Roles Of Gasdermin A3 In Regulating The Expression Of Krt6b And Frizzled 4 In Mouse Skin Epithelium

BackgroundSkin is composed of epidermis and dermis.The mammalian skin and its appendages(e.g.,hair follicle,sebaceous gland and sweat gland)protecting the body from external damage,keaping the homeostasis of the internal environment and maintaining the body temperature.Hair follicle is an important skin appendages,hair follicle morphogenesis begins on embryonic day 12.5(E12.5).Postnatalhair follicle undergoes cycles of anagen,catagen and telogen.Many pathways and factors,especially differentiation associated factors,take part in the hair cycle regulation.Abnormalities in any part may affect the normal circulation of the hair follicle cycle.Keratin is the main component of skin,hair and nails.The type 2 keratinKrt6 b is an important member of the keratin family.Krt6 b plays a role in mature and differentiation of the mammalian epidermal keratinocytes and protects the skin and internal organs of mice and other mammals.Gsdermin A3 belongs to the GSDM family,located on the eleventh mouse chromosome.Mutation of Gsdermin A3 caused thickening mouse skin and abnormal hair follicle development.We found in our previous studythat the number of epidermal cells increased,epidermal and the stratum comeum thickened in Gsdma3 mutant mouse.These are related to the proliferation and differentiation of keratinocytes suggests that Gsdma3 gene may be related to Krt6 b.Wnt/?-catenin pathway is important to regulate the proliferation and differentiation of skin epithelial cells.Our previous study found that Wnt/?-catenin pathway was activated in Gsdma3 mutant mouse,which suggested that the Gsdma3 gene is associated with the Wnt/?-catenin signaling pathway.Frizzled 4 is the receptor of Wnt/?-catenin signaling pathway.It is reported that expression of Krt6 bwas regulated by Frizzled in Wnt/?-catenin pathway.Therefore,the Gsdma3 gene may regulate the expression of Krt6 b through the Wnt/?-catenin signaling pathway.Objectives:1.By analyzing the expression of Krt6 b and Frizzled4 under the condition of Gsdma3 overexpression or downregulation,confirm the effect of Gsdma3 on Krt6 b and Frizzled4 expression.2.By inhibiting the Wnt/?-catenin signaling pathway in mouse skin and observing the Krt6 expression,find out the mechanismof regulation of Krt6 b expression by Gsdma3.Means:1.Analyze the effect of Gsdma3 on the expression of Krt6 b and Frizzled 4,and reveal the relationship between Krt6 b and Frizzled 4in the skin epithelial by virus infection,tissue section,immunofluorescence,RT-PCR,Q-PCR and western blot technique using C57BL/6 mice,Gsdma3 mutant mice(AE mice)and Gsdma3 expression virus,through Wild type mice and Gsdma3 mutation comparison,Wnt signal pathway inhibitor injection in wild type and Gsdma3 mutation mice.2.Analysis of the Gsdma3 gene expression and the changes of Frizzled 4 expression in cultured mouse skin epithelial cells(JB6 cells)using in vitro culture of mouse skin epithelial cells(J6B cells)and Gsdma3 gene over expression of adenovirus,and using viral infection,immune fluorescence,RT-PCR,Q-PCR,WB and other experimental methods.Results:1.By comparing the change between wild type and Gsdma3 mutant mice among different hair follicle cycle stage,we found that expression of Krt6 b was upregulated in Gsdma3 mutant skin epithelium.Gsdma3 restored expression and immunofluorescence found that Krt6 bexpression was inhibited.However,when over express Gsdma3 in depilated wild mice.2.By using immunofluorescence,RT-PCR,Q-PCR and western blot to study the difference in hair follicle cycles between wild type and Gsdma3 mutant mice,we found that expression of Frizzled 4 was upregulated in Gsdma3 mutant mice.By over express Gsdma3 in cultured mouse skin epithelial cells and immunofluorescence,Q-PCR,RT-PCR,and western blot to detected the expression of Frizzled 4,we foung out that Frizzled 4expression was inhibited when Gsdma3 was over expressed.3.Through RT-PCR,Q-PCR and WB to restore Gsdma3 in Gsdma3 mutant mice and over express Gsdma3 in wild type depilation model,we found that expression of Frizzled and Krt6 bare both downregulated.Wnt/ beta-catenin signaling pathway inhibitors injection and immunofluorescence showed that expression of Frizzled and Krt6 bare both downregulated.Conclusion:1.Gsdma3 gene inhibits the expression of Krt6 b in mouse epithelium.2.Gsdma3 inhibits the expression of Wnt/?-catenin pathway receptor Frizzled 4in epithelium.3.In mouse skin epithelium,Gsdma3 inhibits Krt6 bexpression by inhibiting the expression of Frizzled 4.