Wnt5a negatively regulates melanogenesis in primary Arctic fox epidermal melanocytes.

Melanocytes, which are mainly found in the epidermis, are responsible for the melanin of skin and hair, and thereby contribute to the appearance of skin and provide protection from damage by ultraviolet radiation. Our previous study revealed that the Wnt5a, one of the many genes that affect melanin production, might be involved in the coat color seasonal change of the Arctic fox by influencing skin melanogenesis. Although the role of Wnt5a in melanocyte lines and melanoma cells has been extensively studied, its role in primary epidermal melanocytes has not been explored. This study aimed to investigate the role and mechanism of the Wnt5a in influencing melanogenesis in Arctic fox primary epidermal melanocytes. We constructed the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) knockout plasmid targeting exons of the Wnt5a and transfected it into primary epidermal melanocytes. The results of the amplification knockout region assay, RT-qPCR assay, and western blot assay showed the success of Wnt5a knockout. RT-qPCR assay and melanin content assay showed that melanin production in melanocytes was significantly increased after Wnt5a knockout, and melanin-related key genes, such as microphthalmia-associated transcription factor, tyrosinase and tyrosinase-related protein 1, were significantly elevated. In addition, we also found that the expression of the β-catenin gene of the Wnt canonical pathway was significantly elevated after Wnt5a knockout. In conclusion, our results indicate that the Wnt5a plays a negative regulatory role in melanogenesis in primary epidermal melanocytes, and is presumably involved in antagonizing or inhibiting canonical Wnt signaling.