Defining MC1R Regulation in Human Melanocytes by Its Agonist α-Melanocortin and Antagonists Agouti Signaling Protein and β-Defensin 3  Viki B. Swope, Joshua A. Jameson, Kevin L. McFarland, Dorothy M. Supp, William E. Miller, Dennis W. McGraw, Mira A. Patel, Matthew A. Nix, Glenn L. Millhauser, George F. Babcock, Zalfa A. Abdel-Malek  Journal of Investigative Dermatology  Volume 132, Issue 9, Pages 2255-2262 (September 2012) DOI: 10.1038/jid.2012.135 Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Effect of human β-defensin 3 (HBD3) on basal and α-melanocyte-stimulating hormone (α-MSH)–induced cAMP levels, proliferation, and tyrosinase activity. (a) Melanocytes were treated with 0, 1nM α-MSH, or 1, 10, or 100nM HBD3±1nM α-MSH for 45minutes. *Significantly different from control at P<0.01, or #from α-MSH at P<0.05. (b) Melanocytes were treated with 100nM HBD3±1nM α-MSH or 1μM forskolin, or 100nM agouti signaling protein (ASIP)±1nM α-MSH for 45minutes. *Statistically different from control at P<0.001. (c) Melanocytes were treated for 6 days with the same concentrations of α-MSH and/or HDB3 or ASIP as in (b). *Significantly different from control at P<0.05, or #from α-MSH at P<0.001. Data represent mean percentage of control±SEM. Triplicate experiments were performed. Journal of Investigative Dermatology 2012 132, 2255-2262DOI: (10.1038/jid.2012.135) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Regulation of melanocortin 1 receptor (MC1R) gene expression by α-melanocyte-stimulating hormone (α-MSH), agouti signaling protein (ASIP), human β-defensin 3 (HBD3), and UV. Melanocytes were maintained in medium lacking 12-O-tetradecanoylphorbol-13-acetate and bovine pituitary extract overnight, and then treated with 0, 1nM α-MSH, 1μM forskolin, 100nM HBD3 or ASIP, or irradiated with 75 or 105mJcm−2 UV. Total RNA was isolated 8hours after treatment, and equal amounts of RNA from each group were analyzed by quantitative real-time reverse-transcriptase–PCR. Similar results were obtained in two independent experiments using two different melanocyte strains. The data were normalized using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a loading control, and mean relative expression levels are presented±SEM. *Statistically different from control at P<0.001. Journal of Investigative Dermatology 2012 132, 2255-2262DOI: (10.1038/jid.2012.135) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Regulation of cell surface expression of melanocortin 1 receptor (MC1R) by α-melanocyte-stimulating hormone (α-MSH) and UV, as determined by immunostaining for MC1R followed by flow cytometric analysis. (a) Melanocytes were irradiated with increasing doses of UV (0, 20, 50, 75, or 105mJcm−2) and immunostained for MC1R 24hours after exposure. (b) Melanocytes were treated with 0, 1nM α-MSH, 1μM forskolin, or 5ng/ml 12-O-tetradecanoylphorbol-13-acetate (TPA) for 14hours. In (a, b), the data (percentage of control±SEM) represent the combined results of three independent experiments. *Statistically different from control at P<0.05. Journal of Investigative Dermatology 2012 132, 2255-2262DOI: (10.1038/jid.2012.135) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Response to α-melanocyte-stimulating hormone (α-MSH) after pretreatment with agonist or antagonists, and the role of protein kinase A in melanocortin 1 receptor desensitization. Data represent percentage of control±SEM. (a) In 10 independent experiments, melanocytes were pretreated with 0, 1nM α-MSH, 1nM agouti signaling protein (ASIP) or human β-defensin 3 (HBD3), or 1μM forskolin (forsk), and then challenged with α-MSH, as described in the Materials and Methods section. *Statistically different from control, or #from the 20-minute α-MSH treatment at P<0.001. (b) Melanocytes were pretreated with 100nM ASIP or HBD3 and then challenged with 1μM forskolin. (c) Three melanocyte strains were treated for 45minutes, 3hours, or 6hours with 1nM α-MSH. *Statistically different from control, + from the 45-minute α-MSH treatment, or #from the 3-hour α-MSH treatment at P<0.05. (d) Melanocytes were pretreated with 0, or 1nM α-MSH±100nM H-89. This experiment was repeated twice with similar findings. *Statistically different from control or #from the 20-minute α-MSH treatment at P<0.01. Forsk, forskolin; Tx, treatment. Journal of Investigative Dermatology 2012 132, 2255-2262DOI: (10.1038/jid.2012.135) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Western blot analysis. Comparison of basal levels of G protein–coupled receptor kinases (GRKs) 2, 3, 5, and 6, as well as β-arrestin 1, in a panel of cultured human melanocytes with different pigmentary phenotypes (C=lightly pigmented, B=darkly pigmented donor) and MC1R (melanocortin 1 receptor) genotypes using western blotting. Journal of Investigative Dermatology 2012 132, 2255-2262DOI: (10.1038/jid.2012.135) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions