Roles Of Proliferation In Human Immortalized Keratinocytes Induced By NCSTN-TGF-β Signaling In The Pathogenesis Of Acne Inversa

Aims:Patients with acne inversa(AI),which is a chronic inflammatory skin disease,suffer from symptoms of the disorder as well as loss of quality of life.The precise mechanisms of AI have not been known.Loss-of-function mutations of genes encoding components in y-secretase,including NCSTN have been shown to be positively associated with family AI.In AI patients,epidermal cells have increased levels of proliferation and abnormal differentiation,whereas activation of the transforming growth factor(TGF)-βsignaling pathway decreases.However,whether TGF-β signaling is involved in the changes remains unknown.The aim of this project is to test whether TGF-β signaling decreases in HaCaT cells with NCSTN knockdown,and roles of this signaling pathway in proliferation and differentiation NCSTN knockdown HaCaT cellsMethods:HaCaT cells with NCSTN knockdown(referred to as shRNA cells)were used in this study.Proliferation was measured using the CCK8 assay.mRNA levels of genes of interest were assessed using q-PCR.Protein levels and phosphorylation of target proteins were measured by Western blotting.Activation of TGF-β signaling was achieved by application of SRI-011381 in cultured cells.Results:1.shRNA cells had decreased levels of NCSTN in both mRNA and protein levels.2.mRNA levels of genes in the TGF-β signaling pathway decreased.3.Protein levels of TGF-β1,a key ligand in the TGF-β signaling pathway,decreased in shRNA cells.Levels of phosphorylated SMAD2 and SMAD 3 decreased,and inhibitory SMAD7 increased in shRNA cells.Activation of the TGF-β signaling pathway was downregulated in shRNA cells.4.Application of SRI-011381 reversed increased proliferation in shRNA cells,and decreased mRNA expression of Ki-67 and PCNA.5.No effects were observed in differentiation of shRNA cells treated with SRI011381.Conclusion:Knockdown of NCSTN in HaCaT cells causes decreased activation of TGF-βsignaling,which consequently induces increased proliferation.Activation of TGF-βsignaling might be a viable therapeutic option for those with AI.