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Originally published In Press as doi:10.1074/jbc.M610863200 on February 26, 2007

J. Biol. Chem., Vol. 282, Issue 17, 12467-12474, April 27, 2007
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Glutaredoxin Regulates Nuclear Factor {kappa}-B and Intercellular Adhesion Molecule in Müller Cells

MODEL OF DIABETIC RETINOPATHY*

Melissa D. Shelton{ddagger}, Timothy S. Kern{ddagger}§, and John J. Mieyal{ddagger}1

From the Departments of {ddagger}Pharmacology and §Medicine, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106

Received for publication, November 24, 2006 , and in revised form, February 23, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Reversible S-glutathionylation of proteins is a focal point of redox signaling and cellular defense against oxidative stress. This post-translational modification alters protein function, and its reversal (deglutathionylation) is catalyzed specifically and efficiently by glutaredoxin (GRx, thioltransferase), a thioldisulfide oxidoreductase. We hypothesized that changes in glutaredoxin might be important in the development of diabetic retinopathy, a condition characterized by oxidative stress. Indeed, GRx protein and activity were increased in retinal homogenates from streptozotocin-diabetic rats. Also, incubation of rat retinal Müller cells (rMC-1) in normal glucose (5 mM) or diabetic-like glucose (25 mM) medium led to selective upregulation of GRx in contrast to thioredoxin, the other thioldisulfide oxidoreductase system. Under analogous conditions, NF-{kappa}B (p50-p65) translocated to the nucleus, and expression of ICAM-1 (intercellular adhesion molecule-1), a transcriptional product of NF-{kappa}B, increased. Proinflammatory ICAM-1 is increased in diabetic retinae, and it is implicated in pathogenesis of retinopathy. To evaluate the role of GRx in mediating these changes, intracellular GRx content and activity in rMC-1 cells were increased independently under normal glucose via infection with an adenoviral GRx1 construct (Ad-GRx). rMC-1 cells exhibited adenovirus concentration-dependent increases in GRx and corresponding increases in NF-{kappa}B nuclear translocation, NF-{kappa}B luciferase reporter activity, and ICAM-1 expression. Blocking the increase in GRx1 via small interfering RNA in rMC-1 cells in high glucose prevented the increased ICAM-1 expression. These data suggest that redox regulation by glutaredoxin in retinal glial cells is perturbed by hyperglycemia, leading to NF-{kappa}B activation and a pro-inflammatory response. Thus, GRx may represent a novel therapeutic target to inhibit diabetic retinopathy.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Reactive oxygen species are redox signals essential to physiological processes, but they can disrupt normal redox signaling, damage cell components, and irreversibly oxidize cellular proteins when produced in excess (1, 2). Thus, oxidative signals promote protein modifications on redox-sensitive cysteine sulfhydryls in a continuum of oxidative states from redox-activated signal transduction to oxidative stress-induced molecular damage (3). Reversible post-translational modifications such as protein-sulfenic acids (protein-SOH),2 S-nitrosylated proteins (protein-SNO), and S-glutathionylated proteins (protein-SSG) are thought to protect against irreversible oxidation (3, 4), and S-glutathionylation is likely the predominant physiological sulfhydryl modification due to the abundance of cellular glutathione (5) and the ready conversion of cys-SNO and cys-SOH moieties to cys-SSG (6). S-Glutathionylation results in protein-specific functional changes (activation or deactivation), important in regulation of signaling mediators involved in cellular processes. For example, S-glutathionylation activates Ras and leads to downstream phosphorylation of Akt and p38, increased protein synthesis, and cell proliferation (7). In contrast, glutathionylation inactivates PTP1B phosphatase, which consequently amplifies the effects of kinase activation in associated signaling pathways (8, 9). Reversal of S-glutathionylation, i.e. deglutathionylation, is catalyzed specifically and efficiently by the thiol-disulfide oxidoreductase enzyme glutaredoxin (GRx) (10–12). This characteristic has led to the use of GRx as a diagnostic tool, whereby reversal of protein functional changes by GRx, both in biochemical analysis and in cell culture studies, is interpreted as regulation by S-glutathionylation (13–16). Accordingly, alterations in the activity of GRx in cells in the context of diseases that involve oxidative stress would be expected to perturb sulfhydryl homeostasis and redox signaling. With this in mind, we were interested in the status of redox regulation in diabetes, where the hyperglycemia produces a state of chronic oxidative stress leading to a variety of complications such as retinopathy.

Retinas from diabetic animals show numerous abnormalities consistent with oxidative stress (17, 18), and the retinopathy is being interpreted by many investigators as an inflammatory disease (19–21). Increased adhesion of leukocytes to the wall of retinal vessels has been linked to increased vascular permeability and capillary cell death, and each of these has been linked to increased expression of intercellular adhesion molecule-1 (ICAM-1) in retinas of diabetic animals (19, 22). Retinal glial (Müller) cells from diabetic rats also display increased ICAM gene expression (21), suggesting a contribution to the inflammatory response. Müller cells play an essential support role in the retina, interacting with nearly all the other retinal cells, spanning 70% of the width of the retina, acting as metabolic regulators, and storing most of the retinal glutathione content (23–25). Thus, we chose an immortalized rat retinal glial cell line (rMC-1) as the model system for the current study.

ICAM-1 expression is regulated by NF-{kappa}B, a redox-sensitive transcription factor composed of NF-{kappa}B/Rel family protein dimers (p50/p105, p65 (RelA), c-Rel, p52/p100, and RelB) (26, 27), where p50-p65 is the classical and predominant active dimer in most cell types. NF-{kappa}B is activated in retinal glial cells, pericytes, and endothelial cells in diabetes (this study and Refs. 20 and 28, respectively), suggesting that oxidative signals within cells affect transcriptional activity of NF-{kappa}B. In this regard, many proteins that are implicated in the pathway of regulation of NF-{kappa}B activity have been reported to have their functions altered by S-glutathionylation in different contexts. These proteins include p50, p65, IKK, Akt, MEKK-1, and NF-{kappa}B-inducing kinase (29–34, respectively).

The current study was designed to test the hypothesis that the oxidative stress associated with high glucose alters glutaredoxin-regulated redox signaling in retinal glial cells. Here we report that high glucose induces glutaredoxin in retinal Müller cells, with concomitant NF-{kappa}B activation and increased ICAM-1 expression. Overexpression of glutaredoxin in these cells in normal glucose leads to analogous increases in NF-{kappa}B activation and ICAM-1 expression. Conversely, knock-down of GRx1 in cells in high glucose prevents the induction of ICAM-1. These data suggest that redox regulation by glutaredoxin in retinal glial cells is perturbed by hyperglycemia, leading to NF-{kappa}B activation and a pro-inflammatory response.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture—Cell culture supplies were obtained from Invitrogen except where indicated. Rat retinal glial (Müller) cells (rMC-1) were a kind gift from Dr. Vijay Sarthy (Northwestern University, Chicago, IL). Cells were cultured for up to 5 days in high glucose (25 mM) or normal glucose (5 mM) in DMEM with 2% heat-inactivated fetal bovine serum (Fisher, Cellgro MT) and 2 mM glutamine with daily replacements in a humidified 37 °C incubator with 5% CO2. Glucose concentrations in the medium were monitored with a glucose oxidase kit (Pointe Scientific) to ensure that glucose consumption of the cells did not deplete the medium. HEK 293 cells were cultured in high glucose DMEM with 10% fetal bovine serum and 1% penicillin-streptomycin in a humidified 37 °C incubator with 5% CO2.

Animal Retinae—Treatment of animals was in accordance with the Association for Research in Vision and Ophthalmology Resolution on Treatment of Animals in Research and Case Western Reserve University guidelines. Animals were treated with streptozotocin to induce diabetes and with insulin to prevent wasting, as described previously (17). Retinas were excised from rats 10 weeks after induction of streptozotocin-induced diabetes or from non-diabetic control rats and homogenized in 50 mM Tris-HCl, pH 7.4, 10% Nonidet P-40, 0.25% sodium deoxycholate, and 150 mM NaCl.

GRx Activity of Rat Retinal Homogenates—Rat retinal homogenates (0.1–0.2 mg) were assayed for GRx activity via GSH-dependent release of radiolabel (as [35S]GSSG) from the prototype substrate [35S]BSA-SSG as described previously (12, 35).

Cellular Disulfide Reducing Capacity of Intact rMC-1 Cells— Müller cells (50,000–100,000 cells/60-mm dish) were cultured in normal or high glucose medium for 3–5 days and assayed for disulfide reducing capacity (36) with two different cell permeable disulfides. Reduction of bis-(2-hydroxyethyl) disulfide (HEDS) is attributable to total reducing capacity (thioredoxin (TRx) and GRx systems), and lipoate reduction is selective to the TRx system (36). Cells were incubated in 5 ml of medium containing 5 mM HEDS or 5 mM lipoate. Aliquots of the medium were taken at 0, 5, 10, 20, 30, 45, and 60 min and added to separate wells of a 96-well plate containing dithio-bis(2-nitrobenzoic acid (1 mM final) in each well, and absorbance change at 405 nm for each well was monitored in a plate reader. The functional extinction coefficient (6.1 mM–1) was determined from a standard curve for GSH using the dithio-bis(2-nitrobenzoic acid assay with 0.2 ml of total volume in each well and reading absorbance values with a Molecular Devices THERMOmaxTM microplate reader. Data were analyzed with the Molecular Devices SOFTmax® version 2.3.

Propagation and Titration of Adenoviral Constructs in HEK 293 Cells—Adenoviral vector containing the GRx1 cDNA construct (Ad-GRx) and empty vector control construct (Ad-Empty) were created with the CRE-Lox recombination system in collaboration with Dr. Yong Lee (University of Pittsburgh, PA) (37). Subsequently, the adenovirus was propagated and titrated in HEK 293 cells. For propagation, HEK 293 cells were infected with 5 plaque-forming units/cell of adenovirus (Ad-GRx or Ad-Empty). Medium and cells were collected when the cells lifted off the plate (usually after 3–6 days). The cells were lysed via freeze-thaw three times, and then virus was collected by centrifugation at 2,300 x g for 10 min at 4 °C. For adenoviral titration, HEK 293 cells were infected with serial dilutions (0–1014) of stock virus, overlaid with 0.9% low melting point agarose, and incubated until plaques stopped forming (usually 5–7 days). Virus concentration (plaque-forming units/ml) was calculated by dividing the number of plaques by the volume of adenovirus used to infect the cells.

Adenoviral Expression of GRx1 in Müller (rMC-1) Cells— Müller cells (500,000 cells/100-mm dishes) were grown in normal glucose medium for 2 days and infected with various multiplicities of infections (m.o.i. 0–80) of Ad-GRx or Ad-Empty in 1 ml of serum-free DMEM for 1 h. Cells were cultured for 2 days in normal glucose medium and collected in 1% Nonidet P-40 lysis buffer (50 mM Tris, pH 8, 1% Nonidet P-40, and 150 mM NaCl).

Inhibition of Nuclear Translocation of NF-{kappa}B via sn50 in Adenoviral Overexpressing rMC-1 Cells—Müller cells (500,000 cells/100-mm dishes) were grown in normal glucose medium for 2 days, infected with m.o.i. 10 of Ad-GRx in 1 ml of serum-free DMEM for 1 h in the absence or presence of sn50 inhibitor (BIOMOL). Cells were subsequently cultured in normal glucose medium in the absence or presence of sn50 inhibitor and collected in 1% Nonidet P-40 lysis buffer. Control cultures (uninfected and m.o.i. 10 of Ad-Empty) were incubated in parallel in the absence of sn50.

Immunoblotting—Müller cells were collected, lysed in 1% Nonidet P-40 lysis buffer, and centrifuged at 1,500 x g for 5 min. Cleared supernatants were assayed for protein content with the microbicinchoninic acid method (BCA) (Pierce), according to the manufacturer's protocol. Samples were mixed 4:1 with 4x SDS sample buffer (0.5 M Tris-HCl, pH 6.8, 20% glycerol, 10% SDS (w/v), 1% bromphenol blue, and 20 mM dithiothreitol), heated for 15 min at 95 °C, separated by 12% SDS-PAGE, and transferred to Immobilon P membranes (Millipore, Tokyo). Membranes were immunoprobed with the appropriate antibodies: anti-p50 (1:1,000) (ab7971) (AbCam, Cambridge, MA); anti-p65 (1:3,000) (sc372) (Santa Cruz Biotechnology, Santa Cruz, CA); anti-ICAM-1 (1:500) (R&D Systems, Minneapolis, MN); anti-GAPDH (1:10,000) (Chemicon International Inc., Temecula, CA); anti-actin (1:30,000) (Sigma); anti-yy1 (1:1,000) (Santa Cruz Biotechnology); and anti-GRx1 (1:1,000) (generated and purified via an adaptation of the McKinney and Parkinson caprylic acid method (38). Peroxidase-conjugated secondary goat anti-rabbit or anti-mouse antibodies (1:10,000) (Jackson ImmunoResearch Laboratories, West Grove, PA). were used, and Western Lightning chemiluminescence reagent Plus (PerkinElmer Life Sciences) was used according to the manufacturer's protocol. Band intensities were quantified using a Bio-Rad calibrated imaging densitometer GS-710 with Bio-Rad Quantity One software version 4.1.1. Changes in band intensity are reported as ratios relative to loading controls.

Nuclear Extraction—Müller (rMC-1) cells were collected in 1 ml of phosphate-buffered saline, centrifuged for 3 min at 800 x g, and lysed in 300 µl of low salt buffer (20 mM HEPES, pH 7.6, 20% glycerol, 10 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, and 0.1% Triton X-100) for 20 min. Centrifugation at 800 x g for 3 min yielded a cytosolic supernatant. The nuclear pellet was washed twice in phosphate-buffered saline, incubated in 80 µl of high salt buffer (10 mM HEPES, pH 7.6, 10% glycerol, 0.5 M NaCl, 0.7 mM MgCl2, 0.1 mM EDTA, and 0.05% Triton X-100) for 30 min 4 °C, and centrifuged at 16,000 x g for 15 min in 4 °C. Protein content was determined via BCA assay.

NF-{kappa}B Luciferase Reporter Assay—Müller (rMC-1) cells (50,000 cells/well of a 6-well dish) were grown for 2 days and co-transfected for 10–12 h with 1 µg of NF-{kappa}B luciferase (5x) plasmid (Stratagene) and 0.1 µg of Renilla plasmid (Promega) as a control reporter according to the LipofectamineTM reagent protocol (Invitrogen). The binding element for the NF-{kappa}B luciferase plasmid is derived from the consensus NF-{kappa}B binding sequence and contains five repeats of (TGGGACTTTCCGC). 2–4 h after the end of the transfection, cells were infected with Ad-GRx or Ad-Empty for 1 h and collected 8 h later in 1x passive lysis buffer (Promega). NF-{kappa}B activity was assayed via the Dual-Luciferase® reporter assay system (Promega, Madison WI) with the Molecular Devices Lmax luminometer and SOFTmax PRO software. Assay readouts were reported as ratios of firefly luciferase to Renilla luciferase.


Figure 1
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  		FIGURE 1.
Glutaredoxin activity of retinal homogenates from hyperglycemic rats. Using the [35S]BSA-SSG substrate, GRx activity of diabetic retinal homogenate was 2.6 ± 0.1 nmol/min/mg of cellular protein when compared with 1.1 ± 0.1 nmol/min/mg of cellular protein in non-diabetic control rat retinae. (n = 3). *, p < 0.001. See "Experimental Procedures" for details.

 
GRx1 Knockdown via siRNA—Müller (rMC-1) cells (30,000–40,000 cells/well of a 6-well dish) were grown in high glucose (25 mM) for 1 day, transfected with Dharmacon ON-TARGETplus SMARTpool siRNA targeted to rat GRx1 or ON-TARGETplus siCONTROL non-targeting pool siRNA according to manufacturer's instruction for Oligofectamine (Invitrogen), and grown in high glucose for three subsequent days. Cells were lysed in Nonidet P-40 lysis buffer for immunoblotting with antibodies directed toward GRx1, ICAM-1, actin, and GAPDH.

Statistical Analysis—All values and graphs report means ± S.E. (S.E.). Statistical analysis was determined via the Student's t test. Differences displaying p values ≤0.05 were considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
GRx Is Induced in Diabetic Rat Retinae—Retinae from non-diabetic rats and rats diabetic for 10 weeks were homogenized and assayed for GRx activity to assess whether the diabetic condition altered global glutaredoxin activity. In fact, the GRx activity of diabetic rat retinae was increased ~2.5-fold relative to control (Fig. 1). These data reflect the collective change in GRx activity for all cells comprising the retina, although the extent of change in any of the individual cell types is not known. We reasoned that retinal Müller cells comprise a large portion of the total retina and influence the vitality of neighboring cells. Therefore we conducted further studies with this well known in vitro model.

High Glucose Selectively Induces GRx in Müller Cells—To elucidate changes in sulfhydryl homeostasis in the retinal glial (Müller) cells in response to high glucose, the rat Müller cell line (rMC-1) was used, and the cells were cultured under conditions mimicking diabetes (i.e. 25 mM glucose in the medium). The cells were assayed for cellular disulfide reducing capacity with two different disulfide substrates to distinguish the relative contributions of the glutaredoxin and TRx systems in the intact cells (36). Thus, the disulfide reducing capacity of cells is attributable to the two cytosolic thiol-disulfide oxidoreductase enzyme systems, i.e. GRx and TRx and their corresponding reductase systems (GSH, glutathione disulfide reductase, NADPH) and (thioredoxin reductase, NADPH), respectively. Reduction of HEDS is attributable to total reducing capacity (GRx and TRx systems), and lipoate reduction is attributable to the TRx system alone. Therefore changes in the capacity of the respective systems can be distinguished. After 3–5 days of high glucose treatment, the activity of the TRx system (rate of lipoate reduction) of Müller cells was not significantly changed (Fig. 2), and TRx protein was unchanged in Western blot analysis (data not shown). However, the total reducing capacity (rate of HEDS reduction) was increased by nearly 2-fold (Fig. 2), indicating that the change in total disulfide reducing capacity is due to a selective increase in activity of the GRx system. Since this result suggests a selective induction of GRx in the Müller cells in response to high glucose (25 mMD-glucose), we examined the content of GRx1 directly. Consistent with high glucose-induced GRx activity, GRx1 protein expression was increased more than 2-fold according to Western blot analysis of lysates from glucose-treated Müller cells (Fig. 3, A and B). In separate experiments, it was confirmed that no change in GRx1 content occurred when cells were incubated in 25 mML-glucose as a control for increased osmolarity (data not shown).


Figure 2
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  		FIGURE 2.
Effects of high glucose (25 mM) on the disulfide reducing capacity of retinal Müller (rMC-1) cells. The cellular reduction rate of HEDS was 11.6 ± 1.2 nmol thiol/mg of protein/min in normal glucose-treated cells and 19.0 ± 2.5 nmol thiol/mg of protein/min in high glucose-treated cells. The cellular reduction rate of lipoate gave rise to 4.2 ± 0.5 nmol thiol/mg of protein/min under normal glucose and 4.7 ± 0.8 nmol thiol/mg of protein/min under high glucose medium. (n = 10) *, p < 0.02. See "Experimental Procedures" for details.

 
High Glucose Leads to Up-regulation of ICAM-1 in Müller Cells—Analysis of lysates of rMC-1 Müller cells treated with high glucose for 5 days revealed a 3-fold increase in ICAM-1 expression (Fig. 3, C and D).

High Glucose Leads to Increased Nuclear Translocation of NF-{kappa}B (p50 and p65) in Müller Cells—To test whether the observed increase in ICAM-1 production in Müller cells is mediated by NF-{kappa}B, we measured changes in nuclear NF-{kappa}B after incubation in high glucose. The p50 and p65 subunits of NF-{kappa}B in the nucleus increased by about 2–3-fold, whereas cytoplasmic contents were essentially unchanged (Fig. 4, A–C). The concomitant increase in GRx1, NF-{kappa}B translocation, and ICAM-1 expression in response to high glucose suggested that GRx1 might be directly responsible for regulating NF-{kappa}B activity and ICAM-1 expression in Müller cells. Therefore we tested this hypothesis directly.

Infection of Müller Cells in Normal Glucose with Adenovirus Containing cDNA for GRx1 Leads to Increased GRx1 Content and Activity and Concomitant Increase in ICAM-1 Production— We selectively increased GRx activity in Müller cells grown in normal glucose conditions (5 mM) by overexpressing GRx1 using adenovirus containing GRx1 cDNA (Ad-GRx). Infection of the cells with empty vector (Ad-Empty) served as control. Ad-GRx increased cellular GRx1 content and activity in an m.o.i.-dependent fashion (Figs. 5, A and B, and 6, respectively). GRx activity correlated well to GRx protein content at most m.o.i., but cells infected with Ad-GRx at m.o.i. 40 showed an unexplained high amount of GRx1 protein content. Ad-Empty had no effect on either GRx content or activity (Figs. 5, A and B, and 6, respectively). Western blot analysis of lysates from the Ad-GRx infected rMC-1 cells in normal glucose, and not cells infected with empty vector, showed m.o.i.-dependent increases in production of ICAM-1(Fig. 5, C and D). These results indicate that GRx1 regulates ICAM-1 production in Müller cells (see "Discussion"). Similar results were observed in enzyme-linked immunosorbent assays, i.e. Ad-GRx infection of Müller cells increased ICAM-1 expression (data not shown).


Figure 3
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  		FIGURE 3.
Effects of high glucose (25 mM) on GRx1 and ICAM-1 proteins in rMC-1 Müller cells. After 5 days in normal (5 mM) or high (25 mM) glucose medium, Müller cells were lysed and immunoprobed with anti-ICAM-1 (1:500) or anti-GRx1 (1:1,000). Anti-GAPDH (1:10,000) was used as a loading control. High glucose induced GRx1 expression by 2.3-fold (± 0.3) (A and B) and ICAM expression by 3-fold (± 0.7) (C and D)(n = 5). *, p < 0.05. See "Experimental Procedures" for details.

 


Figure 4
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  		FIGURE 4.
Nuclear localization of the NF-{kappa}B subunits p50 and p65 in high glucose-treated rMC-1 Müller cells. After 5 days in normal (5 mM) or high (25 mM) glucose medium, Müller cells were separated into cytoplasmic and nuclear fractions and immunoprobed for anti-p50 (1:1,000) (A and B), anti-p65 (1:3,000) (A and C). Loading controls were actin (1:30,000) in the cytoplasm and YYl (1:1,000) in the nucleus. Nuclear p50 and p65 were increased 2.8-fold (± 0.6) and 1.8-fold (± 0.2), respectively, and cytoplasmic p50 and p65 were not significantly changed (n = 5). *, p < 0.05. See "Experimental Procedures" for details.

 


Figure 5
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  		FIGURE 5.
Western blots of GRx1 and ICAM-1 in rMC-1 Müller cells overexpressing GRx1. Müller cells grown in normal glucose (5 mM) medium and infected with Ad-GRx or Ad-Empty were lysed and immunoprobed with anti-GRx1 (1:1,000) (A and B) and anti-ICAM-1 (1:500) (C and D) antibodies. GAPDH was probed for a loading control, and quantification was based on the ratio of band intensities of GRx1 or ICAM-1 to GAPDH. Ad-GRx increased GRx1 content up to 15-fold (± 4.7)(n = 4) with no effect for Ad-Empty (n = 3). Ad-GRx increased ICAM up to 6.8-fold (± 2.1)(n = 6) with no effect observed after infection with Ad-Empty (n = 4). *, p ≤ 0.05. See "Experimental Procedures" for details.

 


Figure 6
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  		FIGURE 6.
Glutaredoxin activity of rMC-1 Müller cells overexpressing GRx1. Müller cells grown in normal glucose medium (5 mM) were infected with either Ad-GRx or Ad-Empty and assayed for GRx activity using the radiolabeled substrate [35S]BSA-SSG. GRx activity was increased up to 13 nmol/min/mg (± 2.4) (n = 10) after infection with Ad-GRx and was unaffected by infection with Ad-Empty (n = 5). *, p < 0.01. See "Experimental Procedures" for details.

 
Overexpression of GRx1 in Müller Cells in Normal Glucose Increases Nuclear Localization and Gene Transcription Activity of NF-{kappa}B—To test whether GRx1 regulates NF-{kappa}B activity, Müller cells were transfected with Ad-GRx or Ad-Empty, incubated in normal glucose concentrations, and analyzed for NF-{kappa}B nuclear translocation and activity. Overexpression of GRx1 increased nuclear p50 and p65 proteins by about 3–6-fold (Figs. 7, A and B, and 8, A and B). As expected, Ad-Empty did not increase nuclear p50 (Fig. 7, A and B) or nuclear p65 (Fig. 8, A and B). Neither Ad-GRx nor Ad-Empty had a significant effect on abundance of cytoplasmic p50 (Fig. 7, C and D) or cytoplasmic p65 (Fig. 8, C and D).


Figure 7
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  		FIGURE 7.
Nuclear localization of the NF-{kappa}B subunit p50 in rMC-1 Müller cells overexpressing GRx1. Müller cells grown in normal glucose medium (5 mM) and infected with Ad-GRx or Ad-Empty were separated into nuclear and cytoplasmic fractions and immunoprobed for anti-p50 (1:1,000). Actin (1:30,000) and YY1 (1:1,000) were probed as loading controls in the cytoplasm and nucleus, respectively, and quantification was based on the ratio. Ad-GRx increased nuclear p50 by 5.5-fold (± 1.1), and Ad-empty had no effect (A and B)(n = 6). Neither Ad-GRx nor Ad-Empty had a significant effect on abundance of cytoplasmic p50 (C and D)(n = 5). *, p < 0.05. See "Experimental Procedures" for details.

 


Figure 8
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  		FIGURE 8.
Nuclear localization of the NF-{kappa}B subunit p65 in Ad-GRx infected rMC-1 Müller cells. Müller cells grown in normal glucose medium (5 mM) and infected with Ad-GRx or Ad-Empty were separated into nuclear and cytoplasmic fractions and immunoblotted for anti-p65 (1:3,000). Actin (1:30,000) and YY1 (1,1000) were probed as loading controls in the cytoplasm and nucleus, respectively, and quantification was based on the ratio. Ad-GRx increased nuclear p65 by 3.9-fold (± 0.9), and Ad-Empty had no effect (A and B)(n = 6). Neither Ad-GRx nor Ad-Empty had a significant effect on abundance of cytoplasmic p65 (C and D)(n = 5). *, p < 0.05. See "Experimental Procedures" for details.

 
To confirm that increased NF-{kappa}B in the nucleus corresponded to increased NF-{kappa}B transcriptional activity, we assayed for NF-{kappa}B luciferase activity in Müller cells after infections with Ad-GRx or Ad-Empty. Although empty vector had no significant effect, overexpression of GRx1 (Ad-GRx) produced up to 3.5-fold increase in NF-{kappa}B luciferase activity (Fig. 9). These results support the conclusion that GRx1 regulates NF-{kappa}B activity in Müller cells, and consequently, the transcription of ICAM-1.


Figure 9
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  		FIGURE 9.
NF-{kappa}B luciferase activity of Ad-GRx infected rMC-1 Müller cells. Müller cells co-transfected with NF-{kappa}B firefly luciferase and Renilla luciferase plasmids were infected with Ad-GRx or Ad-Empty at m.o.i. 0–40 and assayed for NF-{kappa}B activity with the Dual-Luciferase reporter assay (Promega). NF-{kappa}B luciferase activity was increased by 3.4 (± 0.4) and was unaffected by Ad-Empty (n = 5). *, p < 0.01. See "Experimental Procedures" for details.

 
Sn50 Blocks the GRx-induced Increase in ICAM-1 Production in Ad-GRx Infected rMC-1 Cells—NF-{kappa}B is the most common regulator of ICAM-1 transcription and appears to be the key mediator in GRx-induced ICAM-1 expression in rMC-1 cells. We used an inhibitor of NF-{kappa}B nuclear translocation (sn50) to further test whether signaling to NF-{kappa}B is the major pathway regulating ICAM-1 and whether regulation by GRx1 is exerted on the NF-{kappa}B pathway in the cytosol or the nucleus. Müller cells were transfected with an m.o.i. 10 of Ad-GRx in the absence or presence of sn50 inhibitor in normal glucose concentrations, and the lysates were analyzed for ICAM-1 production (Fig. 10, A and B). Treatment with Sn50 resulted in a dose-dependent decrease in the GRx-induced ICAM-1 production. Cells overexpressing GRx in the presence of 20 µM sn50 contained amounts of ICAM-1 indistinguishable from control cells (no virus (0) and Ad-Empty at m.o.i. 10 (Fig. 10).

Knockdown of GRx1 in Müller Cells in High Glucose Prevents Induction of ICAM-1 Expression—To test the effects of decreasing intracellular GRx in high glucose conditions, Müller cells in diabetic-like concentrations of glucose (25 mM) were transfected with siRNA directed against GRx1 or with siCONTROL. GRx1 was knocked down about 50% in the cells in high glucose, i.e. to an amount similar to that in cells in normal glucose (Fig. 11, A and C). This knockdown of GRx1 was associated with a concomitant decrease in ICAM-1 expression (Fig. 11C). This result suggests that a targeted decrease in GRx activity can prevent increased production of pro-inflammatory ICAM-1 under hyperglycemic conditions (see "Discussion").


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Glutaredoxin in Oxidative Stress and Disease—The regulation of cellular sulfhydryl homeostasis by glutaredoxin typically is considered a protective mechanism, preventing irreversible oxidation of proteins and damage in cells under oxidative stress (3). Thus, changes in glutaredoxin activity could disrupt normal redox signaling and lead to pathological consequences. Indeed, several recent studies have identified changes in glutaredoxin in diseases involving redox perturbations. For example, glutaredoxin is increased in the brains of post-mortem patients with Alzheimer disease, and it was proposed to mediate amyloid beta toxicity (39). Patients with chronic obstructive pulmonary disease were reported to have decreased glutaredoxin with disease progression in alveolar macrophages and lung homogenates but increased glutaredoxin in sputum supernatants (40). In addition, glutaredoxin is elevated in animal models of Parkinson disease, where inactivation of mitochondrial complex I is characteristic of the disease (41). In another context, complex I was reported to be regulated by S-glutathionylation (42). Taken together, these studies implicate glutaredoxin in alterations of redox regulation associated with a variety of diseases involving oxidative stress.


Figure 10
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  		FIGURE 10.
Inhibition of NF-{kappa}B nuclear translocation with sn50 prevents ICAM induction by Ad-GRx in rMC-1 cells. Müller cells infected with either Ad-GRx or Ad-Empty at an m.o.i. of 10 in the presence of sn50 in normal glucose (5 mM) medium were lysed and collected in for immunoblotting of ICAM (1:1000) and actin (1:30,000). ICAM was increased by 2.5-fold (± 0.2) in cells infected with Ad-GRx1 (A and B)(n = 8). *, p < 0.0003. sn50 decreased ICAM-1 production in a dose-dependent fashion in cells infected with Ad-GRx. Cells infected with Ad-GRx in the presence of 20 µM sn50 had similar amounts of ICAM as control cells (no adenovirus and Ad-Empty) and was not significantly different from cells infected with Ad-GRx in the absence of sn50 (n = 8). #, p < 0.0006. See "Experimental Procedures" for details.

 


Figure 11
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  		FIGURE 11.
Western blots of GRx1 and ICAM-1 in rMC-1 Müller cells transfected with siRNA-GRx1 in high glucose. Müller cells transfected with rat GRx1 siRNA or non-targeted siRNA in high glucose (25 mM) medium were lysed and immunoprobed for GRx1 (1:1,000) (A and C) and ICAM-1 (1:500) (B and C). Actin (1:30,000) and GAPDH (1:10,000) were probed as loading controls. GRx1 was decreased by 47% (± 4.9%) (n = 9) and ICAM was decreased by 33% (± 4.5%) (n = 11). *, p ≤ 0.00002. See "Experimental Procedures" for details.

 
The importance of glutaredoxin in regulation of oxidative signal transduction is underscored by the identification of an increasing number of proteins whose cellular functions are modulated by S-glutathionylation, reversible by glutaredoxin (3, 43–45). Our current findings support the interpretation that glutaredoxin regulates the NF-{kappa}B signaling pathway and subsequent ICAM-1 expression in retinal Müller cells under diabetes-like conditions, suggesting that glutaredoxin might play an important role in the hyperglycemia-induced inflammatory response known to occur in the retina in diabetes.

ICAM in Diabetic Retinopathy and Müller Cells, Regulation by Glutaredoxin—ICAM-1 protein expression is increased 2–3-fold in the whole retinae of diabetic rats (20, 22). Also, diabetic mice in which ICAM-1 has been knocked out have decreased adherent leukocytes in the retina and less cell death (19), indicating that ICAM-1 contributes to disease progression in diabetic retinopathy. In Müller cells in particular, isolated from diabetic rats, ICAM-1 gene expression has been reported to be increased about 3-fold (21). Analogously, in our rMC-1 cell culture model, we found that exposure to high glucose led to increased ICAM-1 expression in the Müller cells concomitant with increased expression of glutaredoxin (Fig. 3). Further, the increase in ICAM-1 production could be elicited in normal glucose by adenoviral expression of increased amounts of glutaredoxin (Fig. 5). The magnitude of increase in ICAM-1 corresponds well with that of GRx1 except at the higher m.o.i. where GRx1 content exceeds the amount induced by high glucose. This observation suggests a maximal effect of GRx1 on ICAM-1 production. Overall, the results directly identify a role for glutaredoxin in regulation of ICAM-1 expression in Müller Cells and implicate changes in glutaredoxin in the inflammatory response characteristic of diabetic retinopathy.

The role of ICAM-1 in retinal Müller glial cell function is yet to be fully elucidated, but the contribution of these cells to retinal inflammation is considered likely by analogy to glial cells in other contexts. Glial cells of the central nervous system (e.g. astrocytes) contribute to neuronal inflammation in spinal cord injury, Parkinson disease, and AIDS via ICAM-1 production (46–50). In the studies of recovery after spinal cord injury, selective inhibition of the NF-{kappa}B signaling pathway provided neurological protection (46).

NF-{kappa}B Signaling and Potential Targets for Regulation by S-Glutathionylation and Glutaredoxin—To the best of our knowledge, the current study is the first to show increased activation of NF-{kappa}B in Müller cells linked to corresponding changes in glutaredoxin activity, implicating regulation via reversible glutathionylation of one or more components of the NF-{kappa}B signaling pathway. Inhibiting the nuclear translocation of NF-{kappa}B in rMC-1 cells overexpressing GRx1 blocks the corresponding increase in ICAM production (Fig. 10), suggesting that the target for GRx-regulated S-glutathionylation is a cytoplasmic signaling protein in the NF-{kappa}B pathway, upstream of nuclear p50-p65. Glutaredoxin could regulate NF-{kappa}B activity via the glutathionylation status of upstream mediators in the cytoplasmic NF-{kappa}B signaling pathway or via the glutathionylation status of the NF-{kappa}B subunits (p50 and p65) in the nucleus. The site of GRx regulation may be cell type- and signal-dependent. P50 has been shown to lose DNA binding activity upon S-glutathionylation in vitro (29), and this is likely predictive of modulation of p50 activity in a physiological setting. With pancreatic cancer cells, hypoxia and N-acetylcysteine treatment led to inactivation of p65, and glutaredoxin was shown to restore the p65 transcriptional activity, indicative of p65-SSG formation in situ.3

Typically, inactive NF-{kappa}B is sequestered in the cytoplasm by its inhibitory protein, inhibitor of NF-{kappa} B(I{kappa}B). The phosphorylation of I{kappa}B by a complex of I{kappa}B kinases (IKK{alpha}, IKKbeta, and IKK{gamma}) precedes ubiquitination and degradation of I{kappa}B, releasing NF-{kappa}B for nuclear translocation, where it binds to DNA and activates transcription. Although IKK regulates the phosphorylation of I{kappa}B, mediators in the ubiquitin-protease pathway regulate the degradation of I{kappa}B, both processes contributing to NF-{kappa}B activation.

IKKbeta is the IKK subunit with a primary role in inflammation, and it has redox-sensitive cysteines (51, 52). NF-{kappa}B activity was recently shown to be regulated by S-glutathionylation of IKKbeta in lung epithelial cells (31). S-Glutathionylation has also been reported to inhibit the ubiquitin-activating (E1) and ubiquitin carrier (E2) enzymes (53, 54) and the 20 S proteasome in Saccharomyces cerevisiae (55). The 20 S proteasome constitutes part of the 26 S proteasome that degrades I{kappa}B and cleaves p50 from its p105 precursor (56). The multitude of regulatory sites mediated by S-glutathionylation presents a complex picture, and further studies are needed to distinguish which mediators are most pertinent to regulation of the NF-{kappa}B pathway by glutaredoxin within the context of diabetic retinopathy and the retinal Müller cells.

Regardless of which specific components of the NF-{kappa}B pathway are modulated, our studies show that increases in glutaredoxin activity lead to increased nuclear p50 and p65 subunits of NF-{kappa}BinMüller cells, both in response to high glucose and in response to overexpression of glutaredoxin in normal glucose, and the increases in GRx were similar under these different situations (Figs. 7, 8, 9). Collectively these data support the conclusion that glutaredoxin regulates NF-{kappa}B activity, and concurrently, the production of ICAM-1, and this regulation is altered under high glucose conditions mimicking diabetes.

Glutaredoxin, a Potential Therapeutic Target in Diabetic Retinopathy—When glutaredoxin is overexpressed to mimic induction of the enzyme by high glucose (2–4-fold), the extent of the changes in ICAM-1 and NF-{kappa}B are comparable with changes induced by high glucose in cell culture or induced physiologically in the diabetic animals. These studies suggest that glutaredoxin plays an inflammatory role in the response to diabetes in the retina, most likely through regulation of S-glutathionylation status of redox-sensitive cysteine-containing proteins. Remarkably, knocking down glutaredoxin is an effective means of dampening ICAM-1 production under high glucose (Fig. 11). This finding identifies glutaredoxin as a potential target for pharmacological intervention in diabetic retinopathy. In addition, induction of glutaredoxin in hearts of diabetic rats (30) suggests that inhibition of the enzyme in diabetes might have benefits to other tissues besides the retina that also suffer from complications of diabetes. Additional work is needed to determine the efficacy of glutaredoxin inhibition in combating the tissue pathologies associated with diabetes and possibly other inflammatory diseases.


   FOOTNOTES
 
* This work was supported by NIA, National Institutes of Health Grants R01 AG024413 and P01 AG 15885 (to J. J. M.), a Merit Review grant from the Department of Veteran's Affairs (to J. J. M.), and Visual Sciences Research Training Grant 5 T32 EY07157 from the NEI, National Institutes of Health (to M. D. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Dept. of Pharmacology, School of Medicine Case Western Reserve University, 2109 Adelbert Rd., WRT300-9, Cleveland, OH 44106-4965. Tel.: 216-368-3383; Fax: 216-368-3395; E-mail: JJM5{at}case.edu.

2 The abbreviations used are: protein-SOH, protein-sulfenic acids; protein-SNO, S-nitrosylated proteins; protein-SSG, S-glutathionylated proteins; GRx, glutaredoxin; TRx, thioredoxin; Ad-GRx, adenovirus vector containing GRx1 cDNA construct; Ad-Empty, adenovirus vector-empty construct; DMEM, Dulbecco's modified Eagle's medium; ICAM-1, intercellular adhesion molecule-1; NF-{kappa}B, nuclear factor {kappa} B; I{kappa}B, inhibitor of NF-{kappa} B; IKK, I{kappa}B kinase; m.o.i., multiplicities of infection; BSA, bovine serum albumin; HEDS, bis-(2-hydroxyethyl) disulfide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; siRNA, small interfering RNA. Back

3 S. Qanungo, D. W. Starke, H. V. Pai, J. J. Mieyal, and A. Nieminen, manuscript under review. Back


   ACKNOWLEDGMENTS
 
We thank the Center for AIDS Research for the ICAM-1 enzyme-linked immunosorbent confirmation analyses.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Finkel, T. (2003) Curr. Opin. Cell Biol. 15, 247–254[CrossRef][Medline] [Order article via Infotrieve]
  2. Rhee, S. G. (2006) Science 312, 1882–1883[Abstract/Free Full Text]
  3. Shelton, M. D., Chock, P. B., and Mieyal, J. J. (2005) Antioxid. Redox. Signal. 7, 348–366[CrossRef][Medline] [Order article via Infotrieve]
  4. Mieyal, J. J., Srinivasan, U., Starke, D. W., Gravina, S. A., and Mieyal, P. A. (1995) in Biothiols in Health and Disease (Packer, L., and Cadenas, E., eds) pp. 305–372, Marcel Dekker, Inc., New York
  5. Meister, A., and Anderson, M. E. (1983) Annu. Rev. Biochem. 52, 711–760[CrossRef][Medline] [Order article via Infotrieve]
  6. Thomas, J. A., and Mallis, R. (2003) S-Nitrosylation, and Irreversible Sulfhydryl Oxidation: Roles in Redox Regulation, in Cellular Implications of Redox Signaling (Gitler, C., and Danon, A., eds) pp. 258–264, Imperial College Press, London
  7. Adachi, T., Pimentel, D. R., Heibeck, T., Hou, X., Lee, Y. J., Jiang, B., Ido, Y., and Cohen, R. A. (2004) J. Biol. Chem. 279, 29857–29862[Abstract/Free Full Text]
  8. Barrett, W. C., DeGnore, J. P., Konig, S., Fales, H. M., Keng, Y., Zhong-Yin, Z., Yim, M. B., and Chock, P. B. (1999) Biochemistry 38, 6699–6705[CrossRef][Medline] [Order article via Infotrieve]
  9. Barrett, W. C., DeGnore, J. P., Keng, Y., Zhang, Z., Yim, M. B., and Chock, P. B. (1999) J. Biol. Chem. 274, 34543–34546[Abstract/Free Full Text]
  10. Gravina, S., and Mieyal, J. (1993) Biochemistry 32, 3368–3376[CrossRef][Medline] [Order article via Infotrieve]
  11. Yang, Y., Jao, S., Nanduri, S., Starke, D. W., Mieyal, J. J., and Qin, J. (1998) Biochemistry 37, 17145–17156[CrossRef][Medline] [Order article via Infotrieve]
  12. Chrestensen, C. A., Starke, D. W., and Mieyal, J. J. (2000) J. Biol. Chem. 275, 26556–26565[Abstract/Free Full Text]
  13. Hamnell-Pamment, Y., Lind, C., Palmberg, C., Bergman, T., and Cotgreave, I. A. (2005) Biochem. Biophys. Res. Commun. 332, 362–369[Medline] [Order article via Infotrieve]
  14. Humphries, K. M., Juliano, C., and Taylor, S. S. (2002) J. Biol. Chem. 277, 43505–43511[Abstract/Free Full Text]
  15. Nulton-Persson, A. C., Starke, D. W., Mieyal, J. J., and Szweda, L. I. (2003) Biochemistry 42, 4235–4242[CrossRef][Medline] [Order article via Infotrieve]
  16. Wang, J., Tekle, E., Oubrahim, H., Mieyal, J. J., Stadtman, E. R., and Chock, P. B. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 5103–5106[Abstract/Free Full Text]
  17. Du, Y., Smith, M. A., Miller, C. A., and Kern, T. S. (2002) J. Neurochem. 80, 771–779[CrossRef][Medline] [Order article via Infotrieve]
  18. Kowluru, R. A., Tang, J., and Kern T. S. (2001) Diabetes 50, 1938–1942[Abstract/Free Full Text]
  19. Joussen, A. M., Poulaki, V., Le, M. L., Koizumi, K., Esser, C., Janicki, H., Schraermeyer, U., Kociok, N., Fauser, S., Kirchhof, B., Kern, T. S., and Adamis, A. P. (2004) FASEB J. 18, 1450–1452[Abstract/Free Full Text]
  20. Zheng, L., Szabo, C., and Kern, T. (2004) Diabetes 53, 2960–2967[Abstract/Free Full Text]
  21. Gerhardinger, C., Costa, M. B., Coulombe, M. C., Toth, I., Hoehn, T., and Grosu, P. (2005) Investig. Ophthalmol. Vis. Sci. 46, 349–357[Abstract/Free Full Text]
  22. Joussen, A. M., Poulaki, V., Mitsiades, N., Kirchhof, B., Koizumi, K., Dohmen, S., and Adamis, A. P. (2002) FASEB J. 16, 438–440[Free Full Text]
  23. Pow, D. V., and Crook, D. K. (1995) Neurosci. Lett. 193, 25–28[CrossRef][Medline] [Order article via Infotrieve]
  24. Schutte, M., and Werner, P. (1998) Neurosci. Lett. 246, 53–56[CrossRef][Medline] [Order article via Infotrieve]
  25. Newman, E., and Reichenbach, A. (1996) Trends Neurosci. 19, 307–312[CrossRef][Medline] [Order article via Infotrieve]
  26. Baldwin, A. S. (1996) Annu. Rev. Immunol. 14, 649–681[CrossRef][Medline] [Order article via Infotrieve]
  27. Ledebur, H. C., and Parks, T. P. (1995) J. Biol. Chem. 270, 933–943[Abstract/Free Full Text]
  28. Romeo, G., Liu, W., Asnaghi, V., Kern, T. S., and Lorenzi, M. (2002) Diabetes 51, 2241–2248[Abstract/Free Full Text]
  29. Pineda-Molina, E., Klatt, P., Vazquez, J., Marina, A., Lacoba, M. G., de Perez-Sala, D., and Lamas, S. (2001) Biochemistry 40, 14134–14142[CrossRef][Medline] [Order article via Infotrieve]
  30. Li, X., Xu, Z., Li, S., and Rozanski, G. J. (2005) Am. J. Physiol. 288, H1417–H1424
  31. Reynaert, N. L., Van der Vliet, A., Guala, A. S., McGovern, T., Hristova, M., Pantano, C., Heintz, N. H., Heim, J., Ho, Y., Matthews, D. E., Wouters, E. F. M., and Janssen-Heininger, Y. M. W. (2006) Proc. Natl. Acad. Sci. U. S. A. 103, 13086–13091[Abstract/Free Full Text]
  32. Murata, H., Ihara, Y., Nakamura, H., Yodoi, J., Sumikawa, K., and Kondo, T. (2003) J. Biol. Chem. 278, 50226–50233[Abstract/Free Full Text]
  33. Cross, J. V., and Templeton, D. J. (2004) Biochem. J. 381, 675–683[CrossRef][Medline] [Order article via Infotrieve]
  34. Hirota, K., Matsui, M., Murata, M., Takashima, Y., Cheng, F. S., Itoh, T., Fukuda, K., and Yodoi, J. (2000) Biochem. Biophys. Res. Commun. 274, 177–182[CrossRef][Medline] [Order article via Infotrieve]
  35. Srinivasan, U., Mieyal, P. A., and Mieyal, J. J. (1997) Biochemistry 36, 3199–3206[CrossRef][Medline] [Order article via Infotrieve]
  36. Biaglow, J. E., Donahue, J., Tuttle, S., Held, K., Chrestensen, C., and Mieyal, J. (2000) Anal. Biochem. 281, 77–86[CrossRef][Medline] [Order article via Infotrieve]
  37. Song, J. J., Rhee, J. G., Suntharalingam, M., Walsh, S. A., Spitz, D. R., and Lee, Y. J. (2002) J. Biol. Chem. 277, 46566–46575[Abstract/Free Full Text]
  38. Gravina, S. (1993) Characterization and Kinetic mechanism of Thioltransferase. Ph.D. thesis, Case Western Reserve University, Cleveland, OH
  39. Akterin, S., Cowburn, R. F., Miranda-Vizuete, A., Jimenez, A., Bogdanovid, N., Winblad, B., and Cedazo-Minguez, A. (2006) Cell Death Differ. 13, 1454–1465[CrossRef][Medline] [Order article via Infotrieve]
  40. Peltoniemi, M. J., Rytila, P. H., Harju, T. H., Soini, Y. M., Salmenkivi, K. M., Ruddock, L. W., and Kinnula, V. (2006) Respir. Res. 7, 133–143[CrossRef][Medline] [Order article via Infotrieve]
  41. Kenchappa, R. S., and Ravindranath, V. (2003) FASEB J. 17, 717–719[Abstract/Free Full Text]
  42. Taylor, E. R., Hurrell, F., Shannon, R. J., Tsu-Kung, L., Hirst, J., and Murphy, M. P. (2003) J. Biol. Chem. 278, 19603–19610[Abstract/Free Full Text]
  43. Klatt, P., and Lamas, S. (2000) Eur. J. Biochem. 267, 4928–4944[Medline] [Order article via Infotrieve]
  44. Cotgreave, I. A., and Gerdes, R. G. (1998) Biochem. Biophys. Res. Commun. 242, 1–9[CrossRef][Medline] [Order article via Infotrieve]
  45. Giustarini, D., Rossi, R., Milzani, A., Colombo, R., and Dalle-Donne, D. (2004) J. Cell. Mol. Med. 8, 201–212[Medline] [Order article via Infotrieve]
  46. Bramilla, R., Bracchi-Ricard, V., Hu, W., Frydel, B., Bramwell, A., Karmally, S., Green, E. J., and Bethea, J. R. (2005) J. Exp. Med. 202, 145–156[Abstract/Free Full Text]
  47. Shrikant, P., Benos, D. J., Tang, L. P., and Benveniste, E. N. (1996) J. Immunol. 156, 1307–1314[Abstract]
  48. Shrikant, P., Weber, E., Jilling, T., and Benveniste, E. N. (1995) J. of Immunol. 155, 1489–1501[Abstract]
  49. Sawada, M., Imamura, K., and Nagatsu, T. (2006) J. Neural Transm. Suppl. 70, 373–381[Medline] [Order article via Infotrieve]
  50. Miklossy, J., Doudet, D. D., Schwab, C., Yu, S., McGeer, E. G., and McGeer, P. L. (2006) Exp. Neurol. 197, 275–283[CrossRef][Medline] [Order article via Infotrieve]
  51. Jeon, K., Byun, B., and Jue, D. (2003) Exp. Mol. Med. 35, 61–66[Medline] [Order article via Infotrieve]
  52. Rossi, A., Kapahi, P., Natoli, G., Takahashi, T., Chen, Y., Karin, M., and Santor, M. G. (2000) Nature 403, 103–108[CrossRef][Medline] [Order article via Infotrieve]
  53. Obin, M., Shang, F., Gong, X., Handelman, G., Blumbberg, J., and Taylor, A. (1998) FASEB J. 12, 561–569[Abstract/Free Full Text]
  54. Jahngen-Hodge, J., Obin, M. S., Gong, X., Shang, F., Nowell, T. R. Jr., Gong, J., Abasi, H., Blumberg, J., and Taylor, A. (1997) J. Biol. Chem. 272, 28218–28226[Abstract/Free Full Text]
  55. Demasi, M., Silva, M., and Netto, L. E. S. (2003) J. Biol. Chem. 278, 679–685[Abstract/Free Full Text]
  56. Moorthy, A. K., Savinova, O. V., Ho, J. Q., Wang, V. Y., Vu, D., and Ghosh, G. (2006) EMBO J. 25, 1945–1956[CrossRef][Medline] [Order article via Infotrieve]

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