The Roles And Mechanisms Of FGF7/10 In Regulating The Epidermal Cells Differentiation Into Eccrine Sweat Glands

Objective We had analyze the differential genes of Sprague-Dawley(SD)rat eccrine sweat glands placodes and hair follicle placodes by RNA-seq,and found that the expression of FGF7 and FGF10 in the placodes of the eccrine sweat glands was significantly higher than that in the placodes of the hair follicle.The purpose of this project is to study the roles and mechanisms of FGF7/10 in regulating epidermal cells differentiation into eccrine sweat glands.Methods 1.We were selected E17.5d dorsal skin(present hair follicle placodes)and E19.5d ventral footpad skin(present eccrine sweat glands placodes)of the SD rat to perform immunofluorescence staining.Detected the keratins K4,K5,K7-10,K12,K14,K15,K17-20,K27 and K73 and the reported specific markers of eccrine sweat glands Na+/K+-ATPase(NKA),Na+-K+-2Cl-cotransporter 1(NKCC1)and the markers of hair follicle P-cadherin,Lymphoid enhancer factor 1(LEF1),and LIM Homeobox gene 2(Lhx2).Lhx2/K14 double immunofluorescence staining differentiated the placodes of eccrine sweat glands and hair follicle,and Western blot(WB)experiments detected the differential antigens and some co-expressed antigens of the eccrine sweat glands placodes and hair follicle placodes to further verify the results of immunofluorescence staining.2.Human epidermal cells were culture in nude mice and divided into " human epidermal cells + human epidermal cell 3D culture medium + HC matrigel " group(NC group),"human epidermal cells + human epidermal cell 3D culture medium + HC matrigel + 100ng/m L FGF7 PODS" group(FGF7 group),and "human epidermal cells + human epidermal cell 3D culture medium + HC matrigel + 100ng/m L FGF10 PODS" group(FGF10 group),the "human epidermal cells + human epidermal cell 3D culture medium + HC matrigel + 100ng/m L FGF7 PODS + 100ng/m L FGF10 PODS" group(FGF7+10 group).Cultured for 5 weeks,then sampled for paraffin embedding,HE staining,and immunofluorescence staining to detect the human eccrine sweat gland marker K7 and hair follicle marker K73.Human scalp tissues(This site containing both eccrine sweat glands and hair follicles)were used as positive controls,with 3 samples per group(n=3).3.Human epidermal cells were cultured in nude mice,and divided into NC group,FGF7 group,FGF10 group,and FGF7+10 groups.After 5 weeks of in vivo cultured of nude mice,samples were taken for WB experiment.Detecting of MAPK and AKT signaling pathway genes,such as ERK,p-ERK,P38,p-P38,JNK,p-JNK,AKT,p-AKT,these signaling pathways were significantly higher in the placodes of eccrine sweat gland than in the placodes of hair follicle.Then found out the differential genes in each group.Results 1.Both eccrine sweat glands and hair follicles placodes expressed K4,K5,K14,K15,K17,K18,P-cadherin and LEF1,neither expressed K7,K8,K9,K10,K12,K19,K20,K27,K73,NKA or NKCC1.Hair follicles placodes specifically expressed Lhx2.Combination of Lhx2 and co-expressed antigen K14 through double immunofluorescence staining can distinguish eccrine sweat glands placodes from hair follicles placodes.2.Human epidermal cells cultured in nude mice.The HE staining showed that each group formed organoids.Immunofluorescence staining showed that the organoids in the NC group only expressed the human hair follicle marker K73,but did not express the human eccrine sweat gland marker K7;the FGF7 group,FGF10 group,and FGF7+10 group positively expressed the hair follicle marker K73 and the eccrine sweat gland marker K7.3.The results of WB showed that the expression levels of FGFR1,FGFR2,ERK and p-ERK in the FGF7 group,FGF10 group and FGF7+10 group were higher than the NC group,while not found significant difference expression of AKT,p-AKT,P38,AKT,p-AKT,P38,p-P38,JNK,p-JNK in each groups.Conclusions 1.Lhx2 is a specific marker for hair follicle placodes,and eccrine sweat glands placodes and hair follicle placodes can be distinguished by double immunofluorescence staining of the specific marker Lhx2 and the co-expressed markers,such as K4,K5,K14,K15,K17,K18,P-cadherin and LEF1.2.FGF7 and FGF10 proteins can regulate the human epidermal cells differentiation into eccrine sweat glands.3.ERK1/2 signaling pathway may be involved in FGF7 and FGF10 in the regulation of epidermal cell differentiation to eccrine sweat glands.