Nitric oxide induces apoptosis of human primary melanocytes by regulating calcium homeostasis via VDAC1.

Abstract

Vitiligo is an autoimmune skin disease that originates from damage and loss of melanocytes (MCs). Studies have identified abnormal nitric oxide (NO) metabolism in vitiligo patients, but the mechanisms by which NO contributes to MC loss remain unclear. Skin samples from patients with vitiligo and healthy controls were collected to evaluate the expression of three nitric oxide synthases (NOS) isoforms. Keratinocytes, MCs, and fibroblasts were exposed to a cytokine cocktail (IFN-γ, TNF-α, IL-1β and LPS) to simulate the pro-inflammatory microenvironment of vitiligo. Then NOS activation and the capacity for NO production were examined in each cell type. Sodium nitroprusside (SNP) was used as the NO donor to evaluate its cytotoxic effects on MCs. Cell viability, trypan blue staining rate, and lactate dehydrogenase release were measured following SNP exposure. Ultrastructural changes in MCs were observed using transmission electron microscopy. Apoptosis rate, intracellular calcium concentration, and mitochondrial membrane potential were assessed using flow cytometry. BAPTA-AM was used for intracellular calcium chelation and small interfering RNA was used to silence VDAC1 expression in MCs. Inducible NOS (iNOS) expression was significantly upregulated in patients in the active vitiligo lesions. Under pro-inflammatory conditions, keratinocytes and fibroblasts produced increased NO via iNOS activation. MCs exposed to a high-NO environment displayed dose-dependent cellular damage. SNP-treated MCs showed ultrastructural features of apoptosis, including condensed nuclear chromatin and swollen mitochondria. Flow cytometry and analysis of pro-apoptotic markers confirmed mitochondrial-mediated apoptosis in MCs. Both calcium chelation and VDAC1 silencing alleviated the apoptosis of MCs by restoring calcium homeostasis and mitochondrial membrane potential. NO-induced apoptosis in MCs was mediated by calcium overload and mitochondrial dysfunction. iNOS activation may represent a contributor to melanocyte loss in vitiligo and a potential therapeutic target.