Cutaneous pigmentation is the major photoprotective mechanism against the carcinogenic and aging effects of UV. Epidermal melanocytes synthesize the pigment melanin, in the form of eumelanin or pheomelanin. Synthesis of the photoprotective eumelanin by human melanocytes is regulated mainly by the melanocortins α-melanocortin (α-MSH) and adrenocorticotropic hormone (ACTH), which bind the melanocortin 1 receptor (MC1R) and activate the cAMP pathway that is required for UV-induced tanning. Melanocortins stimulate proliferation and melanogenesis and inhibit UV-induced apoptosis of human melanocytes. Importantly, melanocortins reduce the generation of hydrogen peroxide and enhance repair of DNA photoproducts, independently of pigmentation. MC1R is a major contributor to the diversity of human pigmentation and a melanoma susceptibility gene. Certain allelic variants of this gene, namely R151C, R160W and D294H, are strongly associated with red hair phenotype and increased melanoma susceptibility. Natural expression of two of these variants sensitizes melanocytes to the cytotoxic effect of UV, and increases the burden of DNA damage and oxidative stress. We are designing potent melanocortin analogs that mimic the effects of α-MSH as a strategy to prevent skin cancer, particularly in individuals who express MC1R genotypes that reduce but do not abolish MC1R function, or mutations in other melanoma susceptibility genes, such as p16.
We are privileged to contribute this review to the special issue celebrating Professor Hasan Mukhtar’s 60th birthday. Professor Mukhtar is a pioneer in the field of photobiology and photocarcinogenesis. He has made tremendous contributions to this area of research, and revolutionized our understanding of the signaling pathways of UV and prevention of photocarcinogenesis, using naturally derived compounds, particularly polyphenolic derivatives of green tea. Professor Mukhtar is highly regarded as an accomplished scientist, and equally or even more, as a mentor for a generation of distinguished scientists. While writing this review, the corresponding author could not but reflect on her own mentor, the late Mac E. Hadley, who inspired her to investigate the effects of melanocortins on human melanocytes and human pigmentation, which became the focus of research in her laboratory. Mentors are the role models and the giants in whose footsteps we aspire to walk. They mold our careers, set up the standards, raise the bar, and encourage us to jump scientific hurdles and excel. We follow their lead in science and teaching, and most of all in dignity and caring for students, trainees and humanity that we serve.
Melanin and Photoprotection
Solar UV radiation is the major environmental factor to which humans are exposed. The skin, the largest organ, represents the interface between the environment and the internal organs, and the main target of UV. Acute exposure to UV results in erythema, immune suppression, epidermal thickening and tanning, while chronic exposure results in photocarcinogenesis and photoaging (1–3). The extent of these effects varies among individuals, and is determined to a large extent by melanin content in the cutaneous epidermis. Melanin, the pigment that gives skin and hair their distinctive color, exists in two major forms, the brown/black eumelanin, and the red/yellow pheomelanin, which are synthesized by specialized cells in the epidermis, the melanocytes (4,5). Each melanocyte, regardless of pigmentary phenotype, synthesizes these two forms of melanin in different amounts. Skin pigmentation correlates directly with eumelanin content, which is more abundant in dark than in lightly pigmented skin (6,7). Thus eumelanin, rather than pheomelanin, content accounts for the diversity of human pigmentation. Melanin is considered the major photoprotective mechanism, as it shields the skin from UV rays by reducing their penetration through the epidermal layers and the underlying dermis (8,9).
The increase in pigmentation in response to UV exposure, i.e. the tanning response, is an important risk factor for skin cancer. Numerous epidemiologic studies have concluded that the incidence of skin cancer correlates inversely with skin pigmentation, with the highest incidence in individuals with light skin color who have a poor tanning ability, i.e. skin phototypes I and II (1,10,11). Such individuals typically have low constitutive eumelanin content. The best evidence for the significance of cutaneous pigmentation in determining skin cancer risk is provided by the Celtic population in Australia, with the highest incidence for skin cancer worldwide (12). Eumelanin, compared to pheomelanin, is more photoprotective due to its stability and resistance to degradation by UV (13,14). Ultrastructural studies of light versus dark skin revealed that in the former, melanosomes, the organelles in which melanin is synthesized and deposited, are degraded, leaving only “melanin dust” in the suprabasal layers of the epidermis (15,16). In contrast, melanosomes enriched in eumelanin in dark skin remain intact throughout the epidermis. These melanosomes form supranuclear caps that protect the nuclei from the impinging UV rays (17). Additionally, eumelanin acts as a scavenger for reactive oxygen species (ROS), while pheomelanin itself is thought to generate ROS, particularly singlet oxygen and hydroxyl radicals (13,14,18).
Genetic and Biochemical Regulation of Eumelanin and Pheomelanin Synthesis
The importance of melanin in photoprotection generated interest in understanding the regulation of eumelanin and pheomelanin synthesis by epidermal melanocytes. Valuable lessons were learned from studies on the genetics of mouse coat color. The results of these studies revealed that eumelanin synthesis by melanocytes within the hair follicle is primarily regulated by α-melanocyte stimulating hormone (α-MSH) (19), while pheomelanin synthesis is mainly regulated by agouti signaling protein (ASP), the product of the agouti locus (20–22). Binding of α-MSH to its receptor, the melanocortin 1 receptor (MC1R), activates the synthesis of eumelanin by follicular melanocytes (19,23,24). MC1R is the product of the extension locus, and loss-of-function mutation in its gene, as in the recessive yellow (e/e) mouse, results in a yellow coat color, because of inability of melanocytes to respond to α-MSH by synthesizing eumelanin. ASP is the physiologic antagonist of MC1R, acting as a competitive inhibitor of α-MSH binding to this receptor (25). Temporal expression of ASP results in the agouti phenotype, characterized by hairs with a yellow pheomelanotic band demarcated by a proximal and a distal black eumelanotic band. The yellow band is the outcome of expression of ASP that inhibits α–MSH-induced eumelanin production, allowing only for pheomelanin synthesis. Mutations in the agouti locus that cause overexpression of ASP result in a yellow coat color because of inhibition of eumelanin and the exclusive pheomelanin synthesis (26).
Comparison of the biochemical pathways of eumelanin and pheomelanin synthesis revealed that the former requires higher concentrations of tyrosine, the melanin precursor, as well as higher activity and protein levels of the melanogenic enzymes tyrosinase, and tyrosinase-related proteins (TRP)-1 and -2 (27). Pheomelanin synthesis requires the availability of the amino acid cysteine, which gives it its distinctive red color, and proceeds in the presence of low tyrosine concentrations, low activity and level of tyrosinase, and absence of TRP-1 and TRP-2 (27,28). Based on these findings, pheomelanin synthesis is thought to be the default pathway for melanin synthesis, as its requirements are less stringent than those of eumelanin synthesis.
Melanocortins, the Melanocortin 1 Receptor and Human Pigmentation
While the role of α-MSH as the physiologic regulator of integumental pigmentation of various vertebrate species has been well established (29,30), the role of melanocortin in the physiologic regulation of human pigmentation was widely debated until the early 1990s. Studies carried out by Lerner and McGuire showed that injection of human subjects with melanocortins increased skin darkening (31). Almost three decades later, Levine et al. showed that injection of humans with the potent α-MSH analog, NDP-MSH, resulted in increased pigmentation in the absence of sun exposure (32). These studies clearly demonstrated that melanocortins have pigmentary effects on human skin, but did not prove that melanocytes directly respond to melanocortins. In the early 1990s, it became evident that the genetic regulation of eumelanin and pheomelanin synthesis in humans is similar to that in the mouse, thanks to several breakthroughs. First, the human MC1R gene was cloned from human melanocytes and pharmacologic characterization of this receptor showed that it binds α-MSH and the related melanocortin ACTH with the same affinity (33,34). Second, we demonstrated that cultured human melanocytes respond to these melanocortins with dose-dependent stimulation of melanogenesis and proliferation (35), and others showed that melanocortins increase eumelanin content in cultured human melanocytes (36). Third, we also demonstrated that human melanocytes respond to ASP with inhibition of the effects α-MSH by blocking its binding to MC1R (37). Fourth, other laboratories reported that proopiomelanocortin (POMC) is synthesized and processed into its melanocortin derivatives by various cell types in the skin, including epidermal keratinocytes and melanocytes, strongly suggesting that these factors act as paracrine and autocrine regulators of melanocytes (38,39). Fifth, mutations in the human POMC gene were found to result in red hair phenotype, in addition to various metabolic abnormalities, such as adrenal insufficiency and obesity (40). Sixth, epidemiologic studies from different populations in Northern European countries and Australia revealed that certain allelic variants of MC1R that result in loss of function of the receptor are strongly associated with red hair phenotype (41–46), and that the wild type allele that is predominant in the African continent is associated with dark skin and hair (47). These findings put to rest a longstanding controversy about the role of melanocortins in human pigmentation, and provided unequivocal evidence for the physiologic role of melanocortins in regulating human melanocyte function.
Role of the Main Signaling Pathway of the Activated MC1R in the UV Response
MC1R is a Gs protein-coupled receptor with seven transmembrane domains that is mainly expressed on melanocytes (33). Activation of this receptor by its ligands α-MSH or ACTH results in stimulation of cAMP formation, which leads to increased eumelanin synthesis and proliferation of human melanocytes (35,36,48). The cAMP pathway is considered the major signaling pathway for melanocortins, as factors that increase cAMP by either stimulating its synthesis by activating adenylate cyclase (e.g. forskolin), or blocking its degradation by inhibiting phosphodiesterases (e.g. isobutyl methylxanthine), mimic α-MSH in stimulating melanogenesis and proliferation of human melanocytes (44,49,50). We have reported that activation of the cAMP pathway is pivotal for UV-induced melanogenesis, i.e. tanning response (50). Moreover, treatment with α-MSH enabled UV-irradiated cultural human melanocytes to overcome the growth arrest and enter the S phase. These results clearly indicate that melanocortins and MC1R modulate the response of human melanocytes to UV.
Modulation of the Response of Human Melanocytes to UV by Paracrine and Autocrine Factors
Given that cutaneous pigmentation is the major photoprotective mechanism against the damaging effects of UV, it is important to elucidate how epidermal melanocytes respond to UV. The effects of UV on the skin are direct, in the form of DNA photoproducts, and indirect, being mediated by activating a network of paracrine and autocrine factors that regulate the cell cycle, apoptosis and DNA repair. These factors include the primary cytokines interleukin-1 and tumor necrosis factor-α, the melanocortins α-MSH and ACTH, endothelin-1 and basic fibroblast growth factor that are mainly synthesized by epidermal keratinocytes and regulate the function of human melanocytes (38,39,51–54). Human melanocytes respond directly to UV by induction of DNA photoproducts, and immediately by generation of ROS that result in oxidative DNA damage, such as 8-hydroxydeoxy guanosine (8-OHdG), as well as lipid and protein peroxidation (55–57). Exposure to UV results in growth arrest, a mechanism thought to be necessary to allow cells to undergo DNA repair, as well as apoptosis of cells with extensive damage that exceeds cellular DNA repair capacity, and increased melanin synthesis, i.e. tanning (55). We have shown that α-MSH, ACTH and endothelin-1 rescue human melanocytes from UV-induced apoptosis by activating the Akt survival pathway upstream from the transcription factor CREB, which in turn activates Mitf, the “master regulator” of melanocyte survival and melanin synthesis (56). Mitf is a transcription factor that increases the expression of the antiapoptotic Bcl2, known to be critical for melanocyte survival (58). Treatment of UV-irradiated human melanocytes with either α-MSH and/or endothelin-1 reverses the UV-induced reduction of Bcl2 levels and increases melanocyte survival (56). We surmised that the survival effects of α-MSH and endothelin-1 are the result of enhancement of repair of UV-induced DNA damage and restoration of genomic stability. Indeed, we observed that treatment with α-MSH and/or endothelin-1 reduced the generation of hydrogen peroxide and enhanced the repair of cyclobutane pyrimidine dimers in UV-irradiated human melanocytes. The effects of α-MSH on nucleotide excision repair were corroborated by Bohm et al. (59). Importantly, these newly described effects of α-MSH and endothelin-1 proved to be independent of stimulation of melanogenesis, as they were evident in tyrosinase-negative albino melanocytes that lacked the ability to synthesize melanin (56).
The above results ascribe novel roles for the paracrine factors α-MSH, ACTH and endothelin-1 in the stress response to UV that limit the UV-induced DNA damage by enhancing nucleotide excision repair and reducing oxidative stress. Others have suggested that UV-induced melanogenesis is a response to DNA damage (60,61). We concluded from our findings that melanocytes have immediate as well as latent response to UV. The former involves enhancement of repair of DNA photoproducts and reduction in the generation of ROS to limit the extent of damage and insure genomic stability. The latter response, which might be a consequence of DNA damage, involves increased melanogenesis to protect against subsequent UV exposure. These effects of α-MSH and endothelin-1 are particularly critical for melanocytes, as these cells have a slow proliferation capacity and a long lifespan in the epidermis. Unlike keratinocytes that have a high self-renewal and proliferation capacity, melanocytes are for the most part differentiated cells that reside in the epidermis for decades, and have a poor ability to self-renew or proliferate. Additionally, melanocytes are resistant to apoptosis (62,63), which makes it crucial to insure their genomic stability in order to prevent the accumulation of mutations over time that can potentially lead to malignant transformation to melanoma.
The Signaling Pathway of UV in Cultured Human Melanocytes
We reported that exposure of cultured human melanocytes to UV results in the accumulation of p53 and induction of expression of the p53-dependent cyclin-cdk inhibitor p21 (Waf-1, cip-1) (55,64). These effects are thought to be a response to DNA damage, to arrest cells in the cell cycle in order to allow for DNA repair, which explains the role of p53 as a cell cycle checkpoint and a guardian of the genome (65,66). Others demonstrated that p53, in its capacity as a transcription factor, activates the expression of tyrosinase, the rate-limiting enzyme in the melanin synthetic pathway (67). Recently, it was shown that p53 induces the expression of POMC gene that codes for the melanocortin precursor protein, POMC, in keratinocytes (68). These results implicate p53 in the UV-induced increase in POMC synthesis in the epidermis. In addition to p53, POMC expression was shown to be regulated by the transcription factor USF-1, known to be activated by UV exposure in a p38-dependent manner (69). Based on these findings and our observation that α-MSH and ACTH upregulate MC1R expression and further augment the accumulation of p53 after UV exposure, we propose the following positive feedback loop between p53 and MC1R. Exposure of the skin to UV results in the accumulation of p53 and activation of USF-1, both of which stimulate POMC production and hence increase the levels of α-MSH and ACTH, which upregulate the expression of MC1R and further enhance p53 accumulation. This model illustrates that melanogenesis is the outcome of UV-induced stress, and links the tanning response to the signaling pathway(s) activated by UV, and mediated by p53 and USF-1.
Regulation of MC1R Expression by Paracrine Factors, Including its Physiologic Antagonists and Antagonist
MC1R plays a central role in the regulation of cutaneous pigmentation. We have found that the levels of MC1R mRNA are upregulated upon treatment of human melanocytes with basic fibroblast growth factor or endothelin-1, paracrine factors synthesized by keratinocytes, and their synthesis increased upon UV exposure (70). We have shown that α-MSH interacts synergistically with basic fibroblast growth factor and endothelin-1 to stimulate melanocyte proliferation and survival by augmenting the phosphorylation of the MAP kinases ERK1/2 and inhibiting apoptosis via activation of Akt and Mitf (56,71,72). Interestingly, MC1R mRNA is also increased by the agonists α-MSH and ACTH by a cAMP-dependent mechanism, as forskolin had a similar effect, and markedly reduced by the physiologic MC1R antagonist ASP. We predict that the increase in MC1R mRNA leads to increased expression of MC1R on the cell membrane of melanocytes, and maintains the responsiveness of melanocytes to melanocortins. On the other hand, the observed reduction in MC1R mRNA by ASP represents a mechanism by which this antagonist inhibits the effects of α-MSH on melanocytes, in addition to competitively binding MC1R and blocking its activation by α-MSH.
MC1R—A Major Determinant of the Diversity of Human Pigmentation
Epidemiologic studies have identified more than 70 allelic variants of MC1R, and only two alleles for the Agouti gene in various human populations (46,73). The extensive polymorphism of MC1R led to the conclusion that it is the major contributor to the diversity of human pigmentation. Interestingly, the wild type gene is predominantly expressed in Africa, where photoprotection by eumelanin is mostly needed due to excessive environmental UV exposure (47), and the allelic variants that are associated with red hair are expressed mainly in Northern European countries and the Celtic population in Australia (42,43). The evolution of these variants that resulted in lighter skin and hair color is thought to confer a survival advantage by insuring optimal absorption of the scare solar UV for vitamin D synthesis and optimal bone formation.
MC1R Variants, Tanning Ability and Melanoma Risk
The ability to tan is an important risk factor for melanoma as well as nonmelanoma skin cancers. Overwhelming clinical evidence shows that the incidence of skin cancer, including melanoma, is remarkably higher in individuals with fair skin who burn rather than tan after sun exposure than in those with dark skin and high tanning ability (8,74). Epidemiologic studies have found a strong association between certain MC1R variants and melanoma risk (75–77). These studies concluded that expression of MC1R variants, particularly R151C, R160W and D294H that are strongly linked to red hair phenotype, and termed “R” or “RHC alleles,” results in poor tanning response and compromises the photoprotective effect of the existing melanin. These variants were found to increase the penetrance of CDKN2A mutations, further increasing the risk of melanoma (78). Intriguingly, these variants were not always associated with red hair, as seen in studies on Greek and southern Italian populations, suggesting that the resulting increased melanoma risk is not strictly dependent on skin and hair color (77,79).
Impact of MC1R Genotype on MC1R Function and the UV Response of Melanocytes
We have been interested in elucidating the impact of different naturally expressed MC1R variants on the function of MC1R, the pigmentary phenotype and the response of melanocytes to UV. Comparison of the responses of a panel of human melanocyte cultures, each derived from a single donor, to α-MSH and correlation of this response with the MC1R genotype revealed that expression of the variants R151C, R160W and D294H in the homozygous or compound heterozygous state resulted in loss of the ability of melanocytes to respond to melanocortins by increased cAMP synthesis and stimulation of tyrosinase activity (44). However, these melanocytes responded to forskolin, a direct activator of adenylate cyclase, in these assays, indicating that inability to respond to melanocortins is due to altered MC1R function and not to a defect in its downstream signaling pathway. These results suggest that the above variants represent loss-of-function alleles of the MC1R gene. Melanocytes homozygous or compound heterozygous for these variants exhibited increased sensitivity to UV-induced cytotoxicity, indicating that loss of function of MC1R results in an aberrant response to UV. Expression of the relatively common variant V92M, or R163Q, commonly expressed in Asians (47), did not reduce MC1R function, suggesting that they represent pseudoalleles (44). Analysis of eumelanin and pheomelanin contents of the tested melanocyte cultures revealed that one culture that was compound heterozygous for R151C and D294H has high eumelanin content, which is atypical of melanocytes from a donor with light skin color. This finding is in agreement with the results of epidemiologic studies that R alleles of MC1R are necessary but not sufficient for fair skin and red hair phenotype, and their effects on melanocytes might be independent of pigmentation.
To test experimentally how the MC1R genotype might alter the UV-induced damage response of human melanocytes, we conducted a study in which we compared the UV-dependent induction and repair of cyclobutane pyrimidine dimers, generation of hydrogen peroxide and apoptosis in cultured human melanocytes with different melanin contents and MC1R genotypes (57). We found, as expected, that in melanocyte cultures with functional MC1R, induction of cyclobutane pyrimidine dimers and generation of hydrogen peroxide and apoptosis correlated inversely with melanin, particularly eumelanin content. However, in cultured melanocytes expressing two R alleles, and thus loss-of-function MC1R, these effects were independent of melanin content and markedly higher than in cultured melanocytes with functional MC1R. Comparison of cultures with comparable melanin contents but either functional or nonfunctional MC1R revealed higher cyclobutane pyrimidine dimers and hydrogen peroxide levels, as well as more apoptosis in the latter. Interestingly, one culture with relatively high eumelanin content and loss-of-function MC1R encountered higher levels of cyclobutane pyrimidine dimers than cultures with markedly lower eumelanin content but functional MC1R.
Importantly, cultures with loss-of-function MC1R had lower nucleotide excision repair capacity compared to their counterparts with functional MC1R, regardless of their melanin content. In fact, nucleotide excision repair capacity was independent of pigmentation, as shown in a previous in vivo study (80), but dependent on the MC1R genotype (57). As we have reported earlier that α-MSH enhances nucleotide excision repair and reduces hydrogen peroxide generation and apoptosis following UV irradiation, we expect that melanocytes expressing loss-of-function MC1R have compromised nucleotide excision repair capacity and antioxidant defenses due to their inability to respond to melanocortins (Fig. 1).
DNA Photoproducts, Oxidative DNA Damage and Melanoma
Unlike squamous and basal cell carcinoma in which “UV signature” mutations, e.g. in p53 gene, are commonly found (81), melanoma tumors do not usually express such mutations (82,83). This raised the question about the role of DNA photoproducts in melanoma formation. Recently, it was reported that melanoma patients have a lower nucleotide excision repair capacity than disease-free individuals, and that mutations in the melanoma susceptibility genes p16 and ARF compromise nucleotide excision repair (84,85). These two studies suggested a role for DNA photoproducts in the transformation of melanocytes to melanoma. The low incidence of “UV signature” mutations in melanoma led to the proposal that oxidative DNA damage drives the malignant transformation of melanocytes. The activating V600E mutation in BRAF, which is a common and early event in melanoma tumors (86), is thought to be typical of mutations induced by oxidative stress, and its expression to be increased by carrying MC1R variants (87). The aberrant UV response of melanocytes with loss-of-function MC1R offers an explanation for the increased risk to melanoma in individuals harboring nonfunctional MC1R alleles.
Use of MC1R Analogs for Prevention of Melanoma and Nonmelanoma Skin Cancer
The role of melanocortins in enhancing photoprotection against UV-induced genotoxicity led us to design and test on human melanocytes tetrapeptide α-MSH analogs consisting of His-D-Phe-Arg-Trp sequence with different n-capping groups (88). Of those, two analogs, namely 4-phenylbutyryl and n-pentadecanoyl α-MSH proved to be significantly more potent than the physiological hormone α-MSH in stimulating tyrosinase activity, i.e. melanogenesis. These two analogs mimicked α-MSH in enhancing nucleotide excision repair and inhibiting hydrogen peroxide generation and apoptosis in UV-irradiated human melanocytes. The effects of these analogs were entirely mediated by activating MC1R, as they could be blocked by an analog of ASP, and were absent in melanocytes expressing loss-of-function MC1R. These results indicate that such melanocortin analogs can be developed as a melanoma-preventative strategy. Developing potent melanocortin analogs for melanoma prevention should prove effective, particularly for individuals heterozygous for R MC1R alleles, or who express MC1R alleles that moderately affect receptor function (e.g. V60L), or are carriers of an MC1R variant and CDKN2A mutation. The best known α-MSH analog, NDP- α-MSH, has long been proposed as a sunless tanning agent. However, our novel results, implicating melanocortins in the regulation of nucleotide excision repair and antioxidant responses of human melanocytes, clearly suggest that these hormones are not only for tanning, but also have wider effects on the UV response that exceed the photoprotection by melanin.
Recent advances in the role of MC1R and melanocortins in human pigmentation and the UV response have resulted in a shift of the paradigm that pigmentary phenotype is a reliable predictor for skin cancer risk. The observations that the R alleles of MC1R increase the risk for melanoma and nonmelanoma skin cancers independently of skin and hair pigmentation (75–77), and the findings that these alleles compromise the nucleotide excision repair and the antioxidant defenses of human melanocytes suggest that the MC1R genotype can be used to improve assessment for melanoma and nonmelanoma skin cancer susceptibility. Melanoma is the most deadly form of skin cancers.
Despite increased public education and awareness, the incidence of melanoma continues to rise at an annual rate of 4–5%; the lifetime risk of developing melanoma is 1 out of 52 in men, and 1 out of 77 in women (American Cancer Society Facts and Figures, 2006; Atlanta: American Cancer Society, 2006). With no effective cure for late-stage disease, prevention and early detection of melanoma is crucial for reducing the continuous rise in melanoma incidence and prolonging the patients’ survival. Our strategy to develop potent α-MSH analogs is expected to be effective in melanoma prevention. By augmenting the repair of DNA photoproducts, reducing oxidative stress and stimulating melanogenesis, particularly in high-risk individuals (e.g. those harboring p16 mutations), α-MSH analogs are expected to normalize the UV response of melanocytes and prevent their malignant transformation to melanoma.
Acknowledgement— Supported in part by R01 ES09110 and R01 CA114095 grants (for Z.A.M.).