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J. Biol. Chem., Vol. 277, Issue 37, 33676-33682, September 13, 2002

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Interleukin-22 (IL-22) Activates the JAK/STAT, ERK, JNK, and p38 MAP Kinase Pathways in a Rat Hepatoma Cell Line

PATHWAYS THAT ARE SHARED WITH AND DISTINCT FROM IL-10^*

Diane Lejeune^§, Laure Dumoutier, Stefan Constantinescu, Wiebe Kruijer^¶, Jan Jacob Schuringa^¶, and Jean-Christophe Renauld

From the  Ludwig Institute for Cancer Research, Brussels Branch, and the Experimental Medicine Unit, Université de Louvain, avenue Hippocrate, 74, B-1200 Brussels, Belgium and the ^¶ Department of Genetics, Biological Center, Kerklaan 30, 9751 NN Haren, The Netherlands

Received for publication, April 30, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

IL (interleukin)-22 is an IL-10-related cytokine; its main biological activity known thus far is the induction of acute phase reactants in liver and pancreas. IL-22 signals through a receptor that is composed of two chains from the class II cytokine receptor family: IL-22R (also called ZcytoR11/CRF2-9) and IL-10R (CRF2-4), which is also involved in IL-10 signaling. In this report, we analyzed the signal transduction pathways activated in response to IL-22 in a rat hepatoma cell line, H4IIE. We found that IL-22 induces activation of JAK1 and Tyk2 but not JAK2, as well as phosphorylation of STAT1, STAT3, and STAT5 on tyrosine residues, extending the similarities between IL-22 and IL-10. However our results unraveled some differences between IL-22 and IL-10 signaling. Using antibodies specific for the phosphorylated form of MEK1/2, ERK1/2, p90RSK, JNK, and p38 kinase, we showed that IL-22 activates the three major MAPK pathways. IL-22 also induced serine phosphorylation of STAT3 on Ser⁷²⁷. This effect, which is not shared with IL-10, was only marginally affected by MEK1/2 inhibitors, indicating that other pathways might be involved. Finally, by overexpressing a STAT3 S727A mutant, we showed that serine phosphorylation is required to achieve maximum transactivation of a STAT responsive promoter upon IL-22 stimulation.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

IL-22¹ was originally described as an IL-9-induced gene and was called IL-TIF for IL-10-related T cell-derived inducible factor (1). This cytokine shows 22% amino acid identity with IL-10 and belongs to a family of cytokines with limited homology to IL-10, namely IL-10, IL-22, mda-7/IL-24, IL-19, IL-20, and AK155/IL-26 (2-4). As discovered thus far, IL-22 activities include up-regulation of acute-phase reactants in the liver and hepatoma cells (1) as well as induction of pancreatitis-associated protein (PAP1) in pancreatic acinar cells (5), suggesting a role for this cytokine in inflammatory processes. IL-22 also induces STAT activation in several cell lines such as mesangial cells, lung and intestinal epithelial cells, melanomas, and hepatomas (1, 6).

IL-22 binds at the cell surface to a receptor complex composed of two chains belonging to the class II cytokine receptor family (CRF2): IL-22R and IL-10R (6-8). This family includes receptors for type I and type II IFNs (IFNAR1, IFNAR2, IFNGR1, and IFNGR2), IL-10R, IL-22R/CRF2-9, IL-10R/CRF2-4, IL-20R/CRF2-8, IL-20R/CRF2-11, and tissue factor (9-11). Signaling through the receptors for the IL-10-related cytokines has been poorly investigated so far, with the exception of the IL-10 receptor itself. IL-10 binding to its receptor complex (IL-10R and IL-10R) induces the activation of JAK1 and Tyk2 tyrosine kinases, with JAK1 being associated with IL-10R (12), whereas Tyk2 can be co-immunoprecipitated with IL-10R (13). Activation of these two JAK kinases leads to phosphorylation of three STAT factors: STAT1, STAT3, and STAT5 (12, 14). IL-10 also activates the phosphatidylinositol 3-kinase and p70S6-kinase (15) but not the MAP kinase pathway, which is inhibited by IL-10 in monocytes and dendritic cells (16, 17).

In this report, we show that binding of IL-22 to its surface receptor on rat hepatoma cell line H4IIE induced the rapid activation of JAK1 and Tyk2, leading to phosphorylation of STAT1, STAT3, and STAT5. IL-22 also activated the three major MAPK pathways: the MEK-ERK-RSK, the JNK/SAPK, and the p38 kinase pathways. In addition, IL-22 induced phosphorylation of STAT3 on a serine residue. This further STAT modification was necessary for maximum transactivation and depended only marginally on the ERK pathway.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cell Culture, Reagents, and Cytokines-- H4IIE rat hepatoma cells (from Dr. J.-P. Thissen, University of Louvain, Belgium) were grown in Iscove-Dulbecco's medium supplemented with 10% fetal calf serum, 0.55 mM L-arginine, 0.24 mM L-asparagine, and 1.25 mM L-glutamine. HEK293-EBNA human embryonic kidney cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. 2C4, U4C, (generously provided by Dr. Ian Kerr, Imperial Cancer Research Fund, London, UK) and 2A (generously provided by Dr. George Stark, Cleveland Clinic Research Institute, Cleveland, OH) fibrosarcoma cells (18, 19) were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and with 400 µg/ml geneticin (Invitrogen). Recombinant mouse IL-22 was produced either in Escherichia coli as described previously (6, 20) or by transient transfection of HEK293 cells using the LipofectAMINE method (Invitrogen). Recombinant human IL-10 was purchased from Peprotech Inc. (Rocky Hill, NJ). Recombinant mouse IFN- was provided by Dr. W. Fiers (University of Gent, Belgium). Cytokines were added to the cultures at the following concentrations: 2000 units/ml for rIL-22, 1% for IL-22-containing HEK293 supernatant, 10 ng/ml for IL-10, and 250 units/ml for IFN-.

Western Blots and Inhibitor Treatment-- H4IIE cells were seeded in 6-well plates (Nunc, Rochester, NY) at 5 × 10⁵ cells/well 1 day before stimulation. The next day, cells were stimulated with new medium containing or not containing IL-22. When indicated, cells were preincubated for 1 h with 50 µM PD98059 (Cell Signaling, Beverly, MA) or 10 µM U0126 (Cell Signaling) MEK inhibitors. After various periods of time, cells were lysed in 500 µl of Laemmli buffer (Bio-Rad) and boiled for 3 min before being loading on pre-cast Novex (Carlsbad, CA) SDS-polyacrylamide gels (8 or 14%) and transferred electrophoretically to nitrocellulose membranes (Hybond C; Amersham Biosciences). Membranes were then blocked in 5% nonfat dry milk, washed, and probed using antibodies specific for phosphorylated Tyk2, STAT1-Y701, STAT3-Y705, STAT3-S727, STAT5-Y694, ERK1/2, p90RSK, JNK/SAPK, p38 kinase (Cell Signaling), or MEK1/2 (New England Biolabs, Beverly, MA). Blots were reprobed with anti-Tyk2 (BIOSOURCE, Camarillo, CA) or anti--actin antibodies (Sigma) as a control. A SuperSignal West Pico detection kit (Pierce) was used for detection.

Immunoprecipitation-- Thirty million H4IIE cells were stimulated with mIL-22 (1% HEK293 cell supernatant), IFN- (250 units/ml), or control medium for 5 min, washed, and resuspended in 1 ml of lysis buffer (1% Nonidet P-40, 0.1% deoxycholate, 0.1% SDS, 50 mM Tris, pH 8, 150 mM sodium chloride, 1 mM EDTA, 1 mM sodium vanadate, 1 mM sodium fluoride, and inhibitor mixture (Roche Molecular Biochemicals)). Lysates were homogenized by five passages through a 20-gauge needle, incubated for 45 min on ice, and centrifuged (14,000 × g). 2.5 µg of anti-JAK1 or anti-JAK2 polyclonal antibody (Upstate Biotechnology, Lake Placid, NY) were added to the supernatant and incubated overnight at 4 °C. Lysates were then incubated with protein A-agarose for 2 h. Beads were washed, resuspended in Laemmli buffer (25 µl), and boiled. Proteins were separated in a 8% SDS-PAGE (Novex, Carlsbad, CA) and transferred on a nitrocellulose membrane. The membrane was blocked in 1% bovine serum albumin solution before overnight incubation with 1 µg/ml anti-phosphotyrosine 4G10 (Upstate Biotechnology) antibody. Proteins were detected by chemiluminescence (SuperSignal West Pico; Pierce), and membranes were reprobed with anti-JAK1 or anti-JAK2 antibodies as a control.

Plasmid Construction, Cell Transfection, and Luciferase Assay-- The human IL-22R cDNA was amplified by reverse transcription PCR from the HepG2 hepatoma cell line before cloning into the pEF-BOSpuro expression vector (21). Mouse IL10R cDNA was amplified by reverse transcription PCR from MC9 cells and also cloned into the pEF-BOSpuro expression vector. The pSG5-STAT3 wild-type and the pSG5-STAT3 S727A vectors encoding wild-type or mutated forms of human STAT3 were obtained as described (22). Expression of JAK1 and JAK2 was driven by the pRK5 vector as described by Dr. J. Ihle's group (23).

12.10⁶ H4IIE cells were electroporated (250 V, 200 ohms, 1200 microfarads) with 50 µg of pGRR5-luc (provided by Dr. P. Brennan, Imperial Cancer Research Fund) or 30 µg of pSRE-luc plasmid (Stratagene, La Jolla, CA). The first construct contains five copies of the STAT-binding site of the FcRI gene inserted upstream from a luciferase gene controlled by the TK promoter, and the second one contains repeats of the serum responsive element of the c-fos promoter. 5 µg of another reporter plasmid, pRL-TK (Promega, Madison, WI) encoding renilla luciferase, was co-electroporated as an internal control of the transfection process. Cells were seeded in 12-wells plates at 10⁶/ml. The next day, cells were stimulated for 3 h with 2000 units/ml IL-22 before lysis. When indicated, cells were preincubated 1 h with 50 µM PD98059 or 10 µM U0126 MEK inhibitors. Luciferase assays were performed using the dual luciferase reporter assay kit (Promega).

For stable transfection, mouse IL-10R cDNA was subcloned into the pEF/Myc/Cyto plasmid (Invitrogen) carrying a Geneticin resistance gene. 12.10⁶ H4IIE cells were electroporated (250 V, 200 ohms, 1200 microfarads) with 50 µg of IL-10R cDNA. The next day, cells were cultured with 2 mg/ml Geneticin (Invitrogen) until a bulk population was obtained.

Transient transfection of H4IIE cells with wild-type and mutated STAT3 was performed as follows. 12.10⁶ H4IIE cells were electroporated (250 V, 200 ohms, 1200 microfarads) with 15 µg of pGRR5-luc, 15 µg of either pSG5-STAT3 or pSG5-STAT3 S727A, and 5 µg of pRL-TK vectors. Cells were seeded in 12-well plates at 10⁶/ml. 5 h later, cells were stimulated for 3 h with 2000 units/ml IL-22 before lysis and luciferase assay.

Transient HEK293 cells transfections were carried out as described previously (24). Briefly, cells were seeded in 12-well plates at 4 × 10⁵ cells/well 1 day before transfection. Transfection was carried out using the LipofectAMINE method (Invitrogen), according to the manufacturer's recommendations, with 2 µg of plasmid DNA in a total of: 500 ng of hIL-22R or mIL-10R cDNA, 100 ng of pGRR5, 100 ng of pRL-TK, 1 µg of vector encoding wild-type or mutated forms of STAT3, and empty vector to 2 µg. 5 h after transfection, cells were stimulated with control medium, with IL-22 (2000 units/ml) or human IL-10 (10 ng/ml) for 24 h. Luciferase assays were performed using the dual luciferase reporter assay kit (Promega). The same protocol was used for transient transfection of 2C4, U4C, and 2A fibrosarcoma cells with 500 ng of hIL-22R cDNA, 40 ng of pRK5-JAK1, pRK5-JAK2, or empty vector, 100 ng of pGRR5, and 100 ng of pRL-TK.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

IL-22 Induces Phosphorylation of STAT1, STAT3, and STAT5 in Rat Hepatoma Cells-- In previous reports, we and others (1, 6-8) showed that IL-22 induced STAT1, STAT3, and STAT5 phosphorylation in a variety of cell lines including H4IIE. To further study the kinetics of STATs phosphorylation, we stimulated H4IIE cells with IL-22 for various periods of time. Within 5 min, IL-22 induced tyrosine phosphorylation of STAT1, STAT3, and STAT5 (Fig. 1A). This phosphorylation was transient, decreasing to barely detectable levels for STAT1 and STAT5 after 30 min. However, phosphorylated STAT3 could still be detected at least 1 h after IL-22 stimulation (data not shown). To confirm that IL-22-induced STAT phosphorylation correlated with transcriptional activation, we used luciferase assays with pGRR5-luc reporter plasmid. The pGRR5-luc construct is regulated by five copies of a STAT-binding sequence recognizing at least STAT1, STAT3, and STAT5. As shown in Fig. 1B, when H4IIE cells were electroporated with pGRR5-luc, IL-22 stimulation induced a 35-fold increase in luciferase activity.

View larger version (27K): [in this window] [in a new window]   Fig. 1.   IL-22 induces tyrosine phosphorylation of STAT1, STAT3, and STAT5. A, 4.105 H4IIE cells were stimulated with mIL-22 (1% 293-EBNA cell supernatant) for 5, 15, and 30 min or with a control supernatant for 15 min. Total lysates were analyzed by Western blot with antibodies directed against phosphorylated STAT1, STAT3, and STAT5. The membranes were then reprobed with an anti--actin antibody. Similar results were obtained by stimulating H4IIE cells with E. coli-derived IL-22 (data not shown). B, 12.106 H4IIE cells were electroporated with 50 µg of pGRR5-luc and 5 µg of pRL-TK reporter vectors. The transfected cells were seeded in 12 wells of a 12-well plate. The next day, cells were stimulated with mIL-22 (2000 units/ml) or with control medium for 3 h before a luciferase assay was performed.

IL-22 Activates JAK1 and Tyk2-- As JAK kinases are known to be responsible for STAT phosphorylation in response to cytokines, we next investigated which JAK kinase is activated by IL-22. H4IIE rat hepatoma cells were stimulated with control medium or IL-22, and a Western blot analysis was performed with an anti-phospho-Tyk2 antibody, while immunoprecipitations were used for JAK1 and JAK2. As shown in Fig. 2, A and B, IL-22 stimulation of H4IIE cells induced the rapid phosphorylation of Tyk2 and JAK1 but not of JAK2. As a control for JAK2 immunoprecipitation, H4IIE cells were stimulated with IFN-, which is known to activate this JAK family member as well as JAK1 (25). To further assess the functional role of JAK1 in IL-22 signaling, we transfected JAK1-deficient U4C cells with the IL-22R cDNA together with pGRR5-luc reporter plasmid. As shown in Fig. 2C, IL-22 failed to induce a luciferase activity in U4C cells unless these cells were transfected with JAK1 cDNA. By contrast, parental cells (2C4) or JAK2-deficient cells (2A) were both able to respond to IL-22.

View larger version (41K): [in this window] [in a new window]   Fig. 2.   IL-22 induces phosphorylation of JAK1 and Tyk2 but not JAK2. A, 5.105 H4IIE cells were stimulated with mIL-22 (1% 293-EBNA cell supernatant) for 15, 30, 45, and 60 min or with a control supernatant for 15 min. Cells were lysed, and a Western blot was performed with an anti-phospho-Tyk2 antibody. The membrane was then reprobed with anti-Tyk2 antibody. B, 30.106 H4IIE cells were stimulated with mIL-22 (1% 293-EBNA cell supernatant), with 250 units/ml IFN-, or with control supernatant for 5 min before lysis and immunoprecipitation with antibodies directed against JAK1 or JAK2. Western blot analysis was performed with an anti-phosphotyrosine antibody, and the membranes were then reprobed with anti-JAK1 or anti-JAK2 antibody. Similar results were obtained when H4IIE cells were stimulated with E. coli-derived IL-22 (data not shown). C, 400,000 2C4 parental, U4C JAK1-deficient, or 2A JAK2-deficient cells were seeded in 12-well plates. The next day, cells were transfected with a vector coding for IL-22R together with pGRR5-luc and pRL-TK reporter plasmids and, when mentioned, with a vector coding for JAK1 or JAK2 cDNA. Cells were stimulated with IL-22 (2000 units/ml) or with control medium for 4 h before a luciferase assay was performed.

IL-22 Activates MAPK Pathways-- We next analyzed the ability of IL-22 to activate the MAP kinase pathways. As shown in Fig. 3A, IL-22 induced a sustained phosphorylation of ERK1/2. Two MEK inhibitors, PD98059 and U0126, totally blocked this phosphorylation, suggesting that ERK1/2 phosphorylation results from MEK activation. In line with this result, IL-22 induced the phosphorylation of MEK1/2 (Fig. 3B). p90RSK, a well known substrate of ERK, was also phosphorylated in response to IL-22. To confirm the functional activation of this pathway, we electroporated H4IIE cells with the pSRE-luc reporter plasmid regulated by the serum-responsive element from the c-fos promoter. As shown in Fig. 4, IL-22 stimulation induced a 2.25-fold increase in luciferase activity, which was completely abolished when cells were preincubated with any of the MEK inhibitors. In contrast with IL-22, IL-10 did not activate the ERK MAPK pathway in H4IIE cells transfected with IL-10R cDNA (Fig. 5). In addition to this MEK-ERK-RSK cascade, IL-22 also induced a delayed phosphorylation of JNK/SAPK and p38 MAP kinases, detectable after 30-40 min (Fig. 3B).

View larger version (31K): [in this window] [in a new window]   Fig. 3.   IL-22 induces phosphorylation of several members of the MAPK pathways. A, 5.105 H4IIE cells were seeded in 6-well plates 1 day before stimulation with recombinant mIL-22 (2000 units/ml) for 10, 20, 30, or 40 min or with control medium for 40 min. Where mentioned, cells were preincubated for 1 h with 50 µM PD98059 or 10 µM U0126 MEK1 inhibitors. Total lysates were analyzed by Western blot with an anti-phospho-ERK1/2 antibody. The membranes were then reprobed with an anti--actin antibody. Similar results were obtained by using 1% IL-22-containing 293-EBNA cell supernatant (data not shown). B, 5.105 H4IIE cells were seeded in 6-well plates 1 day before stimulation with mIL-22 (2000 units/ml) for 10, 20, 30, or 40 min or with control medium for 40 min. Total lysates were analyzed by Western blot with antibodies directed against the phosphorylated forms of MEK1/2, p90RSK, JNK/SAPK, and p38 kinase. The membranes were then reprobed with an anti--actin antibody. Similar results were obtained by stimulating H4IIE cells with 1% IL-22-containing 293-EBNA cell supernatant (data not shown).

View larger version (43K): [in this window] [in a new window]   Fig. 4.   IL-22 activates the MAPK pathway in H4IIE cells. 12.106 H4IIE cells were electroporated with 30 µg of pSRE-luc and 5 µg of pRL-TK reporter vectors. The transfected cells were seeded in 12-well plates (106 cells/well). The next day, cells were preincubated for 1 h in the presence of dimethyl sulfoxide (DMSO; 1/1000 final), PD98059 (50 µM final), or U0126 (10 µM final) before stimulation with mIL-22 (2000 units/ml) or control medium. 3 h later, a luciferase assay was performed.

View larger version (17K): [in this window] [in a new window]   Fig. 5.   IL-10 does not induce phosphorylation of ERK. 5.105 H4IIE cells stably transfected with IL-10R were seeded in 6-well plates 1 day before stimulation with IL-10 (10 ng/ml) or mIL-22 (2000 units/ml) for 10, 20, 30, or 40 min or with control medium for 40 min. Total lysates were analyzed by Western blot with an anti-phospho-STAT3 and an anti-phospho-ERK1/2 antibody. The membranes were then reprobed with an anti--actin antibody.

IL-22 Induces STAT3 Serine Phosphorylation by a MAPK-independent Mechanism-- In addition to tyrosine phosphorylation, STAT3 can be phosphorylated on a serine residue in response to cytokines such as IL-6 (22). In our system, IL-22 stimulation of H4IIE cells induced rapid serine phosphorylation of STAT3. Although MAPKs have often been described as mediating STAT3 Ser phosphorylation (22, 26, 27), preincubation of H4IIE cells with MEK inhibitors only slightly delayed but did not inhibit this effect (Fig. 6, A and B).

View larger version (47K): [in this window] [in a new window]   Fig. 6.   IL-22 induces serine phosphorylation of STAT3. 5.105 H4IIE cells were seeded in 6-well plates 1 day before stimulation with mIL-22 (2000 units/ml) for 10, 20, or 30 min or with control medium for 30 min. Where mentioned, cells were preincubated for 1 h with 50 µM PD98059 (A) or 10 µM U0126 MEK1 inhibitors (B). Total lysates were analyzed by Western blot with an antibody directed against the serine-phosphorylated form of STAT3. The membranes were reprobed with an anti-phospho-ERK1/2 antibody and then with an anti--actin antibody.

IL-22-induced STAT3 Serine Phosphorylation Is Necessary for Maximal Transactivation-- To test the functional significance of STAT3 serine phosphorylation, we transfected H4IIE cells with the pGRR5-luc and pRL-TK reporter plasmids together with a plasmid encoding wild-type or a mutated form of STAT3, in which the serine 727 is mutated into alanine, thus preventing phosphorylation (28). As shown in Fig. 7, mutation of Ser⁷²⁷ of STAT3 reduced luciferase induction from an 8-fold to a 4-fold increase upon IL-22 stimulation, strongly suggesting that STAT3 serine phosphorylation is required to achieve maximal transactivation.

View larger version (20K): [in this window] [in a new window]   Fig. 7.   IL-22-induced STAT3 serine phosphorylation is required for maximum transactivation in H4IIE cells. 12.106 H4IIE cells were electroporated with 15 µg of vector coding for STAT3 wild type (wt) or STAT3 mutated on Ser727 residue (S727A) together with 15 µg of pGRR5-luc and 5 µg of pRL-TK reporter vectors. The transfected cells were seeded in 12 wells of a 12-well plate. 5 h later, cells were stimulated with mIL-22 (2000 units/ml) or with control medium for 3 h before a luciferase assay was performed.

To further support this hypothesis, we studied the effect of this STAT3 S727A mutant on IL-10-induced transactivation because IL-10 has never been described to phosphorylate STAT3 on a serine residue. HEK293 cells were transiently transfected with a plasmid encoding either IL-22R or IL-10R cDNA together with a plasmid encoding the wild-type or S727A mutated form of STAT3, pGRR5-luc, and pRL-TK reporter plasmids. As shown in Fig. 8A, IL-22 stimulation of IL-22R-transfected HEK293 cells induced a 6.5-fold increase in luciferase activity. Co-transfection of the STAT3 S727A mutant reduced this induction to 4-fold. By contrast, co-transfection of the STAT3 S727A mutant in cells expressing IL-10R had no effect on IL-10-induced transactivation (Fig. 8A). In line with this result, IL-22, but not IL-10, induced STAT3 serine phosphorylation in these cells, whereas both cytokines induced tyrosine phosphorylation of STAT3 (Fig. 8B).

View larger version (32K): [in this window] [in a new window]   Fig. 8.   STAT3 serine phosphorylation is required for maximal transactivation with IL-22 but not IL-10. 400,000 HEK293 cells were seeded in 12-well plates. The next day, cells were transfected with a vector coding for either IL-22R or IL-10R, together with pGRR5-luc and pRL-TK reporter plasmids and a vector coding for STAT3 wild-type (wt) or STAT3 mutated on Ser727 (S727A). Cells were stimulated with IL-22 (2000 units/ml), IL-10 (10 ng/ml), or control medium for 24 h before a luciferase assay was performed (A). Simultaneously, cells were stimulated with IL-22 (2000 units/ml), IL-10 (10 ng/ml), or control medium for 15 min before lysis. Total lysates were analyzed by Western blot using antibodies detecting either the serine- or tyrosine-phosphorylated form of STAT3 (B). Membranes were then reprobed with an anti--actin antibody.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

IL-22, a new cytokine that is structurally related to IL-10, was originally identified as an IL-9-induced gene and shown to up-regulate the acute phase response in the liver (1, 6). In addition to their sequence homology, IL-22 and IL-10 share one receptor subunit, IL-10R, whereas their functional receptor complex also involves IL-22R for IL-22 and IL-10R for IL-10 (7, 8, 13). In this paper, we have analyzed the signaling pathways activated by IL-22 in rat hepatoma cells. We showed that IL-22 induced the phosphorylation of JAK1 and Tyk2, but not JAK2, and that JAK1 is absolutely required for IL-22 signaling. Because Tyk2 has been shown to be associated with IL-10R (13), these results suggest that JAK1 associates with IL-22R. This observation extends the similarities between IL-22 and IL-10, which also activates JAK1 and Tyk2. Moreover, both cytokines induce the phosphorylation of the same STAT factors (12-14). However, our results unravel some of the differences between IL-22 and IL-10 signaling. First, IL-22 induces the activation of the ERK, JNK, and p38 MAPK pathways that are not activated by IL-10 (2). Secondly, IL-22, but not IL-10, induces serine phosphorylation of STAT3. Thus, despite the structural relationship between IL-22 and IL-10, these cytokines activate overlapping but not identical signaling pathways.

IL-22 activity and signaling pathways are reminiscent of those of IL-6 on hepatocytes. Indeed, IL-22 has been shown to induce acute phase proteins in the liver (6), an effect that has been described to be regulated by IL-6 mainly through STAT3 activation (30-32). Although the IL-6 receptor complex is less related to the IL-22 receptor than the IL-10 receptor complex, IL-6 binding also leads to JAK1 activation and the subsequent phosphorylation of STAT3, STAT1, and STAT5 (33). Moreover, IL-6 also activates the MAPK pathways (34, 35). A synergistic effect between the JAK/STAT and MAPK pathways has been proposed to regulate acute phase protein expression in response to IL-6 (36). Our results suggest that such a synergy can also take place in the regulation of the acute phase response by IL-22.

Accumulating data have stressed the importance of STAT regulation by serine phosphorylation. In this report, we have shown that STAT3 Ser⁷²⁷ phosphorylation is induced upon IL-22 stimulation and is required for maximal transcriptional activation. Similar results were previously reported for IL-6 (22). By contrast, IL-10 signaling does not involve STAT3 Ser⁷²⁷ phosphorylation, indicating that this process is not shared by all STAT3-activating cytokines.

In the case of IL-6, STAT3 serine phosphorylation results from the sequential activation of a Vav-Rac-1-SEK-1/MKK-4-PKC cascade (22, 37). However, we failed to show any IL-22-induced PKC activation (data not shown). STAT serine phosphorylation can also be mediated by MAPKs such as ERK (38), MEKK1 (39), JNK (27), or p38 kinase (40). Interestingly, IL-22-induced STAT3 serine phosphorylation was only slightly delayed but not inhibited by MEK inhibitors PD98059 and U0126, suggesting that this effect could result from several cooperating signaling pathways. This hypothesis is further supported by a recent report showing that both H7 (a PKC inhibitor) and PD98059 inhibitors were required to block STAT3 serine phosphorylation induced by high IL-6 concentrations (41). However, we have failed thus far to identify the kinases responsible for IL-22-induced STAT3 serine phosphorylation by combining signaling pathway inhibitors (data not shown).

This report is the first description of signal transduction by one of the recently described IL-10-related cytokines (IL-22, IL-19, IL-20, mda-7/IL-24, AK155/IL-26; reviewed in Refs. 3 and 4). Our data show both similarities (JAK1, Tyk2, and STAT3 tyrosine phosphorylation) and discrepancies (STAT3 serine phosphorylation, MAPKs activation) between IL-22 and IL-10 signaling. Little is known about the pathways activated by the other members of this family, except that they all induce STAT3 tyrosine phosphorylation (29).² Further studies will be needed to determine whether signaling pathways can be used to define subclassifications within this family.

	ACKNOWLEDGEMENTS

We thank Dr. Ian Kerr (Imperial Cancer Research Fund, London, UK) for the gift of 2C4 and U4C cells, Dr. George Stark (Cleveland Clinic Research Institute, Cleveland, OH) for the gift of 2A cells, Dr. Jean-Paul Thissen for the gift of H4IIE cells, and Dr. P. Brennan (Imperial Cancer Research Fund, London, UK) for the gift of pGRR5-luc plasmid.

	FOOTNOTES

^* This work was supported in part by the Belgian Federal Service for Scientific, Technical and Cultural Affairs and the Actions de Recherche Concertées, Communauté Française de Belgique.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ Research fellow with the Fonds National de la Recherche Scientifique, Belgium.

To whom correspondence should be addressed: Ludwig Institute for Cancer Research, Ave. Hippocrate, 74, B-1200 Brussels, Belgium. Tel.: 32-2-764-74-64; Fax: 32-2-762-94-05; E-mail: renauld@licr.ucl.ac.be.

Published, JBC Papers in Press, June 26, 2002, DOI 10.1074/jbc.M204204200

² L. Dumoutier, unpublished data.

	ABBREVIATIONS

The abbreviations used are: IL-, interleukin-; IL-XR, interleukin-X receptor; mIL-, murine IL-; STAT, signal transducer and activator of transcription; CRF2, class II cytokine receptor family; JAK, Janus kinase; MAP, mitogen-activated protein; MAPK, MAP kinase; ERK, extracellular signal regulated protein kinase; MEK, MAPK/ERK kinase; p90RSK, p90 ribosomal S6 kinase; JNK, c-Jun N-terminal kinase; SAPK, stress-activated protein kinase; IFN, interferon; SEK, SAPK/ERK kinase; MKK, MAPK kinase; MEKK, MEK kinase; PKC, protein kinase C; TK, thymidine kinase; EBNA, Epstein-Barr virus nuclear antigen.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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