UC Irvine

Melanogenesis, the process by which specialized cells within the epidermis synthesize melanin, is an intricate and complicated process that requires the coordinated action of transcriptional, translational and trafficking processes. Melanin, a complex polymer derived from the amino acid tyrosine, is the primary determinant of hair, skin and eye color in humans. It plays a monumental role in the protection of the eyes and skin from solar ultraviolet-radiation through direct absorption mechanisms. Melanin is aberrantly regulated in melasma and vitiligo, two pigmentary disorders that are characterized by areas of skin that are hyper-pigmented and hypo-pigmented, respectively. Although a variety of treatment options exist for these disorders, many of the treatments are not very effective and have significant side effects. Tyrosinase inhibitors are used to treat melasma, and tyrosinase (TYR) is an enzyme that catalyzes the rate-limiting step in melanin synthesis. In this work, we investigate the role of TYR in regulating pigment production in vivo and examine the suitability of TYR as a therapeutic target for hyperpigmentary disorders. We demonstrate that the partial depletion of Tyr in the mouse was not sufficient to induce significant decreases in melanin accumulation in the mouse hair follicle, suggesting that TYR may not be an ideal target for the treatment of hyperpigmentary disorders. To address the need for novel, effective treatments for pigmentary disorders, a genome-wide siRNA functional genomics approach was employed to identify pharmaceutically tractable, single gene loci that impact melanogenesis. The screen uncovered 92 novel regulators of melanogenesis, including Aldehyde dehydrogenase 1A1 (ALDH1A1), which was one of only three genes able to impact TYR expression in three distinct genetic backgrounds. We discovered that a potent catalytic inhibitor of ALDH1A1, cyanamide, was able to inhibit not only the accumulation of pigment, TYR and MITF mRNAs in melanoma cells, but also melanin synthesis in three-dimensional human skin equivalents, suggesting that inhibitors of this enzyme may be ideal to therapeutically treat hyperpigmentation disorders, including melasma. In this work, we have identified how ALDH1A1 regulates melanogenesis. We show that 9-cis retinoic acid, a molecule important in cellular differentiation and the product of ALDH1A1 catalysis in vitamin A metabolism, stimulates the accumulation of pigment, TYR and MITF mRNAs, and TYR protein in melanoma cells and primary human epidermal melanocytes. As the biological effects of 9-cis retinoic acid are mediated by two classes of nuclear receptors, the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs), we searched for and identified a putative RAR/RXR binding site within the promoter of MITF-A, an isoform of MITF not known previously to play a role in melanin synthesis. While rigorous chromatin immunoprecipitation (ChIP) studies demonstrated significant enrichment of both RARα and RXRα upstream of the transcription start site of MITF-A, no enrichment was found in the promoter region of MITF-M, suggesting a novel mechanism in the regulation of melanogenesis. We then went on to show, through luciferase-activity assays, that retinoids can activate the full-length MITF-A promoter, but not a truncated MITF-A promoter that lacks the putative RAR/RXR heterodimer binding site. Lastly, we demonstrate that when used in combination, RARα and RXRα agonists stimulate the accumulation of MITF-A and MITF-M transcripts. Taken together, our data outline a mechanism for how ALDH1A1 regulates melanogenesis. We provide key evidence that targeting this enzyme may be appropriate in the development of novel therapeutics for the treatment of melasma. Finally, we show that retinoids or retinoid-analogs may be ideal in targeting hypopigmentary disorders, including vitiligo.