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The Journal of Immunology, 2003, 171: 257-266.
Copyright © 2003 by The American Association of Immunologists

TNF-{alpha} Induces Tyrosine Phosphorylation and Recruitment of the Src Homology Protein-Tyrosine Phosphatase 2 to the gp130 Signal-Transducing Subunit of the IL-6 Receptor Complex 1

Johannes G. Bode2,*, Jens Schweigart*, Jan Kehrmann*, Christian Ehlting*, Fred Schaper{dagger}, Peter C. Heinrich{dagger} and Dieter Häussinger*

* Klinik für Gastroenterologie, Hepatologie und Infektiologie, Medizinische Klinik der Heinrich Heine Universität, Düsseldorf, Germany; and {dagger} Institut für Biochemie, Universitätsklinikum der Rheinisch-Westfälischen Technischen Hochschule Aachen, Aachen, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Recently, it has been demonstrated that TNF-{alpha} and LPS induce the expression of suppressor of cytokine signaling 3 (SOCS3) and inhibit IL-6-induced STAT3 activation in macrophages. Inhibitor studies suggested that both induction of SOCS3 and inhibition of IL-6-induced STAT3 activation depend on the activation of p38 mitogen-activated protein kinase. Since recruitment of the tyrosine phosphatase Src homology protein tyrosine phosphatase 2 (SHP2) to the signal-transducing receptor subunit gp130 attenuates IL-6-mediated STAT-activation, we were interested in whether TNF-{alpha} also induces the association of SHP2 to the gp130 receptor subunit. In this study we demonstrate that stimulation of macrophages and fibroblast cell lines with TNF-{alpha} causes the recruitment of SHP2 to the gp130 signal-transducing subunit and leads to tyrosine phosphorylation of SHP2 and gp130. In this context the cytoplasmic SHP2/SOCS3 recruitment site of gp130 tyrosine 759 is shown to be important for the inhibitory effects of TNF-{alpha}, since mutation of this residue completely restores IL-6-stimulated activation of STAT3 and, consequently, of a STAT3-dependent promoter. In this respect murine fibroblasts lacking exon 3 of SHP2 are not sensitive to TNF-{alpha}, indicating that functional SHP2 and its recruitment to gp130 are key events in inhibition of IL-6-dependent STAT activation by TNF-{alpha}. Furthermore, activation of p38 mitogen-activated protein kinase is shown to be essential for the inhibitory effect of TNF-{alpha} on IL-6 signaling and TNF-{alpha}-dependent recruitment of SHP2 to gp130.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The inflammatory cascade underlying the immune response of an organism toward pathogens is largely controlled by the actions of different mediators released under inflammatory conditions. Upon activation, blood monocytes and tissue macrophages release a set of primary inflammatory mediators, such as IL-1{beta} and TNF-{alpha}, thereby inducing the synthesis and secretion of several secondary cytokines and chemokines, such as IL-6 and IL-8, by macrophages, monocytes, and local stromal cells. Recruitment of other immune effector cells by chemotaxis then rapidly augments the local inflammatory response to counteract the inflammatory stimulus and to remove cellular debris associated with tissue damage (reviewed in Refs. 1, 2, 3, 4, 5).

In this context IL-6 has been considered a proinflammatory cytokine, since its expression is elevated in inflammatory diseases and is induced by inflammatory stimuli, such as IL-1{beta} and TNF-{alpha} (1, 2, 5). Many of its proinflammatory and immune properties are due to the activation of B cells to produce Abs due to the stimulation of T cells and the induction of chemokine and adhesion molecule expression in endothelial cells (2, 5, 6, 7, 8). However, on macrophages (5, 6, 7), astrocytes (9), and fibroblasts (10) IL-6 has suppressive effects on their inflammatory response and represses the expression of IL-12, IFN-{gamma}, IL-1{beta}, TNF-{alpha}, adhesion molecules, and proteases both in vitro and in vivo (5, 6, 7, 8, 9, 10). In line with this, IL-6 is able to induce the expression of IL-1{beta}R antagonist and soluble TNF receptor p55, antagonizing IL-1{beta} and TNF-{alpha} activities, respectively (11, 12).

Thus, IL-6 mediates both pro- and anti-inflammatory effects. IL-6 is expressed throughout every stage of the inflammatory response, and at least during the onset of inflammation several of its anti-inflammatory properties would be inconvenient (1, 2, 3, 4, 5). Therefore, it is mandatory that molecular mechanisms modulating IL-6 action exist.

IL-6 mediates its biological activities through a receptor complex composed of the specific receptor subunit gp80 and a dimer of the signal-transducing receptor subunit gp130 (2). After ligand binding and dimerization of gp130, tyrosine kinase of the Janus family (Jak),3 Jak1, Jak2, and Tyk2, constitutively associated with gp130, becomes activated by autophosphorylation. The gp130 subsequently tyrosine-phosphorylated on its cytoplasmic tail recruits transcription factors of the STAT family (13, 14) and Src homology protein tyrosine phosphatase 2 (SHP2) (15) via specific phosphotyrosine-SH2 domain interactions involving the tyrosine 759 of the gp130 receptor (16, 17). In turn, these components also become tyrosine phosphorylated. Activated STATs homo- or heterodimerize (18), translocate to the nucleus, and bind to enhancer elements of target genes (19).

The Jak/STAT signal transduction pathway is under negative control by several different mechanisms. The presence of a nuclear phosphatase leading to dephosphorylation and inactivation of activated STATs in the nucleus has been proposed by Haspel et al. (20). On the other hand, Kim and Maniatis (21) demonstrated a proteasome-dependent loss of activated STAT1 in the nucleus. Recently, another group of inhibitors of the Jak/STAT pathway has been described: STAT-binding proteins, known as protein inhibitors of activated STATs (PIAS) (22, 23). Although the PIAS do not contain phosphotyrosine binding domains such as SH2 or protein tyrosine binding domains, they associate with activated, tyrosine-phosphorylated STATs, leading to a loss of STAT-DNA binding activity. The mechanism and regulation of this highly specific interaction of protein inhibitor of activated STATs with activated STAT factors remain to be elucidated. Another new family of inhibitors of cytokine signaling has recently been discovered in three different laboratories. These proteins are referred to as suppressors of cytokine signaling (SOCS) (24), Jak-binding proteins (25), or STAT-induced STAT inhibitors (26). SOCS proteins can also be regarded as feedback inhibitors of cytokine signaling, since they are partially induced by cytokines mediating their own signals via activation of the Jak/STAT cascade. Furthermore, the protein tyrosine phosphatase SHP2 was found to inhibit IL-6 signal transduction. Activation of the IL-6R complex leads to recruitment of SHP2 to tyrosine 759 in gp130 and its subsequent tyrosine phosphorylation (15). SHP2 activation is a crucial event for the induction of the mitogen-activated protein kinase (MAPK) pathway upon IL-6 stimulation (27). Mutation of Tyr759 in gp130 results in enhanced and prolonged STAT1 and STAT3 activation by IL-6-type cytokines and increased gene induction of STAT-dependent genes (28, 29, 30).

In terms of cytokine cross-talk, all these mechanisms can likewise be used by other mediators to influence cytokine signaling via the Jak/STAT signal transduction cascade and therefore represent a potential molecular switch modulating the cellular response toward IL-6, e.g., TNF-{alpha} and LPS act as inhibitors of IL-6 and IFN-{gamma}-mediated STAT-activation, at least in the case of TNF-{alpha} probably through p38MAPK-dependent induction of SOCS3 (31, 32).

Since SHP2 recruitment to the gp130 receptor negatively regulates IL-6-induced STAT activation (33), we asked whether SHP2 might participate in TNF-{alpha}-mediated modulation of IL-6 signal transduction. Using mouse peritoneal RAW 264.7 macrophages and NIH-3T3 fibroblasts, we observed association to gp130 and tyrosine phosphorylation of SHP2 upon stimulation with TNF-{alpha}. Finally, mutation of the SHP2 binding site tyrosine 759 of gp130 results in an insensitivity toward preincubation with TNF-{alpha}. The recruitment of SHP2 to gp130 as a key event for the TNF-{alpha}-induced inhibition of IL-6-dependent STAT activation is further supported by studies in murine fibroblasts lacking functional SHP2.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials

Polymerase was purchased from Roche (Mannheim, Germany); oligonucleotides were obtained from MWG-Biotech (Ebersberg, Germany); recombinant erythropoietin (Epo) was a gift from Drs. J. Burg and K. H. Sellinger (Roche); DMEM, DMEM nutritional mix F-12, OpitMEM, and FCS were obtained from Life Technologies (Eggstein, Germany). Recombinant human IL-6 and soluble IL-6R sgp80 were prepared as previously described (34). The following Abs were used: rabbit polyclonal Ab specifically raised against extracellular signal-regulated kinase 2 (Erk2), SHP2, or gp130 (M20; Santa Cruz Biotechnology, Santa Cruz, CA); rabbit polyclonal Ab specifically raised against STAT3 phosphorylated at tyrosine 705, and mouse mAb specifically recognizing phosphorylated (activated) p42/p44 were obtained from Cell Signal Transduction Technology (Beverly, MA); mAb against STAT3 was purchased from BD Transduction Laboratories (San Diego, CA). For detection of tyrosine phosphorylation, anti-phosphotyrosine mouse mAb 4G10 (Upstate Biotechnology, Lake Placid, NY) was used.

Cultivation and stimulation of cells

RAW 264.7 cells were cultivated in DMEM (1000 mg of glucose/liter) with Glutamax supplemented with 10% heat-inactivated FCS, streptomycin (100 mg/liter), and penicillin (60 mg/L). NIH-3T3 cells were grown in DMEM (4500 mg of glucose/L) with Glutamax supplemented with 10% heat-inactivated FCS, streptomycin (100 mg/L), and penicillin (60 mg/L). 3T3 embryonal fibroblasts were from SHP2 wild-type mice (SHP2-wt) or SHP2 exon 3-deficient mice (SHP2-mut) and were grown in DMEM (4500 mg of glucose/L) with Glutamax, 10% FCS, 100 mg/L streptomycin, and 60 mg/L penicillin (35).

All experiments, except those for the analysis of protein phosphorylation, were performed in the respective culture medium supplemented with 10% FCS. For the analysis of STAT3 and Erk1/2 phosphorylation, medium was changed 12 h before the experiments were performed, and incubation was continued in the respective culture medium supplemented with 0.5% FCS to reduce background activity of Erk-type MAPKs.

Nuclear extracts were prepared as described by Andrews and Faller (36). Protein concentration was determined by protein assay (Bio-Rad, Munich, Germany).

Transfection procedure and reporter gene assay

For reporter gene assay NIH-3T3 or MEF SHP2-wt and MEF SHP2-mut cells were transfected using Lipofectamine 2000 (Invitrogen, San Diego, CA). Briefly, cells were grown in DMEM with 4500 mg of glucose/L supplemented with 10% FCS on 12-well plates toward 80–95% confluence. Cells were preincubated with 1 ml of OptiMEM 1 h before transfection. Lipofectamine 2000 (6 µl) and in total 1.5 µg of DNA were prediluted in 100 µl of OptiMEM each. Diluted reagent and DNA were mixed, incubated for 20 min at room temperature, and then added to the cells. After 16 h medium was changed, and incubation was continued in DMEM with 4500 mg of glucose/liter supplemented with 10% FCS, streptomycin (100 mg/L), and penicillin (60 mg/L). For determination of the transcriptional activation of Elk-1, medium was changed before stimulation to culture medium supplemented with 0.5% FCS to reduce background activity. Cells were stimulated as indicated. Cell lysis and luciferase assays were conducted using the dual luciferase kit (Promega, Madison, WI) as described by the manufacturer. Luciferase activity values were normalized to transfection efficiency monitored by the cotransfected Renilla expression vector (Promega). Error bars are the SD calculated from three independent experiments performed on the same day. The data shown are representative of at least three different experiments with similar results.

For higher transfection efficiency cells were transfected using Lipofectamine 2000 and a modified transfection procedure. In brief, 6 µl of Lipofectamine 2000 were diluted in 250 µl of OptiMEM, and 4 µg of DNA was diluted in 50 µl of OptiMEM. Diluted reagent and DNA were mixed and incubated for 20 min at room temperature. Meanwhile a confluent flask (75 cm2) of NIH-3T3 cells was trypsinized and, after centrifugation, resuspended in 3.5 ml of DMEM with 4500 mg glucose/L supplemented with 10% FCS, then 300 µl of resuspended cells were added to the transfection mixture. One milliliter of DMEM with 4500 mg of glucose/L supplemented with 10% FCS was added, and cells were seeded on a 60-mm culture dish. After a 12-h incubation at 37°C, medium was replaced by DMEM with 4500 mg glucose/L supplemented with 10% FCS, streptomycin (100 mg/L), and penicillin (60 mg/L), and cell culture was continued for another 24 h. Thereafter, experiments were performed as outlined in Results.

DNA constructs

pGL3-{alpha}2M-215Luc contains the promoter region -215 to 18 of the rat {alpha}2-macroglobulin gene fused to the luciferase encoding sequence and was described previously (30). cDNAs for dominant negative p38MAPK mutant tagged with the flag epitope (37) were cloned into the KRSPA expression vector as described by Flory et al. (38). The expression vector pRc/CMV-EG encoding the chimeric EpoR/gp130 receptor (pRc/CMV-EG (YYYYYY)) and the mutants where tyrosine 759 in the cytoplasmic domain of gp130 was exchanged for phenylalanine (pRc/CMV-EG (YFYYYY)) or all tyrosines of the cytoplasmic domain of gp130 were replaced by phenylalanine (pRc/CMV-EG (FFFFFF)) have been described previously (39). The construction of pCBC1-SHP2-wt was described previously (30). For the investigation of Erk-type MAPK activation, the vectors pFR-Luc and pFA2 Elk1 of the PathDetect trans-reporting system (Stratagene, La Jolla, CA) were used. pFR-Luc encodes a sequence of five GAL4 binding elements fused to the luciferase encoding sequence. pFA2-Elk1 represents a fusion trans-activator plasmid that expresses a fusion protein of the activation domain of the transcription factor Elk1 fused with the yeast GAL4 DNA binding domain.

EMSA

EMSAs were performed as described previously (19). The protein-DNA complexes were separated on a 4.5% polyacrylamide gel containing 7.5% glycerol in 0.25-fold TBE (20 mM Tris base, 20 mM boric acid, and 0.5 mM EDTA, pH 8.0) at 20 V/cm for 4 h. Gels were fixed in 10% methanol, 10% acetic acid, and 80% water for 1 h; dried; and autoradiographed. The double-stranded 32P-labeled mutated m67SIE oligonucleotide from the c-Fos promoter (m67SIE, 5'-GATCCGGGAGGGATTTACGGGAAATGCTG-3') (40) was used for EMSA.

Coimmunoprecipitation

For immunoprecipitation cells grown in a 100-mm dish were stimulated with the respective cytokine at the concentrations indicated. Cells were washed twice with PBS supplemented with 0.1 mM Na3VO4 and solubilized in 1 ml of lysis buffer (1% Triton, 20 mM Tris-HCl (pH 7.4), 136 mM NaCl, 2 mM EDTA, 50 mM {beta}-glycerophosphate, 20 mM sodium pyrophosphate, 1 mM Na3VO4, 4 mM benzamidine, 0.2 mM Pefabloc (Roche, Mannheim, Germany), 5 µg/ml aprotinin, 5 µg/ml leupeptin, and 10% glycerol) for 20 min at 4°C. Insoluble material was removed by centrifugation, and the cell lysate was incubated overnight with specific Abs and protein A-Sepharose 4 Fast Flow (Amersham Pharmacia, Freiburg, Germany) (8 mg/ml in lysis buffer) at 4°C. After centrifugation, the Sepharose beads were washed twice with wash buffer (0.1% Triton, 20 mM Tris-HCl (pH 7.4), 136 mM NaCl, 2 mM EDTA, 50 mM {beta}-glycerophosphate, 20 mM sodium pyrophosphate, 1 mM Na3VO4, 4 mM benzamidine, 0.2 mM Pefabloc, 5 µg/ml aprotinin, 5 µg/ml leupeptin, and 10% glycerol). The samples were boiled in gel electrophoresis sample buffer, and the precipitated proteins were separated on an SDS-polyacrylamide (7.5%) gel.

Immunoblotting and immunodetection

The electrophoretically separated proteins were transferred onto polyvinylidene difluoride membranes by the semidry Western blotting method. Nonspecific binding was blocked with 3% nonfatty dry milk powder in TBS-T (20 mM Tris-HCl (pH 7.4), 137 mM NaCl, and 0.1% Tween) overnight at 4°C. For analysis of protein phosphorylation of Erk-type MAPKs or STAT3, nonspecific binding was blocked with 5% BSA in TBS-T. The blots were incubated overnight at 4°C or for 2 h at room temperature with primary Abs at the dilution indicated in TBS-T. After extensive rinsing with TBS-T, blots were incubated with secondary Abs, goat anti-rabbit IgG, or goat anti-mouse IgG conjugated to HRP for 1.5 h. After further rinsing in TBS-T, the immunoblots were developed with the ECL system following the manufacturer’s instructions (Amersham Pharmacia Biotech, Arlington Heights, IL).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
TNF-{alpha} induces recruitment of SHP2 to gp130

The SHP2 recruitment site within gp130 is involved in negative regulation of IL-6-induced STAT activation and gene induction (28, 29, 30). Thus, SHP2 might also play a role in the TNF-{alpha}-mediated modulation of IL-6 signal transduction. Therefore, we tested whether TNF-{alpha} affects SHP2 binding to gp130. Figs. 1 and 2A show SHP2 and gp130 coimmunoprecipitated by either a gp130- or an SHP2-specific Ab from total cell lysates of RAW 264.7 mouse peritoneal macrophages stimulated with TNF-{alpha} for different time periods. Association of gp130 and SHP2 was already detectable after 5 min of stimulation and became pronounced after 20–40 min. This indicates that TNF-{alpha} is able to mediate recruitment of SHP2 to gp130. Association of SHP2 and gp130 was paralleled by TNF-{alpha}-induced tyrosine phosphorylation of both proteins, as shown in Fig. 2, A and B. In contrast to IL-6, TNF-{alpha} did not lead to activation of STAT1 and -3, as shown in Fig. 2C. This indicates that TNF-{alpha} time-dependently induces association of SHP2 to the signal-transducing subunit gp130 paralleled by tyrosine phosphorylation of SHP2 and gp130. The fact that upon stimulation with TNF-{alpha} STAT3 activation was not detectable argues against TNF-{alpha}-induced release of IL-6 leading to subsequent SHP2 recruitment to gp130.



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  		FIGURE 1. TNF-{alpha}-dependent coimmunoprecipitation of gp130 and SHP2. RAW 264.7 cells were stimulated with TNF-{alpha} (10 ng/ml) for the times indicated. Cells were solubilized, and immunoprecipitation was performed using 1 µg of a polyclonal Ab specific for the tyrosine phosphatase SHP2 as described in Materials and Methods. Thereafter, precipitated proteins were separated on an SDS-polyacrylamide (7.5%) gel and blotted onto a polyvinylidene difluoride membrane. Membranes were cut in two parts at the level of ~80 kDa, and the upper part was incubated with polyclonal Ab raised against gp130 (1/2500; upper panel), whereas the lower part was incubated with polyclonal Ab for detection of SHP2 (1/4000; lower panel).

 


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  		FIGURE 2. TNF-{alpha}-dependent tyrosine phosphorylation of SHP2 and gp130. RAW 264.7 cells were stimulated with TNF-{alpha} (10 ng/ml) for the time indicated. As described in Materials and Methods, cell lysates were prepared, and immunoprecipitation and subsequent immunoblotting were performed using 1 µg of a polyclonal Ab specific for the receptor subunit gp130 (A) or the tyrosine phosphatase SHP2 (B). For detection of tyrosine-phosphorylated proteins, membranes were incubated using an mAb specifically raised against phosphotyrosine motifs (4G10; 1/1000; A, upper and third panels; B, upper panel). Blots were stripped. For A, membranes were cut in two parts at the level of ~80 kDa, and the upper part was incubated with polyclonal Ab raised against gp130 (1/1500; second panel), whereas the lower part was incubated with polyclonal Ab for detection of SHP2 (1/4000; last panel). For B, membranes were reprobed with polyclonal Ab specifically raised against SHP2 (1/4000; lower panel). C, For determination of the STAT activation cells were stimulated with TNF-{alpha} (10 ng/ml) or IL-6 (200 U/ml) for the time indicated. Determination of STAT activation was performed after preparation of nuclear extracts. Five micrograms of nuclear protein was mixed with a STAT1/3-specific 32P-labeled oligonucleotide (mutated m67SIE probe of the c-Fos promoter 5'-GAT CCG GGA GGG ATT TAC GGG AA ATG CTG-3'), and EMSAs were performed. The positions of comigrating STAT1/3 heterodimer and STAT3 homodimer from IL-6 stimulated HepG2 cells are indicated by arrows.

 
TNF-{alpha} modulates IL-6-induced association of SHP2 with gp130 and inhibits IL-6-mediated activation of STAT3, whereas activation of Erk is not affected

To further analyze the cross-talk between TNF-{alpha} and IL-6 signaling the effect of TNF-{alpha} on the IL-6-dependent recruitment of SHP2 to the cytoplasmic tail of gp130 was studied in RAW 264.7 macrophages. As shown in Fig. 3A, SHP2 was recruited to gp130 upon stimulation of RAW 264.7 macrophages with IL-6. Recruitment of SHP2 to gp130 was most pronounced after ~20 min of treatment with IL-6 and was paralleled by IL-6-induced tyrosine phosphorylation of gp130 and SHP2. Pretreatment with TNF-{alpha} for 40 min led to maximal complex formation of gp130 and SHP2 after 5 min of IL-6 treatment; this declined thereafter to baseline levels within 40 min. Since this time course (complex formation 40 min post-TNF-{alpha}) resembles that shown in Fig. 1, it is likely that TNF-{alpha} prevents SHP2 recruitment in response to IL-6 treatment, although the preceding TNF-{alpha}-induced complex formation is unaffected.



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  		FIGURE 3. Influence of TNF-{alpha} pretreatment on IL-6-mediated SHP2 recruitment to gp130 and on IL-6-induced STAT3-activation. After a preincubation period of 40 min with or without TNF-{alpha} (10 ng/ml) RAW 264.7 cells were stimulated with IL-6 (200 U/ml) for the times indicated. For A, cells were solubilized, and immunoprecipitation was performed using a polyclonal Ab specific for SHP2 as described in Materials and Methods. For analyses of tyrosine phosphorylation (first and third panels), membranes were incubated using a phosphotyrosine-specific monoclonal Ab (4G10; 1/1000). Blots were stripped, and membranes were cut into two parts at the level of ~80 kDa. The upper part was incubated with polyclonal Ab specific for gp130 (1/2500; second panel), whereas the lower part was incubated with polyclonal Ab raised against SHP2 (1/4000; lower panel). For B, determination of STAT DNA binding from identically treated cells was conducted as described in Fig. 1.

 
Additionally, we analyzed the influence of TNF-{alpha} preincubation on the two major IL-6 downstream signaling events, the activation of STAT factors and of Erk-type MAPKs. It is conspicuous that pretreatment with TNF-{alpha} inhibits IL-6-dependent tyrosine phosphorylation of STAT3 and STAT3 DNA binding (Figs. 4A and 3B), whereas activation of Erk-type MAPKs was not inhibited, but, rather, was accelerated and enhanced (Fig. 4B), particularly during the first 10–20 min, which correlates to the SHP2 recruitment found in macrophages upon costimulation with TNF-{alpha} and IL-6 shown in Fig. 3A. These findings suggest that TNF-{alpha} not only inhibits IL-6 signaling, but modulates IL-6-induced signal transduction.



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  		FIGURE 4. Influence of TNF-{alpha} pretreatment on IL-6-induced phosphorylation of STAT3- or Erk-type MAPK. After a preincubation period of 40 min with or without TNF-{alpha} (10 ng/ml), RAW 264.7 cells were stimulated with IL-6 (200 U/ml), and incubation was continued for the times indicated. Cells were then solubilized for immunoblotting. Tyrosine phosphorylation of STAT3 (A) and tyrosine/threonine phosphorylation of Erk-type MAPK (B) were determined by immunoblot analyses using specific Abs recognizing STAT3 phosphorylated at tyrosine 705 or the activated (phosphorylated) form of Erk1/2 (upper panels). As a loading control, blots where stripped and reprobed with Abs specific for STAT3 or Erk2 (lower panels).

 
Tyrosine 759 of gp130 mediates the inhibition of IL-6-dependent STAT activation by TNF{alpha}

Recruitment of SHP2 and SOCS3 to the gp130 receptor subunit depends on the phosphorylation of the tyrosine residue 759 of gp130 (15, 39, 41). To understand the role of this SHP2/SOCS3 recruitment site in TNF-{alpha}-mediated inhibition of IL-6-induced STAT activation, signaling through a gp130 receptor mutant, where a phenylalanine residue was substituted for the tyrosine residue 759 of the SHP2/SOCS3 recruitment site, was analyzed. To perform such studies NIH-3T3 cells were selected because these cells can easily be transfected. As shown in Fig. 5, A and B, TNF-{alpha} exerts the same effects on IL-6 signal transduction in NIH-3T3 cells as those described for RAW 264.7 macrophages. Preincubation with TNF-{alpha} inhibits IL-6-induced STAT3 activation (Fig. 5A), and TNF-{alpha} again leads to recruitment of SHP2 to gp130 (Fig. 5B), indicating that these cells are suitable for the following experiments.



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  		FIGURE 5. Involvement of tyrosine 759 of the cytoplasmic tail of gp130 in TFN-{alpha}-mediated inhibition of IL-6 signaling. A, NIH-3T3 cells were pretreated for 40 min with TNF-{alpha} (10 ng/ml) and then stimulated with IL-6 (200 U/ml) and soluble gp80 (10 µg/ml) for the times indicated. After preparation of nuclear extracts, STAT activation was analyzed by EMSA as described in Fig. 1 and Materials and Methods. B, After stimulation of NIH-3T3 cells with TNF-{alpha} (10 ng/ml) for the time indicated, cell lysates were prepared, and immunoprecipitation was performed using 1 µg of an Ab specific for SHP2. Subsequent to immunoblotting, membranes were cut into two parts at the level of ~80 kDa, and the upper part was incubated with polyclonal Ab raised against gp130 (1/2500; upper panel), whereas the lower part was incubated with polyclonal SHP2 Ab (1/4000; lower panel). C, NIH-3T3 cells were cotransfected with a reporter gene construct containing the {alpha}2-macroglobulin promoter (-209 to +8) fused to the firefly luciferase gene and expression vectors for chimeric receptors containing the extracellular domain of the Epo receptor and the transmembrane and wild-type (EG(YYYYYY)) or mutated cytoplasmic domains of gp130 where the tyrosine residue 759 of the SHP2/SOCS3 recruitment site (EG(YFYYYY)) or all six tyrosine residues (EG(FFFFFF)) were substituted by phenylalanine. An expression vector for Renilla luciferase was cotransfected for monitoring transfection efficiency. Two days after transfection, cells were preincubated with 10 ng/ml TNF-{alpha} for 40 min and stimulated with or without Epo (7 U/ml) for 10 h as shown. Luciferase activity in cellular extracts of these cells was determined and normalized to the Renilla luciferase activity as outlined in Materials and Methods.

 
For analyzing signaling through mutated receptor constructs we had to avoid stimulation of endogenous wild-type receptors. Therefore, chimeric receptor containing the extracellular domain of the Epo receptor and the transmembrane and wild-type (EG (YYYYYY)) or mutated cytoplasmic domains of gp130 (EG(YFYYYY)) or for control EG(FFFFFF)) were used. These constructs were cotransfected together with a STAT3-responsive {alpha}2-macroglobulin promoter/luciferase reporter into NIH-3T3 fibroblasts.

Stimulation of cells expressing EG(YYYYYY) with Epo led to a significant expression of the reporter. According to the findings for IL-6-induced STAT3 activation, transcriptional activation of the reporter by Epo was inhibited by preincubation with TNF-{alpha} (Fig. 5C). This inhibitory activity of TNF-{alpha} depended on the presence of Y759 in gp130, since its mutation counteracted the inhibitory function of TNF-{alpha} and led to increased IL-6-induced reporter activity. Receptors lacking all cytoplasmic tyrosine motifs (EG(FFFFFF)) did not mediate any significant gene induction.

TNF{alpha}-mediated recruitment of SHP2 to gp130 and inhibition of IL-6-induced STAT activation involve tyrosine kinase activity and activation of p38MAPK

Previous studies suggested that p38MAPK is involved in the inhibition of IL-6-induced STAT activation by proinflammatory mediators such as TNF-{alpha} (31, 42, 43). Thus, it was intriguing to investigate the effect of p38MAPK on TNF-{alpha}-induced SHP2 recruitment to gp130, inhibition of IL-6-induced STAT activation, and gene expression. Until now, the role of p38MAPK for the inhibitory effects of TNF-{alpha} on IL-6-induced STAT3 activation was only demonstrated by studies using low m.w. inhibitors thought to be specific for p38MAPK. In this study we performed a more specific experimental approach using dominant negative p38MAPK (37) to confirm the inhibitory function of p38MAPK.

As shown in Fig. 6 the inhibition of gp130-mediated STAT3 activation (Fig. 6A) and transcriptional activation of the STAT3-responsive reporter gene (Fig. 6B) by TNF-{alpha} pretreatment was completely restored by cotransfection of this inactive mutant of p38MAPK (compare two left panels in Fig. 6, A and B). Thus, these findings provide further evidence that activation of p38MAPK is indeed involved in inhibition of gp130-dependent STAT3 activation by TNF-{alpha}.



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  		FIGURE 6. Involvement of p38MAPK in inhibition of IL-6 signaling by TNF-{alpha}. A, NIH-3T3 cells were transiently cotransfected with either the empty KRSPA vector or with a flag-tagged kinase-deficient mutant of p38MAPK (AF; dominant negative (dn) p38MAPK) and chimeric receptor constructs containing the transmembrane and wild-type (EG(YYYYYY)) or mutated cytoplasmic domains (EG(YFYYYY)) of gp130. After 2 days cells were pretreated for 40 min with TNF-{alpha} as indicated and then stimulated with Epo (7 U/ml) for 30 min. Thereafter, cells were harvested, and nuclear extracts for the assessment of STAT activation were prepared. STAT activation was analyzed by EMSA as outlined in Fig. 2. B, NIH-3T3 cells were transiently cotransfected with either the empty KRSPA (control) vector or with dn p38MAPK and the respective chimeric receptor construct EG(YYYYYY) or EG(YFYYYY). For determination of the transcriptional activation the {alpha}2-macroglobulin promoter reporter gene construct was cotransfected. Transfection efficiency was monitored by cotransfected Renilla luciferase cDNA. Two days after transfection cells were preincubated with 10 ng/ml TNF-{alpha} for 40 min and stimulated with or without Epo (7 U/ml) for 10 h as shown. Luciferase activity in cellular extracts of these cells was determined and normalized to the Renilla luciferase activity as outlined in Materials and Methods.

 
However, the enhanced transcriptional activation achieved by cotransfection of the inactive p38MAPK was not as strong as that observed by mutating tyrosine 759 of gp130 (compare panels 2 and 3 in Fig. 6, A and B). Furthermore, no additional effect of dominant negative p38MAPK was observed in cells expressing EG (YFYYYY) (panels 3 and 4 in Fig. 6B). Thus, these data indicate that besides the inhibitory activity of p38MAPK further inhibitory signals are integrated to gp130-dependent signal transduction via tyrosine 759.

To determine whether TNF-{alpha}-induced recruitment of the protein tyrosine phosphatase SHP2 to gp130 might depend on p38MAPK or tyrosine kinase activity, inhibitor studies were performed in RAW264.7 cells. As shown in the coimmunoprecipitation studies with SHP2-specific Abs in Fig. 7A, inhibition of p38MAPK activation by pretreatment with SB202190, an inhibitor supposed to be specific for p38MAPK, reduces TNF-{alpha}-dependent SHP2 recruitment to gp130. These data suggest that activation of p38MAPK might be involved in the recruitment of SHP2 to gp130 upon stimulation with TNF-{alpha}. On the other hand, pretreatment with the tyrosine kinase inhibitor genistein also inhibited SHP2 recruitment to gp130 upon stimulation with TNF-{alpha}, indicating that tyrosine kinase activity is also required (Fig. 7B).



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  		FIGURE 7. Influence of the activation of p38MAPK or protein tyrosine kinase on TNF-{alpha}-mediated recruitment of SHP2 to gp130. Following a preincubation period for 40 min with SB 202190 (10 µM; A) or 40 min with Genistein (100 µM; B), RAW 264.7 cells were treated with 10 ng/ml TNF-{alpha} as indicated (40 min in A). The extract of total protein was submitted to immunoprecipitation as described in Materials and Methods using 1 µg of Abs specific for the SHP2. After immunoblotting membranes were cut in two parts at the level of ~80 kDa and the upper part was incubated with polyclonal Ab specifically raised against gp130 (1/2500; upper panel), whereas the lower part was incubated with polyclonal Ab specifically raised against SHP2 (1/4000; lower panel).

 
p38MAPK counteracts MAPK activity induced by TNF-{alpha}, but does not play a specific role for IL-6-induced activation of MAPK activity, and the effects of TNF-{alpha} on this

SHP2 have been demonstrated to serve as an adaptor protein linking gp130 signal transduction to Erk-type MAPK signaling (27). We therefore asked whether p38MAPK might also be involved in activation of Erk-1/2 by IL-6, TNF-{alpha}, or TNF-{alpha} plus IL-6. To address this question, reporter gene assays were performed in NIH-3T3 cells using a plasmid that expresses a fusion protein of the activation domain of the transcription factor Elk1 (pFA-Elk1) fused with the yeast GAL4 DNA binding domain. Phosphorylation of the Elk1 activation domain induces transcriptional activation of a reporter plasmid encoding a sequence of five GAL4 binding elements fused to the luciferase-encoding sequence (pFA-Luc).

As shown in Fig. 8 transcriptional activation via the Elk1 trans-activating fusion protein is induced by stimulation of the cotransfected chimeric EG (YYYYYY) receptor with erythropoietin. According to the data shown for Erk-type MAPK activation in Fig. 4, costimulation with TNF-{alpha} or stimulation with TNF-{alpha} alone results in a much stronger expression of luciferase activity. However, cotransfection of an inactive mutant of p38MAPK leads to a significant enhancement of the transcriptional activation of Elk1 after costimulation with TNF-{alpha} plus Epo or stimulation with TNF-{alpha} alone, whereas basal and Epo-induced activation was only slightly affected. Since activation of Elk1 is downstream from Erk/MAPK (44) one can conclude from these data that p38MAPK counteracts MAPK activity induced by TNF-{alpha}, but does not play a specific role for IL-6-induced activation of MAPK activity and the effects of TNF-{alpha} on this. With respect to this, it is interesting to note that p38MAPK is involved in the stress-induced expression of members of the MAPK phosphatase family, thereby counteracting the activation of MAPKs (45, 46, 47). Thus, one might speculate that TNF-{alpha} balances the activation of Erk-1/2 via p38MAPK-dependent activation of members of the MAPK phosphatase family.



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  		FIGURE 8. p38MAPK counteracts MAPK activity induced by TNF-{alpha}, but does not play a specific role for IL-6-induced activation of MAPK activity and the effects of TNF-{alpha} on this. NIH-3T3 cells were cotransfected with the chimeric receptor (EG(YYYYYY), the pFR-Luc plasmid and the pFA2 Elk1 plasmid of the PathDetect trans-reporting system from Stratagene and either the empty KRSPA vector or with a flag tagged kinase-deficient mutant of p38MAPK (AF; dominant negative (dn) p38MAPK). Two days after transfection cells were preincubated with 10 ng/ml TNF-{alpha} for 40 min and stimulated with or without Epo (7 U/ml) for 10 h as shown. Luciferase activity in cellular extracts of these cells was determined and normalized to the Renilla luciferase activity as outlined in Materials and Methods.

 
Involvement of the tyrosine phosphatase SHP2 in inhibition of IL-6-induced STAT activation by TNF{alpha}

As shown in Fig. 9, the expression of SHP2 strongly suppressed STAT activation mediated by the wild-type EpoR/gp130 chimera, indicating the inhibitory potential of SHP2 with respect to gp130-dependent signal transduction. To further reveal the implication of SHP2 in inhibition of IL-6 signaling by TNF-{alpha}, the inhibition of STAT3 and promoter activation was analyzed in murine fibroblasts lacking exon 3 of SHP2. These cells express a mutant SHP2 protein lacking 65 aa within the N-terminal SH2 domain (35). In clear contrast to the corresponding wild-type fibroblasts (SHP2-wt), IL-6-induced STAT3 tyrosine phosphorylation and DNA binding in SHP2-mut cells was almost not affected by preincubation with TNF-{alpha} (Fig. 10, A and B). Accordingly, preincubation of wild-type cells with TNF-{alpha} inhibited gp130-dependent transcriptional activation of the {alpha}2-macroglobulin promoter reporter gene construct, whereas no inhibitory effect of TNF-{alpha} could be observed in cells expressing the SHP2-mutant (Fig. 10C). These data clearly indicate that SHP2 is involved in inhibition of IL-6 signal transduction by TNF-{alpha}.



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  		FIGURE 9. Effects of SHP2 on IL-6-induced STAT-activation. NIH-3T3 cells were cotransfected with either the empty pCBC1 vector or with the expression plasmid pCBC1-SHP2WT (SHP2 wt) encoding for the wild-type protein tyrosine phosphatase SHP2 and the chimeric receptors (EG(YYYYYY) or EG(YFYYYY). After 2 days cells were pretreated for 40 min with TNF-{alpha} as indicated and then stimulated with Epo (7 U/ml) for 30 min. Thereafter cells were harvested, and nuclear extracts for the assessment of STAT activation were prepared. STAT activation was analyzed by EMSA as outlined in Fig. 2.

 


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  		FIGURE 10. Role of SHP2 for the inhibitory effects of TNF-{alpha} on IL-6-signaling. A, Murine fibroblasts lacking exon 3 of SHP2 (SHP2 mut) derived from the respective knockout mice and the corresponding wild-type fibroblasts (SHP2 wt) were pretreated with 10 ng/ml TNF-{alpha} for 40 min and stimulated with 150 U/ml of IL-6 and soluble IL-6 receptor (sgp80; 10 µg/ml) for the times indicated. Thereafter, cells were harvested, and nuclear extracts for the assessment of STAT activation and tyrosine phosphorylation were prepared. STAT activation was analyzed by EMSA as outlined in Fig. 2. B, Tyrosine phosphorylation of STAT3 was assessed by immunoblot analyses using Abs recognizing STAT3 tyrosine phosphorylated at tyrosine 705. As a loading control blots were stripped and reprobed with Abs specific for STAT3 (second panel). C, SHP2 wt and SHP2 mut fibroblasts were cotransfected with the {alpha}2-macroglobulin promoter reporter gene construct and the chimeric receptors EG(YYYYYY). Two days after transfection, cells were preincubated with 10 ng/ml TNF-{alpha} for 40 min and stimulated with or without Epo (7 U/ml) for 10 h as shown. Luciferase activity in cellular extracts of these cells was determined and normalized to the Renilla luciferase activity as outlined in Materials and Methods.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the past it became increasingly evident that the IL-6 signal transduction is substantially modulated by proinflammatory mediators such as TNF-{alpha}, IL-1{beta}, and LPS. It was demonstrated that TNF-{alpha} and LPS inhibit IL-6-induced activation of STAT3 in several types of macrophages, monocytic cell lines, and synovial fibroblasts (31, 42, 43). Furthermore, IL-1{beta} inhibits IL-6-induced signal transduction and expression of acute phase proteins in liver cells (48, 49, 50, 51). The underlying molecular mechanisms for this cytokine cross-talk on the level of signal transduction are scarcely known. Induction of SOCS3 expression, which was at least partially dependent on activation of p38MAPK, has been proposed as a possible mechanism for the inhibitory effects of TNF-{alpha} and LPS on IL-6 signaling (31). On the other hand, several lines of evidence exist that p38MAPK-dependent inhibition of IL-6-induced STAT3 by IL-1{beta} occurs in the absence of new protein synthesis. This indicates that in this context protein synthesis, and thus SOCS expression, is not essential for inhibition of IL-6 signaling by IL-1{beta} (42, 43). Furthermore, analyzing the mechanism underlying the IL-1{beta}-mediated attenuation of IL-6 signal transduction and acute phase protein synthesis, it was observed that IL-1{beta} did not induce SOCS3 gene expression in hepatic parenchymal cells and barely affected IL-6-mediated STAT3 activation, but did affect its binding to IL-6-responsive promoters (51). In these studies NF-{kappa}B was identified as a mediator of IL-1{beta}-dependent negative regulation of IL-6-inducible genes (50, 51). Most likely, in this context NF-{kappa}B exerts its negative regulatory function by counteracting DNA binding of STAT3 at overlapping STAT3/NF-{kappa}B binding sites within the promoter region of the respective gene. This regulatory mechanism represents a very fast and accurate mode by which IL-1{beta} modulates IL-6-induced gene expression of specific target genes (50, 51).

In this paper we identified the recruitment of SHP2 to the cytoplasmic part of the gp130 signal-transducing subunit of the IL-6R complex as another fast-acting mechanism crucial for the TNF-{alpha}-dependent negative regulation of IL-6-mediated STAT3 tyrosine phosphorylation and activation in macrophages and fibroblast cell lines. Inhibitor studies revealed that TNF-{alpha}-induced binding of SHP2 to gp130 might depend on protein tyrosine kinase activity and activation of p38MAPK (Fig. 7). In line with these findings, the expression of a dominant negative p38MAPK completely restores inhibition of IL-6-induced STAT activation by TNF-{alpha}, further indicating the importance of p38MAPK for mediating the effect of TNF-{alpha} on IL-6 signal transduction (Fig. 6). Furthermore, evidence is given that tyrosine phosphorylation of the tyrosine 759 of the cytoplasmic tail of gp130 is a central event for the inhibitory action of TNF-{alpha} on IL-6 signaling (Figs. 5 and 6). Phosphorylation of this tyrosine has been shown to be crucial for the recruitment of both the protein tyrosine phosphatase SHP2 and the SOCS3 to the gp130 signal-transducing subunit (15, 39, 41). Thus, both SHP2 and SOCS3 might contribute to TNF-{alpha}-dependent signal attenuation. Indeed, the expression of SOCS3 occurs upon stimulation of macrophages with TNF-{alpha} (31), and on the other hand, specific disturbance of SHP2 recruitment to gp130 also diminishes the inhibitory activity of TNF-{alpha} (Fig. 10). The individual contributions of SHP2 and SOCS3 in the context of TNF-{alpha} remains to be established. In this respect, it was recently demonstrated that SHP2 and SOCS3, although recruited to the same site of gp130, are able to exert their inhibitory function independently of each other (33). Since SOCS3 protein remains detectable longer than SHP2 tyrosine phosphorylation, and recruitment of SHP2 to gp130 - SHP2 may be important for early signal attenuation, whereas SOCS3 acts later.

As mentioned above, p38MAPK was demonstrated to be involved in the inhibitory action of proinflammatory mediators (31, 42, 43). Particularly, activation of p38MAPK was shown to be essential for the TNF-{alpha}- and LPS-induced expression of SOCS3 (31), but is also involved in the autoregulatory SOCS3 expression induced by IL-6 (52). The induction of SOCS3 has been suggested to be part of the molecular mechanism underlying the inhibitory effects of TNF-{alpha} and LPS. The expression of a dominant negative mutant of p38MAPK completely rescues inhibition of gp130-mediated activation of STAT3 and the STAT3-dependent {alpha}2-macroglobulin promoter (Fig. 6). It is further demonstrated that inhibition of p38MAPK using an inhibitor specific for p38MAPK attenuates TNF-{alpha}-induced recruitment of SHP2 to the gp130 subunit (Fig. 7A). These data indicate that p38MAPK activation by TNF-{alpha} might be involved in the TNF-{alpha}-dependent recruitment of SHP2 to the gp130 receptor subunit. p38MAPK is a serine/threonine protein kinase (53); thus, a direct influence on the tyrosine phosphorylation of gp130 is impossible. We could not detect reliable changes in TNF-{alpha}-induced tyrosine phosphorylation of SHP2 and gp130 upon pretreatment with a p38MAPK-specific inhibitor (data not shown), indicating that an involvement of p38MAPK in regulation of tyrosine phosphorylation of gp130 is unlikely. However, the regulatory effect of p38MAPK on SHP2 recruitment to gp130 might also depend on phosphorylation of additional sites representing a MAPK consensus motif. With respect to this, it is interesting to note that using truncated gp130 receptor constructs, Ahmed et al. (42) restricted the target of the p38MAPK-mediated inhibitory effects on IL-6 signaling to the membrane-proximal 113 aa of the gp130 cytoplasmic tail, containing one consensus phosphorylation site for MAPKs and a serine-rich region (15). Thus, this membrane-proximal region of gp130 might represent another target for the inhibitory effects of p38MAPK on gp130-dependent signaling.

Despite its inhibitory activity on IL-6-induced activation of STAT factors, SHP2 has been further shown to serve as an adaptor protein linking gp130 signal transduction to the Ras/Raf/Erk cascade (27, 54). On the other hand, p38MAPK interferes with TNF-{alpha}-induced recruitment of SHP2 to gp130. Thus, one might expect that inhibition of p38MAPK somehow affects TNF-{alpha}/IL-6-induced Erk activation. As shown in Fig. 8, cotransfection of a dominant negative mutant of p38MAPK leads to enhanced transcriptional activation of Elk-1 in the presence of TNF-{alpha}, but not upon activation of the Epo/gp130 receptor chimera with Epo. Considering that Elk-1 is downstream from Erk/MAPK activation (44), this suggests that p38MAPK activity reduces transcriptional activation of Elk-1 by TNF-{alpha}, but is not specifically involved in IL-6-induced Erk activation and the effect of TNF-{alpha} on it. However, further investigation is required to clarify the interplay between p38MAPK and Erk activation upon costimulation with IL-6 and TNF-{alpha} as well as the role of SHP2 with respect to the activation of Erk-type MAPKs.

As demonstrated in this study, TNF-{alpha}-induced recruitment of SHP2 to the cytoplasmic tail of gp130 is involved in the inhibitory effects of TNF-{alpha} on IL-6 signal transduction via STAT3. The observation that IL-6-induced Erk activation is enhanced rather than inhibited by costimulation with TNF-{alpha} suggests that SHP2 recruitment toward gp130 might function as a kind of molecular switch, inhibiting signal transduction via STAT factors, whereas signaling via Erk-type MAPKs is still admitted or even strengthened.

In some aspects this situation resembles to the COOH-terminal gp130{Delta}STAT knockin mutation that deleted all STAT binding sites. Mice with this gp130{Delta}STAT mutation displayed gastrointestinal ulceration and severe joint disease with features of chronic synovitis, cartilaginous metaplasia, and degradation of the articular cartilage (55). In these animals mitogenic hyper-responsiveness of synovial cells to the leukemia inhibitory factor/IL-6 family of cytokines was related to sustained gp130-mediated SHP2/Ras/Erk activation and the lack of the activation of STAT factors. Thus, the pathologic changes observed in gp130 mice{Delta}STAT are likely to arise from the disturbance of the otherwise balanced activation of the SHP2/Ras/Erk and STAT signaling cascades emanating from gp130. The similarity of the modifications of gp130-dependent signal transduction in gp130{Delta}STAT mice and those presented here are interesting, since TNF-{alpha} also blocks IL-6-induced STAT activation, but not the activation of Erk-type MAPKs (Fig. 4). Thus, one might speculate that TNF-{alpha} provokes a pathophysiological situation in IL-6 signaling similar to that achieved with the COOH-terminal gp130{Delta}STAT knockin mutation that deleted all STAT binding sites.


   Acknowledgments
 
Murine fibroblasts lacking exon 3 of SHP2 were kindly provided by B. G. Neel (Boston, MA). We thank M. Ruhl and J. Matthes for technical assistance.


   Footnotes
 
1 This work was supported by the Deutsche Forschungsgemeinschaft (Bonn, Germany) through the collaborative research center SFB 575 Experimentelle Hepatologie (Düsseldorf, Germany). Back

2 Address correspondence and reprint requests to Dr. Johannes G. Bode, Medizinische Universitätsklinik, Klinik für Gastroenterologie, Hepatologie und Infektiologie, Heinrich Heine Universität, Moorenstrasse 5, D-40225 Düsseldorf, Germany. E-mail address: johannes.bode{at}t-online.de Back

3 Abbreviations used in this paper: Jak, Janus kinase; Erk, extracellular signal-regulated kinase; Epo, erythropoietin; IRF, IFN-regulatory factor; MAPK, mitogen-activated protein kinase; PIAS, protein inhibitor of activated STATs; SHP, SH2-containing protein tyrosine phosphatase; SOCS, suppressor of cytokine signaling; wt, wild type. Back

Received for publication September 19, 2002. Accepted for publication April 18, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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