Glutaredoxin Regulates Nuclear Factor κ-B and Intercellular Adhesion Molecule in Müller Cells
2007; Elsevier BV; Volume: 282; Issue: 17 Linguagem: Inglês
10.1074/jbc.m610863200
ISSN1083-351X
AutoresMelissa D. Shelton, Timothy S. Kern, John J. Mieyal,
Tópico(s)Neutrophil, Myeloperoxidase and Oxidative Mechanisms
ResumoReversible 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-κB (p50-p65) translocated to the nucleus, and expression of ICAM-1 (intercellular adhesion molecule-1), a transcriptional product of NF-κB, 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-κB nuclear translocation, NF-κB 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-κB activation and a pro-inflammatory response. Thus, GRx may represent a novel therapeutic target to inhibit diabetic retinopathy. 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-κB (p50-p65) translocated to the nucleus, and expression of ICAM-1 (intercellular adhesion molecule-1), a transcriptional product of NF-κB, 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-κB nuclear translocation, NF-κB 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-κB activation and a pro-inflammatory response. Thus, GRx may represent a novel therapeutic target to inhibit diabetic retinopathy. 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 (1Finkel T. Curr. Opin. Cell Biol. 2003; 15: 247-254Crossref PubMed Scopus (1213) Google Scholar, 2Rhee S.G. Science. 2006; 312: 1882-1883Crossref PubMed Scopus (1686) Google Scholar). 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 (3Shelton M.D. Chock P.B. Mieyal J.J. Antioxid. Redox. Signal. 2005; 7: 348-366Crossref PubMed Scopus (325) Google Scholar). Reversible post-translational modifications such as protein-sulfenic acids (protein-SOH), 2The 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-κB, nuclear factor κ B; IκB, inhibitor of NF-κ B; IKK, IκB 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. 2The 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-κB, nuclear factor κ B; IκB, inhibitor of NF-κ B; IKK, IκB 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. S-nitrosylated proteins (protein-SNO), and S-glutathionylated proteins (protein-SSG) are thought to protect against irreversible oxidation (3Shelton M.D. Chock P.B. Mieyal J.J. Antioxid. Redox. Signal. 2005; 7: 348-366Crossref PubMed Scopus (325) Google Scholar, 4Mieyal J.J. Srinivasan U. Starke D.W. Gravina S.A. Mieyal P.A. Packer L. Cadenas E. Biothiols in Health and Disease. Marcel Dekker, Inc., New York1995: 305-372Google Scholar), and S-glutathionylation is likely the predominant physiological sulfhydryl modification due to the abundance of cellular glutathione (5Meister A. Anderson M.E. Annu. Rev. Biochem. 1983; 52: 711-760Crossref PubMed Scopus (5947) Google Scholar) and the ready conversion of cys-SNO and cys-SOH moieties to cys-SSG (6Thomas J.A. Mallis R. Gitler C. Danon A. S-Nitrosylation, and Irreversible Sulfhydryl Oxidation: Roles in Redox Regulation, in Cellular Implications of Redox Signaling. Imperial College Press, London2003: 258-264Google Scholar). 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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-κB activation and increased ICAM-1 expression. Overexpression of glutaredoxin in these cells in normal glucose leads to analogous increases in NF-κB 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-κB activation and a pro-inflammatory response. 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 (17Du Y. Smith M.A. Miller C.A. Kern T.S. J. Neurochem. 2002; 80: 771-779Crossref PubMed Scopus (201) Google Scholar). 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 (12Chrestensen C.A. Starke D.W. Mieyal J.J. J. Biol. Chem. 2000; 275: 26556-26565Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar, 35Srinivasan U. Mieyal P.A. Mieyal J.J. Biochemistry. 1997; 36: 3199-3206Crossref PubMed Scopus (108) Google Scholar). 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 (36Biaglow J.E. Donahue J. Tuttle S. Held K. Chrestensen C. Mieyal J. Anal. Biochem. 2000; 281: 77-86Crossref PubMed Scopus (47) Google Scholar) 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 (36Biaglow J.E. Donahue J. Tuttle S. Held K. Chrestensen C. Mieyal J. Anal. Biochem. 2000; 281: 77-86Crossref PubMed Scopus (47) Google Scholar). 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 THERMOmax™ 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) (37Song J.J. Rhee J.G. Suntharalingam M. Walsh S.A. Spitz D.R. Lee Y.J. J. Biol. Chem. 2002; 277: 46566-46575Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar). 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 × 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-κB 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 × 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 4× 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 (38Gravina S. Characterization and Kinetic mechanism of Thioltransferase. 1993; (Ph.D. thesis, Case Western Reserve University, Cleveland, OH)Google Scholar). 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 × 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 × 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 × g for 15 min in 4 °C. Protein content was determined via BCA assay. NF-κB 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-κB luciferase (5×) plasmid (Stratagene) and 0.1 μg of Renilla plasmid (Promega) as a control reporter according to the Lipofectamine™ reagent protocol (Invitrogen). The binding element for the NF-κB luciferase plasmid is derived from the consensus NF-κB 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 1× passive lysis buffer (Promega). NF-κB 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. 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. 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 (36Biaglow J.E. Donahue J. Tuttle S. Held K. Chrestensen C. Mieyal J. Anal. Biochem. 2000; 281: 77-86Crossref PubMed Scopus (47) Google Scholar). 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 3Effects 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT) 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-κB (p50 and p65) in Müller Cells—To test whether the observed increase in ICAM-1 production in Müller cells is mediated by NF-κB, we measured changes in nuclear NF-κB after incubation in high glucose. The p50 and p65 subunits of NF-κB 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-κB translocation, and ICAM-1 expression in response to high glucose suggested that GRx1 might be directly responsible for regulating NF-κB 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 conte
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