Study On IL28RA,Psoriasis Susceptibility Gene,Involved In Proliferation And Differentiation Of HaCaT Cells And Its Molecular Mechanism

BACKGROUND:Psoriasis is a common chronic inflammatory dermatosis,which is charactered with scaly erythema of the skin.The etiology of psoriasis is multifactorial and thought to relate to interactions among environmental,genetic factors and autoimmunity.It is easy to relapse and difficult to cure.The patients are suffered from the pain of physical and mental.However,the exact pathogenesis of psoriasis has not yet been fully elucidated,which is one of the most urgent problems to be solved in the field of dermatology.It is currently thought that the pathogenesis of psoriasis is activation of T cells and dendritic cells,which release IL-23 and IL-12 to activate T cells,Th1 cells and Th22cells that produce large amounts of psoriasis cytokines such as IL-17,IFN-γ,TNF and IL-22,which in turn act on keratinocytes to exacerbate psoriatic inflammation leading to the proliferation,differentiation of keratinocytes,the formation of a clinically visible psoriasis characteristic lesions.As for complex diseases,Genome-wide association analysis is one of the best methods for finding susceptibility genes.A large number of psoriasis susceptible genes have been found by GWAS.IL28RA with immune function is of these susceptibility genes that shared by psoriasis and SLE in the Chinese Han population.In addition,IL28RA had also been reported as the psoriasis susceptibility loci of the European.IL28RA gene localizes in chromosome 1p36.11 region and consists of 9exons.The protein product of this gene belongs to class II cytokine receptor family.IL28RA protein forms a complex with interferon 10 receptor beta（IL10R?）and interacts with IL-28A,IL-28B and IL-29（also known as lambda interferons）which exerts an antiviral and growth-suppressive activity both in vitro and vivo.The function of IL28RA in the pathogenesis of psoriasis has not been reported.Psoriasis is unique to human.Thus,the available nonhuman models of psoriasis usually provide only an approximation of the disease.Application of the TLR7/8 agonist imiquimod induced psoriasis-like skin inflammation in mice to mimic the human psoriasis in the experiments.Key aspects of the pathogenesis of psoriasis,including the activation of plasmacytoid dendritic cells and their dependence on Th17 cells were emphasized in the imiquimod induced annimal model.Object:In this study,constructed human immortalized keratinocyte lines which have stable overexpression and low expression of IL28RA.Then we detected the functional changers such as cell proliferation,differentiation and its signaling pathways.We try to clarify the function of IL28RA in HaCaT cells and speculate a potential role of IL28RA in the pathogenesis of psoriasis.Constructed low-dose imiquimod induced mouse model in vitro,we try to identify the effect of mIL-28A on the skin lesions of mice.Methods:In this study,the expression of IL28RA was detected in normal skin and lesions of psoriasis patients（n=10）by immunohistochemistry and Western Blot.Then,stable overexpression of IL28RA,knock-down and control of the empty cell lines were transfected into HaCaT cells with lipofectamine LIP3000.Cell proliferation was detected by MTS assay and wound healing assay was used to detect cell migration ability.Cell cycle protein and differentiation protein expression were measured by Western Blot.Cell cycle was analyzed by flow cytometry.Due to IL28RA and IL10R?the receptor complex,IL-29 ligand was added to stimulate and then examined its proliferation,cyclins,cell cycle,differentiation markers and the signaling pathways involved respectively;the mRNA expression of differentiation markers was detected by Real-time PCR.Low dose imiquimod（1.56 mg/mouse）were induced psoriasis-like lesions in mice and the other mice were scribbled with low-dose imiquimod（1.56mg/mouse）and the mixure of imiquimod（1.56 mg/mouse）and mIL-28A（100 ng/μl）repectively.Mice were sacrificed after one week.The skins of histopathology were observed by HE staining and the expression of IL28RA of the mice was detected by Western Blot.Results:The expression of IL28RA in the lesions was lower compared with that in normal skin of patients.CyclinB1 expression was down-regulated but CyclinE expression was up-regulated by IL28RA overexpression;When IL-29 was added,CyclinB1 expression was down-regulated while CyclinE expression was up-regulated,compared with the controls.MTS assay and wound healing showed that overexpression of IL28RA inhibited HaCaT cell proliferation,on the contrast,knock-down of IL28RA promoted cell proliferation of HaCaT.Treated with IL-29（100 ng/ml）,the growth of inhibition in the IL28RA overexpression was more significantly than that of the control.However,there was no obvious effect on cell wound healing stimulated with IL-29（100ng/ml）.Overexpression of IL28RA blocked cell cycle in S phase,while stimulation with IL-29（100 ng/ml）,cell cycle were inhibited in G2/M phase by Flow cytometry analysis.The proliferation of HaCaT cells were inhibited by IL-29（100 ng/ml）activating the JAK-STAT signaling pathway and upregulating the phosphorylation of STAT1.The specific inhibitor JAKI can suppress the JAK-STAT signaling pathway.Keratinocyte differentiation markers（Involucrin and Loricrin）were upregulated in IL28RA over-expressed cell lines induced by Ca²⁺,whereas knockdown of IL28RA showed opposite result.Differentiation protein were repressed by IL-29（100 ng/ml）through activating the JAK-STAT signaling pathway.Low-dose imiquimod（1.56mg/mouse）induced psoriatic-like thickening in mice compared to control mice by HE staining.Low dose of imiquimod（1.56 mg/mouse）mixed with mIL-28A（100 ng/μl）which exacerbated the thickening of the skin lesions,indicating that mIL-28A aggravated the inflammation of the lesions.Compared with the control,expression of IL28RA was weaker in the lesions of mice treated with low dose of imiquimod and the mixure of imiquimod（1.56 mg/mouse）and mIL-28A（100 ng/μl）treatment by Western Blot.The expression of IL28RA in the back of mice in each group was similar to that in patients with psoriasis.Conclusion:Compared with non-lesional tissues,the expression of IL28RA protein was lower in the lesions of patients with psoriasis which suggested that its expression might be related to hyperproliferation and abnormal differentiation of psoriatic keratinocytes.Through the IL28RA receptor complex,the proliferation and differentiation of HaCaT were inhibited by IL-29 cytokines by activating the JAK-STAT signaling pathway in vitro experiments.The lesions of mouse skin were aggravated by mIL-28A mixing with low-dose imiquimod.Therefore,we speculated that down-expression of IL28RA in the lesions might be involved in the pathogenesis of psoriasis in Chinese Han population.IL28RA may serve as a potential molecular target and provide a new direction for the treatment of psoriasis.