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

ABSTRACT

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.