Low-concentration hydrogen peroxide can upregulate keratinocyte intracellular calcium and PAR-2 expression in a human keratinocyte–melanocyte co-culture system

Hydrogen peroxide (H₂O₂) may have a biphasic effect on melanin synthesis and melanosome transfer. High H₂O₂ concentrations are involved in impaired melanosome transfer in vitiligo. However, low H₂O₂ concentration promotes the beneficial proliferation and migration of melanocytes. The aim of this study was to explore low H₂O₂ and its mechanism in melanosome transfer, protease-activated receptor-2 (PAR-2) expression and calcium balance. Melanosomes were fluorescein-labeled for clear visualization of their transfer. The expression of protease-activated receptor-2 (PAR-2) in keratinocytes was determined by western blot analysis. Flow cytometry was employed to evaluate the effects of H₂O₂ on calcium levels in keratinocytes. Fluorescence microscopy showed the upregulation of melanosome transfer into keratinocytes following 0.3 mM H₂O₂ treatment in the co-cultures rather than in the untreated control groups, which was associated with higher expression of PAR-2 protein and increased calcium concentration. The addition of a PAR-2 antagonist inhibited the positive activity of H₂O₂ and calcium flow in keratinocytes. When calcium flow was blocked by a calcium chelator, the addition of H₂O₂ did not increase the PAR-2 expression level in keratinocytes, therefore, inhibiting dendrite formation and melanosome transfer. Low H₂O₂ concentration promotes melanosome transfer with increased PAR-2 expression level and calcium concentration in keratinocytes. In addition, the interaction between melanocytes and keratinocytes is more beneficial to enhance calcium levels in keratinocytes which mediate melanin transfer. Moreover, low H₂O₂ concentration promotes dendrite formation, in which extracellular calcium and Par-2 were involved.