Interleukin22 Suppress Renal Cell Carcinoma Growth Figure 7. Lack of apoptosis in A498 cells after rhIL-22 treatment. A. Treatment of A498 cells with 50 ng/ml rhIL-22 for 24 h did not induce apoptosis as measured by annexin V or PI-positive cells. B. Treatment of A498 cells with 50 ng/ml rhIL-22 for 72 h did not induce apoptosis. N = 3. doi:10.1371/journal.pone.0020382.g007 and necessary criteria for clinical treatment demand the development of more effective drugs to combat this disease. IL-22, which in humans is mainly produced by Th22-, Th1- and Th17-cells, is strongly involved in immune regulation and inflammatory responses [19,27,28,30]. IL-22 plays an important role in inflammatory processes through up-regulation of acute phase reactions and of pancreatitis-associated protein [14,15,22,23,31]. However, there is some debate about the effects of IL-22 on tumors. We suppose that IL-22 may play different roles in different tumors and that discrepancies in experimental results may be due to different cell types and diseases. The results of the present study support the idea that IL-22 suppresses the growth of A498 cells both in vivo and in vitro. The functional IL-22 receptor complex consists of two receptor chains, IL-22R and IL-10R2. IL-22R2 is broadly expressed in various tissues [24,32], and the expression of the IL-22R chain determines whether a cell will be a target of IL-22. In our study, we detected measurable expression of IL-22R in A498 cells and showed that IL-22 suppressed the growth of RCC cells in a dose- dependent manner. Similar effects were obtained in A498 xenograft tumors. Although IL-22 is produced by immune cells, unlike other cytokines it does not affect immune cells directly but instead regulates the functions of their target cells, increasing the innate immunity of tissue cells, protecting tissues from damage and enhancing their regeneration. The signal transducer and activator of transcription 1 (STAT1) plays a critical role in carcinogenesis by mediating various biological responses and has been implicated as a tumor suppressor [33]. Both carcinogen-induced and trans- planted tumors are increased in STAT1 knockout mice cpmpared with wild-type control mice [34,35]. Zhu demonstrated that SNPs in the STAT1 gene (homozygotes of the minor alleles at SNP rs867637, rs3771300, or rs2280235) are associated with a risk of developing hepatocellular carcinoma in Chinese people [36]. While, Specific inhibition of ERK1/2 phosphorylation by a variety of natural and synthetic compounds has been shown to be effective in anticancer strategy in the treatment of breast cancer [37,38].To better understand the molecular mechanisms that underlie the anti-RCC tumor effects of IL-22, we assessed the phosphorylation of STAT1 and ERK1/2 in A498 cells after rhIL- 22 treatment. Our western blotting assays showed that p-STAT1 was founded in A498 cells at the 5th minute and reached its peak level 30 minutes after rhIL-22 exposure. While ERK1/2 was dephosphorylated in response to rhIL-22; this effect was maximal 30 min after rhIL-22 exposure. It suggests that rhIL-22 activates STAT1 and inhibits ERK1/2 pathways in A498 cells in a time- and dose-dependent manner and that the biological functions of the receptors for these pathways show time-dependent STAT1 activation and ERK1/2 deactivation evoked by rhIL-22. Concerning the concentration of STAT1 protein in A498 cells after IL-22 exposure was stable independent on the vary dose of IL-22 and longer exposure time, we believe that IL-22 induce STAT1 pathway activating directly rather than act on gene level. Hence, the STAT1-ERK1/2 pathway participates, at least in part, in the inhibition of A498 cell growth that occurs after IL-22 binding to and activation of IL-22R. To further understand the role of STAT1-ERK1/2 pathway in the growth inhibition of A498 cells exposed to IL-22, the expression of STAT1 was deleted in A498 cells by STAT1 specific siRNA. The phosphor- ylation STAT1 was decreased by 87.2% after treated with IL-22 compared with the controls and independent to dephosphorylat- ed ERK1/2 pathway.The results show that the growth inhibition of A498 cells with STAT1 deletion was only partially revised in these cells treated with IL-22 compared with the controls (p.0.05), which indicates that the activation of STAT1 pathway is the major mechanism involved in the growth inhibition of A498 cells rather than dephosphorylation of ERK1/2. Dysfunc- tional STAT1 may contribute to cancer development and progression, while at the same time the ERK1/2 pathway regulates a common set of cell death regulators such as BCL-2, BCL-XL and BIM, indicating that it plays a role in cell cycle control [39–41]. ERK1/2 inhibitors would therefore be expected to function as anti-tumor agents [37,38]. Concerning the lack of contribution of ERK1/2 pathway to the growth inhibition of A498 cells exposed to IL-22, it can be hardly concluded that IL- 22 could serve as a reasonable ERK1/2 inhibitor for A498 cells. (Figure S1: a schematic diagram for STAT1-ERK1/2 pathway work in this procedure.) PLoS ONE | www.plosone.org 5 May 2011 | Volume 6 | Issue 5 | e20382