ABSTRACT
The melanocortin 1 receptor (MC1R), a G(s) protein-coupled receptor, has an important role in human pigmentation. We investigated the regulation of expression and activity of the MC1R in primary human melanocyte cultures. Human ß-defensin 3 (HBD3) acted as an antagonist for MC1R, inhibiting the α-melanocortin (α-melanocyte-stimulating hormone (α-MSH))-induced increase in the activities of adenylate cyclase and tyrosinase, the rate-limiting enzyme for melanogenesis. α-Melanocortin and forskolin, which activate adenylate cyclase, and 12-O-tetradecanoylphorbol-13-acetate, which activates protein kinase C, increased, whereas exposure to UV radiation reduced, MC1R gene and membrane protein expression. Brief treatment with α-MSH resulted in MC1R desensitization, whereas continuous treatment up to 3 hours caused a steady rise in cAMP, suggesting receptor recycling. Pretreatment with agouti signaling protein or HBD3 prohibited responsiveness to α-MSH, but not forskolin, suggesting receptor desensitization by these antagonists. Melanocytes from different donors expressed different levels of the G protein-coupled receptor kinases (GRKs) 2, 3, 5, and 6, as well as ß-arrestin 1. Therefore, in addition to the MC1R genotype, regulation of MC1R expression and activity is expected to affect human pigmentation and the responses to UV.
Subject(s)
Agouti Signaling Protein/pharmacology , Melanocortins/pharmacology , Melanocytes/drug effects , Receptor, Melanocortin, Type 1/agonists , Receptor, Melanocortin, Type 1/antagonists & inhibitors , alpha-MSH/pharmacology , beta-Defensins/pharmacology , Adenylyl Cyclases/metabolism , Arrestins/biosynthesis , Cells, Cultured , Colforsin/pharmacology , G-Protein-Coupled Receptor Kinases/biosynthesis , Humans , Melanocytes/metabolism , Melanocytes/radiation effects , Monophenol Monooxygenase/metabolism , Protein Kinase C/metabolism , Receptor, Melanocortin, Type 1/biosynthesis , Skin Pigmentation/drug effects , Skin Pigmentation/physiology , Skin Pigmentation/radiation effects , Tetradecanoylphorbol Acetate/analogs & derivatives , Tetradecanoylphorbol Acetate/pharmacology , Ultraviolet Rays , beta-Arrestin 1 , beta-ArrestinsABSTRACT
Epidermal melanocytes are skin cells specialized in melanin production. Activation of the melanocortin 1 receptor (MC1R) on melanocytes by α-melanocyte-stimulating hormone (α-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, α-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 α-MSH exerts antioxidant effects in melanocytes. We show that activation of the MC1R by α-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). α-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 α-MSH resulting in reduced phosphorylation of H2AX (γ-H2AX), a marker of DNA damage. Conversely, the p53 inhibitor pifithrin-α or silencing of p53 abolished the effects of α-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.