Activation Of IL-6/MEK/ERK Pathway Promotes The Expression Of LMO4 On Proliferation And Differentiation Of Keratinocyte

Background and Objective Psoriasis is an immune-mediated chronic inflammatory disease of the skin,histologically characterized by(1)epidermal acanthosis,hyperkeratosis,and parakeratosis;(2)dilated capillary network in the papillary dermis;(3)a mixed inflammatory infiltrate including polymorphonuclear cells,as well as intraepidermal collections of neutrophils.The pathogenesis of psoriasis involves a variety of mechanisms,the most important of which are genetic susceptibility and environmental factors that affect the immune system.Currently,most treatment options for psoriasis are associated with immunosuppression.Therefore,it is easy to speculate that a specific defect in the immune system is an important pathogenic principle.There is evidence that cytokines(especially IL-6 and TGF-α)abnormally secreted by immunocompetent cells in the skin of patients play an important role in the inflammatory response of psoriasis.These inflammatory cytokines can stimulate the activation and proliferation of T cells,further promote the secretion of cytokines such as TNF-α,IL-17,and IL-22,which influences the proliferation and differentiation of keratinocytes.Studies have shown that the activated IL-6 pathway will alter the function of the T cells.In the pathogenesis of psoriasis,the abnormal proliferation and differentiation of keratinocytes stimulated by inflammatory factors is the final stage of the development of psoriasis.Therefore,it is particularly important to study the mechanism by which cytokines regulate the proliferation and differentiation of keratinocytes.At present,the study of the signaling pathways by which cytokines function and their target genes are relatively rare.IL-6 is a multifunctional cytokine that plays an important role in immune response,acute phase response,hematopoiesis,and inflammatory responses.In addition,it can also regulate cell proliferation and differentiation.Previous studies have confirmed that IL-6 expression levels are significantly higher in serum and lesion tissues of patients with psoriasis than in normal subjects.It is proved that IL-6 can influence the development of psoriasis by causing a balance disorder between Treg cells and Th17 cells.LMO4,a nuclear transcription factor,is a member of the LMO protein family and plays an important role in cell growth,differentiation,and organ development.Studies have shown that during the development of psoriasis,the cytokine IL-23 can up-regulate the expression of LMO4 gene,thereby promoting the proliferation and differentiation of keratinocytes.Therefore,we hypothesized that IL-6 may also regulate the expression of the LMO4 gene through certain signaling cascades.This study aimed to investigate the effect of cytokine IL-6 on the expression of transcription factor LMO4 in keratinocytes and its molecular mechanism,and provide new ideas for the treatment of psoriasis.Methods 1.Histological level studies Immunohistochemistry was used to detect the difference of LMO4 expression between normal tissues and psoriatic lesions,and the changes of related skin cell marker molecules.At the same time,the level of IL-6 and the activation of its signaling pathway were detected to explore the correlation between IL-6 and psoriasis.2.Cellular level studies Human immortalized keratinocyte cell lines were used as research objects.Western blot,immunofluorescence and EDU cell proliferation assay were used to investigate how IL-6 signaling pathway mediates the expression of LMO4,which influences the proliferation and differentiation of keratinocytes.To further understand the molecular mechanism by which IL-6 regulates LMO4,mutating the transcription factor binding site,analysing the regulation activity by a dual luciferase reporter gene system.3.Animal models A psoriasiform mouse model was established by applying imiquimod ointment to the mice,and the phenotypic changes of the skin tissue were observed.H&E staining was used to analyze the changes of skin tissue structure,Immunohistochemistry was used to detect the expression levels of IL-6 and LMO4 and the activation state of IL-6 signaling pathway,to verify that IL-6 regulates the expression of LMO4,which alters the proliferation and differentiation of keratinocytes.Results 1.Immunohistochemistry results showed that IL-6 and LMO4 expression levels were increased in psoriasis lesions compared with normal skin tissues,and the activation of p-MEK and p-ERK was significantly ascended in the IL-6/MEK/ERK signaling pathway.2.Western Blot and immunofluorescence showed that the expression of K14,K10,IVL and LMO4 increased after keratinocytes were induced by IL-6,suggesting that IL-6induced the differentiation of keratinocytes.Western Blot results showed that the expression of p-MEK,p-ERK and p-p65 in IL-6/MEK/ERK/NF-k B signaling pathway increased significantly,accompanied by the increase of LMO4 expression after IL-6 treatment.The results of cell proliferation assay showed that the proliferation rate of cells was significantly accelerated after IL-6 induction.The luciferase activity of IL-6-treated 293 T cells was significantly increased under the driving of LMO4 gene promoter,while the luciferase activity treated with IL-6 showed no significant difference compared with the control group,after mutation of the NF-k B binding site in promoter.3.The result of psoriasiform mouse model showed that p-MEK and p-ERK were significantly increased in the lesion tissues,accompanied by an increase in LMO4 expression.Conclusion 1.IL-6 can activate the IL-6/MEK/ERK/NF-k B signaling pathway in keratinocyte to regulate the expression of LMO4,thereby altering the proliferation and differentiation of keratinocytes.