Post-Transcriptional Regulation of Melanin Biosynthetic Enzymes by cAMP and Resveratrol in Human Melanocytes  Richard A. Newton, Anthony L. Cook, Donald W. Roberts, J. Helen Leonard, Richard A. Sturm  Journal of Investigative Dermatology  Volume 127, Issue 9, Pages 2216-2227 (September 2007) DOI: 10.1038/sj.jid.5700840 Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Resveratrol and cAMP have opposing effects on tyrosinase activity in NHM. (a) In situ tyrosinase activity determined by incubation of cells in L-DOPA, following 24hours treatment with either vehicle (Ctrl) or 20μg/ml resveratrol (Rsvl). Images were captured under identical conditions using bright field microscopy. Bar=40μm. (b) Time courses for tyrosinase activity measured by both L-DOPA oxidation and tritium release from [3H]tyrosine using lysates obtained from NHM treated for the indicated periods of time with resveratrol. Error bars indicate the average±range of two melanocyte strains. (c) The direct effect of resveratrol on tyrosinase activity was determined by adding resveratrol directly to NHM lysates at an equivalent concentration to that added to cell cultures. Both the L-DOPA oxidation and tritium release assays were used to quantitate the effect. Error bars represent the mean±SEM of three independent experiments. (d) Tyrosinase activity of NHM lysates following treatment of NHM with resveratrol alone, forskolin (Fsk) alone (10μM), or both agents. Values are given as the mean±SEM (n=4) of four different melanocyte strains, normalized for total soluble protein and expressed relative to the activity of vehicle-treated cells, as detailed in materials and methods. (e) Changes in dendritic morphology following the same treatment regime described in (d) were assessed by phase-contrast microscopy, with dendrite length quantified using ImageJ software. Bar= 40μm. All of the above results are representative of at least two independent experiments using at least two melanocyte strains. Journal of Investigative Dermatology 2007 127, 2216-2227DOI: (10.1038/sj.jid.5700840) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Effects of forskolin and resveratrol on MITF levels in NHM. (a) Western-blot analysis of whole-cell lysates prepared from NHM treated for the indicated times with resveratrol alone, forskolin alone, or both agents. Phospho-specific antibodies were used to detect phosphorylated CREB and ERK (p44/p42 MAPK) because these activated forms are involved in the transcriptional upregulation and phosphorylation of MITF, respectively. The monoclonal MITF antibody identified the protein as a doublet, with the upper band previously shown to correspond to MITF that has undergone MAPK phosphorylation (Hemesath et al., 1998). Similar protein levels were verified using a phosphorylation-independent ERK antibody. (b) Q-RT-PCR analysis of MITF RNA levels following the same treatment regime applied for protein analysis. Transcript abundance was normalized against 18S ribosomal RNA and expressed relative to the 3hours control. Each experiment utilized two melanocyte strains and are presented as the mean±SEM (n=4) of two independent experiments. Journal of Investigative Dermatology 2007 127, 2216-2227DOI: (10.1038/sj.jid.5700840) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Effects of forskolin and resveratrol on tyrosinase levels in NHM. (a) Q-RT-PCR analysis of tyrosinase RNA levels following treatment of NHM with resveratrol alone, forskolin alone, or both agents, for the indicated times. Error bars indicate the mean±SEM (n=4), relative to the 3hours control, and were obtained from two melanocyte strains from two independent experiments. (b) Western-blot analysis of tyrosinase protein levels in whole-cell lysates prepared from NHM treated as described above. Similar protein levels were verified using an antibody against GAPDH. Samples were run on two gels (indicated by gap) and blots processed in parallel under identical conditions. Vertical lines indicate that single lanes either side of the forskolin lanes were removed during preparation of the Figure. (c) Fully processed tyrosinase, represented by the uppermost band at approximately 80kDa, was quantified by densitometry of Western-blot autoradiographs. Error bars indicate the mean±SEM (n=4), relative to the untreated controls, and were obtained using three melanocyte strains from two independent experiments. Journal of Investigative Dermatology 2007 127, 2216-2227DOI: (10.1038/sj.jid.5700840) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Endo H sensitivity of tyrosinase from resveratrol and forskolin treated NHM. Cell extracts obtained following 24hours treatment with either resveratrol, forskolin, or both agents were subsequently incubated in the presence or absence of the glycosidases, Endo H and PNGase F. Western-blot analysis for tyrosinase was then used to monitor the extent of digestion. Arrows at approximately 80 and 60kDa indicate bands corresponding to the mature and PNGase F digested forms of tyrosinase, respectively. An Endo H-sensitive band is coincident with the 70kDa marker. Samples were run on two gels (indicated by gap) and blots processed in parallel under identical conditions. Vertical lines indicate removal of two intervening lanes. Identical results were obtained in a second independent experiment. Journal of Investigative Dermatology 2007 127, 2216-2227DOI: (10.1038/sj.jid.5700840) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 DCT, TYRP1, SLC45A2, OCA2, and SLC24A5 expression in NHM following treatment with resveratrol, forskolin, or both agents, for the indicated times. Q-RT-PCR and Western immunoblot analysis was used to determine the levels of mRNA and protein, respectively, for DCT (a, b) and TYRP1 (c, d). (e) Q-RT-PCR was also used to quantify expression of the genes SLC45A2, OCA2, and SLC24A5 that encode the proteins MATP, P-protein, and NCKX5, respectively. Transcript abundance was normalized against 18S ribosomal RNA and expressed relative to the 3hours control. Error bars indicate the mean±SEM (n=4) of two independent experiments, each utilizing two melanocyte strains. Journal of Investigative Dermatology 2007 127, 2216-2227DOI: (10.1038/sj.jid.5700840) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Effect of MITF inhibition on melanogenic enzyme levels in NHM. The cells were transfected with siRNAs that were either directed against MITF or consisted of a commercial negative control sequence. 48hours later, cells were assessed for expression of MITF, tyrosinase, TYRP1, and GAPDH by (a) Q-RT-PCR measurement of mRNA, and (b) Western-blot analysis of protein. (c) Cellular lysates containing tyrosinase were also prepared from identically treated cells then digested with PNGase F to facilitate comparison of core deglycosylated tyrosinase protein levels by Western analysis. (d) Cellular lysates were also assessed for relative tyrosinase activity using the DOPA oxidation assay. Error bars represent the mean±SEM (n=3) of three independent experiments. Journal of Investigative Dermatology 2007 127, 2216-2227DOI: (10.1038/sj.jid.5700840) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions