Mitogen-activated Protein Kinase Pathway and AP-1 Are Activated during cAMP-induced Melanogenesis in B-16 Melanoma Cells

Abstract

In mammalian melanocytes, melanin synthesis is controlled by tyrosinase, the critical enzyme in the melanogenic pathway. We and others showed that the stimulation of melanogenesis by cAMP is due to an increased tyrosinase expression at protein and mRNA levels. However, the molecular events connecting the rise of intracellular cAMP and the increase in tyrosinase activity remain to be elucidated. In this study, using B16 melanoma cells, we showed that cAMPelevating agents stimulated mitogen-activated protein (MAP) kinase, p44mapk . This effect was mediated by the activation of MAP kinase kinase. cAMP-elevating agents induced a translocation of p44 mapk to the nucleus and an activation of the transcription factor AP-1. cAMP-induced AP-1 contained FOS-related antigen-2 in association with JunD, while after phorbol ester stimulation AP-1 complexes consist mainly of JunD/c-Fos heterodimers. In an attempt to connect these molecular events to the control of tyrosinase expression that appears to be the pivotal point of melanogenesis regulation, we hypothesized that following its activation by cAMP, p44 mapk activates AP-1. Then AP-1 could stimulate tyrosinase expression through the interaction with specific DNA sequences present in the mouse tyrosinase promoter.In melanocytes and melanoma cells, melanin synthesis is controlled by a cascade of enzymatic reactions regulated at the level of tyrosinase. This enzyme synthesizes dopaquinone from tyrosine and appears to control the rate-limiting step of melanogenesis. Melanin synthesis is stimulated by a large array of effectors including 1-oleyl-2-acetyl-glycerol (1), ultraviolet B radiations (2), and cAMP-elevating agents (forskolin, IBMX, 1 ␣-MSH) (3-5). Few data are available concerning the molecular mechanisms triggered by these melanogenic agents. Protein kinase C was thought to be involved in the induction of melanogenesis by 1-oleyl-2-acetyl-glycerol and ultraviolet B radiations (6, 7). However, a recent report of Carsberg et al. (8) has shown that the stimulation of melanogenesis by these agents was not affected by RO485, a potent inhibitor of protein kinase C. While the role of protein kinase C in the induction of melanogenesis remains controversial, compelling data have shown that cAMP-elevating agents stimulate melanogenesis in both melanocytes and melanoma cells, indicating that the cAMP pathway plays a key role in the regulation of melanogenesis (3-5). The effect of cAMP on melanogenesis is due to a stimulation of tyrosinase activity. This appears to be the consequence of an augmentation of enzymatic activity of preexisting tyrosinase (4, 9) following post-translational modifications such as (i) phosphorylation or glycosylation (10), (ii) association with an activator (11, 12), and (iii) dissociation from an inhibitor (13). Alternatively, cAMP was shown to increase tyrosinase mRNA (14, 15), resulting in an augmentation of tyrosinase amount, suggesting that cAMP stimulates tyrosinase transcription (16). However, the molecular events connecting the stimulation of t...

Cyclic AMP a Key Messenger in the Regulation of Skin Pigmentation

“…These effects can be mimicked by pharmacological cAMP elevating agents such as forskolin (FK), cholera toxin (CT) or isobutylmethylxanthine (IBMX) (16,17). Taken together, these observations with those made in vivo, corroborate clearly the pivotal role of a-MSH, ACTH and cAMP in the regulation of melanogenesis.…”

Differentiation-inducing activity of lupane triterpenes on a mouse melanoma cell line

“…Some studies demonstrated that B16 cells can be induced to differentiate into mature melanocyte-like cells by treatment with cAMPelevating agents such as a-melanocyte-stimulating hormone (a-MSH, Engalo et al 1995), signal transduction pathway inhibitors (Busca et al 1996;Engalo et al 1998), and mannosylerythritol lipid (Zhao et al 1999). In the differentiation of B16 cells, up-regulation of melanin biosynthesis and dendrite outgrowths is observed; however, information concerning melanoma differentiation inducers is very limited.…”

In B16 melanoma cells, the inhibition of melanogenesis by TPA results from PKC activation and diminution of microphthalmia binding to the M-box of the tyrosinase promoter

“…Noteworthy, in B16 melanoma cells, melanogenesis has been shown to be stimulated by cAMP elevating agents (Englaro et al, 1995) and U.V.B. (Oka and Ichihashi, 1996) indicating that PKC b is not absolutely required for the regulation of melanogenesis.…”

“…radiation. In cultured cells, melanogenesis can be upregulated by a large array of stimuli among which cAMP elevating agents such as aMSH (Fuller and Viskochil, 1979;Jime nez et al, 1988), IBMX and forskolin are the most potent (Englaro et al, 1995;Hearing and Tsukamoto, 1991;Hunt et al, 1994;Wong and Pawelek, 1975). Stimulation of melanogenesis by cAMP elevating agents mainly results from an augmentation of the tyrosine expression.…”