A P value <005 was considered significant Most of the experimen

A P value <0.05 was considered significant. Most of the experiments were repeated in three or four independent trials with similar results, and representative images are included in this article. All other materials and methods are described in the Supporting Materials and Methods. IL-22R1 messenger RNA (mRNA) expression was detected in quiescent and activated mouse HSCs (mHSCs), and these levels were comparable to IL-22R1 mRNA levels in hepatocytes (Fig. 1). IL-22R1 mRNA expression increased further after treatment with IL-22 in cultured

HSCs (Fig. 1B). selleck screening library Expression of IL-10R2 mRNA, which is also required for IL-22 signaling, was detected in HSCs as well as in hepatocytes and Kupffer cells (Fig. 1A). Additionally, western blotting check details analyses revealed the expression of IL-22R1 protein in primary mHSCs, which was slightly increased after IL-22 treatment (Fig. 1C). Fluorescence-activated cell sorting analyses detected IL-22R1 protein expression on the surface of primary mHSCs, and comparable expression levels were observed in HSCs from wild-type (WT) and IL-22TG mice (Supporting Fig. 1A,B). Finally, the expression of

IL-22R1 and IL-10R2 mRNA was also detected in primary human HSCs (hHSCs) from 3 human donors and in the hHSC cell line, LX2 (Fig. 1D). The effects of IL-22 on the signaling pathways in HSCs are shown in Fig. 1E. IL-22 exposure significantly activated STAT3 in all samples, with peak effects observed at 30-60 minutes. Activated STAT3 levels returned to basal levels by 120 minutes. IL-22 also induced extracellular signal-related kinase 1/2 (ERK1/2) medchemexpress activation in primary mHSCs and, to a lesser extent, in hHSCs and LX2 cells. Furthermore, IL-22-dependent STAT3 activation in HSCs was further confirmed by immunostaining for phosphorylated STAT3 (pSTAT3) in the nuclei of HSCs (Supporting Fig. 1C,D). IL-22 has been shown to promote hepatocyte survival and proliferation4; therefore, we examined the potential antiapoptotic and

mitogenic effects of IL-22 on HSCs. The nuclear morphology of HSCs revealed a significant increase in apoptosis after a 4-hour incubation with cycloheximide (CHX) that was markedly reduced in IL-22 pretreated HSCs (Fig. 2A and Supporting Fig. 2). The antiapoptotic function of IL-22 in HSCs was further demonstrated by a reduction in CHX-mediated induction of caspase-3 and -7 activity and cleaved caspase-3 expression in HSCs after IL-22 treatment (Fig. 2A,B). Furthermore, Fig. 2C shows that serum and platelet-derived growth factor (PDGF), but not IL-22 treatment, increased bromodeoxyuridine (BrdU) incorporation in HSCs (Fig. 2C), indicating that IL-22 does not affect HSC proliferation. Finally, the expression of antiapoptotic proteins, such as pSTAT3 and B-cell lymphoma 2 (Bcl-2), was markedly increased, whereas expression of the mitogenic protein, cyclin D1, was slightly elevated in HSCs after IL-22 exposure (Fig. 2D).

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