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EFFECTS OF CADMIUM ON ACTIN
GLUTATHIONYLATION AND FOCAL ADHESIONS
by
Grace Mei Yee Choong
A thesis submitted in conformity with the requirements
for the degree of Master of Science
Graduate Department of Laboratory Medicine and Pathobiology
University of Toronto
© Copyright by Grace Mei Yee Choong 2013
ii
EFFECTS OF CADMIUM ON ACTIN
GLUTATHIONYLATION AND FOCAL ADHESIONS
Grace Mei Yee Choong
Master of Science
Department of Laboratory Medicine and Pathobiology
University of Toronto
2013
ABSTRACT
The toxic metal ion cadmium (Cd2+) is pro-oxidant and specifically disrupts the actin
cytoskeleton in renal mesangial cells. This study investigated the role of Cd2+-mediated
redox modulation of actin through protein S-glutathionylation and the effects of cytoskeletal
changes on focal adhesions (FAs) through a Ca2+/calmodulin dependent-protein kinase II
(CaMK-II) pathway. Only at low concentrations of Cd2+ (0.5-2 μM) was there an increase in
actin glutathionylation, which was a reactive oxygen species-independent, total glutathione-
dependent effect. Immunofluorescence of the cytoskeleton suggests that increases in
glutathionylation levels occurring under low [Cd2+] are protective in vivo. Higher
concentrations ( 10 μM) of Cd2+ resulted in loss of vinculin and focal adhesion kinase
(FAK) from FAs, concomitant with cytoskeletal disruption. Inhibition of CaMK-II preserved
cytoskeletal integrity and focal contacts, while decreasing the migration of FAK-
phosphoTyr925 to a membrane-associated compartment. This study adds further insight into
the Cd2+-mediated effects on the cytoskeleton and FAs.
iii
ACKNOWLEDGEMENTS
First, and foremost, I would like to thank Dr. Douglas M. Templeton for being a
wonderful and patient mentor these past three years. Our scientific and non-scientific
converstions have challenged me and made me think. Most importantly, you have given me
the opportunities to be the best scientist I could be—for that I am eternally grateful.
To Dr. Ying Liu, you have guided and pushed me to take on new challenges inside and
outside the lab. My project started off as your idea, but you gave me the necessary tools to
make it my own. I am very fortunate that I have had someone like you in my life. Thank you.
I am also extremely grateful to Kathy Xiao, who spent a year helping me with the ROS
measurements, and Dr. Zvezdana Popovich for her feedback and help troubleshooting the
glutathione measurements. You both made my time here lively and fun.
I also appreciate the feedback and advice I received from my advisory committee
members, Dr. David Irwin and Dr. Michal Opas. I am grateful to the Opas lab throughout
these years for their moral support, friendship and laughter.
Finally, I am fortunate to have such wonderful and caring parents, who have supported
my decisions, and helped me whenever possible. To my brother, you have been a source of
humour and understanding and helped me put my thoughts into perspective. You have all
been my pillar of support, love and understanding throughout these years. I will never forget
the sacrifices each of you made to allow me to succeed. I owe this degree to all of you.
iv
TABLE OF CONTENTS
ABSTRACT...................................................................................................................ii
ACKNOWLEDGEMENTS ........................................................................................ iii
TABLE OF CONTENTS .............................................................................................iv
ABBREVIATIONS ......................................................................................................ix
LIST OF TABLES .....................................................................................................xiii
LIST OF FIGURES....................................................................................................xiii
1. INTRODUCTION .....................................................................................................1
1.1 Physicochemial properties of cadmium ..............................................................1
1.2 Health effects of cadmium ...................................................................................2
1.2.1 Cadmium exposure .......................................................................................2
1.2.2 Cadmium disposition ....................................................................................3
1.2.3 Cadmium biomarkers ...................................................................................3
1.3 Cadmium toxicity ................................................................................................4
1.3.1 Acute Toxicity ...............................................................................................4
1.3.2 Chronic toxicity.............................................................................................5
1.3.3 Cadmium nephrotoxicity ..............................................................................6
1.3.3.1 Normal renal physiology ........................................................................6
1.3.3.2 Effects of cadmium on the kidney........................................................10
1.4 Molecular mechanisms of cadmium toxicity ....................................................12
1.4.1 Cadmium and signal transduction .............................................................12
1.4.1.1 Calcium/calmodulin-dependent protein kinase (CaMK-II) ...............12
1.4.1.2 Activation of CaMK-II by Cd ..............................................................13
v
1.4.2 Focal contacts ..............................................................................................16
1.4.2.1 The link between the matrix and cytoskeleton....................................16
1.4.2.2 Signal transduction of focal adhesion kinase (FAK)...........................17
1.4.3 Cadmium and the actin cytoskeleton .........................................................20
1.4.3.1 The actin cytoskeleton..........................................................................20
1.4.3.2 Cadmium effects on the actin cytoskeleton .........................................21
1.4.4 Cadmium and reactive oxygen species (ROS)............................................22
1.4.4.1 Sources of ROS.....................................................................................22
1.4.4.2 Antioxidant defense mechanisms.........................................................25
1.4.4.3 Cadmium induction of ROS.................................................................27
1.5 Protein S-glutathionylation ...............................................................................29
1.5.1 Mechanisms of glutathionylation................................................................29
1.5.2 Glutathionylation of proteins......................................................................33
1.6 Hypotheses and objectives.................................................................................33
2. MATERIALS AND METHODS.............................................................................35
2.1 Materials ............................................................................................................35
2.2 Primary Culture of Rat Mesangial Cells ..........................................................36
2.3 Cell treatments...................................................................................................37
2.4 Cellular fractionation ........................................................................................ 37
2.4.1 1% NP-40 Tris whole-cell lysate for detection of glutathionylated
proteins.................................................................................................................39
2.4.2 0.5% NP-40 HEPES whole-cell lysate for detection of FAK ................... 39
2.4.3 Cytoskeletal-cytosolic fractionation ...........................................................41
vi
2.4.4 Membrane-cytosolic fractionation..............................................................41
2.4.5 Nuclear-cytosolic fractionation...................................................................42
2.5 Viability assay ....................................................................................................42
2.6 Western Blotting................................................................................................44
2.7 Immunoprecipitation & Mass Spectrometry....................................................45
2.8 Determination of redox state of -actin with AMS .......................................... 45
2.9 Immunofluorescence..........................................................................................46
2.10 Intracellular reactive oxygen species (ROS) ...................................................46
2.11 Intracellular glutathione using DTNB recycling assay................................47
2.12 Glutaredoxin activity.......................................................................................47
2.13 Effect of actin glutathionylation on polymerization in vitro...........................48
2.14 Reverse-transcriptase polymerase chain reaction (RT-PCR) ........................48
2.15 Statistical analysis............................................................................................49
3. RESULTS ................................................................................................................51
3.1 Cadmium induces actin glutathionylation ........................................................51
3.1.1 Cadmium induces glutathionylation of a 42 kDa protein..........................51
3.1.2 Immunoprecipitation identifies the 42 kDa glutathionylated protein as
actin ......................................................................................................................56
3.2 Factors contributing to actin glutathionylation................................................56
3.2.1 Glutathionylation of actin is not directly dependent on ROS levels..........56
3.2.2 Actin glutathionylation correlates with changes in total glutathione levels
but not the GSH/GSSG ratio...............................................................................59
3.2.3 Inhibition of glutathione synthesis decreases actin glutathionylation.......59
vii
3.2.4 Glutathione levels increase due to increases in antioxidant gene expression
..............................................................................................................................68
3.2.5 Actin glutathionylation correlates with increased activity of glutaredoxin
..............................................................................................................................68
3.3 Functional consequences of actin glutathionylation.........................................73
3.3.1 Glutathionylation inhibits the rate of G-actin polymerization in vitro .....73
3.3.2 Depletion of glutathione changes the redox status of the actin cytoskeleton
..............................................................................................................................73
3.3.3 Glutathionylation of actin precedes Cd-mediated cytoskeletal disruption
..............................................................................................................................76
3.4 Glutathionylated proteins are located in a perinuclear region ........................76
3.5 Cadmium disrupts focal contacts......................................................................79
3.5.1 Cadmium disrupts actin-vinculin contacts.................................................79
3.5.2 CaMK-II is involved in Cd-dependent disruption of focal adhesions .......79
3.6 Cadmium alters FAK localization and activation ............................................83
3.6.1 Focal adhesion kinase (FAK) localization is affected by Cd......................83
3.6.2 Translocation of FAK to the cytoskeletal fraction is stimulated by Cd ....87
3.6.3 Cadmium stimulates site-specific phosphorylation of FAK ......................87
4. DISCUSSION ..........................................................................................................96
4.1 Cadmium and actin glutathionylation ..............................................................96
4.1.1 Background .................................................................................................96
4.1.2 Role of ROS .................................................................................................98
4.1.3 Role of nitrosative stress .............................................................................99
viii
4.1.4 Role of glutathione ......................................................................................99
4.1.5 Cadmium and antioxidant gene expression .............................................101
4.1.6 Cadmium and glutaredoxin......................................................................102
4.1.7 Role of signal transduction ....................................................................... 103 4.2 Effects of Cd on the actin cytoskeleton ........................................................... 103
4.2.1 Effect of actin glutathionylation on actin polymerization ....................... 104
4.2.2 Effect of actin glutathionylation in vivo.................................................... 105
4.3 Localization of glutathionylated proteins .......................................................106
4.4 Cadmium and focal adhesions.........................................................................108
4.4.1 Cadmium effects on focal adhesion localization ......................................108
4.4.2 Cadmium and CaMK-II alter FAK localization......................................109
4.4.3 Cadmium and CaMK-II increase FAK phosphorylation........................110
4.4.4 Differences between cadmium and other toxic metals.............................111
5. SUMMARY & SIGNIFICANCE..........................................................................113
6. FUTURE DIRECTIONS.......................................................................................115
7. REFERENCES ......................................................................................................117
ix
ABBREVIATIONS
ADP Adenosine diphosphate
AIF Apoptosis inducing factor
ARE Antioxidant response element
AMS 4-acetamido-4'maleimidylstilbene 2,2'-disulphonic acid
Arp2/3 Actin-regulated protein 2/3
ATP Adenosine triphosphate
BSA Bovine serum albumin
BSO Buthionine sulfoximine
CaM Calmodulin
CaMK-II Ca2+ /calmodulin-dependent protein kinase II
Cu/Zn SOD Copper/Zinc superoxide dismutase
DAPI 4',6-diamidino-2-phenylindole
DCF 2',7'-Dichlorofluorescein
DMSO Dimethyl sulfoxide
DNase I Deoxyribonuclease I
DTNB 5,5'-dithio-bis-(2-nitrobenzoic acid)
DTT Dithiothreitol
EDTA Ethylenediaminetetraacetic acid
EGFR Epidermal growth factor receptor
EGTA Ethylene glycol tetraacetic acid
ER Endoplasmic reticulum
x
F-actin Filamentous actin
FA Focal adhesion
FAK Focal adhesion kinase
FAT Focal adhesion targeting domain
FERM Band 4.1, ezrin, radixin, moesin homology domain
FBS Fetal bovine serum
G-actin Globular actin
GAPDH Glyceraldehyde-3-phosphate dehydrogenase
GBM Glomerular basement membrane
GCLC Glutathione cysteine ligase catalytic subunit gene
GFR Glomerular filtration rate
Glu-C Staphylococcus aureus serine protease V8 (endopeptidase)
GRase Glutathione reductase
GRx Glutaredoxin
GSH Reduced glutathione
GSH/GSSG Reduced/oxidized glutathione ratio
GSNO Nitrosylated glutathione
GSSG Oxidized glutathione
H2DCF-DA 2',7'-dichlorodihydrofluorescein diacetate
HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
HMOX-1 Heme oxygenase-1 gene
HO-1 Heme oxygenase-1 protein
HRP Horse radish peroxidase
xi
KD Kinase domain of focal adhesion kinase
KN-93 Specific inhibitor of Ca2+ /calmodulin-dependent protein kinase II
LC-MS Liquid-chromatography mass spectrometry
MAPK Mitogen-activated protein kinase
MLCK Myosin-light chain kinase
MnSOD Manganese superoxide dismutase
MT Metallothionein
MTT Thiazolyl blue tetrazolium bromide
NAG N-acetyl-B-glucosaminidase
NADPH Nicotinamide adenine dinucleotide 2'-phosphate reduced
NF B Nuclear factor kappa B
NP-40 Nonidet P-40
Nrf2 Nuclear factor E2-related factor 2
PBS Phosphate-buffered saline
PCT Proximal convoluted tubule
PKC Protein kinase C
PLC- Phospholipase C-
PMSF Phenylmethylsulfonyl fluoride
PRR Proline rich region
PSSG S-glutathionylated protein
PTP1B Protein tyrosine phosphatase 1B
RMC Rat mesangial cells
ROS Reactive oxygen species
xii
RT-PCR Reverse transcriptase polymerase chain reaction
SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis
SERCA Sarco/endoplasmic reticulum calcium ATPase
SF Serum-free
TBS-T Tris-buffered saline containing 0.1% Tween-20
TfR Transferrin receptor
WASP Wiskott-Aldrich syndrome protein
xiii
LIST OF TABLES
Table 1: Effect of cadmium on total glutathione (GSH+GSSG) levels.................... 62
Table 2: Effect of cadmium on oxidized glutathione (GSSG) level ......................... 64
Table 3: Effect of cadmium on GSH/GSSG ratios ................................................... 66
Table 4: Effect of cadmium on glutaredoxin activity............................................... 74
LIST OF FIGURES
Figure 1: Anatomy of the renal nephron.................................................................... 7
Figure 2: CaMK-II signaling pathways and role in cadmium toxicity.................... 14
Figure 3: Focal adhesion kinase structure and signaling cascade ........................... 18
Figure 4: Physiological sources of ROS .................................................................... 23
Figure 5: Glutathione redox cycle............................................................................. 26
Figure 6: Mechanisms of ROS production by Cd .................................................... 28
Figure 7: Mechanisms of protein S-glutathionylation.............................................. 31
Figure 8: Primary cultures of rat mesangial cells display characteristics of smooth
muscle cells ................................................................................................................ 38
Figure 9: Flow chart of subcellular fractionation protocols .................................... 40
Figure 10: Confirmation of the purity of fractions .................................................. 43
Figure 11: Time- and concentration-dependent glutathionylation of a putative actin
protein........................................................................................................................ 52
Figure 12: Viability of rat mesangial cells decreases with increasing concentrations of
cadmium .................................................................................................................... 54
xiv
Figure 13: Diamide increases glutathionylation of the putative actin protein ........ 55
Figure 14: -actin immunoprecipitation identifies actin as the major glutathionylated
protein........................................................................................................................ 57
Figure 15: Effects of Cd2+
on ROS and glutathione levels in mesangial cells.......... 60
Figure 16: Effect of glutathione synthesis on actin glutathionylation ..................... 69
Figure 17: Cadmium promotes the translocation of Nrf2 to the nuclear fraction.. 71
Figure 18: Cadmium increases the expression of antioxidant genes ....................... 72
Figure 19: Effect of glutathionylation of G-actin on actin polymerization in vitro. 75
Figure 20: Depletion of glutathione increases the number of reduced cysteines in -
actin............................................................................................................................ 77
Figure 21: Effects of modulation of glutathione synthesis on F-actin integrity in Cd-
treated cells ................................................................................................................ 78
Figure 22: Glutathionylated proteins are localized in a perinuclear region ........... 80
Figure 23: Cadmium-dependent loss of vinculin from focal contacts ..................... 81
Figure 24: Localization of focal adhesion kinase (FAK) .......................................... 84
Figure 25: Changes in actin dynamics affect FAK localization............................... 86
Figure 26: Cytoskeletal localization of FAK upon cadmium treatment.................. 88
Figure 27: Cadmium-dependent Tyr phosphorylation of FAK............................... 90
Figure 28: Effect of Cd-treatment on localization of FAK-phosphoTyr925 ........... 93
Figure 29: Cell membrane localization of FAK-phosphoTyr925 in Cd-treated cells .
.................................................................................................................................... 95
1
1. INTRODUCTION
1.1 Physicochemial properties of cadmium
Cadmium (Cd) is a highly toxic environmental metal, classified as a known mutagen
and Class I carcinogen (1), causing adverse effects on multiple organ systems (2, 3).
Cadmium is found at low concentrations in the lithosphere, but over the last two hundred
years, Cd has become increasingly concentrated in the biosphere due to mining, smelting,
industrial and agricultural activities (2–5). A particularly concerning feature of Cd is that it
does not biodegrade and has a long biological half-life (10-30 years) in humans (3, 6).
Cadmium belongs to the group 12 elements of the periodic table, which include Zn2+
and Hg2+, and has a complete 4d electron shell, resulting in a very stable divalent cation form
(Cd2+). Its similarity to other physiologically relevant ions, including Ca2+ and Zn2+, allows it
to disrupt multiple pathways, contributing to its toxic effects. Cadmium ion and Ca2+ have
very similar physicochemical properties, having similar charge/radius ratios (Ca2+ = 2.02 C/
Å, Cd2+ = 2.06 C/ Å) and ionic radii (Ca2+ = 0.97 Å, Cd2+ = 0.99 Å) (7). These properties
allow Cd2+ to displace Ca2+ in calcium-binding proteins such as calmodulin (CaM) (8), and
sarcolemmal membranes (9) in vitro, with the potential to affect other Ca2+-dependent
proteins.
Cadmium ion is a “soft ion”. These ions readily bind to inorganic or organic
compounds containing sulfur (sulfhydryl, disulfide, thioether) or nitrogen (amino, imidazole,
histidine, nucleotide base). Therefore, Cd binds with high affinity to thiols, effectively
poisoning them (7, 10). Similar to the effects of Cd2+ on Ca2+, Cd2+ can displace Zn2+ from
2
proteins, including Zn-finger transcription factors, potentially altering gene expression (11,
12).
1.2 Health effects of cadmium
1.2.1 Cadmium exposure
Cadmium toxicity in humans was first described in the nineteenth century in zinc
smelter workers, as zinc ores contain high amounts of Cd (3, 13). Studies on occupational
exposure to Cd have shown that inhalation or ingestion of Cd are linked to the development
of emphysema and renal toxicity (3, 14–16). Cadmium is still commonly used in
electroplating, manufacturing of Li-Cd or Ni-Cd batteries, and in the production of pigments
where occupational exposure remains important (13, 14).
The effects of chronic environmental exposure on the general population are becoming
increasingly important. Cadmium is present within the natural environment and exposure
occurs through consumption of contaminated food or water or inhalation of Cd from
cigarettes (2–4). Cigarettes contain about 1-2 μg of Cd and the average daily consumption of
Cd through food products is 8-25 μg (2). Foods that contain higher levels of Cd include
vegetables and cereals, seafood, and offal products, such as liver. Approximately 10% of Cd
is absorbed through enterocytes, whereas around 50% of Cd inhaled through cigarette smoke
is absorbed into the circulation, explaining the higher Cd load found in smokers (2).
Currently, the recycling of electronic waste (e-waste) allows for new means of
exposure to Cd. E-waste consists of electronic products, such as computers, cell phones,
3
printers, and television sets, all of which contain varying levels of Cd. Although e-waste is
usually treated at its origin, some of it is exported to developing countries, in particular Asia
and Africa (17). Adults and children in these countries who handle, assemble and dismantle
e-waste are at greatest risk of Cd exposure, as these operations are poorly regulated and there
is often little or no personal protection (18).
1.2.2 Cadmium disposition
Cadmium is absorbed through facilitated diffusion or energy-dependent transport, such
as through the divalent metal transporter-1 (DMT-1) (10, 19), though only a small portion of
intestinal Cd is absorbed with the majority of Cd excreted through intestinal sloughing (20).
Following pulmonary or gastrointestinal absorption, Cd2+ is transported to the liver as
inorganic Cd or Cd-bound albumin where it is subsequently taken up and degraded. The
majority of Cd is complexed to proteins, such as metallothionein (MT), or to small peptide
sulfhydryl groups (-SH), such as glutathione (GSH) (20, 21). As Cd2+ builds up over time, a
small portion of liver Cd-MT is released into the blood plasma where it can be filtered
through the renal glomerulus and excreted through the urine. However, Cd has a high affinity
for thiol groups and tends to accumulate in target organs, including the liver and kidney (2,
20), organs which contain high levels of MT (3, 5, 20).
1.2.3 Cadmium biomarkers
The major clinical biomarkers for indication of Cd toxicity include Cd in the blood
and urine (2). The half-life of blood Cd has a fast component of 3-4 months and a slow
component of 10-30 years (22, 23). Blood Cd is a reliable marker for recent Cd exposure
4
and may also be a marker of low-level, long-term exposure due to its relation to Cd-body
burden. On the other hand, urinary Cd concentration better correlates with Cd load in the
kidney (2). Nevertheless, blood Cd and urinary Cd correlate and have been used as markers
of environmental exposure in various epidemiological studies (16, 23, 24). Biomarkers of
renal function are discussed in Section 1.3.3.2.
1.3 Cadmium toxicity
1.3.1 Acute Toxicity
The exact health outcome of Cd toxicity is dependent on the concentration, exposure
time, route of exposure, chemical speciation, and target organ. Acute Cd poisoning is defined
as a short period exposure to Cd, usually within 14 days, according to the U.S. Department
of Health and Human Services (25). Initial studies on acute Cd toxicity investigated
occupationally exposed workers. Symptoms include pulmonary edema (which is often fatal),
hemorrhage, vomiting and diarrhea, fulminant hepatitis, testicular injury and lethality (25).
Acute Cd poisoning through oral ingestion can present with increased salivation, choking or
vomiting, and abdominal pain (13).
Of growing concern is the development of Cd-containing nanoparticles as novel
nanopharmaceuticals. These nanoparticles include Cd-sulfur (Cd-S), Cd-selenium (Cd-Se) or
Cd-tellurium (Cd-Te) polymers encapsulated in coatings to form semiconductor quantum
dots (QDs) (26, 27). Because of their optical and physical properties, QDs are being
developed as new methods of cellular imaging and drug targeting, especially for the
diagnosis and treatment of solid tumours. However, QDs contain a substantial amount of
5
Cd. Studies have shown that QDs persist for several months in animal models and degrade
over time, resulting in increased Cd exposure (26, 28). Very little is known about the health
risks of exposure to Cd nanoparticles and they have been the subject of recent investigations
(29, 30).
1.3.2 Chronic toxicity
Chronic Cd toxicity occurs in individuals who are exposed to Cd for over a year (25).
This is commonly seen in occupationally exposed individuals, who develop lung, kidney,
gastrointestinal tract, ovarian and testicular dysfunction, and bone damage (2, 25).
An example of environmental chronic toxicity of Cd in the human population is the
Itai-Itai disease in Japan. Cadmium contamination of the local water supply used to irrigate
rice paddies resulted in high concentrations of Cd in rice, which was the cause of this disease
(2, 20). The symptoms of the disease include osteomalacia, severe bone pain, anemia, and
gastrointestinal and renal dysfunction. This has been linked to an increase in serum alkaline
phosphatase, and an increase in blood calcium and phosphate levels, with bone
demineralization occurring before the onset of renal injury (31).
For the general population, continual exposure to low-levels of Cd have been linked to
the development of kidney and bone damage, and there have been reports to implicate
increased risk of lung, prostate and kidney cancer (2, 32). Pulmonary edema, respiratory tract
irritation, renal dysfunction, and anemia have also been associated with chronic Cd exposure
(2, 33). One of the major target organs for chronic Cd exposure is the kidney (4, 16, 24, 34).
6
As this study will investigate the effects on the renal mesangial cell, the subsequent
subsections will focus on the renal effects of Cd exposure.
1.3.3 Cadmium nephrotoxicity
1.3.3.1 Normal renal physiology
The major functions of the kidney are to maintain blood volume, blood pressure, blood
osmolarity, and blood pH in addition to filtration and excretion of waste products (35). The
functional unit of the kidney is the nephron (Fig. 1A). Blood enters the glomerulus through
the afferent renal arteriole where plasma is filtered across the glomerulus into the Bowman’s
capsule, becoming plasma filtrate. The filtrate enters the tubular component of the nephron,
passing through the proximal convoluted tubule (PCT), the Loop of Henle and the distal
tubule where it gathers into the collecting duct before exiting the kidney through the ureter
as urine. Each component of the nephron has distinct functions; filtration occurs in the
glomerulus, reabsorption throughout the tubular portion of the nephron, and secretion occurs
in the PCT, distal tubule and collecting ducts (35–37).
The glomerulus functions as the major filtration unit (Fig. 1B), with the glomerular
filtration rate (GFR) being a good indicator of renal function. Plasma is filtered through the
porous fenestrated endothelium resulting in size exclusion of large plasma proteins.
However, smaller plasma proteins, such as albumin, are able to pass through. These proteins
are repelled by the negatively charged glomerular basement membrane (GBM), rich in
proteoglycans including heparan sulfate. Finally, plasma is filtered through the filtration slits
of epithelial podocytes around the mesangium to reach the Bowman’s capsule, entering the
7
Figure 1: Anatomy of the renal nephron. A) Black arrows indicate the flow of blood and
plasma filtrate throughout the nephron. See text for details. Reproduced from Campbell and
Reece (37). B) Cross-section of the glomerulus highlighting the specific cell types involved
in filtration. The mesangial cells (green), which are the focus of this thesis, are located
centrally. These cells remain unprotected by the glomerular basement membrane (pink) and
are in direct contact with toxic substances in the plasma (34). Adapted from Kumar et al.
(36).
8
Figure 1
9
tubular component of the kidney (36, 38, 39).
The mesangial cell (Fig. 1B) of the glomerulus is a perivascular pericyte that is located
in the central portion of the glomerular tuft between capillary loops (34). It maintains the
structural integrity of the glomerulus and can regulate the GFR by altering the luminal
surface area and ultrafiltration coefficient (34, 40, 41). It also has important roles in
generating vasoactive agents, such as prostaglandins, the synthesis and breakdown of matrix
components, phagocytosis of macromolecules such as immune complexes, and modulation
of cell proliferation through production of factors such as interleukin-1 (34, 42). It likely has
a role countering intracapillary pressure, where failure of this function can result in aneurysm
and progression to renal disease (34, 42). The mesangial cell will be the focus of this thesis.
The major function of the PCT is reabsorption of ions and proteins found in the plasma
filtrate and secretion of some materials, in particular drugs. It consists of polarized epithelial
cells that transport ions, and are distinct from cells of the distal segments of the renal tubule.
In terms of ultrastructure, PCT contain two cell types, S1 and S2. S1 cells are taller, have
more interdigitation of cell margins, more mitochondria, more invaginations of basal
plasmalemma, and a taller brush border than S2 cells. S1 cells in all nephrons extend a
variable distance beyond the glomerulus (43). Altered tubular function, by changes to
reabsorption of proteins and ions, is associated with various pathologies including diabetes
(44–46).
10
1.3.3.2 Effects of cadmium on the kidney
The kidney is a major-target organ of Cd toxicity (16, 24, 47) and has been classified
as the critical organ to Cd exposure. The critical organ is defined as one of the first organs to
have reached the critical concentration of a metal under specified circumstances of exposure
for a given population. The critical concentration is the concentration of the metal that causes
adverse functional changes, reversible or irreversible, that occur in the most sensitive cells of
the critical organ (3, 48).
One of the major epidemiological studies on the renal effects of Cd on the general
population was the Cadmibel study conducted in Belgium (16). Several renal biomarkers,
such as urinary calcium, 2-microglobulin and N-acetyl-B-glucosaminidase (NAG) were
positively correlated with urinary Cd, indicating proximal tubule dysfunction. It is estimated
that a urinary Cd excretion of 2 pg/24 h corresponds to a mean renal cortex concentration of
Cd of about 50 parts per million (wet weight) (16). Approximately a third of the total body
burden of Cd is found in the kidney and about 7% of the population has a Cd load that results
in minor renal dysfunction (49). The critical concentration of Cd in the renal cortex is 200
mg/kg with about 10 μg excreted/day (16). Renal effects of Cd exposure are irreversible and
progressive, even after cessation of Cd exposure (50). Overall, Cd first causes minor renal
damage, which can lead to end-stage renal disease, especially if associated with other risk
factors such as diabetes (2, 49).
There are three major ways by which Cd can be transported into the kidney: Cd-MT,
Cd-bound to GSH, and Cd-bound to albumin during synthesis in the liver (3, 21). The most
11
likely method of transport is the Cd-MT form which is filtered across the glomeruli and
enters the kidney (20, 24). Much work has focused on the effects of Cd on the PCT in
mediating toxicity, which has been linked to changes in PCT reabsorption. The S1 segment
of the PCT is a major target of chronic Cd toxicity and mimics the de Toni-Debré-Fanconi
syndrome characterized by defective protein, amino acid, glucose, bicarbonate and
phosphate reabsorption (6). Because the PCT absorbs all proteins that are filtered through
the glomerulus, Cd-bound proteins are reabsorbed into the circulation through various
protein receptors, including megalin and cubilin (10, 19). The Cadmibel study (16, 49)
showed that there was a concentration response between urinary Cd and the extent of
proximal tubular damage. The critical effects of nephrotoxic damage are tubular proteinuria,
aciduria and glucosuria (16, 50).
At higher Cd concentrations, PCT damage can progress to glomerular damage
affecting the GFR and eventually resulting in end-stage renal disease and renal failure (2, 34,
51). Glomerular damage is associated with decreased GFR and creatinine clearance
especially when U-Cd is 1 μg Cd/g creatinine or higher (2). Fewer studies have focused on
the toxic effects of Cd on the glomerulus, which will be the focus of this thesis.
The mesangial cell of the glomerulus is particularly susceptible to the effects of Cd2+
due to lack of an underlying GBM, resulting in direct exposure to toxic substances in the
plasma (Fig. 1B) (34). The IC50 cytotoxicity index of Cd, defined as the Cd concentration
that results in 50% cellular viability, for mesangial cells in vitro is 3.55 ± 1.05 μM (34).
Glomerular damage in humans is indicated by urinary markers of high molecular weight
12
proteins (albumin, immunoglobulin G) and thromboxane B2 (53). Therefore, studying the
mesangial cell in vitro can provide a better understanding of the toxic effects of Cd on the
kidney.
1.4 Molecular mechanisms of cadmium toxicity
Cadmium is a non-essential metal that alters several molecular mechanisms
contributing to its toxic effects. These mechanisms include changes to signal transduction
pathways (5), redox status (47), and cellular morphology (54). The specific mechanisms
important for this study are explored in more detail in the following subsections.
1.4.1 Cadmium and signal transduction
Cadmium activates several signaling cascades resulting in permanent changes in the
levels of second messengers (5), altered gene transcription (55), as well as cell death (56, 57)
and survival pathways (58) . Because of its high affinity for the thiol groups of proteins, Cd
has the ability to inhibit enzymes, such as redox regulating enzymes (47), or enhance the
activation of enzymes through inhibition of phosphatases (59).
1.4.1.1 Calcium/calmodulin-dependent protein kinase (CaMK-II)
The kinase that has been the focus of work in our lab is the Ca2+/calmodulin-dependent
protein kinase II (CaMK-II). It is a ubiquitous enzyme present in all cell types examined and
consists of a family of multifunctional serine/threonine protein kinases (60). Each isoform
( , , , ) of CaMK-II is encoded by a separate gene. The and isoforms are found in
nervous tissues whereas the and isoforms are expressed ubiquitously; the CaMK-II
13
isoform being the primary isoform in mesangial cells (58, 61). It is a calcium effector protein
that leads to downstream signaling events that coordinate and regulate Ca2+-mediated
changes in cellular function (60).
Each isoform has an N-terminal catalytic domain, a C-terminal association domain and
a central autoregulatory domain (Fig. 2A). Regulated by changes to intracellular Ca2+,
CaMK-II binds to CaM via its association domain, resulting in release of the catalytic
domain from the autoregulatory loop (60, 62), which can now form multimeric structures
(62). Subsequently it can become autophosphorylated at Thr-286/287 (depending on the
isoform) resulting in dissociation from calmodulin (CaM) even when Ca2+ levels fall,
forming an autonomously active form (61). Therefore, transient rises of intracellular Ca2+
can lead to prolonged CaMK-II activation (63).
1.4.1.2 Activation of CaMK-II by Cd
In mouse mesangial cells, Cd2+ activates CaMK-II through increased phosphorylation
(57). This activation was biphasic, with increased autonomous activity at 10 μM CdCl2
treatment occurring at 1-5 min and 4-6 h. Cadmium-mediated activation can occur through
redox-dependent or Ca2+-dependent processes (Fig. 2B). Redox modification of Met-
281/282 within the regulatory domain results in activation of CaMK-II in a process that is
very similar to that of autophosphorylation (62). As Cd can increase reactive oxygen species
(ROS) levels (See Section 1.4.4), ROS can activate CaMK-II through two different
mechanisms; direct modification of multiple methionine residues (62) or through inhibition
of phosphatases resulting in prolonged phosphorylation (59). Additionally, Cd2+ stimulates
14
Figure 2: CaMK-II signaling pathways and role in cadmium toxicity. A) CaMK-II is a
Ser/Thr kinase with the majority of isoforms containing a catalytic domain, regulatory
domain, actin binding domain and an association domain (64, 65). Inset is the sequence of
the regulatory domain, highlighting Met-281/282 which are oxidized and the Thr-287 (or
Thr-286 depending on the isoform) autophosphorylation site, both modifications resulting in
release of CaMK-II from its autoregulated form and activation of the catalytic domain.
Figure adapted from Erickson et al. (62). B) CaMK-II activation by Cd2+ has a central role in
mediating changes in signaling pathways. Stimulation of CaMK-II by Cd can occur through
three mechanisms: activation of CaM (8), increases of intracellular Ca2+ through inhibition of
SERCA-channels or stimulation of inositol triphosphate production (5, 66) or increased
ROS production (47). Downstream activation of various MAPKs and EGFR can lead to
enhanced survival. CaMK-II has an actin binding domain which regulates actin bundling and
polymerization (64). Dashed arrow indicates an indirect interaction. Figure adapted from
Xiao et al. (58).
15
Figure 2
16
intracellular Ca2+ release, possibly through stimulation of inositol triphosphate receptors or
inhibition of sarco/endoplasmic reticulum calcium ATPase (SERCA-type Ca2+-ATPase)
channels on the endoplasmic reticulum (ER) (5, 66, 67). This rise in intracellular Ca2+, in
combination with Cd-mediated activation of CaM, an upstream regulator of CaMK-II, can
increase CaMK-II activation (68).
In mesangial cells, Cd2+ is a known regulator of the mitogen-activated protein kinase
(MAPK) pathways, including the Erk, Jnk and p38, all of which are affected by Cd2+ (58,
69). Previous work has shown that activation of CaMK-II contributes to the observed effects
of Cd on MAPKs. Ca2+/calmodulin-dependent protein kinase is also implicated in the
indirect activation of epidermal growth factor receptor (EGFR) (58) and Cd2+-induced
caspase-independent apoptosis (56, 57).
The and isoforms of CaMK-II have been shown to have an actin binding domain
involved in actin bundling in dendritic spines (64, 65) and though it is currently unknown if
the isoform in mesangial cells has a similar function. Recently, Liu and Templeton (70)
showed that in rat mesangial cells (RMC), CaMK-II associated with actin filaments upon
Cd2+ treatment and that this association was abrogated by inhibition of CaMK-II.
1.4.2 Focal contacts
1.4.2.1 The link between the matrix and cytoskeleton
Focal adhesions (FAs) anchor the cytoskeleton to the extracellular matrix and play a
critical role in regulating cell proliferation, apoptosis and migration. Loss of FAs usually
17
results in anoikis, or anchorage-dependent apoptosis (71, 72). Focal adhesions are composed
of several proteins including paxillin, talin, vinculin, -actinin and focal adhesion kinase
(FAK), many of which recruit the cytoskeleton to FAs (Fig. 3B) (73). Both the structural
integrity of the actin scaffold and the intracellular membrane environment determine the
assembly and disassembly of FAs (74). Loss of focal contacts is often associated with
pathologies, including cancer (75).
1.4.2.2 Signal transduction of focal adhesion kinase (FAK)
Focal adhesion kinase is the main non-receptor protein tyrosine kinase involved in
FAs. The major domains are the N-terminal FERM (band 4.1, ezrin, radixin, moesin
homology) domain, a central tyrosine kinase domain (KD), a C-terminal focal adhesion
targeting (FAT) domain, a linker region between FERM and KD, and unstructured proline-
rich regions (Fig. 3A) (73, 75). Under normal conditions, FAK is held in an autoinhibitory
state by the FERM domain which sterically inhibits Src kinase interaction with the activation
domain. Upon integrin clustering, the FERM domain is displaced, allowing FAK to become
autophosphorylated at Tyr-397 resulting in recruitment of Src kinases which phosphorylate
other tyrosine residues leading to subsequent activation of several downstream signaling
cascades involved in FA assembly and disassembly, actin remodeling and cellular migration
(73, 76, 77) (Fig. 3B). To date, very little work has been done on the effects of Cd on focal
contacts.
18
Figure 3: Focal adhesion kinase structure and signaling cascade. A) Domain structure of
FAK consists of an N-terminal FERM domain that binds to various proteins including EGFR
and Arp2/3, an actin nucleating protein. The central kinase domain (KD), containing the
autophosphorylation site Tyr-397, is sterically inhibited by the FERM domain when inactive.
When Tyr-397 is phosphorylated, it recruits other proteins including Src kinase and
phospholipase C (PLC- ). The C-terminal FAT domain contains Tyr-925, when
phosphorylated can recruit actin-binding proteins such as paxillin and talin. Finally, proline
rich regions (PRR) act as docking sites for other proteins. Adapted from Mitra et al. (76). B)
Integrin clustering results in activation of FAK, which acts as a scaffold and kinase for the
recruitment and activation of downstream targets. In particular, Src kinase has a central role
in mediating FAK signaling. Indirect activation of MAPKs results in myosin light chain
kinase phosphorylation (MLCK), calpain activation and FA detachment. Activation of Rho
GTPases has an important role in mediating actin remodeling and FA formation. Dotted
arrow shows an indirect interaction. Adapted from Schneider et al. (78).
19
Figure 3
20
1.4.3 Cadmium and the actin cytoskeleton
1.4.3.1 The actin cytoskeleton
Actin is a major cellular structural protein that is highly conserved across eukaryotic
cells, with important roles in maintaining cellular morphology, motility, proliferation, tissue
repair and protein trafficking (79–81). There are three major isoforms of actin ( , , ), with
the - and - isoforms being the major isoforms in non-muscle cells (80).
Actin exists in a dynamic equilibrium between globular (G-) actin and filamentous (F-)
actin. Polymerization of G-actin into F-actin occurs through several regulated processes
dependent on actin nucleation events and proteins - including profilin, actin-related protein
2/3 (Arp2/3) proteins - that help initiate nucleation events needed to induce
polymerization/depolymerization (82). Actin treadmilling involves the addition of actin
monomers at the barbed (or +) end in an adenosine triphosphate (ATP)-bound state and
dissociation from the pointed (or -) end that is primarily in the adenosine disphosphate
(ADP) state (79, 81).
Various actin binding proteins are involved in actin filament elongation, sequestration,
severing, capping and cross-linking. This process is regulated by the Rho family GTPases
that act as molecular switches, which include Cdc42, Rac and Rho GTPases (83). These
molecular switches often converge on proteins which have a direct effect on the actin
cytoskeleton, including the Arp2/3 complex involved in actin nucleation along with Wiskott-
Aldrich syndrome protein (WASP) proteins (82). Abnormal regulation of actin cytoskeleton
21
is associated with a number of diseases including cancer, neurological disorders, and
cardiomyopathies (79).
1.4.3.2 Cadmium effects on the actin cytoskeleton
It has been shown that Cd2+ contributes to depolymerization of the actin cytoskeleton
in several cell lines, including RMC (54). In RMC, Cd2+ selectively disrupts the F-actin
cytoskeleton without a subsequent increase in G-actin monomers, whereas other similar
divalent cations at equimolar concentrations had no effect (54, 84). This was not due to
changes in Ca2+, as inhibition of Ca2+ release from intracellular stores did not abrogate this
effect. Interestingly, Cd at concentrations >100 μM increased the rate of polymerization of
G-actin monomers in vitro, whereas lower concentrations stabilized filaments (84, 85).
However, when actin was polymerized in the presence of Cd-treated cell lysates, there was a
decrease in the rate of G-actin polymerization indicating that cellular factors may have a role
(84). In mesangial cells, the cellular factors that have been implicated in mediating
cytoskeletal disruption thus far are gelsolin (86) and CaMK-II (70).
Gelsolin is one of a family of actin severing proteins involved in severing and capping
F-actin, favouring depolymerization from the pointed end (81). Gelsolin can bind G-actin
monomer and actin filaments. Cadmium increases the association of gelsolin with actin
filaments in RMC, possibly contributing to the enhanced depolymerization observed in these
cells (70). Cleavage of gelsolin was also enhanced with Cd treatment, possibly activating
different proteins that contribute to its actin severing functions (70, 86).
22
Activation of CaMK-II is also important in mediating cytoskeletal disruption by Cd2+
as antagonism of CaM and inhibition of CaMK-II were shown to prevent Cd2+-dependent
cytoskeletal disruption (87). Ca2+/calmodulin-dependent protein kinase II interacts with both
G-actin and F-actin in RMC and increases the association of a 50 kDa gelsolin fragment with
F-actin (70). The CaMK-II isoform binds to G-actin and inhibits actin polymerization in
vitro, reducing the rate of polymerization, but can also bind to actin filaments facilitating
filament bundling, and increasing filament rigidity (64, 88).
1.4.4 Cadmium and reactive oxygen species (ROS)
1.4.4.1 Sources of ROS
The major ROS include hydrogen peroxide (H2O2), superoxide (O2•-), hydroxyl (OH•),
and hydroperoxide species (ROOH, ROO•) (89, 90), where the source of ROS determines
the exact composition of the radical species formed (91). Pathological increases in ROS can
cause DNA double strand breaks, lipid peroxidation and irreversible oxidation of proteins,
eventually leading to cellular dysfunction and death (89, 91, 92).
Reactive oxygen species can be formed in the cell through several different
mechanisms (Fig. 4). The majority of ROS are produced through metabolic processes, with
oxidative phosphorylation in the electron transport chain of the mitochondria being the
primary source of ROS in aerobic cells (93). Secondary metabolic sources of ROS
production include -oxidation of long chain fatty acids, as a byproduct of cytochrome p450
activity involved in the synthesis and degradation of steroid hormones and retinoic acid, and
a byproduct of purine catabolism by xanthine oxidase. Nicotinamide adenine dinucleotide
23
Figure 4: Physiological sources of ROS. i) The mitochondrial electron transport chain
during cellular respiration; ii) Xanthine oxidase catabolism of purines; iii) NADPH oxidase
conversion of O2 into O2•- or H2O2,; iv) Cytochrome p450 synthesis and degradation of
steroid hormones; v) 5-lipoxygenase enzyme synthesis of leukotrienes. vi) Shuttling of H2O2
through aquaporins across cell membranes during paracrine signaling (91). Reproduced from
Covarrubias et al. (91).
24
Figure 4
25
2'-phosphate reduced (NADPH) is used as an electron donor by NADPH oxidases to convert
O2 into radical species which has a greater role in phagocytic cells, although they are also
found in other tissues (91, 94). Reactive oxygen species can also be transported from the
extracellular space by aquaporins and is also a byproduct of cellular signaling pathways, in
particular those involved in growth factor stimulation (91, 95). Another possible source of
ROS is through Haber-Weiss chemistry or Fenton reaction of Cu2+/Cu+ and Fe3+/Fe2+ (89).
1.4.4.2 Antioxidant defense mechanisms
Several antioxidant defense systems are important in combating ROS production and
include enzymatic and non-enzymatic mechanisms. Manganese-superoxide dismutase
(MnSOD) and copper/zinc-superoxide dismutase (Cu/ZnSOD) scavenge superoxide by
converting superoxide to H2O2 and O2. Catalase, glutathione peroxidases, and peroxiredoxins
are critical in the breakdown of H2O2 into O2 and H2O (89, 91, 93).
The major intracellular antioxidant is the non-protein thiol glutathione (GSH).
Glutathione is a tripeptide antioxidant consisting of -glutamyl-cysteinyl-glycine synthesized
by -glutamylcysteinyl synthetase (91, 96) (Fig. 5). The glutathione redox cycle involves
cycling between reduced glutathione (GSH) and oxidized glutathione (GSSG) through
reduction of GSSG by the enzyme glutathione reductase (97, 98), the GSH/GSSG ratio being
a good indicator of intracellular redox status (99). Several other enzymes use GSH to
detoxify radical species, including glutathione peroxidase and glutathione S-transferase (89) .
Because of these defenses, H2O2 can be tolerated to micromolar concentrations before
26
1.4.4.3 Cadmium induction of ROS
being a good indicator of intracellular redox status (99). Several other enzymes use GSH to
Figure 5: Glutathione redox cycle. The first step in the biosynthesis of GSH involves the
conjugation of the -carbon of glutamate to cysteine, via the enzyme -glutamylcysteinyl
synthetase, which is the rate-limiting step. The final step involves addition of glycine. The
resulting reduced GSH can participate in the glutathione redox cycle. Oxidation of GSH by ROS
converts 2 GSH molecules into GSSG. This process can be catalyzed by enzymes, such as
glutathione peroxidase. Glutathione reductase can use the reductant NADPH to reduce GSSG
back to GSH, completing the cycle (96). Enzymes are in red. Figure adapted from Suttrop et al.
(96).
27
it is lethal (89). Excess production of ROS or inhibition of antioxidant defense systems can
result in an imbalance, leading to oxidative stress.
1.4.4.3 Cadmium induction of ROS
Cadmium exists either in the elemental Cd0 state or as the Cd2+ ion which is very stable
and does not undergo redox cycling under biological conditions. Nevertheless, Cd has been
shown to increase ROS in a variety of culture systems and animal models (47). Cadmium
indirectly increases intracellular ROS by four mechanisms: i) Cd2+ binds with high affinity
to thiol groups resulting in inhibition of redox-regulating enzymes such as catalase (100); ii)
Cd2+ binds to the thiol group of the major intracellular antioxidant glutathione (GSH)
decreasing the ability of GSH to buffer changes in redox state (47); iii) Cd2+ can uncouple
the mitochondrial electron transport chain, resulting in accumulation of ROS and general
mitochondrial dysfunction resulting in the release of pro-oxidative factors, including
cytochrome c (101, 102); and iv) Cd2+ can displace endogenous Fenton metals resulting in
ROS production by Fenton catalysis (2, 3, 6, 47) (Fig. 6). Mitochondrial dysfunction occurs
as Cd binds to protein thiols in the mitochondrial membrane, affecting mitochondrial
permeability transition, inhibiting the respiratory chain reaction and generating ROS (47).
Cadmium also inhibits the mitochondrial complex III, resulting in accumulation of
semiubiquionones that are highly unstable and prone to transferring one electron to O2 to
form superoxide (103).
Though Cd2+ is pro-oxidant, it also upregulates the expression of several antioxidant
defenses, promoting cellular survival. Cadmium induces expression of cysteine-rich MT
28
Figure 6: Mechanisms of ROS production by Cd. There are several indirect mechanisms
of Cd-mediated ROS elevation. i) Antioxidant enzymes have an important role in reducing
free oxygen radicals, such as catalase in peroxisomes, MnSOD in the mitochondria and
Cu/ZnSOD in the cytosol- all of which are inhibited by Cd2+ (100). ii) The major
intracellular antioxidant GSH is depleted by Cd preventing GSH from redox cycling (47). iii)
Cadmium can also displace iron or copper from proteins, which subsequently undergo
Fenton catalysis to form hydroxide or superoxide (47). iv) Disruption of the mitochondria
can uncouple the electron transport chain, resulting in release of ROS (102). See text for
more details.
29
and glutathione (20, 104). This may be due to stabilization of the nuclear factor E2-related
factor 2 (Nrf2) transcription factor by ROS and Cd2+. Nuclear Nrf2, in combination with
small Maf proteins, transactivates the antioxidant response element (ARE) that controls the
expression of phase II enzymes, including those involved in GSH production (55).
Adaptations to Cd have important implications for promoting survival of damaged cells,
allowing cells with DNA damage to proliferate and develop a malignant phenotype (105).
1.5 Protein S-glutathionylation
1.5.1 Mechanisms of glutathionylation
Redox status is now recognized as a means of regulating various proteins and enzymes
under physiological and pathological conditions (106). One prominent modification is
reversible protein S-glutathionylation, a post-translational modification of proteins involving
formation of a mixed disulfide between a protein cysteinyl residue and GSH. This occurs in
response to oxidative stress and may be a mechanism to protect proteins from irreversible
oxidative damage (104, 105). Oxidation of protein thiols can result in the formation of
sulfenic acids (-SOH), which can be further oxidized to sulfinic (-SO2H) or sulfonic acids
(-SO3H) (106). Protein glutathionylation can also occur in the absence of exogenous
oxidative stress, and may be a mechanism for redox regulation of protein function under
physiological conditions (103, 106, 107). Interestingly, increases in protein S-
glutathionylation have been shown to occur in various disease states, including
hyperlipidemia, diabetes mellitus and chronic renal failure (106).
30
The exact mechanisms of protein S-glutathionylation are still the subject of much
debate. They include thiol-disulfide exchange, sulfenic acid intermediates, thiyl radical
intermediates, and S-nitrosylated intermediates (Fig. 7A). Thiol-disulfide exchange depends
on the GSH/GSSG ratio and may occur through an exchange between GSSG and protein this
exchange. More plausible mechanisms involve reactive thiol derivatives including sufenic
acids or thiyl radical intermediates. Sulfenic acids form under physiological conditions, are
highly unstable and rapidly undergo further oxidation to sulfinic or sulfonic acids. Sulfenic
acids are believed to be the major protein intermediates that are readily glutathionylated.
Thiyl radicals are amongst the shortest-lived sulfhydryl derivatives and form
glutathionylated proteins through radical recombination or reaction with a thiolate and O2
(103, 105, 108).
The major enzyme that has been shown to catalyze the reactions between GSH
intermediates and protein S-glutathionylation is glutaredoxin (GRx). Glutaredoxin is part of
the family of thioltransferases and has been shown to be the major deglutathionylating
enzyme (106, 112). Due to the low pKa of the active site-cysteine, GRx may catalyze protein
glutathionyation through stabilization of the glutathione thiyl radical, allowing the active
site-cysteine to become glutathionylated. It is then reduced back to its original state by GSH
(Fig. 7B). Interestingly, under some circumstances, inhibition of GRx resulted in increased
protein glutathionylation (113). Therefore, it is likely that the redox state of the cell
contributes to the glutathionylating or deglutathionylating activity of GRx.
31
Figure 7: Mechanisms of protein S-glutathionylation. A) i) Thiol-disulfide exchange is
dependent on GSH/GSSG ratio and oxidation potential for formation of mixed disulfide
(protein-SSG). ii) Sulfenic acid intermediates (-SOH) and iii) thiyl radical intermediates
(RS•) that form as a result endogenously produced ROS are highly unstable and susceptible
to reduction by GSH. iv) S-nitrosylated intermediates can form GSNO (shown) or protein-
SNO formation. These intermediates are more stable than the oxygen intermediates in ii) and
iii), but have been shown to readily react with a variety of proteins resulting protein-SSG
formation. B) Glutaredoxin mechanism of action involves a first step monothiol-disulfide
exchange between the GRx active site and the glutathionylated sulfur moiety of protein-SSG
resulting in formation of protein-SH. The second step involves reduction of GRx-SSG by
GSH to produce GSSG as the second product, recycling the reduced enzyme (106). Figure
adapted from Mieyl et al. (106).
32
Figure 7
33
1.5.2 Glutathionylation of proteins
Since glutathionylation has been recognized as a post-translational modification,
several proteins have been shown to be regulated in this manner. For the majority of
proteins, protein S-glutathionylation has been shown to be inhibitory, affecting proteins such
as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (114), protein tyrosine phosphatase
1B (PTP1B) (115), nuclear factor kappa B (NF B) (113), and protein kinase C (116). In
contrast, some proteins have been shown to be activated upon glutathionylation, including
matrix metalloproteinase (117) and SERCA (118).
One of the major intracellular proteins that is glutathionylated is actin (97, 107, 119).
Glutathionylation of actin is largely inhibitory, resulting in a reduced efficiency of actin
polymerization, shifting the dynamic equilibrium between G-actin and F-actin towards
depolymerization, resulting in disorganized actin filaments at the cell periphery (107, 120).
It has been proposed that under oxidative stress conditions, actin glutathionylation provides
protection against irreversible oxidative damage at the expense of temporary loss of function.
Alternatively, Sakai et al. (119) have shown that actin glutathionylation may represent a
fine-tuning mechanism of actin dynamics, as glutathionylation was required for proper
migration of polymorphonuclear neutrophils.
1.6 Hypotheses and objectives
The exact molecular mechanisms of Cd cellular toxicity are still not well understood.
This thesis will outline studies conducted on the mechanisms of action of Cd on the actin
cytoskeleton and focal contacts. Firstly, redox modulation of the actin cytoskeleton though
34
protein S-glutathionylation may be enhanced by Cd. Glutathione has a dual role in mediating
Cd toxicity; because Cd has a high affinity for thiol groups, GSH binds to intracellular Cd,
and it can decrease ROS by forming GSSG, thus altering redox status and promoting protein
S-glutathionylation. This may be a contributing factor for the observed Cd cytoskeletal
disruption in RMC. As the integrity of the actin cytoskeleton is intimately linked with FAs,
Cd may also alter FAs. Templeton and Liu (87) showed that Cd caused loss of vinculin at
focal contacts, an effect that is mediated by CaMK-II.
This study attempts to clarify the possible mechanisms of Cd-mediated toxicity on the
mesangial cell cytoskeleton and focal contacts. We hypothesize that: i) Cd-induced oxidative
stress contributes to actin glutathionylation, which may be a potential mechanism of Cd-
mediated actin cytoskeletal disruption, and ii) Cd-mediated disruption of vinculin and focal
contacts is mediated through changes in FAK, with a possible connection to CaMK-II.
The objectives of this thesis were to: i) establish an effect of Cd2+ on actin
glutathionylation and determine the changes in the intracellular redox state that contributes to
these effects; ii) determine how actin glutathionylation has an impact on the integrity of the
actin cytoskeleton; iii) determine if Cd-mediated changes to FAs were due to changes in
FAK localization or activation, and iv) determine if inhibition of CaMK-II can prevent these
Cd-dependent effects.
35
2. MATERIALS AND METHODS
2.1 Materials
Fetal bovine serum (FBS) and RPMI-1640 culture medium were purchased from
Wisent Biocenter (Quebec, Canada). Cadmium chloride (CdCl2), -NADPH, 5,5'-dithio-bis-
(2-nitrobenzoic acid) (DTNB), glutathione reductase from baker’s yeast, GSH, GSSG, 2-
vinylpyridine, triethanolamine, diamide, thiazolyl blue tetrazolium bromide (MTT), 4-
acetamido-4'maleimidylstilbene 2,2'-disulphonic acid (AMS), protease inhibitors
(aproprotinin, leupeptin, pepstatin and phenylmethylsulfonyl fluoride (PMSF)) and the
inhibitor of -glutamylcysteinyl synthetase, buthionine sulfoximine (BSO), were obtained
from Sigma-Aldrich (St. Louis, MO). The inhibitor of CaMK-II, KN93, was purchased from
Calbiochem (Billerica, MA). Probes 2',7'-dichlorodihydrofluorescein diacetate (H2DCF-DA)
and rhodamine-conjugated phalloidin and cytoskeletal inhibitors cytochalasin D and
jasplakinolide were purchased from Molecular Probes (Burlington, ON). 4',6-diamidino-2-
phenylindole (DAPI) was acquired from Vector Laboratories (Burlington, ON). Protein G
Agarose beads were from EMD Millipore (Billerica, MA). Sequencing grade trypsin and
Glu-C were obtained from Roche Applied Science (Indianapolis, IN) and Promega
(Madison, WI), respectively. Taq PCR Master Mix was acquired from MEBEP Bioscience
(Burlington, ON). Ribolock RNase inhibitor, deoxyribonuclease I (DNase I), 10x reaction
buffer with MgCl2, dNTP mixture, RNase free H2O, H Minus M-MulV reverse transcriptase
were purchased from Fermentas (Burlington, ON). RedSafe nucleic acid staining solution for
visualization of PCR products was acquired from Frogga Bio (Toronto, ON).
36
Mouse monoclonal anti-vinculin (#V9139), anti- -actin (#A1978) and anti-vimentin
(#V6630) antibodies were purchased from Sigma-Aldrich. Mouse monoclonal anti-
glutathionylated protein antibody (anti-PSSG) (#101-A) that recognizes glutathione-protein
conjugates was purchased from Virogen (Watertown, MA). Rabbit polyclonal anti-FAK
(#06-543) antibody was obtained from EMD Millipore. Anti-apoptosis inducing factor (AIF)
(#sc-13116), anti-Nrf2 (#sc-365949), and anti-Lamin A (#sc-20680) were purchased from
Santa Cruz (Dallas, TX). Rabbit polyclonal anti-phosphoTyr397-FAK (#ab4803) antibody
was purchased from Abcam (Cambridge, UK). Rabbit polyclonal anti-phosphoTyr925-FAK
(#3284S), anti-GAPDH (#14C10) and HRP-conjugated anti-mouse and anti-rabbit
secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA).
Mouse monoclonal anti-transferrin receptor (TfR) (#136800), anti-secondary Alexa fluor
488-conjugated goat anti-mouse and anti-rabbit antibodies (#A11001, #A11034) were
acquired from Invitrogen (Danvers, MA).
Deproteinization Sample Preparation kit was purchased from BioVision Inc. (Milpitas,
CA). Glutaredoxin Fluorescent Activity Assay kit was bought from Cayman Chemicals
(Burlington, ON). Actin Polymerization Biochem kit was acquired from Cytoskeleton Inc.
(Denver, CO), and a RNeasy Mini kit was purchased from Qiagen (Burlington, ON).
2.2 Primary Culture of Rat Mesangial Cells
Rat mesangial cells were prepared from glomeruli of 100 g male Wistar rats (Charles
River; Saint Constant, Quebec) following the procedure of Wang and Templeton (121). The
decapsulated renal cortex was minced and sieved through graded stainless steel sieves (180,
37
125, 106, 90 μm) and washed with 0.9% saline. Cells were collected from the 106 and 90 μm
sieves and grown in 20% FBS RPMI medium with penicillin G (100 IU/ml), streptomycin
(100 μg/ml). Mesangial cells were subcultured by trypsinization until cells had reached
passage 3, when they were characterized by their morphology and positive staining for
smooth muscle actin (122) (Fig. 8).
2.3 Cell treatments
Cells were cultured in RPMI-1640 medium with 10% FBS, incubated in 5% CO2 at
37°C, and passaged by trypsinization at 5x105 cells per 10 cm culture dish or 2x105 cells per
6 cm dish for glutathionylation experiments, or passaged 1:4 per 10 cm culture dish for FA
experiments. All experiments were conducted on cells between passages 6 and 15. After
cells were attached overnight, they were rendered quiescent by growing in 0.2% FBS serum
for 48 h prior to transfer to serum-free (SF) medium followed by addition of CdCl2 in SF
medium. For inhibitor studies, cells were pre-treated with 50 μM BSO in 0.2% FBS serum
for 16 h, which has been confirmed to effectively decrease glutathione levels in RMC by
approximately 70% (104). In cases where CaMK-II was inhibited, cells were pre-treated for
1 h with 10 μM KN93 before Cd treatments, conditions that were previously optimized by
Liu and Templeton (57).
2.4 Cellular fractionation
Fractionations were optimized for the detection of glutathionylated proteins and FA
proteins. These fractionation methods are designated as 1% NP-40 Tris whole-cell lysate,
38
Fig
ure
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reen
) an
d F
-act
in (
red)
and
ove
rlay
(ye
llow
) co
nfir
ms
that
cul
ture
s ar
e pu
re a
nd d
ispl
ay t
he a
ppro
pria
te
char
acte
rist
ics
of c
ultu
red
smoo
th m
uscl
e ce
lls (
34, 1
22).
Cel
ls d
ispl
ay th
e no
rmal
ste
llat
e sh
apes
of c
ultu
red
mes
angi
al c
ells
. Pan
el
A 2
00 x
mag
nifi
cati
on. P
anel
B 4
00 x
mag
nifi
catio
n.
39
0.5% NP-40 HEPES whole-cell lysate, cytoskeletal-cytosolic fractionation, membrane-
cytosolic fractionation, and nuclear-cytosolic fraction, described in more detail in the
following subsections (Fig. 9).
2.4.1 1% NP-40 Tris whole-cell lysate for detection of glutathionylated proteins
To determine changes in the glutathionylation state of cells after treatment, cells were
washed twice with ice-cold phosphate-buffered saline (PBS) and scraped with 1% Nonidet
P-40 (NP-40) lysis buffer (100 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM
phenylmethylsulfonyl fluoride (PMSF) and 1 μg/ml each of aprotinin, leupeptin, and
pepstatin). Cell lysates were sonicated 3 times for 5 s at 200 W and centrifuged at 16,000 g
for 10 minutes at 4ºC and the supernatant taken as the 1% NP-40 Tris whole-cell lysate.
2.4.2 0.5% NP-40 HEPES whole-cell lysate for detection of FAK
To determine changes to FA proteins in response to CaMK-II, cells were lysed as
indicated by Liu and Templeton (57) to preserve CaMK-II activity. Briefly, cells were
washed twice with chilled PBS and lysed by one freeze-thaw cycle in 50 mM HEPES, pH
7.4, with 0.5% NP-40, containing protease and phosphatase inhibitors (1 mM Na3VO4, 25
mM NaF, 1 mM PMSF, and 1 μg/ml each of aprotinin, leupeptin, and pepstatin). Lysates
were then sonicated twice for 5 s and centrifuged (15,000 g, 15 min). The supernatant was
collected as 0.5% NP-40 HEPES whole-cell lysate.
40
Fig
ure
9:
Flo
w c
hart
of
sub
cell
ula
r fr
act
ion
ati
on
pro
toco
ls. P
roto
cols
are
exp
lain
ed in
mor
e de
tail
in th
e te
xt
41
2.4.3 Cytoskeletal-cytosolic fractionation
To determine changes in the localization of FAK, cytoskeletal fractionation was
performed on cells as described by Liu and Templeton (70). Cells were washed twice with
chilled PBS and lysed with 10 mM Tris-HCl, pH 7.4, with 2 mM MgCl2, 138 mM KCl, and
0.2% Triton X-100, containing protease and phosphatase inhibitors. The lysate was
centrifuged (10,000 g, 15 min) and the supernatant was designated the CK cytosolic fraction.
The detergent-insoluble pellet was washed once with chilled PBS and resuspended in 5 mM
Tris-HCl, pH 8.0, with 0.2 mM CaCl2 and 200 μM ATP, sonicated three times for 5 s and
centrifuged (10,000 g, 5 min). The supernatant was designated the cytoskeletal fraction.
2.4.4 Membrane-cytosolic fractionation
Differential detergent fractionation was used to isolate subcellular fractions according
to Bierderbick et al. (123). Briefly, cells were washed twice with chilled PBS and pelleted
(400 g, 5 min). The cell pellets were resuspended in 0.007% Digitonin in 5 mM Tris-HCl,
pH 7.4, containing 250 mM sucrose, 1 mM EDTA, 1 mM EGTA, 1.5 mM MgCl2 and
protease inhibitors. The suspension was agitated for 8 min on ice and centrifuged (1,800 g, 8
min), and the supernatant was further clarified (15,000 g, 20 min) and designated the M
cytosolic fraction. The pellet was washed twice with chilled PBS and resuspended in 20 mM
Tris-HCl, pH 7.4, with 2 mM MgCl2, 138 mM KCl, and 0.5% Triton X-100, containing
protease and phosphatase inhibitors, and incubated on ice for 30 min. The suspension was
centrifuged (8,000 g, 10 min) and the supernatant collected as the membrane fraction. The
cytosolic fraction was shown to be free of the mitochondrial marker apoptosis inducing
42
factor (AIF), and the membrane protein transferrin receptor (TfR), two proteins that were
found in the membrane fraction (Fig. 10A).
2.4.5 Nuclear-cytosolic fractionation
The fractionation was followed as described in Chen and Shaikh (55). Briefly, cells
were washed twice with ice-cold PBS followed by scraping and pelleted at 1,000 g for 5 min.
The pellet was resuspended in 0.2% NP-40 lysis buffer (10 mM HEPES-NaOH pH 7.9, 10
mM KCl, 1 mM EDTA, 1 mM PMSF and 1 μg/ml each of aprotinin, leupeptin, and
pepstatin). The suspension was kept on ice for 15 min with occasional vortexing and
centrifuged at 14,000 g for 1 min and the supernatant designated as the N cytosolic fraction.
The pellet was washed once with ice-cold 1x PBS and resuspended in nuclear buffer (20 mM
HEPES-NaOH, pH 7.9, 420 mM NaCl, 1 mM EDTA). After gentle shaking for 30 min at
4°C, the suspension was centrifuged at 14,000 g for 15 min. The supernatant was designated
the nuclear fraction. The purity of the cytosolic fraction and nuclear fractions were
determined using GAPDH and lamin A respectively (Fig. 10B)
2.5 Viability assay
For the MTT (Thiazolyl Blue) assay, 1 103 cells were seeded in 96-well plates. After
treatment, cells were washed with SF medium, and 1 mg/ml MTT in PBS was added and
incubated at 37°C for 1 h. After the incubation, cells were washed twice with warm PBS
followed by incubation in DMSO at room temperature under agitation for 5 min. Optical
density was measured at 560 and 670 nm, where values at 670 nm were subtracted from
those at 560 nm.
43
Figure 10: Confirmation of the purity of fractions. A) Membrane fractionation as
described in Section 2.4.4. TfR and AIF were primarily localized to the membrane fraction
and were absent from the cytosolic fraction. B) Nuclear fractionation, as described in Section
2.4.5, resulted in a clean cytosolic fraction, only containing GAPDH and clean nuclear
fraction with only Lamin A. Each lane indicates independent preparations of cells treated
under different conditions.
44
2.6 Western Blotting
For the detection of glutathionylated proteins, protein concentrations were determined
using the Bradford assay and 5 μg protein were mixed with 2 x SDS without dithiothreitrol
(DTT). Samples were heated for 5 min at 95°C and resolved on an 8% SDS-PAGE gel.
Proteins were transferred to nitrocellulose membranes and membranes were blocked in Tris-
buffered saline containing 0.1% Tween-20 (TBS-T) and 5% non-fat milk for 1 h and
incubated overnight at 4ºC with primary antibody.
For detection of FA proteins, equal amounts of protein were mixed with 5 x SDS
loading buffer, heated for 5 min at 95°C and resolved on 8% SDS-PAGE gels. Proteins were
transferred to nitrocellulose membranes and blocked with 5% BSA for 1 h, prior to
incubation with primary antibody overnight at 4°C.
Antibody dilutions were as follows: anti-vinculin (1:10,000); anti-FAK (1:2,000); anti-
phosphoTyr397-FAK (1:1,000); anti-phosphoTyr925-FAK (1:2,000); anti-TfR (1:10,000);
anti-AIF (1:1,000), anti-PSSG (1:5,000), and anti-Nrf2 (1:1,000). Anti- -actin antibody
(1:10,000), anti-vimentin (1:10,000), anti-GAPDH (1:1,000), and Lamin A (1:2,000) were
used as loading controls. Membranes were washed with TBS-T and incubated with HRP-
conjugated secondary antibody (1:10,000) in 1% non-fat milk in TBS-T for 1 h. Blots were
visualized using enhanced chemiluminescence. Film was scanned and band intensities were
analyzed using ImageJ software (NIH, Bethesda, MD).
45
2.7 Immunoprecipitation & Mass Spectrometry
Samples containing 200-300 μg of protein (1% NP-40 Tris whole-cell lysate) were
incubated overnight at 4ºC with 2 μg/sample of anti- -actin antibody. Immune complexes
were isolated by the addition of Protein G Agarose beads (4 h, 4ºC) and washed 3 times with
1% NP-40 Tris lysis buffer with PMSF and finally washed once with ice-cold PBS. Samples
were processed and loaded onto gels for SDS-PAGE and subsequently transferred to a
nitrocellulose membrane for Western Blot analysis. Coomassie blue-stained bands around 42
kDa were excised and destained by sequential washing with 50 mM ammonium bicarbonate
(NH4HCO3) and acetonitrile. Gel pieces were shrunk with 50% acetonitrile and 25 mM
NH4HCO3 and digested with trypsin or Glu-C (6.6 ng/ml in 50 mM NH4HCO3 and 5 mM
CaCl2) overnight at 37°C. For sequential digestion, gel pieces were digested first with Glu-C
overnight followed by trypsin digestion for 3 h at 37°C. Trypsin cleaves proteins at the C-
terminus of lysine and arginine residues, whereas Glu-C cleaves at the C-terminus of
glutamic acid residues. The peptides were extracted sequentially with 25 mM NH4HCO3, 5%
formic acid, and acetonitrile. Pooled extracts were evaporated by SpeedVac and dissolved in
0.1% formic acid for liquid chromatography mass spectrometry (LC-MS) without further
clean up.
2.8 Determination of redox state of -actin with AMS
Cells were lysed as in 2.4.1 and lysates were incubated with AMS according to the
protocol by Papp et al. (124). Briefly, 20 μg of cell lysates were incubated with 20 mM AMS
for 30 min at 37°C. The redox-sensitive agent AMS binds to free thiol groups of proteins
resulting in greater retention of the proteins during gel electrophoresis. These AMS-labeled
46
proteins were separated on 12% SDS-PAGE gels, transferred to nitrocellulose membranes
and processed for -actin immunoblot as described in Section 2.6.
2.9 Immunofluorescence
Cells were seeded on 12 mm cover-slips, grown overnight and starved for 48 h with
0.2% FBS in RPMI-1640. After treatment with CdCl2, cells were washed with chilled PBS,
fixed with 4% paraformaldehyde, and permeabilized with 100 mM PIPES, pH 6.9,
containing 0.5% Triton X-100 lysis buffer, 1 mM EGTA, and 4% polyethyleneglycol 8000.
Permeabilized cells were blocked in 5% BSA/PBS and incubated overnight with primary
antibody (1:30 or 1:50) at 4°C. Cells were washed 3 times with 0.2% BSA/PBS followed by
incubation with rhodamine-phalloidin (1:100) and Alexa fluror 488-conjugated secondary
antibody (1:30) for 1 h at room temperature. Coverslips were mounted on glass slides with
mounting medium containing DAPI to stain the nuclei. Appropriate primary antibody and
secondary antibody controls were performed, confirming the specificity of the antibody
interactions (data not shown). Images were taken using a Nikon fluorescent microscope.
2.10 Intracellular reactive oxygen species (ROS)
Intracellular H2O2 was measured with H2DCF-DA. The H2DCF-DA probe was
oxidized rapidly to highly fluorescent dichlorofluorescein (DCF) by H2O2 (125). Cells were
treated for the desired time period with CdCl2 and washed once with SF medium, followed
by incubation with 5 μM H2DCF-DA for 1 h at 37°C. Cells were collected by trypsinization
and the fluorescence intensity of DCF was analyzed immediately in an Epics Elite flow
cytometer (Beckman Coulter).
47
2.11 Intracellular glutathione using DTNB recycling assay
Glutathione was measured as stated in Rahman et al. (126) with some modifications.
Briefly, cells were collected as in Section 2.4.1 (1% NP-40 Tris whole-cell lysis buffer) and
deproteinized using the Deproteinization Sample Preparation kit (perchloric acid, followed
by neutralization with a potassium hydroxide solution). For measurement of total glutathione
(GSSG + GSH), 1.68 mM DTNB in the presence of 1 U GRase and 0.90 mM NADPH in
potassium phosphate buffer, pH 7.5, was incubated with samples and changes of absorbance
monitored at 405 nm for 2 minutes. Values were corrected by subtracting the baseline slope
and values were determined from a standard curve derived from reduced GSH.
Oxidized glutathione was measured similarly as total glutathione, though 1 M 2-
vinylpyridine in 100% ethanol was added to the deproteinized cell lysates to quench reduced
GSH. After 1 hour incubation, triethanolamine was added to remove excess 2-vinylpyridine
prior to measurements (126). All samples were measured in duplicate for total glutathione
and GSSG quantification. Based on these values, the GSH values were calculated as follows:
Total glutathione – 2 x GSSG. Subsequently, the GSH/GSSG ratios were determined.
2.12 Glutaredoxin activity
Glutaredoxin activity was measured kinetically using a Glutaredoxin Fluorescent
Activity Assay kit. Briefly, cells were scraped into PBS and centrifuged at 1,000 g for 10 min
to pellet cells. Cells were resuspended in 10 mM Tris-EDTA, pH 8.0, and sonicated three
times at 200 W for 5 s. Samples were normalized to protein amount prior to GRx
48
measurements and measured kinetically over 60 min at ex=520 nm and em=540 nm
(FLUOstar OPTIMA microtiter plate reader).
2.13 Effect of actin glutathionylation on polymerization in vitro
Actin polymerization was analyzed using an Actin Polymerization Biochem kit based
on an increase in pyrene fluorescence with ex=355 nm and em=405 nm. Actin
glutathionylation was induced by 0.5 mM diamide in the presence of 2 mM GSH according
to the protocol of Dalle-Donne et al. (111). Polymerization was initiated by addition of
polymerization buffer (final concentration: 12.5 mM KCl, 0.5 mM MgCl2, and 0.25 mM
ATP). The change of pyrene fluorescence was measured over 60 min at 37°C.
Glutathionylation status was confirmed by trichloroacetic acid precipitation followed by
immunoblot with anti-PSSG.
2.14 Reverse-transcriptase polymerase chain reaction (RT-PCR)
Cells were collected and total RNA purified as described in the Qiagen RNeasy kit.
Preparation of DNA-free RNA was achieved by reaction with DNase I in 1x reaction buffer
with MgCl2 for 30 min at 37°C. The amount of RNA was measured using a Nanodrop
spectrophotometer to determine the concentration and purity of the samples. All samples
were considered pure, with A260/A280 values approximately equal to 2.0. Total RNA was
reverse transcribed in the presence of a deoxyribonucleotide triphosphate (dNTP) mix,
RNase inhibitor and hexameric primer for 5 min at 25°C followed by addition of reverse
transcriptase enzyme and incubation for 10 min at 25°C. The mixture was finally incubated
49
at 42°C for 60 min. The reaction was stopped by heating the mixture to 70°C for 10 min,
after which complementary DNA (cDNA) was obtained.
Polymerase chain reaction (PCR) on isolated cDNA amplified two genes known to be
under the control of Nrf2, glutathione cysteine ligase catalytic subunit (GCLC) and
hemeoxygenase-1 (HMOX-1). Amplification of GAPDH was used as a loading control. The
primer sequences (127, 128) are as follows:
GCLC:
(F) 5'-CCTTCTGGCACAGCACATTG-3' (R) 5'-TAAGACGGCATCTCGCTCCT-3'
HMOX-1:
(F) 5'-AGCATGTCCCAGGATTTGTC-3' (R) 5'-AAGGCGGTCTTAGCCTCTTC-3'
GAPDH:
(F) 5'-TTCACCACCATGGAGAAGGC-3' (R) 5'-GGCATGGACTGTGGTATGA-3'
The samples were heated to 94°C for 5 min and subsequent cycles were performed at
three temperature steps as follows: 94°C for 30 sec, 60°C 30 sec, 72°C 1 min. A total of 30
cycles were completed followed by 72°C for 10 min for extension. Products from the PCR
were run on 1% agarose gels with RedSafe to stain the nucleic acids and images were taken
using the Biorad GelDoc imaging system. Bands were scanned and quantified using Image J
software as in Section 2.6.
2.15 Statistical analysis
Multiple measurements are reported as mean±S.D. Pairwise comparisons were
50
performed with Student’s unpaired t-test. When multiple comparisons were made, analysis
was by two-way ANOVA, followed either by Dunnett’s post hoc test comparing the
measured value to that of a specified control treatment, or by Tukey’s test for multiple
internal comparisons.Significance was reported as a P-value for measurements. Calculations
were performed with InStat Software (GraphPad, San Diego, CA).
51
3. RESULTS
3.1 Cadmium induces actin glutathionylation
3.1.1 Cadmium induces glutathionylation of a 42 kDa protein
Subconfluent cultures of quiescent RMC were treated with varying concentrations of
CdCl2 in SF medium over different time points, and 1% NP-40 Tris whole-cell lysates were
examined for protein glutathionylation by Western blotting with anti-PSSG, specific for
glutathione-protein complexes. Cadmium induces significant glutathionylation of a 42 kDa
protein as early as 3 h at 0.5 μM CdCl2, with maximal levels of glutathionylation occurring
with exposure to 2 μM CdCl2 at each time from 3-16 h (Fig. 11). A direct comparison of
band intensities at different times cannot be performed as these represent different exposure
times for independent experiments. Subsequent experiments used incubation with 2 μM
CdCl2 for 6 h to induce maximal glutathionylation for that time point. The viability of cells
as determined using the MTT assay was unaffected under these conditions (Fig. 12).
Treatment with diamide, a thiol-specific oxidant, also induces glutathionylation of the same
42 kDa protein, with a second band appearing at higher concentrations of diamide in addition
to the appearance of high molecular weight glutathionylated proteins (Fig. 13; compare Cd2+
treatment at 6 h in Fig. 11). These bands are eliminated upon reduction with DTT. Vimentin,
a characteristic component of intermediate filaments, is stably expressed in cultured smooth
muscle cells (129). It is shown to be an appropriate loading control, especially under non-
reducing conditions, where higher concentrations of diamide result in decreasing amounts of
-actin, precluding its use as a control.
52
Figure 11: Time- and concentration-dependent glutathionylation of a putative actin
protein. Quiescent cultures of mesangial cells were held in serum-free (SF) medium or
exposed to the indicated concentrations of CdCl2 up to 20 M, for the indicated times of 1, 3,
6, 8, or 16 h. Glutathionylated protein (PSSG) detected with an anti-PSSG antibody is
shown in relation to molecular weight markers of 40 and 50 kDa, and a band off-scale
stained with an anti-vimentin antibody is shown as a gel-loading control. Blots are of
representative gels taken at different exposure times and the histograms show mean ± s.d. of
densitometric scans of between n = 4-6 independent experiments, with the ratio of
PSSG/vimentin at each time point normalized to 2 M CdCl2. Band intensities at 1 h were
for longer exposure times than at other time points. Significant increases above SF controls
are indicated (*p<0.05, **p<0.01).
53
Figure 11
54
Fig
ure
12:
Via
bil
ity o
f ra
t m
esan
gia
l ce
lls
dec
rease
s w
ith
in
crea
sin
g c
on
cen
trati
on
s of
cad
miu
m. V
iabi
lity
was
ass
esse
d us
ing
the
MT
T a
ssay
(se
e S
ecti
on 2
.5).
The
re i
s a
conc
entr
atio
n-de
pend
ent
decr
ease
in
viab
ilit
y, a
t co
ncen
trat
ions
gre
ater
tha
n 2 μ
M C
dCl 2
trea
tmen
t for
6 h
. The
gra
ph s
how
s m
ean
± s.
d. o
f A=
560
nm
, nor
mal
ized
to S
F c
ontr
ol (0
μM
CdC
l 2)
at 6
h ta
ken
at 1
00%
. Gra
ph w
as
plot
ted
wit
h no
n-li
near
reg
ress
ion
of d
ata
from
n =
7 in
depe
nden
t ex
peri
men
ts.
Sig
nifi
cant
dec
reas
es b
elow
SF
con
trol
are
indi
cate
d
(*p<
0.05
).
55
Figure 13: Diamide increases glutathionylation of the putative actin protein. Treatment
of cells with the thiol-oxidizing agent diamide caused an increase in the Western blot signal
detected as in Fig. 11 by an anti-PSSG (first 4 lanes). In the last 4 lanes, the samples were
treated with DTT before electrophoresis, and the glutahionylated signal disappears. Both -
actin and vimentin levels are shown as gel-loading controls.
56
3.1.2 Immunoprecipitation identifies the 42 kDa glutathionylated protein as actin
Actin is an abundant cellular protein and a known target of glutathionylation (97, 107).
Since the major glutathionylated protein migrated at 42 kDa, close to the molecular weight
of actin, we hypothesized that it was actin. To confirm this, immunoprecipitation with an
anti- -actin antibody was performed on 1% NP-40 Tris whole-cell lysate from cells treated
with 2 μM CdCl2 in SF medium followed by electrophoresis and immunoblotting with anti-
PSSG. The increase of glutathionylation seen in whole-cell lysate with Cd treatment
compared to cells treated with SF medium alone remained following immunoprecipitation
and the precipitated protein appeared at the same molecular weight as the unknown 42 kDa
band (Fig. 14). A non-specific IgG antibody control and a protein G-agarose beads control
did not precipitate any glutathionylated proteins at 42 kDa (Fig. 14B). To determine if the
protein was actin, bands at 42 kDa were excised from the gel and digested with trypsin, Glu-
C, or a combination of trypsin and Glu-C. The major protein was identified as -actin by LC-
MS. Only a single cysteine (Cys-217) was identified when tryspin and Glu-C were used in
combination, resulting in 80% sequence coverage (Fig. 14). This cysteine contains a putative
sulfenic acid modification, although it was not identified as being glutathionylated. We
subsequently refer to this band as glutathionylated actin.
3.2 Factors contributing to actin glutathionylation
3.2.1 Glutathionylation of actin is not directly dependent on ROS levels
Cadmium is pro-oxidant by virtue of its ability to inhibit redox regulating enzymes
such as catalase and by binding with high affinity to thiol groups (2, 3, 47). Cells were
57
Figure 14: -actin immunoprecipitation identifies actin as the major glutathionylated
protein. A) Cell lysates collected from SF medium or 2 μM CdCl2 conditions were incubated
overnight with 2 μg anti- -actin antibody followed by electrophoresis and immunoblot with
anti-PSSG to confirm changes in glutathionylation status. -actin was used as a gel loading
control. Gels were stained with Coomassie blue dye and the major bands at 42 kDa were
excised as indicated by the boxed region. Gel bands were digested overnight by individual
enzymes, or consecutive days with first Glu-C overnight followed by a 3 h trypsin digestion
and analyzed using liquid-chromatography mass spectrometry. The major band identified
from 3 independent digestions was -actin. Digestion conditions, percentage sequence
coverage, and identified peptides in red are shown. The identified Cys-217 is highlighted in
yellow and identified to be modified as a sulfenic acid, when trypsin and Glu-C were used
for digestion. B) Negative controls to show the specificity of the immunoprecipitation are
shown using a non-specific IgG antibody control and a protein G agarose beads control. Both
conditions were unable to precipitate a glutathionylated protein at 42 kDa.
58
Fig
ure
14
59
treated with CdCl2 for different time periods followed by measurement of ROS using DCF
fluorescence. There were significant increases (p<0.01) in ROS levels at 20 μM CdCl2 for 3
h and at 10 μM and 20 μM CdCl2 for 6 h (Fig. 15A). In contrast, ROS levels were
significantly decreased at 6 h when cells were treated with 2 μM CdCl2. Interestingly,
increases in ROS did not correlate with the glutathionylation status of actin, which was
unaffected under high ROS conditions and significantly increased when ROS levels were
low or unchanged.
3.2.2 Actin glutathionylation correlates with changes in total glutathione levels but not
the GSH/GSSG ratio
To determine whether changes to glutathione levels could explain the change of
glutathionylation in response to Cd, total glutathione (GSH + GSSG) levels were measured.
Treatment of cells with varying concentrations of CdCl2 resulted in significant increases of
total glutathione by 6 h at 0.5 μM and 2 μM CdCl2 compared to the SF control, returning to
control levels at higher Cd concentrations (Fig. 15B, Table 1). A similar pattern was
observed when measuring GSSG (Table 2), although there was no change in the GSH/GSSG
ratios at any concentration across different times (Table 3).
3.2.3 Inhibition of glutathione synthesis decreases actin glutathionylation
To determine if glutathione synthesis is required for an increase in glutathionylation,
RMC were cultured overnight with BSO, a specific inhibitor of -glutamylcysteinyl
synthetase, effectively inhibiting glutathione synthesis (104). Cells treated with 2 μM CdCl2
60
Figure 15: Effects of Cd2+
on ROS and glutathione levels in mesangial cells. A) Levels
of ROS measured by DCF fluorescence following treatment with the indicated
concentrations of CdCl2 in SF cultures. All experiments were done in triplicate and values
are mean ± s.d., normalized to the signal in arbitrary fluorescent units in the absence of Cd2+
taken as 100%. Cadmium exposure was for 1 h (open circles), 3 h (filled circles), or 6 h
(squares). Significant differences from Cd-free conditions at the same time point are
indicated: *p<0.05; **p<0.01. B) Absolute levels of total glutathione (GSH + GSSG) were
measured under the same conditions of Cd2+ exposure as in panel A and the same symbols
are used. Total glutathione levels at 1, 3, 6, 8, and 16 h are tabulated in Table 1.
61
Figure 15
62
Table 1: Effect of cadmium on total glutathione (GSH+GSSG) levels. Rat mesangial cells
were treated with indicated CdCl2 concentrations over 1, 3, 6, 8, and 16 h. Cells were
collected, lysed with 1% NP-40 Tris lysis buffer and deprotenized. Total glutathione was
assayed as stated in Section 2.11. All measurements are from at least 4 independent
experiments with mean and SD indicated. The P values were determined by ANOVA
Dunnett’s post hoc test compared to SF control at each time. NS indicates not significant.
63
Time (h)
n
[CdCl2]
(μM)
Mean
(nmol/mg
protein)
SD
P vs SF
SF
11.8
2.06
-
0.5 13.1 2.84 (NS) 2 13.6 3.79 (NS)
10 16.9 7.13 (NS)
1
4
20 16.5 3.43 (NS)
SF
23.5
4.54
-
0.5 30.3 6.28 (NS) 2 33.8 6.37 (NS)
10 37.3 9.96 p<0.05
3
5
20 27.1 9.02 (NS)
SF
21.5
7.47
-
0.5 36.1 9.67 p<0.05 2 43.1 14.8 p<0.01
10 30.1 12.9 (NS)
6
9
20 29.3 12.5 (NS)
SF
23.1
7.34
-
0.5 43.1 10.3 (NS) 2 49.9 13.1 p<0.05
10 46.9 18.8 p<0.05
8
6
20 37.9 19.4 (NS)
SF
20.9
4.28
-
0.5 45.2 20.4 (NS) 2 56.0 31.2 p<0.05
10 38.0 9.34 (NS)
16
4
20 23.6 2.00 (NS)
Table 1
64
Table 2: Effect of cadmium on oxidized glutathione (GSSG) levels. Rat mesangial cells
were treated with indicated Cd concentrations over 1, 3, 6, 8, and 16 h. Cells were collected,
lysed with 1% NP-40 Tris lysis buffer and deprotenized as described in Section 2.11. All
measurements are from at least 4 independent experiments with mean and SD indicated. The
P values were determined by ANOVA Dunnett’s post hoc test compared to SF control at
each time. NS indicates not significant.
65
Time (h)
n
Treatment
(μM)
Mean
(nmol/mg
protein)
SD
P vs SF
SF
0.460
0.174
-
0.5 0.514 0.385 (NS) 2 0.454 0.436 (NS)
10 0.562 0.423 (NS)
1
4
20 0.299 0.288 (NS)
SF
0.249
0.0858
-
0.5 0.473 0.335 (NS) 2 0.366 0.159 (NS)
10 0.413 0.276 (NS)
3
5
20 0.389 0.215 (NS)
SF
0.356
0.225
-
0.5 0.635 0.379 (NS) 2 1.02 0.946 (NS)
10 0.596 0.547 (NS)
6
9
20 0.454 0.215 (NS)
SF
0.433
0.354
-
0.5 1.20 0.679 (NS) 2 2.30 1.45 P<0.05
10 1.59 1.49 (NS)
8
6
20 1.13 0.900 (NS)
SF
0.792
0.769
-
0.5 2.61 1.63 (NS) 2 2.74 1.37 (NS)
10 2.37 0.369 (NS)
16
4
20 1.39 0.987 (NS)
Table 2
66
Table 3: Effect of cadmium on GSH/GSSG ratios. Cells were treated and collected as in
Tables 1 and 2. The GSH value was calculated by subtracting the GSSG value from the total
glutathione value. All measurements are from at least 4 independent experiments with mean
and SD indicated. The P values were determined by ANOVA Dunnett’s post hoc test
compared to SF control at each time. NS indicates not significant.
67
Time (h)
n
Treatment
(μM)
Mean
SD
P vs SF
SF
27.9
12.6
-
0.5 36.8 23.1 (NS) 2 62.4 52.4 (NS)
10 54.6 50.7 (NS)
1
4
20 73.6 86.7 (NS)
SF
86.0
18.4
-
0.5 85.5 54.1 (NS) 2 99.8 54.7 (NS)
10 111 86.0 (NS)
3
5
20 76.6 40.0 (NS)
SF
114
124
-
0.5 74.3 39.4 (NS) 2 75.4 54.1 (NS)
10 87.9 65.6 (NS)
6
9
20 76.1 41.4 (NS)
SF
103
98.6
-
0.5 53.4 44.5 (NS) 2 47.9 40.7 (NS)
10 48.1 33.4 (NS)
8
6
20 48.2 38.7 (NS)
SF
8.74
9.45
-
0.5 6.98 7.10 (NS) 2 6.96 5.07 (NS)
10 4.06 1.24 (NS)
16
4
20 8.96 10.4 (NS)
Table 3
68
for 6 h showed a significant increase (p<0.001) in actin glutathionylation that was
completely abrogated by pre-treatment with BSO (Fig. 16).
3.2.4 Glutathione levels increase due to increases in antioxidant gene expression
A major transcription factor that controls antioxidant gene expression is Nrf2 (55).
Upon exposure to ROS or electrophiles, Nrf2 is stabilized resulting in its translocation into
the nucleus and binding to the ARE, which controls the expression of a number of phase II
enzymes, including those needed for glutathione synthesis (130, 131). Treatment of RMC
with 2 μM CdCl2 and 10 μM CdCl2 for 6 h induced nuclear translocation of Nrf2, though the
increase at 10 μM CdCl2 was not significant (Fig. 17), despite being conditions that increase
ROS levels (Fig. 15A).
To confirm that genes under the control of Nrf2 are upregulated upon Cd2+ exposure,
RT-PCR was used to measure changes in mRNA levels of GCLC, which produces -
glutamylcysteinyl synthetase, and heme oxygenase-1 gene (HMOX-1), which produces HO-
1 protein, an important enzyme involved in the breakdown of heme (132). There was a
significant increase in expression of both genes with 2 μM and 10 μM CdCl2 treatments at 6
h (Fig. 18).
3.2.5 Actin glutathionylation correlates with increased activity of glutaredoxin
Glutaredoxin (GRx) is a major deglutathionylating enzyme and non-specific inhibition
of GRx with Cd has been shown to increase protein S-glutathionylation in several cell types
(119, 120, 133, 134). To determine if inhibition of GRx by Cd contributes to increased actin
69
Figure 16: Effect of glutathione synthesis on actin glutathionylation. Cells in SF
medium were either unexposed or exposed to 2 M CdCl2 for 6 h with or without
pretreatment with the glutathione synthesis inhibitor, BSO (see Section 2.3). A)
Representative Western blots of PSSG and vimentin loading controls. B) Values (mean ±
s.d.) of densitometric scans from three independent experiments such as depicted in A),
taken as a PSSG/vimentin ratio and normalized to the mean value at 2 M CdCl2 in the
absence of BSO treatment taken as 100%. *p<0.001 vs. 100% normalization signal.
70
Figure 16
71
Figure 17: Cadmium promotes the translocation of Nrf2 to the nuclear fraction. Rat
mesangial cells were treated in SF medium, with or without CdCl2 for 6 h. Cells were
fractionated as described in Section 2.4.5. A) Representative Western Blots of Nrf2 (57 kDa)
cytosolic and nuclear fractions are shown. Lamin A and GAPDH indicate the purity of the
nuclear and cytosolic fractions, respectively. No polyubiquitinated band (100 kDa) was
found. B) Values (mean ± s.d.) of densitometric scans of the nuclear fraction from four
independent experiments, normalized to the mean value in SF medium alone, taken as 100%.
*p<0.05 vs. 100% normalization signal.
72
Figure 18: Cadmium increases the expression of antioxidant genes. Quiescent cultures of
mesangial cells were held in SF medium or exposed to the indicated concentrations of CdCl2.
Reverse-transcriptase PCR was used to determine changes in mRNA expression of HMOX-1
and GCLC as in Methods (Section 2.14). A) Representative gel scans of PCR products with
GAPDH amplification used as a loading control. B) Values (mean ± s.d.) of densitometric
scans from four independent experiments such as depicted in A), normalized to the mean
value at 10 M CdCl2 taken as 100%. *p<0.01 vs. SF medium alone.
73
glutathionylation, RMC were treated with varying concentrations of Cd. In fact, GRx activity
was increased by 2 μM CdCl2 exposure for 6 h and remained unchanged at higher
concentrations (Table 4).
3.3 Functional consequences of actin glutathionylation
3.3.1 Glutathionylation inhibits the rate of G-actin polymerization in vitro
Pyrene-labeled actin was incubated with diamide and GSH to induce glutathionylation,
and polymerization was monitored over time. The glutathionylation status was confirmed to
be preserved after the polymerization experiments, by trichloroacetic acid precipitation
followed by immunoblot with anti-PSGG (Fig. 19A). There was a 30% decline in the initial
rate of polymerization of glutathionylated actin measured over 60 min compared to a control
(p<0.05; Fig. 19B, C).
3.3.2 Depletion of glutathione changes the redox status of the actin cytoskeleton
As glutathionylation oxidizes free thiols, Cd-induced actin gluathionylation can alter
the redox state of actin. To determine how the redox state of the actin cytoskeleton changes
with Cd treatment, 4-acetamido-4'maleimidylstilbene 2,2'-disulphonic acid (AMS) was used.
AMS binds to reduced cysteines, causing a molecular weight shift of the protein during gel
electrophoresis (124), with larger shifts indicating that the protein is in a more reduced state.
The addition of AMS to cell lysates resulted in a small upward shift in molecular weight,
without any appreciable shift between and Cd treatments (Lanes 2, 3), possibly indicating
that 2 μM CdCl2 treatment for 6 h does not drastically alter the redox state of -actin.
74
Table 4: Effect of cadmium on glutaredoxin activity. Rat mesangial cells were treated
with the indicated concentrations of CdCl2 for 6 h and harvested for measurement of total
glutaredoxin activity as described in Section 2.12. Activity is expressed relative to that in SF
controls without CdCl2, taken at 100%. Values are mean ± s.d., n=4. * Different from SF
control, p<0.05.
[CdCl2] (μM) GRx activity (%)
0
100
2 145 ± 25*
10 111 ± 25
20 140 ± 27
75
Fig
ure
19
: E
ffec
t o
f g
luta
thio
ny
lati
on
of
G-a
ctin
on
act
in p
oly
mer
iza
tio
n in
vit
ro.
Pol
ymer
izat
ion
of p
yren
e-la
bele
d G
-act
in w
as
carr
ied
out i
n v
itro
and
mon
itor
ed a
s an
incr
ease
in p
yren
e fl
uore
scen
ce, a
s de
scri
bed
in S
ecti
on 2
.13.
A) W
este
rn b
lots
of n
ativ
e G
-act
in
(lef
t lan
e) a
nd g
luta
thio
nyla
ted
G-a
ctin
(rig
ht la
ne) p
robe
d w
ith
anti-
PS
SG
ant
ibod
y. B
) Tim
e-de
pend
ence
of i
ncre
ase
in fl
uore
scen
ce
of a
ctin
(fi
lled
cir
cles
) or
glu
tath
iony
late
d ac
tin
(ope
n ci
rcle
s) u
nder
con
diti
ons
init
iati
ng p
olym
eriz
atio
n at
t =
0.
Lin
ear
regr
essi
on
anal
ysis
was
per
form
ed u
sing
Gra
phP
ad.
Val
ues
are
mea
n ±
s.d,
n =
3.
C)
Gra
ph o
f li
near
reg
ress
ion
anal
ysis
of
the
init
ial
rate
of
poly
mer
izat
ion
from
par
t B. T
he r
ate
of p
olym
eriz
atio
n of
glu
tath
iony
late
d ac
tin
is s
igni
fica
ntly
dec
reas
ed (
p<0.
05) c
ompa
red
to G
-
acti
n co
ntro
l (n=
3).
76
When cells were pre-treated with BSO to decrease actin glutathionylation, there was a large
upward shift of actin, though no difference was observed between SF medium control and
Cd treatments (Lanes 5, 6) (Fig. 20), indicating -actin is in a more reduced state when
glutathione is depleted.
3.3.3 Glutathionylation of actin precedes Cd-mediated cytoskeletal disruption
To determine if actin glutathionylation contributes to Cd-mediated cytoskeletal
disruption, cells were stained with rhodamine-conjugated phalloidin (Fig. 21A). As
previously described (70, 86), the F-actin cytoskeleton was disrupted by 10 μM CdCl2 (Fig.
21C). However, with 2 μM CdCl2 treatment for 6 h, i.e., conditions leading to increased actin
glutathionylation, the cytoskeleton remained intact (Fig. 21B). When cells were pre-treated
with BSO to decrease actin glutathionylation the cytoskeleton was unaffected (Fig. 21D).
However, when cells were co-treated with BSO and CdCl2, the cells were smaller in size and
F-actin was partially disrupted (Fig. 21E), suggesting that glutathionylation may afford
some protection against Cd-induced effects on actin filaments.
3.4 Glutathionylated proteins are located in a perinuclear region
As conditions in this study were optimized to investigate changes to actin
glutathionylation, determining the change to general protein glutathionylation may provide
additional insight into the effects of Cd on glutathionylation. Cells were stained with anti-
PSSG after treatment with Cd to induce glutathionylation and BSO to deplete GSH. The
anti-PSSG was present in a perinuclear region, and was visible in the cytosol. Despite
increases in the glutathionylation of actin by Cd, Cd treatment did not greatly alter the
77
Figure 20: Depletion of glutathione increases the number of reduced cysteines in -
actin. Cells were treated with or without 2 M CdCl2 or pre-treated with BSO as in Fig. 16.
Cell lysates were incubated with or without AMS to induce molecular weight shift as
described in Section 2.8. Equal amounts of proteins were run on a 12% non-reducing SDS-
polyacrylamide gel and immunoblotted with anti- -actin.
78
Fig
ure
21
: E
ffec
ts o
f m
od
ula
tio
n o
f g
luta
thio
ne
syn
thes
is o
n F
-act
in in
tegri
ty in
Cd
-tre
ate
d c
ells
. A
ll p
anel
s sho
w m
esan
gial
cel
ls a
t
400
x m
agni
fica
tion
, wit
h F
-act
in s
tain
ed b
y rh
odam
ine-
phal
loid
in (r
ed)
and
nucl
ei s
tain
ed w
ith
DA
PI (
blue
). C
adm
ium
trea
tmen
t was
for 6
h a
t 2
M o
r 10
M.
A) Q
uies
cent
cel
ls w
itho
ut e
ithe
r Cd2+
or B
SO
trea
tmen
t. B
) C
ells
trea
ted
wit
h 2 μ
M C
d2+ a
lone
or
C) 1
0 μ
M
Cd2+
alo
ne.
D)
Cel
ls tr
eate
d w
ith
BS
O a
lone
. E
) C
ells
trea
ted
wit
h 2 μ
M C
dCl 2
or
F)
10 μ
M C
dCl 2
fol
low
ing
BS
O p
retr
eatm
ent.
79
glutathionylation of proteins. Unexpectedly, when cells were treated with BSO to deplete
GSH, there was an increase in glutathionylation of proteins located in a perinuclear region.
Furthermore, when cells were treated with both BSO and Cd, there was an increased
presence of glutathionylated proteins localized within vacuoles (Fig. 22).
3.5 Cadmium disrupts focal contacts
The following work was completed in conjunction with Dr. Ying Liu in a follow-up
study to her original observations showing loss of actin-vinculin contacts upon Cd treatment
in RMC (87), and has now been published as Choong et al. (135).
3.5.1 Cadmium disrupts actin-vinculin contacts
Vinculin is an integral component in the assembly of the FA complex, serving to
anchor F-actin to the contacts, and it might be expected that Cd-induced disruption of the
actin filament would destabilize the complex. In preliminary studies (70), Cd treatment lead
to a loss of localization of vinculin at contact surfaces. This is demonstrated again here by
dual staining of vinculin and F-actin (Fig. 23). At 10 M CdCl2, actin filaments are less
prominent and vinculin localization to FAs is diminished (Fig. 23G-I). With 40 M Cd,
FAs as revealed by vinculin staining are all but absent (Fig. 23M-O).
3.5.2 CaMK-II is involved in Cd-dependent disruption of focal adhesions
Inhibition of CaMK-II has been shown to prevent cytoskeletal disruption and protect
against Cd-induced apoptosis (136), translocation of CaMK-II to the cytoskeleton (70), and
vinculin delocalization (87). We investigated more fully the role of CaMK-II in focal
80
Fig
ure
22:
Glu
tath
ion
yla
ted
pro
tein
s are
loca
lize
d i
n a
per
inu
clea
r r
egio
n.
All
pan
els
show
mes
angi
al c
ells
at
400
x
mag
nifi
cati
on,
wit
h A
lexa
488
flu
or-c
onju
gate
d an
ti-P
SS
G a
ntib
ody
(gre
en)
and
nucl
ei s
tain
ed w
ith D
AP
I (b
lue)
. C
adm
ium
trea
tmen
t was
for 6
h a
t 2
M.
A) U
ntre
ated
qui
esce
nt c
ells
. B) C
ells
trea
ted
wit
h C
d2+. C
) Cel
ls tr
eate
d w
ith
BS
O a
lone
. D
) Cel
ls
trea
ted
wit
h C
d2+ fo
llow
ing
BS
O p
retr
eatm
ent.
Arr
ows
indi
cate
glu
tath
iony
late
d va
cuol
es.
81
Figure 23: Cadmium-dependent loss of vinculin from focal contacts. Subconfluent
cultures of RMC were incubated in SF medium (panels A-C) or treated with CdCl2 at either
10 μM (panels G-I, J-L) or 40 μM (panels M-O, P-R) for 6 h. Cells treated with 10 μM of the
CaMK-II inhibitor, KN93, alone (panels D-F) or with KN93 in combination with Cd (panels
J-L, P-R). Cells were stained with rhodamine-conjugated phalloidin (red, right column) to
visualize F-actin or Alexa fluor 488-conjugated anti-vinculin antibody (green, middle
columns). Red and green channel overlays (left column) show localization of the vinculin-
rich contacts at the ends of actin filaments, lost upon Cd treatment (panels G, M), an effect
that was abrogated with inhibition of CaMK-II (panels J, P). Nuclei are stained blue with
DAPI in the overlays. All micrographs are 400 x magnification.
82
Figure 23
83
contact disruption. A 6 h exposure at 10 M CdCl2 was chosen as the basal condition, as it
showed extensive loss of vinculin from FAs (Fig. 23). The CaMK-II inhibitor, KN93, was
largely protective of vinculin localization to FAs (Fig. 23J-L). Even at a much higher
concentration of Cd (40 M) that caused extensive disruption of actin filaments and vinculin
localization (Fig. 23M-O), KN93 preserved both F-actin integrity and localization of
vinculin to the ends of the filaments (Fig. 23P-R).
3.6 Cadmium alters FAK localization and activation
3.6.1 Focal adhesion kinase (FAK) localization is affected by Cd
Focal adhesion kinase (FAK) is a key signaling molecule involved in the recruitment,
assembly, and consequent signaling of the FA complex (74, 76). Total FAK localization to
the anchored ends of actin filaments mirrors that of vinculin (Fig. 24A), and is likewise
disrupted by 6 h Cd treatment at 10 M (Fig. 24G) and 40 M (Fig. 24M) where KN93 is
protective at both concentrations (Fig. 24J,P).
To demonstrate further the role of F-actin in maintaining localized vinculin contacts,
cytochalasin D was used to disrupt the actin cytoskeleton. After 1 h in 500 nM cytochalasin
D, localization of FAK to the contacts was disrupted (Fig. 25E). Interestingly, the F-actin
stabilizing agent, jasplakinolide (50 nM), also resulted in loss of FAK localization to FAs
(Fig. 25H), suggesting F-actin polymerization-depolymerization dynamics are more
important than filament integrity in sustaining total FAK localization.
84
Figure 24: Localization of focal adhesion kinase (FAK). Cells were stained with
rhodamine-phalloidin (red, right column), Alexa Fluor 488-anti-FAK antibody (green,
middle column) and DAPI (blue), with the three-colour overlays are shown (left column).
Control cells in SF medium (Panels A-C), or 10 μM KN93 in SF medium alone (Panels D-F)
shows that FAK localization mirrors that of vinculin in contacts at the termini of actin
filaments, as in Fig. 22. After treatment with 10 μM CdCl2 (Panels G-I) and 40 μM CdCl2
(Panels P-R) for 6 h, less FAK is localized in contacts. In the presence of 10 μM CdCl2 and
10 μM KN93, localization of FAK to the contacts is preserved (Panels J-K). Protection by
KN93 persists even at 40 μM CdCl2 (Panels P-R). All micrographs are 400 x magnification.
85
Figure 24
86
Figure 25: Changes in actin dynamics affect FAK localization. Cells were stained with
rhodamine-phalloidin to visualize actin filaments (red, right column), Alexa Fluor 488-anti-
FAK antibody (green, middle column) and DAPI (blue), with the three-colour overlays are
shown (left column). Treatment of RMC with cytochalasin D (500 nM, panels D-F) and
jasplakinolide (50 nM, panels G-I) caused loss of FAK from the contacts compared to
treatment with SF medium alone (panels A-C). All micrographs are 400 x magnification.
87
3.6.2 Translocation of FAK to the cytoskeletal fraction is stimulated by Cd
To determine more quantitatively whether Cd favours loss or redistribution of vinculin
or FAK, we performed Western blotting of cytosolic and cytoskeletal fractions of Cd-treated
cells. There were no discernible changes in vinculin in either compartment after treatment
with up to 40 M CdCl2 (data not shown, but see Choong et al. (135)), suggesting that
disruption of the focal contacts does not result in degradation or significant
recompartmentalization of vinculin. However, with increasing Cd concentration up to 40
M, there is a concentration-dependent increase in total FAK in the cytoskeletal
compartment (Fig. 26A), indicating enhanced association of FAK with actin filaments
despite its loss from the termini of the filaments. This translocation of total FAK to the
cytoskeletal fraction was abrogated by KN93 (Fig. 26B).
3.6.3 Cadmium stimulates site-specific phosphorylation of FAK
Because inhibition of CaMK-II maintained FAK in the FA and suppressed its Cd-
dependent translocation to the cytoskeletal fraction, we examined the possible role of Cd-
dependent phosphorylation of FAK in this phenomenon. Activation of FAK results in its
autophosphorylation at Tyr-397, whereas subsequent recruitment of Src to the FA complex
achieves additional phosphorylation on Tyr-925 (74, 137). Cadmium (10 M) does not
stimulate significant phosphorylation at Tyr-397, but does induce a rapid (by 30 sec) and
sustained (up to at least 6 h) phosphorylation of Tyr-925 (Fig. 27A). KN93 is without effect
on phosphorylation at either site (Fig. 27B), indicating that the effect of CaMK-II on
preservation of the FAK content of the FA is not due to a direct effect on FAK as a substrate
for a CaMK-II-dependent kinase cascade. However, KN93 does protect against loss of
88
Figure 26: Cytoskeletal localization of FAK upon cadmium treatment. Western blots of
FAK from cytosolic (left side) and cytoskeletal (right side) fractions are indicated.
Immunoblots of -actin are included as protein loading controls. The histograms under each
set of blots are the mean ± s.d. of densitometric scans of the FAK signals from several
independent experiments, representative blots being shown. Signals are expressed relative to
the fractions from SF cells taken as 100%. A) Cells were either held in SF conditions or
treated for 6 h with 10, 20, or 40 μM CdCl2. The signals from n=4 independent experiments
are quantitated; * indicates a significant increase above SF control (p<0.05). B) Cells were
treated with 40 μM CdCl2 without (Cd 40) or with (Cd 40 + KN) KN93. Values from n=5
independent experiments show an increase in cytoskeletal localization of FAK with Cd
treatment (** p<0.01) that is abrogated by KN93 (* p<0.05).
89
Figure 26
90
Figure 27: Cadmium-dependent Tyr phosphorylation of FAK. Western blots are shown
with antibodies to total FAK and phospho-specific antibodies to FAK phosphorylated at
Tyr397 (pY-397) and Tyr925 (pY-925), with anti- -actin as a protein loading control. A)
Signal as a function time with 10 μM CdCl2 treatment. The blots are representative of two
experiments and the histogram shows the results of densitometry of the ratio pY-925 FAK
blot to total FAK, normalized to time 0 as 100%. B) Lanes from the left show SF control
RMC not treated with Cd, cells treated for 6 h with 10 μM CdCl2 (Cd), and cells treated with
10 μM CdCl2 in the presence of 10 μM KN93 (Cd + KN). The right-most lane shows cells
treated with KN93 alone, to rule out possible increases in phosphorylation brought about by
the inhibitor alone. The histogram shows the ratio of pY-925 FAK to total FAK as in Panel
A, normalized to the SF control taken as 100%.
91
Figure 27 Figure 27
92
FAK-phosphoTyr-925 from FAs (Fig. 28), indicating further that CaMK-II, while
influencing cytoskeletal assembly, does not act on FAs through FAK phosphorylation. The
actin cytoskeletal inhibitors cytochalasin D and jasplakinolide caused loss of FAK-
phosphoTyr-925 from FAs upon immunostaining (Fig. 28N, Q), which likely reflect
changes to total FAK localization (Fig. 24). However, Cd2+ significantly increases the
association of FAK-phosphoTyr-925 with a cell membrane fraction, with no change in total
FAK (Fig. 29A). KN93 attenuates the Cd-dependent increase in membrane-associated
FAK-phosphoTyr-925 (Fig. 29B).
93
Figure 28: Effect of Cd-treatment on localization of FAK-phosphoTyr925. Overlay
immunofluorescent staining (left column), rhodamine phalloidin (red, right column), Alexa
fluor 488-anti-FAK-phosphoTyr925 (green, middle column), and DAPI (blue) are shown
(400 x magnification). Control cells in SF medium (panels A-C) or cells in SF medium and
10 μM KN93 (panels D-F) show localization that is similar to total FAK. Cells treated for 6
h with 10 μM CdCl2, showing significant loss of F-actin and less prominent localization of
FAK-phosphoTyr925 to focal contacts (panels G-I). Cells treated for 6 h with 10 μM CdCl2
in the presence of 10 μM KN93, showing preservation of F-actin and FAK-phosphoTyr925
in focal contacts (panels J-L). Treatment with 500 nM cytochalasin D showed disruption of
F-actin and (Panels M-O) and treatment with 25 nM jasplakinolide stabilized F-actin (Panels
P-R), though both resulted in loss of FAK-phosphoTyr925 localization as similarly shown
in Fig. 24.
94
Figure 28
95
Figure 29: Cell membrane localization of FAK-phosphoTyr925 in Cd-treated cells. Cell
membrane fractions were prepared as described in Section 2.4.4. Western blots of the
membrane fraction were performed with antibodies to FAK and FAK-phosphoTyr925, as
well as -actin as a protein loading control. Cells were held under SF medium, or treated
with 10 μM CdCl2 in the absence (Cd) or presence (Cd + KN) of 10 μM KN93 for 6 h.
Representative blots are depicted (A) and the histogram (B) shows the results of the ratio of
FAK-phosphoTyr925 to total FAK from densitometric scans (mean ± s.d., n=3) from
independent experiments, normalized to the SF control taken as 100%. Significant
differences determined by ANOVA and Tukey’s post hoc tests are indicated by * (p<0.001).
96
4. DISCUSSION
Cadmium has pleiotropic effects on cells, including initiation of oxidative stress (47),
cytoskeletal disruption (54), mutagenesis (138), and both inhibition and induction of
apoptosis (56, 58, 139). Determining how Cd affects the cytoskeleton in mesangial cells may
provide more insight into the mechanisms of toxicity, as these changes can alter glomerular
structure eventually leading to renal failure (140). This study investigated two related
mechanisms: redox modulation of actin through protein S-glutathionylation, and focal
contact disruption through alterations in FAK and CaMK-II signaling and changes to the
actin cytoskeleton.
4.1 Cadmium and actin glutathionylation
4.1.1 Background
Redox modulation of protein sulfhydryl groups is an important regulatory mechanism
of protein function and signal transduction pathways, under both physiological and
pathological conditions (106, 119, 141, 142). In particular, protein S-glutathionylation has
been shown to regulate several proteins, including NF B (98), PTPB1 (115), and actin (97).
Actin was identified as one of the major cellular proteins that is glutathionylated (97, 107); it
is constitutively glutathionylated, with actin glutathionylation increasing under conditions of
oxidative stress, such as occurs in Friedrich’s Ataxia (142) and ischemia-reperfusion injury
(141). We have shown that Cd2+ increases actin glutathionylation in a concentration-
dependent manner that is maximal at treatment of cultured cells with approximately 2 μM
CdCl2, decreasing back to basal levels at higher concentrations (Figs. 11, 14). This is in
97
agreement with a previous study in which 5 μM Cd2+ increased actin glutathionylation in
haemocytes, the leukocytes of mussels (143).
Actin was the major protein that was glutathionylated upon Cd2+ and diamide
treatments under the conditions used in this study. Only when high concentrations of diamide
(250 μM) were used, was there an appearance of high molecular weight glutathionylated
proteins >170 kDa (Fig. 13). These proteins were not identified, but may be disulfide-
bonded protein aggregates that form under diamide-induced oxidative conditions. This may
also be the explanation for the variable levels of -actin that occur under strong oxidative
conditions, as actin may form higher molecular weight aggregates that are not resolved
during electrophoresis under non-reducing conditions. The presence of a single major protein
that is glutathionylated by Cd is unexpected as several other proteins have been found to be
glutathionylated with Cd treatment (133, 134). It has been proposed that the accessibility of
the cysteines rather than their pKa values determines whether the protein is glutathionylated
(106). Therefore, it is possible that the abundance of actin, in combination with easily
accessible cysteines, results in actin being the major glutathionylated protein in mesangial
cells under these specific conditions.
When actin is highly glutathionylated following treatments with either Cd or diamide,
a second glutathionylated band located at 42 kDa appears. These separate bands on Western
blot were indistinct on Coomassie-stained gels prior to excision and digestion for mass
spectrometry. As actin was the major protein that was identified by LC-MS following three
separate digestion methods, it is possible that these bands represent different isoforms of
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actin as , , and isoform sequences were all identified by mass spectrometry.
Alternatively, they may represent different levels of actin glutathionylation, as any of the 6
cysteines (or 5 in the case of -actin) are available for glutathionylation (80, 108).
4.1.2 Role of ROS
The precise mechanism of actin glutathionylation in vivo is still a subject of much
debate. There has been evidence to support a sulfenic acid intermediate (108) rather than a
thiol-disulfide exchange mechanism (111). Other mechanisms have also been proposed,
including formation of thiyl radicals or through nitrosylation of glutathione or free cysteine
residues (106), with many of the proposed mechanisms involving oxidative or nitrosative
changes. Because Cd2+ is pro-oxidant, we attempted to clarify the mechanism of Cd-
mediated actin glutathionylation by considering the role of ROS. Here we have shown that
Cd-dependent ROS production does not lead to increased actin glutathionylation (Fig. 15A),
bringing into question the suggestion that oxidative stress is responsible for increased protein
S-glutathionylation (141, 142).
Interestingly, actin glutathionylation is not always dependent on levels of intracellular
ROS. This is supported by the observation that treatment of A431 cells with exogenous H2O2
increased actin glutathionylation, but when cells were treated with EGF, the corresponding
rise in intracellular ROS enhanced deglutathionylation (97).Therefore, other mechanisms
may be of greater importance in Cd-induced actin glutathionylation.
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4.1.3 Role of nitrosative stress
Reactive nitrogen species (RNS) include nitric oxide (NO)-related species (NO•, NO+,
NO•-, ONOO-) that are produced by NO synthase (144). Nitric oxide is an important second
messenger, with physiological and pathophysiological functions that include endothelium-
dependent vasodilation, inflammation and sepsis (114). Increases in nitrosative stress,
through increased production of nitrosative species, is a commonly accepted mechanism of
protein S-glutathionylation (111, 140). Dailianis et al. (143) have implicated NO synthase,
necessary for the production of NO, in Cd-mediated actin glutathionylation. As this study
used the fluorescent sensor DCF, which recognizes multiple radical species (125), it is not
possible to implicate the role of nitrosative stress in this study.
The role of Cd in mediating nitrosative stress is controversial, with the majority of
work showing that Cd decreases NO production in several cell types (5, 146), though low-
level Cd exposure has also been shown to increase NO production in macrophages (147,
148). As mesangial cells are phagocytic and express NO synthase (149, 150), nitrosative
stress remains an unexplored possibility in mediating increases in actin glutathionylation.
4.1.4 Role of glutathione
Since the intracellular redox state, as defined by the GSH/GSSG ratio, may be more
important than actual ROS levels in mediating actin glutathionylation, we investigated the
changes in cellular glutathione after Cd2+ exposure. Cadmium at 0.5 and 2 μM for 6 h
increased total glutathione in RMC (Fig. 15B, Table 1). Glutathione levels were also
increased over time, in agreement with previous studies on RMC (104). However, this effect
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was not sustained at higher concentrations of Cd. The biphasic effect of Cd on total
glutathione mirrored the changes in actin glutathionylation seen over a similar range of Cd
concentrations. The lack of increase in total glutathione at higher Cd2+ concentrations may be
due to depletion of GSH at these concentrations or to preferential export of GSSG from the
cell in response to ROS accumulation (5, 151), although we did not measure the GSSG
levels in the culture medium. Cellular GSSG levels were also increased by Cd2+ but did not
affect the GSH/GSSG ratios. This effect is likely due to the rise in total glutathione levels,
which proportionally increases GSSG levels, resulting in no change in the GSH/GSSG ratio.
As a decrease in the GSH/GSSG ratio is believed to contribute to the thiol-disulfide
exchange mechanism of protein S-glutathionylation, this is not likely to be the mechanism
involved in Cd-mediated actin glutathionylation.
The ROS-independent, glutathione-dependent effect of Cd2+ on actin glutathionylation
is in agreement with in vitro studies of -actin glutathionylation that showed an increase in
glutathionylation upon treatment with GSH in the absence of the thiol-specific oxidant,
diamide (108). This occurred through formation of a sulfenic acid intermediate, which can
form under physiological conditions, accounting for a basal level of actin glutathionylation.
Formation of sulfenic acid derivatives of the reactive cysteines on actin, in combination with
increased GSH concentrations, likely contribute to the observed increases in actin
glutathionylation at low levels of Cd2+. However, at higher Cd2+ concentrations, rises in
intracellular ROS may be opposed by the release of GSH from glutathionylated actin,
resulting in the observed decrease in actin glutathionylation.
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Interestingly, Johansson and Lundberg (108) showed that -actin is preferentially
glutathionylated in the presence of GSSG. As RMC are smooth muscle-like cells that express
-actin, no distinction between the different isoforms of actin was investigated. As
immunoprecipitation experiments focused on -actin, it is not possible from our results to
determine if -actin or -actin have differing effects and rates of glutathionylation in vivo.
Further experiments are needed to help clarify the importance of glutathionylation of either
isoform in the maintenance of mesangial cell morphology.
4.1.5 Cadmium and antioxidant gene expression
Short-term exposure to Cd2+ appears to decrease the activities of catalase, superoxide
dismutase, glutathione reductase, glutathione peroxidase and glutathione-S-transferase in
vivo (152). However, when Cd2+ concentrations are elevated or exposure time is extended,
there was an increase in activities of those enzymes. Similarly, in mesangial cells, an
increase of glutathione levels, and the upregulation of GCLC and HMOX-1 genes were
observed (Figs. 15B, 17) (104).
Increases in glutathione levels and antioxidant gene expression may be linked to the
transcription factor Nrf2, which is stabilized in the presence of electrophiles or ROS (131,
153). Cadmium has also been shown to stabilize Nrf2, leading to upregulation of phase II
enzymes, including -glutamylcyteinyl synthetase (encoded by the GCLC gene) needed for
the biosynthesis of glutathione to buffer ROS (55, 154). These observations are consistent
with those presented in this study. However, Nrf2 translocation was only increased with 2
μM CdCl2 treatment, a concentration at which ROS were significantly decreased, and was
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not increased by 10 μM CdCl2 when ROS were increased, bringing into question the role of
Cd-induced ROS in this process. Stewart et al. (154) have shown that Cd decreased Nrf2
degradation through the ubiquitin-proteasomal pathway, resulting in a longer protein half-life
that promoted nuclear translocation, which may explain the lack of a ROS-dependent effect
on Nrf2 translocation and gene expression. Nevertheless, further studies are needed to
determine if Cd plays a direct role in Nrf2-dependent upregulation of antioxidant gene
expression.
4.1.6 Cadmium and glutaredoxin
Glutaredoxin, also known as thioltransferase, is a member of a family of
oxidoreductases that specifically catalyzes the reduction of glutathionylated protein mixed
disulfides (109, 155). Cadmium is a non-specific inhibitor of GRx, with an IC50 for Cd2+ of 2
μM; inhibition results in indirect increases in protein S-glutathionylation (98, 119, 120, 134).
As the effects of Cd on GRx are non-specific, and Cd is known to affect various signaling
cascades, other studies sought to implicate GRx more directly. Knockdown of GRx results in
an increase in actin glutathionylation, while overexpression of GRx leads to decreased actin
glutathionylation (97, 119, 120). While this should be considered in the present study, we
have nevertheless shown that Cd2+ increases GRx activity at 2 μM Cd2+ (Table 4), the
concentration of maximal actin glutathionylation. Although 2 μM Cd2+ was used in this
study, the resultant intracellular Cd2+ concentrations are expected to be in the picomolar
range (54), a value less than the Cd2+ IC50 of GRx. As GRx catalysis establishes an
equilibrium between glutathionylated and deglutathionylated forms of the protein, the Cd-
mediated increase in total glutathione (GSH + GSSG) concentration may shift the
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equilibrium towards a more glutathionylated state, in agreement with previous work showing
that GRx activity directly correlates with the glutathionylation status of the p65 subunit of
NF B (113). A study on pea seeds during germination showed an increase in GRx activity
and expression with Cd2+ treatment (156). Therefore, while it is possible that low Cd2+
exposure increases GRx expression in RMC, leading to increased enzymatic activity, the
consequences for actin glutathionylation warrant further investigation.
4.1.7 Role of signal transduction
A previous study investigating the effects of Cd on actin glutathionylation had
implicated the activation of the kinases PI3K and PKC, as well as adenylate cyclase (143)-
enzymes that have previously been shown to be activated by Cd2+ (5, 58). In contrast, growth
factor signaling has been implicated in deglutathionylation of actin (97). In RMC, Cd2+
rapidly activates EGFR at 5 min, and the PI3K substrate Akt at 15 min (58). Although the
present study did not examine actin glutathionylation at such early time points, EGFR
activation by Cd may have an important role in changing actin glutathionylation status at
these times (97). Therefore, a ROS-independent mechanism of actin glutathionylation by Cd
may occur through activation of different signaling cascades as well as by changes in redox
state.
4.2 Effects of Cd on the actin cytoskeleton
The actin cytoskeleton has a major role in maintaining cellular morphology,
proliferation and migration. In mesangial cells, Cd has been shown to disrupt the actin
cytoskeleton at 10 μM CdCl2 treatment for 6 h (Figs. 21, 23) and 8 h (54). This effect was
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specific when compared to other divalent cations, including Cu2+, Hg2+, Mn2+, Co2+, Ni2+,
and Zn2+ (54). However, Cd2+ increases actin polymerization in vitro (54, 85). Only when
Cd-treated cell lysates were used to monitor changes in actin was there a decrease in the rate
of polymerization and an increase in F-actin depolymerization (84), indicating a role for
cellular factors in mediating this effect.
4.2.1 Effect of actin glutathionylation on actin polymerization
Actin glutathionylation alters actin dynamics and leads to cytoskeletal disorganization
(97, 107, 119). We have shown that actin glutathionylation leads to inhibition of the rate of
polymerization in vitro (Fig. 19), an effect similarly shown in other studies (97, 107). In
addition, glutathionylated actin has several altered properties compared to unmodified actin:
increased hydrophobicity, more rapid ATP exchange, and decreased cleavage by subtilisin,
possibly suggesting that glutathionylated actin is more stable (107).
The altered actin structure with the addition of a glutathione moiety has been
previously studied to determine how glutathionylation affects actin polymerization. Previous
in vitro studies have idenfitied the site of actin glutathionylation as Cys-374, the penultimate
cysteine in the protein (97, 107). This is a highly reactive cysteine in -actin (97, 107), as is
Cys-272 (80), although no studies to date have shown that Cys-272 is glutathionylated. The
structure of the C-terminal region has a role in intermonomer interactions and maintenance
of F-actin stability. Removal of the last two or three C-terminal residues (including Cys-374)
resulted in the destabilization of actin filaments (157). Modification at Cys-374 results in
steric inhibition of addition of G-actin polymers on the C-terminal end of the monomer,
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contributing to the decrease in the observed rate of actin polymerization (107). With the
digestion methods used in this study, we were unable to identify the site(s) of actin
glutathionylation in vivo (Fig. 14); such identification might have provided more insight into
the effects of Cd-mediated actin glutathionylation. Although Cys-217 was shown in this
study to be modified to a sulfenic acid, a known precursor to glutathionylation, it is not
possible to conclude whether this cysteine residue was in fact modified in vivo, or is an
artifact of protein preparation. Thus, glutathionylation of actin may represent an important
tradeoff between actin function, through polymerization into filaments and protection of
actin against irreversible oxidative damage.
4.2.2 Effect of actin glutathionylation in vivo
Several studies have attempted to clarify the effects of actin glutathionylation in vivo.
Pastore et al. (142) have shown increased cytoskeletal disorganization in skin fibroblasts
from patients with Friedrich’s ataxia that was correlated with increased actin
glutathionylation. In contrast, we have shown that when glutathionylation is increased by 2
μM Cd2+, or conversely decreased by BSO, there is no change in the integrity of the actin
cytoskeleton in RMC (Fig. 21). Only under conditions where glutathione synthesis was
inhibited and cells were subsequently treated with 2 μM Cd2+ was the F-actin cytoskeleton
disrupted, indicating that actin glutathionylation may be a protective mechanism against Cd-
mediated cytoskeletal disruption. Additionally, Cd exposure does not appear to affect the
overall redox status of the actin cytoskeleton, as there was no difference in the shift in
molecular weight of the actin protein when treated with the thiol-binding agent AMS (Fig.
20), indicating that the amount of total actin that is glutathionylated is very small. Therefore,
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we can conclude that Cd-mediated glutathionylation of the actin cytoskeleton does not
contribute to cytoskeletal disruption, though stabilization of a small population of actin may
represent an important adaptive mechanism against Cd-toxicity.
Determining the functional effects of actin glutathionylation can provide insight into
the role of Cd-mediated actin glutathionylation. When GRx was knocked down or knocked
out to increase actin glutathionylation, the cytoskeleton was reorganized around the cell’s
periphery, and this was accompanied by membrane ruffling (120). In contrast, when GRx
was overexpressed in a neutrophil cell line, there was an increase in F-actin, a defect in actin
depolymerization, and an increase in the number of pseudopodia (119). In the same study,
when actin was mutated from Cys-374 to Ala-374 to prevent actin glutathionylation,
directional migration was impaired when compared to wildtype cells. Therefore, in addition
to protection against irreversible oxidative damage, glutathionylation of actin may have a
physiological role in mediating actin dynamics, potentially altering focal contacts, cellular
contractility and cytoskeletal integrity in mesangial cells.
4.3 Localization of glutathionylated proteins
Protein S-glutathionylation is recognized as a post-translational modification and is a
tightly regulated process, due to its specificity, sensitivity and reversibility (106, 107). To
determine if Cd increases the glutathionylation of proteins other than actin, cells were stained
with anti-PSSG antibody (Fig. 22). There was no difference in the intensity of the stain or
localization of glutathionylated proteins following Cd treatment when compared to the SF
medium control. However, when intracellular GSH was depleted, there was an intense stain
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in the perinuclear region, with the appearance of large glutathionylated vacuoles when cells
were also treated with Cd (Fig. 22C-D). These data at first appear to contradict previous
work showing a decrease in actin glutathionylation with BSO treatment (Fig. 16). However,
the increased intensity in this perinuclear region may instead indicate changes in the
localization of glutathionylated proteins to a membrane-bound organelle, possibly the
endoplasmic reticulum (ER). Alternatively, as BSO only inhibits de novo glutathione
synthesis, residual GSH may modify proteins to protect them from oxidative damage.
The ER is a highly oxidized environment, and there is a large pool of GSH present to
ensure proper protein folding under these oxidative conditions (158). In fact, approximately
50% of all ER proteins are glutathionylated (159), an interesting result, given that actin was
the major protein glutathionylated under these conditions. It is possible that glutathionylation
of proteins represents an intermediate stage to protect free cysteine residues prior to the
formation of native disulfide bonds in the ER (158). The increased presence of
glutathionylated proteins in a perinuclear region (Fig. 22C) when de novo glutathione
synthesis was inhibited may be due to preferential import of intracellular GSH to the ER
under oxidative stress conditions, or to increased retention of misfolded proteins, that are
subsequently glutathionylated, within the ER. When cells are also treated with Cd, this may
exacerbate toxicity, resulting in ER fragmentation as shown by the increased presence of
glutathionylated vacuoles (Fig. 22D). Fragmentation of the ER has been shown to result
from increases in oxidative stress (160) or a rise in intracellular Ca2+ (161), both cellular
characteristics that are affected by inhibition of glutathione synthesis and the presence of
Cd2+ (5, 162). This observation may indicate an adaptation to Cd exposure, or an alternative
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cell death pathway, that combines oxidative stress and Cd, with a potential link to Ca2+
signaling.
4.4 Cadmium and focal adhesions
4.4.1 Cadmium effects on focal adhesion localization
Focal adhesions have an important role in mediating cellular contact to the
extracellular matrix environment, preventing cellular detachment-induced cell death
(anoikis), mechanosensing of the surrounding environment, and alterations in signal
transduction (73, 163). Focal adhesions are important in maintaining mesangial cell
morphology, allowing for cell attachment, contraction, survival and proliferation (164).
Previously, Templeton and Liu (87) observed that Cd caused a loss of localization of
vinculin to focal contacts, an effect that was dependent on CaMK-II. In this study, we further
investigated the mechanism of this effect. Cadmium disrupts focal contacts by disrupting the
localization of the structural protein vinculin and the tyrosine kinase FAK. This phenomenon
appears to be dependent on actin dynamics, because disrupting F-actin using cytochalasin D
or stabilizing it with jasplakinolide both resulted in disruption of focal contacts (Figs. 23-25).
This is in agreement with previous data showing a change in focal contacts with the addition
of cytochalasin B and mycalolide B, both agents that disrupt the F-actin cytoskeleton (165).
Moreover, treatment of cells with 1 μM Cd2+ caused a significant decrease in cell surface
area, indicating a loss of FAs (34). Because Cd disrupts normal actin dynamics leading to
increased G-actin and a more depolymerized state (54, 84), this depolymerization may be a
potential mechanism for Cd-mediated disruption of FAs.
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Cadmium activates CaMK-II and increases the association of both CaMK-II and
cleaved gelsolin with actin, which promotes cytoskeletal disruption (70). CaMK-II is
autophosphorylated upon binding of Ca2+/CaM, generating an active CaMK-II species
independent of Ca2+/CaM (60, 166). Inhibition of CaMK-II with KN93 maintained
cytoskeletal integrity and focal contacts upon Cd treatment (Figs. 22-23). Chen et al. (166)
have shown that CaMK-II inhibition prevents the decrease in cell spreading associated with
Cd2+ treatment in neuronal cells, an observation also in agreement with Szabo et al. (167)
who showed an increase in cell spreading and focal contacts in fibroblasts upon CaMK-II
inhibition. As CaMK-II has been implicated in mediating cytoskeletal disruption in
mesangial cells, inhibition by KN93 could abrogate this effect by restoring normal actin
dynamics.
4.4.2 Cadmium and CaMK-II alter FAK localization
Focal adhesion kinase has also been shown to regulate focal contacts and the actin
cytoskeleton, as FAK-null cells have larger focal contacts, increased Rho kinase activity,
and more cortical actin distribution (71, 137). To study the mechanism of the effects of Cd
and CaMK-II on FA integrity, we investigated the changes in FAK levels and its localization
in mesangial cells. Previous work by Siu et al. (168) showed a decrease in total FAK levels
with Cd treatment, and this was associated with a disruption of the rat blood-testis barrier.
However, in whole cell lysates, we found that FAK levels were unchanged by Cd treatment
(Fig. 27A). This discrepancy may be due to differences in tissue type and model systems
between the studies.
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We next sought to determine how subcellular localization of FAK is affected by Cd.
Cadmium stimulates FAK translocation to the cytoskeleton in a concentration-dependent
manner, an effect that is dependent on CaMK-II (Fig. 26). This result apparently contradicts
the current paradigm of focal adhesion formation, as previous studies have shown that focal
contact formation results in FAK-actin association (169). However, in activated platelets,
FAK becomes associated with the cytoskeleton upon aggregation, resulting in FAK cleavage
and loss of kinase activity (170). Given that FAK cleavage was not detected in the present
study, this is unlikely to be an effect of Cd-mediated cytoskeletal translocation. Whether the
cytoskeletal translocation of FAK in mesangial cells by Cd has a functional downstream
effect or is a consequential effect associated with Cd-mediated cell death remains to be
investigated.
4.4.3 Cadmium and CaMK-II increase FAK phosphorylation
Cadmium increases the phosphorylation of several other kinases in mesangial cells,
including p38, Erk1/2, Src, and CaMK-II (57, 58). Focal adhesion kinase becomes
autophosphorylated at Tyr-397, resulting in recruitment of Src which subsequently
phosphorylates other tyrosine residues on FAK (74, 137). Under basal conditions, FAK was
constitutively autophosphorylated at Tyr-397, which remained unchanged with Cd treatment
(Fig. 27). This result is in agreement with other studies that have shown constitutive
phosphorylation of Tyr-397 in several cell types (171, 172). In contrast, Cd causes a rapid
increase in phosphorylation of Tyr-925 beginning at 30 s and remaining sustained over time,
an effect that is independent of CaMK-II (Fig. 27). This is believed to occur by inhibition of
phosphatases through oxidative stress (59), though the rapid rise in phosphorylation likely
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involves activation of another signaling cascade as Cd alone would seem to be insufficient to
induce oxidative stress at such short time periods. Interestingly, Tyr-925 is usually
unphosphorylated in vivo, as mutating Tyr-925 had no effect on the overall tyrosine
phosphorylation status of FAK (172). Additionally, phosphorylation of FAK at Tyr-925
leads to increased FA turnover (173). Therefore, it is likely that the loss of the balance
between phosphorylated and unphosphorylated FAK in mesangial cells treated with Cd
contributes to the observed disruption of FAs.
Focal adhesion kinase is found primarily in the cytosol. However, it must translocate
to the plasma membrane to mediate focal contact assembly and disassembly in response to
changes in extracellular signaling (165). Phosphorylation of FAK at Tyr-925 has been
shown to be enhanced in a membrane-targeted FAK fusion protein which is primarily
excluded from FAs (172). In agreement with this, we found that Cd increases the presence
of FAK-phosphoTyr-925 in the membrane fraction, while not affecting total FAK levels
(Fig. 29). Interestingly, FAK-phosphoTyr-925 in the membrane fraction was decreased by
inhibition of CaMK-II, although CaMK-II inhibition has no effect on total FAK
phosphorylation. This effect may be due to a Cd-mediated CaMK-II-dependent influence on
actin dynamics that facilitates release of FAK-phosphoTyr-925 from FAs, resulting in its
localization to other membrane domains.
4.4.4 Differences between cadmium and other toxic metals
Other toxic metals, such as arsenic and lead, have also been shown to cause changes in
focal contacts. The toxic mechanisms of arsenic are somewhat similar to those of Cd, in that
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both poison thiol groups and impose an oxidative stress on cells (174, 175). Although ROS
have been implicated in FAK regulation, this does not always appear to be an important
factor. Sodium arsenite, a toxic species of arsenic, decreased FAK phosphorylation, resulting
in decreased cell migration with FA and cytoskeletal disruption in myoblasts (174).
Similarly, lead caused a decrease in tyrosine phosphorylation of FAK, while total FAK
levels were unchanged (175). This apparent difference between the effects on FAK
phosphorylation of Cd versus arsenic and lead may occur due to an effect of Cd2+ on Ca2+
signaling. Shinohara et al. (176) have shown that CaM, but not CaMK-II, is required for
tyrosine phosphorylation of FAK. As our study did not investigate changes to FAK tyrosine
phosphorylation upon CaM antagonism, it is possible CaM activation is responsible for
increased FAK tyrosine phosphorylation by Cd. Furthermore, a local rise in intracellular
Ca2+ has been implicated in increased focal adhesion disassembly (177), potentially
indicating that a Cd-induced rise in intracellular Ca2+, in combination with activation of CaM
and CaMK-II, mediates the Cd-dependent disruption of FAs.
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5. SUMMARY & SIGNIFICANCE
This study examines the effects of Cd on redox regulation of actin and FA disruption,
with a major focus on the actin cytoskeleton. We have shown that at low-level ( 2 μM) Cd2+
exposure for 6 h, there was an increase in actin glutathionylation. This process was
independent of ROS induction, but dependent on total glutathione levels, indicating that
glutathione metabolism rather than the direct effects of ROS on thiol groups mediates actin
glutathionylation. The rise in GSH levels may be linked to the upregulation of the enzyme
needed for the biosynthesis of GSH through a Nrf2-dependent pathway. This study also
highlights the importance total GSH levels in mediating
glutathionylation/deglutathionylation by the enzyme GRx. In contrast to several studies that
have shown Cd-mediated inhibition of GRx contributes to increased protein
glutathionylation (97, 119, 133), we have shown the opposite; that Cd-mediated increases in
GRx activity are directly correlated with increases in actin glutathionylation. Although actin
glutathionylation decreased the rate of G-actin polymerization in vitro, this effect was not
seen in vivo, and instead it may be a protective mechanism against Cd-mediated cytoskeletal
disruption.
Focal adhesions link the actin cytoskeleton to the extracellular matrix environment.
Because Cd can disrupt the actin cytoskeleton at higher concentrations ( 10 μM), we
investigated the effects of Cd on FAs. Cadmium caused a loss of vinculin and FAK from
FAs which may be related to actin dynamics, as both disruption and stabilization of actin
filaments disrupt the FAs. Cadmium also increased the translocation of FAK to a
cytoskeletal fraction and increased FAK-phosphoTyr-925 association with a membrane
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fraction, and both these effects were dependent upon activation of CaMK-II. CaMK-II-
dependent disruption of the actin cytoskeleton may allow for diffusion of FAK out of FAs,
which may be facilitated by a Cd-dependent phosphorylation of FAK at Tyr-925 that results
in sequestration of the FAK-phosphoTyr-925 in another membrane domain. The effects of
Cd on FAs may represent a previously unidentified target of toxicity that requires further
investigation.
The majority of biomarkers, such as 2-microglobulin and NAG, are indicative of
renal damage brought upon by acute or chronic exposure to Cd (2, 3). Therefore, it is
important to develop biomarkers for low-level Cd exposure in order to prevent the onset of
irreverisible renal damage in humans. Actin glutathionylation may be a good candidate
biomarker, as it occurs upon low-level exposure to Cd and does not indicate any major
change in cellular function.
In conclusion, this study has shown that low-level Cd exposure results in increases in
actin glutathionylation and glutathione in mesangial cells which may protect the
cytoskeleton, whereas at higher concentrations of Cd, disruption of FAs is linked to changes
in the actin cytoskeleton and increased phosphorylation of FAK within a membrane fraction.
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6. FUTURE DIRECTIONS
This study suggests several possible lines of future investigation. As actin is the major
protein that is glutathionylated by Cd, further clarification of the functional effects of actin
glutathionylation is warranted. These effects may be studied by expressing a fluorescently-
tagged Cys-374 mutant of actin, which cannot be glutathionylated, in vivo and determining
how this mutant responds to Cd exposure. Although there is potential for confounding effects
due to the presence of native actin, Sakai et al. (119) have shown that a Cys-374 mutated
actin had a dominant negative effect. Some parameters that would be of interest include actin
localization, presence of F-actin, F-actin structure, and cellular contractility. Additionally,
the role of GRx in this process should be more thoroughly investigated by knockdown and
overexpression of GRx. Further studies can also determine the effect of Cd on GRx activity
through increased gene expression or decreased GRx degradation.
Determining if, and under what conditions, other proteins are glutationylated by Cd
may provide additional insight into the mechanisms of glutathionylation and its role in
protein function, cell survival, or apoptosis. These proteins may be glutathionylated under
different conditions from those used in this study, as proteins have different rates of
glutathionylation under different conditions (106). The formation of glutathionylated
vacuoles under conditions of both GSH depletion and Cd treatment should be further
explored to determine molecular mechanisms and downstream consequences of this effect,
such as its effect on cell death or survival pathways (58). The vacuoles may indicate ER
fragmentation which can be confirmed by colocalization with an ER marker, such as protein
disulfide isomerase (159).
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Another avenue of investigation should be the role of Nrf2 in mediating antioxidant
gene expression and actin glutathionylation. Electrophoretic mobility shift assays or
luciferase assays could be used to confirm the role of Nrf2 in the increase of antioxidant gene
expression by Cd shown in this study. To investigate the role of Nrf2 in mediating actin
glutathionylation through increased -glutamycysteinyl synthetase expression, knockdown of
Nrf2 could implicate its role in this process. Additional experiments could be performed to
determine the upstream mechanisms that stabilize Nrf2, through ROS induction or
alternative pathways. As Nrf2 binds to the inhibitory protein Keap1 in the absence of
electrophilic stimuli (130), the effects of Cd on Keap1 should also be investigated. An added
level of complexity is that the Nrf2-Keap1 inhibitory complex has been previously shown to
bind to the actin cytoskeleton (178), which may also contribute to Nrf2 stabilization.
Finally, a follow-up study to investigate the exact role of Cd in mediating CaMK-II
and FAK signaling will contribute to the knowledge about these signaling pathways. Since
CaMK-II is a Ser/Thr kinase, it does not have a direct role in mediating FAK
phosphorylation. Nevertheless, CaMK-II affects phosphorylation of FAK-Tyr-925 in a
membrane fraction, which may have an important role in loss of focal contacts. This may be
linked to Cd activation of CaMK-II and Src kinases (74) that increases FAK-phosphoTyr-
925 or its localization to a membrane fraction. Mutation of Tyr-925 in FAK could determine
the functional consequences of this Cd-dependent effect. As CaMK-II and FAK have been
linked to survival and pro-apoptotic signals (58, 71), determining the factor(s) that mediate
cross-talk between both kinases may further elucidate mechanisms of Cd toxicity.
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7. REFERENCES
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