The significance of melanotropic hormones as physiologic regulators of cutaneous pigmentation in humans is still controversial. Until recently, no direct effect for melanotropins could be demonstrated on human melanocytes.Here we present conclusive evidence that a!-melanotropin (a-melanocyte-stimulating hormone, ai-MSH) and the related hormone corticotropin (adrenocorticotropic hormone, ACTH) stimulate the proliferation and melanogenesis of human melanocytes maintained in culture in a growth medium lacking any AMP inducer. The minimal effective dose of either hormone is 0.1 nM. In time-course experiments, the increase in cell number and tyrosinase activity became evident after one treatment of the melanocytes with 100 nM a-MSH for 48 hr. The mitogenic effect gradually increased to 50-270S% above control, depending on the individual melanocyte strain, with continuous treatment with 100 nM a-MSH for 8 days, whereas the melanogenic effect became maximal (70-450% increase above control) after 4 days oftreatment. Western blot analysis of tyrosinase and the tyrosinase-related proteins TRP-1 and TRP-2 revealed that a-MSH increased the expression of those three melanogenic proteins. This was not accompanied by any change in their mRNA levels after brief (1.5-24 hr) or prolonged (6 days) treatment with 100 nM a-MSH, suggesting that the increased expression of these melanogenic proteins was due to posttranscriptional events. These results demonstrate both mitogenic and melanogenic effects of a-MSH and ACTH on human melanocytes. That both hormones are effective at subnanomolar concentrations, combined with the presence of melanotropin receptors on human melanocytes, strongly suggests that these melanotropins play a physiologic role in regulating human cutaneous pigmentation.a-Melanotropin (a melanocyte-stimulating hormone, a-MSH) is the physiologic hormone that regulates integumental pigmentation of many vertebrate species. For example, a-MSH induces rapid skin darkening in amphibians and reptiles and stimulates follicular eumelanogenesis in the mouse (1,2). In addition to the pigmentary effects, other functions for a-MSH and related melanotropins have been described, such as the antagonistic interaction with interleukin 1 (3, 4) and trophic effects on neurons (5, 6

The melanocortin 1 receptor gene is a main determinant of human pigmentation, and a melanoma susceptibility gene, because its variants that are strongly associated with red hair color increase melanoma risk. To test experimentally the association between melanocortin 1 receptor genotype and melanoma susceptibility, we compared the responses of primary human melanocyte cultures naturally expressing different melanocortin 1 receptor variants to α-melanocortin and ultraviolet radiation. We found that expression of 2 red hair variants abolished the response to α-melanocortin and its photoprotective effects, evidenced by lack of functional coupling of the receptor, and absence of reduction in ultraviolet radiation-induced hydrogen peroxide generation or enhancement of repair of DNA photoproducts, respectively. These variants had different heterozygous effects on receptor function. Microarray data confirmed the observed differences in responses of melanocytes with functional vs. nonfunctional receptor to α-melanocortin and ultraviolet radiation, and identified DNA repair and antioxidant genes that are modulated by α-melanocortin. Our findings highlight the molecular mechanisms by which the melanocortin 1 receptor genotype controls genomic stability of and the mutagenic effect of ultraviolet radiation on human melanocytes.

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Epidermal melanocytes are skin cells specialized in melanin production. Activation of the melanocortin 1 receptor (MC1R) on melanocytes by a-melanocyte-stimulating hormone (a-MSH) induces synthesis of the brown/black pigment eumelanin that confers photoprotection from solar UV radiation (UVR). Contrary to keratinocytes, melanocytes are slow proliferating cells that persist in the skin for decades, in an environment with high levels of UVR-induced reactive oxygen species (ROS). We previously reported that in addition to its role in pigmentation, a-MSH also reduces oxidative stress and enhances the repair of DNA photoproducts in melanocytes, independent of melanin synthesis. Given the significance of ROS in carcinogenesis, here we investigated the mechanisms by which a-MSH exerts antioxidant effects in melanocytes. We show that activation of the MC1R by a-MSH contributes to phosphorylation of p53 on serine 15, a known requirement for stabilization and activation of p53, a major sensor of DNA damage. This effect is mediated by the cAMP/PKA pathway and by the activation of phosphoinositide 3-kinase (PI3K) ATR and DNA protein kinase (DNA-PK). a-MSH increases the levels of 8-oxoguanine DNA glycosylase (OGG1) and apurinic apyrimidinic endonuclease 1 (APE-1/Ref-1), enzymes essential for base excision repair. Nutlin-3, an HDM2 inhibitor, mimicked the effects of a-MSH resulting in reduced phosphorylation of H2AX (g-H2AX), a marker of DNA damage. Conversely, the p53 inhibitor pifithrin-a or silencing of p53 abolished the effects of a-MSH and augmented oxidative stress. These results show that p53 is an important target of the downstream MC1R signaling that reduces oxidative stress and possibly malignant transformation of melanocytes. Mol Cancer Res; 10(6); 778-86. Ó2012 AACR. IntroductionThe melanocortin 1 receptor (MC1R) is best known for its role in stimulating eumelanin synthesis in melanocytes (MC). In the skin, presence of eumelanin is considered the major photoprotective factor against UV radiation (UVR)-induced DNA damage and carcinogenesis. Eumelanin creates not only a physical barrier against the deep penetration of UVR into the epidermal and dermal layers (1) but also reduces oxidative stress by scavenging free radicals (2-4).