Nitric oxide enhances melanogenesis of alpaca skin melanocytes in vitro by activating the MITF phosphorylation

Ultraviolet (UV) B radiation can cause skin-tanning via the synthesis of melanin which is synthesized by specific tyrosinase and tyrosinase-related enzymes expressed in melanocytes. It is reported that several melanogenic factors are released from keratinocytes and other cells surrounding melanocytes in the skin following UV radiation. Some of them are reported to up-regulate tyrosinase gene expression through a different pathway, but most regulate tyrosinase via microphthalmia-associated transcription factor (MITF). It is unknown whether an NO-induced pathway regulates melanogenesis via MITF in vitro. In this study, we investigated this problem because it is important for our understanding of how to enhance the coat color of alpaca. We set up three groups for experiments using alpaca melanocytes: the control cultures were allowed a total of 5 days growth; the UV group cultures were also allowed 5 days of growth like the control group, but were then irradiated once everyday with 312 mJ/cm(2) of UVB; the UV + L-NAME group was the same as the UV group, but with the addition of 300 μM L-NAME every 6 h. To determine the NO inhibition effect, NO product was measured. To determine the effect of NO on MITF, the expression levels of the MITF gene and protein were measured by immunofluorescence, quantitative real-time PCR and western immunoblotting. To determine the influence of NO on MITF phosphorylation, phosphorylated MITF protein (p-MITF) was measured by western immunoblotting. To determine the effect of NO on melanogenesis, the melanin content was measured. The results provide exciting new evidence that NO can enhance melanogenesis in alpaca skin melanocytes by stimulating MITF phosphorylation.