Non-Keratinocyte Snap29 Influences Epidermal Differentiation and Hair Follicle Formation in Mice

melanin through the regulation of signalling molecules associated with the melanogenesis both in vitro and in vivo model systems. Experimental design A detailed description of the materials and methods is provided in the supplementary section, see Data S1. Results The cytotoxicity of AA (Fig. 1a) in the melan-a murine melanocytes was determined. AA did not exhibit a significant cytotoxicity (% survival >86%, Figure S1a) and a morphological change (Figure S1b) when treated with up to 20 lM AA. However, as depicted in Fig. 1b, the numbers of pigmented cells was markedly reduced in AA-treated cells. Therefore, further mechanistic studies were performed with the test concentrations of AA up to 20 lM in cultured cells. When primarily determined the tyrosinase inhibition by AA using a mushroom TYR in a cell-free system, AA slightly inhibited the enzyme activity (Fig. 1c). However, AA significantly inhibited the melanin synthesis in a concentration-dependent manner with the IC50 value of 11.5 lM (Fig. 1d, e) in melan-a cells, and the inhibitory activity was comparable to that of a-arbutin, a well-known skin-whitening agent. We next elucidated the mechanism of action in the inhibition of melanin synthesis by AA with the analysis of melanogenesisassociated biomarkers in melanocytes. AA effectively downregulated the melanogenesis-related mRNA expressions of MITF (Figure S2a) and TYR (Figure S2b) compared to those of control cells. AA also significantly suppressed the protein levels of TYR, TRP-1 and TRP-2 expressions (Fig. 2a). The expressions of c-Kit, a relevant upstream regulator of MITF, and a transcription factor SOX10 which acts on promoter region of MITF, were also downregulated by AA (Fig. 2b). The expressions of MITF, TYR and TRP-1 were also suppressed by AA with time exposure until 8 h (Figure S2c). The downregulation of MITF by AA was further confirmed using an immunocytochemical analysis (Fig. 2c). The expressions of MEK1/2 and ERK which involved in melanogenesis pathway (7), and p21, a cofactor of MITF in melanoma cells (8) and also downstream target of MITF (9), were also suppressed by AA (Figure S2d). AA also significantly inhibited the promoter activities of both MITF and TYR (Fig. 2d, e), suggesting that AA affects both the expression and activity of MITF in the melanogenesis. The antimelanogenesis activity of AA was further confirmed in in vivo zebrafish embryo model systems (Fig. 2f). Conclusions In this study, americanin A, a natural lignan, was identified as an effective inhibitor of melanin synthesis by the downregulation of MITF and TYR expressions without affecting the cytotoxicity in melanocytes. Taken together, these findings suggest that the antimelanogenic activity of AA may be attributable to serve as a plausible candidate for a skin-whitening agent through the regulation of MITF signalling pathway. Acknowledgements The wild-type zebrafishes were kindly provided by Dr. Kyu-Won Kim and Dr. Jae-Hak Park (Seoul National University), and this study was supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (Grant Number: HN14C0088). Author contributions Y. S., H. J. P., J.-Y. H., S. S. K. and S. K. L. conceived and designed the study. Y. S. and E. J. J. performed the experiments. Y. S. and S. K. L. wrote and revised the manuscript. All authors read and approved the final version of the manuscript. Conflict of interests The authors have declared no conflicting interest. Supporting Information