Melanosomes undergo maturation in four stages, and the matured melanosomes are transported to neighboring keratinocytes through a protein complex consisting of Myo Va, Rab27a, and Mlph. Dysfunctions in these proteins hinder melanosome transport, leading to the accumulation of melanosomes in the nuclear region of melanocytes and resulting in vitiligo or hypopigmentation. However, the regulatory mechanisms governing Myo Va expression in melanocytes are still largely unknown.
Previous studies have shown that MNQO causes melanosome aggregation and reduces key proteins involved in melanosome transport in melanocytes [12]. Additionally, BMP-2 has been shown to upregulate Myo Va expression in vascular smooth muscle cells [13]. Thus, we investigated whether BMP-2 is an upstream regulator of Myo Va expression with MNQO treatment.
Knockdown of BMP-2 decreased Myo Va expression in melanocytes (Fig. 1A, B). MNQO treatment reduced BMP-2 and Myo Va expression in melanocytes (Fig. 1C, D). Consequently, we aimed to elucidate Myo Va expression in BMP-2-induced melanocytes, unveiling a novel role for BMP-2 in stimulating Myo Va gene expression in melanocytes. Finally, BMP-2 induces the expression of Myo Va in melanocytes by Smad signaling pathway (Fig. 2).
We confirmed that BMP-2 increased the phosphorylated Smad protein in a concentration-dependent manner in melanocytes (Fig. 2A). BMP-2 also expressed phosphorylated Smad protein at 2 hours (Fig. 2B). Smad1 knockdown via siSmad1 transfection significantly reduced Myo Va expression, which was increased by BMP-2 (Fig. 2C). We sought to ascertain a direct role of Smad signaling in BMP2-induced Myo Va activation. Smad-binding elements reported so far contain a 5-bp consensus of CAGAC or GTCT, which are usually GC-enriched [14, 15]. We confirmed that the Myo Va promoter contains an SBE site associated with Smad-mediated transcriptional activity (Fig. 2D). The direct binding of Smad 1/5/8 phosphorylated at the Myo Va promoter containing SBE was confirmed by ChIP assay, with enhanced binding upon BMP-2 treatment (Fig. 2E).
In summary, our findings reveal a novel mechanism by which BMP-2 in the Smad signaling pathway regulates Myo Va expression in melanocytes. Further investigation is necessary to further investigate regulatory role of BMP-2 in melanosome transport, such as Mlph and Rab27a, and to determine if there is a new mechanism underlying BMP-2 regulation of Myo Va gene expression. Also, these data will be helpful in future treatment of hyperpigmentation and albinism.