Investigation That The Related Mechanism Of IL-22 Effects On HB-EGF And TIG3 Expression In Keratinocytes

Psoriasis is a common skin disorder characterized by the development of erythematous,scaling plaques on the skin and a wide spectrum of clinical presentations. It has psoriasis vulgaris, psoriasis arthropathica, erythroderma psoriaticum and psoriasis pustulosa four forms. The pathogenesis of psoriasis is not yet clear.Present study suggests that the primarily pathogenesis of psoriasis is the immune response mediatted by T cells in keratinocytes as a target.Th22 cells, a new subset of CD4+ T-helper, is characterized by secretion of IL-22 but not IL-17 or IFN-γ. Abnormal expression of Th22 cells was seen in peripheral blood of psoriasis. IL-22, which is the most important functional cytokine of Th22 cells plays an important role in autoimmune diseases. IL-22 expression is elevated in the plasma and skin lesions and has a strong positive association with the severity of disease in patients with psoriasis.Our previous studies have shown that heparin-binding epidermal growth factor-like growth factor(HB-EGF) plays an important role in psoriatic epidermis hyperproliferation. The previous studies indicate that IL-22 could upregulate the expression of HB-EGF in cultured Ha Ca T cells,but the mechanism is unknown. Mitogen-activated protein kinase(MAPK-ERK1/2) pathway and activator of transcription pathway(JAK/STAT) signaling pathways play an important role in the pathogenesis of psoriasis. Tazarotene-induced gene 3(TIG3) as a tumor suppressor gene plays an important role in the proliferation of keratinocytes. The expression of TIG3 is downregulated in psoriatic lesions.Therefore, we hypothesize that IL-22 upregulates the expression of HB-EGF in keratinocytes may through MAPK-ERK1/2 and JAK/STAT signaling pathways. IL-22 may downregulate the expression of TIG3 in psoriasis lesions. IL-22 takes part in the pathogenesis of psoriasis mainly through upregulating the HB-EGF expression and inhibiting the TIG3 expression. In this experiment, we study the mechanism of IL-22 upregulating HB-EGF expression and the effects of IL-22 on TIG3 expression in Ha Ca T cells.Objective To investigate the mechanism of IL-22 effects on HB-EGF expression and TIG3 expression in keratinocytes.Methods 1.Firstly, at a 60% confluence, HaCa T cells were exposed to different concentrations(12.5, 25, 50 and 100μg/L) of IL-22 for 24 h.We collected the total cellular protein.The Western-blot was used to detect the signaling pathways proteins. Secondly, for inhibitor experiments, cells were pre-treated with AG490 or PD98059 for 2h before stimulation with 50 μg/L IL-22. Both the total cellular protein and total m RNA were collected.Western blot, ELISA method were used to detect the protain level of HB-EGF and real-time quantitative reverse transcription-polymerase chain reaction(RT-PCR) was used to detect the m RNA level of HB-EGF in Ha Ca T cells. Statistical significance was evaluated by one-way analysis of variance(ANOVA) followed by Dunnett-t test. A p value of <0.05 was considered statistically significant. 2. The mice model of psoriasis-like were created by application of imiquimod sustained on the back skin of mice for six days. Then we injected 1μg IL-22 subcutaneous. After 48 hours, the psoriasis-like lesions on the back of mice was taken to detect the expression of HB-EGF by immunohistochemistry. 3.At a 60% confluence, Ha Ca T cells were exposed to different concentrations(12.5, 25, 50 and 100μg/L) of IL-22 for 24 h. For inhibitor experiments, cells were pre-treated with AG490 or PD98059 for 2h before stimulation with 50 μg/L IL-22. The immunofluorescence was use to detect the position of the expression of TIG3 in the cells by assay protein.Both the total cellular protein and the total m RNA were collected.Western blot, ELISA method were used to detect the protain level of TIG3 and RT-PCR was used to detect the m RNA level of TIG3 in Ha Ca T cells. Statistical significance was evaluated by one-way analysis of variance(ANOVA) followed by Dunnett-t test. A p value of <0.05 was considered statistically significant.Results 1. Western blot analysis showed that IL-22 treatment promoted the phosphorylation of JAK2, STAT3 and ERK1/2 in Ha Ca T cells, compared to untreated control. The total levels of JAK2, STAT3 and ERK1/2 protein remained unchanged after IL-22 treatment. The inhibition of cell signaling by PD98059 and AG490 significantly blocked the upregulation of HB-EGF by IL-22(50 μg/L), compared to exposure to IL-22 alone. 2. The imiquimod successfully induced psoriasis lesions in Bal B/c mice, IL-22 could upregulate the HB-EGF experssion of normal Bal B/c mice and mice model of psoriasis-like back skin. 3. IL-22 exposure resulted in a significant(p < 0.05) reduction in the m RNA level of TIG3 in Ha Ca T cells. Moreover, such reduction was in a concentration-dependent manner. Western blot and ELISA analysis confirmed the downregulation of TIG3 at the protein level in IL-22-treated Ha Ca T cells.TIG3 protein displayed a predominant cytoplasmic localization in Ha Ca T cells and this distribution pattern was not altered upon IL-22 treatment. However, TIG3 immunofluorescence intensity was decreased in IL-22-treated cells.Moreover, pre-treatment with PD98059 or AG490 significantly(p<0.05) blocked the release of TIG3 from Ha Ca T cells induced by IL-22.Conclusions IL-22 can activate the MAPK-ERK1/2 and JAK2/STAT3 signaling pathway. The effects of IL-22 on HB-EGF expression in Ha Ca T cells may related with MAPK-ERK1/2 and JAK2/STAT3 signaling pathways.IL-22 can upregulate the HB-EGF experssion of normal Bal B/c mice and mice model of psoriasis-like back skin.IL-22 can suppress the expression and secretion of TIG3 in Ha Ca T cells. Both MAPK-ERK1/2 and JAK2/STAT3 signaling pathways are involved in the downregulation of TIG3 by IL-22. These findings warrant further exploration of the IL-22/TIG3 axis in the pathogenesis of skin diseases including psoriasis.