Stimulation of tyrosinase in human melanocytes by pro-opiomelanocortin-derived peptides S D McLeod, C Smith and R S Mason Department of Physiology, F13, University of Sydney, Sydney, New South Wales 2006, Australia (Requests for offprints should be addressed to R S Mason) Abstract Human melanocytes, maintained on bovine corneal endothelium-derived extracellular matrix for at least 4 days in the absence of phorbol 12-myristate 13-acetate (PMA) and cholera toxin (CT), displayed increased tyrosinase activity when exposed to several pro-opiomelanocortin- derived (POMC) peptides. Melanocytes from 9 of 14 donors showed significantly increased tyrosinase activity after treatment with adrenocorticotropic hormone (ACTH; mean increase 320\m=+-\107(s.e.m.)% of control, P<0\m=.\005), while melanocytes from 8 of 13 donors in- creased tyrosinase in the presence of diacetyl-melanocyte stimulating hormone (di-MSH; mean increase 223\m=+-\31 (s.e.m.)% of control, P<0\m=.\005). Maximal increases in tyrosinase were seen after treatment with 10\m=-\10m ACTH and with 10\m=-\6 m di-MSH. In two cell cultures which showed tyrosinase stimulation, melanin synthesis was simi- larly increased in the presence of added POMC peptides. PMA but not CT increased tyrosinase activity in melano- cytes cultured under these conditions. In the presence of staurosporine, an inhibitor of protein kinase C (PKC), the magnitude of the increase in tyrosinase due to PMA, ACTH and di-MSH was significantly reduced. These results indicate that tyrosinase activity in melanocytes from most human donors, under appropriate conditions, is susceptible to the stimulatory effects of POMC peptides, that ACTH is considerably more potent than di-MSH in this test system and that in human cells the PKC pathway may be important in modulating melanogenesis. Journal of Endocrinology (1995) 146, 439\p=n-\447 Introduction The classical stimulatory melanotropins of vertebrates belong to a family of polypeptides which descend from the large common precursor protein pro-opiomelanocortin (POMC). POMC-derived peptides of the human pitui¬ tary gland and skin include adrenocorticotropic hormone (ACTH) and alpha-melanocyte-stimulating hormone (tx- MSH) (Kock et al. 1990), both of which can produce significant skin darkening when administered to humans (Levine et al. 1991, Griego & Levine 1992). Hyper- pigmentation is also characteristic of some patients with disorders where serum concentrations of ACTH and a-MSH are elevated (Brown & Doe 1978). Whilst skin pigmentation can be increased by POMC- derived peptides in vivo, it has been remarkably difficult to demonstrate direct effects of these peptides on human melanocytes in culture (Iwata et al. 1990, Pawelek 1990, Yaar & Gilchrest 1991). As discussed by Pawelek (1990) and Gordon & Gilchrest (1990), a contributing factor to the problem probably lies in the culture system for melanocytes. In the presence of proliferation-inducing agents such as phorbol 12-myristate 13-acetate (PMA) and cholera toxin (CT), which are also potent stimulators of intracellular signal pathways, it might be expected to be difficult to demonstrate effects of added putative modula¬ tors like POMC peptides, which are also likely to act through these pathways. To date, there is no evidence that melanogenesis is mediated principally via the cyclic 3',5'-adenosine mono- phosphate (cAMP) pathway (Gordon & Gilchrest 1990, Pawelek 1990). There is, however, recent data to indicate that at least some melanogenic signals are transmitted via the 1,2-diacylglycerol-protein kinase C (PKC) pathway (Gordon & Gilchrest 1989, Oka et al. 1990, Pawelek 1990, Park et al. 1991, Buffey et al. 1992, Maeda et al. 1992, Park et al. 1992, 1993). Synthetic diacylglycerol analogues augment melanin synthesis and content in normal human melanocytes and influence melanocyte morphology in vitro (Gordon & Gilchrest 1989, Friedman et al. 1990«, Maeda et al. 1992). Previous studies from this laboratory showed that melanocytes can be maintained for several days in a healthy but relatively quiescent state when cultured on a bovine corneal endothelium-derived extracellular matrix (ECM) in the absence of PMA and CT (Ranson et al. 1988a). We now report that under these conditions, melanocyte tyrosinase activity is stimulated by POMC peptides, and that evidence is consistent with a PKC- Downloaded from Bioscientifica.com at 12/01/2018 12:31:30PM via free access