members like Bax, Bid and Bad are sequestered in the cytosol prior to a death signal (49). In the mitochondrial membrane, Bad interacts with and antagonises anti-apoptotic Bcl-2 and/or Bcl-XL (50). The anti-apoptotic Bcl-2 family members inhibit apoptosis by binding Bax and Bak, sequestering tBid and Apaf-1, and preventing mitochondrial release of cytochrome c and Smac/DIABLO (51-54). ROS can also damage the mitochondria directly, by oxidizing mtDNA, proteins and lipids or by further increasing lipid peroxidation-induced damage of mitochondria. It has been described that increased permeability of the mitochondrial membranes can also lead to necrosis. The decision between necrotic and apoptotic cell death may depend on the cellular concentration of ATP. Thus, the mitochondrial permeability transition (MPT) mediates both necrosis and apoptosis, but when the MPT affects most mitochondria, a marked depletion of ATP can develop, leading to necrotic cell death. When the MPT occurs without severe ATP depletion, apoptosis develops, which may be followed by secondary necrosis if ATP is eventually depleted (55). The balance between pro-apoptotic and anti-apoptotic pathways determines the outcome of cell death upon a stimulus. As long as protective proteins are available, the balance will be in favour of cell survival. Thus, understanding of cell survival mechanisms is essential to find therapeutic interventions for liver diseases associated with oxidative stress. Survival pathways in liver diseases Among the main signalling pathways that are activated in response to oxidant injury are the mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinase (ERK1/2), c-jun amino-terminal kinase (JNK) and p38 MAPK, the phosphoinositide 3-kinase (PI3K)/Akt pathway, the nuclear factor kappa B (NF-κB) signalling system and the enzyme heme oxygenase. In general, heme oxygenase, ERK1/2 and p38 MAPK, PI3K and NF-κB signalling pathways are considered to be pro-survival during oxidative stress-induced injury, whereas activation of JNK MAPK is usually linked to apoptosis. Hence, ROS-induced activation of survival pathways may attenuate their toxicity. Nuclear factor kappa B (NF-κB) signalling pathway NF-κB is an important regulator of the balance between cell survival and cell death. It is an ubiquitous heterodimeric transcription factor that is sequestered in the cytoplasm by proteins of the IκB family (56). IκB is regulated by a protein complex that includes two kinases IKKα and IKKβ, both capable of phosphorylating IκB and a regulatory subunit IKK gamma


19

(NEMO). Phosphorylation and degradation of IκB frees NF-κB and exposes a nuclear localization sequence, leading to the translocation of NF-κB to the nucleus. Once in the nucleus, NF-κB binds to κB binding sites in promoters of target genes, inducing their transcription. NF-κB is activated by inflammatory cytokines, such as TNF-α and IL-1β, oxidative stress (57), endotoxin (LPS) (58), protein kinase C (PKC) and phosphatidylinositol-3 (PI3K). NF-κB signalling pathway has been described to antagonise hepatocyte cell death by influencing the balance between pro- and anti-apoptotic signals. It has been postulated that NF-κB inhibits TNF-α-induced accumulation of ROS that normally mediate prolonged c-Jun N-terminal kinase (JNK) activation and cell death (59). Indeed, inhibition of NF-κB activity induces apoptosis in hepatocytes, suggesting its role in the transcription of anti-apoptotic genes (60). Mitogen-activated protein kinases (MAPKs) signalling pathways The MAPK cascade includes a mitogen-activated protein kinase kinase kinase (MAPKKK), mitogen-activated protein kinase kinase (MAPKK) and MAPK. In the large MAPK family three subgroups have been identified: the c-Jun N-terminal kinase (JNK), p38 MAPK and the extracellular signal-regulated kinase (ERK1/2), which have been shown to be activated by oxidative stress and affect cell survival. ERK1/2 and p38 MAPK have been associated to cell survival, whereas JNK has been linked to cell death. The balance between ERK1/2, p38 and JNK activation is crucial in the determination between cell death and cell survival (fig. 3). It has been described that inhibition of JNK activation, using specific inhibitors or dominant-negative mutants for JNK, suppresses apoptosis. Several studies have shown the important role of JNK in signal transduction of oxidative stress-induced apoptotic cell death. Furthermore, many studies have described an effect of JNK at the level of the mitochondria triggering the mitochondrial death pathway, including phosphorylation and activation of pro-apoptotic bcl-2 family members and cytochrome c release (61-67). In this thesis the role of MAPK signalling pathways, in cell survival and cell death, has been studied in detail using specific inhibitors.

P

Grb

cytoplasm

nucleus

E-Box

p90RSK cMyc ELK1SRE

MLKs/TAK/ASK1

CRE

ATF-2

SAPK/JNK

SEK1/MKK4/7

AP-1

fosjun

SOS

c/B-RafMAPKKK

ERK1/2

MEK1/2MAPKK

MAPK

ASK1/MEKK1/4

CytokinesOxidative Stress

P

p38

MKK3/6

GF

PPPPP PPPP

Ras

P

GF

GrbSOSRas

Plasma membrane

P

Grb

cytoplasm

nucleus

E-Box

p90RSK cMyc ELK1SRESRE

MLKs/TAK/ASK1

CRE

ATF-2

SAPK/JNK

SEK1/MKK4/7

AP-1

fosjun

SOS

c/B-RafMAPKKK

ERK1/2

MEK1/2MAPKK

MAPK

ASK1/MEKK1/4

CytokinesOxidative Stress

P

p38

MKK3/6

GF

PPPPP PPPP

Ras

P

GF

GrbSOSRas

Plasma membrane

Figure 3. Schematic overview of the MAPK signalling pathways. See text for details.

Chapter 2

20

Phosphoinositide 3-kinase (PI3K)/Akt pathway signalling

The PI3-kinase family is a superfamily subdivided in three different classes of enzymes that are linked to different cellular functions, including cell survival (FIG). Class I enzymes have been characterised and subdivided into two groups of PI3-kinases, class IA and IB. The catalytic subunit of class IA interacts with adaptor proteins and is involved in activation by growth factor receptors (e.g. the epidermal growth factor receptor: EGF-R), while class IB is required for G-protein-coupled receptor systems (68). Class I PI3-kinase reside mainly in the cytosol until recruited into active signalling complexes at the plasma membrane, where they are involved in the generation of 3’-phosphorylated phosphoinositides, that function as signalling intermediates in signal transduction cascades. Targets of PI3K, such as the serine kinase Akt, also known as protein kinase B, have been associated with the inhibition of apoptosis in a variety of ways (69, 70). The PI3K/Akt pathway transduces survival signals through phosphorylation processes and regulates pro- and anti-apoptotic factors, such as BAD, caspase-9, and IKKα. It has been reported that Akt activates ERK1/2, NF-κB and inhibits JNK and Bax phosphorylation and thus protects against mitochondria disruption and apoptosis (71, 72). In these studies a cross talk between pro- and anti-apoptotic signalling pathways is described, such as PI3K/Akt and JNK MAPK pathways, modulating the balance between cell survival and cell death. Src family signalling Src-family protein-tyrosine kinases are intermediate regulatory proteins that play important roles in differentiation, motility, proliferation and survival. Src activates the anti-apoptotic PI3K/Akt pathway in human colon tumor cell lines (73). In addition, Src increases Bcl-xl expression in rat intestinal epithelial cells (74). Heme oxygenase Heme oxygenase (HO) catalyzes the oxidation of heme to form equimolar amounts of ferrous iron, carbon monoxide (CO) and biliverdin, which is rapidly converted into bilirubin by NAD(P)H:biliverdin reductase. Three different isoforms of HO have been described (75). These isozymes are products of different genes and differ in their tissue distribution and molecular properties. The HO-2 isoform is constitutively expressed and is present in high levels in brain and testes. HO-3 has a catalytic activity and function as a heme-binding protein. In contrast, HO-1 is ubiquitously distributed and highly inducible by a variety of different stimuli, most of them associated with oxidative stress (76). HO-1 may act as an inducible defence system against oxidative stress, e.g. in models of inflammation, ischemia-reperfusion, hypoxia and hyperoxia-mediated injury (77). In the liver, HO-1 induction protects against ischemia/reperfusion injury (78, 79) and endotoxemia (80, 81). In addition, overexpression of HO-1 by gene transfer has been shown to protect against hyperoxia induced by lung injury (82) and from immune-mediated apoptotic liver damage in mice (83). However, the mechanisms by which HO-1 mediates cytoprotection have not been elucidated yet. Protective effects of both biliverdin and CO have been reported and several studies suggest that biliverdin protects against oxidative stress by acting as an anti-oxidant in different models of liver injury (84, 85). Although there has been a lot of recent interest in the


21

role of carbon monoxide as signalling molecule (86, 87), little is known about the biological actions of CO. In this thesis, we investigate the regulation of HO-1 in vivo (model of acute inflammation and chronic cholestasis) and in vitro in primary cultures of rat hepatocytes. In addition, we examine whether HO-1 and its product CO have a protective role against oxidative stress-induced apoptosis in primary hepatocytes. Clinical relevance: oxidative stress in human liver disorders Most chronic liver diseases are accompanied by oxidative stress. This induces liver injury and loss of liver function. Non-alcoholic fatty liver diseases (NAFLD), also known as hepatic steatosis or fatty liver, refer to the clinical condition of accumulation of fat in the liver. This is associated with insulin resistance and usually is accompanied by obesity and diabetes. Insulin resistance increases lipolysis of adipose tissue, resulting in increased influx of free fatty acids to the liver. In addition, insulin resistance induces de novo triglyceride synthesis in the liver and inhibits fatty acid oxidation, promoting triglyceride accumulation. Insulin also influences glucose metabolism. Insulin induces storage of glucose in the liver, inhibits hepatic glucose production and stimulates uptake of glucose by muscle and adipose tissue. Due to oxidative stress and lipid peroxidation, hepatic steatosis can cause inflammation and hepatocellular damage, leading to non-alcoholic steatohepatitis (NASH), which can progress to fibrosis (88). NAFLD can progress to cirrhosis and hepatocellular carcinoma (89). Strategies to treat NAFLD aim to improve insulin resistance and protect the liver from oxidative stress-induced secondary injury. The pathogenesis of NASH remains unclear, although it has been proposed that the excessive intrahepatic lipid accumulation and production of free radicals result in membrane and DNA damage (90-92). Oxidative stress has been identified as a feature of experimental models of fibrosis and cirrhosis (bile duct ligation; CCl4-intoxication). The oxidative stress responsive gene HO-1 is induced in many conditions of liver fibrosis and cirrhosis, both experimental and human (93). Antioxidants have been shown in some studies to be partially beneficial in the treatment of fibrosis and/or cirrhosis (94). Accumulation of bile acids in the liver during cholestasis stimulate ROS production and apoptosis, which can be prevented by antioxidants and TUDCA (95). Additionally, ROS play an important role in the pathogenesis of cholestasis as a consequence of biliary obstruction (96-99). Many studies have reported a beneficial effect of antioxidants in cholestasis, for example, N-acetylcysteine in biliary obstructed rats (100), melatonin prevents oxidative stress and hepatocytes cell death in cholestasis (101) and vitamin E (102) or resveratrol (103). It has been described that cholestasis causes liver injury by mechanisms involving mitochondrial oxidative stress. Gene delivery of mitochondrial MnSOD blocks formation of oxygen radicals and reduces liver injury induced by cholestasis (104). In addition, UDCA partially prevents hepatic and mitochondrial glutathione depletion and oxidation resulting from chronic cholestasis. Thus, UDCA treatment enhances the antioxidant defence mediated by glutathione and prevents cell injury in animal with secondary biliary cirrhosis (105). Additionally,

Chapter 2

22

obstructive cholestasis during pregnancy causes oxidative stress and apoptosis in rat placenta, which can be prevented by treatment with UDCA. Hepatitis C virus infection is characterized by oxidative stress caused by chronic inflammation, and causes hepatitis that can progress to fibrosis, steatosis and liver failure (106). Oxidative stress has been implicated in the pathogenesis of acute liver failure, which represents a serious clinical problem associated with high morbidity and mortality and very limited treatment options. HO-1, an enzyme induced by oxidative stress, has been shown to be induced in hepatocytes of acute liver failure patients (107). Additionally, the antioxidant N-acetylcysteine, decreases oxidative stress, hepatocytes necrosis and acute hepatic failure induced by carbon tetrachloride in rats (108). In conclusion, under oxidative stress conditions, the mode of cell death is dependent on the variety of ROS and the cell type. Since in chronic liver diseases hepatocytes are exposed to various ROS, it is of clinical importance to elucidate the mode and mechanism of cell death in hepatocytes exposed to oxidative stress. This may provide tools that may develop into future therapeutic targets for liver disorders. Reference List 1. Okuda M, Li K, Beard MR, Showalter LA, Scholle F, Lemon SM, et al. Mitochondrial injury, oxidative

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CHAPTER 3

Superoxide anions and hydrogen peroxide induce

hepatocyte death by different mechanisms:

involvement of JNK and ERK MAP kinases.

Laura Conde de la Rosa1, Marieke H. Schoemaker1, Titia E.Vrenken1, Manon

Buist-Homan1, Rick Havinga2, Peter L.M. Jansen3, Han Moshage.

1Center for Liver, Digestive and Metabolic Diseases, University Medical Center Groningen,

Groningen, The Netherlands, 2Laboratory of Pediatrics, University Medical Center

Groningen, 3Department of Gastroenterology and Hepatology, Academic Medical Center

Amsterdam, The Netherlands.

Journal of Hepatology 44 (2006) 918-929

Chapter 3

28

Abstract. In liver diseases, reactive oxygen species (ROS) are involved in cell death and liver injury, but the mechanisms are not completely elucidated. Aim: To elucidate the mechanisms of hepatocyte cell death induced by the ROS superoxide anions and hydrogen peroxide. Primary cultures of hepatocytes were exposed to the superoxide anion donor menadione (10-50µmol/L) or H2O2 (1-5 mmol/L). Hepatocytes were also treated with caspases and MAPKs inhibitors, superoxide dismutase (PEG-SOD) and SNAP, a nitric oxide donor. Apoptosis was determined by measuring caspase-9, -6, -3 activation and cleaved PARP, and necrotic cell death by Sytox Green staining. Results: 1) Menadione (50µmol/L) induces JNK phosphorylation, caspase-9, -6, -3 activation, PARP cleavage and apoptosis. Superoxide anions-induced apoptosis is dependent on JNK activity. Menadione (50µmol/L) induces the phosphorylation of ERK1/2 and this attenuates cell death. 2) H2O2 increases necrotic cell death at high concentration or when H2O2 detoxification is impaired. H2O2 does not activate MAPKs signalling. 3) PEG-SOD prevents ERK1/2-, JNK- phosphorylation, caspase activation and apoptosis induced by menadione. Glutathione depletion increases menadione-induced apoptosis. 4) SNAP abolishes menadione-induced apoptosis but increases necrotic cell death. Conclusions: in normal hepatocytes, superoxide anions-induced caspase activation and apoptosis is dependent on JNK activity and totally abolished by superoxide scavengers.

ROS-induced cell death in primary hepatocytes

29

Introduction

Reactive oxygen species (ROS) such as superoxide anions (O2.-

), hydrogen peroxide (H2O2)

and hydroxyl radicals (HO.) are produced during normal intracellular metabolism, e.g. mitochondrial metabolism (1;2), and the action of P450 cytochromes (3) and superoxide dismutases (4) and by inflammatory cells. Oxidative stress is the inappropriate exposure to ROS and results from the imbalance between prooxidants and antioxidants leading to cell damage and tissue injury. ROS generation is increased in many pathological situations (5-7), including chronic viral hepatitis (8), alcoholic hepatitis, non-alcoholic fatty liver disease (NAFLD) (9) and chronic cholestasis. In liver diseases, excess of ROS can induce cell death by either apoptosis or necrosis. Apoptosis, or programmed cell death (10-12), is an active process characterized by cell shrinkage, chromatin condensation, formation of apoptotic bodies and activation of caspases (13-15). In contrast, necrosis is passive and associated with ATP depletion, rupture of the plasma membrane and spilling of the cellular content eliciting inflammation (12). Hepatocytes are equipped with several enzymatic antioxidant defences, including superoxide dismutases, that converts superoxide anions into H2O2; catalase and glutathione peroxidase (GPx) that decompose H2O2 into water (16). In addition, a variety of non-enzymatic antioxidant defences exist, including glutathione (17;18) and vitamins A, C and E. Finally, ROS-induced activation of survival pathways such as MAPK (19) and NF-κB, may attenuate their toxicity. The mode of oxidative stress-induced cell death is dependent on the variety of ROS and the cell type. Many studies have investigated the role of ROS on cell death in transformed hepatoma cells, although it is known that cell lines differ significantly from normal hepatocytes in many respects and in primary hepatocytes (20;21), however without making a distinction between necrotic and apoptotic cell death. Since in chronic liver diseases non-transformed hepatocytes are exposed to various ROS, it is of clinical importance to elucidate the mode and mechanism of cell death in normal hepatocytes. The aim of this study was to elucidate and compare the mechanisms of cell death induced by superoxide anions and H2O2 in normal, non-transformed hepatocytes. Furthermore, the role of MAPK signalling pathways, ROS detoxification mechanisms and different ROS scavengers were investigated in ROS-induced cell death. Materials and Methods Animals. Specified pathogen-free male Wistar rats (220-250 g) were purchased from Harlan (Zeist, The Netherlands). They were housed under standard laboratory conditions with free access to standard laboratory chow and water. Experiments were performed following the guidelines of the local Committee for Care and Use of laboratory animals. Rat hepatocyte isolation. Hepatocytes were isolated as described previously (22) and cultured in William’s E medium (Life Technologies Ltd; Breda, The Netherlands) supplemented with 50µg/mL gentamicin

Chapter 3

30

(BioWhittaker, Verviers, Belgium) without the addition of hormones or growth factors. During the attachment period (4hrs) 50nmol/L dexamethasone (Sigma, St Louis, USA) and 5% fetal calf serum (Life Technologies Ltd.) were added to the medium. Cells were cultured in a humidified incubator at 37°C and 5% CO2. Hepatocyte viability was always more than 93% and purity more than 95% as determined by trypan blue staining. Experimental design. Experiments were started 24hrs after isolation of hepatocytes. Monolayer cultures were exposed to the commonly used intracellular superoxide anions-donors menadione (2-methyl-1,4-naphthoquinone, Sigma) (23) and paraquat (50mmol/L, Sigma); and the extracellular superoxide generator hypoxanthine (0.5mmol/L) / xanthine oxidase (20µU/ml) (HX/XO, Roche Diagnostics), or H2O2 (MERCK, Haarlem, The Netherlands) for the indicated time. Hepatocytes were also treated with caspase-9 inhibitor (Z-LEHD-FMK, 0.2µmol/L), caspase-6 inhibitor (Z-VEID-FMK, 0.2µmol/L) and caspase-3 inhibitor (Z-DEVD-FMK, 0.05µmol/L), all obtained from R & D Systems, Abingdon, UK; and tauroursodeoxycholic acid (TUDCA, 50µmol/L). Depletion of intracellular reduced glutathione, was achieved using D,L-buthionine-(S,R)-sulfoximine (BSO, 200µmol/L, Sigma). Glutathione methylester (GSH-MEE; 5mmol/L; Calbiochem) was used as a donor of glutathione. Glutathione assay was performed as describe previously (24). Signal transduction pathways were blocked using MEK inhibitor U0126 at 10 µmol/L to inhibit activation of ERK1/2 (Promega, Madison, USA), p38 inhibitor (SB203580, 10µmol/L) (Biomol, Plymouth Meeting, USA) and JNK inhibitor (SP600125, 10µmol/L, MERCK). H2O2 detoxification was inhibiting using the catalase inhibitor 3-amino-1,2,4-triazole (3-AT; 20mmol/L) and the GPx inhibitor mercaptosuccinic acid (MS; 10mmol/L, Sigma). All inhibitors were added 30 minutes before the addition of menadione or H2O2. Recombinant, replication-deficient adenovirus Ad5IκBAA was used to inhibit NF-κB activation as described previously and Ad5LacZ was used as control virus (25). Hepatocytes were exposed to adenoviruses at a multiplicity of infection of 10, 15 hours before exposure to superoxide anions or cytokine mixture (26). Each experimental condition was performed in triplicate wells. Each experiment was performed, at least three times, using hepatocytes from different isolations. Caspase enzyme activity assays. Caspase-3 enzyme activity was assayed as described previously (27). Caspase-6 activity was assayed according to the manufacturer’s instructions (BioVision). Sytox Green and acridine orange nuclear stainings. To estimate necrotic cell death, hepatocytes were incubated 15 minutes with SYTOX green (Molecular Probes, Eugene, USA) nucleic acid stain, which penetrates cells with compromised plasma membranes but does not cross the membranes of viable cells or apoptotic bodies. Fluorescent nuclei were visualized using an Olympus CKX41 microscope at 450-490nm. Morphology of nuclei was examined with acridine orange as described previously (27).


31

Western Blot analysis. Western blot analysis of cell lysates was performed by SDS-PAGE followed by transfer to Hybond ECL nitrocellulose membrane (Amersham). An antibody against GAPDH (Calbiochem) and Ponceau S staining were used to ensure equal protein loading and electrophoretic transfer. Caspase cleavage was detected using polyclonal rabbit antibodies against cleaved caspase-9, -6, and -3. Poly(ADP-ribose) polymerase (PARP) cleavage was detected with rabbit anti-PARP polyclonal antibody. PARP is a substrate of caspase-3 yielding a product of 89kDa and it is considered a late marker for apoptosis. After Western blot analysis for p-p38 (rabbit polyclonal), p-ERK1/2 (monoclonal), and p-JNK (monoclonal, Santa Cruz Biotechnology), blots were stripped using 0.1 % SDS in PBS/Tween at 65°C for 30 minutes and incubated with antibodies against total-p38, total-ERK1/2 or total-JNK. Unless indicated otherwise, all antibodies were obtained from Cell Signalling Technology, and used at 1:1000 dilution. Statistical analysis. All numerical results are reported as the mean of at least 3 independent experiments ± standard deviation. A Mann-Whitney test was used to determine the significance of differences between experimental groups. Statistical difference was accepted at P<0.05. Results Superoxide anions, but not hydrogen peroxide, induce apoptosis in primary rat hepatocytes. To investigate whether ROS induce apoptotic cell death in primary rat hepatocytes, cells were exposed to superoxide anion donors or H2O2. Menadione increases caspase-3 activity, peaking between 9 and 12hrs, and leads to PARP cleavage (Fig. 1A, B). Based on caspase-3 staining and nuclear morphology we estimate that at least 90% of cells were apoptotic. Lower concentrations of menadione did not induce caspase-3 activity, PARP cleavage or nuclear condensation (Fig. 2A, C).

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H2O21 mM kDa

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controlH2O2 (1mM)Menadione 50µ M

Cleaved PARP

Control

89

A

B Men 50 µM

H2O21 mM kDa

GAPDH36

Figure 1. The superoxide anion-donor menadione, but not hydrogen peroxide, induces caspase-3 activity and PARP-cleavage in primary rat hepatocytes. Cells were exposed to menadione (Men, 50µmol/L) or hydrogen peroxide (H2O2, 1 mmol/L) for different time points. Controls: untreated cells. A) Caspase-3 activity assay. B) Western blot analysis against cleaved PARP (89 kDa) and GAPDH. Data represent mean of at least 3 independent experiments with n=3 per condition.

Chapter 3

32

To verify these findings, we used paraquat and HX/XO as alternative superoxide-generators to reproduce the results obtained with menadione. Both paraquat and HX/XO induced caspase-3 activation and PARP cleavage and apoptosis in a time dependent manner, peaking at 4 and 9hrs respectively (Fig. 2B). In contrast, H2O2 is not apoptotic in primary hepatocytes at concentrations up to 5mmol/L (Fig. 2C, D). Of note, caspase-3 activity always correlated with the level of cleaved caspase-3 and cleaved PARP.

05000

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4 hrs 6 hrs 9 hrs

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ConParaquat50mM HX/XO

GAPDH

kDaAControl

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Cleaved PARP

89

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Control

B

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4 hrs 6 hrs 9 hrs



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ConParaquat50mM HX/XO

GAPDH

kDaAControl

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36 GAPDH

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Control

B

Control

Menadione25 µmol/L

Menadione10 µmol/L

Menadione30 µmol/L

Menadione50 µmol/L H2O2 C Control

Menadione25 µmol/L

Menadione10 µmol/L

Menadione30 µmol/L

Menadione50 µmol/L H2O2 Control

Menadione25 µmol/L

Menadione10 µmol/L

Menadione30 µmol/L

Menadione50 µmol/L H2O2 H2O2 C

Figure 2. A) Menadione induces caspase-3 activity and PARP cleavage only at 50µmol/L. Hepatocytes were incubated or not (controls) with different concentrations of menadione (Men) for 9hrs. B) Caspase-3 activity and Western blot. Paraquat (50mmol/L) and hypoxanthine (0.5mmol/L) /xanthine oxidase (20µU/ml) (HX/XO) induce apoptosis, PARP and caspase-3 cleavage peaking at 4 and 9 hrs respectively. C) Menadione, but not hydrogen peroxide, induces nuclear condensation (apoptosis indicator), as determined by acridine orange staining, only at 50µmol/L. Hepatocytes were treated with different concentrations of menadione (9hrs) or H2O2 (1-4mmol/L, 6hrs). Original magnification: 1000x. D) H2O2 does not induce caspase-3 activity in primary hepatocytes at concentrations up to 5mmol/L.

Menadione (50µmol/L) induced only minimal necrotic cell death as determined by Sytox Green staining (Fig. 3A). At concentrations above 100µmol/L menadione, all hepatocytes became necrotic and detached. H2O2 at 1 mmol/L did not induce necrotic cell death but becomes necrotic above 5mmol/L (Fig. 3B).

0

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Con H2O2 1mM

H2O22.5mM

H2O24mM

H2O25mM

casp

ase-

3 ac

tivity

(AFU

)

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5000

10000

15000

20000

Con H2O2 1mM

H2O22.5mM

H2O24mM

H2O25mM

casp

ase-

3 ac

tivity

(AFU

)

D


33

Menadione

± 5% necrotic nuclei

Control

± 1%necrotic nuclei

>95% necrotic nuclei

Positive controlMenadione


Control


>95% necrotic nuclei

Positive control

H2O2 1 mM H2O2 2.5 mM H2O2 5 mMControl





H2O2 1 mM H2O2 2.5 mM H2O2 5 mMControl





Figure 3. Sytox Green nuclear staining of necrotic cells. A) Menadione (Men, 50µmol/L, 9hrs) slightly increases necrotic cell death in primary hepatocytes (magnification: 40x). B) H2O2 induces necrotic cell death only at high concentrations. Hepatocytes were exposed to increasing concentrations of H2O2 (1-5mmol/L, 6hrs). Upper panel phase contrast and fluorescence; lower panel fluorescence only. Magnification: 40x.

To further investigate whether H2O2 plays a role in cell death, experiments were performed in the presence of inhibitors of the H2O2 detoxification enzymes. In the presence of the catalase inhibitor 3-AT or the GPx inhibitor MS, caspase-3 activity level was only slightly increased (Fig. 4A), whereas necrotic cell death was highly increased when catalase was inhibited and only slightly increased when GPx was inhibited (Fig. 4B). In contrast, 3-AT treatment had no effect on menadione-induced apoptosis or necrotic cell death (Fig. 4C). Taken together, these observations indicate that 50µmol/L menadione induces apoptosis in normal primary rat hepatocytes, whereas H2O2 causes necrotic cell death at high concentrations, or when H2O2 detoxification mechanisms are blocked.

A

B

Chapter 3

34

Con H2O2 H2O2+MS

H2O2+3AT

H2O2+3AT+MS

3AT 3AT+MS

MSca

spas

e-3

activ

ity (A

FU

)

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Con H2O2 H2O2+MS

H2O2+3AT

H2O2+3AT+MS

3AT 3AT+MS

MSca

spas

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activ

ity (A

FU

)

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H2O2Control H2O2+ MS H2O2+ 3AT H2O2+ 3AT+MS

3AT MS MS+ 3AT



±20% necrotic nuclei


±100% Necrotic nuclei

H2O2Control H2O2+ MS H2O2+ 3AT H2O2+ 3AT+MS

3AT MS MS+ 3AT





±100% Necrotic nuclei

05000

100001500020000250003000035000

Con Men Men + 3ATcasp

ase-

3 ac

tivity

(AF

U)

control Menadione Men + 3AT 3AT

3AT





05000

100001500020000250003000035000

Con Men Men + 3ATcasp

ase-

3 ac

tivity

(AF

U)

control Menadione Men + 3AT 3AT

3AT





Figure 4. A) Hydrogen peroxide (H2O2) slightly increases caspase-3 activity in primary hepatocytes when H2O2 detoxification is impaired. Cells were exposed to H2O2 (1mmol/L) for 6hrs in the presence of the catalase inhibitor 3-AT and/or GPx inhibitor MS. B) H2O2 (1mmol/L) induces massive necrotic cell death in hepatocytes with impaired H2O2 detoxification, as determined by Sytox Green nuclear staining. Magnification: 40x. C) The catalase inhibitor 3-AT does not affect menadione-induced apoptosis (upper graph) or necrotic cell death (lower pictures). Upper panels phase contrast and fluorescence; lower panels fluorescence only.

A

B

C


35

Superoxide anions-induced apoptosis is dependent on caspase activation. Since apoptosis is usually associated with activation of caspases, we analyzed whether superoxide anions-induced apoptosis is mediated by caspase activation. Cleaved caspase-9, -6, and -3 were detected 9hrs after treatment with 50µmol/L menadione, but not at lower concentrations. In contrast, H2O2 did not induce caspase-9, -6, or -3 activation in primary hepatocytes (Fig. 5A). Superoxide anions-induced apoptosis is completely dependent on caspase activation, since treatment with caspase-9, -6, and -3 inhibitors totally abolished superoxide anions-induced apoptosis (caspase-3 activity and PARP-cleavage) (Fig. 5B). Furthermore, caspase-9 inhibition blocked caspase-6 and -3 activation (Fig 5C), suggesting that caspase-9 is upstream of caspase-6 and -3, and that the mitochondria are involved in superoxide anions-induced apoptosis.

Cleaved caspase-9

Cleaved caspase-3

Cleaved caspase-6

ConMen

50 µMH2O2

(1mM)Men 25 µM kDa

38

17

18

36 GAPDH

36 GAPDH

Cleaved caspase-9

Cleaved caspase-3

Cleaved caspase-6

ConMen

50 µMH2O2

(1mM)Men 25 µM kDa

38

17

18

36 GAPDH

36 GAPDH

0

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Con Menadione 50 µM

casp-9

inh+

casp-6

inh+

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inh+

Cas

pase

-3 a

ctiv

ity (A

FU)

ConMen 50 µM

Men + c9 inh

Men + c6 inh

Men + c3 inh

Cleaved PARP89

kDa

GAPDH36

casp-9

inh

casp-6

inh

casp-3

inh

Cleaved caspase-3Con

Men 50 µM

Men + c9 inh

Cleaved caspase-6

kDa17

18

GAPDH36

GAPDH36

0

5000

10000

15000

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25000

Con Menadione 50 µM

casp-9

inh+

casp-6

inh+

casp-3

inh+

Cas

pase

-3 a

ctiv

ity (A

FU)

ConMen 50 µM

Men + c9 inh

Men + c6 inh

Men + c3 inh

Cleaved PARP89

kDa

GAPDH36

casp-9

inh

casp-6

inh

casp-3

inh

Cleaved caspase-3Con

Men 50 µM

Men + c9 inh

Cleaved caspase-6

kDa17

18

GAPDH36

GAPDH36

Figure 5. Superoxide anions-induced apoptosis is dependent on caspase activation. Cells were incubated with menadione (Men, 50 and 25µmol/L, 9hrs) or hydrogen peroxide (H2O2, 1 mmol/L, 6hrs). A) Western Blot analysis of caspase activation. Only 50µmol/L menadione, but not 25µmol/L menadione or H2O2, induces caspase-9, -6, and -3 activation. B) Addition of caspase-9, -6, and -3 inhibitors before and during menadione (50µmol/L) treatment, abolishes caspase-3 activity and PARP-cleavage. C) Caspase-9 inhibitor blocks caspase-6 and -3 cleavage.

A

C

B

Chapter 3

36

Involvement of MAP-kinase signal transduction pathways in superoxide anions-induced apoptosis. Menadione (50µmol/L) induced a marked increase in ERK1/2 phosphorylation within 2hrs and JNK phosphorylation within 1hr that persisted for 4hrs. In contrast, 25µmol/L menadione did not induce p-ERK1/2 and only slightly induced p-JNK, whereas H2O2 did not induce p-ERK1/2 or p-JNK. These results indicate that superoxide anions induce JNK and ERK1/2 phosphorylation only at concentrations which induce apoptosis. Neither menadione nor H2O2 induced phosphorylation of p38 MAPK. No significant changes were detected in total-ERK, total-JNK or total-p38 after exposure to menadione or H2O2 (Fig. 6A). Next, the effect of MAPK inhibitors was determined after exposure to menadione or H2O2. The MEK1/2 inhibitor U0126 markedly inhibited ERK1/2 phosphorylation and increased caspase-3 activity after treatment with menadione at 50µmol/L. In contrast, inhibition of ERK-phosphorylation did not sensitize hepatocytes to apoptosis induced by a non-toxic concentration of menadione (25µmol/L). Our data indicate that ERK1/2 MAPK, when phosphorylated, mediates protection against superoxide anions-induced apoptosis. Inhibition of the p38 MAPK pathway had no effect on menadione-induced apoptosis, indicating that p38 is not involved in superoxide anions-induced apoptosis (Fig. 6B). On the other hand, the JNK inhibitor SP600125 (28) blocked caspase-9, -6 and -3 processing (Fig. 6C), caspase-6 activity and apoptosis (Fig 6D) induced by 50µmol/L menadione. These results indicate that superoxide anions-induced apoptosis is dependent on JNK activity. None of the MAPK inhibitors sensitized hepatocytes to death after exposure to H2O2, suggesting that MAPK are not involved in the protection against H2O2 (not shown). Role of NF-κκκκB in superoxide anions-induced apoptosis. It has been proposed that ROS may activate NF-κB in various cell types. In our study, inhibition of NF-κB pathway by recombinant adenovirus expressing dominant negative IκB did not influence superoxide anions-induced apoptosis (Fig. 7A). Functionality of this virus was demonstrated by sensitizing hepatocytes to cytokine-induced apoptosis (Fig 7B).


37

A

p-JNK (1hr)

p-ERK (2hr) 4442

4656

38 p-p38 (2hr)

kDaConMen

25 µMH2O2 (1mM)

Men 50 µM

t-ERK (2hr) 4442

t-JNK (1hr) 4656

A

p-JNK (1hr)

p-ERK (2hr) 4442

4656

38 p-p38 (2hr)

kDaConMen

25 µMH2O2 (1mM)

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t-ERK (2hr) 4442

t-JNK (1hr) 4656

p-JNK (1hr)

p-ERK (2hr) 4442

4656

38 p-p38 (2hr)

kDaConMen

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t-JNK (1hr) 4656

B

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+ U0

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+ SBM

en 50

µM

+ SB

P< 0.05

n.s.

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ConMen 50 µM

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t-ERK

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+ SBM

en 50

µM

+ SB

P< 0.05

n.s.

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ConMen 50 µM

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4442

t-ERK

C

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kDa17

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GAPDH36

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ControlMen 50 µM

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kDa17

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GAPDH36

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Figure 6. A) Phosphorylation of MAP kinases. Menadione 50µmol/L, but not menadione 25µmol/L or hydrogen peroxide (H2O2), induces p-JNK and p-ERK1/2 after 1hr and 2hrs respectively; MAPK p38 was not phosphorylated by menadione or H2O2. B) ERK activation attenuates menadione-induced apoptosis. Western blot demonstrates functionality of U0126, used as ERK inhibitor. Inhibition of ERK phosphorylation increases menadione-induced caspase-3 activity (p<0.05; statistically significant according to Mann-Whitney U-test). C) Caspase-9, -6 and -3 activation and D) Menadione-induced caspase-6 activity and apoptosis are dependent on JNK activity. Cells were exposed to menadione with or without JNK inhibitor SP600125 (SP).

Chapter 3

38

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AdIκBAA

+

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+

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+

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A

B

Role of glutathione in menadione-induced apoptosis. To provide a more mechanistic interpretation of superoxide action, we evaluated the role of glutathione in menadione-induced apoptosis. Menadione treatment reduced glutathione level approximately 10% compared to control level. Depletion of intracellular reduced glutathione (GSH) was achieved using BSO which inhibits γ-gluyamyl-cysteinyl-synthetase, the rate limiting step in GSH biosynthesis. Addition of BSO, 12 hrs before and during menadione treatment, reduced GSH levels more than 80% and further increased superoxide anions-induced caspase-3 activity in primary hepatocytes. In contrast, addition of the glutathione, donor GSH-MEE slightly inhibited menadione-induced apoptosis. Western blot against active caspase-3 confirmed the data obtained by caspase-3 activity assay (Fig. 8 A, B).

Con MenMen

+BSOMen

+GSH-MEE

GAPDH36

Cleaved casp-317

0

10000

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Contro

lM

en 50

µM

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+ B

SO

BSO 200 µM

Men

+ G

SH_M

EE GSH-M

EE 5 mM

casp

ase-

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(AF

U)

A

BCon Men

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Cleaved casp-317

Con MenMen

+BSOMen

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Figure 8. A) Caspase-3 activity assay. Addition of BSO (200µmol/L) , 12 hrs before and during menadione treatment (9hrs), further increased superoxide anions-induced caspase-3 activity in primary hepatocytes. In contrast, addition of GSH-MEE (5mmol/L), a donor of glutathione, slightly inhibited menadione-induced apoptosis. B) Western blot against active caspase-3.

Figure 7. NF-κB-activation is not involved in menadione-induced caspase-3 activation. A) Menadione-induced caspase-3 activity (50µmol/L, 9hrs) is not changed by the inhibitor of NF-κB pathway Ad5IκBAA or Ad5LacZ (virus control). B) Functionality of the NF-κB inhibitor Ad5IκBAA is demonstrated by increased caspase-3 activity after exposure to cytokines (CM: m-TNF-α (20ng/ml), h-Interleukin-1β (10ng/ml) and r-Interferon-γ (10ng/ml)).


39

Scavengers of superoxide anions prevent menadione-induced apoptosis To investigate whether superoxide anions are directly involved in menadione-induced apoptosis in primary hepatocytes, the role of different superoxide anion scavengers was examined. Hepatocytes were treated 30 min before and during the addition of menadione with the nitric oxide donor SNAP or the cell-permeable superoxide dismutase mimic PEG-SOD (29). Exposure to SNAP significantly reduced menadione-induced apoptosis (fig 9A) but dramatically increased necrotic cell death in hepatocytes (Fig 9B). In contrast, PEG-SOD reduced menadione-induced caspase-3 and -6 activities (Fig 10A) but did not increase necrotic cell death (Fig. 10B). Moreover, PEG-SOD prevented superoxide anions-induced caspase-9, -6, and -3 activation and nuclear condensation (Fig. 10C, D). Additionally, PEG-SOD blocked JNK and ERK1/2 phosphorylation induced by 50µmol/L menadione (Fig. 10E).

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Figure 9. Nitric oxide blocks caspase-3 activity induced by superoxide anions but increases necrotic cell death. Hepatocytes were incubated with menadione (Men, 50µmol/L, 9hrs) with or without the nitric oxide donor SNAP. A) Caspase-3 activity (apoptosis indicator). B) Sytox Green nucleic acid staining (necrotic cell death indicator). Original magnification: 40x.

These results indicate that superoxide anion scavengers protect against superoxide anions-induced apoptosis, suggesting that menadione-induced apoptosis is directly dependent on superoxide anions production. Finally, although highly effective against bile acid-induced apoptosis (27), the anti-apoptotic bile acid tauroursodeoxycholic acid, added before, at the same time or after menadione, did not protect against menadione-induced apoptosis (not shown).

Chapter 3

40

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50 µMMen

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p-ERK (2 hr) 4442

4654

t-JNK (1 hr) 4654

t-ERK (2 hr) 4442

Figure 10. Superoxide dismutase prevents menadione-induced caspase-6 and -3 activity, processing of caspases and phosphorylation of JNK and ERK pathways but does not increase necrotic cell death. PEG-SOD (100U/ml) was added 30 min before menadione (Men, 50µmol/L) and cells were harvested 9 hrs after menadione addition. A) Caspase-6 and -3 activity (apoptosis). B) Sytox Green nucleic acid staining (necrotic cell death indicator). C) Western blot analysis for cleaved caspase-9, -6, and -3. D) Nuclear condensation determined by acridine orange staining. Magnification: 1000x. E) Western blot analysis for phospho-JNK and phospho-ERK.

A

B

E


41

Discussion

In this study we investigated the role of ROS in primary rat hepatocyte cell death. We demonstrate that the superoxide anion donor menadione mainly induces apoptosis and to a much lesser extent necrotic cell death in primary hepatocytes. In other studies, generation of superoxide in Sprague Dawley rats in vivo (30) and in isolated perfused rat liver (31) causes limited toxicity and only necrotic cell death. Several differences may explain these contrasting results: 1) different rat strains differ with respect to the susceptibility to oxidant injury, 2) Site of ROS formation: diquat was added to the perfusion medium whereas in our study, superoxide-generators were added directly to the culture medium, which may result in different uptake and final intracellular concentration of the ROS-donor in vivo and in vitro. 3) Mode of cell death: in the previous in vivo studies, only LDH/AST/ALT leakage was used as a determinant of cell death, however apoptosis was not specifically analyzed. In our experiments, we observed that paraquat as well as HX/XO also induced apoptosis. Furthermore, we show that H2O2 at concentrations below 5mmol/L does not induce apoptosis or necrotic cell death in normal primary hepatocytes. Only at high concentration or when H2O2 detoxification (catalase and GPx) is impaired, H2O2 turns into a necrotic compound. Inhibition of catalase sensitizes hepatocytes to necrotic cell death much more than inhibition of GPx. This suggests that catalase is the principal H2O2 detoxification mechanism in hepatocytes. The high efficiency of detoxification, especially by catalase, explains why H2O2 is not or only moderately toxic at concentrations up to 4 mmol/L. A previous study (31) concluded that catalase contributes to only 20% of H2O2 detoxification. It is difficult to compare this in vivo study using diquat to generate H2O2, with our data using H2O2 added to the medium. In addition, the extent of catalase inhibition using a single intraperitoneal injection of 3-aminotriazole has not been verified. In our studies we use defined concentrations of H2O2 and 3-aminotriazole and, we observed a strong effect after inhibiting catalase. Other studies showed divergent effects of H2O2, reporting both necrotic and apoptotic cell death in hepatoma cell lines (32;33). Apparently, the mode of cell death is dependent on the cell type, with hepatoma cell lines yielding different results than normal non-transformed hepatocytes. Indeed, it is well known that tumour cells exhibit increased intrinsic oxidative stress and have adapted to this situation by altered oxidative stress defence mechanisms (34;35). Differences in the sensitivity to apoptotic stimuli between transformed hepatoma cells and normal non-transformed hepatocytes have been reported (36;37). Therefore, results obtained using hepatoma cell lines cannot be extrapolated to normal, non-transformed hepatocytes. Induction of apoptosis by 50µmol/L menadione, as determined by PARP-cleavage and nuclear condensation, consistently correlates with processing and activation of caspase-9, -6 and -3, and inhibitors of these caspases prevent PARP-cleavage. The induction of apoptosis and caspase activation by menadione is in accordance with previous studies (33). In contrast with previous studies, we demonstrate that menadione-induced apoptosis is totally dependent on caspase activation, as shown by cleaved PARP. Our finding that caspase-6 and caspase-9 are activated by superoxide anions also represents a novel finding. We demonstrate that caspase-9 activation is upstream of caspase-6 and -3 activation. In type II cells, apoptosis is dependent on the release of pro-apoptotic factors from the mitochondria, that activate caspase-

Chapter 3

42

9 and subsequently caspase-3 (38-40). Our demonstration of superoxide anions-induced caspase-9 activation indicates disruption of the mitochondria and release of pro-apoptotic factors (40). Exposure to ROS has been shown to activate signal transduction pathways, which may modulate cell death (27;41;42). Our study shows that only apoptotic concentrations of menadione induces the phosphorylation of ERK1/2 MAPK and this activation attenuates cell death, since inhibition of ERK1/2 enhances superoxide anions-induced apoptosis. However, activation of ERK1/2 is clearly not sufficient to prevent menadione-induced apoptosis, since superoxide anions also activate pro-apoptotic pathways, e.g. JNK, that overrule the protective ERK1/2 pathway. In contrast, non-toxic concentrations of superoxide anions or H2O2 do not phosphorylate ERK1/2 and, as expected, inhibition of ERK1/2 in these conditions does not sensitize primary hepatocytes to cell death. This is in accordance with previous reports, in which a protective effect of ERK1/2 activation against bile acid (27) and superoxide anions-induced apoptosis has been reported, although in the latter study a sensitizing effect of ERK1/2 inhibition to low concentrations of menadione was observed (43). The explanation for this discrepancy could be that in the RALA cell line, ERK1/2 phosphorylation was also observed at low menadione concentrations, whereas in our study ERK1/2 phosphorylation was only observed at 50µmol/L. In our study, we demonstrate that JNK phosphorylation and apoptosis are induced only with 50µmol/L menadione but not by H2O2. Furthermore, we show that the inhibition of JNK blocks menadione-induced apoptosis, indicating that superoxide anions-induced apoptosis is dependent on JNK pro-apoptotic activity. The mechanisms by which JNK exert its pro-apoptotic properties are not completely understood. The fact that JNK inhibition blocks caspase-9 activation indicates that JNK triggers the mitochondrial pathway after menadione treatment. In many studies, an effect of JNK has been described at the level of the mitochondria triggering the mitochondria death pathway, including phosphorylation and activation of pro-apoptotic bcl-2 family members (43-49). The p38 MAPK pathway is not involved in menadione-induced apoptosis. Although the NF-κB pathway is involved in protection against cytokine-induced apoptosis in primary rat hepatocytes (50;51), we demonstrate that the NF-κB pathway is not involved in the protection against superoxide anions-induced apoptosis in hepatocytes. We also evaluated the anti-apoptotic potential of several compounds that are considered to have therapeutic applications and demonstrate remarkable differences between different scavengers. Nitric oxide blocks apoptosis but increases necrotic cell death. This switch in the balance between apoptosis and necrotic cell death could be caused since NO. is highly reactive with superoxide anions leading to the formation of the cytotoxic compound peroxynitrite (ONOO-) (52). Hence, the metabolism of ROS can switch the balance between apoptosis and necrotic cell death. Therefore, recent therapeutic strategies aimed at donating NO in liver diseases (53), should take into account the possibility of ROS generation in these diseases. In contrast, PEG-SOD totally inhibits caspase activation, abolishes apoptosis and does not increase necrotic cell death. In support, depleting the intracellular antioxidant glutathione increases and repleting glutathione decreases superoxide anions-induced


43

apoptosis. Interestingly, and in contrast to previous studies using a hepatoma cell line (54), inhibition of catalase had no effect on superoxide anions-induced apoptosis, whereas PEG-SOD is completely protective. These data demonstrate that superoxide anions produced by menadione directly induce apoptosis in primary hepatocytes. Furthermore, PEG-SOD is able to block the phosphorylation of both JNK and ERK1/2, indicating that PEG-SOD completely scavenges superoxide anions. Indeed, PEG-SOD represents a potential strategy in therapy in liver diseases caused by oxidative stress, as shown in studies in which overexpression of superoxide dismutases inhibit apoptosis in alcohol-induced liver injury in rats (55;56). Finally, the anti-apoptotic bile acid tauroursodeoxycholic acid, which inhibits apoptosis induced by toxic bile acids (27), did not attenuate superoxide anions-induced apoptosis, indicating that the mechanism of bile acid and superoxide anions-induced apoptosis are different. In summary, this study demonstrates major differences in the mechanisms and mode of cell death induced by superoxide anions and H2O2 in primary, non-transformed hepatocytes and provides evidence for an anti-apoptotic role for ERK and a pro-apoptotic role for JNK MAP-kinases. Furthermore, we demonstrate that it is imperative to evaluate the consequences of any intervention in cell death on both apoptotic and necrotic cell death. Chronic liver diseases, including alcoholic hepatitis, NAFLD, cholestasis and chronic viral hepatitis are almost invariably accompanied by exposure of hepatocytes to ROS and this exposure may contribute to liver damage. Therefore, knowledge about the mode of cell death and the mechanisms involved in ROS-induced damage to normal hepatocytes may contribute to the development of novel therapies. Acknowledgements This work is supported by grants from the Dutch Digestive Diseases Foundation and the J.K. de Cock Foundation Groningen. Part of this work was presented at the annual meeting of the American Association for the Study of Liver Diseases, Boston 2003.

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CHAPTER 4

Heme oxygenase-derived carbon monoxide prevents

apoptosis in primary hepatocytes via inhibition of

JNK activation

Laura Conde de la Rosa1, Titia E.Vrenken1, Rebekka A. Hannivoort1, Manon

Buist-Homan1, Rick Havinga2, Dirk-Jan Slebos3, Henk F. Kauffman3, Peter

L.M. Jansen4, Han Moshage.1

1Center for Liver, Digestive and Metabolic Diseases, 2Laboratory of Pediatrics, 3Dept. of

Pulmonary Diseases and Lung Transplantation, University Medical Center Groningen,

Groningen, The Netherlands. 4Department of Gastroenterology and Hepatology, Academic

Medical Center Amsterdam, The Netherlands.

Submitted for publication

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Abstract Oxidative stress is thought to be a key element in inflammatory and metabolic liver diseases. Heme oxygenase-1 (HO-1) may serve as a major oxidative stress protective mechanism. This enzyme causes the degradation of heme and the production of carbon monoxide (CO), free iron and biliverdin. CO is an important intracellular messenger that may play a role in the cytoprotective effect of HO-1. Aim: To investigate whether HO-1 and CO protect primary hepatocytes against oxidative stress-induced apoptosis. Methods: Bile duct ligation was used as model of chronic liver disease. In vitro, primary hepatocytes were exposed to the superoxide anion-donor menadione in a normal and a CO containing atmosphere. Apoptosis was determined by measuring caspase-9,-6,-3 activity and PARP cleavage, and necrosis by Sytox Green staining. Results: 1) HO-1 is induced in chronic cholestatic liver disease. 2) Superoxide anions induce HO-1 activity time and dose dependently. 3) HO-1 overexpression inhibits superoxide anion-induced apoptosis. 4) CO blocks superoxide anion-induced JNK-phosphorylation, caspase-9, -6, -3 activation and abolishes apoptosis, but does not increase necrosis. Conclusion: HO-1-derived CO protects primary hepatocytes against superoxide anion-induced apoptosis partially via inhibition of JNK-activity. In future therapy, CO could represent an important candidate for treatment of liver diseases.

.

HO-1 derived CO protects against apoptosis via JNK inhibition

49

Introduction Chronic liver diseases, such as non-alcoholic steatohepatitis (NASH), chronic cholestasis and alcoholic and chronic viral hepatitis, are almost invariably accompanied by exposure to reactive oxygen species (ROS) (1-3), which can contribute to hepatocyte cell death by either apoptosis or necrosis, leading to liver injury. Caspases (4, 5) represent the central executioners of apoptosis, or programmed cell death (6, 7), which is an active process characterized by cell shrinkage, chromatin condensation and formation of apoptotic bodies. In contrast, necrosis is a passive process and associated with ATP depletion, rupture of the plasma membrane and spilling of the cellular content eliciting inflammation (6). Heme oxygenase-1 (HO-1) catalyzes the oxidation of heme to form equimolar amounts of ferrous iron, carbon monoxide (CO) and biliverdin, which is rapidly converted into bilirubin by NAD(P)H:biliverdin reductase. HO-1 can be induced by a variety of different stimuli, most of them associated with oxidative stress (8). HO-1 acts as an inducible defence against oxidative stress, e.g. in models of inflammation, ischemia-reperfusion, hypoxia and hyperoxia-mediated injury (9). In the liver, HO-1 induction protected against ischemia/reperfusion injury (10, 11) and endotoxemia (12, 13). In addition, overexpression of HO-1 by gene transfer has been shown to protect against immune-mediated apoptotic liver damage in mice (14) and from CYP2E1-dependent toxicity in HepG2 cells (15). However, the mechanisms by which HO-1 mediates cytoprotection are not elucidated yet. Protective effects of both biliverdin and CO have been reported and several papers suggest that biliverdin protects against oxidative stress by acting as an anti-oxidant in different models of liver injury (16, 17). Less is known about the biological actions of CO. Although CO is known to be toxic and lethal at massive doses, recently, interest has emerged regarding the role of CO as a signalling and regulatory molecule in cellular processes (18, 19). Indeed, a cytoprotective effect of CO has been demonstrated in several conditions associated with apoptosis. In the present study, our aim was to investigate the regulation of HO-1 in vivo (model of acute inflammation and chronic cholestasis) and in vitro in primary cultures of rat hepatocytes. In addition, we investigate whether CO contributes to the HO-1 protective effect against oxidative stress-induced apoptosis in primary hepatocytes, and we examine the mechanisms involved in the process. Materials and Methods Animals Specified pathogen-free male Wistar rats (220-250 g) were purchased from Harlan (Zeist, The Netherlands). They were housed under standard laboratory conditions with free access to standard laboratory chow and water. Experiments were performed following the guidelines of the local Committee for Care and Use of laboratory animals.

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Animal models Bile Duct ligation Male Wistar rats were anaesthetized and subjected to bile duct ligation (BDL) as a model of chronic cholestasis (20). At the indicated time points after BDL, rats (n= 4 per group) were sacrificed, livers were perfused with saline and removed. Control rats (n= 4) received a sham operation (SHAM) or not (control). Model of acute inflammation Rats were injected intraperitoneally (IP) with 5 mg/kg body weight lipopolysaccharide (LPS serotype 0127:B8; Sigma, St. Louis, MO), or the same volume of phosphate-buffered saline (PBS, control group, n= 4 for each experimental group), with or without diethylmaleate (DEM: 4mmol/kg bodyweight, Sigma) 30 minutes prior to LPS or PBS injection. LPS and DEM were dissolved in sterile PBS. Rats were anaesthetized with pentobarbital (60 mg/kg, IP) 6hrs after LPS injection. Livers were perfused with saline and snap-frozen in liquid nitrogen until further use. Rat hepatocytes isolation. Hepatocytes were isolated as described previously (21) and cultured in William’s E medium (Invitrogen; Breda, The Netherlands) supplemented with 50µg/mL gentamicin (Invitrogen, Carlsboad, California, USA) without the addition of hormones or growth factors. During the attachment period (4hrs) 50nmol/L dexamethasone (Sigma) and 5% fetal calf serum (Invitrogen) were added to the medium. Cells were cultured in a humidified incubator at 37°C and 5% CO2. Hepatocyte viability was always more than 90% and purity more than 95% as determined by trypan blue staining. Experimental design. Experiments were started 24hrs after isolation of hepatocytes. Cells were exposed to the intracellular superoxide anion-donor menadione (2-methyl-1,4-naphthoquinone, 50µmol/L Sigma) (22), the bile acids glycochenodeoxycholic acid (GCDCA, Calbiochem, La Jolla, California, USA), tauroursodeoxycholic acid (TUDCA, Calbiochem) or taurochenodeoxycholic acid (TCDCA, Calbiochem) at 50µmol/L , hydrogen peroxide (H2O2 , 1mmol/L, MERCK, Haarlem, The Netherlands) or cytokine mixture (23) composed of 20ng/ml murine TNF-α (R&D System, UK), 10ng/ml human interleukin-1β (R&D System, UK) and 100U/ml rat interferon-γ (Life Technologies Ltd) for the indicated time. Activation of ERK1/2 MAPK was inhibited using the MEK inhibitor U0126 at 10µmol/L (Promega, Madison, USA). 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ, 50µmol/L, Sigma) was used to inhibit soluble guanylate cyclase (sGC). Depletion of intracellular reduced glutathione, was achieved using D,L-buthionine-(S,R)-sulfoximine (BSO, 200µmol/L, Sigma) and diethylmaleate (DEM, 1mmol/L, Sigma).

Hepatocytes were exposed to the adenoviruses Ad5HO-1, Ad5IκBAA or Ad5LacZ (multiplicity of infection of 10) 15 hours before exposure to the superoxide anion-donor menadione or cytokine mixture.


51

Parallel experiments were performed in a normal and a carbon monoxide containing culture atmosphere. Each experimental condition was performed in triplicate wells. Each experiment was performed, at least three times, using hepatocytes from different isolations. Carbon monoxide exposure CO at a concentration of 1% (10000 parts per million) in compressed air was mixed with compressed air containing 5% CO2 before being delivered into the culture incubator, yielding a final concentration of 400 ppm (parts per million). CO. The incubator was humidified and maintained at 37°C. A CO analyzer was used to determine CO levels in the chamber. After the chamber had stabilized, no oscillations were measured in the CO concentration. Caspase enzyme activity assays Caspase-3 enzyme activity was assayed as described previously (24). Sytox Green nuclear stainings Rupture of the plasma membrane distinguishes necrotic from apoptotic cell death. To estimate necrosis, hepatocytes were incubated 15 minutes with SYTOX green (Molecular Probes, Eugene, USA) nucleic acid stain, which penetrates cells with compromised plasma membranes but does not cross the membranes of viable cells or apoptotic bodies. Fluorescent nuclei were visualized using an Olympus CKX41 microscope at 450-490 nm. RNA isolation and reverse-transcriptase polymerase chain reaction (RT-PCR). RNA was isolated using Tri-reagent (Sigma) according to the manufacturer’s instructions. Reverse transcription was performed on 5µg of total RNA using random primers in a final volume of 75µl (Reverse Transcription System, Sigma). PCR was performed as described previously (23). PCR primers are listed in table 1. GAPDH (glyceraldehyde 3-phosphate dehydrogenase) was used as internal control. Each PCR product was loaded on a 2% agarose gel and stained with ethidium bromide. Quantitative Real-Time PCR. Reverse transcription was performed on 2.5µg of total RNA using random primers in a final volume of 50µl. Real time detection was performed on the ABI PRISM 7700 (PE Applied Biosystems) initialized by 10 min at 95°C, followed by 40 cycles of 15 sec at 95°C, and 1 min at 60°C. Each sample was analyzed in duplicate. 18S mRNA levels were used as an endogenous control. Real time-PCR primers and probes are listed in table 1.

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CAG CTC CTC AAA CAG CTC AAT GTT GAG C

CTG GTC TTT GTG TTC CTC TGT CAG (antisense)

HO-1 CAC AGG GTG ACA GAA GAG GTC AA (sense)

CCA ATT ACA GGG CCT CGA AA (antisense)

CGC GCA AAT TAC CCA CTC CCG A (probe)

CGG CTA CCA CAT CCA AGG A (sense)18S

Primers (rat) Real Time-PCR: Primers and probe 5’� 3’

CAC GCA TAT ACC GGC TAC CT (sense)AAG GCG GTC TTA GCC TCT TC (antisense)

HO-1

CCA TCA CCA TCT TCC AGG AG (sense)CCT GCT TCA CCA CCT TCT TG (antisense)

GAPDH

Primers (rat) Classic PCR: Primers 5’� 3’

CAG CTC CTC AAA CAG CTC AAT GTT GAG C

CTG GTC TTT GTG TTC CTC TGT CAG (antisense)

HO-1 CAC AGG GTG ACA GAA GAG GTC AA (sense)

CCA ATT ACA GGG CCT CGA AA (antisense)

CGC GCA AAT TAC CCA CTC CCG A (probe)

CGG CTA CCA CAT CCA AGG A (sense)18S

Primers (rat) Real Time-PCR: Primers and probe 5’� 3’

CAC GCA TAT ACC GGC TAC CT (sense)AAG GCG GTC TTA GCC TCT TC (antisense)

HO-1

CCA TCA CCA TCT TCC AGG AG (sense)CCT GCT TCA CCA CCT TCT TG (antisense)

GAPDH

Primers (rat) Classic PCR: Primers 5’� 3’

Table 1. Oligonucleotide primers and probes used for classic- and Real time-PCR

Western Blot analysis. Western blot analysis of cell lysates was performed by SDS-PAGE followed by transfer to Hybond ECL nitrocellulose membrane (Amersham). An antibody against GAPDH (Calbiochem) and Ponceau S staining were used to ensure equal protein loading and electrophoretic transfer. Caspase cleavage was detected using polyclonal rabbit antibodies against cleaved caspase-9, -6, and -3. A monoclonal antibody was used to detect HO-1 (Stressgen). Poly(ADP-ribose) polymerase (PARP) cleavage was detected with rabbit anti-PARP polyclonal antibody. PARP (116 kDa) is a substrate of caspase-3 yielding a product of 89 kDa and it is considered a late marker for apoptosis. After Western blot analysis for p-ERK1/2 and p-JNK (monoclonal, Santa Cruz Biotechnology), blots were stripped using 0.1 % SDS in PBS/Tween-20 at 65°C for 30 minutes and incubated with antibodies against, total-ERK1/2 or total-JNK. All antibodies were obtained from Cell Signalling Technology (Beverly, MA), unless indicated otherwise, and used at 1:1000 dilution. Adenoviral constructs. Recombinant, replication-deficient adenovirus Ad5IκBAA was used to inhibit NF-κB activation as described previously (25). Ad5LacZ was used as a control virus, which contains the E.coli β-galactosidase gene. The Ad5HO-1 adenovirus was a kind gift of prof. Augustine Choi, University of Pittsburg, Pittsburg, USA and has been described before (26, 27). Statistical analysis. All data are expressed as the mean of at least 3 independent experiments ± standard deviation. Statistical significance was determined by the Mann-Whitney test; p<0.05 was considered statistically significant.

HO-derived CO protects against apoptosis via JNK inhibition

53

Results Regulation of HO-1 in vivo and in vitro To investigate the regulation of HO-1 in vivo, models of chronic cholestatic liver disease (BDL) and acute inflammation were used. Chronic cholestasis increased HO-1 mRNA level about 6-fold compared with control level in a time dependent manner (Fig 1). In contrast, acute inflammation does not increase HO-1 expression, although iNOS expression was highly elevated in the same livers at protein and at mRNA level as previously demonstrated (28). However, DEM-induced oxidative stress, superposed on acute inflammation, strongly increases HO-1 mRNA level in acute inflammation in vivo. Induction of oxidative stress with DEM alone also increases HO-1 mRNA in the liver (Fig. 2).

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Figure 1. HO-1 expression is induced in chronic cholestasis. Rats were subjected to bile duct ligation (BDL) as a model of cholestasis. At indicated time points after BDL, rats (n=4 per group) were sacrificed, liver were perfused with saline and removed. Control rats received a sham operation (SHAM) or not (Con). HO-1 expression is induced in BDL at protein level (as determined by Western blot, upper bands) and at mRNA level (as determined by Real time-PCR, lower graph). GAPDH (glyceraldehyde 3-phosphate dehydrogenase) was used as a protein loading control.

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Figure 2. Acute inflammation does not induce HO-1 expression in the liver. Rats were injected intraperitoneally with 5mg/kg body weight lipopolysaccharide or the same volume of phosphate-buffered saline (PBS, control group, Con) with or without diethylmaleate (DEM: 4mmol/kg body weight), 30 min prior to LPS or PBS injection. After 6hrs of LPS injection, rats were anaesthetized with pentobarbital (60 mg/kg, IP). However, DEM-induced oxidative stress, superposed on acute inflammation, strongly increases HO-1 mRNA level in acute inflammation in vivo, as determined by Real time-PCR. In the figure, a representative set of a group (n=4) is shown.

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To confirm our in vivo data we investigated the regulation of HO-1 expression in vitro in primary hepatocytes. Concerning bile acids, GCDCA, TUDCA and TCDCA induce HO-1 expression in vitro after 4hrs (Fig. 3).

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The superoxide anion-donor menadione induces HO-1 expression in a time (Fig. 4A) and concentration dependent manner (Fig. 4B).

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Figure 4. The intracellular superoxide anion-donor menadione induces HO-1 expression in a time- and concentration-dependent manner in vitro. Cells were exposed to menadione (Men, 50µmol/L) and harvested at different time points (A) or exposed to different concentrations of menadione for 9hrs (B,C). HO-1 expression was determined at A) protein level by Western blot. GAPDH (glyceraldehyde 3-phosphate dehydrogenase) was used as a protein loading control and at B) mRNA level by Real time-PCR (9hrs). 18S mRNA levels were used as an endogenous control.

In contrast, cytokine mixture slightly induces HO-1 expression in vitro (Fig. 5), although iNOS expression was highly induced at protein and mRNA level under the same circumstances as previously shown (28).

Figure 3. Bile acids increase HO-1 expression in vitro. Hepatocytes were exposed to the bile acids taurochenodeoxycholic (TCDCA), tauroursodeoxycholic acid (TUDCA) or glycochenodeoxycholic acid (GCDCA) at 50µmol/L for 4 hrs. Bile acids increase HO-1 mRNA level as determined by Real time-PCR.


55

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Figure 5. Cytokines slightly induces HO-1 expression in primary hepatocytes in vitro. D,L-buthionine-(S,R)-sulfoximine (BSO, 200µmol/L) was added 15hrs before and 30 min before the start of the experiment. Diethylmaleate (DEM, 1mol/L) was added 30 min before the start of the experiment. Then, cells were exposed to cytokine mixture (CM: 20ng/ml murine TNF-α, 10ng/ml human interleukin-1β , 100U/ml rat interferon-γ) for 10 hrs. HO-1 mRNA level was increased by BSO and DEM but only a minor increase was observed after exposure to CM as determined by quantitative real time-PCR.

Exposure of hepatocytes to H2O2 does not induce HO-1 expression in hepatocytes (Fig. 6). Recently, we have shown that depletion of glutathione, using D,L-buthionine-(S,R)-sulfoximine (BSO) which inhibits γ-glutamyl-cysteinyl-synthetase, the rate limiting step in GSH biosynthesis, exacerbates menadione-induced apoptosis (25). In this study, we observed that depletion of the antioxidant GSH, using BSO or DEM, further enhances the induction of HO-1 mRNA and HO-1 protein level in vivo (Fig. 2) and in vitro (Fig. 5, 6) in hepatocytes in all conditions tested.

A

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Figure 6. Heme oxygenase induction in hepatocytes exposed to oxidative stress. D,L-buthionine-(S,R)-sulfoximine (BSO, 200µmol/L) was added 15hrs before and 30 min before the start of the experiment. Diethylmaleate (DEM, 1mol/L) was added 30 min before the start of the experiment. After that, cells were exposed to hydrogen peroxide (H2O2, 1mmol/L, 6hrs) or the superoxide anion-donor menadione (Men, 50µmol/L, 9hrs). A) HO-1 mRNA level was increased by the superoxide anion-donor menadione, BSO and DEM but not by hydrogen peroxide as determined by quantitative real time-PCR. B) At protein level, HO-1 expression is induced by menadione and BSO as determined by Western blot.

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Heme Oxygenase-1 overexpression protects against oxidative stress To investigate whether HO-1 plays a role in the protection against oxidative stress, primary rat hepatocytes were treated with Ad5HO-1 15 hrs before exposure to menadione. As shown in Fig. 7A, Ad5HO-1 infection induced overexpression of HO-1 mRNA in hepatocytes at the time of menadione exposure. Overexpression of HO-1 protected against menadione-induced caspase-3 activity and processing compared with control and Ad5LacZ infected hepatocytes (Fig 7B). Although metalloporphyrins like hemin or chromium mesoporphyrin are widely used to induce or reduce expression of HO-1 respectively, in our hands it showed serious side-effects. This confirms recent reports that metalloporphyrins induce oxidative stress themselves and therefore cannot be used in combination with menadione (29). In addition, it has recently been reported that metalloporphyrins interfere with caspase activity, independent of its HO-1 inducing effect (30). To avoid these confounding side effects, we opted to use an adenoviral construct to achieve overexpression of HO-1.

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Figure 7. Overexpression of HO-1, using the adenovirus Ad5HO-1, leads to protection of primary hepatocytes against oxidative stress-induced apoptosis. Cells were exposed to Ad5HO-1 or Ad5LacZ (virus control) 15hrs before the addition of menadione (Men, 50µmol/L, 9hrs). A) Real time-PCR. Ad5HO-1 highly induces HO-1 expression (up to 70-fold) compared with Ad5LacZ and control. B) Overexpression of HO-1 prevents capase-3 activity (graph) and processing (Western blot, upper panel).

A

B


57

Carbon monoxide protects primary rat hepatocytes against oxidative stress-induced apoptosis. Since HO-1 overexpression protects primary hepatocytes against superoxide anions-induced apoptosis, we investigated whether CO contributes to the protective effect of HO-1 expression. We first demonstrated that CO suppresses menadione-induced PARP-cleavage and apoptosis in primary hepatocytes (Fig 8A, B), indicating that CO has a protective role against superoxide anions-induced apoptosis. Furthermore, we previously showed that menadione-induced apoptosis is dependent on caspase activation in primary hepatocytes (25). Because of the important role of caspases in apoptosis, we analysed the effect of carbon monoxide on caspases activation. Our results demonstrate that CO inhibits menadione-induced caspase-9, -6 and -3 activation (Fig. 9).

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CO did not induce necrosis in primary hepatocytes (fig. 10), suggesting that CO is protective against oxidative stress-induced apoptosis. Next, the mechanism by which CO inhibits apoptosis was examined.

Figure 8. Carbon monoxide prevents oxidative stress-induced apoptosis in hepatocytes. Cells were incubated with menadione (Men, 50µmol/L, 9hrs) or not (Con, untreated cells) in the presence and absence of carbon monoxide (CO, 400 parts per million, 9hrs). A) Western blot: CO inhibits PARP (Poly (ADP-ribose) polymerase)-cleavage induced by menadione. GAPDH (glyceraldehyde 3-phosphate dehydrogenase) was used as a protein loading control. B) Caspase-3 activity assay: Carbon monoxide blocks menadione-induced apoptosis (expressed as capase-3 activity).

Figure 9. Carbon monoxide prevents menadione-induced caspase activation in hepatocytes. Cells were incubated with menadione (Men, 50µmol/L, 9hrs) and without (Con, untreated cells) in the presence and absence of carbon monoxide (CO, 400 parts per million, 9hrs). Western blot: CO inhibits casapse-9, -6 and -3 cleavage induced by menadione. GAPDH was used as a protein loading control

Chapter 4

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Positivecontrol

CO atmosphereNormal atmosphere

Control Menadione Control Menadione

Positivecontrol

CO atmosphereNormal atmosphere

Control Menadione Control Menadione

Figure 10. Carbon monoxide does not induce necrotic cell death. Hepatocytes were incubated with menadione (Men, 50µmol/L, 9hrs) and without (Con, untreated cells) in the presence and absence of carbon monoxide (CO, 400ppm, 9hrs). Sytox Green nucleic staining: Necrotic cells represent less than 2% of total cells in all condition tested. Upper panel phase contrast and fluorescence; lower panel fluorescence only. Magnification: 40x

The protective effect of CO is not dependent on soluble Guanylate Cyclase To investigate whether sGC is involved in the protective effect of CO against menadione-induced apoptosis in primary hepatocytes, cells were exposed to the sGC inhibitor ODQ with or without menadione in the presence of carbon monoxide. The ability of CO to inhibit menadione-induced apoptosis was not reversed by ODQ (Fig. 11), indicating that the antiapoptotic effect of CO is not dependent on the activation of the soluble guanylate cyclase or the generation of cGMP. In the absence of CO, ODQ had no effect on primary hepatocytes.

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Figure 11. The protective effect of carbon monoxide is not dependent on the activation of soluble guanylate cyclase. Cells were exposed to ODQ (1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one, a sGC inhibitor) with or without menadione (Men, 50µmol/L, 9hrs) in the presence of carbon monoxide (CO, 400ppm, 9hrs). Caspase-3 activity assay: The ability of CO to inhibit menadione-induced apoptosis was not reversed by ODQ.


59

05000

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Figure 12. NF-κB pathway is not involved in the protective effect of CO against oxidative stress-induced apoptosis. Caspase-3 activity assay: inhibition of the NF-κB pathway, using the Ad5dnIκB 15hrs before menadione addition, did not reverse the protective effect of carbon monoxide (CO, 400ppm, 9hrs) against menadione-induced apoptosis.

NF-κκκκB pathway is not involved in the protective effect of CO against menadione-induced apoptosis. In our study, inhibition of the NF-κB pathway by recombinant adenovirus expressing dominant negative IκB did not reverse the protective effect of CO against superoxide anions-induced apoptosis (Fig.12). Functionality of the virus was demonstrated by sensitizing hepatocytes to cytokine-induced apoptosis as described previously (25). Carbon monoxide blocks phosphorylation of the pro-apoptotic JNK MAPK. In a previous study, we reported that menadione-induced caspase activation and apoptosis were dependent on JNK activity in primary hepatocytes (25). Having demonstrated that CO prevents caspase-9, -6 and -3 activation, we also investigated the effect of CO on MAPK-phosphorylation. As shown in fig. 13A, CO induces ERK-phosphorylation at 2hrs. To further investigate the role of ERK in the protection against menadione-induced apoptosis, hepatocytes were exposed to CO in the presence of ERK1/2 inhibitor (U0126). Under these circumstances, the protective effect of CO was not reversed (Fig. 13B). On the other hand, CO does not induce JNK-phosphorylation, but prevents menadione-induced JNK-phosphorylation (Fig. 13A). Since inhibition of JNK activity, using the inhibitor SP600125, prevents menadione-induced caspase activation and apoptosis (25), our results indicate that the protective effect of CO against superoxide anions-induced apoptosis is mainly via inhibition of JNK activation.

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ControlCO +

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Figure 13. Carbon monoxide induces ERK1/2- and inhibits JNK-phosphorylation. Cells were exposed to menadione (Men, 50µmol/L, 1-2 hrs (A) or 9hrs (B)) in the presence and absence of carbon monoxide (CO, 400ppm, 1-2 hrs (A) or 9hrs (B)). A) Western blot were performed against phosphorylated-JNK, total-JNK, phosphorylated-ERK1/2 and total-ERK1/2. Total-JNK and total-ERK were used as loading controls. CO induces p-ERK1/2 at 2hrs. In contrast, CO prevents menadione-induced JNK phosphorylation (1hr). B) Caspase-3 activity assay: Cells were exposed to the ERK1/2 inhibitor U0126, 30 min before and during the incubation with menadione (Men, 50µmol/L, 9hrs) in the presence of CO (400ppm). Inhibition of EKR1/2 activation does not prevent the protective effect of CO.

Discussion Previous studies have shown the importance of HO-1 expression in mediating antioxidant and anti-apoptotic effects (31-34). In addition, induction of HO-1 is cytoprotective both in vitro and in vivo. HO-1 has been shown to protect against ischemia/reperfusion injury (10, 11), inflammation (14), immune mediated liver injury (17), endotoxemia (13), endotoxic shock (35) and against CYP2E1-dependent cytotoxicity (15). In this study, we demonstrate that HO-1 expression is induced in different models of oxidative stress in vivo and in vitro in the liver, including chronic cholestasis. A novel finding in our study is that bile acids, both pro-apoptotic and anti-apoptotic, induce HO-1 expression. Whether this is a direct effect or acts via bile acid-induced ROS generation remains to be elucidated. Interestingly, acute inflammation, induced by endotoxin administration, is a very weak inducer of HO-1, this despite the fact that NF-kB-regulated genes like iNOS are highly induced (28). It is remarkable that HO-1 expression is not induced by H2O2 in vitro. As we described before (25), hepatocytes have highly efficient H2O2 detoxification mechanisms and therefore it is likely that this molecule is hardly sensed by hepatocytes. This also correlates with our previous finding that 1mmol/L H2O2 does not induce cell death. Although, the superoxide anion-donor menadione induces HO-1 expression in primary hepatocytes, this induction does not prevent apoptosis, probably because HO-1 induction is not high and/or rapid enough. However, overexpression of HO-1, using the adenovirus Ad5HO-1, 15hrs before the addition of menadione leads to protection against oxidative stress-induced apoptosis. Metalloporphyrins like hemin or chromium mesoporphyrin are widely used as inducers or inhibitors of HO-1, respectively. However, in our hands it showed serious

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side-effects, confirming recent reports that describe aspecific effects of these compounds, in particular when studying phenomena related to oxidative stress and apoptosis (29, 30). In this report we study whether CO could be involved in the protective effect of HO-1 against superoxide anion-induced apoptosis in primary hepatocytes. Previously, we demonstrated that superoxide anions-induced apoptosis is dependent on caspase activation and JNK pro-apoptotic activity, since caspase inhibitors and the JNK inhibitor SP600125 abolished menadione-induced apoptosis (25). In the present study we provide evidence that CO exerts a protective role against superoxide anion-induced apoptosis. We describe that CO, exogenously administrated, is a potent inhibitor of caspase activation, PARP cleavage and apoptosis in primary hepatocytes exposed to the superoxide anion-donor menadione. The finding that CO prevents apoptosis in primary hepatocytes is in accordance with studies in which cell death was induced by glucose-deprivation in BNL CL.2 hepatoma cells or by Fas (36) or TNF-α in hepatocytes (37). In addition, a protective effect of CO has been demonstrated for different cell types (33, 38, 39). In contrast, a pro-apoptotic effect of CO has been observed for murine thymocytes and bovine pulmonary arterial endothelial cells (40, 41). These data indicate that CO may regulate apoptosis and cytotoxicity in a cell-specific manner. Previously, we demonstrated that apoptotic cell death can be switched to necrotic cell death under certain conditions (25). In this report, we prove that CO indeed protects hepatocytes exposed to oxidative stress and does not switch apoptotic into necrotic cell death. CO may exert its effects via various mechanisms. Activation of soluble guanylate cyclase (sGC) by CO has been well documented. However, in our study, we demonstrate that the mechanism by which CO prevents superoxide anions-induced apoptosis in primary hepatocytes is not dependent on sGC, since ODQ, a soluble guanylate cyclase inhibitor, did not reverse the anti-apoptotic effect of CO. Previous studies are contradictory concerning the role of sGC in the anti-apoptotic effect of CO, both confirming (33) and contradicting (39) our findings. Again, different signalling pathways may be involved in the protective effect of CO in different cell types. In addition, we found evidence that the NF-κB pathway is not involved in the protective effect of CO against superoxide anions-induced apoptosis in hepatocytes. Functionality of the virus was demonstrated by sensitizing hepatocytes to cytokine-induced apoptosis (25). Superoxide anion-induced apoptosis is dependent on JNK pro-apoptotic activity, since inhibition of JNK activity, using the inhibitor SP600125, prevents menadione-induced caspase activation and apoptosis in primary hepatocytes (25), although the mechanisms by which JNK exert its pro-apoptotic properties are not completely understood. In many studies, an effect of JNK has been described at the level of the mitochondria triggering the mitochondrial death pathway, including phosphorylation and activation of pro-apoptotic bcl-2 family members (42-48). In the current study, we demonstrate that CO prevents JNK-phosphorylation and its pro-apoptotic activity. The fact that CO inhibits caspase-9 activation suggests that CO prevents

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activation of the death mitochondrial pathway and subsequent apoptosis. It has been shown that CO blocks mitochondrial cytochrome c release (39, 49), although the mechanism is unknown. A similar inhibitory effect on JNK-phosphorylation by CO has been described in a model of sepsis in vivo (50). We also show that CO differentially modulates pro- and anti-apoptotic MAPK pathways. On one hand, CO prevents JNK-phosphorylation and its pro-apoptotic activity. Therefore, our results indicate that the protective effect of CO against superoxide anions-induced apoptosis is at least in part via inhibition of JNK activation. On the other hand, CO induces ERK 1/2 MAPK-phosphorylation. ERK1/2-activity is anti-apoptotic and its activation attenuates cell death, since inhibition of ERK1/2 enhances superoxide anions-induced apoptosis (25). Hence, CO-induced ERK1/2-phosphorylation further increases the protection against superoxide anions-induced apoptosis. CO has been shown to regulate MAPK signalling pathways in a variety of cell types in previous studies (49, 51). In addition, it has been reported that CO mediates protection against CYP2E1-dependent cytotoxicity in part via inhibition of CYP2E1 activity (15). In this study, we focus on the mechanism of the protective action of CO. It has been described that biliverdin, other product of HO-1, is also protective against oxidative stress-induce injury in part due to its anti-oxidant properties (16). In addition, a cooperative protective effect of biliverdin and carbon monoxide has been shown in immune-mediated liver injury in mice (17). In conclusion, HO-1 is induced by oxidative stress in primary hepatocytes in vivo and in vitro. HO-1 protects primary hepatocytes against superoxide anions-induced apoptosis, and this protection may be mediated in part via CO production and CO inhibition of JNK activation. CO inhibits the activity of the pro-apoptotic JNK MAPK and, in addition, induces ERK1/2 MAPK anti-apoptotic activity. Modulation of HO-1 expression, or the levels of its product CO, may become an important therapeutic target in liver disorders caused by exposure to excessive oxidative stress. Reference List 1. Reid AE. Nonalcoholic steatohepatitis. Gastroenterology 2001 Sep;121(3):710-723. 2. Bomzon A, Holt S, Moore K. Bile acids, oxidative stress, and renal function in biliary obstruction.

Semin Nephrol 1997 Nov;17(6):549-562. 3. Loguercio C, Federico A. Oxidative stress in viral and alcoholic hepatitis. Free Radic Biol Med 2003

Jan 1;34(1):1-10. 4. Thornberry NA, Lazebnik Y. Caspases: enemies within. Science 1998 Aug 28;281(5381):1312-1316. 5. Riedl SJ, Shi Y. Molecular mechanisms of caspase regulation during apoptosis. Nat Rev Mol Cell Biol

2004 Nov;5(11):897-907. 6. Nieminen AL. Apoptosis and necrosis in health and disease: role of mitochondria. Int Rev Cytol

2003;224:29-55. 7. Granville DJ, Carthy CM, Hunt DW, McManus BM. Apoptosis: molecular aspects of cell death and

disease. Lab Invest 1998 Aug;78(8):893-913. 8. Bauer M, Bauer I. Heme oxygenase-1: redox regulation and role in the hepatic response to oxidative

stress. Antioxid Redox Signal 2002 Oct;4(5):749-758.


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9. Ryter SW, Otterbein LE, Morse D, Choi AM. Heme oxygenase/carbon monoxide signaling pathways: regulation and functional significance. Mol Cell Biochem 2002 May;234-235(1-2):249-263.






15. Gong P, Cederbaum AI, Nieto N. Heme oxygenase-1 protects HepG2 cells against cytochrome P450 2E1-dependent toxicity. Free Radic Biol Med 2004 Feb 1;36(3):307-318.



18. Maines MD. The heme oxygenase system: a regulator of second messenger gases. Annu Rev Pharmacol Toxicol 1997;37:517-554.

19. Verma A, Hirsch DJ, Glatt CE, Ronnett GV, Snyder SH. Carbon monoxide: a putative neural messenger. Science 1993 Jan 15;259(5093):381-384.

20. Kountouras J, Billing BH, Scheuer PJ. Prolonged bile duct obstruction: a new experimental model for cirrhosis in the rat. Br J Exp Pathol 1984 Jun;65(3):305-311.

21. Moshage H, Casini A, Lieber CS. Acetaldehyde selectively stimulates collagen production in cultured rat liver fat-storing cells but not in hepatocytes. Hepatology 1990 Sep;12(3 Pt 1):511-518.

22. Thor H, Smith MT, Hartzell P, Bellomo G, Jewell SA, Orrenius S. The metabolism of menadione (2-methyl-1,4-naphthoquinone) by isolated hepatocytes. A study of the implications of oxidative stress in intact cells. J Biol Chem 1982 Oct 25;257(20):12419-12425.

23. Schoemaker MH, Gommans WM, de la Rosa LC, Homan M, Klok P, Trautwein C, et al. Resistance of rat hepatocytes against bile acid-induced apoptosis in cholestatic liver injury is due to nuclear factor-kappa B activation. J Hepatol 2003 Aug;39(2):153-161.

24. Schoemaker MH, Conde de la Rosa L, Buist-Homan M, Vrenken TE, Havinga R, Poelstra K, et al. Tauroursodeoxycholic acid protects rat hepatocytes from bile acid-induced apoptosis via activation of survival pathways. Hepatology 2004 Jun;39(6):1563-1573.

25. Conde de la Rosa L, Schoemaker MH, Vrenken TE, Buist-Homan M, Havinga R, Jansen PL, et al. Superoxide anions and hydrogen peroxide induce hepatocyte death by different mechanisms: involvement of JNK and ERK MAP kinases. J Hepatol 2005 Sep 12.

26. Fallaux FJ, Kranenburg O, Cramer SJ, Houweling A, Van Ormondt H, Hoeben RC, et al. Characterization of 911: a new helper cell line for the titration and propagation of early region 1-deleted adenoviral vectors. Hum Gene Ther 1996 Jan 20;7(2):215-222.

27. McGrory WJ, Bautista DS, Graham FL. A simple technique for the rescue of early region I mutations into infectious human adenovirus type 5. Virology 1988 Apr;163(2):614-617.

28. Vos TA, van Goor H, Tuyt L, Jager-Krikken A, Leuvenink R, Kuipers F, et al. Expression of inducible nitric oxide synthase in endotoxemic rat hepatocytes is dependent on the cellular glutathione status. Hepatology 1999 Feb;29(2):421-426.

29. Erario MA, Gonzales S, Noriega GO, Tomaro ML. Bilirubin and ferritin as protectors against hemin-induced oxidative stress in rat liver. Cell Mol Biol (Noisy -le-grand) 2002 Dec;48(8):877-884.

30. Blumenthal SB, Kiemer AK, Tiegs G, Seyfried S, Holtje M, Brandt B, et al. Metalloporphyrins inactivate caspase-3 and -8. FASEB J 2005 Aug;19(10):1272-1279.

31. Otterbein LE, Choi AM. Heme oxygenase: colors of defense against cellular stress. Am J Physiol Lung Cell Mol Physiol 2000 Dec;279(6):L1029-L1037.

32. Poss KD, Tonegawa S. Reduced stress defense in heme oxygenase 1-deficient cells. Proc Natl Acad Sci U S A 1997 Sep 30;94(20):10925-10930.

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33. Brouard S, Otterbein LE, Anrather J, Tobiasch E, Bach FH, Choi AM, et al. Carbon monoxide generated by heme oxygenase 1 suppresses endothelial cell apoptosis. J Exp Med 2000 Oct 2;192(7):1015-1026.

34. Petrache I, Otterbein LE, Alam J, Wiegand GW, Choi AM. Heme oxygenase-1 inhibits TNF-alpha-induced apoptosis in cultured fibroblasts. Am J Physiol Lung Cell Mol Physiol 2000 Feb;278(2):L312-L319.

35. Sarady JK, Zuckerbraun BS, Bilban M, Wagner O, Usheva A, Liu F, et al. Carbon monoxide protection against endotoxic shock involves reciprocal effects on iNOS in the lung and liver. FASEB J 2004 May;18(7):854-856.

36. Choi BM, Pae HO, Kim YM, Chung HT. Nitric oxide-mediated cytoprotection of hepatocytes from glucose deprivation-induced cytotoxicity: involvement of heme oxygenase-1. Hepatology 2003 Apr;37(4):810-823.

37. Zuckerbraun BS, Billiar TR, Otterbein SL, Kim PK, Liu F, Choi AM, et al. Carbon monoxide protects against liver failure through nitric oxide-induced heme oxygenase 1. J Exp Med 2003 Dec 1;198(11):1707-1716.

38. Zhang X, Shan P, Otterbein LE, Alam J, Flavell RA, Davis RJ, et al. Carbon monoxide inhibition of apoptosis during ischemia-reperfusion lung injury is dependent on the p38 mitogen-activated protein kinase pathway and involves caspase 3. J Biol Chem 2003 Jan 10;278(2):1248-1258.

39. Liu XM, Chapman GB, Peyton KJ, Schafer AI, Durante W. Carbon monoxide inhibits apoptosis in vascular smooth muscle cells. Cardiovasc Res 2002 Aug 1;55(2):396-405.

40. Thom SR, Fisher D, Xu YA, Notarfrancesco K, Ischiropoulos H. Adaptive responses and apoptosis in endothelial cells exposed to carbon monoxide. Proc Natl Acad Sci U S A 2000 Feb 1;97(3):1305-1310.

41. Turcanu V, Dhouib M, Gendrault JL, Poindron P. Carbon monoxide induces murine thymocyte apoptosis by a free radical-mediated mechanism. Cell Biol Toxicol 1998 Feb;14(1):47-54.


43. Davis RJ. Signal transduction by the JNK group of MAP kinases. Cell 2000 Oct 13;103(2):239-252. 44. Tournier C, Hess P, Yang DD, Xu J, Turner TK, Nimnual A, et al. Requirement of JNK for stress-

induced activation of the cytochrome c-mediated death pathway. Science 2000 May 5;288(5467):870-874.


46. Lei K, Nimnual A, Zong WX, Kennedy NJ, Flavell RA, Thompson CB, et al. The Bax subfamily of Bcl2-related proteins is essential for apoptotic signal transduction by c-Jun NH(2)-terminal kinase. Mol Cell Biol 2002 Jul;22(13):4929-4942.



49. Zhang X, Shan P, Alam J, Davis RJ, Flavell RA, Lee PJ. Carbon monoxide modulates Fas/Fas ligand, caspases, and Bcl-2 family proteins via the p38alpha mitogen-activated protein kinase pathway during ischemia-reperfusion lung injury. J Biol Chem 2003 Jun 13;278(24):22061-22070.

50. Morse D, Pischke SE, Zhou Z, Davis RJ, Flavell RA, Loop T, et al. Suppression of inflammatory cytokine production by carbon monoxide involves the JNK pathway and AP-1. J Biol Chem 2003 Sep 26;278(39):36993-36998.

51. Tsui TY, Siu YT, Schlitt HJ, Fan ST. Heme oxygenase-1-derived carbon monoxide stimulates adenosine triphosphate generation in human hepatocyte. Biochem Biophys Res Commun 2005 Oct 28;336(3):898-902.

CHAPTER 5

Metformin protects primary rat hepatocytes against

oxidative stress-induced apoptosis

Laura Conde de la Rosa1, Titia E. Vrenken1, Manon Buist-Homan1,

Peter L. Jansen2 and Han Moshage1.

1Center for Liver, Digestive and Metabolic Diseases, University Medical Center Groningen,

Groningen, The Netherlands. 2Department of Gastroenterology and Hepatology, Academic

Medical Center Amsterdam, The Netherlands.

In preparation

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Abstract Recent studies suggest that oxidative stress and insulin resistance are important in the pathogenesis of nonalcoholic fatty liver disease (NAFLD) and the pathophysiology of diabetes complications. Metformin has been shown to be hepatoprotective in the insulin-resistant and leptin-deficient ob/ob mouse model of NAFLD. However, the mechanism of its protective effect has not been elucidated. Aim: To investigate the protective effect of metformin against oxidative stress-induced apoptosis. Primary hepatocytes were exposed to the superoxide anion-donor menadione and/or metformin. Apoptosis was determined by measuring caspase activity and PARP-cleavage, and necrosis was measured by Sytox Green nuclear staining. Results: 1) Metformin inhibits menadione-induced caspase-9,-6,-3 activation, PARP-cleavage and apoptosis in a concentration dependent manner. 2) Metformin increases menadione-induced HO-1 expression and inhibits JNK-phosphorylation. 3) Metformin does not induce necrosis in primary hepatocytes. In conclusion, metformin protects hepatocytes against superoxide anions-induced caspase activation, PARP-cleavage and apoptosis. The anti-apoptotic effect of metformin is in part dependent on HO-1 overexpression and inhibition of JNK activation. Our results elucidate a novel mechanism of metformin in the protection against oxidative stress-induced liver injury.

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Introduction Oxidative stress may induce cell death (apoptosis and/or necrosis) in hepatocytes (1-3). Apoptosis, or programmed cell death (4-6), is an active process characterized by cell shrinkage, chromatin condensation, caspases activation (7-9) and formation of apoptotic bodies. In contrast, necrosis is passive and associated with ATP depletion, rupture of the plasma membrane and spilling of the cellular content eliciting inflammation (5). Recent studies suggest that oxidative stress and insulin resistance are important in the pathogenesis of non-alcoholic fatty liver disease (NAFLD) (10, 11) and the pathophysiology of diabetes complications (12-19). Metformin has been shown to be hepatoprotective in the insulin-resistant and leptin-deficient ob/ob mouse model of NAFLD (20). Metformin also prevents endotoxin-induced liver injury after partial hepatectomy (21) and protects against carbon tetrachloride hepatotoxicity in mice (22). In addition, metformin has been proposed to be beneficial in patients with NASH (non-alcoholic steatohepatitis) (23). Apart from its insulin-sensitizing mode of action, the mechanism involved in the protective effect of metformin has not been fully elucidated yet. In this study we investigate whether metformin protects against oxidative stress-induced apoptosis in primary rat hepatocytes. We demonstrate that metformin protects primary rat hepatocytes against superoxide anions-induced apoptosis. The protective effect of metformin is in part dependent on HO-1 overexpression and inhibition of JNK activation. Materials and Methods Animals Specified pathogen-free male Wistar rats (220-250 g) were purchased from Harlan (Zeist, The Netherlands). They were housed under standard laboratory conditions with free access to standard laboratory chow and water. Experiments were performed following the guidelines of the local Committee for Care and Use of laboratory animals. Rat hepatocyte isolation. Hepatocytes were isolated as described previously (24) and cultured in William’s E medium (Invitrogen; Breda, The Netherlands) supplemented with 50µg/mL gentamicin (Invitrogen) without the addition of hormones or growth factors. During the attachment period (4hrs) 50nmol/L dexamethasone (Sigma) and 5% fetal calf serum (Invitrogen) were added to the medium. Cells were cultured in a humidified incubator at 37°C and 5% CO2. Hepatocyte viability was always more than 90% and purity more than 95% as determined by Trypan Blue exclusion assay. All the experiments were performed in the absence of insulin. Experimental design. Experiments were started 24hrs after isolation of hepatocytes. Cells were exposed to the intracellular superoxide anion-donor menadione (2-methyl-1,4-naphthoquinone, 50µmol/L Sigma) (25) for the indicated time and/or metformin (0.1-0.5mmol/L), added 10 min before menadione treatment.

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Caspase enzyme activity assays Caspase-3 enzyme activity was assayed as described previously (26). Caspase-6 activity was assayed according to the manufacturer’s instructions (BioVision). Glutathione determination Total glutathione (the sum of oxidized and reduced glutathione) was determined in liver homogenates and cell lysates by the enzymatic recycling procedure using GSH reductase and 5,5’-dithiobis 2-nitrobenzoic acid as previously described (27). Values were normalized for protein content of the samples. Sytox Green nuclear stainings Rupture of the plasma membrane distinguishes necrotic from apoptotic cell death. To estimate necrotic cell death, hepatocytes were incubated 15 minutes with SYTOX green (Molecular Probes, Eugene, USA) nucleic acid stain, which penetrates cells with compromised plasma membranes but does not cross the membranes of viable cells or apoptotic bodies. Fluorescent nuclei were visualized using an Olympus CKX41 microscope at 450-490 nm. Western Blot analysis. Western blot analysis of cell lysates was performed by SDS-PAGE followed by transfer to Hybond ECL nitrocellulose membrane (Amersham). An antibody against GAPDH (Calbiochem) and Ponceau S staining were used to ensure equal protein loading and electrophoretic transfer. Caspase cleavage was detected using polyclonal rabbit antibodies against cleaved caspase-9, -6, and -3. Poly(ADP-ribose) polymerase (PARP) cleavage was detected with rabbit anti-PARP polyclonal antibody. PARP (116 kDa) is a substrate of caspase-3 yielding a product of 89 kDa and it is considered a late marker for apoptosis. After Western blot analysis for p-JNK (monoclonal, Santa Cruz Biotechnology), blots were stripped using 0.1 % SDS in PBS/Tween-20 at 65°C for 30 minutes and incubated with antibodies against total-JNK. Antibodies were obtained from Cell Signalling Technology (Beverly, MA) and used at 1:1000 dilution. RNA isolation and reverse-transcriptase polymerase chain reaction (RT-PCR). RNA was isolated using the Tri-reagent (Sigma) according to the manufacturer’s instructions. Reverse transcription was performed on 5µg of total RNA using random primers in a final volume of 75µl (Reverse Transcription System, Sigma). Quantitative Real-Time PCR. Reverse transcription was performed on 2.5µg of total RNA using random primers in a final volume of 50µl (Reverse Transcription System, Sigma). Real time detection was performed on the ABI PRISM 7700 (PE Applied Biosystems) initialized by 10 min at 95°C, followed by 40 cycles of 15sec at 95°C, and 1 min at 60°C. Each sample was analyzed in duplicate. 18S mRNA levels were used as an endogenous control. Real time-PCR primers and probes for HO-1 were previously described (chapter 4).


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Statistical analysis. All data are expressed as the mean of at least 3 independent experiments ± standard deviation. Statistical significance was determined by the Mann-Whitney test; p<0.05 was considered statistically significant. Results Metformin protects against menadione-induced caspase activation, PARP cleavage and apoptosis. We investigated whether metformin exerts a protective effect against oxidative stress-induced apoptosis. As shown in figure 1A, metformin prevents superoxide anions-induced apoptosis (expressed as caspase-3 activity) in a concentration dependent manner in primary hepatocytes. In addition, metformin dose-dependently inhibits menadione-induced PARP-cleavage, which is considered a late marker for apoptosis (Fig. 1B), suggesting a protective role of metformin against superoxide anions-induced apoptosis.

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Figure 1. Metformin prevents superoxide anions-induced apoptosis and PARP cleavage in a concentration dependent manner. Primary hepatocytes were exposed to metformin (Met, 0.1mmol/L and 0.5mmol/L) 10min before and during menadione (Men, 50µmol/L, 9hrs) treatment. Control (con, untreated cells). A) Caspase-3 activity assay. B) Western Blot was performed against native (116 kDa) and cleaved (89 kDa) PARP. GAPDH was used as a loading control.

In a previous study we showed that menadione-induced apoptosis is dependent on caspase activation in primary hepatocytes (28). Because of the important role of caspases in apoptosis, we analysed whether metformin has an effect on caspase activation. Metformin prevents menadione-induced capase-6 activity (Fig. 2A) and caspase-9, -6 and -3 processing in a dose dependent manner in primary rat hepatocytes (Fig. 2B). Metformin did not induce necrosis in primary hepatocytes (Fig. 3), indicating that metformin is protective against oxidative stress-induced apoptosis and does not switch the balance between apoptosis and necrosis. Next, we examined the mechanism involved in the protective effect of metformin against superoxide anions-induced apoptosis.

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Figure 2. Metformin blocks menadione-induced casapse-9, -6 and -3 activation in a dose dependent manner in primary rat hepatocytes. Cells were incubated with the superoxide anion-donor menadione (men, 50µmol/L, 9hrs) or not (control, untreated cells) in the presence and absence of metformin (Met, 0.1mmol/L and 0.5mmol/L, added 10min before and during menadione treatment). A) Caspase-6 activity assay: Metformin dose-dependently prevents caspase-6 activation. B) Western Blot: Metformin inhibits caspase-9, -6 and -3 processing and cleavage induced by the superoxide anion-donor menadione in a concentration dependent manner. GAPDH was using as a loading control.

Figure 3. Metformin does not induce necrotic cell death in primary hepatocytes. Cells were incubated with menadione (Men, 50µmol/L, 9hrs) and without (Con, untreated cells) in the presence and absence of metformin (Met, 0.1 mmol/L and 0.5mmol/L, added 10min before menadione exposure). Sytox Green nucleic staining: Necrotic cells represent less than 2% from total cells in all condition tested. Upper panel phase contrast and fluorescence, lower panel fluorescence only. Magnification 40x.

Metformin reduces superoxide anions-induced JNK phosphorylation Menadione-induced apoptosis is dependent on JNK activity, since inhibition of JNK activation abolished superoxide anions-induced apoptosis, demonstrating an important pro-apoptotic role for JNK in primary hepatocytes (28) Metformin does not induce JNK-phosphorylation, but reduces menadione-induced JNK-phosphorylation (Fig. 4). These results suggest that the protective effect of metformin against superoxide anions-induced apoptosis is in part via inhibition of JNK activation.

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Metformin upregulates menadione-induced HO-1 expression Heme Oxygenase-1 has been described to be cytoprotective against oxidative stress. Previously, we showed that the superoxide anion donor menadione induces HO-1 expression in a time and concentration dependent manner, but this is not sufficient to prevent apoptosis. Overexpression of HO-1, using the adenovirus Ad5HO-1, leads to protection against oxidative stress-induced apoptosis in primary hepatocytes (chapter 4). In this study, we investigate the effect of metformin on HO-1 expression. As shown in Fig. 5, metformin further increases menadione-induced HO-1 expression. Remarkably, metformin alone does not induce HO-1 expression in primary hepatocytes.

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Metformin does not modulate the anti-oxidant glutathione level To investigate whether metformin modulates the content of the anti-oxidant glutathione, we determined total glutathione content in our experimental conditions. We demonstrate that menadione slightly (10% compared to control level) reduces glutathione in primary hepatocytes (28). Furthermore, glutathione content is not modulated by metformin, suggesting that the antioxidant GSH is not involved in the protective effect of metformin against superoxide anions-induced apoptosis.

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Figure 6. Metformin does not modulate total glutathione levels. Cells were exposed to menadione (Men, 50µmol/L, 9 hrs) or not (control, untreated cells) in the presence and absence of metformin (Met, 0.1-0.5mmol/L, added 10 min before menadione treatment). As a positive control to decrease total glutathione, D,L-buthionine-(S,R)-sulfoximine (BSO, 200µmol/L) was added 15 hrs before and 30 min before the start of the experiment.

Discussion As an insulin sensitizing agent, metformin is used in the treatment of type 2 diabetes (29-31), a disorder that has been associated with oxidative stress (13-19). In addition, metformin has been demonstrated to be hepatoprotective (20-23). In this study, we document that metformin protects primary rat hepatocytes against superoxide anions-induced caspase-9, -6 and -3 activation, PARP cleavage and apoptosis in a concentration dependent manner. Our results are in accordance with recent studies, in which metformin prevents high glucose-induced endothelial cell death (32) and ameliorates functional defects, caspase activation and apoptosis in pancreatic islets from type 2 diabetic patients (15). In contrast, it has been described that exposure to metformin for more than 24hrs induces apoptosis (33). Moreover, metformin inhibits caspase-9 activation suggesting that metformin prevents disruption of mitochondria and subsequent apoptosis (34-36). In endothelial cells, it has been shown that metformin blocks the mitochondrial permeability transition and cytochrome c released induced by high glucose-derived oxidative stress in endothelial cells (32). In a previous study, we demonstrated that, under certain conditions, inhibition of apoptosis may induce necrosis (28). In the present report, we provide evidence that metformin indeed protects hepatocytes exposed to oxidative stress but does not cause necrosis. We reported that superoxide anions-induced apoptosis is dependent on JNK pro-apoptotic activity, since inhibition of JNK activity, using the inhibitor SP600125, prevents menadione-induced caspase activation and apoptosis in primary hepatocytes (28). Concerning the involvement of JNK in the protective effect of metformin against superxide anions-induced apoptosis, we demonstrate that metformin reduces JNK-phosphorylation and its pro-apoptotic activity. Therefore, our results indicate that the protective effect of metformin against superoxide anions-induced apoptosis is at least in part via inhibition of JNK activation. JNK has been described to be a crucial mediator of insulin resistance and a critical element in the pathogenesis of fatty liver disease and type 2 diabetes (37, 38). JNK activity has been reported to be highly elevated in diabetes, due to the presence of inflammation (TNF-α), free fatty acids and oxidative stress, all features associated with diabetes and obesity. JNK can

% Total glutathione content


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directly phosphorylate insulin receptor substrate (IRS)-1 inducing defects in insulin signalling and consequently insulin resistance (39). Thus, it has been reported that JNK-deficient obese mice are protected against insulin resistance and defective insulin receptor signalling (38). In addition, dominant-negative JNK isoform in the livers of obese animal results in increases insulin sensitivity and glucose homeostasis. Furthermore, exogenous expression of JNK in adult liver results in insulin resistance in mice (40). Metformin-induced inhibition of JNK phosphorylation could be related to its well-known properties as an insulin sensitizing agent. However, in our study, experiments were performed in the absence of insulin. Therefore, it is unlikely that the protective effect of metformin is related to early events in insulin signal transduction. Nevertheless, it cannot be excluded that metformin interferes in down-stream steps of insulin signal transduction, e.g. inhibition of JNK activation The mechanisms by which JNK exert its pro-apoptotic properties are not completely understood. An effect of JNK has been described at the level of the mitochondria triggering the mitochondrial death pathway, including phosphorylation and activation of pro-apoptotic bcl-2 family members (41-47). Our result correlates with a previous study, in which basal protein expression of JNK was reduced after metformin treatment in Wistar rats (48). In contrast, exposure to metformin for more than 24hrs induces JNK phosphorylation that leads to apoptosis (33). Heme oxygenase-1 (HO-1) has been shown to be protective (49-55). Previously, we demonstrated that overexpression of HO-1, using the adenovirus Ad5HO-1, leads to protection against oxidative stress-induced apoptosis in primary hepatocytes. In this report we document that metformin further increases menadione-induced HO-1 expression. Remarkably, metformin alone does not induce HO-1 expression. We also demonstrate that total glutathione content is not modulated by metformin, suggesting that prevention of GSH depletion is not involved in the protective effect of metformin against superoxide anions-induced apoptosis. However, it should be taken into account that in our study we did not make a distinction between reduced and oxidized GSH. Additional studies are required to investigate whether the ratio of reduced to oxidized GSH increased due to the presence of metformin. In conclusion, in this study we demonstrate a protective role of metformin against superoxide anions-induced apoptosis and we report that metformin causes an increase in menadione-induced HO-1 upregulation and a reduction of JNK activation. Due to the central role of JNK in pathogenesis of oxidative stress-mediated liver diseases, modulation JNK activity by metformin represents a promising and attractive target for the treatment of these disorders. Acknowledgements This work is supported by grants from the Dutch Digestive Diseases Foundation and the J.K. de Cock Foundation Groningen. Part of this work was presented at the annual meeting of the American Association for the Study of Liver Diseases 2005, San Francisco, California.

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Semin Nephrol 1997 Nov;17(6):549-562. 3. Loguercio C, Federico A. Oxidative stress in viral and alcoholic hepatitis. Free Radic Biol Med 2003

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disease. Hepatology 2005 Nov;42(5):987-1000. 11. Machado M, Cortez-Pinto H. Non-alcoholic fatty liver disease and insulin resistance. Eur J

Gastroenterol Hepatol 2005 Aug;17(8):823-826. 12. Du X, Matsumura T, Edelstein D, Rossetti L, Zsengeller Z, Szabo C, et al. Inhibition of GAPDH

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13. Evans JL, Goldfine ID, Maddux BA, Grodsky GM. Are oxidative stress-activated signaling pathways mediators of insulin resistance and beta-cell dysfunction? Diabetes 2003 Jan;52(1):1-8.

14. Maechler P, Wollheim CB. Mitochondrial function in normal and diabetic beta-cells. Nature 2001 Dec 13;414(6865):807-812.

15. Marchetti P, Del Guerra S, Marselli L, Lupi R, Masini M, Pollera M, et al. Pancreatic islets from type 2 diabetic patients have functional defects and increased apoptosis that are ameliorated by metformin. J Clin Endocrinol Metab 2004 Nov;89(11):5535-5541.

16. Nishikawa T, Edelstein D, Du XL, Yamagishi S, Matsumura T, Kaneda Y, et al. Normalizing mitochondrial superoxide production blocks three pathways of hyperglycaemic damage. Nature 2000 Apr 13;404(6779):787-790.

17. Rosen P, Nawroth PP, King G, Moller W, Tritschler HJ, Packer L. The role of oxidative stress in the onset and progression of diabetes and its complications: a summary of a Congress Series sponsored by UNESCO-MCBN, the American Diabetes Association and the German Diabetes Society. Diabetes Metab Res Rev 2001 May;17(3):189-212.

18. Sakuraba H, Mizukami H, Yagihashi N, Wada R, Hanyu C, Yagihashi S. Reduced beta-cell mass and expression of oxidative stress-related DNA damage in the islet of Japanese Type II diabetic patients. Diabetologia 2002 Jan;45(1):85-96.

19. Wiernsperger NF. Oxidative stress: the special case of diabetes. Biofactors 2003;19(1-2):11-18. 20. Lin HZ, Yang SQ, Chuckaree C, Kuhajda F, Ronnet G, Diehl AM. Metformin reverses fatty liver

disease in obese, leptin-deficient mice. Nat Med 2000 Sep;6(9):998-1003. 21. Bergheim I, Luyendyk JP, Steele C, Russell GK, Guo L, Roth RA, et al. Metformin prevents endotoxin-

induced liver injury after partial hepatectomy. J Pharmacol Exp Ther 2006 Mar;316(3):1053-1061. 22. Poon MK, Chiu PY, Mak DH, Ko KM. Metformin protects against carbon tetrachloride hepatotoxicity

in mice. J Pharmacol Sci 2003 Dec;93(4):501-504. 23. Uygun A, Kadayifci A, Isik AT, Ozgurtas T, Deveci S, Tuzun A, et al. Metformin in the treatment of

patients with non-alcoholic steatohepatitis. Aliment Pharmacol Ther 2004 Mar 1;19(5):537-544. 24. Moshage H, Casini A, Lieber CS. Acetaldehyde selectively stimulates collagen production in cultured

rat liver fat-storing cells but not in hepatocytes. Hepatology 1990 Sep;12(3 Pt 1):511-518. 25. Thor H, Smith MT, Hartzell P, Bellomo G, Jewell SA, Orrenius S. The metabolism of menadione (2-

methyl-1,4-naphthoquinone) by isolated hepatocytes. A study of the implications of oxidative stress in intact cells. J Biol Chem 1982 Oct 25;257(20):12419-12425.


27. Griffith OW. Determination of glutathione and glutathione disulfide using glutathione reductase and 2-vinylpyridine. Anal Biochem 1980 Jul 15;106(1):207-212.


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28. Conde de la Rosa L, Schoemaker MH, Vrenken TE, Buist-Homan M, Havinga R, Jansen PL, et al. Superoxide anions and hydrogen peroxide induce hepatocyte death by different mechanisms: Involvement of JNK and ERK MAP kinases. J Hepatol 2006 May;44(5):918-929.

29. Knowler WC, Barrett-Connor E, Fowler SE, Hamman RF, Lachin JM, Walker EA, et al. Reduction in the incidence of type 2 diabetes with lifestyle intervention or metformin. N Engl J Med 2002 Feb 7;346(6):393-403.

30. Inzucchi SE, Maggs DG, Spollett GR, Page SL, Rife FS, Walton V, et al. Efficacy and metabolic effects of metformin and troglitazone in type II diabetes mellitus. N Engl J Med 1998 Mar 26;338(13):867-872.

31. Effect of intensive blood-glucose control with metformin on complications in overweight patients with type 2 diabetes (UKPDS 34). UK Prospective Diabetes Study (UKPDS) Group. Lancet 1998 Sep 12;352(9131):854-865.

32. Detaille D, Guigas B, Chauvin C, Batandier C, Fontaine E, Wiernsperger N, et al. Metformin prevents high-glucose-induced endothelial cell death through a mitochondrial permeability transition-dependent process. Diabetes 2005 Jul;54(7):2179-2187.

33. Kefas BA, Cai Y, Kerckhofs K, Ling Z, Martens G, Heimberg H, et al. Metformin-induced stimulation of AMP-activated protein kinase in beta-cells impairs their glucose responsiveness and can lead to apoptosis. Biochem Pharmacol 2004 Aug 1;68(3):409-416.

34. Wang X. The expanding role of mitochondria in apoptosis. Genes Dev 2001 Nov 15;15(22):2922-2933. 35. Green DR, Reed JC. Mitochondria and apoptosis. Science 1998 Aug 28;281(5381):1309-1312. 36. Saleh A, Srinivasula SM, Acharya S, Fishel R, Alnemri ES. Cytochrome c and dATP-mediated

oligomerization of Apaf-1 is a prerequisite for procaspase-9 activation. J Biol Chem 1999 Jun 18;274(25):17941-17945.

37. Wellen KE, Hotamisligil GS. Inflammation, stress, and diabetes. J Clin Invest 2005 May;115(5):1111-1119.

38. Hirosumi J, Tuncman G, Chang L, Gorgun CZ, Uysal KT, Maeda K, et al. A central role for JNK in obesity and insulin resistance. Nature 2002 Nov 21;420(6913):333-336.

39. Aguirre V, Uchida T, Yenush L, Davis R, White MF. The c-Jun NH(2)-terminal kinase promotes insulin resistance during association with insulin receptor substrate-1 and phosphorylation of Ser(307). J Biol Chem 2000 Mar 24;275(12):9047-9054.

40. Nakatani Y, Kaneto H, Kawamori D, Hatazaki M, Miyatsuka T, Matsuoka TA, et al. Modulation of the JNK pathway in liver affects insulin resistance status. J Biol Chem 2004 Oct 29;279(44):45803-45809.


42. Davis RJ. Signal transduction by the JNK group of MAP kinases. Cell 2000 Oct 13;103(2):239-252. 43. Tournier C, Hess P, Yang DD, Xu J, Turner TK, Nimnual A, et al. Requirement of JNK for stress-

induced activation of the cytochrome c-mediated death pathway. Science 2000 May 5;288(5467):870-874.





48. Cleasby ME, Dzamko N, Hegarty BD, Cooney GJ, Kraegen EW, Ye JM. Metformin prevents the development of acute lipid-induced insulin resistance in the rat through altered hepatic signaling mechanisms. Diabetes 2004 Dec;53(12):3258-3266.


50. Ryter SW, Alam J, Choi AM. Heme oxygenase-1/carbon monoxide: from basic science to therapeutic applications. Physiol Rev 2006 Apr;86(2):583-650.

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52. Otterbein LE, Kolls JK, Mantell LL, Cook JL, Alam J, Choi AM. Exogenous administration of heme oxygenase-1 by gene transfer provides protection against hyperoxia-induced lung injury. J Clin Invest 1999 Apr;103(7):1047-1054.




CHAPTER 6

TUDCA protects rat hepatocytes from

GCDCA-induced mitochondria-controlled

apoptosis via activation of survival pathways

Marieke H. Schoemaker1, Laura Conde de la Rosa1, Manon Homan1, Titia E.

Vrenken1, Rick Havinga2, Klaas Poelstra1, Hidde J. Haisma3, Peter L.M. Jansen1

and Han Moshage1.

Center for Liver, Digestive and Metabolic Diseases1, Laboratory of Pediatrics2, Department

of Therapeutic Gene Modulation3, Groningen University Institute for Drug Exploration, The

Netherlands.

Hepatology 2004;39:1563-1573.

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Abstract Ursodeoxycholic acid (UDCA) is used in the treatment of cholestatic liver diseases, but its mechanism of action is not well defined yet. The aim of this study is to explore the protective mechanisms of the taurine-conjugate of UDCA (tauroursodeoxycholic acid (TUDCA)) against glycochenodeoxycholic acid (GCDCA)-induced apoptosis in primary cultures of rat hepatocytes. Hepatocytes were exposed to GCDCA, TUDCA, the glyco-conjugate of UDCA (GUDCA) and taurochenodeoxycholic acid (TCDCA). The PI3-kinase (PI3K) pathway and NF-κB were inhibited using LY 294002, and adenoviral overexpression of dominant negative IκB, respectively. The role of p38 and ERK MAP kinase pathways were investigated using the inhibitors SB 203580 and U0 126, and Western blot analysis. Transcription was blocked by actinomycin D. Apoptosis was determined by measuring caspase-3, -9 and -8 activity using fluorimetric enzyme detection, Western blot analysis, immunocytochemistry and nuclear morphology. Our results demonstrated that uptake of GCDCA is needed for apoptosis induction. TUDCA, but not TCDCA and GUDCA, rapidly inhibited, but did not delay, apoptosis at all time points tested. However, the protective effect of TUDCA was independent of its inhibition of caspase-8. Up to six hours pre-incubation with TUDCA prior to GCDCA clearly decreased GCDCA-induced apoptosis. Up to 1.5 hours after exposure with GCDCA, addition of TUDCA was still protective. This protection was dependent on activation of p38, ERK MAP kinases and PI3-kinase pathways, but independent of competition on the cell membrane, NF-κB activation and transcription. In conclusion: TUDCA contributes to the protection against GCDCA-induced mitochondria-controlled apoptosis by activating survival pathways.

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Introduction Cholestatic liver diseases are characterized by accumulation of toxic bile acids, e.g. glycochenodeoxycholic acid (GCDCA), causing damage to hepatocytes and cholangiocytes. Ursodeoxycholic acid (UDCA) is used as a treatment for patients with chronic cholestatic liver diseases. In primary biliary cirrhosis, doses of 13 to 15 mg/kg/day of UDCA decrease serum liver enzymes, improve liver histology, and delay the time to liver transplantation or death for up to 4 years. (1) (2) However, the mechanisms of the beneficial effect of UDCA in these conditions remain unclear. From in vitro and in vivo studies it is postulated that UDCA protects cholangiocytes against membrane damage induced by hydrophobic bile acids. (3) (4) Furthermore, it has been demonstrated that UDCA stimulates biliary secretion of bile acids and other toxic compounds. (5) (6) In addition, anti-apoptotic effects of UDCA have been described, such as the inhibition of the mitochondrial membrane permeability transition in hepatocytes,(7) (8) leading to prevention of the mitochondrial release of cytochrome c.(9) A recent study has suggested that TUDCA does inhibit apoptosis by preventing the binding of Bax to mitochondria. (10) Hepatocytes are exposed to many pro-apoptotic compounds. Therefore, anti-apoptotic signalling pathways are important to limit programmed cell death. The anti-apoptotic action of UDCA may in part be due to activation of these anti-apoptotic pathways. A major survival pathway in hepatocytes is the activation of the transcription factor NF-κB. Activation of NF-κB-regulated survival genes causes inhibition of apoptosis. (11) (12). Although we have shown that NF-κB is not activated by bile acids (13), its role in the protection of UDCA against bile acid-induced apoptosis is not clear. Furthermore, other cell survival pathways, like the activation of mitogen-activated protein kinases (MAPK), could be involved in the anti-apoptotic action of UDCA. These kinases are involved in regulation of cell proliferation, differentiation and apoptosis and are comprised of at least three different pathways: ERK, p38 and JNK. (14) Although, it is postulated that inhibition of ERK enhances UDCA-induced apoptosis, (15) little is known about the role of MAP kinases in the protection of taurine-conjugated UDCA against GCDCA-induced apoptosis. Another important survival pathway is the phosphatidylinositol-3 kinase (PI3K) pathway. This kinase cascade results in activation of a number of cellular intermediates of which Akt seems to be one of the most important survival factors. (16) The mechanisms by which PI3K/Akt promote cell survival are diverse and its role in the protection of UDCA against GCDCA-induced apoptosis has not been explored yet. In rats, the taurine conjugate of UDCA predominates compared to glycine-conjugated UDCA.(17;18) Furthermore, TUDCA may be of benefit for patients suffering from primary biliary cirrhosis (19). Therefore, we have investigated the anti-apoptotic actions of TUDCA in primary rat hepatocytes. Materials and methods Animals Specified pathogen-free male Wistar rats (220-250 g) were purchased from Harlan, Zeist, the Netherlands. They were housed under standard laboratory conditions with free access to standard laboratory chow and water. Each experiment was performed following the guidelines of the local Committee for Care and Use of Laboratory Animals.

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Hepatocyte isolation Hepatocytes were isolated as described previously (20). Cell viability was consistently more than 90 % as determined by trypan blue exclusion. Isolated hepatocytes were cultured in William’s medium E (Life Technologies Ltd., Breda, The Netherlands) supplemented with 50 µg/ml gentamycin (BioWhittaker, Verviers, Belgium) without the addition of hormones or growth factors. During the attachment period (4 hours) 50 nmol/L dexamethasone (Sigma, St Louis, MO) and 5 % fetal calf serum (Life Technologies Ltd.) were added to the medium. Experimental design Experiments were started twenty-four hours after isolation. Hepatocytes were exposed to 50 µmol/L GCDCA (Calbiochem, La Jolla, CA), 50 µmol/L TUDCA (Calbiochem), 50 µmol/L glyco-ursodeoxycholic acid (GUDCA, Calbiochem), or 50 µmol/L TCDCA (Calbiochem) for 4 hours or the indicated time period. In some experiments, hepatocytes were exposed to 20 ng/ml recombinant mouse tumor necrosis factor α (TNFα, R&D Systems, Abingdon, United Kingdom) or a mixture of cytokines as described previously (11). Signal transduction or apoptosis pathways were specifically inhibited with the following compounds: 50 µmol/L of caspase-8 inhibitor Ac-IETD-CHO or the caspase-3 inhibitor Ac-DEVD-CHO (Biomol, Plymouth Meeting, USA), 10 µmol/L of the p38 inhibitor SB 203580 (Biomol), 10 µmol/L of the ERK1/2 inhibitor U0126 (Promega, Madison, USA), 50 µmol/L PI3-kinase inhibitor LY 294002 (Sigma-Aldrich), and 200 ng/ml of the transcriptional inhibitor actinomycin-D (Roche Diagnostics, Almere, The Netherlands). All inhibitors were added 30 minutes prior to bile acids or cytokines. Hepatocytes received adenovirus (MOI of 10) fifteen hours prior to exposure of bile acids. Each experimental condition was performed in triplicate wells. Each experiment was performed at least three times, using hepatocytes from different isolations.Cells were harvested at the indicated times after the addition of bile acids as described previously (11). HepG2 cell experiments The human hepatoma cell line HepG2 and a stable derivative expressing rNtcp were cultured as described before (21;22). Cells were incubated with indicated amounts of GCDCA or 1 µg/ml anti-Fas antibody (clone nr. 7C11; Immunotech, Marseille, France) for 4 hours followed by harvesting in hypotonic cell lysis buffer (11). Adenoviral constructs Adenoviral constructs have been described previously (23).

Caspase-3 and caspase-8 enzyme activity assay Hepatocytes were scraped and cell lysates were obtained by three cycles of freezing (-80 °C) and thawing (37 °C) followed by centrifugation for 5 minutes at 13.000 g. Caspase-3 and caspase-8 enzyme activities were assayed as described previously (11,13).


81

Nuclear staining Morphological features of apoptotic nuclei were demonstrated with acridine orange. Cells were seeded on glass coverslides and treated as indicated. These coverslides were fixed in methanol for 5 minutes, air-dried and rinsed twice in phosphate buffer saline before incubating in acridine orange (1:1000) for 15 minutes in the dark. Fluorescent nuclei were visualized using a Leica confocal laser scanning microscope. Immunocytochemistry and uptake of fluorescent bile acids Analysis of active caspase-9 was performed on hepatocytes cultured on coverslips and exposed to 50 µmol/L bile acids for 4 hours. Coverslips were washed in phosphate buffer saline, fixed in 4 % paraformaldehyde for 10 minutes followed by incubation in 1 % Triton-X100 for 5 minutes. Antibody against active caspase-9 was used at a dilution of 1:50 for 30-60 minutes. Fluorexein isothiocyanate-conjugated goat-anti-rabbit Ig (Molecular Probes, Eugene, Oregon, USA) was added at a dilution of 1:600 for 45 minutes. The uptake of bile acids was demonstrated using the fluorescent bile acid cholyl lysyl fluorescein (CLF) (24). Cells were incubated with 2 µmol/L CLF at 37 °C for 15 minutes. All slides were evaluated on a Leica confocal laser scanning microscope. Western blot analysis Western blot analysis of cell lysates was performed using polyclonal rabbit antibodies against cleaved caspase-9 and phosphorylated p38 MAP, and monoclonal antibody against phosphorylated ERK1/2 (p44/42) MAP kinase (Cell Signaling Technology, Beverly, MA) at a dilution of 1:1000. Hepatocytes exposed to 50 µmol/L deoxycholic acid (DCA) served as positive control for phophorylated ERK1/2(25). In addition, activated neutrophil extracts (kindly provided by Dr. Gwenny Fuhler, Department of Haematology, University Hospital Groningen) were used to confirm detection of phosphorylated ERK1/2 MAP kinase. For the detection of caspase-9 and phospho-p38, horse radish-peroxidase conjugated swine-anti-rabbit Ig was used as a secondary antibody at a dilution of 1:2000. Phospho-ERK1/2 was detected with horse radish-peroxidase rabbit-anti-mouse Ig (1:2000). Each lane contained the lysate of 150000 cells. Equal loading was demonstrated by Ponceau-S staining. After Western blot analysis of phosphorylated p38 and ERK1/2 MAP kinases, blots were stripped using 0.1 % SDS at 65 °C for 30 minutes and incubated with 1:1000 antibody against total p38 MAP kinase or total ERK1/2 MAP kinase (Santa Cruz Biotechnology, Santa Cruz, USA). Western blot analysis for iNOS was performed as described before (26). Statistical analysis Results are presented as the mean of at least 3 independent experiments ± standard deviation. A Mann-Whitney test was used to determine the significance of differences between two experimental groups. A P value of less than 0.05 (P < 0.05) was considered to be statistically significant.

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Results Ntcp is required for GCDCA-induced mitochondria-controlled apoptosis To investigate the protective mechanisms of TUDCA against GCDCA-induced apoptosis, we first determined whether GCDCA needs to be taken up by cells to induce apoptosis. Previous studies have postulated death receptor activation by toxic bile acids (27-29). However, strong distinction between the role of the bile acid uptake transporter Ntcp and death receptor-mediated apoptosis has not been made yet. Recently, we have shown that GCDCA induced apoptosis in a mitochondrial-controlled manner in primary rat hepatocytes, which was FADD-independent. (13) In the present study, we examined whether Ntcp is required for GCDCA-induced apoptosis. For this purpose, we exposed Ntcp-negative and Ntcp-positive HepG2 cells(21) to different concentrations of GCDCA. Both cell lines do express Fas death receptor. Anti-Fas antibody served as positive control. As shown in Figure 1A, only HepG2 cells expressing Ntcp on their cell membrane are sensitive to GCDCA-induced apoptosis, which increased with increasing amounts of GCDCA. In contrast, anti-Fas antibody induced apoptosis in both Ntcp-positive and Ntcp-negative cell lines to the same extent. These data indicate that GCDCA first needs to be taken up by Ntcp before the onset of apoptosis and that ligand-dependent death receptor activation is not involved. To exclude HepG2-specific artifacts, primary hepatocytes were exposed to GCDCA at 24 hours and 72 hours after isolation. Ntcp was expressed at high levels 24 hours after isolation (data not shown) but decreased in hepatocytes maintained in primary cultures for 72 hours, thereby confirming previous data.(30) In addition, uptake of bile acids in these hepatocytes decreased in time, as shown with the fluorescent bile acid CLF (Fig 1C). Accumulation of CLF was only detected in bile canaliculi of hepatocytes cultured for 24 hours, as previously reported.(24) At this time point, GCDCA strongly induced caspase-3 activity. In contrast, GCDCA did not induce caspase-3 activity anymore in hepatocytes cultured for 72 hours (Fig 1B). Induction of caspase-3 activity by TNF-α in the presence of actinomycin-D remained unaffected. All together, these data provide evidence that Ntcp is required for GCDCA-induced apoptosis in primary hepatocytes.


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TUDCA inhibits but does not delay GCDCA-induced caspase-3 activity and apoptotic nuclear morphology GCDCA-induced caspase-3 activity in hepatocytes peaks around 4 hours. (13) Therefore, at this time-point, we investigated the effect of TUDCA on GCDCA-induced caspase-3 activity in primary hepatocytes. TUDCA itself did not induce caspase-3 activation (Fig 2a) confirming previous data. (13) TUDCA, but not TCDCA and GUDCA, inhibited GCDCA-induced caspase-3 activity for 70 % as shown in Figure 1a. A concentration-dependent curve displayed that the minimal concentration exerting the maximal protective effect is 50 µM of TUDCA (Fig 2b). To demonstrate that TUDCA inhibits but does not delay GCDCA-induced caspase-3 activity, a time course study was performed. Two to 15 hours after addition of GCDCA + TUDCA, caspase-3 activity was inhibited significantly at all time-points (Fig 2c). Nuclei staining was performed with acridine orange confirming that GCDCA-induced activation of caspase-3 activity results in apoptosis. Nuclear fragmentation and condensation were observed 4 hours after the addition of GCDCA, which increased after 8 hours and persisted up to 15 hours. After 8 hours, 30 to 40 percent of hepatocytes displayed apoptotic nuclei, which was inhibited in the presence of TUDCA to 5 percent (Fig 2d). Cytokine-

Figure 1. Ntcp is required for glycochenodeoxycholic acid (GCDCA)-induced apoptosis. (A) Ntcp positive and negative HepG2 cells were treated with different amounts of GCDCA and 1 µg/µl anti-Fas antibody (positive control) for 4 hours. Only in Ntcp-positive HepG2 cells, caspase-3 activity increased with increasing amounts of GCDCA. *P<0.05 for GCDCA 100 µM and anti-Fas antibody vs. control. (B) Caspase-3 activity in primary rat hepatocytes exposed to 50 µmol/L GCDCA or 20 ng/ml TNF-α in the presence of actinomycin-D (ActD) at 24 hours and 72 hours after isolation. (C) Uptake of bile acids demonstrated by fluorescence microscopy. Primary hepatocytes were exposed to 2 µmol/L of the fluorescent bile acid cholyl lysyl fluorescein (CLF) for 15 minutes. Accumulation is observed in the bile canaliculus between adjacent hepatocytes at 24 hours but not 72 hours after isolation. (Original magnification is 400x).

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exposed hepatocytes in which transcription was blocked with actinomycin-D served as positive control.

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Figure 2. Tauroursodeoxycholic acid (TUDCA), but not taurochenodeoxycholic acid (TCDCA) and glycoursodeoxycholic acid (GUDCA), inhibits glucochenodeoxycholic acide (GCDCA)-induced caspase-3 activity and nuclear fragmentation. (A) Primary rat hepatocytes were stimulated for 4 hours with 50 µmol/L of GCDCA, 50 µmol/L of TUDCA, 50 µmol/L of TCDCA, 50 µmol/L of GUDCA, or a combination thereof.. Caspase-3 activity is presented as percentage of GCDCA alone. Data represent mean of at least 3 independent experiments with n = 3 per condition. *P < 0.05 for GCDCA + TUDCA vs. GCDCA alone. (B) Caspase-3 activity. Primary rat hepatocytes were stimulated for 4 hours with 50 µmol/L of GCDCA and different amounts of TUDCA, as indicated in the figure. *P<0.05 for GCDCA + 50, 25, and 10 µM TUDCA vs. GCDCA alone. (C) Time course study. TUDCA (50 µmol/L) significantly inhibits GCDCA-induced caspase-3 activity at all indicated time-points. Representative data of 3 independent experiments are shown.. (D) Nuclear morphology in hepatocytes as determined by acridine orange staining. Cells were treated for 8 hours as indicated in the figure. Treatment with 50 µmol/L GCDCA induces nuclear condensation and fragmentation, which persists for at least 15 hours and is blocked with 50 µmol/L TUDCA. Hepatocytes treated with cytokinemix (CM) + actinomycin-D (ActD) for 15 hours served as positive control

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The protective action of TUDCA depends on the inhibition of caspase-9 activation

Because we previously noticed that GCDCA induces apoptosis in a mitochondria-controlled manner (13), the effects of TUDCA on caspase-9 and caspase-8 were investigated. As shown in Figure 3, TUDCA also prevented GCDCA-induced activation of caspase-9. Both Western blot (Fig 3A) and immunocytochemistry (Fig 3B) demonstrated this effect.

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Although TUDCA inhibited GCDCA-induced caspase-8 activity for 50 % (Fig 4B), we have previously shown that caspase-8 inhibition does not inhibit GCDCA-induced caspase-3 activity. (13) In contrast, the caspase-3 inhibitor Ac-DEVD-CHO and the caspase-9 inhibitor Ac-LEHD-CHO inhibited GCDCA-induced activation of both caspase-3 (Fig 4A) and caspase-8 (Fig 4B). These data demonstrate that inhibition of caspase-9 is more important in the TUDCA-mediated protection against GCDCA-induced apoptosis than caspase-8 inhibition. In contrast to TUDCA, TCDCA and GUDCA did not inhibit GCDCA-induced caspase-9 Activity (Fig 3) and caspase-8 activity (Fig 4B).

Figure 3. Tauroursodeoxycholic acid (TUDCA), but not taurochenodeoxycholic acid (TCDCA) or glycoursodeoxycholic acid (GUDCA), prevents glycochenodeoxycholic acid (GCDCA)-induced activation of caspase-9. Primary rat hepatocytes were exposed for 4 hours to 50 µmol/L of GCDCA, 50 µmol/L of TUDCA or both, plus TCDCA or GUDCA. (A) Western blot analysis on cell lysates for active caspase-9. (B) Immunocytochemistry for active caspase-9 (original magnification is X 400). AFU, arbitrary fluorescent units

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Figure 4. Caspase-3 and caspase-8 activation in glycochenodeoxycholic acid (GCDCA)-exposed primary rat hepatocytes in the presence of bile acids and caspase inhibitors. Representative data of 3 independent experiments are shown with n = 3 per condition. (A) Caspase-3 activity. Cells were treated for 4 hours with 50 µmol/L of (GCDCA) plus 50 µmol/L caspase-8 inhibitor, 50 µmol/L caspase-9 inhibitor, or 50 µmol/L caspase-3 inhibitor. *P<0.05 for GCDCA + caspase-9 inhibitor and GCDCA + caspase-3 inhibitor vs. GCDCA. (B) Caspase-8 activity. Cells were treated for 4 hours with 50 µmol/L of GCDCA plus 50 µmol/L tauroursodeoxycholic acid (TUDCA), 50 µmol/L taurochenodeoxycholic acid (TCDCA), or 50 µmol/L glycoursodeoxycholic acid (GUDCA), 50 µmol/L caspase-8 inhibitor, 50 µmol/L caspase-9 inhibitor, or 50 µmol/L caspase-3 inhibitor. *P<0.05 for GCDCA + TUDCA vs. GCDCA. AFU, arbitrary fluorescent units. TUDCA does not compete with GCDCA for uptake at the cell membrane Next, we investigated whether TUDCA needs to be present at the same time with GCDCA, before GCDCA or after the addition of GCDCA, to exert its protective effect. For this purpose, we pre-incubated hepatocytes with TUDCA for 9, 6 and 3 hours, washed these cells and exposed them to GCDCA in fresh medium for 4 hours. Six and 3 hours pre-incubation with TUDCA significantly inhibited GCDCA-induced caspase-3 activity for 50 % (Fig 5A). Pre-incubation with TUDCA for 3 hours also inhibited GCDCA-induced caspase-9 activation (Fig 5B). Furthermore, exposure to TUDCA up to 90 min after the addition of GCDCA still exerted protection against GCDCA-induced apoptosis (Fig 5C). At this time point, GCDCA-induced apoptosis had not reached its maximum yet (Fig 5D), confirming previous results.(13) These data indicate that the protective effect of TUDCA is not due to competition with GCDCA for uptake at the cell membrane. In addition, these data imply that the anti-apoptotic actions of TUDCA are very rapidly induced since simultaneous addition or addition of TUDCA after GCDCA is still able to prevent apoptosis in these cells. This could mean that signalling cascades are activated by TUDCA.

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Figure 5. Tauroursodeoxycholic acid (TUDCA) does not compete with glycochenodeoxycholic acid (GCDCA) for uptake at the cell membrane. (A) Pre-incubation with TUDCA inhibits GCDCA-induced apoptosis. Primary rat hepatocytes were exposed to 50 µmol/L of TUDCA for 9 (-9 hr), 6 (-6 hr) and 3 (-3 hr) hours after which cells were washed and exposed to 50 µmol/L of GCDCA for 4 hours. Cells were also incubated for 4 hours with GCDCA alone or with simultaneous addition of TUDCA (simultaneous). Caspase-3 activity is presented as percentage of GCDCA alone. *P<0.05 for -6 hours, -3 hours and simultaneous incubation of TUDCA + GCDCA vs. GCDCA alone (control). (B) Western blot analysis for active caspase-9 on cell lysates of hepatocytes pre-incubated for 3 hours with TUDCA followed by addition of GCDCA in refreshed medium for 4 hours. (C) Caspase-3 activity. Primary rat hepatocytes were incubated with 50 µmol/L of GCDCA for 4 hours (only GCDCA). Simultaneously (0-4 hr) or half an hour (0.5-4 hr), 1 hour (1-4 hr), 2 hours (2-4 hr), or 3 hours (3-4 hr) after the addition of GCDCA, 50 µmol/L TUDCA was added. All cells were harvested 4 hours after the addition of GCDCA. Representative data of 3 independent experiments are shown with n = 3 per condition. *P<0.05 for (0-4 hr) of TUDCA, (0.5-4 hr) of TUDCA, (1-4 hr) of TUDCA vs. only GCDCA. (D) Time course study of (50 µmol/L) GCDCA-induced caspase-3 activity. Representative data of 3 independent experiments are shown with n = 3 per condition.

NF-κκκκB is not involved in the protection of TUDCA against GCDCA-induced apoptosis Previous results demonstrated that activation of the transcription factor NF-κB resulted in the transcription of survival genes protecting hepatocytes against apoptosis (11) (12). However, TUDCA does not activate NF-κB and does not induce the expression of NF-κB-regulated anti-apoptotic genes (13). Because NF-κB can be activated indirectly, we investigated the role of NF-κB activation in relation to the anti-apoptotic mechanisms of TUDCA. Therefore, primary hepatocytes were infected with recombinant adenovirus expressing dominant negative IκB preventing NF-κB activation. Functionality of this virus was demonstrated by EMSA and sensitizing hepatocytes to cytokine-induced apoptosis (data not shown). As presented in Figure 6, inhibition of the NF-κB survival pathway does not significantly reduce the protective effect of TUDCA against GCDCA-induced caspase-3 activity.

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Figure 6. NF-κB is not involved in the protection of Tauroursodeoxycholic acid (TUDCA) against glycochenodeoxycholic acid (GCDCA)-induced apoptosis. Primary rat hepatocytes were exposed for 4 hours to 50 µmol/L of GCDCA, 10 µmol/L of TUDCA or both, 15 hours after receiving 10 plaque-forming units/cell of recombinant adenovirus inhibiting NF-κB activation (Ad5IkBAA). Ad5LacZ served as control virus. Protection of TUDCA against GCDCA-induced caspase-3 activity did not change significantly. Caspase-3 activity is presented as percentage of GCDCA alone. Data represent mean of at least 3 independent experiments with n = 3 per condition.

Anti-apoptotic action of TUDCA depends on the activation of p38 MAP kinase, ERK MAP kinase, and PI3 kinase, whereas gene transcription is not involved To investigate whether PI3 kinase and MAP kinases pathways are involved in the anti-apoptotic effects of TUDCA, specific inhibitors of PI3K (LY 294002), p38 MAPK (SB 203580) and ERK1/2 MAP kinase (U0126) were used. Dimethyl sulfoxide was used as a solvent for inhibitors but did not have an effect itself (data not shown). Experiments with inhibitors in control hepatocytes and in hepatocytes exposed to TUDCA or GCDCA were included as well. Hepatocytes exposed to inhibitors of MAPK pathways in the absence of bile acids demonstrated caspase-3 values around control level (Fig 7A). Caspase-3 activity increased slightly when PI3 kinase was blocked in control hepatocytes, and this effect was enhanced in combination with MAPK inhibitors. This pattern was similar for hepatocytes exposed to inhibitors in the presence of TUDCA (Fig 7A).


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Figure 7. Tauroursodeoxycholic acid (TUDCA) protects against glycochenodeoxycholic acid (GCDCA)-induced apoptosis by activation of the p38 mitogen-activated protein dinase (MAPk), ERK1/2 MAPK and the PI3K pathway. (A) Caspase-3 activity in primary rat hepatocytes treated as indicated in the figure with 50 µmol/L of GCDCA, 50 µmol/L of TUDCA or both with or without inhibitors of ERK MAPK (10 µmol/L of U0126; UO), p38 MAPK(10 µmol/L of SB 203580; SB), PI3K (50 µmol/L of LY 294002; LY) or a combination thereof. Caspase-3 activity is presented as percentage of GCDCA alone. Data represent mean of at least 3 independent experiments with n = 3 per condition. �P<0.05 for control + LY, control + SB + LY, and for control + UO + LY vs. control. �P<0.05 for TUDCA + SB+LY vs TUDCA and for TUDCA + UO+LY vs TUDCA. �P<0.05 for GCDCA + LY vs GCDCA and for GCDCA + SB+LY vs. GCDCA and for GCDCA + UO+LY vs. GCDCA. �P<0.05 for GCDCA+TUDCA vs. GCDCA. #P<0.05 for GCDCA+TUDCA + inhibitors vs. GCDCA+TUDCA

GCDCA-induced apoptosis was slightly, but not significantly, enhanced with inhibitors of p38 or ERK MAP kinases (Fig 7A). In contrast, inhibition of PI3K pathway aggravated GCDCA-induced caspase-3 activity significantly. In the presence of both MAPK and PI3K inhibitors, exposure to GCDCA significantly increased caspase-3 values compared to GCDCA alone. The protective effect of TUDCA against GCDCA-induced apoptosis was partially, but significantly abolished by inhibition of p38 MAPK pathway (Fig 7A). In support, TUDCA activated p38, which was blocked by SB 203580 as shown in Figure 7B. Western blot demonstrated equal presence of total p38 MAP kinase in all lanes (Fig 7B). Blocking of the PI3 kinase survival cascade resulted in abrogation of TUDCA protection against apoptosis as well (Fig 7A). The protective effect of TUDCA against GCDCA-induced apoptosis was completely abolished when both p38 MAP kinase and PI3 kinase pathways were blocked (Fig 7B).

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Figure 7 (B+C). Tauroursodeoxycholic acid (TUDCA) protects against glycochenodeoxycholic acid (GCDCA)-induced apoptosis by activation of the p38 mitogen-activated protein dinase (MAPk), ERK1/2 MAPK and the PI3K pathway. (B) Western blot analysis for phosphorylated p38 MAPK in control lysates (con) and cell lysates of TUDCA-exposed hepatocytes (20 and/or 60 minutes) with or without p38 MAPK inhibitor (SB). The same blots for total p38 MAP kinase are presented. (C) Western blot analysis for phosphorylated ERK1/2 MAPKin cell lysates of TUDCA-exposed hepatocytes (30 minutes) with and without 10 µmol/L of ERK1/2 inhibitor U0126, and control lysates (con). Positive control was 50 µmol/L of deoxycholic acid (DCA) for ERK1/2 phosphorylation in hepatocytes, whereas an activated neutrophil extract was included for Western blot detection of ERK1/2 (pos. con). Upper panel presents same blot for total ERK1/2 MAPK.

Next, the ERK1/2 MAP kinase was investigated. As shown in Figure 7A, specific inhibition of ERK1/2 MAP kinase using U0126 significantly prevented the protective effect of TUDCA against GCDCA-induced apoptosis. In addition, Western blot demonstrated that TUDCA activates ERK1/2, which can be blocked with U0126 (Fig 7C). The bile acid deoxycholic acid (DCA) was included as positive control for hepatocytes, which was confirmed with a neutrophil extract displaying high level of phopho-ERK1/2. U0126 also inhibited DCA-mediated ERK1/2 phosphorylation (data not shown). Total ERK1/2 MAP kinase was equally present in all lanes (Fig 7c). Inhibition of both ERK1/2 MAP kinase and PI3 kinase abolished the protective effect of TUDCA against GCDCA-induced apoptosis completely (Fig 7A). Finally, the role of trancription in the protection of TUDCA against bile acid-induced apoptosis was studied. Inhibition of transcription using actinomycin D, at a dose sensitizing hepatocytes to cytokine-induced apoptosis (Fig 2D) had no influence on the protective effect of TUDCA (Fig 8). Blocking of transcription was confirmed with Western blot for cytokine-induced transcription of inducible nitric oxide synthase (iNOS) (Fig 8 inset) (11).


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Figure 8 Transcription-independent protection of tauroursodeoxycholic acid (TUDCA) against glycochenodeoxycholic acid (GCDCA)-induced apoptosis. Caspase-3 activity in rat hepatocytes treated with 50 µmol/L GCDCA, 50 µmol/L of TUDCA or both with or without 200 ng/ml of transcription inhibitor actinomycin-D (ActD) for 4 hours. ActD did not significantly change the protective effect of TUDCA. Cytokine-exposed hepatocytes (CM) for 6 hours served as positive control for ActD. Inset: 200 ng/mL ActD blocks transcription. Western blot for cytokine (CM)-induced expression of inducible nitric oxide synthase (iNOS), 11 hours after exposure. See Materials and Methods for details. RLU, relative light units.

Discussion In this study, we investigated the protective mechanisms of taurine-conjugated UDCA against GCDCA-induced apoptosis in primary rat hepatocytes. We have previously shown that GCDCA induces apoptosis in a mitochondria-dependent manner, in which FADD is not involved and caspase-8 activation is not initially required. (13) In the present study, we investigated this further by blocking caspase-9. Indeed, peptide inhibitors of caspase-9 and caspas-3 blocked caspase-8 activation. Although these caspase inhibitors could have overlapping inhibitory effects, previous results with the human homologue of Inhibitor of Apoptosis protein1 (HIAP1) demonstrated that overexpression of HIAP1 inhibited GCDCA-induced apoptosis. HIAP1 exclusively inhibits caspase-3 and caspase-9 activation, but not caspase-8 (31). These data indicate that GCDCA induces apoptosis in a mitochondria-controlled manner. In the present study, we demonstrated that TUDCA inhibits, but does not delay GCDCA-induced caspase-9 and caspase-3 activity and the formation of apoptotic nuclei. Furthermore, we have shown that Ntcp is required for GCDCA-induced apoptosis in primary hepatocytes, indicating that ligand-dependent death receptor activation is not likely to occur. This report demonstrates that the anti-apoptotic action of TUDCA against GCDCA-induced apoptosis is not due to a direct competitive effect on the cell membrane. For example, TUDCA could compete with GCDCA for uptake by the bile acid importer Ntcp, thus preventing the uptake of pro-apoptotic GCDCA into hepatocytes. Pre-incubation for several hours with TUDCA followed by removal of TUDCA inhibits GCDCA-induced caspase-3 and caspase-9 activity,

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indicating the activation of survival pathways. Nine hour pre-incubation with TUDCA does not protect cells against GCDCA, likely because of quenching of survival signals. Overall, TUDCA appears to protect mitochondria from GCDCA-induced injury by preventing GCDCA-induced caspase-9 activation rather than by preventing death receptor-mediated apoptosis. The inhibitory effect on GCDCA-induced apoptosis is exclusively exerted by TUDCA. Our results demonstrate that taurine-conjugated CDCA does not inhibit GCDCA-induced caspase-3, caspase-9 and caspase-8 activity. This is in contrast to others showing inhibition of Fas-mediated caspase-8 activation by TCDCA-induced PI3-kinase. (32) However, the regulation of cell survival and apoptosis in a hepatoma cell line and primary non-transformed hepatocytes differs and this may explain the different results. GUDCA could be clinically relevant since in humans, contrary to rats, glycine-conjugated UDCA is much more abundant compared to taurine-conjugated UDCA (17;18). On the other hand, TUDCA has been reported to be beneficial in humans.(19) Nevertheless, TUDCA abundance in rats could explain why GUDCA does not inhibit GCDCA-induced apoptosis in rat hepatocytes in vitro. Primary human hepatocytes should give more information about the protective effect of taurine- and glycine-conjugated UDCA against bile acid-induced apoptosis. Our data demonstrate that the p38 and ERK MAP kinase pathways and the PI3 kinase pathway are involved in the protection of TUDCA against GCDCA-induced apoptosis. Because blocking of both PI3 and MAP kinases exaggerates caspase-3 activity compared to blocking a single pathway, these pathways are partly redundant. Moreover, inhibition of the PI3 kinase pathway alone or in combination with MAP kinase inhibitors aggravates GCDCA-induced apoptosis and sensitizes hepatocytes slightly to TUDCA. Indeed, we have demonstrated that in addition to TUDCA (Fig 7B), GCDCA activates p38 and ERK MAP kinases (data not shown). These results are important for the interpretation of the protective effects of TUDCA. Blocking of protective kinases induces higher caspase-3 activity in GCDCA-exposed hepatocytes compared to GCDCA + TUDCA-exposed hepatocytes, implying additional protective effects of TUDCA, e.g. direct alterations of the mitochondrial membrane environment. Evidence for this was given recently by showing that TUDCA stabilizes the lipid and protein structure of mitochondrial outer membranes, thus inhibiting Bax binding to the outer membrane (10). Because simultaneous addition of TUDCA and GCDCA, or addition of TUDCA after GCDCA blocks apoptosis in hepatocytes, TUDCA very rapidly exerts its protective effect. Indeed, no transcription or NF-κB activation is needed for protection, indicating that survival signaling involves post-translational mechanisms exerted within 30 minutes as shown by Western blot (Fig 7B, C). TUDCA protection against GCDCA-induced apoptosis can only be exerted up to 1,5 hours after GCDCA addition. An explanation could be that in this time frame, GCDCA-induced apoptosis has not reached its maximum yet, whereas after 2 hours, apoptosis has proceeded beyond the point that TUDCA can be protective. Further evidence for rapid anti-apoptotic mechanisms of TUDCA is inferred from our previous results using cytokine-mediated protection against GCDCA. Cytokines do not protect against bile acid-


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induced apoptosis after the addition of GCDCA, since their protective mechanism depends on NF-κB-mediated transcription with a time frame of hours instead of minutes (13). Our PI3K data supports other reports, although in these studies unconjugated UDCA was found to be apoptotic in itself (15). Activation of Akt protects against apoptosis via several mechanisms, including the phosphorylation of the pro-apoptotic Bcl-2 family member Bad, which can no longer associate with and inhibit anti-apoptotic Bcl-XL. (33) (34) Akt activation also results in the phosphorylation and inactivation of caspase-9 (35) and may suppress pro-apoptotic Bax translocation to the mitochondria.(36) It is reported that bile acids activate the ERK1/2 MAP kinase pathway via activation of the EGF receptor. (15) (25;37;38) The exact mechanism is still unclear, but mitochondrial-derived reactive oxygen species may be involved as recently suggested(37;39), and TUDCA prevents the generation of ROS.(10) The inhibition of Bax relocation to the mitochondria could also be mediated by ERK1/2 MAP kinase.(40) Since TUDCA prevents Bax-induced membrane perturbation(10), TUDCA-activation of ERK1/2 MAP kinase and PI3 kinase could be a mechanism to act on Bax. All together, our data fit very well with mitochondria-controlled bile acid-induced apoptosis in primary hepatocytes and provide more information about the link between TUDCA-activated survival pathways and mitochondria. Interestingly, our data are in contrast to a recent study describing that the protection of TUDCA against TLCS-induced apoptosis is p38 MAP kinase-, ERK1/2 MAP kinase- and PI3 kinase-independent, and depends on inhibition of Fas trafficking and caspase-8 activation.(41) The authors suggest that TUDCA does inhibit TLCS-induced apoptosis upstream of caspase-8 activation. However, they also suggest that TUDCA inhibits TLCS-triggered mitochondrial ROS formation (41), which is needed for Fas trafficking.(39) Therefore, TUDCA may inhibit TLCS-induced apoptosis in a mitochondria-controlled manner. Alternatively, GCDCA and TLCS may induce apoptosis via different mechanisms, which could explain the discrepancies between the protective mechanisms of TUDCA against these bile acids. The activation of the p38 MAP kinase pathway by TUDCA in order to inhibit GCDCA-induced stress in rat hepatocytes is in line with the activation of p38 MAP kinases during other forms of environmental stress. (14) Several reports describe the involvement of these kinases in mRNA stabilization (42). Since we demonstrated that the protective effect of TUDCA is at a post-transcriptional level, this mechanism could be present in primary rat hepatocytes as well. Although, the NF-κB pathway is involved in protection against cytokine-induced stress in primary rat hepatocytes (11) (12), we did not find evidence that the NF-κB pathway is involved in the protective action of TUDCA against GCDCA-induced apoptosis. Bile acids do not directly activate NF-κB (13). Moreover, inhibition of the NF-κB pathway did not change the protective action of TUDCA. In summary, we have shown that the anti-apoptotic effect of TUDCA against GCDCA-induced apoptosis in primary rat hepatocytes is independent of caspase-8 inhibition, but is due to activation of p38, ERK MAP kinases and PI3 kinase survival pathways. Furthermore, we have demonstrated that TUDCA protects against GCDCA-induced mitochondrial injury in

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primary rat hepatocytes. Our data provide more information about the mechanism of action of UDCA in cholestatic liver diseases. Acknowledgements Supported by grants from the Netherlands Digestive Diseases Foundation (WS99-28), the J.K. de Cock Foundation and an unrestricted grant from Tramedico B.V. the Netherlands and the Falk Foundation, Germany. Part of this work was presented at the annual meeting of the American Association for the Study of Liver Diseases, Boston, 2002.

Reference List 1. Poupon RE, Lindor KD, Cauch-Dudek K, Dickson ER, Poupon R, Heathcote EJ. Combined analysis of

randomized controlled trials of ursodeoxycholic acid in primary biliary cirrhosis. Gastroenterology 1997; 113(3):884-890.

2. Paumgartner G, Beuers U. Ursodeoxycholic acid in cholestatic liver disease: mechanisms of action and therapeutic use revisited. Hepatology 2002; 36(3):525-531.

3. Guldutuna S, Zimmer G, Imhof M, Bhatti S, You T, Leuschner U. Molecular aspects of membrane stabilization by ursodeoxycholate [see comment]. Gastroenterology 1993; 104(6):1736-1744.

4. Van Nieuwkerk CM, Elferink RP, Groen AK, Ottenhoff R, Tytgat GN, Dingemans KP et al. Effects of Ursodeoxycholate and cholate feeding on liver disease in FVB mice with a disrupted mdr2 P-glycoprotein gene. Gastroenterology 1996; 111(1):165-171.

5. Beuers U, Bilzer M, Chittattu A, Kullak-Ublick GA, Keppler D, Paumgartner G et al. Tauroursodeoxycholic acid inserts the apical conjugate export pump, Mrp2, into canalicular membranes and stimulates organic anion secretion by protein kinase C-dependent mechanisms in cholestatic rat liver. Hepatology 2001; 33(5):1206-1216.

6. Kurz AK, Graf D, Schmitt M, Vom DS, Haussinger D. Tauroursodesoxycholate-induced choleresis involves p38(MAPK) activation and translocation of the bile salt export pump in rats. Gastroenterology 2001; 121(2):407-419.

7. Botla R, Spivey JR, Aguilar H, Bronk SF, Gores GJ. Ursodeoxycholate (UDCA) inhibits the mitochondrial membrane permeability transition induced by glycochenodeoxycholate: a mechanism of UDCA cytoprotection. J Pharmacol Exp Ther 1995; 272(2):930-938.

8. Rodrigues CM, Fan G, Ma X, Kren BT, Steer CJ. A novel role for ursodeoxycholic acid in inhibiting apoptosis by modulating mitochondrial membrane perturbation. J Clin Invest 1998; 101(12):2790-2799.

9. Rodrigues CM, Ma X, Linehan-Stieers C, Fan G, Kren BT, Steer CJ. Ursodeoxycholic acid prevents cytochrome c release in apoptosis by inhibiting mitochondrial membrane depolarization and channel formation. Cell Death Differ 1999; 6(9):842-854.

10. Rodrigues CM, Sola S, Sharpe JC, Moura JJ, Steer CJ. Tauroursodeoxycholic acid prevents bax-induced membrane perturbation and cytochrome C release in isolated mitochondria. Biochemistry 2003; 42(10):3070-3080.

11. Schoemaker MH, Ros JE, Homan M, Trautwein C, Liston P, Poelstra K et al. Cytokine regulation of pro- and anti-apoptotic genes in rat hepatocytes: NF-kappaB-regulated inhibitor of apoptosis protein 2 (cIAP2) prevents apoptosis. J Hepatol 2002; 36(6):742-750.

12. Hatano E, Bennett BL, Manning AM, Qian T, Lemasters JJ, Brenner DA. NF-kappaB stimulates inducible nitric oxide synthase to protect mouse hepatocytes from TNF-alpha- and Fas-mediated apoptosis. Gastroenterology 2001; 120(5):1251-1262.

13. Schoemaker MH, Gommans WM, Conde de la Rosa L, Homan M, Trautwein C, Van Goor H et al. Resistance of rat hepatocytes against bile acid-induced apoptosis in cholestatic liver injury is due to NF-kB activation. J.Hepatol. 39(2), 153-161. 2003.

14. Johnson GL, Lapadat R. Mitogen-activated protein kinase pathways mediated by ERK, JNK, and p38 protein kinases. Science 2002; 298(5600):1911-1912.

15. Qiao L, Yacoub A, Studer E, Gupta S, Pei XY, Grant S et al. Inhibition of the MAPK and PI3K pathways enhances UDCA-induced apoptosis in primary rodent hepatocytes. Hepatology 2002; 35(4):779-789.


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16. Datta SR, Brunet A, Greenberg ME. Cellular survival: a play in three Akts. Genes Dev 1999; 13(22):2905-2927.

17. Perwaiz S, Tuchweber B, Mignault D, Gilat T, Yousef IM. Determination of bile acids in biological fluids by liquid chromatography-electrospray tandem mass spectrometry. J Lipid Res 2001; 42(1):114-119.

18. Combes B, Carithers RL, Jr., Maddrey WC, Munoz S, Garcia-Tsao G, Bonner GF et al. Biliary bile acids in primary biliary cirrhosis: effect of ursodeoxycholic acid. Hepatology 1999; 29(6):1649-1654.

19. Invernizzi P, Setchell KD, Crosignani A, Battezzati PM, Larghi A, O'Connell NC et al. Differences in the metabolism and disposition of ursodeoxycholic acid and of its taurine-conjugated species in patients with primary biliary cirrhosis. Hepatology 1999; 29(2):320-327.

20. Moshage H, Casini A, Lieber CS. Acetaldehyde selectively stimulates collagen production in cultured rat liver fat-storing cells but not in hepatocytes. Hepatology 1990; 12(3 Pt 1):511-518.

21. Plass JR, Mol O, Heegsma J, Geuken M, Faber KN, Jansen PL et al. Farnesoid X receptor and bile salts are involved in transcriptional regulation of the gene encoding the human bile salt export pump. Hepatology 2002; 35(3):589-596.

22. Kullak-Ublick GA, Ismair MG, Kubitz R, Schmitt M, Haussinger D, Stieger B et al. Stable expression and functional characterization of a Na+-taurocholate cotransporting green fluorescent protein in human hepatoblastoma HepG2 cells. Cytotechnology 34, 1-9. 2000.

23. Iimuro Y, Nishiura T, Hellerbrand C, Behrns KE, Schoonhoven R, Grisham JW et al. NFkappaB prevents apoptosis and liver dysfunction during liver regeneration [published erratum appears in J Clin Invest 1998 Apr 1;101(7):1541]. J Clin Invest 1998; 101(4):802-811.

24. Mills CO, Milkiewicz P, Muller M, Roma MG, Havinga R, Coleman R et al. Different pathways of canalicular secretion of sulfated and non-sulfated fluorescent bile acids: a study in isolated hepatocyte couplets and TR- rats. J Hepatol 1999; 31(4):678-684.

25. Qiao L, Studer E, Leach K, McKinstry R, Gupta S, Decker R et al. Deoxycholic acid (DCA) causes ligand-independent activation of epidermal growth factor receptor (EGFR) and FAS receptor in primary hepatocytes: inhibition of EGFR/mitogen-activated protein kinase-signaling module enhances DCA-induced apoptosis. Mol Biol Cell 2001; 12(9):2629-2645.

26. Vos TA, Gouw AS, Klok PA, Havinga R, Van Goor H, Huitema S et al. Differential effects of nitric oxide synthase inhibitors on endotoxin-induced liver damage in rats. Gastroenterology 1997; 113(4):1323-1333.

27. Miyoshi H, Rust C, Roberts PJ, Burgart LJ, Gores GJ. Hepatocyte apoptosis after bile duct ligation in the mouse involves Fas. Gastroenterology 1999; 117(3):669-677.

28. Faubion WA, Guicciardi ME, Miyoshi H, Bronk SF, Roberts PJ, Svingen PA et al. Toxic bile salts induce rodent hepatocyte apoptosis via direct activation of Fas. J Clin Invest 1999; 103(1):137-145.

29. Higuchi H, Bronk SF, Takikawa Y, Werneburg N, Takimoto R, El Deiry W et al. The bile acid glycochenodeoxycholate induces trail-receptor 2/DR5 expression and apoptosis. J Biol Chem 2001; 276(42):38610-38618.

30. Rippin SJ, Hagenbuch B, Meier PJ, Stieger B. Cholestatic expression pattern of sinusoidal and canalicular organic anion transport systems in primary cultured rat hepatocytes. Hepatology 2001; 33(4):776-782.

31. Deveraux QL, Roy N, Stennicke HR, Van Arsdale T, Zhou Q, Srinivasula SM et al. IAPs block apoptotic events induced by caspase-8 and cytochrome c by direct inhibition of distinct caspases. EMBO J 1998; 17(8):2215-2223.

32. Takikawa Y, Miyoshi H, Rust C, Roberts P, Siegel R, Mandal PK et al. The bile acid-activated phosphatidylinositol 3-kinase pathway inhibits Fas apoptosis upstream of bid in rodent hepatocytes. Gastroenterology 2001; 120(7):1810-1817.

33. Zha J, Harada H, Osipov K, Jockel J, Waksman G, Korsmeyer SJ. BH3 domain of BAD is required for heterodimerization with BCL-XL and pro-apoptotic activity. J Biol Chem 1997; 272(39):24101-24104.

34. Muslin AJ, Tanner JW, Allen PM, Shaw AS. Interaction of 14-3-3 with signaling proteins is mediated by the recognition of phosphoserine. Cell 1996; 84(6):889-897.

35. Cardone MH, Roy N, Stennicke HR, Salvesen GS, Franke TF, Stanbridge E et al. Regulation of cell death protease caspase-9 by phosphorylation. Science 1998; 282(5392):1318-1321.

36. Tsuruta F, Masuyama N, Gotoh Y. The phosphatidylinositol 3-kinase (PI3K)-Akt pathway suppresses Bax translocation to mitochondria. J Biol Chem 2002; 277(16):14040-14047.

37. Rao YP, Studer EJ, Stravitz RT, Gupta S, Qiao L, Dent P et al. Activation of the Raf-1/MEK/ERK cascade by bile acids occurs via the epidermal growth factor receptor in primary rat hepatocytes. Hepatology 2002; 35(2):307-314.

38. Yoon JH, Higuchi H, Werneburg NW, Kaufmann SH, Gores GJ. Bile acids induce cyclooxygenase-2 expression via the epidermal growth factor receptor in a human cholangiocarcinoma cell line. Gastroenterology 2002; 122(4):985-993.

39. Reinehr R, Graf D, Haussinger D. Bile salt-induced hepatocyte apoptosis involves epidermal growth factor receptor-dependent CD95 tyrosine phosphorylation. Gastroenterology 2003; 125(3):839-853.

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40. Zhang XD, Borrow JM, Zhang XY, Nguyen T, Hersey P. Activation of ERK1/2 protects melanoma cells from TRAIL-induced apoptosis by inhibiting Smac/DIABLO release from mitochondria. Oncogene 2003; 22(19):2869-2881.

41. Graf D, Kurz AK, Fischer R, Reinehr R, Haussinger D. Taurolithocholic acid-3 sulfate induces CD95 trafficking and apoptosis in a c-Jun N-terminal kinase-dependent manner. Gastroenterology 2002; 122(5):1411-1427.

42. Reunanen N, Li SP, Ahonen M, Foschi M, Han J, Kahari VM. Activation of p38 alpha MAPK enhances collagenase-1 (matrix metalloproteinase (MMP)-1) and stromelysin-1 (MMP-3) expression by mRNA stabilization. J Biol Chem 2002; 277(35):32360-32368.

CHAPTER 7

Summary, general discussion

and perspectives

Chapter 7

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Oxidative stress has been defined as the inappropriate exposure to reactive oxygen species (ROS) and results from the imbalance between prooxidants and antioxidants leading to cell damage and tissue injury. ROS, including superoxide anions (O2

.-), hydrogen peroxide (H2O2) and hydroxyl radicals (HO.), are produced during normal intracellular oxygen metabolism and from exogenous substances. Hepatocytes are equipped with highly efficient detoxification mechanisms to defend themselves and prevent ROS-induced cell damage. Superoxide anions are efficiently detoxified to hydrogen peroxide by Cu/Zn or Mn- superoxide dismutase (SOD). Hydrogen peroxide in turn is converted into water by catalase or glutathione peroxidase (1). Glutathione is present in high amounts in hepatocytes (2, 3) and represents an efficient mechanism to detoxify ROS (4). In spite of the highly efficient detoxification mechanisms, over-exposure to high level of ROS results in oxidative stress, resulting in cell death. Under oxidative stress conditions, the mode of cell death (apoptosis or necrosis) is primarily dependent on the variety of ROS and the cell type. In chronic liver diseases, such as alcoholic and viral hepatitis, NASH and cholestasis, hepatocytes are invariably exposed to oxidative stress caused by diverse reactive oxygen species, which induces cell damage and subsequently hepatocyte cell death and loss of liver function. Therefore, it is important to know the mechanisms that lead to ROS-induced hepatocyte death, because a more complete understanding of the cellular response to oxidative stress could be of significant relevance to comprehend and treat liver diseases. Chapter 3 describes major differences in mechanisms and mode of cell death (apoptosis and necrosis) induced by distinct reactive oxygen species in primary hepatocytes. On one hand, H2O2 does not induce apoptosis or necrotic cell death in normal primary hepatocytes at concentrations below 5mmol/L. Only higher concentrations or when H2O2 detoxification is impaired, turns H2O2 into a necrotic compound. The high efficiency of H2O2 detoxification, especially catalase, explains why H2O2 is hardly sensed by hepatocytes, being only moderately toxic at concentrations up to 4 mmol/L. Other studies showed divergent effects of H2O2, reporting both necrotic and apoptotic cell death in hepatoma cell lines (5, 6). Indeed, the mode of cell death is dependent on the cell type, with hepatoma cell lines yielding different results than normal non-transformed hepatocytes. Additionally, it is well known that tumour cells exhibit increased intrinsic oxidative stress and have adapted to this situation by altered oxidative stress defence mechanisms (7, 8). Differences in the sensitivity to apoptotic stimuli between transformed hepatoma cells and normal non-transformed hepatocytes have been reported (9, 10). Therefore, results obtained using hepatoma cell lines cannot be extrapolated to normal, non-transformed hepatocytes. Exposure to ROS has been shown to activate signal transduction pathways, which may modulate cell death (11-13). However, we have demonstrated that hydrogen peroxide does not modulate ERK or JNK MAPK. This could be due to the fact that hydrogen peroxide is hardly sensed by the hepatocytes. On the other hand, we have shown that the superoxide anions-donor menadione mainly induces apoptosis and to a much lesser extent necrotic cell death in primary hepatocytes.

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Superoxide anions-induced PARP cleavage and apoptosis are dependent on caspase activation, since inhibitors of caspases prevent PARP-cleavage and subsequently apoptosis. Additionally, we demonstrate that caspase-9 activation is upstream of caspase-6 and -3 activation. In type II cells, apoptosis is dependent on the release of pro-apoptotic factors from the mitochondria, that activate caspase-9 and subsequently caspase-3 (14-16). Our demonstration of superoxide anions-induced caspase-9 activation indicates disruption of the mitochondria and release of pro-apoptotic factors (16). We have reported that superoxide anions activate JNK and ERK1/2 MAPK in hepatocytes. In addition, we have demonstrated that superoxide anions-induced apoptosis is dependent on JNK activity, since inhibition of JNK activation abolishes menadione-induced apoptosis, providing evidence for a pro-apoptotic role of JNK. The mechanisms by which JNK exert its pro-apoptotic properties are not completely understood. The fact that JNK inhibition blocks caspase-9 activation indicates that JNK triggers the mitochondrial pathway after menadione treatment. Indeed, many studies have described an effect of JNK at the level of the mitochondria, triggering the mitochondria death pathway, including phosphorylation and activation of pro-apoptotic bcl-2 family members (17-23). Superoxide anions-induced ERK MAPK phosphorylation attenuates cell death, since inhibition of ERK1/2 enhances superoxide anions-induced apoptosis, indicating an anti-apoptotic role of ERK1/2 against apoptosis. However, activation of ERK1/2 is clearly not sufficient to prevent menadione-induced apoptosis, since superoxide anions also activate the JNK pro-apoptotic pathway, which overrules the protective ERK1/2 pathway, suggesting that JNK plays a more determinant and central role in apoptosis. Again, there are discrepancies between primary hepatocytes and cell lines, since in our study ERK1/2 phosphorylation was only observed at 50µmol/L whereas ERK1/2 phosphorylation was observed also at lower menadione concentrations in the RALA cell line (17), indicating the importance of using primary hepatocytes to mimic normal hepatocytes and to extrapolate to liver diseases. Although the NF-κB pathway is involved in protection against cytokine-induced apoptosis in primary rat hepatocytes (24, 25), we demonstrate that the NF-κB pathway is not involved in the protection against superoxide anions-induced apoptosis in hepatocytes. In chapter 3, we demonstrate remarkable differences between different ROS scavengers with therapeutic applications. PEG-SOD totally inhibits caspase activation, abolishes apoptosis and does not increase necrotic cell death. Indeed, PEG-SOD represents a potential strategy in the therapy of liver diseases caused by oxidative stress, as shown in studies in which overexpression of superoxide dismutases inhibit apoptosis in alcohol-induced liver injury in rats (26, 27). In contrast, nitric oxide blocks superoxide anions-induced apoptosis but increases necrotic cell death, switching the balance between apoptotic and necrotic cell death. Hence, the metabolism of ROS can switch the balance between apoptotic and necrotic cell death. Therefore, future therapeutic strategies, aimed at donating NO to modulate cell death and treat liver diseases (28), should take into account the possibility of ROS generation and a switch in the mode of cell death in these diseases. Indeed, we demonstrate that it is imperative to evaluate the consequences of any intervention in both apoptotic and necrotic cell death.

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In support, increasing the concentration of the intracellular antioxidant glutathione attenuates superoxide anions-induced apoptosis and vice versa. Finally, the anti-apoptotic bile acid tauroursodeoxycholic acid (TUDCA), which inhibits apoptosis induced by toxic bile acid (chapter 6), did not attenuate superoxide anions-induced apoptosis, indicating that the mechanism of bile acid and superoxide anions-induced apoptosis are different. Thus, oxidative stress activates both pro- and anti-apoptotic pathways in hepatocytes. Cell survival then depends on the balance between pro- and anti-apoptotic pathways. In conditions of moderate oxidative stress, the cell will survive. However, under intense oxidative stress conditions, pro-apoptotic pathways will prevail and switch the balance to cell death. Knowledge about different ROS involved in liver diseases is needed to specifically interfere and treat these disorders using the appropriate therapeutic tools that modulate crucial pathways involved in the process and do not change the mode of cell death. Many studies have demonstrated that HO-1, which decomposes heme into ferrous iron, CO and biliverdin, acts an inducible defence against oxidative stress and mediates antioxidant and anti-apoptotic effects (29-32). In the liver, HO-1 induction protected against ischemia/reperfusion injury (33, 34), endotoxemia (35, 36), inflammation (37), immune mediated liver injury (38), endotoxic shock (39) and against CYP2E1-dependent cytotoxicity (40). However, the mechanisms by which HO-1 mediates cytoprotection are not elucidated yet. Chapter 4 describes in detail the regulation of HO-1 in vivo and in vitro and the contribution CO to the protective effect of HO-1 against apoptosis, indicating the mechanisms involved in the process. Interestingly, HO-1 was only slightly induced in acute inflammation, induced by endotoxin administration, although in this model, NF-κB-regulated genes like iNOS are highly induced as previously shown (41). We have observed a similar reciprocal regulation of AP-1 responsive genes like HO-1 and NF-κB responsive genes like iNOS in intestinal epithelial cells (42). However, HO-1 induction was much more pronounced in this model when anti-oxidants like glutathione were depleted, both in vivo and in vitro. Nevertheless, under these circumstances cell death occurs due to the simultaneous excess of ROS and impairment of antioxidant defences. These results demonstrate that acute inflammation is not accompanied by extensive oxidative stress and consumption of antioxidants. It is remarkable that HO-1 expression is not induced by H2O2 in vitro, probably because hepatocytes hardly sense this molecule due to the highly efficient H2O2 detoxification mechanisms that they posses. The fact that both pro-apoptotic and anti-apoptotic bile acids induce HO-1 expression represents a novel finding in our study. Whether this is a direct effect or indirect via bile acid-induced ROS generation remains to be elucidated. HO-1 is induced by the superoxide anion-donor menadione, although this does not prevent apoptosis in hepatocytes, likely because HO-1 induction is not high and/or rapid enough.


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However, overexpression of HO-1, using the adenovirus Ad5HO-1, 15hrs before the addition of menadione leads to protection against oxidative stress-induced apoptosis. We demonstrate that CO exerts a protective role against superoxide anions-induced apoptosis. Indeed, CO is a potent inhibitor of caspase activation, PARP cleavage and apoptosis in primary hepatocytes. CO has been shown to be protective in other studies (31, 43, 44). In contrast, a pro-apoptotic effect of CO has been also described (45, 46). These data suggest that CO may regulate apoptosis and cytotoxicity in a cell-specific manner. CO inhibits caspase-9 activation, suggesting that CO prevents disruption of mitochondria and subsequent apoptosis. It has been shown that CO blocks mitochondrial cytochrome c release (44, 47), although the mechanism remains unknown. We have shown that CO prevents JNK-phosphorylation and its pro-apoptotic activity, indicating that the protective effect of CO against superoxide anions-induced apoptosis is at least in part via inhibition of JNK activation. Many studies have described an effect of JNK on the mitochondrial death pathway and activation of pro-apoptotic bcl-2 family members (17-23). Furthermore, we show that CO differentially modulates pro- and anti-apoptotic MAPK pathways. CO prevents JNK-phosphorylation and its pro-apoptotic activity. In addition, CO induces ERK1/2 MAPK-phosphorylation and its anti-apoptotic activity, which further increases the protection against superoxide anions-induced apoptosis.

Plasma membraneOxidative Stress

JNK p-JNK

Cyt c

Casp-9

Casp-3

APOPTOSIS

ERK

+nucleus

AP-1 HO-1 CO

Biliverdin

SURVIVAL

Plasma membraneOxidative Stress

JNK p-JNK

Cyt c

Casp-9

Casp-3

APOPTOSIS

ERK

+nucleus

AP-1 HO-1 CO

Biliverdin

SURVIVAL Figure 1. Schematic overview of the protective mechanisms of carbon monoxide against superoxide anions-induced apoptosis in primary hepatocytes.

In addition, we have shown that the protective effect of CO against superoxide anions-induced apoptosis is not mediated by soluble guanylate cyclase (sGC), p38 or the NF-κB pathway in primary hepatocytes as suggested by other studies (44) (48). It has also been reported that CO mediates protection against CYP2E1-dependent cytotoxicity via inhibition of CYP2E1 activity (40). Collectively, these data indicate that CO can exert anti-apoptotic effects via different mechanisms depending on the cell type and stimulus.

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Biliverdin, the other product of HO-1, is also protective against oxidative stress-induced injury, in part due to its anti-oxidant properties (49). A cooperative protective effect of biliverdin and carbon monoxide has been shown in immune-mediated liver injury in mice (38). Finally, we prove that CO indeed protects hepatocytes exposed to oxidative stress and does not switch apoptotic into necrotic cell death. Thus, modulation of HO-1 expression, or the levels of its product CO, may become an important therapeutic target in liver disorders caused by exposure to excessive oxidative stress. In chapter 5 of this thesis, we described a novel approach, using metformin, to prevent superoxide anions-induced apoptosis in hepatocytes and we introduce the mechanisms involved in this process. Since oxidative stress and insulin resistance are important in the pathogenesis of NAFLD (50, 51) and the pathophysiology of diabetes complications (52-59), and since metformin, an insulin sensitizing agent used in type 2 diabetes, has been shown to be hepatoprotective in many models including NAFLD (60) and NASH (61), we examined whether metformin has a protective effect against oxidative stress-induced apoptosis in primary rat hepatocytes. We report a protective effect of metformin against superoxide anions-induced caspase-9, -6 and -3 activation, PARP cleavage and apoptosis in primary hepatocytes. Metformin-induced inhibition of caspase-9 suggests that metformin prevents disruption of mitochondria and subsequent apoptosis (14-16). In endothelial cells, it has been shown that metformin blocks the mitochondrial permeability transition and cytochrome c release induced by high glucose-derived oxidative stress in endothelial cells (62). In support, we demonstrate that metformin reduces JNK-phosphorylation and its pro-apoptotic activity, which includes the mitochondrial death pathway activation and phosphorylation of pro-apoptotic bcl-2 family members. JNK has been described as a crucial mediator in insulin resistance and a critical element in the pathogenesis of fatty liver disease and type 2 diabetes (63, 64). JNK activity has been reported to be highly elevated in diabetes, due to the presence of inflammation (TNF-α), free fatty acids and oxidative stress, all features associated with diabetes and obesity. JNK can directly phosphorylate insulin receptor substrate (IRS)-1 inducing defects in insulin signalling and consequently insulin resistance (65). Thus, it has been reported that JNK-deficient obese mice are protected against insulin resistance and defective insulin receptor signalling (64). In addition, the presence of a dominant-negative JNK isoform in the livers of obese mice results in increased insulin sensitivity and glucose homeostasis. Furthermore, exogenous expression of JNK in adult liver results in insulin resistance in mice (66). Metformin-induced inhibition of JNK phosphorylation could be related to its well-known properties as an insulin sensitizing agent. However, in our study, experiments were performed in the absence of insulin. Therefore, it is unlikely that the protective effect of metformin is related to early events in insulin signal transduction. Nevertheless, it cannot be excluded that metformin interferes in down-stream steps of insulin signal transduction, e.g. inhibition of JNK activation.


103

Our result correlates with a previous study in vivo, in which basal protein expression of JNK was reduced after metformin treatment in Wistar rats (67). In contrast, exposure to metformin for more than 24hrs induces JNK phosphorylation that leads to apoptosis (68). Additional experiments are needed to further delineate the mechanisms of action of metformin. In addition, we document that metformin further increases menadione-induced HO-1 expression, which is known to be protective against superoxide anions-induced apoptosis, suggesting that HO-1 contributes to the anti-apoptotic effect of metformin. Remarkably, metformin alone does not induce HO-1 expression in primary hepatocytes. We provide evidence that metformin indeed protects hepatocytes exposed to oxidative stress and does not switch apoptotic into necrotic cell death. In this study, we propose interesting and novel mechanisms involved in the protective effect of metformin against superoxide anions-induced apoptosis. However, we do not exclude the possibility that the protective effect of metformin is also partially mediated by other survival pathways, such as ERK1/2 or p38 MAPK, c-Src, AMPK, PI3K/Akt or mTOR (mammalian target of rapamycin). Metformin-induced AMPK activation has been well documented, including in primary rat hepatocytes (69). A recent study demonstrates that metformin reduces apoptosis induced by high-glucose in an AMPK-dependent manner in endothelial cells (70). In contrast, it has also been suggested that metformin promotes Bax translocation to the mitochondria via AMPK and p38 activation, thus enhancing apoptosis (71). Finally, we demonstrate that total glutathione content is not modulated by metformin, suggesting that GSH is not involved in the protective effect of metformin against superoxide anions-induced apoptosis. However, it should be taken into account that in our study we did not make a distinction between reduced and oxidized GSH. Additional studies are required to investigate whether the ratio of reduced to oxidized GSH increased due to the presence of metformin.

Menadione

Apoptosis

MetforminJNK

caspases

HO-1 Survival

+

Menadione

Apoptosis

MetforminJNK

caspases

HO-1 Survival

+

Figure 2. Schematic overview of the protective mechanisms of metformin against superoxide anions-induced apoptosis in primary hepatocytes.

For the first time, a study demonstrates that HO-1 induction and JNK inhibition are at least in part involved in the protective effect of metformin against oxidative stress-induced apoptosis. Due to the central role of JNK in the pathogenesis of oxidative stress-mediated diseases, including diabetes and most chronic liver diseases, modulation of JNK activity represents a promising and attractive target for the treatment of these disorders.

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In chapter 6, we have shown that exposure to glycochenodeoxycholic acid (GCDCA) induces apoptosis in hepatocytes in vitro. However, not all bile acids are toxic to the cells. We have demonstrated that neither the taurine-conjugated bile acid chenodeoxycholic acid (TCDCA) nor the taurine-conjugated ursodeoxycholic acid (TUDCA) induce apoptosis in hepatocytes. Instead TUDCA, but not TCDCA, prevents GCDCA-induced apoptosis. Unconjugated UDCA is used as a treatment of patients with chronic liver diseases, therefore, we investigated the mechanism involved in TUDCA anti-apoptotic properties. In rats, UDCA is conjugated predominantly to taurine (TUDCA) rather than to glycine (GUDCA), therefore in our study, we have used TUDCA. In addition, TUDCA treatment of patients suffering from primary biliary cirrhosis may even be more beneficial than UDCA treatment, which can not be fully explained by reconjugation to GUDCA (72). We have shown that one to three hours preincubation with TUDCA is protective against GCDCA-induced apoptosis, and TUDCA can be added one hour after GCDCA without losing its antiapoptotic properties, suggesting that a competitive effect of TUDCA for GCDCA-uptake by Ntcp on the cell membrane is not likely. Finally, we can not exclude a direct effect of TUDCA on the mitochondrial membrane. It has been reported that TUDCA inhibits binding of pro-apoptotoic Bax to the mitochondria (73). Additionally, TUDCA prevents permeabilization of the mitochondrial membrane, suggesting that TUDCA has an important role in the stabilization of the mitochondrial membrane during bile acid-induced apoptosis. Conclusions Knowledge about the cellular mechanisms controlling death of liver cells is of clinical and scientific relevance to identify targets for the development of novel therapeutics to treat liver diseases. This thesis describes the mechanisms of oxidative stress induced cell death in hepatocytes, showing that different reactive oxygen species induce distinct modes of cell death and the possibility to switch the balance between apoptosis and necrosis. In addition, we have identified new strategies to prevent apoptotic cell death in hepatocytes. Overall, we can conclude that JNK plays an important role in hepatocyte apoptosis, and should therefore be considered a potential target to interfere with cell death and to treat liver disorders. In addition, it is important to determine the exact contribution of various ROS species to liver injury, since preventing cell death will depend on the species of ROS involved.


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CHAPTER 8

Nederlandse Samenvatting

Written in English by Laura Conde de la Rosa

Translated and adapted by Han Moshage

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110

Oxidative stress wordt gedefinieerd als de bovenmatige blootstelling aan reactieve zuurstofverbindingen (Engels: reactive oxygen species; afgekort als ROS). Oxidatieve stress is het gevolg van een verstoorde balans tussen pro-oxidanten en anti-oxidanten en kan leiden tot cel- en weefselschade. ROS zoals waterstofperoxide (H2O2), superoxide anionen (O2

.-) en hydroxylradicalen (HO.) worden gegenereerd in metabool actieve cellen. Daarnaast kunnen cellen ook worden blootgesteld aan extern geproduceerde ROS. Hepatocyten, de parenchymale levercellen, zijn uitgerust met efficiënte detoxificatie-mechanismen voor ROS, zodat schade aan cellen wordt voorkomen of beperkt. Superoxide anionen worden omgezet in waterstofperoxide door superoxide dismutases (SOD), waarna het gevormde waterstofperoxide wordt omgezet in water en zuurstof door catalase en/of glutathionperoxidase. Glutathion is een belangrijk anti-oxidant en is in hoge concentraties aanwezig in hepatocyten. Ondanks de aanwezigheid van efficiënte detoxificatie mechanismen leidt bovenmatige of langdurige blootstelling aan ROS tot oxidatieve stress en celschade en/of celdood. De wijze van celdood, apoptose (geprogrammeerde celdood) of necrose, is afhankelijk van het celtype en de specifieke reactieve zuurstofverbinding waaraan de cel wordt blootgesteld. Apoptose is een actief proces dat gekenmerkt wordt door celkrimp, condensatie van chromatine, activatie van caspases en de vorming van apoptotische lichaampjes. Necrose is daarentegen een passief proces en wordt gekarakteriseerd door ATP-depletie, beschadiging van het plasmamembraan en lekkage van de celinhoud naar de circulatie met als gevolg ontsteking. Chronische leverziekten zoals alcoholische en virale hepatitis, NASH (niet-alcoholische steatohepatitis) en cholestase gaan vrijwel zonder uitzondering gepaard met oxidatieve stress, veroorzaakt door blootstelling aan verschillende reactieve zuurstofverbindingen. Dit leidt tot sterfte van hepatocyten en op termijn tot vermindering van leverfunctie en zelfs leverfalen. Om deze reden is het belangrijk de precieze mechanismen op te helderen van celschade en celdood geïnduceerd door ROS, zodat gerichte interventies kunnen worden ontwikkeld om deze celschade en celdood te voorkomen of te verminderen. Hoofdstuk 3 beschrijft de mechanismen van celdood geïnduceerd door verschillende ROS in hepatocyten. Waterstofperoxide veroorzaakt geen celdood van hepatocyten, tenzij de concentratie extreem hoog is (> 5mmol/L) of de detoxificatie van waterstofperoxide is geremd. Onder die omstandigheden veroorzaakt waterstofperoxide necrotische celdood. De effciënte wijze waarop waterstofperoxide onschadelijk wordt gemaakt in de hepatocyt door o.a. catalase, verklaart waarom waterstofperoxide nauwelijks door de hepatocyt wordt “waargenomen”. Blootstelling aan ROS activeert verschillende signaaltransductieroutes welke celdood kunnen moduleren. Twee belangrijke signaaltransductiemoleculen in dit verband zijn ERK en JNK MAP-kinases. Echter, waterstofperoxide bleek deze belangrijke signaaltransductieroutes niet te activeren, hetgeen in overeenstemming is met de gedachte dat waterstofperoxide niet of nauwelijks wordt “waargenomen” door de hepatocyt. Superoxide anionen, in onze studies gegenereerd door de superoxide anion donor menadion, induceren voornamelijk apoptotische celdood en niet of nauwelijks necrotische celdood van hepatocyten. Apoptose geïnduceerd

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door superoxide anionen is afhankelijk van de activiteit van caspases en de activiteit van het signaaltransductiemolecuul JNK. Remming van JNK remt activatie van caspase-9 en apoptose, hetgeen aangeeft dat JNK-activiteit een pro-apoptotische werking heeft en de mitochondriële route naar apoptose activeert. Superoxide anionen activeren ook het signaaltransductiemolecuul ERK1/2. Remming van ERK1/2 vergroot superoxide anion geïnduceerde celdood, hetgeen aangeeft dat ERK1/2-activiteit een anti-apoptotische werking heeft, maar niet voldoende om apoptose te voorkomen. JNK-activiteit is in dit geval dominant t.o.v. ERK1/2-activiteit. Het effect van anti-oxidanten en/of “scavengers” (“opruimers”) van ROS op oxidatieve stress geïnduceerde celdood is opmerkelijk variabel: de plasmamembraan-permeabele ROS-scavenger PEG-SOD remt superoxide anion geïnduceerde celdood volledig, zonder effecten op necrotische celdood. PEG-SOD is daarom een aantrekkelijke kandidaat voor anti-oxidanttherapie van leverziekten veroorzaakt door oxidatieve stress. Stikstofoxide (NO•) daarentegen remt superoxide anion geïnduceerde celdood eveneens, maar leidt tot een sterke toename van necrotische celdood. Deze bevinding toont aan dat NO• niet geschikt is als therapie voor oxidatieve stress gemediëerde leverziekten. Samenvattend: oxidatieve stress activeert zowel pro- als anti-apoptotische signaaltransductie-routes. Celoverleving is afhankelijk van de balans tussen deze pro- en anti-apoptotische routes. Onder condities van “gematigde” oxidatieve stress zullen cellen overleven, terwijl in condities van extreme oxidatieve stress de balans zal doorslaan naar apoptose. Hoofdstuk 4 beschrijft het beschermend effect van koolmonoxide (CO), één van de producten van het enzym heme-oxygenase-1 (HO-1), tegen superoxide anion geïnduceerde apoptose van hepatocyten. HO-1 wordt geïnduceerd onder condities van oxidatieve stress. Echter, HO-1 wordt niet geïnduceerd tijdens acute ontsteking, tenzij anti-oxidanten zoals glutathion gedepleteerd zijn. Dit wijst er op dat HO-1 een sensor is van oxidatieve stress en dat acute (endotoxine) geïnduceerde ontsteking niet gepaard gaat met oxidatieve stress. Hoewel HO-1 geïnduceerd wordt door superoxide anionen, voorkomt de normale inductie van HO-1 niet apoptose geïnduceert door superoxide anionen. Een mogelijke verklaring hiervoor is dat de inductie van HO-1 door superoxide anionen onvoldoende en/of te traag is. Overexpressie van HO-1 d.m.v. een adenovirus voorafgaand aan blootstelling aan superoxide anionen beschermt echter wel tegen apoptotische celdood. CO (koolmonoxide), bleek in staat de activering van caspases, PARP-splitsing en apoptose van hepatocyten blootgesteld aan superoxide anionen te remmen. Nader onderzoek wees uit dat CO de activatie van pro-apoptotisch JNK door superoxide anionen remt, terwijl de activatie van het anti-apoptotische ERK1/2 juist gestimuleerd wordt door CO. Bovendien veroorzaakt CO geen verschuiving naar necrotische celdood van hepatocyten blootgesteld aan superoxide anionen. Samenvattend: modulatie van de expressie van HO-1, of het HO-1-product CO, beschermt tegen oxidatieve stress geïnduceerde apoptotische celdood van hepatocyten en kan dus een belangrijk therapeutisch aangrijpingspunt vormen voor de behandeling van leverziekten veroorzaakt door oxidatieve stress. In Hoofdstuk 5 van dit proefschrift wordt een nieuwe benadering voor het voorkomen van superoxide anion geïnduceerde apoptose van hepatocyten gepresenteerd: metformine. Tevens

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wordt het mechanisme van het beschermend effect van metformine onderzocht. De reden voor het onderzoeken van het effect van metformine op superoxide anion geïnduceerde apoptose is dat oxidatieve stress en insuline-resistentie een belangrijke rol spelen in de pathogenese van NAFLD (“non-alcoholic fatty liver disease”) en de pathofysiologie van complicaties van diabetes mellitus type II. Metformine wordt gebruikt in de behandeling van patiënten met diabetes mellitus type II als een insulinegevoeligheid verhogend geneesmiddel. Omdat uit diverse studies bleek dat metformine een hepatoprotectief effect heeft bij NASH en NAFLD, onderzochten wij het effect van metformine op superoxide anion geïnduceerde celdood van hepatocyten. Uit onze studies bleek dat metformine superoxide anion geïnduceerde activering van caspases, PARP-splitsing en apoptose volledig remt. Bovendien konden wij vaststellen dat metformine de activiteit van het pro-apoptotische signaaltransductiemolecuul JNK remt. Uit andere studies was reeds bekend dat JNK-activatie een belangrijke rol speelt bij insuline- resistentie en in de pathogenese van steatohepatitis en diabetes mellitus type II. Tenslotte konden wij ook aantonen dat metformine de superoxide anion-geïnduceerde expressie van het anti-apoptotische enzym HO-1 vergroot. Metformine leidde niet tot een toename van superoxide anion geïnduceerde necrotische celdood. Samenvattend: metformine beschermt tegen apoptose geïnduceert door superoxde anionen door verrmindering van de activiteit van het pro-apoptotische signaaltransductiemolecuul JNK en door een toename van de expressie van het anti-apoptotische enzym HO-1. Niet uitgesloten moet worden dat metformine nog additionele beschermende effecten heeft. Vanwege de centrale rol die JNK speelt in de pathogenese van oxidatieve stress gemediëerde ziekten zoals diabetes mellitus type II en de meeste chronische leverziekten vormt modulatie van JNK-activiteit een aantrekkelijk aangrijpingspunt voor de behandeling van deze ziekten. In Hoofdstuk 6 hebben wij aangetoond dat verschillende galzuren verschillende effecten hebben op hepatocyten. Het galzuur glycochenodeoxycholzuur (GCDCA), maar niet het taurine-geconjugeerde galzuur chenodeoxycholzuur (TCDCA) of het taurine-geconjugeerde galzuur ursodeoxycholzuur (TUDCA), induceert apoptose van hepatocyten in vitro. TUDCA beschermt juist tegen GCDCA-geïnduceerde apoptose. De ongeconjugeerde vorm van TUDCA, UDCA wordt gebruikt in de behandeling van patiënten met chronische leverziekten. Het is daarom van belang om het mechanisme van het anti-apoptotische effect van TUDCA te bestuderen. Bovendien lijkt bij de behandeling van patiënten met primaire biliaire cirrose TUDCA een gunstiger effect te hebben dan UDCA, hetgeen niet volledig verklaard kan worden door reconjugatie van UDCA tot GUDCA. Het anti-apoptotische galzuur TUDCA beschermt echer niet tegen apoptose geïnduceerd door superoxide anionen, hetgeen aantoont dat de mechanismen van galzuur (i.c. GCDCA) en superoxide anion geïnduceerde apoptose verschillend zijn. Conclusies Inzicht in de mechanismen die de dood van levercellen reguleren is zowel van klinisch als wetenschappelijk belang. Deze inzichten kunnen leiden tot de identificatie van nieuwe “leads” voor de ontwikkeling therapeutica voor de behandeling van leverziekten. Dit proefschrift beschrijft de mechanismen die betrokken zijn bij oxidatieve stress geïnduceerde celdood van

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hepatocyten. Aangetoond werd dat verschillende reactieve zuurstofverbindingen op verschillende wijzen celdood veroorzaken. Daarnaast werd aangetoond dat er een delicate balans kan bestaan tussen apoptose en necrose en dat deze balans kan verschuiven door interventie. Hiermee moet rekening worden gehouden bij de ontwikkeling van interventies gericht op het remmen van één vorm van celdood. Tenslotte hebben wij verschillende nieuwe “targets” geïdentificeerd om apoptotische celdood van hepatocyten te voorkomen. Samenvattend kan gezegd worden dat JNK een belangrijke rol speelt in de regulatie van apoptose van hepatocyten. Om die reden vormt JNK een belangrijk aangrijpingspunt om te interveniëren in celdood van hepatocyten als onderdeel van een therapie voor leverziekten. Andere veelbelovende aangrijpingspunten zijn het enzym heme-oxygenase-1 en metformine. Vervolgonderzoek, gericht op de ontwikkeling van nieuwe therapeutica voor leverziekten, dient zich onder anderen te richten op het vaststellen van welke reactieve zuurstofverbindingen van belang zijn bij specifieke leverziekten en de relatieve bijdrage van deze verbindingen in de pathogenese van leverziekten.

CHAPTER 9

Sumario en español

Mecanismos de muerte celular inducidos por

estrés oxidativo en hepatocitos.

Objetivos para una intervención de protección

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Radical libre es aquella especie química que contiene uno o más electrones desapareados en su capa de valencia, lo que los hace muy reactivos. Los radicales libres son compuestos prooxidantes generalmente derivados del oxígeno, por lo que se denominan especies reactivas del oxigeno (ROS). Existen distintas clases de ROS, entre las que destacan el anión superóxido (O2

.-), peroxido de hidrógeno (H2O2) y radicales oxidrilo (HO.). Los aniones superóxido proceden de la reducción univalente del oxígeno molecular, principalmente en la cadena de transporte mitocondrial. No poseen gran reactividad pero a partir de estos aniones, se generan gran cantidad de especies reactivas derivadas del oxígeno (ROS) que son muy reactivas y dañinas. Las especies reactivas del oxígeno surgen continuamente como consecuencia del metabolismo normal de las células al utilizar el oxígeno, y se consideran productos no deseados e inevitables, formados a partir de reacciones químicas imprescindibles para la vida. También pueden originarse a partir de fuentes externas, como por ejemplo la polución ambiental, el humo del tabaco, etc. Una vez formadas, las especies reactivas del oxígeno dañan biomoléculas como proteínas, membranas lipídicas y ácidos nucleicos, elementos esenciales para las células, produciendo su deterioro, envejecimiento y muerte. Como sistema de protección frente a estos eventos, las células están equipadas con antioxidantes que funcionan a modo de mecanismos de detoxificación de ROS para evitar daños celulares y en órganos. Entre los principales mecanismos de detoxificacion de ROS destacan sistemas enzimáticos, como son la enzima superóxido dismutasa (Mn/Cu,Zn-SOD), que detoxifica eficientemente los aniones superóxido a peróxido de hidrógeno, las enzimas glutatión peroxidase (GPx) y catalasa, que a su vez transforman el peróxido de hidrógeno en agua. Sistemas no enzimáticos de detoxificacion son el glutatión, la bilirrubina y las vitaminas. A pesar de los mecanismos sumamente eficientes de detoxificación que poseen las células, la exposición continuada a altos niveles de ROS tiene como resultado una situación de estrés para las células. Cuando se produce un desequilibrio entre sustancias prooxidantes (dañinas) y antioxidantes (protectoras), a favor de las primeras, se produce una situación de estrés oxidativo. El estrés oxidativo ha sido definido como la exposición inadecuada a especies reactivas del oxígeno (ROS) y es el resultado del desequilibrio entre prooxidantes y antioxidantes que conduce al daño y muerte de la célula y del tejido. Muchos desórdenes, como por ejemplo, enfermedades degenerativas del sistema nervioso, enfermedades cardiovasculares y hepáticas, diabetes y envejecimiento, están caracterizadas por la presencia de estrés oxidativo, que daña las células y tejidos y agrava y contribuye a la patología de estas enfermedades. Bajo condiciones de estrés oxidativo, y dependiendo de la variedad de ROS presente, la muerte de la célula puede seguir dos procesos diferentes, denominados apoptosis y necrosis. La necrosis es pasiva (no dependiente de energía) y asociada con el agotamiento de ATP, ruptura de la membrana plasmática que rodea a la célula y vertido del contenido celular al medio exterior, lo que afecta a células vecinas y produce inflamación. Por otro lado, la apoptosis, o muerte programada de la célula, es un proceso activo (dependiente de energía en forma de ATP) caracterizado por la reducción de la célula en

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pequeños fragmentos (cuerpos apoptóticos) rodeados de una membrana plasmática intacta, la condensación del núcleo de la célula y la activación de caspasas, sin afectar a células vecinas. Existen dos rutas principales que conducen a la muerte por apoptosis. La vía extrínseca, iniciada por la unión de ligandos específicos a ciertos receptores situados en la membrana que rodea la célula, lo que produce la activación de la caspasa-8, la cual comienza la cascada de activación de otras moléculas que conducen a la muerte celular. La otra vía es la intrínseca, iniciada a nivel mitocondrial, con la ruptura de la membrana que rodea la mitocondria y formación de poros, a través de los cuales se produce la liberación de factores (citocromo c) que activan la caspasa-9, que consecuentemente inicia la cascada que conduce a la muerte por apoptosis.

FADDCaspasa-8

Caspasa-3

APOPTOSIS

Receptor de muerte (TNF-R, Fas-R)

Caspasa-9ROS

Membrana plamatica

Estres oxidativo (ROS)

SUPERVIVENCIA

nucleoNF-κB

NF-κB

IκB

p38

MAPK

ERK

PI3-K

AktJNK

Cit c

BID

tBID

Mitocondria Poro mitocondrial

FADDCaspasa-8

Caspasa-3

APOPTOSIS

Receptor de muerte (TNF-R, Fas-R)

Caspasa-9ROS

Membrana plamatica

Estres oxidativo (ROS)

SUPERVIVENCIA

nucleoNF-κB

NF-κB

IκB

p38

MAPK

ERK

PI3-K

AktJNKJNK

Cit c

BID

tBID

Mitocondria Poro mitocondrial

Figura 1. Esquema de las rutas de señalización inducidas por el estrés oxidativo.

La apoptosis es un proceso controlado, considerado como un suicidio de la célula. Posee un papel fundamental en el mantenimiento del equilibrio entre muerte y proliferación de las células, e interviene en importantes procesos fisiológicos como la embriogénesis, renovación de tejidos y el desarrollo y mantenimiento del sistema inmune. Así, la apoptosis actúa como un sistema de calidad y de reparación, eliminando células anormales y defectuosas. Por lo tanto, existe una larga lista de enfermedades asociadas con alteraciones y desregulación de la apoptosis. Factores que aumentan el proceso de la apoptosis contribuyen al origen de enfermedades neurodegenerativas (Parkinson, Alzheimer, esclerosis multiple), enfermedades del sistema inmune (artritis reumatoide, hepatitis), diabetes, ateroesclerosis y enfermedades hepáticas. Factores que disminuyen la apoptosis contribuyen al surgimiento y desarrollo de tumores y cánceres. En enfermedades hepáticas crónicas, como por ejemplo la hepatitis alcohólica y vírica, esteatohepatitis y colestasis, los hepatocitos (las células del hígado) están invariablemente expuestos a estrés oxidativo, que los daña y produce su muerte celular (apoptosis o necrosis) y la pérdida de la función de hígado, llegando a provocar la muerte del individuo. Por lo tanto, es importante conocer los mecanismos por los que las especies reactivas del oxigeno (ROS) conducen a la muerte de las células del hígado (hepatocitos). Un

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entendimiento más completo de la respuesta celular al estrés oxidativo podría ser de gran utilidad para entender y tratar las enfermedades hepáticas relacionadas con el estrés oxidativo. Por este motivo, esta tesis doctoral está enfocada al estudio de los mecanismos que conducen a la muerte de hepatocitos expuestos a estrés oxidativo y a posibles puntos y vías de intervención para tratar y prevenir enfermedades hepáticas. El capítulo 3 describe diferencias importantes en los mecanismos y el modo de muerte (apoptosis y necrosis) del hepatocito, inducidos por distintas especies reactivas del oxígeno (anión superóxido y peróxido de hidrógeno). Por un lado, el peróxido de hidrógeno (H2O2, a concentraciones menores de 5mmol/L) no produce la apoptosis o necrosis de los hepatocitos. Solamente concentraciones más altas o cuando se deterioran los sistemas de detoxificacion, el peróxido de hidrógeno actúa como una sustancia necrótica. La elevada eficacia de los hepatocitos en la eliminación de H2O2, especialmente mediante la enzima catalasa, explica porqué el H2O2 apenas es detectado por estas células, siendo sólo moderadamente tóxico para los hepatocitos. Ha sido demostrado que la exposición a ROS activa rutas de señalización, las cuales influencian el equilibrio entre la muerte de la célula y su supervivencia. Entre la rutas de señalización activadas por el estrés oxidativo, destacan la vía de las protein kinasas activadas por mitógenos (MAPK), entre las que se encuentran la kinasa extracelular reguladora (ERK1/2), la kinasa amino-terminal c-jun (JNK) y la kinasa p38. También destacan la vía de la fosfoinositol-3-kinasa (PI3K/Akt), el factor nuclear-kappa B (NF-κB) y la enzima Heme oxigenasa-1 (HO-1). Sin embargo, en este estudio, hemos mostrado que el peróxido de hidrógeno no activa las rutas ERK1/2 o JNK MAPK. Esto podría ser debido al hecho de que el peróxido de hidrógeno es solo ligeramente detectado por los hepatocitos. En cambio, en esta tesis demostramos que menadione, un donante de aniones superóxido, induce principalmente apoptosis en hepatocitos. Además, menadione induce la activación de caspasas y las rutas de señalización ERK1/2 y JNK MAPK. La apoptosis inducida por menadione es dependiente de la activación de las caspasas y la actividad de JNK MAPK, lo que aporta evidencias de un papel pro-apoptótico de JNK en hepatocitos expuestos a estrés oxidativo. La inhibición de la ruta JNK bloquea la activación de la caspasa-9, indicando que JNK provoca la disrupción de la mitocondria y la formación de poros en su membrana, activando así la vía intrínseca que conduce a la muerte apoptótica. La fosforilación y activación de ERK1/2 MAPK producida por menadione atenúa la muerte de los hepatocitos, indicando un papel anti-apoptótico de ERK1/2 contra la apoptosis. Sin embargo, la activación de ERK1/2 es claramente insuficiente para prevenir la apoptosis inducida por menadione, sugiriendo que JNK desempeña un papel más determinante y más central en la apoptosis. En esta tesis, demostramos diferencias notables entre diversas sustancias con usos terapéuticos que eliminan las especies reactivas del oxígeno (ROS). PEG-SOD (superóxido dismutasa) inhibe totalmente la activación de las caspasas, suprime apoptosis y no aumenta muerte necrótica de la célula. De hecho, PEG-SOD representa una novedosa estrategia para tratar las enfermedades del hígado causadas por estrés oxidativo. En cambio, la exposición de

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los hepatocitos a óxido nítrico bloquea la apoptosis inducida por menadione, pero provoca la muerte necrótica de las células, indicando un cambio en el modo de muerte de los hepatocitos. Con estos resultados demostramos que es imprescindible evaluar las consecuencias de cualquier intervención para evitar la muerte celular, teniendo en cuenta la posibilidad de modificar el equilibrio entre apoptosis y necrosis. El estrés oxidativo activa rutas a favor y en contra de la apoptosis en hepatocitos. Así, la supervivencia de la célula depende del equilibrio entre las rutas que activan procesos a favor y en contra de la muerte celular. En condiciones de estrés oxidativo moderado, la célula sobrevivirá. Sin embargo, bajo condiciones de intenso estrés oxidativo, las rutas que favorecen la apoptosis prevalecerán y cambiarán el equilibrio provocando la muerte de la célula. El conocimiento sobre diversos ROS implicados en las enfermedades del hígado es necesario para interferir y tratar específicamente estos desórdenes usando las herramientas terapéuticas apropiadas que modulen las rutas implicadas en el proceso y no cambien la manera en la que muere la célula. El capítulo 4 describe una intervención protectora contra la apoptosis inducida por aniones superóxido (menadione), mediante la sobreexpresión de la enzima heme oxigenasa-1 (HO-1) y el monóxido de carbono (uno de los productos del metabolismo de esta enzima). HO-1, encargada de la degradación del grupo heme a monóxido de carbono, bilirrubina e hierro, ha sido identificada como un sistema protector contra el estrés oxidativo, aunque los mecanismos que intervienen en este efecto protector no han sido identificados en su totalidad. Menadione induce la expresión de HO-1, aunque esto no previene la apoptosis en los hepatocitos, probablemente porque la expresión de HO-1 no es lo suficientemente alta o rápida. Sin embargo, la sobre-expresión de HO-1, usando el adenovirus Ad5HO-1, 15hrs antes de la adición de menadione promueve la protección contra apoptosis en hepatocitos inducida por el estrés oxidativo. Hemos demostrado que el monóxido de carbono (CO) es un potente inhibidor de la activación de las caspasas, la activación de PARP (marcador de apoptosis) y la apoptosis en hepatocitos. Además, aportamos evidencias de que el CO previene a fosforilación y activación de JNK y su actividad promotora de apoptosis, indicando que el efecto protector del CO contra la apoptosis es, al menos en parte, debido a la inhibición de JNK. Adicionalmente, demostramos que el CO modula rutas de señalización tanto a favor como en contra de la apoptosis. El monóxido de carbono previene la activación de la ruta JNK y su efecto a favor de la apoptosis. Al mismo tiempo, el CO activa la ruta ERK1/2 MAPK y su efecto en contra de la apoptosis, aumentando así su efecto protector.

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Membrana PlasmaticaEstres oxidativo

JNK p-JNK

Cyt c

Casp-9

Casp-3

APOPTOSIS

ERK

+nucleo

AP-1 HO-1 CO

Biliverdina

SUPERVIVENCIA

Formacion poro mitocondrial

Liberacion

Activacion

Mitocondria

Bloqueo

Membrana PlasmaticaEstres oxidativo

JNK p-JNK

Cyt c

Casp-9

Casp-3

APOPTOSIS

ERK

+nucleo

AP-1 HO-1 CO

Biliverdina

SUPERVIVENCIA

Formacion poro mitocondrial

Liberacion

Activacion

Mitocondria

Bloqueo

Figura 2. Resumen esquemático del mecanismo protector de la enzima Heme oxigenasa-1 (HO-1) y el monóxido de carbono (CO) en la prevención de la apoptosis inducida por el estres oxidativo.

Finalmente, indicamos que el CO protege contra la apoptosis en hepatocitos expuestos a estrés oxidativo y no cambia el modo de muerte celular, al no inducir necrosis. De este modo, la modulación de la expresión de la enzima HO-1 o de los niveles de CO, podría convertirse en un blanco terapéutico importante en los desórdenes del hígado causados por la exposición al estrés oxidativo. En el capítulo 5, aportamos una estrategia novedosa para prevenir la apoptosis inducida por aniones superóxido, usando metformin como elemento protector de hepatocitos expuestos a estrés oxidativo. Además, introducimos los mecanismos implicados en este proceso. El estrés oxidativo y la resistencia a la insulina son factores importantes en la patología de enfermedades hepáticas y en las complicaciones asociadas con la diabetes. Se ha demostrado que metformin, un agente de sensibilización a la insulina utilizado para tratar diabetes tipo 2, es un protector hepático en muchos modelos in vivo. En esta tesis, examinamos el efecto protector del agente metformin contra la apoptosis en hepatocitos expuestos a menadione, un donante de aniones superóxido. Este estudio muestra que metformin protege los hepatocitos contra la activación de caspasas y la apoptosis inducida por aniones superóxido. En respaldo de estos resultados, también demostramos que metformin reduce la activación de la ruta JNK y de su actividad a favor de la apoptosis. Esta actividad pro-apoptosis de JNK incluye la activación de la vía intrínseca mitocondrial que conduce a la muerte del hepatocito. De hecho, JNK ha sido descrito como un mediador crucial en la resistencia a la insulina y un elemento importante en la patogénesis de la enfermedad del hígado graso y de la diabetes tipo 2. En esta tesis documentamos que metformin aumenta la expresión de la enzima Heme oxigenasa-1 inducida por aniones superóxido, lo cual tiene un efecto protector contra la apoptosis inducida por menadione, como anteriormente hemos indicado. De este modo, sugerimos que HO-1 contribuye al efecto anti-apoptótico de metformin (figura 3). También proporcionamos evidencias de que metformin protege contra la apoptosis y al mismo tiempo no induce necrosis en hepatocitos, indicando la realidad de su naturaleza protectora.

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Menadione(ROS)

APOPTOSIS

MetforminJNK

caspases

HO-1 Supervivencia

+Inhibición

Menadione(ROS)

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MetforminJNK

caspases

HO-1 Supervivencia

+Inhibición

Figure 3. Resumen esquemático de los efectos protectores del agente metformin contra la apoptosis inducida por el donante de aniones superóxido menadione. Esta tesis demuestra que la inducción HO-1 y la inhibición de JNK están, al menos en parte, implicadas en el efecto protector de metformin contra la apoptosis inducida por estrés oxidativo. Sin embargo, no excluimos la posibilidad de que en la actividad protectora de metformin parcialmente también intervengan otras rutas de señalización protectoras. Debido al papel central de JNK en la patogénesis de enfermedades asociadas con el estrés oxidativo, incluyendo la diabetes y la mayoría de las enfermedades crónicas del hígado, la modulación de la actividad de JNK representa un objetivo prometedor y atractivo para el tratamiento de estos desórdenes. En el capítulo 6, exponemos otra manera de inducir apoptosis en hepatocitos y como protegerlos de ésta. Hemos demostrado que el ácido glycochenodeoxycholic (GCDCA), pero no el ácido taurochenodeoxycholic (TCDCA) o ácido ursodeoxycholic taurino-conjugado (TUDCA), induce apoptosis en hepatocitos in vitro. Sin embargo, TUDCA previene la apoptosis inducida por GCDCA. En la actualidad, UDCA es utilizado en pacientes para el tratamiento de enfermedades crónicas del hígado. Finalmente, TUDCA no inhibe la apoptosis inducida por aniones superóxido (menadione), indicando que los mecanismos de muerte celular (apoptosis) inducidos por aniones superóxido y GCDCA son diferentes. Conclusiones El conocimiento sobre los mecanismos que controlan la muerte de las células del hígado es de importancia clínica y científica para identificar y desarrollar nuevas herramientas terapéuticas para el tratamiento de enfermedades hepáticas. Esta tesis describe el mecanismo que conduce a la muerte de hepatocitos expuestos a estrés oxidativo, demostrando que diversas especies reactivas del oxígeno inducen diferentes modos de muerte celular. También identificamos nuevos objetivos y estrategias concretas para posibles intervenciones de protección contra la apoptosis en hepatocitos. Además, discutimos la posibilidad de modificar el equilibrio entre apoptosis y necrosis durante una intervención para prevenir un tipo muerte celular determinada. En resumen, podemos concluir que JNK desempeña un papel importante en la apoptosis de los hepatocitos, por lo tanto, puede ser considerado un blanco potencial para interferir con la

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muerte celular y para el tratamiento de enfermedades del hígado. Finalmente, futuros estudios dirigidos a la identificación de nuevas estrategias para tratar y prevenir enfermedades del hígado deben centrarse en el tipo de especies reactivas del oxígeno formadas en estos desórdenes y en la contribución relativa del modo de muerte celular a la patogénesis de estas enfermedades asociadas con el estrés oxidativo.

Acknowledgements/ Agradecimientos

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It looks incredible but it is real. After four years and a half, my book is finished and here I am thanking all the people who made possible that I could finish my PhD and my booklet. The first person I would like to thank is my promotor and supervisor Prof. Dr. Han Moshage. Four years and six months ago, you gave me the opportunity to get a PhD position in your group (and I know that the first impression I gave you in the interview job was not my best!). Han, you trusted me and you gave me the chance to learn many things, go to a lot of international congresses and taste the feeling of being a scientist. Thank you very much! The second person I thank is my other promotor: Prof. Dr. Peter LM Jansen. Thank you very much Peter for all your help and your concern about my project and my future in science. I really appreciate everything you have done for me during these years. Next in the list is Klaas Nico Faber. Thanks for your enthusiasms, your support during my PhD and all the comments and suggestions I got from you. Of course I thank to the members of the reading committee: Klaas Poelstra, Folkert Kuipers and José C Fernández Checa (muchas gracias José por formar parte de este proyecto, por tu visita a Groningen y por tu inmensa amabilidad. Gracias también a Carmen García Ruiz, por aquella charla tan especial en mi primer congreso en Boston). Very important is to thank the people from my AIO room: Aldo (we all miss you here!), Janine (Thank you for the nice dinners and chats at the kantine and for choosing me as your paranimf, you made me feel very special. It was an honour. I will never forget it), Thierry (thanks a lot for your help, your comments and suggestions and the long and nice chats we had, and for the delicious tons of chocolate with nuts that you found for me!), Antonella (thanks for being there all these years. You have been my colleague, housemate, friend and the person before me in all the queues we have done together. Thanks a lot!), Marijke (thanks for your sweetness and for helping me so much), Anja, Robert, Baukje (we miss you a lot!), Hilde, Anke and Harmen (good luck to the three of you with your projects!). My colleagues: Rebekka (you have been one of my best friends here. Thanks for the quality time we spent together, and as I used to tell you, my life was much easier having you around! I really miss you a lot!!), Manon (thanks!, greetings to Marcus and Anna), Titia, Jannes, Krzysztof (a nice trio of smokers!), Sandra (good luck for the future!), Lisette (thanks for being my paranimf. Good luck!), Fiona (my dear Fiona, I will miss you a lot!), Mariska, Janette, Martijn (thanks for your kindness), Axel, Joost, Lu (Good luck!), Hans (We miss you in Groningen!), Marieke, Jenny, Jacqueline, Gerard and all the students (Karen (thanks!), Sytze thanks for the emails after all these years and best regards to your bunnies!). I also thank to: Yan (thanks for all the chats we had together and being such a nice person. Good luck for the future! I hope you will not forget about me in China, you have big plans there. Tell me everything!), Jelske (It was fun to be your half orange paranimf in Janine’s promotion), Maaike, Thomas (thanks for the French music!), Rick (thanks! for all the isolations you did, from which I got very nice hepatocytes for my experiments), Fjodor (the chocolate specialist. Thanks for the primers and the kilos of Ritter Sport!), Hester, Leonie, Esther, Annike W, Niels, Uwe, Torsten, Annike K, Jaap (thanks for all the Buenos dias and


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buenas tardes you said to me), Dirk-Jan, Henkjan, Han R, Frans, Tinneke, Vincent, Renze, Juul, Nicolette, Janny, Jan Peter, Sabine, Victor, Gloria, Elena and Eric, Magdalena and Daan. Un saludo a Jordi Muntané y mi agradecimiento por lo aprendido durante el tiempo que trabajé en su grupo en la Unidad de Investigación del Hospital Reina Sofia de Córdoba. Mi sincero agradecimiento a Natalia Nieto, por estar siempre ahí, por todo tu apoyo y por ofrecerme la oportunidad de trabajar contigo en uno de los mejores hospitales del mundo. A mis amigos: David y Elena (Elenita, una de las personas más especiales que conozco. Chicos, mucha suerte en el futuro!), Jose y Rafi (por hacer de vuestra casa la nuestra y por vuestra amistad. Jose, tus cuentos son magníficos! Besos a Irene), Lindo (fuente ilimitada de ideas y frases geniales), Paco y Yolanda. Esas mañanas de navidad y tardes de verano en el Correo con una cervecita fresca al solecito cordobés en vuestra compañía son inolvidables, muchas gracias!. A Jose Galván y Cristina (por vuestra amistad, se os echa de menos!), Paco y Maribel (por los estupendos días en la Rambla y por ser unos amigos estupendos), Fran Marcos y Sophie (gracias por las visitas a Leiden, por acogernos en vuestra casa y por las charlas por Internet. Besos para Rafa), Santi y Celia (por las maravillosas vacaciones juntos y por ser tan buenos amigos), Antonio y Angie, y David Quesada. Iker e Itziar (gracias por venir a Córdoba y por los días que pasamos en Bilbao). I thank my friends in Leiden: Matt and Anne (for all those concerts together and dinners, chats and the rest at your place. Thanks for your visit to Córdoba, it was very special to have your there), Jim and Eniko (thanks for the dinners at your place and for being always so friendly), and Duncan (wonderful dinners and chats together). Thanks for the time we spent together. I will miss you all a lot.

A los profesores que contribuyeron a mi formación, haciendo posible el llegar hasta aquí.

A mi familia, papá y mamá (Ángel y Paqui, por vuestro apoyo y cariño incondicional), a mi hermana Susana (por ser la mejor de las hermanas, por poder contártelo todo y estar siempre ahí. Besitos a tu Raúl), a mi abu Encarna (una abuela con mucha marcha. Me encantan tus cartas, las galletitas y los pestiños), a mis tíos Joaquín y Pilar, Carmen y Juan y mis primos, Carmen y Maria, Anai y Pablo. A mi querido Blacky (por todos tus años de compañía y cariño a la familia. Siento perderme los últimos años de tu vida y no poder estar ahí para ayudarte). A Flaush, por acompañarme las tardes lluviosas mientras escribía la tesis.

A mi otra familia: M Ángeles (gracias por todas las llamadas a Groningen) y Juan Francisco (mil gracias por todo!), Juan Fran (mucha suerte en Pittsburg!), Jorge y Jaime, por todos estos años siendo una más en la familia.

Muchas gracias Raúl, por apoyarme y aconsejarme siempre, por todos esos viajes a Groningen, por las comiditas tan ricas, por poner música en mi vida, por hacerme sentir especial y por querer compartir el futuro conmigo.

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Laura Conde de la Rosa was born the 23rd February 1976 in Córdoba, Spain. She started the studies of Biology at the University of Córdoba in 1994. After finishing Biology, in 1999 she started the studies of Enology (wine production and cultivation of the grapevine) at the University of Córdoba. Then in 2001, she had the opportunity to collaborate in a project with Dr. Jordi Muntané Relat at the department of Gastroenterology and Hepatology, in the Research Unit of the Reina Sophia University Hospital in Cordoba. In March 2002, she got a PhD position under the supervision of Prof. Han Moshage at the department of Gastroenterology and Hepatology, University Medical Center Groningen. The project, resulting in this thesis, was financially supported by the Ubbo Emmius program from the University of Groningen. In January 2007 she will start working as a Postdoc in a project led by Prof. Scott Friedman and supervised by Dr. Natalia Nieto, at the department of Medicine, Liver diseases division, Mount Sinai School of Medicine in New York, USA.


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List of publications: L. Conde de la Rosa, MH. Schoemaker, TE. Vrenken, M. Buist-Homan, R. Havinga, PLM. Jansen, H. Moshage. “Superoxide anions and hydrogen peroxide induce hepatocyte death by different mechanisms: involvement of JNK and ERK MAP kinases”. Journal of Hepatology 44 (2006) 918-929. L. Conde de la Rosa, TE.Vrenken, RA. Hannivoort, M. Buist-Homan, R. Havinga, D.J Slebos, HF. Kauffman, PLM. Jansen, H. Moshage.“Heme oxygenase-1 derived carbon monoxide protects primary hepatocytes against oxidative stress-induced apoptosis via inhibition of JNK activation” Submitted for publication. L. Conde de la Rosa, TE. Vrenken, M. Buist-Homan, H. Moshage. “Metformin protects against oxidative stress induced apoptosis in primary hepatocytes” In preparation MH.Schoemaker, L. Conde de la Rosa, M. Buist-Homan, TE. Vrenken, R. Havinga, K. Poelstra, HJ. Haisma, PLM.Jansen, H. Moshage.“Tauroursodeoxycholic acid protects rat hepatocytes from bile acid-induced apoptosis via activation of survival pathways”. Hepatology. 2004 Jun 39(6):1563-73. MH. Schoemaker, WM. Gommans, L. Conde de la Rosa, M. Homan, P. Klok, C. Trautwein, H. van Goor, K. Poelstra, HJ. Haisma, PLM Jansen, H. Moshage.“Resistance of rat hepatocytes against bile acid-induced apoptosis in cholestatic liver injury is due to nuclear factor-kappa B activation”. Journal of Hepatology. 2003 Aug:39(2):153-61. Presentations: Poster presentations - Early GUIDE Summer Meeting June 2003, 2004, 2006. - 54th American Association for the study of liver diseases (AASLD). Oct 2003, Boston. - Apoptosis 2003: from signaling pathways to therapeutic tools. Luxemburg 2003. - 56th AASLD Annual Meeting. , November 2005, San Francisco Posters: 2. - 57th AASLD Annual Meeting. October 2006. Boston. - Falk symposium: XIX international Bile Acid Meeting. October 2006. Freiburg, Germany. Oral presentations - Early GUIDE summer meeting. June 2003. Groningen. The Netherlands - Early GUIDE summer meeting. June 2006. Groningen, The Netherlands - Najaarsvergadering van de Nederlandse vereniging voor Gastroenterologie. Oct 2003. Veldhoven, The Netherlands - Najaarsvergadering van de Nederlandse vereniging voor Gastroenterologie. Oct 2004 - Najaarsvergadering van de Nederlandse vereniging voor Gastroenterologie. Oct 2005 - Meeting of the European Club for Liver Cell Biology. May 2005, Switzerland. - Najaarsvergadering van de Nederlandse vereniging voor Gastroenterologie. Oct 2006.


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Abstracts Conde de la Rosa L, Vrenken TE, Buist-Homan M, et al. Apoptosis induced by reactive oxygen species in primary rat hepatocytes is prevented by carbon monoxide via JNK inhibition Hepatology 42 (4): 638A-638A Suppl. 1 Oct 2005 Conde de la Rosa L, Schoemaker M, Vrenken T, et al. Carbon monoxide protects hepatocytes against oxidative stress-induced apoptosis European Journal of Gastroenterology and Hepatology 17 (1): A50-A51 Jan 2005 Conde de la Rosa L, Buist-Homan M, Moshage H, et al. Oxidative stress-induced apoptosis is inhibited by metformin via an ERK and SRC dependent pathway in rat hepatocytes Hepatology 42 (4): 635A-636A Suppl. 1 Oct 2005 Conde de la Rosa L, Schoemaker MH, Homan M, et al. Reactive oxygen species induce hepatocytes cell death via different mechanisms: a balance between apoptosis and necrosis. Hepatology 38 (4): 866 Suppl. 1 Oct 2003 Schoemaker MH, Conde de la Rosa L, Buist-Homan M, et al. Tauroursodeoxycholic acid protects rat hepatocytes from bile acids-induced apoptosis via activation of survival pathways Hepatology 39 (6): 1563-1573 JUN 2004 Vrenken TE, Conde de la Rosa L, Buist-Hornan M, et al. Caspase-6 is involved in bile acid-induced apoptosis European Journal of Gastroenterology and Hepatology 17 (1): A39-A39 Jan 2005 Vrenken TE, Conde de la Rosa L, Buist-Homan M, et al. Metformin protects rat hepatocytes against bile acid and cytokine-induced apoptosis Hepatology 42 (4): 500A-500A Suppl. 1 Oct 2005 Vrenken TE, Conde de la Rosa L, Buist-Homan M, et al. Caspase-6 is involved in bile acid-induced hepatocyte apoptosis Hepatology 40 (4): 566A-567A Suppl. 1 Oct 2004 van der Weide K, Gerrits A, Conde de la Rosa L, et al. Overexpression of NF-kappa B-regulated BCL-2 family member A1/BFL-1 prevents bile acid induced apoptosis in human hepatoma cells Hepatology 42 (4): 499A-500A Suppl. 1 Oct 2005

Documents

University of Groningen Mechanisms of oxidative stress ......Mechanisms of oxidative stress-induced cell death in hepatocytes: Targets for protective intervention Laura Conde de la