Suppressors of Cytokine Signaling Regulate Angiotensin IIâActivated ...

Suppressors of Cytokine Signaling Regulate Angiotensin II–Activated Janus Kinase-Signal Transducers and Activators of Transcription Pathway in Renal Cells Purificacioń Hernańdez-Vargas,* Oscar Lo´pez-Franco,* Guillermo Sanjuań,* Mońica Rupe´rez,* Guadalupe Ortiz-Munõz,* Yusuke Suzuki,‡ Pablo Aguado-Roncero,† Gloria Pe´rez-Tejerizo,† Julia Blanco,§ Jesu´s Egido,* Marta Ruiz-Ortega,* and Carmen Go´mez-Guerrero* *Renal and Vascular Research Laboratory, †Pediatric Surgery Department, Fundacioń Jimeńez Dıáz, Autonoma University, Madrid, Spain; ‡Division of Nephrology, Department of Internal Medicine, Juntendo University, Tokyo, Japan; and §Department of Pathology, Hospital Clıńico San Carlos, Madrid, Spain Suppressors of cytokine signaling (SOCS) family is constituted by cytokine-inducible proteins that modulate receptor signal transduction via tyrosine kinases, mainly the Janus kinase-signal transducers and activators of transcription (JAK-STAT) pathway. Differential SOCS expression was noted in renal cells that were incubated with inflammatory stimuli, but the role of SOCS in the pathogenesis of renal diseases is not yet well defined. Because angiotensin II (Ang II) plays a key role in renal disease, SOCS proteins were studied as a novel mechanism involved in the negative regulation of Ang II–mediated processes. Systemic Ang II infusion for 3 d increased the renal mRNA expression of SOCS-3 and SOCS-1. SOCS protein synthesis was found in glomerular mesangial area and tubules. In cultured mesangial cells and tubular epithelial cells, Ang II induced a rapid and transient SOCS-3 and SOCS-1 expression in parallel with JAK2 and STAT1 activation. In both cell types, overexpression of SOCS proteins prevented the STAT activation in response to Ang II. SOCS expression observed in Ang II–infused rats and in Ang II–stimulated cells was significantly inhibited by treatment with AT1 but not AT2 receptor antagonist and was attenuated in mesangial cells from AT1a-deficient mice, demonstrating the implication of AT1 in those responses. In SOCS-3 knockdown studies, antisense oligonucleotides inhibited the expression of SOCS-3 and increased the Ang II–induced STAT activation and c-Fos/c-Jun expression, then resulting in a more severe renal damage. These results suggest that SOCS proteins may act as negative regulators of Ang II signaling in renal cells and implicate SOCS as important modulators of renal damage. J Am Soc Nephrol 16: 1673–1683, 2005. doi: 10.1681/ASN.2004050374

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he Janus kinase-signal transducers and activators of transcription (JAK-STAT) pathway is an essential intracellular mechanism of cytokine actions and constitutes a link between activation of cell surface receptors and nuclear transcriptional event (1,2). Control of the magnitude and duration of cytokine signaling is essential to prevent tissue damage. In this sense, recent studies have shown that JAK-STAT signaling can be regulated at many steps through different mechanisms. Receptor internalization, tyrosine phosphatases, and members of the protein inhibitors of activated STAT family all contribute to this negative regulatory network (2). Recently, the discovery of the suppressors of cytokine signaling (SOCS) proteins has defined an important additional mechanism for the negative regulation of the JAK-STAT pathway (3–5). SOCS family is constituted by eight cytokine-inducible pro-

Received May 11, 2004. Accepted February 28, 2005. Published online ahead of print. Publication date available at www.jasn.org. Address correspondence to: Dr. Carmen Go´mez-Guerrero, Renal and Vascular Research Laboratory, Fundacioń Jimeńez Dıáz, Avda Reyes Cato´licos 2, Madrid, Spain 28040. Phone: 34-91-5504800; Fax: 34-91-5442636; E-mail: [email protected] Copyright © 2005 by the American Society of Nephrology

teins (SOCS-1 to SOCS-7 and CIS), each of which contains a central Src-homology 2 domain and a C-terminal SOCS box, and its expression is under the transcriptional control of STAT (5). SOCS can inhibit cytokine signal transduction through several mechanisms. CIS and SOCS-2 bind to receptor sites, blocking the recruitment and activation of STAT (4,5). SOCS-1 binds to the kinase domain of JAK1–3 then suppresses the JAK catalytic activity, SOCS-3 can interact with cytokine receptor or with target sequences in JAK and STAT, whereas little is known about the functions of the other SOCS family members (2–5). In addition to cytokine receptors, SOCS proteins interact with insulin, leptin, growth hormone, and chemokine receptors, suggesting that this family may have a broader range of action than originally thought (2,5,6). Angiotensin II (Ang II) is a pleiotropic vasoactive peptide that binds to two specific receptor subtypes, AT1 and AT2 (7,8). Only a single AT1 receptor gene is present in humans, but this receptor is encoded by at least two distinct genes (AT1a and AT1b) in rodents, with 96% homology (9). AT1 contributes to chronic diseases, such as hypertension, renal injury, atherosclerosis, and cardiac hypertrophy, by promoting cell growth, inISSN: 1046-6673/1606-1673

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flammatory responses, and fibrosis. AT2 causes cardioprotection, vasodilation, renal natriuresis, cell growth inhibition, and renal inflammatory cell infiltration (7,8,10,11). In addition, some Ang II responses could be mediated by both AT1 and AT2 receptors, among them nitric oxide (NO) release, collagen synthesis, and NF-␬B activation (8,11,12). The Ang II signal transduction is mediated by G proteins, which activate several intracellular pathways, including Ca2⫹/ protein kinase C (PKC), mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase, and numerous tyrosine kinases, such as Src, Pyk2, p130Cas, and focal adhesion kinase (9,10). Ang II also activates nuclear transcription factors, including NF-␬B, activating protein-1, cyclic adenosine monophosphate response element binding protein, and nuclear factor of activated T cells (12,13,14). In addition, similar to classical cytokine receptors, Ang II stimulates members of the JAK family (JAK2 and TYK2), leading to the tyrosine phosphorylation of STAT transcription factors (15–17). Activation of STAT (STAT1, 2, 3, and 5) has been implicated in the cell proliferation induced by cytokines and Ang II in several cell types, including mesangial cells (MC) (18 –21). Although the molecular mechanisms involved in Ang II– induced renal damage have been studied extensively, less attention has been devoted toward an understanding of the negative regulators that terminate Ang II signaling. In this work, we examined the role of SOCS as new proteins implicated in the negative regulation pathway of Ang II–mediated processes in the kidney. For that purpose, we studied the effects of Ang II on the renal expression of SOCS, both in vivo (experimental model of Ang II infusion) and in vitro (cultured MC and tubular cells stimulated with Ang II). We also analyzed whether SOCS modulate the activation of JAK-STAT pathway induced by Ang II, as well as the Ang II receptor subtype involved in these processes.

Materials and Methods Reagents Ang II, PD123319, and cycloheximide were purchased from Sigma Chemicals (St. Louis, MO). Losartan was provided by Merck Sharp and Dome (Madrid, Spain). Antibodies against SOCS-3, P-JAK2, P-STAT1, c-Fos, and c-Jun were from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit anti–SOCS-1 and mAb anti-tubulin were from Zymed Laboratories (South San Francisco, CA) and Sigma, respectively. Secondary antibodies and ECL system were purchased from Amersham (Buckinghamshire, UK). Human and mouse IFN-␥ and IL-6 were from Immunegenex Corporation (Los Angeles, CA) and Peprotech Ec. (London, UK), respectively. The pGAS-Luc and pISRE-Luc reporter vectors were obtained from BD Biosciences (Erembodegem, Belgium). The SOCS expression vectors (S3wt and S1wt) cloned in the p513HA plasmid were a gift from Dr. H. Boeuf (IGBMC, Illkirch Cedex, France). Oligodeoxynucleotides (ODN) for human SOCS-3 (antisense, 5⬘-CGGGAAACTTGCTGTGGGTGACCAT-3⬘; sense, 5⬘-ATGGTCACCCACAGCAAGTTTCCCG-3⬘) were synthesized as phosphorothionated (for in vitro studies) and FITC-labeled or unmodified (for in vivo transfections) by Metabion (Marinsried, Germany).

Experimental Design Systemic infusion of Ang II was done into Wistar rats (subcutaneously by osmotic minipumps; Alza Corp., Palo Alto, CA), at the dose of

J Am Soc Nephrol 16: 1673–1683, 2005 50 ng/kg per min (n ⫽ 8) (12). Pharmacologic blockade of AT1 and AT2 was done with Losartan (10 mg/kg per d; drinking water; n ⫽ 8) and PD123319 (30 mg/kg per d; subcutaneous osmotic minipumps; n ⫽ 4) from 24 h before Ang II infusion (12). Control animals (saline-infused; n ⫽ 8) were also studied. Animals were killed after 3 d, and the renal tissue samples were removed and processed further for histologic and RNA studies. All studies were performed in accordance with the European Union normative. The in vivo ODN transfection of rat kidneys was performed by injection into the left renal vein, as described (22). In brief, rats were anesthetized and the left kidney was exposed by midline incision. SOCS-3 antisense and sense ODN (200 ␮g) were complexed with jetPEI transfection reagent (N/P ratio ⫽ 5; Qbiogene, Montreal, Quebec, Canada) in 5% glucose solution and injected into the left renal vein using a 24-gauge intravenous catheter, after the vein was clamped. Complexes were kept in contact with the kidney for 7 min, before circulation was restored. For assessing the transfection efficiency and localization of ODN, FITC-labeled ODN was injected into the left kidney, as described. FITC-ODN–transfected left kidneys and contralateral right kidneys were removed 7 min after injection, and 4-␮mthick cryostat sections of unfixed snap-frozen specimens were examined by fluorescence microscopy (n ⫽ 6). For studying the effects of SOCS-3 ODN transfer on Ang II–mediated responses, rats received an injection of sense ODN (n ⫽ 5) or antisense ODN (n ⫽ 6) via the left renal vein 24 h before Ang II infusion by osmotic minipumps, and the transfected and contralateral kidneys were harvested after 3 d.

Histopathologic Studies For histologic analysis, kidney sections fixed with 4% buffered paraformaldehyde were embedded in paraffin, and 5-␮m-thick sections were stained with Masson’s trichrome. Immunohistochemistry in renal samples was performed by incubation with antibodies against SOCS, c-Fos, and P-STAT1 (2, 3, and 10 ␮g/ml, respectively) followed by peroxidase-conjugated secondary antibodies. Samples were developed with 3,3⬘-diaminobenzidine tetrahydrochloride and counterstained with hematoxylin (except for P-STAT1). Staining in the glomerular and tubular areas was examined in a blind manner (20 fields from each animal) and was graded semiquantitatively on a scale from 0 to 3.

Cell Cultures MC were obtained from human and mouse kidneys and cultured in RPMI 1640 with 25 mM HEPES (pH 7.4), supplemented with 10% FCS, 100 U/ml penicillin, 100 mg/ml streptomycin, and 2 mM glutamine (all from Life Technologies, Paisley, Scotland, UK) as described previously (14,23). Mice used were male wild-type (WT, C57BL6) and AT1a-deficient mice (AT1⫺/⫺), with the same genetic background (24). MC were characterized by phase contrast microscopy and immunostaining (desmin and vimentin positive, factor VIII and cytokeratin negative). Murine proximal tubuloepithelial cells (MCT) (12) were cultured in RPMI 1640 with 10% FCS. MC and MCT were made quiescent by incubation in medium without FCS during 24 and 48 h, respectively, and then stimulated with Ang II or cytokines. For inhibition studies, cells were incubated with cycloheximide (5 ⫻ 10⫺5 M), losartan (10⫺6 M), and PD123319 (10⫺5 M) for 60 min or with SOCS-3 ODN (0.5 to 3 ⫻ 10⫺6 M) for 24 h before stimulation.

Analysis of mRNA Expression Total RNA from cultured cells or renal cortex pieces was extracted with the Tryzol reagent (Life Technologies), and the SOCS-3 and SOCS-1 mRNA expression was analyzed by reverse transcription–PCR with specific primers for rat (provided by Dr. H. Brady, Dublin, Ire-

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land) (25), human, and mouse (designed according to Genebank sequences) (26). One microgram of RNA was reverse transcribed, and the PCR reaction that contained 20 pmol of primers, 0.5 ␮Ci [␣32P]dCTP (3000 Ci/mmol; Amersham) and 3 U of TaqDNA was performed with annealing temperatures of 54°C (rat SOCS-3, ⫺1), 58°C (human and mouse SOCS-3), and 65°C (human and mouse SOCS-1). PCR products were analyzed on 4% polyacrylamide/urea gels, bands densitometered and corrected by glyceraldehyde-3-phosphate dehydrogenase. In some cases, SOCS PCR products were purified and used as cDNA probes in Northern blot analysis, using 28S as internal control.

Western Blot Cells were lysed in cold buffer (10 mM Tris [pH 7.4], 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 0.5% NP-40, 0.2 mM Na3VO4, 10 mM NaF, 0.2 mM PMSF, and protease inhibitors cocktail), and 25 ␮g was loaded on SDS-PAGE gels, then transferred and immunoblotted with antibodies against SOCS-3, SOCS-1, P-JAK2, P-STAT1, c-Jun, and c-Fos. After membranes were visualized with the ECL system, they were reblotted with anti-tubulin as loading control.

Transient Transfection and Luciferase Assay Cells (5 ⫻ 104) on 24-well plates were transfected for 24 h with luciferase plasmids (pGAS-Luc, pISRE-Luc) and pRL-TK vector (Renilla gene) at a 10:1 ratio, by using the FuGENE reagent (Roche, Barcelona, Spain) (26). Under these conditions, transfection efficiency ranged from 50 to 70% in MC and MCT. In some experiments, cells were co-transfected with SOCS expression vectors (S1wt, S3wt). For knockdown studies, cells were pretreated during 24 h with ODN before transfection. Transfected cells were stimulated for 24 h in triplicate, and luciferase activity in cleared lysates was assayed using a luminometer. Firefly luciferase activity was normalized for total protein content and variations in transfection efficiency (Renilla activity).

Statistical Analyses Results are expressed as mean ⫾ SD and analyzed by ANOVA and Tukey-Kramer tests using Instat (Graphpad Software, San Diego, CA). P ⬍ 0.05 was considered significant.

Results Ang II Induces Expression of SOCS Proteins in the Kidney For analyzing the effects of Ang II on SOCS expression in the kidney, Wistar rats received an infusion of Ang II (50 ng/kg per min) and were killed after 3 d. The pathologic lesions caused by Ang II infusion, including leukocyte infiltration, mesangial matrix expansion, and tubular damage, have been previously described in detail (12,27). Systemic Ang II infusion markedly increased the renal mRNA expression of SOCS-1 and SOCS-3 (Figure 1A). The histologic distribution of SOCS proteins was evaluated by immunohistochemistry. Control animals express very low levels of SOCS (Figure 1, B and C). In Ang II–infused rats, SOCS-3 and SOCS-1 proteins increased, mainly in glomerular mesangial area and proximal tubules (Figure 1, D and E). Semiquantitative score of SOCS staining revealed significant differences after Ang II infusion (Figure 1, F and G).

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expression of SOCS-1 and SOCS-3, peaking at approximately 2 h. The positive control (IFN-␥⫹IL-6) caused a higher SOCS-3 expression (MC, 4.8 ⫾ 0.6; MCT, 2.8 ⫾ 0.3 n-fold versus basal at 2 h; n ⫽ 3). MC preincubation with 50 mM cycloheximide did not affect the Ang II–induced SOCS-3 mRNA (3.4 ⫾ 0.7 versus 3.2 ⫾ 0.4, n-fold versus basal at 2 h; P ⬎ 0.05; n ⫽ 3), indicating that de novo protein synthesis is not required for SOCS-3 gene induction. The synthesis of SOCS proteins in Ang II–stimulated cells was analyzed by Western blot. In MC (Figure 2C), Ang II caused a transient production of SOCS-1 and SOCS-3, being maximal at 1.5 to 2 h. In MCT (Figure 2D), SOCS proteins increased within 30 min of Ang II stimulation, peaked at 1 to 2 h, then returned to baseline.

Ang II Activates the JAK-STAT Pathway in MC and MCT We next analyzed the Ang II–mediated signals, focusing on JAK-STAT, the classical pathway modulated by SOCS (2,5). In human MC (Figure 3A), Ang II caused a rapid (5 min) and transient (peak at 1 h; 3.5 ⫾ 0.6-fold increase) tyrosine phosphorylation of JAK2 and STAT1, demonstrating kinetics consistent with previous information (20,21). The STAT transcriptional activity was assayed in MC that were transfected with the pGAS-Luc and pISRE-Luc plasmids, which contain STAT1/ STAT1 and STAT1/STAT2 binding sites, respectively. Ang II incubation elicited the expression of the two STAT-driven reporter vectors (3.3 ⫾ 0.14 and 2.8 ⫾ 0.1, n-fold versus basal; Figure 3C). In MCT, Ang II induced a rapid tyrosine phosphorylation of JAK2, peaking at 1 h (2.0 ⫾ 0.2-fold increase) and decreasing to basal after 4 h. Furthermore, STAT1 tyrosine phosphorylation occurred at approximately 1 h (2.2 ⫾ 0.2-fold increase) and remain stimulated even after 4 h (Figure 3B). In the reporter assay, Ang II dose-dependently increased the STAT transcriptional activity of pISRE-Luc (Figure 3D) and pGAS-Luc (data not shown) plasmids.

SOCS Expression Inhibits the Ang II–Induced STAT1 Activation in Renal Cells For studying whether SOCS proteins can modulate the Ang II signaling pathways, MC and MCT were transiently transfected with expression vectors for SOCS-1 (S1wt) and SOCS-3 (S3wt) or with empty plasmid (p513HA). In human MC, SOCS overexpression prevented the Ang II–induced STAT1 tyrosine phosphorylation (Figure 4A). Furthermore, co-transfection with S3wt vector inhibited the luciferase activity of pGAS-Luc and pISRE-Luc plasmids induced by Ang II and the positive control (IFN-␥⫹IL-6; Figure 4B). Similarly, the Ang II–induced STAT reporter activity in MCT was impaired by S1wt or S3wt co-transfection (76 and 74% inhibition versus empty plasmid; Figure 4C). These results indicate that SOCS are regulators of Ang II– and cytokine-induced STAT activation in renal cells.

Involvement of AT1 in the Renal Expression of SOCS Ang II Increases SOCS Expression in Cultured Renal Cells The direct effect of Ang II on SOCS induction was assessed in cultured MC and MCT. Treatment of MC (Figure 2A) and MCT (Figure 2B) with 10⫺7 M Ang II induced a time-response mRNA

The Ang II receptor type involved in the induction of SOCS expression in the kidney was determined by using specific antagonists of AT1 (losartan) and AT2 (PD123319). In vivo, the increased SOCS-3 mRNA expression observed in Ang II–in-

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Figure 1. Effect of angiotensin II (Ang II) infusion on the renal expression of suppressors of cytokine signaling (SOCS). (A) The renal mRNA expression of SOCS-1 and SOCS-3 was analyzed in control (f) and Ang II–infused rats (䡺) by reverse transcription– PCR (RT-PCR). Quantification of mRNA expression was performed by densitometry, and values were corrected by the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression. (B through G) Histologic distribution of SOCS proteins in renal tissues. Control animals showed a low SOCS-1 (B) and SOCS-3 (C) staining. Ang II infusion increased SOCS-1 (D) and SOCS-3 (E) synthesis in glomeruli and tubules (indicated by arrows). Representative micrographs of animals killed at day 3. Semiquantitative score of SOCS-1 (F) and SOCS-3 (G) in control (f) and Ang II–infused rats (䡺) was graded in a scale from 0 to 3. Data are expressed as mean ⫾ SD of eight animals analyzed in duplicate (*P ⬍ 0.05 versus control rats). Magnification, ⫻200.

fused rats was significantly prevented by losartan but not by PD123319 (Figure 5A). Similar effects on SOCS-1 mRNA expression were obtained (n-fold versus control: Ang II, 2.1 ⫾ 0.2; losartan, 0.8 ⫾ 0.3 [P ⬍ 0.01]; PD123319, 2.2 ⫾ 0.6 [P ⬎ 0.05]). In MCT, preincubation with losartan significantly inhibited the SOCS-3 and SOCS-1 protein expression and the STAT1 tyrosine phosphorylation in response to Ang II, whereas no effect was observed with PD123319 (Figure 5B). AT1 blockade also decreased the JAK2 tyrosine phosphorylation (% inhibition at 1 h: losartan, 50 ⫾ 7, P ⬍ 0.01; PD123319, 5 ⫾ 2, P ⬎ 0.05; n ⫽ 5). In human MC, the Ang II–induced SOCS-3

and SOCS-1 mRNA expression was significantly inhibited by losartan (66 ⫾ 13 and 48 ⫾ 12% inhibition, respectively; P ⬍ 0.05; n ⫽ 3) but not by PD123319 (15 ⫾ 2 and 7 ⫾ 6% inhibition; P ⬎ 0.05; n ⫽ 3). The involvement of AT1 in SOCS expression was corroborated by using MC from AT1⫺/⫺ mice, which presents a deficiency in AT1a (24). Moreover, previous papers described that murine AT1a presents a similar tissue distribution than human AT1, whereas AT1b is not expressed in renal cells (28). Compared with WT, MC from AT1⫺/⫺ mice expressed very low levels of SOCS-3 and SOCS-1 mRNA in response to Ang II, as

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Figure 2. Ang II induces SOCS-1 and SOCS-3 expression in cultured renal cells. Quiescent human mesangial cells (MC) (A and C) and murine proximal tubuloepithelial cells (MCT) (B and D) were stimulated with 10⫺7 M Ang II for the indicated times. (A and B) SOCS-1 (䡺) and SOCS-3 (f) mRNA expression was analyzed by RT-PCR. Densitometered SOCS bands were corrected by GAPDH expression. (C and D) SOCS-1 (䡺) and SOCS-3 (f) protein synthesis was assessed by immunoblotting, using tubulin as loading control. Data of densitometric analysis are expressed with respect to basal and are mean ⫾ SD of two to six experiments (*P ⬍ 0.05 versus basal).

determined by reverse transcription–PCR (Figure 6A) and confirmed by Northern blot (SOCS-3, n-fold versus basal at 3 h: WT, 1.6 ⫾ 0.2; AT1⫺/⫺, 1.0 ⫾ 0.1; P ⬍ 0.05; n ⫽ 3). Similarly, SOCS protein expression and STAT1 phosphorylation were attenuated in AT1⫺/⫺ MC (Figure 6B).

Effect of SOCS-3 Antisense ODN on Ang II–Mediated Responses in Renal Cells As an alternative approach to examine the role of SOCS-3 in Ang II–stimulated STAT activation, knockdown studies were done using SOCS-3 antisense ODN, which was designed to hybridize to SOCS-3 mRNA at the translation start site, blocking translation and leading to decreased protein expression (26). MC preincubation with SOCS-3 antisense ODN diminished the Ang II–induced SOCS-3 production (Figure 7A). In MCT, antisense ODN caused a significant increase in the STAT luciferase activity stimulated by both Ang II and the positive control (IFN-␥⫹IL-6), whereas no effect was observed with sense ODN (Figure 7B). As a functional consequence of SOCS-3

inhibition, we analyzed differences in the protein levels of c-Jun and c-Fos, early inducible genes expressed by cultured renal cells and in animals exposed to Ang II (29,30). In MC, the expression of c-Jun protein after 1 h of stimulation with Ang II was dose-dependently increased by SOCS-3 antisense ODN (Figure 7A). In other experiments, MCT were preincubated with antisense and sense ODN, then transfected with S3wt expression vector and stimulated with Ang II. As shown in Figure 7C, the inhibitory effect of SOCS-3 overexpression on the Ang II–induced STAT1 phosphorylation and c-Fos expression was reversed by increasing concentrations of antisense ODN but not by sense ODN, indicating the specificity of antisense ODN in the SOCS-3 protein expression reduction. To explore further whether SOCS-3 suppression has a functional consequence for Ang II–mediated responses in vivo, we examined the effect of SOCS-3 antisense ODN transfer in rat kidney. We first investigated the validity of the gene transfer method and the cell type that incorporated the ODN by inject-

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Figure 3. Activation of Janus kinase-signal transducers and activators of transcription (JAK-STAT) pathway by Ang II in renal cells. Whole lysates from Ang II–stimulated MC (A) and MCT (B) were electrophoresed and immunoblotted for tyrosine phosphorylated JAK2 and STAT1, using tubulin expression as loading control. Gels are representative of four experiments. (C) MC were transiently transfected with control plasmid (pGL2-Luc) or the STAT-responsive luciferase reporter vectors (pGAS-Luc and pIRSE-Luc) and then stimulated for 24 h with 10⫺7 M Ang II or positive control (IFN-␥⫹IL-6, 100 U/ml each). (D) MCT that were transfected with STAT reporter vector (pISRE-Luc) were incubated with IFN-␥⫹IL-6 or increasing concentrations of Ang II. Data of relative luciferase units (Firefly/Renilla/mg protein) are mean ⫾ SD of two to four experiments in triplicate (*P ⬍ 0.05 versus basal).

ing FITC-labeled antisense ODN into the left kidney of normal rats. Green fluorescence was mostly accumulated in the nuclei of glomerular and tubular cells, with occasional signals in

interstitial cells (Figure 8A). No signal was detected in the contralateral kidney (Figure 8B). For examining whether antisense ODN can suppress the Ang

Figure 4. SOCS overexpression inhibits the JAK-STAT activation in Ang II–stimulated renal cells. (A) MC were transfected with SOCS-1 (S1wt) and SOCS-3 (S3wt) expression vectors or with the empty plasmid (p513HA) and then stimulated with 10⫺7 M Ang II for 1 h. Cell lysates then were immunoblotted with P-STAT1, SOCS-1, SOCS-3, and tubulin antibodies. Representative of five experiments. (B) MC were co-transfected with SOCS-3 expression vector (S3wt) and STAT-luciferase reporter vectors (pGAS-Luc or pISRE-Luc) and then stimulated for 24 h with Ang II or IFN-␥⫹IL-6. (C) MCT were co-transfected with pISRE-Luc plasmid and the SOCS expression vectors and then stimulated with IFN-␥⫹IL-6 or Ang II. Luciferase activity was assayed as described, and data are expressed as n-fold versus basal conditions. Mean ⫾ SD of three to five experiments (*P ⬍ 0.05 versus basal; #P ⬍ 0.05 versus empty plasmid).

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Figure 5. Effects of Ang II receptor antagonists on the renal expression of SOCS. (A) Rats were treated for 24 h with PD123329 (PD) or losartan (Los) and then infused with Ang II for an additional 3 d. Representative RT-PCR experiment showing SOCS-3 mRNA expression of two different animals from each group. SOCS-3 bands were quantified by densitometry and corrected by GAPDH expression, and data are expressed as mean ⫾ SD of four to eight animals (*P ⬍ 0.05 versus control; #P ⬍ 0.05 versus Ang II infusion). (B) MCT were pretreated with PD123319 (1 ⫻ 10⫺5 M) or losartan (1 ⫻ 10⫺6 M) before stimulation with 10⫺7 M Ang II for 1 h. SOCS proteins and tyrosine phosphorylated STAT1 levels were analyzed by Western blot. Data of densitometric analysis are expressed with respect to basal and are mean ⫾ SD of four to seven experiments (*P ⬍ 0.05 versus basal; #P ⬍ 0.05 versus Ang II).

II–induced SOCS expression, rats were transfected with SOCS-3 antisense or sense ODN via the left renal vein, 24 h before Ang II infusion. The SOCS-3 protein expression decreased in the antisense ODN left kidney (Figure 8C) and did not appreciably change in the contralateral kidneys (data not shown) and in sense ODN transfected rats (Figure 8D). Blocking the Ang II–induced endogenous SOCS-3 expression by antisense ODN had a functional consequence for Ang II–mediated signal transduction, as evidenced by the stronger nuclear staining of phosphorylated STAT1 (Figure 8E) and the increased c-Fos protein expression (Figure 8G) when compared with the Ang II effects in sense ODN

transfected kidneys (Figure 8, F and H). Moreover, in vivo SOCS-3 knockdown exacerbated the Ang II–induced renal damage, examined in Masson’s trichrome-stained sections of antisense (Figure 8I) or sense (Figure 8J) ODN transfected kidneys. Ang II infusion in antisense ODN transfected left kidneys mainly caused tubular atrophy, interstitial and periglomerular fibrosis, and infiltration (tubulointerstitial score: antisense ODN, 2.8 ⫾ 0.8; contralateral, 0.8 ⫾ 0.3; P ⬍ 0.05; Figure 8I). There was no statistically significant difference in the renal score between the control groups that were infused with Ang II (contralateral antisense, left sense, and contralateral sense; data not shown).

Figure 6. Implication of AT1 in the mesangial expression of SOCS. MC from wild-type (WT; 䡺) and AT1a-deficient mice (AT1⫺/⫺; f) were incubated with 10⫺7M Ang II for the indicated periods. (A) SOCS-3 and SOCS-1 mRNA expression was analyzed by RT-PCR and corrected by GAPDH expression. (B) SOCS-1 protein synthesis and STAT1 tyrosine phosphorylation were analyzed by Western blot. Data are expressed with respect to basal conditions and are mean ⫾ SD of three experiments (*P ⬍ 0.05 versus basal; #P ⬍ 0.05 versus WT).

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Figure 7. Inhibition of SOCS-3 by antisense oligodeoxynucleotides (ODN) in cultured MC and MCT. (A) Human MC were treated with SOCS-3 antisense ODN (0.5 and 1 ␮M) during 24 h and then stimulated with 10⫺7 M Ang II for 1 h. SOCS-3, c-Jun, and tubulin (loading control) expressions were analyzed by Western blot. Densitometry data of c-Jun bands (n-fold versus basal) are mean ⫾ SD of three experiments. (B) MCT were preincubated with antisense and sense ODN and subsequently transfected with the pISRE-Luc plasmid. Cells then were incubated for 24 h with Ang II, and luciferase activity was assayed. Mean ⫾ SD of four experiments (*P ⬍ 0.05 versus basal; #P ⬍ 0.05 versus Ang II). (C) MCT that were treated with antisense or sense ODN (0.5 to 3 ␮M) were transfected with S3wt expression vector and then stimulated with Ang II for 1 h. P-STAT1 and c-Fos protein levels were analyzed by Western blot. Gels are representative of three experiments.

Discussion The molecular mechanisms of Ang II signaling have been well studied, and multiple pathways such as the JAK-STAT have been implicated (10,17). Although not totally defined, the regulatory mechanisms of Ang II signal transduction comprise receptor internalization, G protein uncoupling, desensitization by PKC activation, and tyrosine phosphorylation control (9,10,17). The regulation of tyrosine phosphorylation by protein tyrosine phosphatases seems to be a key event for Ang II– induced inflammatory responses in different cell types (31,32). Recently, members of the SOCS family have been identified as negative regulators of signal transduction by temporarily inhibiting the JAK-STAT pathway (3–5). In this work, we have focused on two members of the SOCS family (SOCS-3 and SOCS-1) and analyzed their role in the regulation of Ang II signaling in the kidney. We demonstrate the induction of SOCS proteins by Ang II in renal cells and the implication of SOCS in the control of AT1 signal transduction. Ang II is a pleiotropic vasoactive peptide that not only controls cardiovascular and renal homeostasis but also promotes cell growth, migration, inflammation, and fibrosis (7,8,11). Previous papers have described that systemic infusion of Ang II into normal animals causes renal expression of cytokines (TNF-␣ and IL-6), chemokines (monocyte chemoattractant protein-1 and RANTES), growth-related factors (PDGF, endothelin-1, and c-Fos), fibrotic mediators (TGF-␤ and connective

tissue growth factor), and NO synthase (7,27,29,33–35). Ang II also induces in vivo the renal activation of several transduction pathways, including MAPK, Ras family, calcium/calmodulin protein kinase, NF-␬B, and activating protein-1 (12,17,36). In general, all of these responses correlate with inflammatory cell infiltration and glomerular and tubular lesions, indicating that Ang II acts as a true cytokine regulating many factors that contribute to inflammation and fibrosis in renal diseases (7,8). In this article, we show that systemic infusion of Ang II into rats for 3 d increased the renal expression of SOCS-3 and SOCS-1 (gene and protein), suggesting that the SOCS family is involved in the Ang II actions in the kidney. Our data are in accordance with a recent report describing that Ang II induces SOCS-3 expression in the heart (37). Because both SOCS-3 and SOCS-1 proteins are produced mainly by glomerular and tubular cells after Ang II infusion, we analyzed their expression in cultured renal cells. SOCS-3 and SOCS-1 were rapid and transiently expressed in both MC and MCT that were stimulated with Ang II in parallel with the activation of JAK2 and STAT1. Moreover, SOCS expression was resistant to the protein synthesis inhibitor cycloheximide, indicating that they are immediate-early genes induced by Ang II. Several groups, including us, have previously described the differential SOCS expression in MC that were stimulated with cytokines (25,38) and immune complexes (26), but this is the first report to describe the SOCS expression in tubular cells and

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Figure 8. In vivo effects of SOCS-3 antisense ODN on Ang II–mediated responses. (A and B) Distribution of FITC-labeled SOCS-3 antisense ODN injected into the kidney. Representative photomicrographs of transfected left kidney (A) showing FITCpositive nuclei in glomerular and tubular cells, whereas no fluorescence was observed in the contralateral right kidney (B). Rats received either SOCS-3 antisense (C, E, G, and I) or SOCS-3 sense (D, F, H, and J) ODN via the left renal vein, and 24 h later, Ang II was systemically infused during 3 d. (C and D) Immunohistochemical detection of Ang II–induced SOCS-3 revealed an important reduction by antisense ODN transfection (C) compared with sense ODN group (D). (E and F) Activation of STAT1 after Ang II infusion was analyzed by immunohistochemistry with anti–P-STAT1 antibodies. The number of positive nuclei in glomerular (arrows) and tubular cells (arrowheads) was higher in antisense ODN (E) than in sense ODN (F)


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the potential implication of SOCS in the Ang II– caused tubulointerstitial damage. The molecular mechanisms and cellular responses of the different Ang II receptors are not completely defined. Signaling pathways activated by AT1 receptor are coupled to multiple, specific signaling cascades, including phospholipases (C, A2, and D), PKC, nonreceptor tyrosine kinases (Src, JAK, focal adhesion kinase, Pyk2, and p130Cas), phosphatidylinositol 3-kinase, MAPK (ERK1/2, JNK, and p38), small G proteins, and reactive oxygen species (9,10,17). AT1 receptor can also induce transactivation of other receptors, such as endothelial growth factor and PDGF receptors (9). AT2 signaling comprises activation of protein phosphatases, NO-cGMP system, phospholipase A2, and sphingolipid-derived ceramide and can negatively regulate AT1 and cytokine receptors (10,17,39). These intracellular signaling cascades elicited by Ang II do not function independently and are actively engaged in cross-talk (10). In this work, we analyzed which Ang II receptor is implicated in the induction of SOCS expression by using two specific Ang II receptor antagonists (losartan and PD123319). We have observed that only AT1 blockade significantly reduced the renal SOCS expression in Ang II–infused rats. Similarly, in cultured renal cells, both SOCS expression and JAK-STAT activation induced by Ang II were inhibited with losartan but not with PD123319. The involvement of AT1 receptor on the renal expression of SOCS was confirmed by using cultured MC from AT1⫺/⫺ mice, which failed to respond to Ang II activation, as shown by impaired SOCS expression and STAT1 activation. To elucidate the role of SOCS proteins in Ang II signaling in renal cells, we analyzed the effects of their overexpression and inhibition on the JAK-STAT pathway regulation. In MC and MCT, transfection with S3wt and S1wt expression vectors inhibited the Ang II–stimulated STAT1 tyrosine phosphorylation and STAT-luciferase activity. Moreover, reduced expression of SOCS-3 protein by antisense ODN leads to an increase in the magnitude of Ang II responses, including STAT activation and c-Fos/c-Jun expression. These results indicate that SOCS proteins are negative regulators of Ang II–induced JAK-STAT activation, although this is not the only mechanism involved in the control of this signal transduction pathway, as described previously in other cell types (1,2). In vascular smooth muscle cells, JAK-STAT activation by Ang II may be regulated by protein phosphatases SHP-1 and SHP-2. It is interesting that whereas SHP-1 mediates JAK2 dephosphorylation, SHP-2 exerts a positive regulation (31,32). Then, it is feasible that both

rats. (G and H) Renal c-Fos protein expression induced by Ang II was also increased by antisense ODN (G) when compared with the control sense ODN (H). (I and J) Differences in Ang II–induced renal damage between sense and antisense treated kidneys were examined in Masson’s trichrome-stained sections. Ang II infusion in SOCS-3 knockdown rats (I) caused more severe tubular atrophy, increased leukocyte infiltration, and even the appearance of interstitial and periglomerular fibrosis when compared with sense ODN transfected rats (J). Magnification, ⫻200.

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SOCS and phosphatases may be involved in the negative regulation of Ang II signal transduction in renal cells. Consistently, Bartoe et al. (40) recently demonstrated that both SOCS-3 and SHP-2 act independently as negative regulators of leukemia inhibitory factor in neuronal cells. In addition to JAK-STAT, SOCS family can regulate other transcription pathways. Recent papers have described that SOCS-3 and SOCS-1 can inhibit nuclear factor of activated T cells activation through the association with calcineurin, sustain MAPK activation through the interaction with Ras, and mediate NF-␬B activation by inhibiting tyrosine kinase pathways (41– 43). In heart, SOCS-3 inhibits three independent signaling pathways, STAT3, ERK1/2, and Akt, thus constituting a negative feedback circuit for myocyte hypertrophy and survival (44). Whether the same occurs in Ang II signaling in the kidney is the aim of future studies. Evidence is emerging for the involvement of SOCS proteins in inflammatory diseases, such as rheumatoid arthritis, cerebral ischemia, heart failure, and renal injury (2,6,25,26,44 – 47). In this sense, inhibition of SOCS-1 or SOCS-3 expression enhanced the tissue damage (46,47), whereas SOCS-3 gene expression effectively reduced the disease progression (44,45). Consistently, our study shows that local suppression of SOCS-3 resulted in a more severe renal damage, and antisense ODN transfected kidneys exhibited a greater response to Ang II (STAT activation and c-Fos expression). On that basis, we can speculate that induction of SOCS proteins in the kidney may be a therapeutic strategy for treating Ang II–related renal diseases. On the whole, our results indicate that SOCS proteins may play a regulatory role in signal transduction pathways of Ang II and implicate SOCS as important modulators of Ang II– mediated renal diseases. Moreover, both SOCS-3 and SOCS-1 proteins participate in the control of AT1 receptor signal transduction. The negative regulatory role of SOCS proteins could be important in preventing the potentially deleterious chronic activation of downstream signaling pathways of Ang II in glomerular and tubular cells during renal injury.

Acknowledgments This work was supported by grants from Fondo Investigaciones Sanitarias (FIS00/0111, PI02/0539, and PI02/0513), Comunidad de Madrid (CAM 08.4/0014/2001 and CAM 08.4/0021/2003 1), and European Union (QLRT-2001-01215). O.L.-F. and M.R. are fellows from Fondo Investigaciones Sanitarias, and P.H.-V. is a fellow from Comunidad de Madrid. Part of these data were presented at the 36th Annual Meeting of American Society of Nephrology, November 2003, San Diego, CA. We especially thank Drs. T. Sugaya, H. Boeuf, and H. Brady for the AT1⫺/⫺ mice, SOCS expression vectors, and primers, respectively. We are also grateful to Drs. J.L. Martin-Ventura and A. Kuhn for helpful comments and corrections on the manuscript.

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Suppressors of Cytokine Signaling Regulate Angiotensin IIâActivated ...

Suppressors of Cytokine Signaling Regulate Angiotensin IIâActivated ...

Suppressors of Cytokine Signaling Regulate Angiotensin IIâActivated ...

Suppressors of Cytokine Signaling Regulate Angiotensin IIâActivated ...