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Interleukin-22 (IL-22) is a member of the IL-10 family of cytokines produced by activated T cells and is involved in several tissue responses. IL-22 signals through a heterodimeric receptor composed of IL-22 receptor 1 (IL-22R1) and IL-10R2, and the intracellular signaling pathways mediated by IL-22 receptor are not completely known. Here we investigate the effect of Src homology-2 containing protein-tyrosine phosphatase (Shp2) on IL-22 signaling pathway using SW480 colon cancer cells as a model. The results show that IL-22 induces IL-22R1 phosphorylation, and Shp2 is recruited to the tyrosine phosphorylated IL-22R1 upon IL-22 stimulation. Furthermore, Tyr251 and Tyr301 of IL-22R1 are required for Shp2 binding to the IL-22R1. Shp2 binding to IL-22R1 and Shp2 protein tyrosine phosphatase activity are required for activation of MAP kinases and signal transducer and activator of transcription (STAT3) phosphorylation by IL-22. These results reveal a critical role of Shp2 in IL-22 mediated signal transduction pathways.
Interleukin-22 (IL-22) is a T-cell-derived cytokine belonging to a family of cytokines structurally related to IL-10 (Wolk and Sabat, 2006). IL-22 is known to regulate local tissue inflammation and could exert its effects on non-immune cells (Wolk et al., 2002; Wolk et al., 2004; Wolk and Sabat, 2006). It is known that IL-22 mediates signal transduction through a receptor complex composed of two chains, the tissue-specific receptor component IL-22R1 and the relatively ubiquitous receptor component IL-10R2 (Wolk and Sabat, 2006). IL-22R1 is expressed in liver, kidney, pancreas and skin, but not in peripheral blood mononuclear cells (Kotenko et al., 2001). Although IL-22R1 is expressed in various cancer cell lines such as C170, HepG2, A549 and SW480 cells (Wolk et al., 2002), little is known about the role of IL-22 in regulation of cancer cells.
Previous studies have found that, upon binding to its receptor, IL-22 activates JAK1 and Tyk2 tyrosine kinases, which leads to phosphorylation of STAT proteins (Dumoutier et al., 2000; Wolk et al., 2002; Wolk et al., 2006). In addition to the Jak-STAT pathway, IL-22 was also found to activate MAP kinase pathways in several cell lines (Lejeune et al., 2002; Brand et al., 2006). However, the molecular mechanism leading to MAP kinase activation by IL-22 in these cell lines was unclear. Shp2 is known to have a positive effect on MAP kinase activation upon stimulation by several cytokines. Shp2 positively regulates IL-2-induced MAPK activation in transfected murine fibroblasts NIH-3T3 cells and in cutaneous T cell lymphoma cells (Gadina et al., 1998; Lundin et al., 2002). IL-6-induced association of Shp2 with the adapter protein, Grb2-associated binder-1 (Gab1), also leads to activation of the MAPK cascade and direct interaction of Shp2 with proline-rich tyrosine kinase 2 (Pyk2) has been reported (Chauhan et al., 2000; Bard-Chapeau et al., 2006).
In this report, we show that Shp2 is recruited to IL-22R1 upon IL-22 stimulation in SW480 colon cancer cells. We identify that Tyr251 and Tyr301 of IL-22R1 are required for Shp2 binding and IL-22-induced extracellular signal regulated (Erk) MAP kinase activation. Furthermore, Shp2-IL-22R1 interaction perturbs tyrosine and serine phosphorylation of signal transducer and activator of transcription (STAT3) and cell proliferation in response to IL-22. These results demonstrate that Shp2 is involved in IL-22 signaling leading to Erk and STAT3 activation.
Previous studies have shown that IL-22-induces activation of STAT3 and MAP kinase pathways in rat hepatoma cells and intestinal epithelial cells (Lejeune et al., 2002; Brand et al., 2006). In this report, SW480 cells were used as a model to investigate IL-22 signaling because this cell line is known to express IL-22R1. Results showed that both tyrosine and serine phosphorylation of STAT3 were induced upon exposure to IL-22, and IL-22 also activated Erk1/2 and Src as measured by phospho-specific antibodies (Figure 1A). To confirm that IL-22-induced STAT3 and Erk phosphorylation correlated with transcriptional activation, luciferase assays were performed. As shown in Figure 1B, IL-22 stimulation induced both Erk reporter gene (upper panel) and STAT3 reporter gene (lower panel).
Since Shp2 has been found to mediate Erk1/2 activation by several other cytokines, we were interested in whether Shp2 plays a role in IL-22 signaling. Results showed that IL-22R1 was tyrosine-phosphorylated upon stimulation of cells with the cytokine (Figure 1C). Examination of the cytoplasmic region of IL-22R1 reveals the presence of two potential Shp2 SH2 domain-binding sites (Y251XXV and Y301XXI motifs). Therefore, the possibility of Shp2 binding to IL-22R1 in IL-22-stimulated cells was determined. As predicated, we found that Shp2 was recruited to the IL-22R1 after IL-22 stimulation in SW480 cells (Figure 1C).
A previous study indicated that human embryonic kidney (HEK)293 cells do not express endogenous IL-22R1, and overexpression of IL-22R1 in HEK293 cells renders them sensitive to IL-22 (Bleicher et al., 2008). To analyze the interaction between IL-22R1 and Shp2, we transiently expressed a Flag-tagged IL-22R1 in HEK293 cells with or without co-expression of HA-tagged Shp2. As shown in Figure 2A, the exogenous IL-22R1 was tyrosine phosphorylated, and both the endogenous and the exogenous HA-Shp2 were recruited to the IL-22R1 upon IL-22 stimulation (Figure 2A–C).
Next, we made single and double Tyr251/Tyr301 site mutants of IL-22R1, and expressed these mutants in HEK293 cells. All transfectants had a similar cell surface expression of IL-22R1, ruling out the possibility that these mutations affect the stability or trafficking of the protein (Figure 3A). As shown in Figure 3B, tyrosine phosphorylation signal of IL-22R1 was minimal in the absence of IL-22, and IL-22-induced robustic tyrosine phosphorylation of the Flag-IL-22R1 in HEK293 cells, which correlated with recruitment of Shp2 to the IL-22R1 receptor. IL-22R1-Y251F, IL-22R1-Y301F or IL-22R1-FF (double mutants) had reduced IL-22-induced receptor tyrosine phosphorylation and Shp2 binding to the receptor (Figure 3B). This observation indicates that both Tyr251 and Tyr301 are required for Shp2 binding to IL-22R1 in response to IL-22.
To determine whether Tyr251 and Tyr301 of IL-22R1 are required for IL-22-stimulated Erk2 activation, IL-22R1-Y251F, IL-22R1-Y301F, IL-22R1-FF, and the wild-type IL-22R1, respectively, were co-expressed with an HA-tagged Erk2 in HEK 293 cells. IL-22-stimulated Erk2 activation was examined. As shown in Figure 3C (upper panels), IL-22 treatment markedly activated Erk2 in HEK293 cells that expressed the wild-type IL-22R1, but not in cells expressing IL-22R1-Y251F, IL-22R1-Y301F or double mutant IL-22R1-FF receptors. A luciferase assay using an Erk reporter gene was also determined. As shown in Figure 3C (lower panel), activation of SRE-luciferase activity was increased 5.5-fold in response to IL-22 stimulation, whereas much weaker activation was observed in IL-22R1 mutants expression cells. Thus, both Tyr251 and Tyr301 are necessary for IL-22R1 to mediate the IL-22-stimulated Erk2 activation.
To further analyze the function of IL-22R1-Shp2 interaction in the activation of MAP kinase and STAT3 signaling pathways in response to IL-22, we generated Flp-In-293 cell lines stably expressing the wild-type or the double Tyr251/Tyr301 mutant IL-22R1-FF receptor. Previous studies have shown that the catalytic Cys-459 to Ser mutant of Shp2 (Shp2-C459S) had dominant negative effect on endogenous Shp2 tyrosine phosphatase activity (Chan et al., 2008). Therefore, we checked whether the tyrosine phosphatase activity of Shp2 is required for Erk2 activation in response to IL-22. The Flag-tagged wild-type Shp2 (Shp2-Flag) or a dominant negative Flag-tagged Shp2-C459S mutant (Shp2-CS-Flag) were transiently expressed with HA-Erk2 in Flp-In-293/IL-22R1 stable cell lines (Figure 3D), Erk2 was immunoprecipitated and immunoprecipitated phosphorylated Erk1/2 was immunobloted. As shown in Figure 3D, Erk2 was activated in the cells expressing wild-type Shp2 in response to IL-22, but not in Shp2-C459S overexpression cells. These data suggest that the protein-tyrosine phosphatase (PTP) activity of Shp2 is required for IL-22-induced Erk2 activation.
Next, Shp2 PTP inhibitor NSC87877 was used to further test the above effect. NSC87877 was recently identified as a potent Shp2 PTP inhibitor for its binding to the catalytic cleft of Shp2 PTP (Chen et al., 2006). Serum-starved SW480 cells were preincubated with or without NSC-87877 and then stimulated with IL-22 or mock treated. As shown in Figure 3E, NSC87877 completely inhibited Erk1/2 activation 10 min later after IL-22 treatment.
Using the pair of Flp-In-293 cell lines expressing the wild-type (Flp-In-293/IL-22R1) and Shp2-binding defective IL-22R1 receptor (Flp-In-293/IL-22R1-FF) cells, the role of Shp2-IL-22R1 interaction in IL-22-induced activation of STAT3 and Erk, p38 and Jnk MAP kinases were examined by immunoblotting. Flp-In-293/IL-22R1 and Flp-In-293/IL-22R1-FF cells were treated with IL-22, respectively, and the activating phosphorylation of STAT3, Erk1/2, p38 and Jnk were analyzed at various time points after IL-22 treatment. As shown in Figure 4A, same cell surface expression of human wild-type IL-22R1 (hIL-22R) and mutant IL-22R1 (hIL-22R-FF) were expressed in both stable cell lines. Maximal tyrosine phosphorylation of STAT3 occurred within 10 min after IL-22 stimulation in Flp-In-293/IL-22R1 cells (Figure 4B), but STAT3 tyrosine phosphorylation was delayed for 30 min in Flp-In-293/IL-22R1-FF cells (Figure 4C). Furthermore, a marked effect of mutant IL-22R1 receptor on STAT3 serine phosphorylation was also observed in Flp-In-293/IL-22R1 cells. IL-22-induced STAT3 serine phosphorylation in Flp-In-293/IL-22R1 cells (Figure 4B), but was completely blocked in Flp-In-293/IL-22R1-FF cells (Figure 4C). These results suggest that Shp2 binding to IL-22R1 is essential for IL-22 to induce both tyrosine and serine STAT3 phosphorylation.
We next examined the activation of three major MAP kinases in Flp-In-293/IL-22R1 and Flp-In-293/IL-22R1-FF cells in response to IL-22. As shown in Figure 4B, IL-22 activated Erk1/2, p38 and Jnk with a similar kinetics that reached the maximal activation in 10 min in Flp-In-293/IL-22R1 cells, but a delayed (to >60 min) Erk and Jnk activation in Flp-In-293/IL-22R1-FF cells, and a smaller effect on p38 was detected (Figure 4C). The Erk MAP kinase pathway is known to be involved in regulation of cell proliferation (Johnson and Lapadat, 2002; Zhang and Liu, 2002). As data in this report have shown that IL-22 activates MAP kinase pathway in both SW480 and Flp-In-293/IL-22R1 cells, and IL-22R1-FF delays this activation, the role of IL-22R1-Shp2 interaction on cell proliferation was further examined. Results showed that IL-22 increased SW480 cell proliferation, but the IL-22-stimulated cell proliferation was blocked by a Shp2 PTP inhibitor, NSC87877 (Figure 4D). IL-22-stimulated cell proliferation was also studied by using Flp-In-293/IL-22R1 and Flp-In-293/IL-22R1-FF cells, and a lower level of cell proliferation was observed in Flp-In-293/IL-22R1-FF cells treated with IL-22 compared with Flp-In-293/IL-22R1 cells (Figure 4E). These results suggest that Shp2 binding to IL-22R1 contributes to the proliferative effect of IL-22.
Previous reports showed that IL-22 activates Erk and Jnk MAP kinases in several cell lines including hepatic cells and intestinal epithelial cells (Lejeune et al., 2002; Brand et al., 2006), however, the detailed mechanism for IL-22-induced MAPK activation in these cell lines is mostly unclear. Here we show for the first time that the protein tyrosine phosphatase Shp2 is associated with IL-22R1 upon stimulation with IL-22, and the Shp2-IL-22R1 interaction mediates Erk activation induced by IL-22.
Shp2 is known to play a positive role in activation of the MAPK pathway induced by growth factors such as EGF (Cunnick et al., 2002). Besides, it also regulates cytokine-evoked Erk activation (Cunnick et al., 2002; Neel et al., 2003). Previous observations indicated that Shp2 upregulated IL-2-induced activation of MAPK in both cutaneous T cell lymphoma and NIH3T3 cells (Gadina et al., 1998; Lundin et al., 2002), whereas defective Shp2 inhibited activation of MAPK in response to IL-2. Shp2 also binds to pY759 of the human gp130 receptor subunit after IL-6 induction, and IL-6-induced activation of the MAPK cascade is blocked by mutation of Tyr759 on gp130 (Lehmann et al., 2003). In this report IL-22R1-Shp2 interaction in response to IL-22 in SW480 cells and HEK293 cells was shown, and the role of IL-22R1-Shp2 interaction in the activation of Erk MAP kinase was further investigated. We provided evidence that mutations of either Tyr251 or Tyr301 to phenylalanine in IL-22R1 abolished the binding of Shp2 to IL-22R1 in cells treated with IL-22. Because Shp2 contains two SH2 domains that are arranged in tandem in the N-terminal portion of Shp2, the requirement of both Tyr251 and Tyr301 for IL-22R1-Shp2 association suggests that both phosphotyrosines may simultaneously interact with the tandem SH2 domains of Shp2. Thus, both Tyr251 and Tyr301 in IL-22R1 are required for Shp2 binding to IL-22R1 though this notion needs to be examined further. We previously have shown that as a major Gab1-binding partner in EGF-stimulated cells, Shp2 interacts with Gab1 in a specific orientation, in which the N-SH2 domain of Shp2 binds the Tyr627 site in Gab1, while the C-SH2 domain binds to Tyr659 site, and both Tyr627 and Tyr659 of Gab1 are required for EGF-stimulated Erk2 activation (Cunnick et al., 2001). In this study, we show that both Tyr251 and Tyr301 of IL-22R1 are required for IL-22-stimulated Erk2 activation in HEK293 cells. Surprisingly, Erk1/2 and Jnk activation in response to IL-22 were delayed in cell lines stably expressing Shp2-binding defective IL-22R1 mutants, whereas IL-22-induced activation of p38 was slightly affected. These observations need to be further investigated. So far, how IL-22R1-Shp2 interaction contributes to the activation of the MAP kinase pathway induced by IL-22 is unclear. Our previous studies have shown that Src is a downstream effector of Shp2 that mediates Erk activation by EGF (Cunnick et al., 2001; Cunnick et al., 2002; Ren et al., 2004). In this research, we also detected that IL-22-induced Src activation in SW480 cells, and activation of Erk1/2 happened at almost the same time point as Src was activated (10 min), the role for Src in the activation of Erk induced by IL-22 remains to be elucidated.
Shp2 may also affect JAK/STAT pathways (Xu and Qu, 2003). Shp2 upregulates JAK2 activity induced by prolactin though Shp2 was reported to negatively regulate the IFN-induced JAK1/STAT pathway (Ali et al., 2003; Baron and Davignon, 2008; Tsai et al., 2009). Consisting with previous studies in human hepatocytes and rat hepatoma cells, IL-22-induced rapid tyrosine phosphorylation of STAT3 in cell lines stably expressing WT IL-22R1, whereas tyrosine phosphorylation of STAT3 was delayed in cell lines stably expressing Shp2-binding defective IL-22R1. This observation is consistent with the data reported by Dumoutier et al. (2009), which show that mutation of all cytoplasmic tyrosine residues of the IL-22R1 only partially affects STAT3 activation and tyrosine phosphorylation of STAT3 is only abolished by both mutating all tyrosines, and deleting the C-terminal domain of the receptor. Interestingly, serine phosphorylation of STAT3 in cell lines stably expressing Shp2-binding defective IL-22R1 was completely inhibited rather than delayed, the detailed mechanism for this phenomenon and the physiological significance remain to be clarified.
Induction of cell growth by IL-22 is observed in some cell types. For instance, IL-22 induces proliferation of IL-22R1-transfected BaF3 cells (Bleicher et al., 2008), and another experiment in HT-29 cells showed that at concentrations of 10 and 100 ng/ml IL-22 significantly increased cell proliferation, whereas there is a trend for an antiproliferation activity using higher concentrations of IL-22 (Brand et al., 2006). Whether Shp2-IL-22R1 interaction plays a role in cell proliferation induced by IL-22 is investigated in this research. Our results indicate that proliferation of cell lines stably expressing mutant IL-22R1 induced by IL-22 was greatly inhibited. Furthermore, we observed the Shp2 PTP inhibitor NSC87877 almost completely inhibited IL-22-induced proliferation of SW480 cells. These results suggest that association of Shp2 to IL-22R1 is required for cell proliferation induced by IL-22.
In summary, we presented evidence that Shp2 is recruited to IL-22R1 upon stimulation with IL-22. Furthermore, both tyrosine 251 and tyrosine 301 of IL-22R1 are required for Shp2 binding to IL-22R1 and for IL-22-stimulated Erk2 activation. Moreover, Shp2-IL-22R1 interaction contributes to tyrosine and serine phosphorylation of STAT3 and cell proliferation in response to IL-22. Considering IL-22R1 is expressed in many important tissues such as kidney, liver and skin, our data indicate a critical role for Shp2-IL-22R1 interaction in related tissue responses.
Recombinant human IL-22 was from Cytolab. Antibodies to active Erk1/2 and total Erk were from Promega, antibodies to β-actin and FLAG tag (M2) were from sigma, anti-HA (haemagglutinin) antibody was from Covance Research Products, antibodies against phosphotyrosine (4G10), Jak1, Tyk2 and Shp2 were from Upstate Biotechnology, antibody to IL-22R1 was from BD, phospho-specific antibodies to p38 MAP kinase, c-jun amino-terminus kinase (Jnk), Src homology 2 (Src), phospho-Tyk2 and phospho-STAT3 were from Cell Signaling Technology. Shp2 inhibitor NSC87877 was from Merck. Flp-In™-293 cells, a pcDNA5/FRT/TOPO Expression kit, Zeocin™, hygromycinB, recombinant human EGF and Lipofectamine were purchased from Invitrogen. WST-1 cell proliferation assay kit was from Roche Applied Science.
Plasmids expressing Flag-Shp2 and Flag-Shp2-Cys459Ser, HA-Erk2 and HA-Shp2 were previously described (Cunnick et al., 2002). An IL-22R1 expression plasmid was kindly provided by Dr Kotenko and was subcloned into a pcDNA3.1-FLAG vector. Mutagenesis was performed by changing tyrosine into phenylalanine residues and subcloning into the pcDNA5/FRT/TO, respectively.
HEK293 and human colorectal cancer SW480 cells were grown in Dubecco's Modified Eagle Medium containing 10% fetal calf serum in a CO2 incubator at 37 °C. Transfection was performed with lipofectamine according to the manufacturer's instructions. To establish stable cell lines for expression of Flag-IL-22R1 and Flag-IL-22R1 mutants, Flp-In-293 cells were cultured in DMEM containing 10% fetal bovine serum and 100 µg/ml Zerocin, wild type or mutant IL-22R1 plasmids and the Flp recombinase expression plasmid pOG44 were transfected into Flp-In-293 cells according to the manufacturer's instructions. Single colonies resistant to hygromycin B were picked and subcultured. The positive colonies were selected by immunobloting using an anti-FLAG antibody. Cell surface expression of the different IL-22R1 variants was checked by flow cytometry using an anti-FLAG monoclonal antibody.
For luciferase assays, SW480 cells were electroporated (250 V, 200 ohms, 1200 µF) with 30 µg of pSRE-Luc or STAT3-Luc. Three micrograms of pRL-TK (Promega) encoding renilla luciferase reporter were co-electroporated as an internal control of the transfection process. Otherwise, 0.9 µg of various IL-22R1 plasmid constructs, combined with 0.9 µg of pSRE-Luc or STAT3-Luc and 0.2 µg of pRL-TK were co-transfected into HEK293 cells. The cells were seeded in 24-wells plates at 10 000 cells and starved for 16 h. Cells were then stimulated with 100 ng/ml IL-22 before lysis, luciferase activity was measured using a luminometer. For each experiment, luciferase activity was determined and normalized using the corresponding internal renilla luciferase control activities, and results were shown as fold increases by the mean ± SEM from at least three separate experiments.
Immunoprecipitation (IP) and immunoblot (IB) analyses were performed essentially as described previously (Meng et al., 2005), and all IP and IB experiments were performed twice.
Cells were seeded onto 96-well plates at a density of 10 000 cells per well and were allowed to attach overnight, then stimulated with IL-22 or with cytokine-free medium (negative control) for 48 h. The cell proliferation rate was determined by WST-1 assay according to the manufacturer's instructions.
This work was supported by the Natural Science Foundation of Jiangsu Province (BK2006071), and in part by grants from National Institutes of Health (R01CA077467), National Natural Science Foundation of China (30772523), National Basic Research Program of China (2009CB918401), the Shanghai projects (07SG28, 08ZZ23, 08PJ14042), and the Dr. Tsai-fan Yu Research Endowment Fund.