The Mechanism By Which Cathepsin B Cleaves GSDMA To Regulate The Proliferation And Differentiation Of Keratinocytes

GSDMA is a member of the GSDMs family that is mainly expressed in the epithelium of the gastrointestinal tract and the skin.Although it has shown that the mutation of Gsdma3 at 1124 G makes hair follicle stem cells unable to differentiate into sebaceous gland and deregulates the proliferation and differentiation of keratinocyte in the skin,the underlying mechanism by which GSDMA regulates keratinocyte proliferation and differentiation remains unkown.To address this,we first analyzed the expression of GSDMA during the differentiation of primary human keratinocytes(NEHKs)induced by high concentration of calcium,and found that the expression of GSDMA was significantly increased in the differentiated keratinocyte.We then used CRISP/Cas9 to generate GSDMA^–/–Ha Cat.The deficiency of GSDMA inhibited the proliferation of keratinocytes.While the overexpression of N-GSDMA and C-GSDMA,but not GSDMA in Ha Cat promoted the keratinocyte differentiation.Altogether,these data demonstrate that GSDMA regulates keratinocyte proliferation and differentiation.The above data suggest that N-GSDMA,C-GSDMA and GSDMA might exert different functions in keratinocyte proliferation and differentiation.However,how GSDMA is cleaved under physiological condition is unclear.We then screened which enzyme could cleave GSDMA and found that cathepsin B(CSTB),highly expressed in epidermal keratinocytes around wounded skin,cleaved GSDMA,while the inhibition of CSTB activity by its inhibitor CA 074Me inhibited the cleavage activity of CSTB on GSDMA.These data suggest that CSTB cleaves GSDMA to regulate keratinocyte proliferation and differentiation.Taken together,our data demonstrate that GSDMA regulates keratinocyte proliferation and differentiation and CSTB cleaves GSDMA to release C-GSDMA,and N-GSDMA.Both C-GSDMA and N-GSDMA promote keratinocyte differentiation.The increase of GSDMA during keratinocyte differentiation might be a feedback response;however,the underlying mechanism needs further investigation.