Invited Review MC1R, Eumelanin and Pheomelanin: their role in determining the susceptibility to skin cancer

Abstract

Skin pigmentation is due to the accumulation of two types of melanin granules in the keratinocytes. Besides being the most potent blocker of ultraviolet radiation (UVR), the role of melanin in photo-protection is complex. This is because one type of melanin called eumelanin is UV absorbent whereas the other, pheomelanin, is photo-unstable and may even promote carcinogenesis. Skin hyperpigmentation may be caused by stress or exposure to sunlight, which stimulates the release of α-melanocyte stimulating hormone (α-MSH) from damaged keratinocytes. Melanocortin 1 receptor (MC1R) is a key signaling molecule on melanocytes that responds to α-MSH by inducing expression of enzymes responsible for eumelanin synthesis. Persons with red hair have mutations in the MC1R causing its inactivation; this leads to a paucity of eumelanin production and makes red-heads more susceptible to skin cancer. Apart from its effects on melanin production, the α-MSH/MC1R signaling is also a potent anti-inflammatory pathway and has been shown to promote anti-melanoma immunity. This review will focus on the role of MC1R in terms of its regulation of melanogenesis and influence on the immune system with respect to skin cancer susceptibility.

INTRODUCTION

Skin, the largest organ of our body is organized into two primary layers-the epidermis and the dermis. Since epidermis serves as a point of contact with the external environment, various biological and physical characteristics of this layer determine its ability to resist repeated exposure to various damaging environmental stimuli like ultraviolet radiation (UVR). The carcinogenic action of UVA which makes up approximately 95% of the terrestrial UV spectrum, is mainly through indirect damage caused by the generation of reactive oxygen species (ROS), while UVB, which makes up less than 5% of the terrestrial spectrum, is a potent mutagen that directly alters nucleotide structure in DNA (1, 2). Therefore, many endogenous mechanisms have evolved to protect, decrease and/or repair UVR-mediated damage. Some of these mechanisms include increasing epidermal thickness, cytokeratin in suprabasal layers of keratinocytes, normal shedding of epidermal squamous layers, DNA repair activity, apoptosis of damaged cells, enhanced activity of antioxidant enzymes and, last but not least, skin pigmentation (3, 4). Skin pigmentation is due to the accumulation of melanin granules in keratinocytes, which act as a natural sunscreen that potently absorb UVR (5), thereby blocking its ability to penetrate into the deeper layers skin where proliferating cells reside. However, its mechanism of photo-protection is more complex than a simple correlation to photo-absorbtion. Melanin is also a potent antioxidant and free-radical scavenger. Further, melanogenesis is enhanced after UVR exposure and thus might have other roles as well. Epidemiological studies indicate that light skinned individuals (Fitzpatrick Scale type I & II) are more prone to UVR-induced damage and tumor formation than darker skin individuals (3). The percentage of the two types of melanin pigment, eumelanin and pheomelanin, is also important in establishing the susceptibility to DNA damage and tumor development. This review will highlight the importance of different melanin types and their role in the development of skin cancer, particularly melanoma.

MELANIN

Skin pigmentation has considerable cosmetic importance. The mechanism of protection by melanin is not fully understood, but it is among the most important factors of UV sensitivity and melanoma risk. Melanin is ubiquitously found throughout the animal kingdom (6) except in arachnids (7) and usually serves in protection against environmental stressors. Although melanin is present in epidermal keratinocytes, it is not synthesized in these cells. This function is performed by melanocytes, which are abundantly present in the lowest layer of epidermis, and at a lower frequency in the dermis. Pigment produced by melanocytes is transferred to keratinocytes through cellular organelles called melanosomes and occurs with the help of melanocytic dendrites (8). In fact, a single melanocyte can transfer melanin to as many as forty keratinocytes (4). The close contact with keratinocytes leads to the tight regulation of melanin synthesis and transfer and can be dictated by subtle variations in the keratinocyte homeostasis. The synthesis of melanin takes place inside melanosomes, following transfer of the enzyme tyrosinase, which oxidizes tyrosine to dihydroxyphenylalanine (DOPA) and to dopaquinone (DQ). Further, modification together with the formation of protein complexes eventually results in melanin deposition onto a fibrillar scaffold composed of modified PMEL17 (also known as gp100) molecules (an important marker of melanocytes)(9). Large bio-aggregates composed of homo or hetero-units of pheo and eu-melanin compounds are formed by oxidation and cyclization of the initial substrate, tyrosine. Melanin can be classified into at least three basic types: eumelanin, pheomelanin, and neuromelanin (10). The function of neuromelanin, which is found in the brain, is not clear and will not be reviewed here. We will focus on the skin melanins; eumelanin, which is prevalent in individuals with black and brown hair and pheomelanin (yellow-reddish) which is responsible for red hair and freckles (4, 11). Both melanins have a common organization of repeating units linked by carbon bonds; however, the pigments differ from each other in terms of chemical, structural and physical properties (4).

Pheomelanin and Eumelanin

Pheomelanin, consists mainly of sulfur-containing benzothiazine and benzothiazole derivatives (Figure 1a). L-cysteine is the chief source of sulfur and is, therefore, essential for the pheomelanin synthesis (12). Eumelanin, on the other hand is an extremely heterogeneous polymer comprising of 5,6-dihydroxyindole (DHI) and or 5,6-dihydroxyindole-2-carboxylic acid (DHICA) units (Figure 1b).

Figure 1. Building blocks of Pheomelanin (a) and Eumelanin (b).

Benzothiazine and Benzothiazol (a) are the building blocks of pheomelanin, they polymerize to form Pheomelanin. In case of eumelanin 5,6-dihydroxyindole and 5,6-dihydroxyindole-2-carboxylic acid shown in (b) polymerize to form eumelanin. The structures were drawn using ChemSketch.

Synthesis and regulation

The biosynthetic pathways for pheomelanin and melanin synthesis have been well characterized over the years. Tyrosine is the common precursor for both eumelanin and pheomelanin production. It is first converted to an extremely reactive intermediate DOPA-Quinone (DQ) by the action of the enzyme Tyrosinase (Tyr). Tyrosinase enzymes are found in almost every species on the planet; however, the sequence and length assessment shows that they are highly heterogeneous and diverse. All tyrosinases are highly conserved around the active site (13); in fact, the overall understanding of tyrosinase has been possible by analyzing the catalytic properties of mushroom tyrosinases (14). All tyrosinases use copper as a cofactor (15, 16), but substrate specificity can vary between species (17, 18). Tyrosinases are bi-functional enzymes that catalyze the hydroxylation of monophenols and subsequent oxidation of diphenols to quinones (19). The rate-limiting step in the melanin biosynthesis was thought to be the conversion of tyrosine to DOPA and then its further oxidation to DQ. For many years, DOPA was thought to be an important precursor in the melanin biosynthesis. Inside the melanosomes, the DQ cyclizes to form leucodopachrome and then spontaneously converts to an orange colored intermediate known as dopachrome. As shown by in vitro studies, dopachrome loses its carboxylic acid, forming 5,6-dihydroxyindole (DHI), whose polymerization forms eumelanin (20, 21). Recent studies have shown that DOPA is not only produced initially from tyrosine but is also produced by the reduction of DQ and fed into the pathway (22). Dopachrome can also be rearranged by enzymes called tyrosine -related protein 1 (TRP1) and tyrosine -related protein 2 (TRP2), also called as dopachrome tautomerase (DCT), to form a carboxylated intermediate DHICA (as shown in Figure 2). It was demonstrated that tyrosinase along with the two tyrosinase-related proteins forms a multi-enzyme complex within melanocytes facilitating their physiological interactions (23) and disruption of one of the enzymes due to mutations severely affects pigmentation (16).

Figure 2. Biosynthetic pathways involved in Eumelanin and Pheomelanin.

TYR is involved in both eumelanin and pheomelanin synthesis, while TRP1, DCT (also called TRP2) are only involved in eumelanin (not pheomelanin) synthesis. Synthesis of eumelanin requires higher O2 consumption than pheomelanin. The structures were drawn using ChemSketch. MC1R, melanocortin 1 receptor; ASIP, agouti signaling protein; α-MSH, alpha-melanocyte stimulating hormone.

In the pheomelanin synthesis, there is a critical role of sulfhydryl groups. Once DQ is formed the same way as in eumelanin synthesis, it immediately reacts with sulfhydryl groups to form cysteinyl-DOPA and then quinone, which is then further converted to benzothiazine and benzothiazol. These products polymerize to produce pheomelanin (24-26).

Melanogenesis produces mixtures of eumelanin and pheomelanin at different mixed ratios. The ratio is determined by tyrosinase activity and the substrate concentrations of tyrosine and cysteine (27). Analysis of uveal melanosomes reveals an inner and outer core made from pheomelanin coated with eumelanin, at different inner and outer core thicknesses depending on eye color; however, the structure of epidermal melanin granules is not yet known (27, 28). In addition to mixed melanogenesis, it is also proposed that melanosome biochemistry becomes committed to either eumelanogenesis or pheomelanogenesis, and these discrete types of granules coexist within the same cell at different ratios (29, 30, 10). (Diagrammed as discrete melanosomes in Figure 2, for simplicity). The density of melanocytes in all types of skin is similar and constant; it is the distribution and amount of melanin that determines the color of skin (4). Eumelanin production is regulated primarily by MC1R signaling. Two different MC1R ligands have opposing activities – α-MSH is an agonist, while agouti signaling protein (ASP in mice, ASIP in human) is an antagonist. (Figure 2) (31-33). Binding of α-MSH to MC1R activates adenylate cyclase and ultimately leads to the generation of secondary messenger cAMP signaling cascade for the transcription of enzymes necessary for eumelanin production (4). ASP inhibits eumelanin synthesis by directly affecting the binding of α-MSH and the downstream expression of microphthalmia-associated transcription factor (MITF), necessary for promoter activation of all the three enzymes (Tyr, TRP1 and TRP2), thus promoting pheomelanin synthesis (34, 35). Others have also suggested that MITF is involved only in the activation of TRP1 and Tyr, not TRP2 (36).

Keratinocytes and their role in melanin production

As discussed earlier, keratinocytes and melanocytes form a tight niche, with a single melanocyte reaching out to as many as 40 keratinocytes. After UVR exposure, keratinocytes secrete a wide range of cytokines and chemokines that influence eumelanin production. Keratinocytes secrete α-MSH (37) and adrenocorticotropic hormone (ACTH) upon UVR exposure, which signals melanocytes to increase melanogenesis. Their receptors activate adenylate cyclase through G-proteins, which in turn switches on cAMP signaling. cAMP leads to the phosphorylation of cAMP response element binding protein (CREB) through protein kinase A (PKA). CREB later binds to the cAMP response element present in the promoter of MITF (38, 39). Once MITF expression is induced it can be phosphorylated by a wide variety of kinases (as shown in Figure 3). Once activated MITF up-regulates TYR, TRP1 and TRP2 gene expression, required for eumelanin. Other factors secreted by keratinocytes, such as prostaglandin E2 (PGE2), endothelin-1 (ET-1), fibroblast growth factor (FGF), granulocyte macrophage colony stimulating factor (GM-CSF), steel factor (SLF), stem cell factor (SCF), leukemia inhibitory factor (LIF) and hepatocyte growth factor (HGF), influence the melanocyte differentiation, growth, proliferation and melanogenesis (4). Keratinocytes secrete abundant amounts of prostaglandin E2 (PGE2) upon UVR, which has been shown to stimulate the formation of dendrites in melanocytes. The dendritogenesis has been thought to be cAMP independent and mediated through Phospholipase C (4, 40). Others have also shown that PGE2 activates tyrosinase activity independent of MC1R through cAMP synthesis (41). Further, PGE2 signaling through EP4 and EP3 has been shown to stimulate or inhibit cAMP signaling respectively (41). ET-1 has been shown to activate dendrites, induce melanin production in melanocytes and also aid in melanocyte migration (42). ET-1 also aids in IL-1 production which can induce tyrosinase expression in melanocytes. The downstream signaling is through MAPK, cAMP and phosphokinase C pathways (43, 44, 4). FGF and SCF secreted by keratinocytes are also involved in regulating the proliferation, migration and melanogenesis/dendritogenesis of human epidermal melanocytes in normal skin as well as in UV irradiated skin (4, 45, 46). GM-CSF induces proliferation and differentiation of melanocytes and expression of tyrosinase, TRP1 and DCT (4, 47). HGF, LIF and SLF also control proliferation of melanoblasts and melanocytes (48-50). Thus the crosstalk between keratinocytes and melanocytes during steady state or in response to UVR-induced stress is important to melanocyte proliferation, pigment production and melanin granule distribution.

Figure 3. MC1R signaling and its downstream effectors.

Keratinocyte mediators activate MITF which induces expression of enzymes for eumelanin production. Other factors secreted by keratinocytes can also induce MITF phosphorylation and induction of eumelanin synthesizing enzymes. ASP can act at different points of the pathway and inhibit α-MSH binding, can inhibit MITF phosphorylation and can also block cAMP synthesis and thus reduce eumelanin synthesis.

PHEOMELANIN AND ITS ROLE IN UVR INDUCED SKIN CANCER

Individuals with light skin color are 70 times more prone to develop skin cancer as compared to individuals with dark skin (51). Thus, there exists a reciprocal relationship between melanin content in the skin and incidence of skin cancer. These beneficial effects of melanin are mainly due to the presence of eumelanin that serves to scatter and absorb 50-75% of UVR and scavenge the UV-generated free radicals, which protect against UVR-damage in deeper layers (3). In some light skinned individuals, there is an increased rate of eumelanin turnover in keratinocytes due to increased degradation by lysosomal enzymes, hence photoprotection in these individuals is diminished (52, 3).

Beneficial effects of melanin is largely contributed by eumelanin

Since the beneficial effects of melanin are attributed mainly to eumelanin, the question that comes to mind is whether higher susceptibility to skin cancer is due to the reduced eumelanin in the skin or whether pheomelanin has an active role in increasing the risk factor? It was shown that the melanocytes derived from light skinned individuals compared to dark skin – derived melanocytes produce more pheomelanin in the presence of L-tyrosine (53). Evidence suggests there is higher pheomelanin content in atypical nevi and melanomas compared to normal melanocytes from the same patient; however, melanoma cells had lower pheomelanin than atypical nevi (54). Although not well understood why melanoma cells have less pigment, some investigators suggest that the high division rate of melanoma cells dilutes the pigment (54), or the process selects for this trait, as its loss may have a direct impact on promoting aggressive behavior. Mice that have activating BRAF mutations are susceptible to a low rate of spontaneous melanoma development. When the MC1R inactivating mutation is introduced, eumelanin is no longer produced, resulting in pheomelanin-rich red fur and a concomitant increase in spontaneous melanoma (55). Since this model does not depend on exposure to carcinogens, such as UVR, the mechanism of increased melanomagenesis was proposed to be due to increased oxidative damage generated by pheomelanin (55). Interestingly, when the tyrosinase gene was further deleted in these mutant mice the resulting loss of pheomelanin production abrogated melanoma susceptibility, even when activating BRAF and in-activating MC1R mutations remain. Thus, the presence of pheomelanin is strongly implicated in the pathogenesis of melanoma development. Although UVR remains an important contributor to melanomagenesis, this study addresses how nevi and melanomas may develop in sun unexposed areas.

Pheomelanin function is not fully understood. Pheomelanin melanosomes are generally small and oval, in contrast to the larger elongated shape of eumelanin containing melanosomes. Therefore, it is thought that pheomelanin may have a role in preparing the melanosome for eumelanin synthesis, and may act as a template for eumelanin polymerization and deposition (27, 56). The increased frequency of blonde or red-haired individuals at higher latitudes suggests that favoring pheomelanin over eumelanin production results in increased UVR penetration permitting more efficient UV-dependent transformation of circulating precursors in the skin, which is the essential first step of vitamin D synthesis.

Mechanism of pheomelanin induced tumorigenesis

Some of the mechanisms by which pheomelanin promotes carcinogenesis may be related to increased ROS production that is associated with its synthesis. Pheomelanin synthesis requires high amounts of antioxidants, which in turn depletes the scavengers of ROS such as glutathione, making melanocytes more vulnerable to ROS-related damage (57). Pheomelanin generation utilizes cysteine delivered by glutathione. Therefore, pheomelanin synthesis can reduce glutathione stores and make melanocytes more susceptible to DNA damage and genetic instability. Many studies have shown a correlation between pheomelanin and glutathione depletion and oxidative stress (57-59). The mechanism of pheomelanin-dependent ROS formation has been reported to occur with or without UVR (57, 60). It is thought that the sulfur in pheomelanin’s aromatic ring lowers its ionization potential, making it a less stable, and more efficient for free radical generation, in comparison to eumelanin (60, 61, 26, 57). Many studies have put forth the mechanisms by which pheomelanin-associated ROS is generated. It is not well understood how pheomelanin produces ROS and whether it can induce ROS production without electromagnetic energy input. Some reports suggest that zinc, which is excessively present in red hair can assist pheomelanin to produce higher levels of ROS, even in the presence of visible light (57, 62). Zinc enhances oxygen consumption and superoxide production by pheomelanin following irradiation with UVA and visible light. Pheomelanin photoreactivity is higher in the presence of zinc as compared its absence. This enhanced photoreactivity is due to zinc-induced changes in pheomelanin structure.

Further, nuclear radiation and noise have been shown to mediate ROS production in birds and pigs with high amounts of pheomelanin, which indicates that pheomelanin does not require electromagnetic energy to produce ROS (63, 64, 57). Various studies have shown that pheomelanin generates ROS, however, pheomelanin detection in the nuclei of melanoma cells is controversial and hence its ability to directly damage genomic DNA is debatable (57). On the other hand, studies by Maresca et al. reported that pheomelanin generated ROS can damage DNA bases present in the cytosol (2). It can amplify the effects of UVA-induced ROS generation by acting as a photosensitizing agent, negatively affecting catalase enzyme function. Apart from the modification and inactivation of antioxidant enzymes, simple amplification of ROS in the cytosol robs pheomelanin-containing cells of intracellular antioxidants, such as super oxide dismutase, leaving the cell susceptible to increased DNA damage (1).

Effects on the immune system

Another mechanism by which pheomelanin can promote tumor development is through its indirect effects on the immune system. Interestingly, studies in various animals and birds suggest there are some effects of melanin on immune responses. We know that myeloid-derived suppressor cells (MDSC) are induced after UVR and are potent inhibitors of T-cell–mediated antitumor immunity. Their inhibitory activity is attributed to the production of arginase, ROS, inducible nitric oxide synthase, etc. They can also block T-cell activation by limiting the availability of cysteine (65). Cysteine is an essential amino acid for T-cell activation because T cells lack the enzyme Cystathionine-β-synthase that converts methionine to cysteine (65). Therefore, T-cells require cysteine at the site of action. Hence, in tissues where pheomelanin is abundant, cysteine may be limiting, and the ability of local T cells to eradicate mutated cells may be compromised. Further, increased ROS concentrations, associated with the pheomelanin, inhibits CD8+T cell function, indicated by reduced expression of CD3 ζ as well as IFN-γ (66). Other mechanisms involve nitration and nitrosylation of components of the T cell receptor (TCR) signaling complex, thereby inhibiting T cell activation(67). ROS is potent in differentiating macrophages to the M2 phenotype, associated with tumor associated macrophage (TAMs) (68). All these events are summarized in Figure 4.

Figure 4. The immune system in light and dark colored individuals.

In dark colored individuals after UVR, there is balanced inflammation, less ROS production, higher T-cells activity (higher IFN-γ responses) that can remove mutated cells. Further, high MC1R signaling in endothelial cells down regulates E-selectin, vascular cell adhesion molecule (VCAM) and intercellular adhesion molecule (ICAM) and less infiltration of macrophages and neutrophils. Proinflammatory and protumorigenic cytokine levels are lower. All this leads to microenvironment favorable to antitumor immunity. The light skinned individuals have high inflammatory response, high ROS generation and reduced cysteine stores which leads to T-cell inhibition. Together with high neutrophil and macrophages infiltration leads to an environment that is conducive for mutated cells to propagate and lead to tumor development.

Because genes that control melanin synthesis are highly conserved and are diverse in function, they also affect many other, non-melanogenic processes. The effects of pheomelanin and eumelanin on the immune system can be widely seen across phylogeny. For example, melanin-based coloration of the barn swallow (Hirundo rustica) belly feathers have an effect on their acquired immunity (69). Swallows with more pheomelanin in their belly feathers exhibited weaker humoral immune responses than the swallows with more eumelanin in their feathers. In studies of different owl species, it was similarly observed that those with higher eumelanin content in their feathers responded better to experimental immune challenges and demonstrated enhanced T-cell and B-cell reponses as compared to their pale counterparts (52, 53, (70). Collectively these studies suggest that eumelanin possesses immune activating properties or, alternatively, it protects against UV-induced immune suppression. The contribution by MC1R signaling is not clear.

Another important point to make here is that in humans sunlight is the major source of vitamin D. Since vitamin D is cutaneously produced after exposure to UVR light its synthesis is influenced by a variety of factors, including skin pigmentation (71). As eumelanin absorbs UVR, it has a profound effect on the synthesis of the vitamin D precursor formation from 7-dihydrocholesterol (71). Thus, in higher latitudes (where the sun’s rays are less potent), people with dark skin are prone to a deficiency of vitamin D. Vitamin D has numerous effects on the immune system. It has been shown that human monocytes produce antimicrobial peptides after vitamin D exposure (72). Others have shown that it not only inhibits B cell differentiation, proliferation and immunoglobulin secretion (73, 74), but also suppresses T cell proliferation (75). It leads to a shift in T cell differentiation from a Th1 to a Th2 phenotype (76, 77) and facilitates the development of suppressive regulatory T cells (Tregs) (78). Vitamin D treated dendritic cells produce increased levels of IL-10 (a potent suppressor cytokine) and reduced levels of IL-12 (which activates immune cells as well as DNA repair in skin cells) (79, 80).

MC1R AND ITS ROLE IN SKIN CANCER

The melanocortin-1 receptor (MC1R) is a G-protein coupled 7-membrane spanning receptor (GPCR) that was originally described on cells of melanocytic origin, but is also present on keratinocytes, fibroblasts and most immune cells, suggesting that it can influence innate and adaptive immunity (81, 82). MC1R function is important in regulating the amount of eumelanin pigment produced after UVR, and it is activated by a class of peptide hormones called melanocortins (MC), derivatives of proopiomelanocortins (POMCs) produced in the pituitary gland. Among various MCs, α-MSH is responsible for pigmentation in humans and coat color in mice (83, 84). MC1R belongs to a five-member subfamily of GPCRs that mediate the physiologic actions of MCs by activating G-proteins that, in turn, activate the cyclic AMP signaling pathway. The human and mouse MC1R genes were cloned in 1992 (85, 86). The cloning of the other mammalian MC1Rs shows that this gene is highly conserved in mammals. As shown in Supplementary Figure 1, an alignment of MC1R protein sequences from different organisms are highly similar between the different mammalian species. Bird or reptile sequences have lower identity, but conservation at the arginine and cysteine residues indicate their importance in MC1R function (Figure 5 and Supplementary Figure 1). Indeed, mutations at these highly conserved residues lead to MC1R inactivation, and are found in red-headed individuals (Figures 6). Because MC1R is resistant to crystal formation, its protein structure has been deduced using information from modeling of its secondary and tertiary structures in comparison to bacteriorhodopsin, the prototype molecule for the class A subfamily of GPCRs, of which MC1R is a member (87-91). MC1R has a typical seven transmembrane structure, with an N-terminus that extends extracellularly, a C-terminus that is intracellularly located, with a series of intracellular and extracellular loops in between (Figure 6).

Figure 5. Phylogenetic relationships and % identity of MC1Rs from various organisms.

(a) Phylogenies were estimated using the MEGA 5 software. Numbers at the nodes indicate the percentage of replicate trees in which the associated species clustered together in the bootstrap test (1000 replicates). (b) Percent identity across various species is shown in the table. Red color indicates higher identity between species and blue indicates less identity. The sequences were Homo sapiens (Human, NP_002377); Pan troglodytes (Chimpanzee, JAA33119); Gorilla gorilla (Gorilla,AAX82901); Pongo pygmaeus (Orangutan, AAP30961); Papio anubis (Baboon, NP_001158061); Bos taurus (Cow, NP_776533); Balaenoptera physalus (Fin whale, ACR56643); Equus caballus (Horse, NP_001108006); Mus musculus (Mouse:C57BL/b, NP_032585); Sonora mutabilis (Mexican snake, AGA83481.1); Corvus corone (crow, ACA64022). The accession number from the rest of the mouse strains are Balb/c, (BAG85190); DBAF1, (CAA46589); C3H/HeJ, (ERS076383).

Figure 6. The MC1R receptor.

The MC1R receptors and the alleles that are associated with red hair and light skinned (the RHC phenotype) (shown in red). RHC alleles (designated R) show odds ratios for red hair in the range of 50 to 120 (strong RHC alleles). These are the frequent R151C, R160W and D294H variants and the rare D84E and R142H alleles. The weaker RHC alleles (designated r) are V60L, V92M, R163Q, have odd ratios for red hair ranging roughly from 2 to 6 as discussed elsewhere (91). Cysteine residues are shown in blue. Most of these form important disulphide bonds.

The MC1R gene has a high number of allelic polymorphisms, mainly in Caucasian populations (92). Recently it has been documented that there are 57 nonsynonymous and 25 synonymous polymorphisms in different populations (93). The N-terminus consists of many residues that are glycosylated. There are also cysteine residues, which are highly conserved in all MC1R alleles, because, when mutated to glycine or alanine leads to receptor inactivation (94, 95). Among the three extracellular loops, the third loop harbors a number of proline and the cysteine residues that are conserved and along with residues from loop 2 are critical in ligand binding (91, 94). Loop 1 has many residues that are prone to mutations. There are four intracellular loops, with potential sites for binding of G-proteins and motifs for phosphorylation. Mutations in these loops can occur at key positions that lead to loss of function. Other polymorphic variants in transmembrane regions also result in loss of function. These include Arg142His, Arg151Cys, Arg160Trp, and Asp294His substitutions, which are strongly associated with red hair phenotype, poor tanning ability, and, significantly, associated with melanoma and possibly non-melanoma skin cancer (96-100). A number of alleles, such as Val60Leu, Val92Met, and Arg163Gln substitutions, are thought to have lower penetrance for red hair phenotype but are vital for MC1R function. The cytosolic tail is short and has a cysteine residue that is a potential site for acylation. Information covering MC1R structure and its associated mutations is presented in greater detail, reviewed by Garcia-Borren et al. (91).

MC1R and immune system

Some of the immune regulating properties of eu- and pheo-melanin were noted above. Since MC1R signaling is central to eu and pheo-melanin regulation, it is important to determine whether MC1R induces its effects independent of melanin production. Using animal models, it has been shown that MC1R and other MCRs like MC3R and MC5R can influence inflammatory processes and the presence of MC1R on most immune cells suggests that it can affect both adaptive and innate responses (101-103). Further, α-MSH, originally thought to be produced solely by the pituitary gland, is also secreted by most immune cells (81). While α-MSH exerts its immune modulation and anti-inflammatory activity through activation of MC1R, the receptor and ligand are not exclusive to each other. Other ligands like ACTH can also stimulate MC1R while α-MSH can activate other MCRs. α-MSH exerts a wide range of activities that includes anti-inflammatory effects and immuno-modulation through MC1R signaling on macrophages and neutrophils (2, 104). MC1R expression can be induced on most immune cells. Its expression level can be altered on monocytes following lipopolysaccharide (LPS) (105). Stimulation of MC1R on endothelial cells leads to down-regulation of adhesion molecules: E-selectin, vascular cell adhesion molecule (VCAM) and intercellular adhesion molecule (ICAM) (106). Further MC1R stimulation can also reduce TNF-α production in endothelial cells (107) and production of cytokines such as IL-1 (IL1), TNF-α, and IL-6 and nitric oxide in monocytes (108, 109, 81). This decrease in cytokine production is reportedly due to inhibition of NFκB activation (104, 110-112) and IκBα degradation (113). Thus, the reduced inflammatory responses in tissues may be partly due to direct reduction of inflammatory cytokines and down regulation of adhesion molecules on endothelial cells, which leads to a reduction in inflammatory cell influx (Figure 4). Thus, the anti-inflammatory activities of MC1R signaling during UVR might play an important role in preventing UVR-induced carcinogenesis. It is noteworthy that mice lacking the MC1R have a normal immune system, but how they respond to external stressors needs further investigation (114, 115).

α-MSH/MC1R signaling induces tolerogenic dendritic cells that were shown to expand regulatory T-cells (Tregs) in vitro, as well as in vivo (116). The α-MSH stimulated DCs induced Tregs that were functional, as they inhibited the proliferation and cytokine secretion of T-helper-17 (Th17) cells from individuals with psoriasis. Further, α-MSH has been shown to down-regulate CD86, a major T-cell costimulatory molecule on monocytes (105). Peripheral blood monocytes stimulated with α-MSH increase IL-10 transcription and secretion (117). IL-10 is a highly immunosuppressive cytokine and an excellent inducer of regulatory T-cells (118), which are often associated with tumor-induced immune suppression. A minority of studies report that IL-10 possesses anti-tumor properties under some conditions (119). Taking these findings together, it can be hypothesized that MC1R has an important role in supporting an immunosuppressive environment which may facilitate UV-induced tumorigenesis.

On the other hand, stimulation of MC1R on certain immune cells can lead to a reduction in tumor development in mouse model systems. Loser et. al. showed that when α-MSH treated CD8+T-cells were transferred into mice, they became resistant to developing allergic contact hypersensitivity responses, but at the same time, maintained melanoma-specific CTL activity, as demonstrated by the expression of CTL—related genes and specific cytolytic activity in vitro and in vivo (102).Thus, it can be argued that MC1R maintains a balance of controlled inflammation that facilitates the maintenance of a tumor free environment.

AGOUTI SIGNALING PROTEIN (ASP)

As discussed earlier ASP works as an antagonist to α-MSH. The human ASIP (132 aa) and mouse ASP (131aa) protein, share 85% homology. ASP favors the synthesis of pheomelanin. It also inhibits melanoblast differentiation. The opposing effects of ASP to that of α-MSH is due to its antagonistic competitive binding to the MC1R (120), leading to reduce cAMP production. This leads to the inhibition of MITF expression, which requires cAMP-dependent transcription factors CRE and CREB. Further, it can block MITF downstream events of gene activation through binding to M-box sequences in the promoters of tyrosinase, TPR1, TRP2 genes (34). Although studies addressing whether an interaction of MC1R and ASP could further enhance the pigmentation pattern to pheomelanin and increase susceptibility to melanoma needs further investigation, there are studies in human populations showing polymorphisms in ASP that are closely associated with redhead color and skin sensitivity to sun or melanoma susceptibility (121-123).

Various mouse models have been developed that have mutations in different genes along the eumelanin biosynthetic pathway. Also in mice where the agouti gene has alleles that lead to a gain of ASP function, which block MC1R signaling, have a coat with higher pheomelanin levels. In wild-type agouti (A/A) mice like C3H stains, a shift from the synthesis of eumelanin to pheomelanin and back to eumelanin occurs within individual melanocytes. This results in a characteristic subapical band of yellow pigmentation on an otherwise black or brown hair (124). Many different mutations have been described at the agouti locus. There is a phenotypic spectrum of alleles that produce mice with different colors of fur. At one of this spectrum is the recessive allele, extreme non-agouti (a^e/a^e) that produce only black pigment, and on the other end are the top dominant lethal yellow (A^Y/+), allele that produce only yellow pigment (124). Some alleles also produce altered phenotypes in terms of the pigment deposition pattern. Analysis of eumelanin and pheomelanin content in C3H versus B6 strains has shown that C3H mice have lower eumelanin to pheomelanin ratio compared to C57/BL6 mice (125-128)(Table 1). The eumelanin/pheomelanin ratio is one order higher in C57BL/6 mice compared to agouti strains like C3H. Similar differences in the ratio can be found between the black human hair versus the red or blonde hair as shown (129-131) (Table 1). All these mouse strains, are wildtype in nature, and are well suited for investigating melanoma initiation, as they are not altered with exogenous gene manipulation and mimic differences in human skin and hair color. Some variations between the ratios of eu and pheo-melanin in different reports and in different human populations was seen, which might be due to the method of extractions used and sensitivity of assay done.

TABLE I. Eumelanin and pheomelanin ratio in hair samples of mice and human.

Mouse strain	Fur Color (genotype)	Eumelanin (%)	Pheomelanin (%)	Ratio Eu/pheo	Reference
C3H/HeJ	Brown (A/A)	77.6	22.4	3.5	(125)
B10	Black (a/a)	97.4	2.6	37.4	(126)
C57BL/6	Black (a/a)	97	3	32.3	(127)
C3H/HeSnJ	Brown (A/A)	93	7	13.3	(128)
B10	Brown (A/A)	60.3	39.7	1.51	(126)
Agouti	Brown (A/A)	47.2	42.8	1.1	(127)

Human type	Hair Color	Eumelanin (%)	Pheomelanin (%)	Ratio Eu/pheo	Reference
Red head	Red	3	97	0.03	(129)
Blond	Yellow	13	87	0.15	(130)
Black	Black	70	30	2.3	(130)
Black	Black	78	22	3.5	(131)

In summary, there seems to be a correlation of strains with high pheomelanin content, such as agouti strains of mice, along with other mutant strains that develop yellow coat color, with susceptibility to melanoma development. It is well known that C3H/HeN mice are more susceptible than C57/BL6 mice to tumor development induced by a number of carcinogenic protocols. Others have shown that C3H/HeN mice treated with a combination of ethanol, aloe emodin, and UV radiation develop high numbers of primary cutaneous melanomas, though they didn’t do a comparison with the standard B6 mouse strain (132). UVR is the major factor responsible for melanoma development, but because melanomas also develop in sun-unexposed areas, other factors are also involved. We have shown that exposure the polyaromatic carcinogen compound 7,12-Dimethylbenz(a)anthracene (DMBA) and the promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) results in development of nevi in C3H/HeN mice (133). When we compared the incidence of nevi generated in C3H/HeN mice with B6, we found that more tumors developed in C3H/HeN and each lesion was larger when compared to lesion development in B6. Although the UVR protocol needs to analyzed, our personal observation is that C3H/HeN mice also develop UVB induced tumors faster than B6 mice. It must be emphasized, however, that these observations are only correlative, and further work needs to be done to tease out the susceptibility genes.

CONCLUSION

In conclusion, what we have learned is that in addition to the appreciated importance of MC1R-αMSH interactions in determining the fate of melanin synthesis and UVR susceptibility, the presence of the MC1R receptor on immune cells indicates a second, less well-understood role as an immune modulator. Information regarding pheomelanin function and whether it possesses any beneficial biological activities is lacking, and should be further investigated.

Supplementary Material

Supp FigureS1

Supplementary Figure 1. Sequences of proteins of MC1R from different species were retrieved from pubmed and aligned. The accession numbers are given in figure 5. Red color and blue shows 100% and 0% conservation. Each black box represents the transmembrane sequence. The red boxes represent the alleles that are associated with red hair and light skin. Most of these were conserved in all species except valine 92 and Arginine 163. It is interesting to note that baboon has V92M change which is present in red-headed human individuals. Cysteine residues (shown in blue) form important disulphide bonds. All the cysteine residues were found to be conserved through all the species aligned indicating their importance in MC1R. Only the snake and crow showed more differences than other species. Each black box represents the transmembrane segment.