Nicastrin mutations in familial acne inversa impact keratinocyte proliferation and differentiation through the Notch and phosphoinositide 3-kinase/AKT signalling pathways

BACKGROUND: Acne inversa (AI) is a chronic inflammatory skin disease with an autosomal dominant inheritance pattern. Mutations of the gene encoding nicastrin (NCSTN), a cofactor subunit of γ-secretase, are responsible for familial AI. However, whether deficiency of nicastrin is functionally implicated in the biological behaviours of human keratinocytes and related molecular mechanisms remains unknown. OBJECTIVES: To study alterations of biological traits and related signalling pathways modulated by nicastrin knockdown in keratinocytes. METHODS: A human immortalized keratinocyte cell line (HaCaT) was treated with efficient small interfering (si)RNA-targeted NCSTN. Cell proliferation was measured by CCK-8 assay; cell-cycle and cell apoptosis analyses were detected by flow cytometry. Microarray analysis was applied to uncover impacts of NCSTN silencing on whole-genome expression of HaCaT cells. Altered signalling pathways were further confirmed by real-time polymerase chain reaction, Western blotting and immunohistochemistry in both HaCaT cells and lesions of a patient with AI with NCSTN mutation. RESULTS: NCSTN knockdown in HaCaT cells impaired γ-secretase activity, leading to increased cell proliferation and S-phase population. Microarray data also showed that numerous genes and pathways implicated in proliferation and differentiation of keratinocytes were statistically changed. Among these genes, expression levels of several Notch pathway molecules, known as γ-secretase substrates, were validated to be significantly attenuated in both nicastrin-silencing HaCaT cells and the lesion of the patient. Furthermore, a remarkable elevation of expression of phosphoinositide 3-kinase (PI3K), AKT and its activated form pAKT was illustrated in siRNA-treated HaCaT cells. CONCLUSIONS: Deficiency of the NCSTN in familial AI may regulate proliferation and differentiation of keratinocytes mainly through the Notch and PI3K/AKT signalling pathways.