Protease Inhibitor A2ML1 Regulates The Differentiation And Adhesion Of Skin Cells In Healthy Humans And Patients With Basal Cell Carcinoma Via KLK7

ObjectiveThe balance between proteases and protease inhibitors is important to maintain the normal barrier function of human epidermis.Alpha-2-Macroglobulin Like 1(A2ML1)is a member of the serine protease inhibitors family.It is predominately expressed in the highly differentiated keratinocytes,however,it is not clear whether affects epidermal differentiation and epidermal barrier function.In this study,we investigated A2ML1 expression in basal cell carcinoma compared to normal skin biopsies.Additionally,we investigated that A2ML1 regulating the differentiation process and adhesion levels by interacting with serine protease KLK7.Methods 1.Performed immunohistochemical staining in paraffin-embedded sections from normal healthy human's skin tissue(n=11)and from basal cell carcinoma patients' skin tissues,then detected and compared the differences in A2ML1 expression and distribution between these two groups.2.Co-IP and western blotting were used to investigate the expression and interaction of A2ML1 and KLK7,Chymotrypsin in undifferentiated and differentiated HaCaT cells.3.A2ML1 expression was inhibited in the undifferentiated and differentiated HaCaT cells by si RNA,m RNA expression of the KLK5,KLK7,adhesion molecules(DSC-1,DSG-1,FN-1,Ecad-1/2),and differentiation labeled markers(IVL,FLG-1)were evaluated by q RT-PCR,and the protein variation of KLK7,adhesion-related proteins(E-cad-1,FN-1,DSC-1),and differentiationlabeled markers(IVL,FLG-1,CDSN)protein were detected by western blotting.4.High-and low-A2ML1 expression-samples were selected from the basal cell skin tissues,together with normal healthy skin tissues to evaluate the correlation of A2ML1 to KLK7,DSC-1,E-cad-1,FN-1,KRT-10,CDSN and FLG-1.Results 1.A2ML1 expressed in the granular layer and stratum corneum of the normal epidermis tissues.And the expression of A2ML1 in the epidermis of BCC skin tissues was significantly reduced or almost absent.2.KLK7 and Chymotrypsin are expressed in undifferentiated and differentiated keratinocytes.A2ML1 expression is very limited in undifferentiated cells,but significantly increased in differentiated cells.What's more,endogenous A2ML1 interacts with KLK7 and Chymotrypsin.3.After A2ML1 knockdown in the undifferentiated and differentiated HaCaT,the m RNA of KLK5 was reduced,but it wasn't obviously regulated before differentiation,the m RNA of KLK7 was not affected by the variation of A2ML1 before or after HaCaT differentiation.In the predifferentiated HaCaT,common differentiation markers and adhesion molecules are almost out control of A2ML1 while in the differentiated HaCaT cell line,DSC-1,E-cad-1,E-cad-2,DSG-1,FN-1,together with IVL and FLG-1 showed different degrees of decline relative to the control group,after being transfected with si-A2ML1 of No.3 and No.4 actually.For the protein level,except for that FLG-1 is weakly regulated by A2ML1 after HaCaT differentiation,DSC-1,FN-1,E-cad-1,CDSN,and IVL are affected to different extents by A2ML1 knockdown before or after differentiation.4.KLK7,differentiation markers(CDSN,FLG-1,KRT-10)and adhesion factors(DSC-1,FN-1,Ecad-1)in the skin tissues among the healthy people,the A2ML1-high-expressed BCC patients and the A2ML1-low-expressed BCC patients showed a significant differences in the expression level and distribution fields.Conclusion 1.There is a significant difference in the expression and distribution range of A2ML1 between normal skin tissue and basal cell carcinoma skin tissue.2.A2ML1 in the HaCaT cells could interact with the serine proteases such as KLK7 and Chymotrypsin.3.A2ML1 took part in the regulation process of differentiation and adhesion in HaCaT cells.4.The expression level of A2ML1 in healthy normal skin and basal cell carcinoma skin tissue is correlated with the degree of differentiation and adhesion of skin cells.