In mouse follicular melanocytes, the switch between eumelanin and pheomelanin synthesis is regulated by the extension locus, which encodes the melanocortin-1 receptor (MC1R) and the agouti locus, which encodes a novel paracrine-signaling molecule that inhibits binding of melanocortins to the MC1R. Human melanocytes express the MC1R and respond to melanotropins with increased proliferation and eumelanogenesis, but a potential role for the human homolog of agouti-signaling protein, ASIP, in human pigmentation has not been investigated. Here we report that ASIP blocked the binding of alpha-melanocyte-stimulating hormone (alpha-MSH) to the MC1R and inhibited the effects of alpha-MSH on human melanocytes. Treatment of human melanocytes with 1 nM-10 nM recombinant mouse or human ASIP blocked the stimulatory effects of alpha-MSH on cAMP accumulation, tyrosinase activity, and cell proliferation. In the absence of exogenous alpha-MSH, ASIP inhibited basal levels of tyrosinase activity and cell proliferation and reduced the level of immunoreactive tyrosinase-related protein-1 (TRP-1) without significantly altering the level of immunoreactive tyrosinase. In addition, ASIP blocked the stimulatory effects of forskolin or dibutyryl cAMP, agents that act downstream from the MC1R, on tyrosinase activity and cell proliferation. These results demonstrate that the functional relationship between the agouti and MC1R gene products is similar in mice and humans and suggest a potential physiologic role for ASIP in regulation of human pigmentation.

the implications of these variants on the function of the The cloning and characterization of the human melanocortin-1 receptor (MC1R) and the demonstration that normal human MC1R revealed the following. Human melanocytes homelanocytes respond to the melanocortins, a-melanocyte stimmozygous for Arg160Trp mutation in the MC1R demonulating hormone (a-MSH) and adrenocorticotrophic hormone strated a significantly reduced response to a-MSH. Also, this culture responded poorly to ASP and exhibited an exagger-(ACTH), with increased proliferation and eumelanogenesis ated cytotoxic response to UVR. Another culture, which was had put an end to a long-standing controversy about the role homozygous for Val92Met mutation in the MC1R, demonof melanocortins in regulating human cutaneous pigmentation. strated a normal response to a-MSH. Heterozygous mutaWe have shown that a-MSH and ACTH bind the human tions that are frequently expressed in various melanocyte MC1R with equal affinity, and are equipotent in their mitocultures did not disrupt MC1R function. These results begin genic and melanogenic effects on human melanocytes. We also to elucidate the significance of MC1R variants in the function showed that the activation of the MC1R is important for the of the receptor. Our data emphasize the significance of a melanogenic response of human melanocytes to ultraviolet normally functioning MC1R in the response of melanocytes to radiation (UVR). The MC1R is also the principal mediator of melanocortins, ASP, and UVR. the inhibitory effects of agouti signaling protein (ASP) on melanogenesis. Expression of the MC1R is subject to regulation by its own ligands a-MSH and ACTH, as well as by Key words: Melanocortin 1 receptor, Human melanocytes, Ultraviolet radiation, Melanocortins, Agouti signaling protein, UVR and endothelin-1. Recent studies that we conducted on the expression of MC1R variants by human melanocytes and Eumelanin, Pheomelanin that have an extensive amino acid sequence homology. These peptides include a-melanocyte stimulating hormone (a-MSH), b-MSH, g-MSH, and adrenocorticotrophic hormone (ACTH), all of which are derived from a large precursor peptide, pro-opiomelanocortin (POMC) (1). The 4-10 carboxy teminus amino acid residues of a-MSH, b-MSH,

In normal human melanocytes various mitogens activate the mitogen-activated protein kinases ERK1/2 and the downstream transcription factor CREB (Ca2+/cAMP response element binding protein). Endothelin-1, basic fibroblast growth factor, and alpha-melanotropin interact synergistically to stimulate human melanocyte proliferation. The former two mitogens phosphorylated ERK1/2, its substrate p90rsk, and CREB. Alpha-melanotropin, forskolin, or dibutyryl cAMP failed to phosphorylate any of those targets, however. The concomitant presence of endothelin-1, basic fibroblast growth factor, and alpha-melanotropin significantly potentiated CREB phosphorylation. The mitogen-induced phosphorylation of p90rsk and CREB was dependent on ERK1/2 activation, and was mediated by intracellular calcium mobilization and by protein kinase C and tyrosine kinase activation, but not by activation of the cAMP-dependent protein kinase A. Exposure of melanocytes to ultraviolet radiation B resulted in the phosphorylation of the stress-induced mitogen- activated protein kinases p38 and JNK/SAPK, but not ERK1/2. Ultraviolet radiation B induced the phosphorylation of CREB via a pathway that was partially dependent on p38, but had no effect on p90rsk or ERK1/2. Therefore, in human melanocytes, CREB is a common downstream target for distinct effectors that are involved in either mitogenic signaling or stress signaling initiated by ultraviolet radiation B.

One of the mutagenic and carcinogenic heterocyclic amines (HCAs), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), is present in cooked foods and we are chronically exposed to this compound in our daily life. To study the role of HCAs in human carcinogenesis, we analyzed MeIQx-DNA adducts in 38 DNA samples obtained from surgical and autopsy specimens by the 32P-postlabeling method under adduct-intensification conditions with the modification of additional digestion with nuclease P1 and phosphodiesterase I after 32P-labeling at 5'-hydroxyl termini. This modified 32P-postlabeling method can detect N2-(deoxyguanosin-8-yl)-2-amino-3,8-dimethylimidazo- [4,5-f]quinoxaline 5'-monophosphate (5'-pdG-C8-MeIQx) at levels down to 1/10(10) nucleotides. The DNA samples from colon and rectum surgical specimens and a kidney taken at autopsy were found to contain an adduct spot corresponding to that of standard 5'-pdG-C8-MeIQx on TLC at levels of 14,18 and 1.8 per 10(10) nucleotides, respectively. Each adduct spot was extracted from TLC and identified to be 5'-pdG-C8-MeIQx by HPLC. Thus, MeIQx-DNA adducts actually exist in human tissues and this adduct formation may be involved in human cancer development.

No abstract

The cloning of the melanocortin-1 receptor (MC1R) gene from human melanocytes and the demonstration that these cells respond to the melanocortins alpha-melanocyte stimulating hormone (alpha-MSH) and adrenocorticotropic hormone (ACTH) with increased proliferation and melanogenesis have renewed the interest in investigation the physiological role of these hormones in regulating human pigmentation. Alpha-melanocyte stimulating hormone and ACTH are both synthesized in the human epidermis, and their synthesis is upregulated by exposure to ultraviolet radiation (UVR). Activation of the MC1R by ligand binding results in stimulation of cAMP formation, which is a principal mechanism for inducing melanogenesis. The increase in cAMP is required for the pigmentary response of human melanocytes to UVR, and for allowing them to overcome the UVR-induced G1 arrest. Treatment of human melanocytes with alpha-MSH increases eumelanin synthesis, an effect that is expected to enhance photoprotection of the skin. Population studies have revealed more than 20 allelic variants of the MC1R gene. Some of these variants are overexpressed in individuals with skin type I or II, red hair, and poor tanning ability. Future studies will aim at further exploration of the role of these variants in MC1R function, and in determining constitutive human pigmentation, the response to sun exposure, and possibly the susceptibility to skin cancer.

␣ -Melanocyte stimulating hormone ( ␣ -MSH) is known to be the main physiologic regulator for integumental pigmentation of various vertebrate species. However, the role of ␣ -MSH and related melanocortins in the regulation of human cutaneous pigmentation is only beginning to be understood. Cloning of the melanocortin-1 receptor (MC1R), and the feasibility of establishing normal human epidermal melanocyte cultures have made it possible to demonstrate direct and specific biological effects of ␣ -MSH on these cells. It is now recognized that both ␣ -MSH and ACTH have similar mitogenic and melanogenic effects on human epidermal melanocytes. These effects are mediated by binding of these hormones to the specific MC1R that recognizes them both with similar affinity. Human MC1R is homologous to its mouse counterpart in that its activation leads to stimulation of eumelanin synthesis. MC1R is also the binding site for agouti signaling protein (ASP), the product of the agouti locus. Human epidermal melanocytes respond to purified recombinant mouse or human ASP, with a reduction in basal tyrosinase activity, and complete abrogation of the mitogenic and melanogenic effects of ␣ -MSH. These results suggest that ASP induces pheomelanin synthesis by competing with ␣ -MSH for binding to the MC1R. This receptor seems to be subject to regulation by a variety of paracrine and/or autocrine factors that are synthesized in response to exposure of the skin to ultraviolet radiation (UVR). Activation of MC1R seems to be pivotal for UV-induced melanogenesis, since stimulation of the cAMP pathway plays a key role in the melanogenic response of human epidermal melanocytes. The melanogenic response to UVR might be influenced by the presence of allelic variants of the MC1R gene. Allelic variants have been identified and shown to be associated with red hair, poor tanning ability, and possibly melanoma. The possible influence of these variants on the function of the MC1R needs to be investigated, in order to understand the physiological consequence of these mutations. Also, the interaction of ␣ -MSH with other factors that are known to affect pigmentation needs to be better understood in order to define the role possible of this hormone and its receptor in acquired human cutaneous hyper-or hypopigmentation.

No abstract