Chie Sakai

Modulation of murine melanocyte function in vitro by agouti signal protein

Molecular and biochemical mechanisms that switch melanocytes between the production of eumelanin or pheomelanin involve the opposing action of two intercellular signaling molecules, α‐melanocyte‐stimulating hormone (MSH) and agouti signal protein (ASP). In this study, we have characterized the physiological effects of ASP on eumelanogenic melanocytes in culture. Following exposure of black melan‐a murine melanocytes to purified recombinant ASP in vitro, pigmentation was markedly inhibited and the production of eumelanosomes was decreased significantly. Melanosomes that were produced became pheomelanosome‐like in structure, and chemical analysis showed that eumelanin production was significantly decreased. Melanocytes treated with ASP also exhibited time‐ and dose‐dependent decreases in melanogenic gene expression, including those encoding tyrosinase and tyrosinase‐related proteins 1 and 2. Conversely, melanocytes exposed to MSH exhibited an increase in tyrosinase gene expression and function. Simultaneous addition of ASP and MSH at approximately equimolar concentrations produced responses similar to those elicited by the hormone alone. These results demonstrate that eumelanogenic melanocytes can be induced in culture by ASP to exhibit features characteristic of pheomelanogenesis in vivo. Our data are consistent with the hypothesis that the effects of ASP on melanocytes are not mediated solely by inhibition of MSH binding to its receptor, and provide a cell culture model to identify novel factors whose presence is required for pheomelanogenesis.

Human tyrosinase related protein‐1 (TRP‐1) does not function as a DHICA oxidase activity in contrast to murine TRP‐1

Abstract: Tyrosinase related protein‐1 is a melanocyte specific protein and a member of the tyrosinase gene family which also includes tyrosinase and TRP‐2 (DOPAchrome tautomerase). In murine melanocytes, TRP‐1 functions as a 5,6‐dihydroxyindole‐2‐carboxylic acid [DHICA] oxidase during the biosynthetic conversion of tyrosine to eumelanin and mutations affecting TRP‐1 result in the synthesis of brown rather than black pelage coloration. In this study, we examined the putative DHICA oxidase activity of TRP‐1 in human melanocytes using several approaches. We first utilized a line of cultured melanocytes established from a patient with a form of oculocutaneous albinism completely lacking expression of TRP‐1 (OCA3). This line of melanocytes endogenously exhibited the same amount of DHICA oxidase activity as control melanocytes expressing TRP‐1. In other experiments, cultured human fibroblasts were transfected with a cDNA for TRP‐1, in either the sense or antisense direction, or with the retroviral vector alone. TRP‐1 expression was induced in fibroblasts transfected with the TRP‐1 cDNA in the sense direction only. Although TRP‐1 was expressed by sense‐transfected cells, there was no significant DHICA oxidase activity above controls. These results demonstrate that human TRP‐1 does not use DHICA as a substrate for oxidation.

Pigment production in murine melanoma cell is regulated by tyrosinase, tyrosinase-related protein 1 (TRP1), dopachrome tautomerase(TRP2), and a melanogenic inhibitor

Using antibodies that recognize either tyrosinase, tyrosinase-related protein-1 (TRP1), or tyrosinase-related protein-2 (TRP2, DOPAchrome tautomerase), the quantities of those melanogenic enzymes were analyzed in five melanoma cell lines that possess various degrees of melanin production. All cells except JB/MS-W increased melanin production four to 30 times after 4 d of melanocyte-stimulating hormone (MSH) treatment. Melanin production by JB/MS-W cells was always under background, with or without MSH treatment. There was a positive correlation between quantities and synthetic rates of those melanogenic enzymes and their melanin formation or DOPAchrome tautomerase activities. The activity of a heat-resistant melanogenic inhibitory factor was also analyzed. The results showed, surprisingly, that pigmented cells showed higher levels of melanogenic inhibitors activity. Tyrosinase activity was increased dramatically whereas the level of melanogenic inhibitor was remarkably decreased following MSH treatment. Interestingly, melanogenic inhibitor derived from JB/MS-W cells suppressed not only tyrosinase but also DOPAchrome tautomerase, another enzyme functional in melanin production. These results clearly suggest that melanin production is regulated by a subtle balance between the activities of these enzymes and other factors such as the melanogenic inhibitor.