Mechanism of Ca2+ overload caused by STIM1/ORAI1 activation of store-operated Ca2+ entry (SOCE) in hydrogen peroxide-induced mitochondrial damage and apoptosis in human primary melanocytes.

Abstract

Background: Vitiligo is a common depigmentation disorder. Oxidative stress in melanocytes is thought to be the primary cause of vitiligo. Imbalances in cellular calcium ion (Ca²⁺) levels may be associated with the onset and progression of various diseases through a process that has been linked to oxidative stress. The purpose of this study was to investigate the regulatory mechanism by which Ca²⁺ levels change in normal human melanocytes (NHMs) under oxidative stress, thereby providing new insights and potential clinical therapeutic targets for the pathogenesis and treatment of vitiligo.

Methods and results: Single-cell RNA sequencing data from vitiligo patients were analyzed using bioinformatics techniques. NHMs were treated with hydrogen peroxide (H₂O₂), store-operated Ca²⁺ entry (SOCE) blocker BTP2, and SOCE agonist cyclopiazonic acid. Flow cytometry was used to detect Ca²⁺ levels, apoptosis rates, intra-mitochondrial reactive oxygen species (ROS) levels, and mitochondrial membrane potential (MMP) damage. The expression levels of target proteins were detected using immunofluorescence, quantitative real-time PCR, and western blotting. We found that H₂O₂-induced oxidative stress resulted in significantly increased intracellular Ca²⁺ levels, upregulation of stromal interaction molecule 1 (STIM1) and calcium release-activated calcium channel protein (ORAI1), and mitochondrial dysfunction. Inhibition of SOCE and small interfering RNA-mediated silencing of STIM1/ORAI1 expression lowered mitochondrial levels of ROS and oxidative stress-induced intracellular Ca²⁺ overload and restored MMP, ultimately terminating oxidative stress-induced apoptosis.

Conclusions: Oxidative stress upregulates STIM1/ORAI1 expression, leading to melanocyte apoptosis via increased Ca²⁺ influx, whereas inhibition of SOCE protects melanocytes against oxidative stress-induced damage.