Suppression of melanin synthesis by Americanin A in melan‐a cells via regulation of microphthalmia‐associated transcription factor

Abnormal skin pigmentations are caused by an accumulation of skin colouring pigment melanin. The mammalian pigmentation is a complex process that is finely regulated by various factors including microphthalmia-associated transcription factor (MITF), tyrosinase-related protein (TRP)-1, -2 and tyrosinase (TYR). The critical step in the melanin synthesis in humans is the hydroxylation of L-tyrosine to 3,4-dihydroxy-phenylalanine (L-DOPA) and the sequential oxidation of L-DOPA to dopaquinone. TYR catalyses these biochemical reaction steps in melanocytes and thus is known to be a key regulator enzyme in melanogenesis 1. In addition, the transcription of TYR is regulated by the MITF in melanocytes 2. Therefore, the TYR and MITF are considered crucial molecular targets in the screening of inhibitors of melanin synthesis. Natural products have been served as potential sources in the development of skin-whitening agents with the inhibition of melanin hyperpigmentation 3. In our programme of searching for the inhibitors of melanin hyperpigmentation from natural sources, many plant extracts were found to be active with the inhibition of melanin synthesis and modulation of MITF. In this study, we suggest americanin A (AA), an active principle of the seeds of Phytolacca americana (Phytolaccaceae), is a potential candidate in the inhibition of melanin synthesis. Previous findings showed the biological activities of AA with the anti-inflammatory 4 and antioxidant effects 5. The inhibition of TYR was also reported 6, but the detailed mechanism of action remains to be elucidated. Herein, we investigated the transcriptional regulation and signal transduction pathways in the melanogenesis by AA in cultured melanocytes. What is the mechanism of action related to the antimelanogenic activity of americanin A (AA), a natural lignan? In this study, we elucidated the inhibitory activity of AA in the production of melanin through the regulation of signalling molecules associated with the melanogenesis both in vitro and in vivo model systems. A detailed description of the materials and methods is provided in the supplementary section, see Data S1. 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 μm 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 μm 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 μm (Fig. 1d, e) in melan-a cells, and the inhibitory activity was comparable to that of α-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 melanogenesis-associated 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). 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. 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). 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. The authors have declared no conflicting interest. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. 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