Pym-5 promotes MIFT-dependent melanogenesis in vitro.

The hallmark of differentiated melanocytes is the presence of melanin pigment, and there are multiple targets of MITF involved in melanin synthesis. To this end, we next investigated whether Pym-5 could affect melanogenesis. Here, forskolin was applied as a reference compound that induces melanogenesis. In parallel with AT7519, CHIR and forskolin, Pym-5 increased the levels of limiting enzymes in melanin biosynthesis, including tyrosinase (TYR), tyrosinase-related protein 1 (TYPR1), and tyrosinase-related protein 2 (TYRP2) in two murine melanoma cells (Fig. 3A). Furthermore, Pym-5 augmented the tyrosinase activity (Fig. 3B) and promoted melanin production in B16 and B16BL6 cells (Fig. 3C). Next, we ascertained the effects of Pym-5 by a pharmacological inhibitor of the MITF molecular pathway that inhibits melanogenesis [[16]Faloon P.W. Bennion M. Weiner W.S. Smith R.A. Wurst J. Weiwer M. Hartland C. Mosher C.M. Johnston S. Porubsky P. Neuenswander B. Dandapani S. Munoz B. Schoenen F.J. Metkar S. Haq R. Fisher D.E. Aube J. Palmer M. Schreiber S.L. Small A. Molecule inhibitor of the MITF molecular pathway, probe reports from the NIH molecular libraries program.]. ML-329 treatment alone reduced the expression of MITF, and Pym-5-induced expression of MITF was partially abrogated by ML-329 (Fig. 3D). Moreover, ML-329 attenuated Pym-5-induced transcription levels of MITF and its target gene TYR (Fig. 3E), as well as melanin production (Fig. 3F) in two melanoma cells, indicating that MITF was essential for Pym-5-mediated melanogenesis. Taken together, Pym-5 can activate MITF-dependent metabolic pathway in melanin biosynthesis.