Regulation of heme oxygenase-1 expression through the phosphatidylinositol 3 kinase ... · 2003-12-19 · Regulation of heme oxygenase-1 expression through the phosphatidylinositol

Regulation of heme oxygenase-1 expression through the

phosphatidylinositol 3 kinase/Akt pathway and the Nrf2 transcription factor

in response to the antioxidant phytochemical, carnosol.

Daniel Martin1, Ana I. Rojo1, Marta Salinas1, Raquel Diaz1,3, German Gallardo3,

Jawed Alam2, Carlos M. Ruiz de Galarreta3, and Antonio Cuadrado1*.

1 Instituto de Investigaciones Biomédicas and Departamento de Bioquímica

Facultad de Medicina, Universidad Autónoma de Madrid. 28029 Madrid, Spain,

2Department of Biochemistry and Molecular Biology, Louisiana State University

Health Science Center, New Orleans, Louisiana 70112. USA, 3 Departamento de

Bioquímica, Facultad de Medicina, Universidad de las Palmas de Gran Canaria,

35016 Gran Canaria, Spain.

Running title: Carnosol induction of HO-1 through PI3K and Nrf2.

Keywords: heme oxygenase, carnosol, Nrf2, PI3K, Akt, AREs.

* To whom correspondence should addressed to:

Dr. Antonio Cuadrado Departamento de Bioquímica, Universidad Autónoma de Madrid Arzobispo Morcillo 4 28029 Madrid Spain. Tel: 3491 497 5327 FAX 3491 585 4401 E-mail: [email protected]

Copyright 2003 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on December 19, 2003 as Manuscript M309660200 by guest on February 26, 2020

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ABSTRACT

The phosphatidylinositol 3 kinase (PI3K)/Akt pathway elicits a survival

signal against multiple apoptotic insults. In addition, phase II enzymes, such as

heme oxygenase-1 (HO-1), protect cells against diverse toxins and oxidative

stress. In this report, we describe a link between these defense systems at the

level of transcriptional regulation of the antioxidant enzyme HO-1. The herb-

derived phenol, carnosol, induced HO-1 expression at both messenger RNA and

protein levels. Luciferase reporter assays indicated that carnosol targets the

mouse ho1 promoter at two enhancer regions comprising the antioxidant

response elements (AREs). Moreover, carnosol increased the nuclear levels of

Nrf2, a transcription factor governing AREs. Electrophoretic mobility shift assays

and luciferase reporter assays with a dominant negative Nrf2 mutant, indicated

that carnosol increases the binding of Nrf2 to ARE and induces Nrf2-dependent

activation of the ho1 promoter. While investigating the signaling pathways

responsible for HO-1 induction, we observed that carnosol activates the ERK,

p38 and JNK pathways as well as the survival pathway driven by PI3K. Inhibition

of PI3K reduced the increase in Nrf2 protein levels and activation of the ho1

promoter. Expression of active PI3K-CAAX was sufficient to activate AREs. The

use of dominant negative mutants of PKCζ and Akt1, two downstream kinases

from PI3K, demonstrated a requirement of active Akt1 but not PKCζ. Moreover,

the long-term antioxidant effect of carnosol was partially blocked by PI3K or HO-

1 inhibitors, further demonstrating that carnosol attenuates oxidative stress

through a pathway that involves PI3K and HO-1.

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INTRODUCTION

High levels of reactive oxygen species (ROS) cause damage to cells and

are involved in several human pathologies including neurodegenerative disorders

and cancer (1, 2). Therefore, the use of compounds with anti-oxidant properties

may help prevent or alleviate diseases in which oxidative stress is a primary

cause (3). Carnosol, a diterpene derived from the herb rosemary, is a

representative member of a family of plant-derived phenols, which also include

curcumin, carnosic acid, phenylethyl isothiocyanate, epigallocatechin gallate and

other green tea polyphenols. These bioactive phytochemicals exhibit Michael

acceptor function and therefore behave as antioxidants (4). In addition, being

themselves xenobiotic compounds, they activate a xenobiotic response in the

target cells affecting the expression of phase II enzymes such as

NAD(P)H:quinone oxidoreductase, aldo-keto reductase, glutathione S-

transferase, gamma-glutamylcysteine synthetase, glutathione synthetase or

heme oxygenase-1 (HO-1) (5, 6, 7).

Heme oxygenase isozymes, HO-1 and HO-2, catalyze the stepwise

degradation of heme to release free iron and equimolar concentrations of carbon

monoxide (CO) and the linear tetrapyrrol, biliverdin, which is converted to

bilirubin by the enzyme biliverdin reductase (8). The HO-1 isozyme is a phase II

enzyme that is transcriptionally regulated by a large variety of stimuli. These

include its substrate, heme (9, 10), oxidative stress (11, 12), the signaling

proteins NGF, TNF-α, interleukin1β and interferon -γ (13, 14, 15) and phenolic

compounds such as curcumin and caffeic acid (16). Many reports have


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demonstrated the potent antioxidant activity of the heme-derived metabolites

generated by HO-1 catalysis, biliverdin and bilirubin, and the cytoprotective

actions of CO on vascular endothelium and nerve cells (17, 18, 19, 20).

Moreover, both HO-1-defficient mice (21) and a human case of genetic HO-1

deficiency (22) exhibit a serious impairment of iron metabolism leading to liver

and kidney oxidative damage and inflammation. Therefore, it is now widely

accepted that induction of HO-1 expression represents an adaptive response that

increases cell resistance to oxidative injury.

The signaling mechanisms used to activate transcription of phase II genes

such as HO-1 are poorly defined. Most studies have focused on the activation of

the mitogen-activated protein kinases (MAPKs) related to cell growth and stress

response. In vertebrates, the three major MAPK pathways are represented by

kinase cascades leading to activation of extracellular signal-regulated kinase

(ERK), c-jun N-terminal kinase (JNK) and p38 (for a review see 23). All three

pathways appear to be involved to some extent in the up-regulation of HO-1

expression in response to diverse stimuli. For instance, in rat hepatocytes,

sodium arsenite appears to regulate HO-1 expression via activation of Ras and

the JNK pathway but not the ERK pathway (24). On the other hand, arsenite

induces HO-1 expression via ERK and p38 pathways in LMH chicken hepatoma

cells (25). Ischemia-reperfusion in the lung activates HO-1 through JNK and p38

(26). Finally, regarding phytophenols, curcumin up-regulates HO-1 through the

p38 pathway in porcine renal epithelial cells (27).


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However, the role of the survival signaling pathway represented by

PI3K/Akt on the regulation of HO-1 expression is still not defined. An initial study

by Lee et al (28) suggested a role of PI3K in regulation of the phase II enzyme,

NADPH:quinone oxidoreductase. In addition, we previously showed that NGF

induces the expression of HO-1 by a still unknown PI3K-dependent mechanism

(13).

It is accepted that induction of HO-1 expression is mediated through cis-

regulatory DNA sequences located at their promoter region. Among the large

variety of putative regulatory sites found in the promoter 5’-flanking region of

human, rat, mouse and chicken HO-1 genes (29), most investigations have

focused on those which exhibit inducible response to oxidative signals (30).

Several AP-1 binding sites have been shown to be involved in the transcriptional

induction of HO-1 by MAPKs, cyclic nucleotides, protein phosphatase inhibitors

and the polyphenol curcumin (31, 32, 33).

More recently, a new class of AP-1 related site has been shown to

mediate oxidative stress responsiveness of phase II genes. These regions are

termed stress-response elements (StREs) or antioxidant response elements

(AREs) and are tightly regulated by the redox-sensitive transcription factor Nrf2

(NF-E2-related factor 2) (34). Nrf2 is a member of the Cap’n’Collar family of

transcription factors (35). Under non-stimulated conditions Nrf2 is sequestered in

the cytoplasm by binding to Keap1, an actin binding protein (36). Several stimuli,

which include oxidative stress, lead to the disruption of this complex freeing Nrf2

for translocation to the nucleus and dimerization with basic leucine zipper


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transcription factors such as Maf and Jun family members (37). The mechanism

by which Nrf2 is liberated from the Nrf2-Keap1 complex remains controversial but

probably involves phosphorylation and stabilization of the Nrf2 protein (38, 39)

which otherwise has a half life of just 30 min and is degraded via the ubiquitin-

proteasome pathway (39, 40, 41).

In this study we have analyzed the effect of carnosol on expression of the

antioxidant enzyme HO-1 and we have dissected the signaling pathways leading

to HO-1 regulation. Our results indicate that carnosol activates HO-1 expression

probably by increasing Nrf2 protein levels in a PI3K/Akt-dependent manner.

Therefore, in addition to its intrinsic antioxidant nature, carnosol activates the

PI3K/Akt survival pathway and up-regulates expression of the antioxidant

enzyme HO-1.

EXPERIMENTAL PROCEDURES

Cell culture, transfections and reagents. Rat phaeochromocytoma PC12

cells were seeded on poly D-lysine coated plates and grown in Dulbecco’s

modified Eagle medium (DMEM) supplemented with 7.5 % fetal bovine serum

(FBS), 7.5 % heat-inactivated horse serum (HS) and 80 µg/ml gentamicin. Sub-

confluent cell cultures were maintained overnight (16 h) in DMEM medium

supplemented with 1% FBS and 1% HS, an experimental condition that does not

compromise viability in our PC12 cells (13, 42). For luciferase assays, transient

transfections of PC12 cells were performed with the expression vectors pGL3bv,

pHO1-15Luc, pHO1-15-Luc?E1, pHO1-15Luc?E2, pHO1-15Luc(?E1+?E2),

pEF-Nrf2(DN), pEF18-MafG(DN) (43), pCEFL(X-)Akt1(K179M) (42), pcDNA3-


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HA-PKCζ-DN (kindly provided by Dr. J. Moscat, Centro de Biología Molecular,

Madrid), pcDNA3 and pcDNA3-ß-gal. Lipofectamine Reagent (Invitrogen,

Carlsbad, CA, USA), was used according to manufacturer instructions. NGF and

carnosol were purchased from Calbiochem (San Diego, CA, USA), and hemin,

LY294002, PD98059, SB203580, SP600125 and MG132 from Sigma-Aldrich (St

Louis, Mo, USA) These drugs were dissolved in DMSO and used to a final

concentration of vehicle below 0.2%.

Analysis of mRNA levels by semi-quantitative reverse transcriptase-PCR.

Cells were plated on 60 mm dishes and total cellular RNA was extracted using

TRIzol reagent (Invitrogen). Equal amounts (1 µg) of RNA from the different

treatments were reverse-transcribed (75 min, 42 ºC) using 5 units of AMV-RT

(Promega, Madison, WI, USA) in the presence of 20 units of RNAsin (Promega).

Amplification of cDNA was performed in 25 µl of PCR buffer (10 mM Tris-HCl, 50

mM KCl, 5 mM MgCl2, 0.1 % Triton X-100, pH 9.0) containing 0.6 units of Taq

DNA polymerase (Promega), and 30 pmol of synthetic gene-specific primers for

HO-1 (forward, 5’-AAGGCTTTAAGCTGGTGATGG-3’; and reverse, 5’-

AGCGGTGTCTGGGATGAACTA-3’). To ensure that equal amounts of cDNA

were added to the PCR, the ß-actin gene was amplified using synthetic

oligonucleotides (forward 5’-TGTTTGAGACCTTCAACACC-3’; and reverse 5’-

CGCTCATTGCCGATAGTGAT-3’). After an initial denaturation step (4 min, 94

ºC), amplification of each cDNA was performed at the minimal number of cycles

that allowed detection of basal HO-1 mRNA levels in control cells (data not

shown); 19 cycles for ß-actin and 24 cycles for HO-1, using a thermal profile of 1


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min at 94 ºC (denaturalization), 1 min at 58 ºC (annealing), and 1 min at 72 ºC

(elongation). The amplified PCR products were resolved by 1.8% agarose gel

electrophoresis and stained with ethidium bromide.

Real-time quantitative PCR. Quantitative PCR was performed with 20 ng

cDNA obtained as indicated above, in 25 µl containing 0.5 µM primers (HO-1

forward: 5’-GCCTGCTAGCCTGGTTCAAG-3’, HO-1 reverse: 5’-

AGCGGTGTCTGGGATGAACTA-3’, β-actin forward: 5’-

GCCTCACTGTCCACCTTCCA-3’, β-actin reverse: 5’-

CCCGGCCTGAGTAGCATGA-3’) and the nucleotides, buffer and Taq

polymerase included in SYBR Green I Mastermix (PE Applied Biosystems).

Amplification was conducted in an ABI Prism 7700 sequence detection system.

PCR conditions were: 50 ºC for 2 min, 95 ºC for 10 min, 40 cycles at 94 ºC for 15

s, and 60 ºC for 1 min. HO-1 expression was estimated in duplicate samples and

was normalized with β-actin expression levels.

Preparation of nuclear extracts. 5x106 PC12 cells were washed three

times with cold phosphate-buffered saline and harvested by centrifugation at

1100 rpm during 10 min. The cell pellet was carefully resuspended in three pellet

volumes of cold buffer A (20 mM HEPES pH 7.0, 0.15 mM EDTA, 0.015 mM

EGTA, 10 mM KCl, 1% Nonidet-40, 1 µg/ml leupeptin, 1 mM

phenylmethylsulfonyl fluoride, 20 mM NaF, 1 mM sodium pyrophosphate, 1 mM

Na3VO4). The homogenate was then centrifuged at 500 g for 20 min and the

nuclear pellet was resuspended in five pellet volumes of cold buffer B (10 mM

HEPES pH 8.0, 25 % glycerol, 0.1 M NaCl, 0.1 mM EDTA, 1 µg/ml leupeptin, 1


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mM phenylmethylsulfonyl fluoride, 20 mM NaF, 1 mM sodium pyrophosphate, 1

mM Na3VO4). After centrifugation at 500 g for 20 min, nuclei were resuspended

in two pellet volumes of hypertonic cold buffer C (10 mM HEPES pH 8.0, 25%

glycerol, 0.4 M NaCl, 0.1 mM EDTA, 1 µg/ml leupeptin, 1 mM

phenylmethylsulfonyl fluoride, 20 mM NaF, 1 mM sodium pyrophosphate, 1 mM

Na3VO4), and incubated during 30 min at 4 ºC on a rotating wheel. Nuclear

debris was removed by centrifugation at 900 g for 20 min at 4ºC; one part of the

supernatant was resolved in SDS-PAGE, and submitted to immunoblot analysis

using anti-Nrf2 and anti-Sp1 antibodies. The rest of the supernatant was used for

EMSA. Protein concentration was determined with the Dc Protein Assay kit

(BioRad, Hercules, CA, USA).

Electrophoretic mobility shift assays (EMSAs). The double stranded wild-

type oligonucleotide used as Nrf2 probe was 5’-TTTTATGCTGTGTCATGGTT-3’.

Complementary strand was annealed by incubation at a concentration of 250

ng/µl in STE buffer (100 mM NaCl, 10 mM Tris pH8.0, 1 mM EDTA), at 80 ºC for

2 min. The mixture was then slowly cooled down to 4 ºC with a thermal profile of

1 ºC/min in a thermal incubator. Annealed oligonucleotides were diluted to 25

ng/µl in STE buffer. The 5’ end labeling was performed with T4 polynucleotide

kinase (Promega), using 25 ng of double-stranded oligonucleotide and 25 µCi of

[?32P]-ATP (3000 Ci/mmol, Amersham Biosciences, Little Chalfont,

Buckinghamshire, England). The labeled probes were purified using a G-25 spin

column (Amersham Biosciences). The binding reaction mixture contained 5 µg of

nuclear extract in buffer containing 40 mM HEPES pH 8.0, 50 mM KCl, 0.05 %


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Nonidet-40, 1% dithiothreitol and 10 µg/ml poly(dI)-poly(dC), in a total volume of

20 µl. Anti-Nrf2 antibody (2 µg) or unlabeled competitor probe (25 ng) was added

as indicated, and the reaction was incubated for 1 h at 25 ºC. Labeled DNA (0.25

ng) was added to the mixture and submitted to an additional 20 min incubation at

25 ºC. Samples were resolved at 4 ºC, in a 5 % non-denaturing poly-acrylamide

gel in 0.5x Tris-Borate/EDTA. After electrophoresis, the gel was dried and

autoradiographed.

Immunoblotting. 30 µg cell lysate were resolved in SDS-PAGE and

transferred to Immobilon-P membranes (Millipore, Billerica, MA, USA). Blots

were analyzed with the appropriate antibodies: anti-Akt1C20 (1:500), anti-ERK2

(1:1000), anti-p38 (1:1000), anti-Nrf2 (1:16000), anti-Sp1 (1:1000) (Santa Cruz

Biotechnology, Santa Cruz, CA, USA), anti-phosphoAkt1S473 (1:1000), anti-

phospho-ERK2 (1:1000), anti-phospho-p38 (1:1000) (Cell Signaling Technology,

Inc, Beverly, Ma, USA), anti-phospho-JNK (1:1000) (Upstate-Biotech, Lake

Placid, NY, USA), anti-PDI (Protein disulfide isomerase, kind gift of Dr. González

Castaño, Universidad Autónoma de Madrid, Spain), anti-HO-1 (1:1000) or anti-

HO-2 (1:1000) (Stressgene, Victoria, British Columbia, Canada). Appropriate

peroxidase-conjugated secondary antibodies (1:10000) were used to detect the

proteins of interest by enhanced chemiluminiscence.

Flow cytometry. A FACScan flow cytometer (Becton-Dickinson, San Jose,

CA, USA) was used to analyze intracellular ROS with the fluorescence probe

2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) (Molecular Probes, Leiden,

Netherland) that passively diffuses into the cell and is cleaved and oxidized to


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2',7'-dichloro fluorescein (DCF) (BP 530/25 nm). Cells were submitted to

carnosol, Zn-protoporphyrin IX (ZnPPIX), LY294002 (as indicated for each

experiment) and H2O2 (5 min) and then detached mechanically from the plates,

washed once with cold PBS and analyzed immediately. Three independent

samples of 10.000 cells were analyzed for each experimental condition.

Statistics. Student’s t test was used to assess differences between groups.

A p value <0.05 was considered significant. All luciferase and flow cytometry

experiments were performed at least three times using triplicate or quadruplicate

samples per group. The values in graphs correspond to the average of at least

three samples + SEM. Densitometric analyses were performed with the NIH

Image V2.51 software on representative immunoblots from experiments that

were repeated 2-5 times.

RESULTS

Carnosol induces expression of HO-1.

We investigated the possibility that carnosol, a natural diterpene derived

from the herb rosemary, might alter the expression of the antioxidant enzyme

HO-1. PC12 cells were maintained for 16 h in Dubeccos modified Eagle’s

medium supplemented with 7.5% fetal bovine serum plus 7.5% horse serum

(high serum medium) or with 1% fetal bovine serum plus 1% horse serum

(thereafter low serum). Under both growing conditions cells retained a similar

viability as determined by cytometric analysis of propidium iodide incorporation

and annexin-V PE staining, at least within the time-frame of these experiments

(data not shown and 42). Then, cells were stimulated with 10 µM carnosol for 6 h


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or an equivalent volume of DMSO vehicle (final concentration less than 0.2%)

and analyzed for the amount of HO-1 protein. In agreement with a recent study

(44), cells grown in low serum exhibited a slight decrease in HO-1 protein levels

as compared to complete serum-growing cells (Fig 1A). Interestingly, carnosol

induced a significant increase of HO-1 protein that was more evident in cells

submitted to low serum conditions. To further study the effect of carnosol on

induction of HO-1 expression, PC12 cells were maintained in low serum for 16 h

and then treated for 6 h with carnosol concentrations ranging from 1 to 100 µM.

Real-time PCR (Fig. 1B) indicated that carnosol induces a maximal increase

(over 200 fold) of HO-1 mRNA at 10 µM. Moreover, as shown in Fig. 1C, semi-

quantitative PCR also evidenced a dose-dependent increase in HO-1 mRNA

levels. This increase was not as high as that found with real-time PCR because

amplicon size and primers were different (see Experimental Procedures) and

because, in order to detect basal mRNA levels in the carnosol-untreated cells, it

was necessary to increase PCR cycles over the linear range in the carnosol-

induced cells. In addition, carnosol also produced a parallel increase in HO-1

protein levels, while HO-2 levels corresponding to the other isozyme

constitutively expressed in these cells, remained without significant alteration (Fig.

1D). Consistent with these data, 10 µM carnosol also induced a time-dependent

increase in HO-1 mRNA levels which was apparent as early as 2 h after

treatment and was followed by a robust increase in HO-1 protein levels at 4 h

(Fig. 1E and 1F). Similar results were obtained in other cell lines of human and

mouse origin (data not shown). Therefore, these results suggest that carnosol


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specifically up-regulates expression of the inducible heme oxygenase isozyme,

HO-1.

Carnosol activates HO-1 through the antioxidant response elements

(AREs)

The mouse ho1 promoter contains two AREs that might represent

potential targets of regulation by carnosol. To investigate this possibility, PC12

cells were transfected with luciferase expression vectors carrying the wild type 15

kb mouse promoter or deletion mutant versions lacking either the proximal ARE

(∆E1) or distal ARE (∆E2) or both (∆E1+∆E2) regulatory sequences. Cells were

maintained in low serum for 16 h and then stimulated with 10 µM carnosol for 16

h. As shown in Fig. 2, deletion of ARE sequences E1 or E2 resulted in 30% and

36% decrease in carnosol induction, respectively. Interestingly, deletion of both

AREs abolished the response to carnosol. Therefore, these results suggest that

carnosol activates HO-1 expression through both AREs.

Carnosol increases the levels of the Nrf2 transcription factor.

Several transcription factors have been reported to interact with AREs.

However, considering that induction of HO-1 expression by several inducers

requires de novo protein synthesis (45, 46) we analyzed the possibility that this

molecule would operate on Nrf2, a transcription factor of very short half life (39,

40, 41) that regulates AREs. PC12 cells were maintained in medium with low

serum for 16 h and then stimulated for 3 h with the carnosol concentrations

indicated in Fig. 3A. As a control, cells were also treated with the proteasome

inhibitor MG132. In agreement with previous studies, blockage of proteolysis with


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MG132 resulted in a large accumulation of Nrf2 protein within 3 h, even in the

absence of carnosol. Interestingly, carnosol increased Nrf2 levels in a dose-

dependent manner with maximal effect at 10 µM in our PC12 cells. Additionally,

we analyzed the time-course of Nrf2 accumulation in the presence of 10 µM

carnosol. As shown in Fig. 3B, the accumulation of Nrf2 protein levels was

already detectable after 30 min in the presence of 10 µM carnosol and increased

for at least 3 h during treatment. Of note, the combined treatment with the

proteasome inhibitor MG132 and carnosol did not significantly increase Nrf2

levels over those obtained with each drug separately, suggesting that carnosol

prevents degradation of Nrf2 via the ubiquitin-proteasome pathway.

Next, we analyzed the nuclear levels of carnosol-induced Nrf2 protein. As

shown in Fig. 4A, both MG132 and carnosol induced a strong accumulation of

Nrf2 in the nucleus, while the levels of the house keeping transcription factor Sp1

were stable. Moreover, we investigated whether the carnosol-induced increase of

Nrf2 at the nucleus was sufficient to promote binding of protein complexes

containing this transcription factor to the ARE (E1 sequence) corresponding to

the mouse ho1 promoter. EMSA reactions were performed with nuclear extracts

from cells exposed to carnosol or MG132. As shown in Fig. 4B, among the

several retarded complexes formed, one of them was significantly increased in

intensity upon treatment with carnosol or MG132. Moreover, when the EMSA

reaction was conducted in the presence of an anti-Nrf2 antibody, the intensity of

this complex dropped dramatically, indicating the presence of Nrf2 in the complex.

Taken together these results indicate that carnosol increases both total and


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nuclear protein levels of Nrf2 resulting in increased binding to the ARE promoter

sequence of HO-1.

To verify the functional relevance of Nrf2 binding to the ho1 ARE, PC12

cells were co-transfected with pHO1-15-Luc and expression vectors for dominant

negative mutants of either Nrf2 or one of its dimerization partners, MafG. As

shown in Fig. 4C, the carnosol-induced activation of the ho1 promoter was

significantly decreased in the presence of dominant negative Nrf2 or MafG or

both mutants. These results further suggest that Nrf2 mediates the carnosol-

induced activation of the ho1 promoter.

Carnosol activates the canonical MAPK cascades and the PI3K/Akt

pathway.

In order to identify the signaling pathways used by carnosol to induce HO-

1 expression, we first analyzed the effect of this phytophenol on the three MAPK

kinase cascades, leading to activation of ERK, p38 and JNK, and on the survival

pathway represented by the PI3K and Akt kinases. The activation of these

pathways was analyzed with activation-specific antibodies that selectively

recognize the active and phosphorylated form of ERK, p38, JNK and Akt (see

Experimental Procedures). Cells under low serum conditions were treated with

10 µM carnosol for the indicated times and then cell lysates were immunoblotted

with the anti-phospho-specific antibodies. As shown in Fig. 5, carnosol activated

the three MAP kinase cascades as well as the PI3K/Akt pathway with different

kinetics. Maximal activation ERK was observed after 30 min stimulation while

maximal activation of Akt, JNK and p38 required at least 1 h. Moreover, ERK


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activation was more transient. Therefore, we analyzed which of these pathways

might be involved in channeling the stimulus for HO-1 up-regulation. Low serum

growing cells were pre-incubated for 15 min with inhibitors of each pathway

LY294002 (PI3K), PD98059 (MEK), SB203580 (p38) and SP600125 (JNK), and

then stimulated with 10 µM carnosol for 6 h. The effectiveness of these inhibitors

was confirmed in carnosol-treated cells with antibodies specific for phospho-ERK,

phospho-Jun, phospho-ATF2 and phospho-Akt1 (data not shown). As shown in

Fig. 6A, HO-1 protein levels increased following incubation with 10 µM carnosol,

as expected. Inhibition of the ERK and JNK pathways had little or no effect on

HO-1 protein levels, suggesting that these MAP kinases are not required for HO-

1 up-regulation by carnosol. Curiously, inhibition of both PI3K and, to a lower

extent, p38 pathways dramatically reduced the capacity of carnosol to increase

HO-1 protein levels.

To further confirm these observations, we analyzed the effect of carnosol

on the upstream regulatory sequences of the mouse ho1 gene. PC12 cells were

co-transfected with expression vectors for β-galactosidase, used for

normalization, and pHO1-15-Luc. After serum-starvation, cells were pre-treated

with the MAP kinase and the PI3K inhibitors for 15 min and then incubated with

10 µM carnosol for 16 h. As shown in Fig. 6B, carnosol increased luciferase

activity, consistent with the notion that it activates HO-1 expression. Moreover,

the stimulation of this promoter fragment was significantly reduced in the

presence of the p38 and the PI3K inhibitors while remained unaltered in the

presence of the ERK and JNK inhibitors. Therefore, these observations on the


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mouse promoter correlate with the induction of the endogenous rat HO-1 protein

and strongly suggest that carnosol requires PI3K and, to a lower extent, p38

kinases to induce HO-1 expression in PC12 cells.

Since other groups have described the involvement of the p38 in

activation of HO-1 in the presence of polyphenols (27), we focused our study on

the role of PI3K. Cells maintained in low-serum medium were pretreated with

several concentrations of the PI3K inhibitor, LY294002, and then submitted to 10

µM carnosol for 6 h. As a control for the effectiveness and specificity of the

inhibitor, we monitored the activation of the PI3K downstream effector Akt1, after

30 min of carnosol addition, with activation specific anti-phospho-Akt antibodies.

As shown Fig. 7A, LY294002 concentrations in the low micromolar range

successfully inhibited carnosol-induced activation of Akt and also blocked, at

least partially, the increase in HO-1 and Nrf2 protein levels. Therefore, these

results indicate that PI3K is required to induce HO-1 expression and suggest that

the Nrf2 transcription factor may mediate at least in part this response.

Then, we analyzed whether PI3K alone was sufficient to activate the ho1

promoter, and, in such case, which was the role of AREs. We co-transfected

PC12 cells with pcDNA3-β-gal, an expression vector for constitutively active PI3K

(PI3K-CAAX), and the luciferase expression vectors pHO1-15-Luc or deletion

mutants ∆E1, ∆E2 or (∆E1+∆E2). As shown in Fig. 7B, active PI3K was sufficient

to induce the activity of the ho1 wild type promoter. However, this activation was

significantly decreased on the deletion mutants lacking the E1 or E2 sites and

was drastically reduced in the double mutant (E1+E2). In addition, we analyzed


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the requirement of Nrf2 for PI3K-induced activation of the ho1 promoter. PC12

cells were co-transfected with pHO1-15-Luc, pcDNA3-β-gal, and PI3K-CAAX

together with the expression vectors for dominant negative Nrf2 and MafG

mutants. In several independent experiments, the stimulatory effect of PI3K-

CAAX on the ho1 promoter was smaller in this experimental setting, implying co-

transfection of four plasmids, as compared to the previous experiments

performed by co-transfection of two or three plasmids. Nevertheless, as shown in

Fig. 7C, dominant negative Nrf2 and MafG alone or in combination significantly

blocked PI3K-induced activation of the ho1 promoter. These results indicate that

PI3K is sufficient to activate the ho1 promoter through the AREs in a Nrf2-

dependent manner.

We also analyzed the contribution of kinases regulated by PI3K to the

carnosol-induced up-regulation of HO-1. PC12 cells were transfected with the

expression vector pHO1-15-Luc and HA-tagged, dominant negative versions of

the protein kinases Akt1 and PKCζ, two well known effectors of PI3K. Over-

expression of these proteins was determined by immunoblot analysis with anti-

HA antibodies (Fig. 8C). As shown in Fig. 8A, transfection with dominant

negative PKCζ (K281W) did not have a significant effect on the response to

carnosol. By contrast, as shown in Fig. 8B, dominant negative Akt1(K179M),

significantly blocked the induction of HO-1. Moreover, co-transfection of both

expression vectors inhibited carnosol induction to the same extent as dominant

negative Akt1 alone (data not shown). These results suggest that Akt1, rather

than PKCζ, is responsible for at least part of the carnosol induced up-regulation


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of HO-1. Moreover, as shown in Fig. 8D, PC12 cells, stably transfected with

active PI3K and Akt1 versions, displayed increased HO-1 protein levels, further

indicating that the PI3K/Akt axis is sufficient to up-regulate HO-1 expression.

Finally, we studied whether carnosol attenuates oxidative stress through

the induction of HO-1 expression an the relevance of the PI3K pathway in such

induction. PC12 cells, maintained in low serum for 16 h, were preloaded (1h at

37ºC) with 8 µM 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA), a probe

that passively diffuses into the cells and is cleaved and oxidized in the

intracellular environment to the green fluorescence emitting compound, 2',7'-

dichlorofluorescein (DCF) (47). Then, cells were treated with DMSO vehicle

(control) or carnosol for 30 min, and finally exposed to 250 µM H2O2 for 5 min.

Then, cells were immediately analyzed by flow cytometry. As shown in Fig. 9A

and 9B, micromolar concentrations of carnosol dose-dependently prevented the

intracellular oxidation of the fluorescent probe despite the presence of H2O2.

Moreover, as shown in Fig. 9C, 10 µM carnosol fully prevented the oxidation of

H2DCFDA by H2O2 concentrations ranging from 50 µM to 500 µM. Since the

length of carnosol incubation (just 30 min) was probably insufficient to elicit a

significant effect on gene expression, these results support the intrinsic

antioxidant nature of this phytochemical, in agreement with other studies (48).

Then, we analyzed the long-term effect of carnosol, after 6 h-incubation, a

time consistent with the induction of HO-1 expression through the PI3K/Akt

pathway described in this study. The antioxidant effect of carnosol was compared

in the presence of the HO-1 inhibitor, ZnPPIX, and the PI3K inhibitor, LY294002.


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PC12 cells, maintained in low-serum medium for 16 h, were pre-treated with

vehicle, 10 µM LY294002 or 100 µM ZnPPIX, as indicated in Fig. 10A and 10B,

and then incubated with 10 µM carnosol for 30 min or 6 h. Finally, cells were

challenged with 250 µM H2O2 for 5 min and immediately analyzed by flow

cytometry. As shown in Fig. 10A, the HO-1 inhibitor (ZnPPIX) did not significantly

alter the antioxidant effect of carnosol after 30 min incubation. By contrast,

ZnPPIX partially, but not completely, blocked the long-term antioxidant effect of

carnosol after 6 h incubation. Similarly, as shown in Fig. 10B, inhibition of PI3K

(10 µM LY294002) did not significantly alter the antioxidant effect of carnosol

after 30 min but partially reduced its antioxidant effect at 6 h incubation. These

results indicate that induction of HO-1 by carnosol through the PI3K pathway is

essential to elicit at least part of its long-term antioxidant effects (but see

Discussion).

DISCUSSION

In this study, we show the involvement of the PI3K/Akt survival pathway in

the up-regulation of HO-1, a prototypical phase II enzyme, in response to the

herb-derived diterpene, carnosol. We have found that carnosol up-regulates HO-

1 expression by targeting the ARE sequences found in the mouse ho1 gene

promoter. The regulation of AREs by carnosol was mediated, at least in part,

through an increase in Nrf2 protein levels in a PI3K- and Akt- dependent manner.

To our knowledge this is the first study reporting the activation and the relevance

of the PI3K/Akt1 pathway to the induction of phase II enzymes by food

phytophenols with therapeutic potential.


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Most studies on the regulation of phase II gene expression have focused

on the role of the MAPK pathways. Our results indicate that, although the ERK,

p38 and JNK pathways are activated by carnosol, they do not participate equally

in the induction HO-1 in PC12 cells. Thus, ERK and JNK were dispensable for

HO-1 up-regulation while inhibition of p38 significantly reduced the response to

carnosol. The role of MAPKs in ARE activation remains controversial. For

instance, in agreement with our data, dominant negative mutants of JNK did not

prevent cadmium-induction of the mouse ho1 promoter (43). However, in rat

hepatocytes, the glutathione depletor pherone and arsenite promoted a JNK-

dependent induction of ho1 gene expression (24, 49). Balogun et al (27)

described the effect of curcumin on induction of HO-1 expression by promoting

inactivation of the Nrf2-Keap1 complex in a p38-dependent manner and

subsequent binding of Nrf2 to the AREs. Accordingly, arsenate induced a p38-

dependent activation of the ho1 promoter in the LMH chicken hepatoma cell line

(50) but inhibition of p38 had no effect on cadmium, arsenate and hemin-

dependent HO-1 mRNA induction in HeLa cells (51). Our results are consistent

with the requirement of p38 for full activation of the ho1 promoter by carnosol

since a generic inhibitor of p38 significantly reduced Nrf2 protein levels (data not

shown) and HO-1 protein levels and promoter activity in response to carnosol.

Finally, the ERK pathway was dispensable of HO-1 induction in our system and

in rat hepatoma cells challenged with arsenite (24) but, it was necessary in the

arsenite-induced response in LMH cells (50). One possible interpretation for

these diverging observations may stem from the diverse assortment and intensity


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of the signaling pathways activated by different inducers in different cell types. In

this regard, it is interesting to note that carnosol failed to induce HO-1 at the

highest concentration tested (100 µM) despite the fact that this concentration

yielded the strongest activation of the MAPK pathways (data not shown),

therefore suggesting that specificity may rely not only on the nature of the

inducer and cell type but also on the relative potencies of these pathways.

We found that carnosol increases the levels of Nrf2 transcription factor in

the nucleus and its binding to the AREs. The mechanisms leading to nuclear

translocation of Nrf2 are poorly defined but certainly include the release from

Keap1 at the cytosol. However, since the half life of this transcription factor is

very short (39, 40, 41), these mechanisms must necessarily rely on the

stabilization of Nrf2 protein. Our results demonstrate that, indeed, carnosol

increases the levels of Nrf2 as does the proteasome inhibitor MG132, suggesting

that this phytochemical blocks Nrf2 degradation. The increase in Nrf2 protein

levels induced by carnosol was partially dependent of the activation of PI3K since

LY294002 concentrations that inhibited PI3K and the subsequent

phosphorylation and activation of Akt1 decreased the carnosol-induced

accumulation Nrf2 protein. However, inhibition of PI3K did not fully prevent the

carnosol-induced increase in Nrf2 and HO-1 protein levels, suggesting that other

elements, probably p38 and other PI3K-independent pathways, are also involved

in carnosol signaling to Nrf2 and HO-1. Interestingly, active PI3K was sufficient to

activate the ho1 promoter through AREs and constitutive expression of active

PI3K or Akt yielded cells with increased HO-1 levels.


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In search for the downstream kinases that might be responsible for the

increase Nrf2 protein levels we analyzed the effect of PKCζ. A dominant negative

version of this kinase only produced a slight decrease in the carnosol-induced

activation of the ho1 promoter, suggesting a very minor role of this atypical PKC

on HO-1 transcriptional regulation. However, a recent study by Numazawa et al

(52) has indicated that PKCι, another member of the atypical PKCs,

phosphorylates Nrf2 at Ser 40, leading to its release from Keap1 and nuclear

translocation. Whether the dispensability of PKCζ is due to a lack of involvement

in the regulation of Nrf2 or to redundancy with other atypical PKCs, such as PKCι,

remains to be determined.

On the other hand, a dominant negative mutant of Akt1 significantly

reversed carnosol-induced activation of the ho-1 gene promoter. Contrary to

PKCι, direct phosphorylation of Nrf2 or Keap1 by Akt seems unlikely because

these proteins do not have a consensus phosphorylation sequence for Akt. Since

Nrf2 is regulated primarily at the level of stability we propose that Akt might be

acting on the ubiquitin ligases involved in its tagging for degradation. In fact, at

least two ubiquitin ligases, Mdm2 (53) and BRCA1/BARD1 (54) have been

reported to be substrates of Akt.

Interestingly, the activation of Akt by carnosol was a transient event that

subsided by 2 h. However, Nrf2 protein levels increased beyond 3 h. Therefore,

transient activation of Akt must result in persistent activation/inhibition of effector

substrates that regulate Nrf2, resulting in increased protein levels. Thus,

phosphorylation of the Nrf2-specific ubiquitin ligase might result in its persistent


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inhibition leading to sustained increase in Nrf2 protein levels. Alternatively, the

transient activation of Akt might activate transcription of stable proteins that

contribute to increase Nrf2 protein levels.

Plant-derived phenols exhibit strong antioxidant properties due to Michael

reaction acceptor function. Indeed, we have observed the intrinsic antioxidant

nature of carnosol since 30 min pre-treatment with this drug completely abolished

the oxidant effect of as much as 500 µM H2O2, as determined by the blockage in

the conversion of H2DCFDA to its oxidized, green fluorescent form, DCF. More

importantly for this study, inhibition of the PI3K pathway, leading to activation of

Akt and up-regulation of HO-1 expression, impaired the long-term, but not the

short-term, antioxidant function of carnosol, therefore evidencing the relevance of

this pathway for carnosol to confer persistent antioxidant protection. Interestingly,

a fraction of about half of the antioxidant effect of carnosol was insensitive to

inhibition of PI3K and HO-1 at 6 h. This observation is consistent with the

possibility that at this time there is still enough non-metabolized carnosol to

maintain its intrinsic antioxidant effect. In fact, the PI3K and HO-1 inhibitors

dropped H2DCFDA oxidation to similar levels as those observed at 30 min.

However, we favor the non-exclusive hypothesis that carnosol also up-regulates

other intracellular antioxidant systems that do not belong to the PI3K/HO-1

module and that also contribute to the long-term antioxidant properties of this

compound. In fact, preliminary microarray data from mouse neuroblastoma cells

indicates that carnosol also up-regulates the expression of mitochondrial, Mn-

dependent superoxide dismutase and glutathione peroxidase (data not shown).


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A corollary of this study is that pharmacological activation of the PI3K/Akt

pathway by this food-related compound, leading to increase of Nrf2 and HO-1,

efficiently protects cells from oxidative stress and should be evaluated as a new

therapeutic approach in degenerative processes, such as Alzheimer’s and

Parkinson diseases, that correlate with oxidative damage.

ACKNOWLEDGEMENTS

We thank Ms Beatriz Palacios for her excellent technical assistance. This

work was supported by grants SAF2001-0545 from Ministerio de Ciencia y

Tecnología (AC), 08.5/0048/2001 from Comunidad Autónoma de Madrid (AC),

FEDER 97/0602 (CMRG) and PI-2002/168 from Comunidad Autónoma de

Canarias (CMRG).

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LEGEND TO FIGURES

Figure 1.- Carnosol induces HO-1 expression. A. Effect of serum and

carnosol on the protein levels of HO-1 and HO-2 isoenzymes. PC12 cells were

maintained for 16 h in DMEM supplemented with 1% fetal calf serum and 1%

horse serum (low serum) or in 7.5% fetal calf serum and 7.5% horse serum (high

serum) and then stimulated with vehicle (DMSO) or 10 µM carnosol for 6 h.

Upper panel, immunoblot with anti-HO-1 antibodies; middle panel, immunoblot

with anti-HO-2 antibodies; lower panel, densitometric quantification of relative

HO-1 protein levels. B. Real-time PCR determination of HO-1 mRNA levels in the

presence of carnosol. PC12 cells, maintained for 16 h in low serum conditions,

were incubated with the indicated concentrations of carnosol and analyzed as

indicated in Experimental Procedures. C. Semi-quantitative reverse-transcriptase

PCR showing a dose-dependent induction of HO-1 mRNA by carnosol. Upper

panel, HO-1 mRNA; middle panel, β-actin mRNA used for normalization; lower

panel, densitometric quantification of relative HO-1 mRNA levels. D. Dose-

dependent induction of HO-1 protein by carnosol. Upper panel, immunoblot with

anti-HO-1 antibodies; middle panel, immunoblot with anti-HO-2 antibodies; lower

panel, densitometric quantification of relative HO-1 protein levels. For C and D,

PC12 cells, maintained in low serum of 16 h, were treated with the indicated

carnosol concentrations for 6 h, or with 50 µM hemin, a well-established induced

of HO-1, and then analyzed for HO-1 mRNA and protein levels. E. Time-course

of HO-1 mRNA expression by carnosol. Upper panel, HO-1 mRNA; middle panel,

β-actin mRNA used for normalization; lower panel, densitometric quantification of


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relative HO-1 mRNA levels. F. Time-course of HO-1 protein expression by

carnosol. Upper panel, immunoblot with anti-HO-1 antibodies; middle panel,

immunoblot with anti-HO-2 antibodies; lower panel: densitometric quantification

of relative HO-1 protein levels. For E and F, PC12 cells were maintained in low

serum for 16 h, and then submitted to 10 µM carnosol for the indicated times or

with 50 µM hemin for 6 h.

Figure 2.- Carnosol targets the mouse ho1 gene promoter at the two

AREs. PC12 cells were transfected with expression vectors for luciferase under

the control the 15 kb 5’ promoter region of mouse ho1 (WT) or the same

promoter with deletions of either E1 (∆E1) or E2 (∆E2) or both regions (∆E1+∆E2)

comprising the AREs. In addition, cells were co-transfected with pcDNA3-β-gal

for normalization. 16 h after transfection, cells were maintained in low serum for

16 h and then stimulated with 10 µM carnosol for additional 16 h. Then cells were

lysed and analyzed for luciferase and β-galactosidase activity.

Figure 3.- Carnosol increases the levels of Nrf2 transcription factor. Dose-

effect (A) and time-course (B) of carnosol-induced increase of Nrf2 protein levels.

Upper panels: blots with anti-Nrf2 antibodies; middle panels: blots with anti-

protein disulfide isomerase (PDI) used for normalization; lower panels:

densitometric quantification of relative Nrf2 levels. In A, Low serum growing

PC12 cells were treated for 3 h with the indicated carnosol concentrations, or

with MG132 (20 µM) or both MG132 (20 µM) and carnosol (10 µM) as indicated.

In B PC12 cells, maintained in low serum for 16 h, were treated with 10 µM


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carnosol for the indicated times or with MG132 or with MG132 plus carnosol for 3

h.

Figure 4.- Carnosol stimulates activation of ho1 promoter AREs by Nrf2. A.

Increase of Nrf2 protein levels in nuclear extracts of cells treated with 10 µM

carnosol or 20 µM MG132 for 3h. B. EMSA reactions using a double strand

oligonucleotide corresponding to the mouse ho1 ARE and nuclear extracts from

cells treated with 10 µM carnosol or 20 µM MG132. The last two lanes

correspond to cell extracts from carnosol-treated cells pre-incubated without (-)

or with (+) anti-Nrf2 antibody. The arrow points the position of the complex that is

not formed in the presence of the anti-Nrf2 antibody. C. Requirement of Nrf2 and

MafG for activation of the mouse ho1 promoter by carnosol. PC12 cells were co-

transfected with pHO1-15-Luc, pcDNA-β-gal, and expression vectors for

dominant negative versions of Nrf2 or MafG, as indicated. Following 16 h after

transfection cells were kept in low serum for 16 h and then stimulated with 10 µM

carnosol. After 6 h cells were lysed and analyzed for luciferase and β-

galactosidase activity.

Figure 5.- Carnosol activates the MAPK and PI3K pathways. PC12 cells in

low serum were stimulated with 10 µM carnosol for the indicated times and then

immunoblotted with activation-specific antibodies that recognize the

phosphorylated kinases (P-Akt1, P-ERK2, P-p38 and P-JNK). Parallel

immunoblots were analyzed for total kinase levels with the indicated antibodies

(Akt1, ERK, p38, and JNK). Lower graphics correspond to a densitometric


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quantification of P-Akt, P-ERK2, P-JNK and P-p38 levels relative to total kinase

levels.

Figure 6.- Effect of MAPK and PI3K inhibitors on induction of HO-1 by

carnosol. A. Upper panel: immunoblot with anti-HO-1 antibodies. Lower panel:

immunoblot with anti-PDI antibodies used for normalization. PC12 cells were

maintained in low serum for 16 h and then were pre-incubated with 40 µM

LY294002 (LY), 50 µM PD98059 (PD), 5 µM SB203580 (SB), and 10 µM

SP600125 (SP) for 15 min. Then, cells were submitted to 10 µM carnosol for 6 h.

B. Carnosol induced luciferase activity in the mouse ho1 promoter in the

presence of the MAPK and PI3K inhibitors. PC12 cells were co-transfected with

pHO1-15-Luc and pcDNA3-β-gal vectors. After 16 h cells were transferred to low

serum in the presence of the inhibitors and then stimulated with 10 µM carnosol

for 16 h. Then cells were lysed and analyzed for luciferase and β-galactosidase

activity.

Figure 7.- The PI3K/Akt axis is involved in the carnosol-induced increase

in Nrf2 protein levels and on ho1 promoter activation. A. Inhibition of PI3K

prevents the increase of HO-1 and Nrf2 protein levels in response to carnosol.

PC12 cells, maintained in low serum for 16 h, were pre-incubated with the

indicated LY294002 concentrations for 15 min and then with 10 µM carnosol for

either 30 min (immunoblot with P-Akt) or 6 h (immunoblots with HO-1, Nrf2 and

PDI). B. PI3K induces HO-1 expression through regulation of AREs. PC12 cells

were transfected with pHO1-15-Luc, pHO-∆E1-Luc, pHO1-∆E2-Luc and pHO1-

(∆E1+∆E2) together with either empty vector or expression vector for active PI3K


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Martin et al, 2003

34

(PI3K-CAAX). In addition, cells were co-transfected with pcDNA3-β-gal for

normalization. Following 16h from transfection, cells were transferred to low

serum medium for 16 h and analyzed for luciferase and β-galactosidase activity.

C. Induction of HO-1 expression by PI3K is attenuated by interfering with Nrf2

function. PC12 cells were co-transfected with pHO1-15-Luc, empty vector

(pcDNA3), PI3K-CAAX, pcDNA-β-gal, and expression vectors for dominant

negative versions of Nrf2 or MafG, as indicated. Following 16 h after transfection

cells were kept in low serum for 16 h and then stimulated with 10 µM carnosol.

After 6 h cells were lysed and analyzed for luciferase and β-galactosidase activity.

Figure 8.- Carnosol requires Akt1 but not PKCζ to activate the ho1

promoter. Cells were transfected with pHO1-15-Luc and the indicated vectors for

expression of dominant negative Nrf2 mutant or the indicated amounts in A of

dominant negative PKCζ (PKCζ (DN)) or in B of dominant negative Akt1

(Akt1(DN)). Cells were also transfected with pcDNA3-β-gal for normalization.

Following 16 h from transfection, cells were maintained in low serum medium for

16 h and analyzed for luciferase and β-galactosidase activity. C. Detection of HA-

tagged dominant negative versions of PKCζ and Akt1 by immunoblot with anti-

HA antibodies. Cells were treated in parallel with the luciferase experiments

indicated in A and B, were lysed and immunoprecipitated with anti-HA antibodies.

The immune-complexes were resolved in PAGE and immunoblotted with anti-HA

antibodies. The lower band (IgG H) corresponds to the immunoglobulin heavy

chain of the anti-HA antibody used during immunoprecipitation. D. Immunoblots

showing HO-1 protein levels in cells stably transfected with empty vector


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Martin et al, 2003

35

(pcDNA3), membrane-anchored active PI3K (PI3K-CAAX), and membrane-

anchored active myristylated Akt1 (myr-Akt1). Upper panel immunoblot with anti-

HO-1 antibodies. Lower panel, immunoblot with anti PDI antibodies.

Figure 9. - Carnosol prevents oxidative stress. PC12 cells were

maintained in low serum for 16 h, and then loaded for 1 h with 8 µM H2DCFDA

and simultaneously treated with vehicle (controls) or 10 µM carnosol for 30 min.

Finally cells were exposed to 250 µM H2O2 for 5 min and immediately analyzed

by flow cytometry. A. A representative sample of 10.000 cells is shown for

vehicle-treated cells (control), or cells treated with H2O2 alone or in combination

with carnosol. B. Dose-dependent, antioxidant effect of carnosol. PC12 cells

were loaded with H2DCFDA and treated with DMSO vehicle (control) or the

indicated carnosol concentrations for 30 min. During the last 5 min cells were

exposed to 250 µM H2O2, as indicated, and rapidly used for flow cytometry

analysis. C. Carnosol-mediated antioxidant protection against micromolar

concentrations of H2O2. PC12 cells were loaded with H2DCFDA as indicated

above, incubated for 30 min with vehicle or 10 µM carnosol and treated for the

last 5 min with increasing concentrations of H2O2, as shown.

Figure 10.- HO-1 and PI3K inhibitors attenuate long- but not short-term

antioxidant protection of carnosol. A. Effect of the HO-1 inhibitor, ZnPPIX, on the

antioxidant activity of carnosol. B. Effect of the PI3K inhibitor LY294002 on the

antioxidant activity of carnosol. PC12 cells were maintained in low serum for 16 h

and then loaded for 1 h with H2DCFDA. Then, cells were pre-pretreated with

either 100 µM ZnPPIX (A) or 10 µM LY294002 (B) for 15 min and submitted to


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Martin et al, 2003

36

carnosol for 30 min and 6 h as indicated. During the last 5 min of carnosol

incubation, cells were challenged with 250 µM H2O2 and then analyzed by flow

cytometry.


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DC

carnosol ( M)µ

0

2

12

4

6

hemin100 10030µM: 50

carnosol

HO-1

HO-2

3

0 10020 40 60 80

carnosol ( M)µ

0

heminµM: 50

carnosol

HO-1

β-actin

12345

100 100303

0 10020 40 60 80

Figure 1

FE

time (h):

10 M carnosolµ

HO-1

β-actin

10 420.5 6

time (h)

HO-1

HO-2

0

15

36

129

time (h):

10 M carnosolµ

10 420.5 6

time (h)

250

200

150

100

2

00 3 10 30 100

carnosol Mµ

B

HO-1

HO-2

high serumlow serum

vehicle:carnosol:

--

+-

-+

--

+-

-+

A


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18

16

14

12

10

8

6

4

0

untreated

p

∆E1 ∆E2 ∆(E1+E2)WT pGL3bv

HO1-15-Luc

carnosol

Figure 2


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carnosol ( M)µ

0

Nrf2

PDI

12345

0 10 20 30

1010 3 30µM:

carnosol

6

Nrf2

PDI

10.50 2hours:

carnosol

3

Time (h)

012345

0 1 2 3

6

A B

Figure 3


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- +

α-Nrf2

B

Nrf2

Sp1

A

0

4

2

6

8

10

12

14

16

carnosoluntreated

Nrf2(DN):

MafG(DN):

pHO1-15-Luc:

- -

- -

+ +

+

- -

+ +

+

- -

+ +

+

+ +

+

C

Figure 4

+

+ +

+


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ERK2Akt

P-Akt1 P-ERK2

P-p38

p38

P-JNK

P-JNK

60100 120min: 30 60100 120min: 30

60100 120min: 30 60100 120min: 30

Time (min)

0123456

30 60 1200 30 60 1200 30 60 1200 30 60 1200

P-Akt P-ERK2

JNK

P-p38

Figure 5


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HO-1

PDI

Carnosol: - - -+ + + - -+ +

LY PD SB SP

A untreatedcarnosol

18

16

14

12

10

8

6

4

0LY PD SB SP-

B

Figure 6


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A

vectorPI3K-CAAX

p

∆E1 ∆E2 ∆(E1+E2)WT pGL3bv

HO1-15-Luc

25

20

15

10

5

2.5

0

B

Figure 7

HO1

Nrf2

P-Akt1

PDI

- + + + + -+Carnosol:LY ( M):µ - - 103 30 100 100

3

2

1

0

C

pcDNA3

vectorPI3K-CAAX

Nrf2(DN) MafG(DN)Nrf2 (DN)

+MafG(DN)


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12

10

8

6

4

0

untreatedcarnosol

12

10

8

6

4

0

untreatedcarnosol

A B

Akt1(DN)

PKC (DN)ζ

IgG H

PKC (DN)ζ Akt1(DN)

C

Figure 8

PDI

HO-1

D


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100

010 10 10 10

0 1 2 3

H DCFDA fluorescence2

controlH OH O + carnosol

2 2

2 2

10

5

1 3 10 30

Carnosol ( M)µ

0

H O + carnosol2 2

50

H O ( M)µ2 2

10

5

0125 250 500

H O + carnosol2 2H O2 2

A

B

C

Figure 9


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-+- -

- -

- -

+-

-

-

+

+-

-

+

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+

+-

-

-

+-

+

-

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+

+

0

5

10

15

carnosol 30 min:

carnosol 6 h:

ZnPPIX:

H O :22

+-

-

-

-

-

-

-

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-

-

+

+-

-

+

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+

+-

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-

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+

-

+-

+

+

0

5

10

15

carnosol 30 min:

carnosol 6 h:

LY294002:

H O :22

A

B

Figure 10


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Carlos M. Ruiz de Galarreta and Antonio CuadradoDaniel Martin, Ana I. Rojo, Marta Salinas, Raquel Diaz, German Gallardo, Jawed Alam,

antioxidant phytochemical, carnosolkinase/Akt pathway and the Nrf2 transcription factor in response to the

Regulation of heme oxygenase-1 expression through the phosphatidylinositol 3

published online December 19, 2003J. Biol. Chem.

10.1074/jbc.M309660200Access the most updated version of this article at doi:

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Regulation of heme oxygenase-1 expression through the phosphatidylinositol 3 kinase ... · 2003-12-19 · Regulation of heme oxygenase-1 expression through the phosphatidylinositol