MC1R,the cAMP pathway,and the response to solar UV: extending the horizon beyond pigmentation

The melanocortin 1 receptor (MC1R) is a G protein‐coupled receptor crucial for the regulation of melanocyte proliferation and function. Upon binding melanocortins, MC1R activates several signaling cascades, notably the cAMP pathway leading to synthesis of photoprotective eumelanin. Polymorphisms in the MC1R gene are a major source of normal variation of human hair color and skin pigmentation, response to ultraviolet radiation (UVR), and skin cancer susceptibility. The identification of a surprisingly high number of MC1R natural variants strongly associated with pigmentary phenotypes and increased skin cancer risk has prompted research on the functional properties of the wild‐type receptor and frequent mutant alleles. We summarize current knowledge on MC1R structural and functional properties, as well as on its intracellular trafficking and signaling. We also review the current knowledge about the function of MC1R as a skin cancer, particularly melanoma, susceptibility gene and how it modulates the response of melanocytes to UVR.     

Regulation of eumelanin/pheomelanin synthesis and visible pigmentation in melanocytes by ligands of the melanocortin 1 receptor

The production of melanin in the hair and skin is tightly regulated by the melanocortin 1 receptor (MC1R) whose activation is controlled by two secreted ligands, alpha-melanocyte stimulating hormone (alphaMSH) and agouti signal protein (ASP). As melanin is extremely stable, lasting years in biological tissues, the mechanism underlying the relatively rapid decrease in visible pigmentation elicited by ASP is of obvious interest. In this study, the effects of ASP and alphaMSH on the regulation of melanin synthesis and on visible pigmentation were assessed in normal murine melanocytes and were compared with the quick depigmenting effect of the tyrosinase inhibitor, phenylthiourea (PTU). alphaMSH increased pheomelanin levels prior to increasing eumelanin content over 4 days of treatment. Conversely, ASP switched off the pigment synthesis pathway, reducing eu- and pheo-melanin synthesis within 1 day of treatment that was proportional to the decrease in tyrosinase protein level and activity. These results demonstrate that the visible depigmentation of melanocytes induced by ASP does not require the degradation of existing melanin but rather is due to the dilution of existing melanin by melanocyte turnover, which emphasizes the importance of pigment distribution to visible color.     

Melanin content and MC1R function independently affect UVR-induced DNA damage in cultured human melanocytes

Malignant transformation of melanocytes leads to melanoma, the most fatal form of skin cancer. Ultraviolet radiation (UVR)-induced DNA photoproducts play an important role in melanomagenesis. Cutaneous melanin content represents a major photoprotective mechanism against UVR-induced DNA damage, and generally correlates inversely with the risk of skin cancer, including melanoma. Melanoma risk is also determined by susceptibility genes, one of which is the melanocortin 1 receptor (MC1R) gene. Certain MC1R alleles are strongly associated with melanoma. We hereby present experimental evidence for the role of two melanoma risk factors, constitutive pigmentation, as assessed by total melanin, eumelanin and pheomelanin contents, and MC1R genotype and function, in determining the induction and repair of DNA photoproducts in cultured human melanocytes after irradiation with increasing doses of UVR. We found that total melanin and eumelanin contents (MC and EC) correlated inversely with the extent of UVR-induced growth arrest, apoptosis and induction of cyclobutane pyrimidine dimers (CPD), but not with hydrogen peroxide release in melanocytes expressing functional MC1R. In comparison, melanocytes with loss-of-function MC1R, regardless of their MC or EC, sustained more UVR-induced apoptosis and CPD, and exhibited reduced CPD repair. Therefore, MC, mainly EC, and MC1R function are independent determinants of UVR-induced DNA damage in melanocytes.     

Melanin content and MC1R function independently affect UVR‐induced DNA damage in cultured human melanocytes

Malignant transformation of melanocytes leads to melanoma, the most fatal form of skin cancer. Ultraviolet radiation (UVR)‐induced DNA photoproducts play an important role in melanomagenesis. Cutaneous melanin content represents a major photoprotective mechanism against UVR‐induced DNA damage, and generally correlates inversely with the risk of skin cancer, including melanoma. Melanoma risk is also determined by susceptibility genes, one of which is the melanocortin 1 receptor (MC1R) gene. Certain MC1R alleles are strongly associated with melanoma. We hereby present experimental evidence for the role of two melanoma risk factors, constitutive pigmentation, as assessed by total melanin, eumelanin and pheomelanin contents, and MC1R genotype and function, in determining the induction and repair of DNA photoproducts in cultured human melanocytes after irradiation with increasing doses of UVR. We found that total melanin and eumelanin contents (MC and EC) correlated inversely with the extent of UVR‐induced growth arrest, apoptosis and induction of cyclobutane pyrimidine dimers (CPD), but not with hydrogen peroxide release in melanocytes expressing functional MC1R. In comparison, melanocytes with loss‐of‐function MC1R, regardless of their MC or EC, sustained more UVR‐induced apoptosis and CPD, and exhibited reduced CPD repair. Therefore, MC, mainly EC, and MC1R function are independent determinants of UVR‐induced DNA damage in melanocytes.     

The genetics of sun sensitivity in humans

Humans vary >100-fold in their sensitivity to the harmful effects of ultraviolet radiation. The main determinants of sensitivity are melanin pigmentation and less-well-characterized differences in skin inflammation and repair processes. Pigmentation has a high heritability, but susceptibility to cancers of the skin, a key marker of sun sensitivity, is less heritable. Despite a large number of murine coat-color mutations, only one gene in humans, the melanocortin 1 receptor (MC1R), is known to account for substantial variation in skin and hair color and in skin cancer incidence. MC1R encodes a 317-amino acid G-coupled receptor that controls the relative amounts of the two major melanin classes, eumelanin and pheomelanin. Most persons with red hair are homozygous for alleles of the MC1R gene that show varying degrees of diminished function. More than 65 human MC1R alleles with nonsynonymous changes have been identified, and current evidence suggests that many of them vary in their physiological activity, such that a graded series of responses can be achieved on the basis of (i) dosage effects (of one or two alleles) and (ii) individual differences in the pharmacological profile in response to ligand. Thus, a single locus, identified within a Mendelian framework, can contribute significantly to human pigmentary variation.     

Loss-of-function variants of the human melanocortin-1 receptor gene in melanoma cells define structural determinants of receptor function.

The alpha-melanocyte-stimulating hormone (alphaMSH) receptor (MC1R) is a major determinant of mammalian skin and hair pigmentation. Binding of alphaMSH to MC1R in human melanocytes stimulates cell proliferation and synthesis of photoprotective eumelanin pigments. Certain MC1R alleles have been associated with increased risk of melanoma. This can be theoretically considered on two grounds. First, gain-of-function mutations may stimulate proliferation, thus promoting dysplastic lesions. Second, and opposite, loss-of-function mutations may decrease eumelanin contents, and impair protection against the carcinogenic effects of UV light, thus predisposing to skin cancers. To test these possibilities, we sequenced the MC1R gene from seven human melanoma cell (HMC) lines and three giant congenital nevus cell (GCNC) cultures. Four HMC lines and two GCNC cultures contained MC1R allelic variants. These were the known loss-of-function Arg142His and Arg151Cys alleles and a new variant, Leu93Arg. Moreover, impaired response to a superpotent alphaMSH analog was demonstrated for the cell line carrying the Leu93Arg allele and for a HMC line homozygous for wild-type MC1R. Functional analysis in heterologous cells stably or transiently expressing this variant demonstrated that Leu93Arg is a loss-of-function mutation abolishing agonist binding. These results, together with site-directed mutagenesis of the vicinal Glu94, demonstrate that the MC1R second transmembrane fragment is critical for agonist binding and maintenance of a resting conformation, whereas the second intracellular loop is essential for coupling to the cAMP system. Therefore, loss-of-function, but not activating MC1R mutations are common in HMC. Their study provides important clues to understand MC1R structure-function relationships.     

Alpha-melanocyte-stimulating hormone suppresses antigen-induced lymphocyte proliferation in humans independently of melanocortin 1 receptor gene status

Studies in mice indicate that alpha-melanocyte-stimulating hormone (alphaMSH) is immunosuppressive, but it is not known whether alphaMSH suppresses human immune responses to exogenous Ags. Human PBMCs, including monocytes, express the melanocortin 1 receptor (MC1R), and it is thought that the ability of alphaMSH to alter monocyte-costimulatory molecule expression and IL-10 release is mediated by this receptor. However, the MC1R gene is polymorphic, and certain MC1R variants compromise receptor signaling via cAMP, resulting in red hair and fair skin. Here, we have investigated whether alphaMSH can suppress Ag-induced lymphocyte proliferation in humans and whether these effects are dependent on MC1R genotype. alphaMSH suppressed streptokinase-streptodornase-induced lymphocyte proliferation, with maximal inhibition at 10(-13)-10(-11) M alphaMSH. Anti-IL-10 Abs failed to prevent suppression by alphaMSH, indicating that it was not due to MC1R-mediated IL-10 release by monocytes. Despite variability in the degree of suppression between subjects, similar degrees of alphaMSH-induced immunosuppression were seen in individuals with wild-type, heterozygous variant, and homozygous/compound heterozygous variant MC1R alleles. RT-PCR of streptokinase-streptodornase-stimulated PBMCs for all five melanocortin receptors demonstrated MC1R expression by monocytes/macrophages, MC1R and MC3R expression by B lymphocytes, but no melanocortin receptor expression by T lymphocytes. In addition, alphaMSH did not significantly inhibit anti-CD3 Ab-induced lymphocyte proliferation, whereas alphaMSH and related analogs (SHU9119 and MTII) inhibited Ag-induced lymphocyte proliferation in monocyte-depleted and B lymphocyte-depleted assays. These findings demonstrate that alphaMSH, acting probably via MC1R on monocytes and B lymphocytes, and possibly also via MC3R on B lymphocytes, has immunosuppressive effects in humans but that suppression of Ag-induced lymphocyte proliferation by alphaMSH is independent of MC1R gene status.     

Melanocortin 1 receptor (MC1R), pigmentary characteristics and sun exposure: Findings from a case–control study of diffuse large B-cell and follicular lymphoma

The relationship between skin cancer and non-Hodgkin lymphoma (NHL) suggests common genetic, host or environmental causes. Ultraviolet radiation (UVR), pigmentary characteristics have been linked with both malignancies, and for skin cancer, the melanocortin 1 receptor (MC1R) which influences pigmentation has also been implicated. This paper reports on the relationship between MC1R, skin, hair and eye colour, time spent outdoors, and diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL). Persons carrying MC1R homozygote variant alleles at R151C, R160W, D294H and D84E were more likely to have fair skin, red hair and to spend less time outdoors than those who did not. The variant allele at V92M was associated with FL (odds ratio (OR) = 1.61, 95% confidence interval (CI) 1.08–2.39) and the r:wild type genotype with DLBCL (OR = 0.58, 95% CI 0.38–0.89). Interactions between MC1R genotypes and skin colour influenced DLBCL risk; the RR genotype increased risk in individuals with medium or dark skin, based on 5 cases and no controls, but decreased risk among those of fair skin. On the whole, DLBCL and FL risk were not related to genetic variation in MC1R, pigmentation or time spent outdoors.     

Diversity of pigmentation in cultured human melanocytes is due to differences in the type as well as quantity of melanin

Cultured human melanocytes differ tremendously in visual pigmentation, and recapitulate the pigmentary phenotype of the donor's skin. This diversity arises from variation in type as well as quantity of melanin produced. Here, we measured contents of eumelanin (EM) and pheomelanin (PM) in 60 primary human melanocyte cultures (51 neonatal and nine adults), and correlated some of these values with the respective activity and protein levels of tyrosinase, and the melanocortin-1 receptor (MC1R) genotype. Melanocytes were classified into four phenotypes (L, L+, D, D+) as depicted by visual pigmentation using light microscopy, and by the pigmentary phenotype of the donor's skin. There were large differences in total melanin (TM) and EM, which increased progressively for L, L+, D and D+ melanocytes. TM content, the sum of EM and PM, showed a good correlation with TM measured spectrophotometrically, and with the activity and protein levels of tyrosinase. Log EM/PM ratio did not correlate with MC1R genotype. We conclude that: (i) EM consistently correlates with the visual phenotype; (ii) lighter melanocytes tend to be more pheomelanic in composition than darker melanocytes; (iii) in adult melanocyte cultures, EM correlates with the ethnic background of the donors (African-American > Indian > Caucasian); and (iv) MC1R loss-of-function mutations do not necessarily alter the phenotype of cultured melanocytes.     

Alpha-melanocyte-stimulating hormone suppresses oxidative stress through a p53-mediated signaling pathway in human melanocytes

Epidermal melanocytes are skin cells specialized in melanin production. Activation of the melanocortin 1 receptor (MC1R) on melanocytes by α-melanocyte-stimulating hormone (α-MSH) induces synthesis of the brown/black pigment eumelanin that confers photoprotection from solar UV radiation (UVR). Contrary to keratinocytes, melanocytes are slow proliferating cells that persist in the skin for decades, in an environment with high levels of UVR-induced reactive oxygen species (ROS). We previously reported that in addition to its role in pigmentation, α-MSH also reduces oxidative stress and enhances the repair of DNA photoproducts in melanocytes, independent of melanin synthesis. Given the significance of ROS in carcinogenesis, here we investigated the mechanisms by which α-MSH exerts antioxidant effects in melanocytes. We show that activation of the MC1R by α-MSH contributes to phosphorylation of p53 on serine 15, a known requirement for stabilization and activation of p53, a major sensor of DNA damage. This effect is mediated by the cAMP/PKA pathway and by the activation of phosphoinositide 3-kinase (PI3K) ATR and DNA protein kinase (DNA-PK). α-MSH increases the levels of 8-oxoguanine DNA glycosylase (OGG1) and apurinic apyrimidinic endonuclease 1 (APE-1/Ref-1), enzymes essential for base excision repair. Nutlin-3, an HDM2 inhibitor, mimicked the effects of α-MSH resulting in reduced phosphorylation of H2AX (γ-H2AX), a marker of DNA damage. Conversely, the p53 inhibitor pifithrin-α or silencing of p53 abolished the effects of α-MSH and augmented oxidative stress. These results show that p53 is an important target of the downstream MC1R signaling that reduces oxidative stress and possibly malignant transformation of melanocytes.     

Melanocyte function and its control by melanocortin peptides.

Melanocytes are cells of neural crest origin. In the human epidermis, they form a close association with keratinocytes via their dendrites. Melanocytes are well known for their role in skin pigmentation, and their ability to produce and distribute melanin has been studied extensively. One of the factors that regulates melanocytes and skin pigmentation is the locally produced melanocortin peptide alpha-MSH. The effects of alpha-MSH on melanogenesis are mediated via the MC-1R and tyrosinase, the rate-limiting enzyme in the melanogenesis pathway. Binding of alpha-MSH to its receptor increases tyrosinase activity and eumelanin production, which accounts for the skin-darkening effect of alpha-MSH. Other alpha-MSH-related melanocortin peptides, such as ACTH1-17 and desacetylated alpha-MSH, are also agonists at the MC-1R and could regulate melanocyte function. Recent evidence shows that melanocytes have other functions in the skin in addition to their ability to produce melanin. They are able to secrete a wide range of signal molecules, including cytokines, POMC peptides, catecholamines, and NO in response to UV irradiation and other stimuli. Potential targets of these secretory products are keratinocytes, lymphocytes, fibroblasts, mast cells, and endothelial cells, all of which express receptors for these signal molecules. Melanocytes may therefore act as important local regulators of a range of skin cells. It has been shown that alpha-MSH regulates NO production from melanocytes, and it is possible that the melanocortins regulate the release of other signalling molecules from melanocytes. Therefore, the melanocortin signaling system is one of the important regulators of skin homeostasis.     

Evidence for variable selective pressures at MC1R

It is widely assumed that genes that influence variation in skin and hair pigmentation are under selection. To date, the melanocortin 1 receptor (MC1R) is the only gene identified that explains substantial phenotypic variance in human pigmentation. Here we investigate MC1R polymorphism in several populations, for evidence of selection. We conclude that MC1R is under strong functional constraint in Africa, where any diversion from eumelanin production (black pigmentation) appears to be evolutionarily deleterious. Although many of the MC1R amino acid variants observed in non-African populations do affect MC1R function and contribute to high levels of MC1R diversity in Europeans, we found no evidence, in either the magnitude or the patterns of diversity, for its enhancement by selection; rather, our analyses show that levels of MC1R polymorphism simply reflect neutral expectations under relaxation of strong functional constraint outside Africa.     

Worldwide polymorphism at the MC1R locus and normal pigmentation variation in humans

While there have been many advances in our understanding of the genetics of pathological skin pigmentation in humans, our knowledge about what determines variation in normal skin color is still incomplete. Variation in one gene, melanocortin 1 receptor (MC1R), has been associated with red hair and fair skin in Europeans. However, this gene might also play an important role in shaping pigmentation of other human populations, where it experiences different selective pressures. Below we review what is currently known about polymorphism and selection at the MC1R coding and promoter regions in human populations, the pattern of MC1R evolution in nonhuman primates, and the interaction of MC1R with other genes.     

High polymorphism at the human melanocortin 1 receptor locus

Variation in human skin/hair pigmentation is due to varied amounts of eumelanin (brown/black melanins) and phaeomelanin (red/yellow melanins) produced by the melanocytes. The melanocortin 1 receptor (MC1R) is a regulator of eu- and phaeomelanin production in the melanocytes, and MC1R mutations causing coat color changes are known in many mammals. We have sequenced the MC1R gene in 121 individuals sampled from world populations with an emphasis on Asian populations. We found variation at five nonsynonymous sites (resulting in the variants Arg67Gln, Asp84Glu, Val92Met, Arg151Cys, and Arg163Gln), but at only one synonymous site (A942G). Interestingly, the human consensus protein sequence is observed in all 25 African individuals studied, but at lower frequencies in the other populations examined, especially in East and Southeast Asians. The Arg163Gln variant is absent in the Africans studied, almost absent in Europeans, and at a low frequency (7%) in Indians, but is at an exceptionally high frequency (70%) in East and Southeast Asians. The MC1R gene in common and pygmy chimpanzees, gorilla, orangutan, and baboon was sequenced to study the evolution of MC1R. The ancestral human MC1R sequence is identical to the human consensus protein sequence, while MC1R varies considerably among higher primates. A comparison of the rates of substitution in genes in the melanocortin receptor family indicates that MC1R has evolved the fastest. In addition, the nucleotide diversity at the MC1R locus is shown to be several times higher than the average nucleotide diversity in human populations, possibly due to diversifying selection.     

Skin phototype: a new perspective

Cutaneous phototype is considered mainly related to cutaneous pigmentation and to the eumelanin/pheomelanin ratio, which is mostly genetically determined by the melanocortin 1 receptor (MC1R) polymorphisms. However, data in literature indicate that, in addition to stimulation of eumelanin synthesis, the MC1R signalling activates antioxidant, DNA repair and survival pathways. New emerging aspects regarding photoprotection and skin phototypes are going beyond those features connected to the melanin content in the skin. Important new findings link the MC1R to nuclear receptors activation, shedding light on new extra‐melanogenic effects dependent on the α‐melanocyte‐stimulating hormone (α‐MSH) activity and new ways through which such functions are modulated. These evidences indicate that several factors including melanin play a part in defining the basis for individual sun sensitivity, suggesting that the cutaneous phototype represents a ‘biochemical fingerprint’.     

The Role of Melanocortin‐1 Receptor Polymorphism in Skin Cancer Risk Phenotypes

We have examined melanocortin‐1 receptor (MC1R) variant allele frequencies in the general population and in a collection of adolescent dizygotic and monozygotic twins to determine statistical associations of pigmentation phenotypes with increased skin cancer risk. This included hair and skin color, freckling, mole count and sun exposed skin reflectance. Nine variants were studied and designated as either strong R (OR = 63; 95% CI 32–140) or weak r (OR = 5; 95% CI 3–11) red hair alleles. Penetrance of each MC1R variant allele was consistent with an allelic model where effects were multiplicative for red hair but additive for skin reflectance. To assess the interaction of the brown eye color gene BEY2/OCA2 on the phenotypic effects of variant MC1R alleles we imputed OCA2 genotype in the twin collection. A modifying effect of OCA2 on MC1R variant alleles was seen on constitutive skin color, freckling and mole count. In order to study the individual effects of these variants on pigmentation phenotype we have established a series of human primary melanocyte strains genotyped for the MC1R receptor. These include strains which are MC1R wild‐type consensus, variant heterozygotes, and homozygotes for strong R alleles Arg151Cys and Arg160Trp. Ultrastructural analysis demonstrated that only consensus strains contained stage III and IV melanosomes in their terminal dendrites whereas Arg151Cys and Arg160Trp homozygous strains contained only immature stage I and II melanosomes. Such genetic association studies combined with the functional analysis of MC1R variant alleles in melanocytic cells should provide a link in understanding the association between pigmentary phototypes and skin cancer risk.     

Agonist-independent, high constitutive activity of the human melanocortin 1 receptor

The melanocortins (alpha-melanocyte-stimulating hormone and adrenocorticotropin) act on epidermal melanocytes to increase melanogenesis, the eumelanin/pheomelanin ratio and dendricity. These actions are mediated by the heptahelical melanocortin 1 receptor (MC1R), positively coupled to adenylyl cyclase. Gain-of-function mouse Mc1r alleles are associated with a dark, eumelanic coat. Conversely, loss-of-function variants, or overexpression of agouti, a natural melanocortin antagonist, yield yellow, pheomelanic furs. In humans, loss-of-function MC1R variants are associated with fair skin, poor tanning, propensity to freckle and increased skin cancer risk. Therefore, MC1R is a key regulator of mammalian pigmentation. Several observations such as induction of constitutive pigmentation in amelanotic mouse melanoma cells following expression of MC1R indicate that the receptor might display agonist-independent activity. We report a systematic and comparative study of MC1R and Mc1r constitutive activity. We show that expression of MC1R in heterologous systems leads to an agonist-independent increase in cyclic adenosine monophophate (cAMP). Basal signalling is a function of receptor expression and is two to fourfold higher for MC1R than for Mc1r. Moreover, it is observed in human melanoma cells over-expressing the MC1R. Constitutive signalling is abolished or reduced by point mutations of MC1R impairing the response to agonists, and is only doubled by the Lys94Glu mutation, mimicking the constitutively active mouse E(so-3J) allele. Stable or transient expression of wild-type MC1R, but not of loss-of-function mutants, potently stimulates forskolin activation of adenylyl cyclase, a common feature of constitutively active Gs-coupled receptors. Therefore, human MC1R displays a strong agonist-independent constitutive activity.     

Cyclic analogs of alpha-melanocyte-stimulating hormone (alphaMSH) with high agonist potency and selectivity at human melanocortin receptor 1b

Alpha-melanotropin (alphaMSH), Ac-Ser1-Tyr2-Ser3-Met4-Glu5-His6-Phe7-Arg8-Trp9-Gly10-Lys11-Pro12-Val13-NH2,(1) has been long recognized as an important physiological regulator of skin and hair pigmentation in mammals. Binding of this peptide to the melanocortin receptor 1 (MC1R) leads to activation of tyrosinase, the key enzyme of the melanin biosynthesis pathway. In this study, interactions of the human MC1bR (an isoform of the receptor 1a) with the synthetic cyclic analogs of alphaMSH were studied. These ligands were analogs of MTII, Ac-Nle4-cyclo-(Asp5-His6-D-Phe7-Arg8-Trp9-Lys10)-NH2, a potent pan-agonist at the human melanocortin receptors (hMC1,3-5R). In the structure of MTII, the His6-D-Phe7-Arg8-Trp9 segment has been recognized as "essential" for molecular recognition at the human melanocortin receptors (hMC1,3-5R). Herein, the role of the Trp9 in the ligand interactions with the hMC1b,3-5R has been reevaluated. Analogs with various amino acids in place of Trp9 were synthesized and tested in vitro in receptor affinity binding and cAMP functional assays at human melanocortin receptors 1b, 3, 4 and 5 (hMC1b,3-5R). Several of the new peptides were high potency agonists (partial) at hMC1bR (EC50 from 0.5 to 20 nM) and largely inactive at hMC3-5R. The bulky aromatic side chain in position 9, such as that in Trp, was found not to be essential to agonism (partial) of the studied peptides at hMC1bR.