The melanocortin 1 receptor and the UV response of human melanocytes--a shift in paradigm

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 alpha-melanocortin (alpha-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 alpha-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.     

MC1R Gene Polymorphism Affects Skin Color and Phenotypic Features Related to Sun Sensitivity in a Population of French Adult Women

The melanocortin-1 receptor ( MC1R ) gene is known to play a major role in skin and hair pigmentation and to be highly polymorphic in Caucasians. This study was performed to investigate the relationships between MC1R gene polymorphisms and skin color in a large sample of French middle-aged Caucasian women. The codons 60 to 265 and the codon 294 of the MC1R gene were sequenced in 488 women. The skin color was measured on the inner side of the forearm using a spectrophotometric instrument. Fifteen variants were identified: Arg151Cys, Arg160Trp, Arg142His, Asp294His, Ile155Thr, Asp84Glu, Val60Leu, Val92Met, Arg163Gln, Ser83Pro, Thr95Met, Pro256Ser, Val265Ile, Ala166Ala and Gln233Gln. Women carrying Arg151Cys, Asp294His, Arg160Trp and Asp84Glu variants had a significantly higher reflectance in the red region, which indicates a lower level of functional melanin. This association was the most pronounced for women carrying Asp84Glu. In contrast, no significant difference was observed for other variants. Moreover, associations between MC1R polymorphisms and the risks of experiencing sunburn and of having freckles were found independently of skin color. Our findings support the hypothesis that MC1R polymorphisms do not necessarily alter the skin color but should sensitize the skin to UV-induced DNA damage.     

The Arg160Trp allele of melanocortin-1 receptor gene might protect against vitiligo

Melanocortin-1 receptor (MC1R) and agouti signaling protein (ASIP) play pivotal roles in the regulation of human pigmentation. We aimed to study whether single nucleotide polymorphisms (SNPs) of the MC1R and ASIP genes contribute to the pathogenesis of the polygenic pigment skin disorder, vitiligo. The PCR-amplified, full-length MC1R gene was studied with sequence analysis, and the 3' untranslated region (3' UTR) SNP of ASIP was detected using restriction fragment length polymorphism. The allele frequency of the ASIP SNP did not show any difference between the skin type, hair color and eye color-matched 97 vitiligo patients and the 59 healthy control individuals. As one of the MC1R polymorphisms showed significantly higher incidence among fair-skinned individuals (Fitzpatrick I+II, n=140) than among dark-skinned individuals (Fitzpatrick III+IV, n=90), both vitiligo patients and controls were divided into two groups and the frequency of the MC1R alleles was studied separately in fair-skinned and dark-skinned subgroups of diseased and healthy groups. C478T, one of the MC1R SNPs studied in 108 fair-skinned vitiligo patients and in 70 fair-skinned healthy control individuals, showed a significant difference (P=0.0262, odds ratio [95% confidence interval]=3.6 [0.0046-0.1003]) in allele frequency between the two groups: the allele frequency was higher in the control group, suggesting protection against vitiligo. Computer prediction of antigenicity has revealed that the Arg160Trp amino acid change caused by this SNP results in a decrease in antigenicity of the affected peptide epitope.     

MC1R,Eumelanin and Pheomelanin: Their Role in Determining the Susceptibility to Skin Cancer

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, the role of melanin in photoprotection is complex. This is because one type of melanin called eumelanin is UV absorbent, whereas the other, pheomelanin, is photounstable 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 antimelanoma 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.     

Minimization of MC1R selectivity by modification of the core structure of alpha-MSH-ND

BACKGROUND: Melanocortin, through its distinct receptor subtypes, has many different effects. Receptor-selective ligands are required to reduce the undesirable effects of melanocortin. To investigate which conformation is preferable to a given melanocortin receptor subtype, a structural and functional analysis of the ligand-receptor interactions was made by studying the biological activity, the nuclear magnetic resonance structures, and the patterns of the ligand-receptor interaction for each receptor subtype by homology modeling analysis. RESULTS: Among the several analogues examined, [Gln(6)]alpha-melanocyte-stimulating hormone (MSH)-ND was found to have 10000 times less biological activity than alpha-MSH-ND for the MC1R, whereas, the potencies of both oligopeptides were comparable in both the melanocortin-3 receptor (MC3R) and MC4R. [Gln(6)]alpha-MSH-ND exhibited a type I' beta-turn that was similar to the type I beta-turn structure of alpha-MSH-ND. However, a remarkable structural difference was observed with respect to the side chain orientations of the sixth and seventh residues of [Gln(6)]alpha-MSH-ND, which were found to be mirror images of alpha-MSH-ND. By homology modeling analysis, the His(6) of alpha-MSH-ND was found to interact with the TM2 regions of all three receptors (Glu(94) of MC1R, Glu(94) of MC3R, and Glu(100) of MC4R), but [Gln(6)]alpha-MSH-ND did not. The phenyl ring of the D-Phe(7) residue of [Gln(6)]alpha-MSH-ND revealed an interaction with the TM3 regions of both the MC3R and MC4R (Ser(122) of MC3R or Ser(127) of MC4R). However, in the MC1R, these serine residues corresponded to Val(122), which contains two methyl groups that induce steric hindrance with D-Phe(7) of [Gln(6)]alpha-MSH-ND. This is a possible explanation for the biological activity of [Gln(6)]alpha-MSH-ND for the MC1R being significantly lower than that for either the MC3R or MC4R. CONCLUSIONS: Minimization of the MC1R selectivity whilst preserving its comparable potency for both the MC3R and MC4R could be achieved by modifying the D-Phe(7) orientation of alpha-MSH-ND, while maintaining the 'type I beta-turn'-like structure.     

Skin pigmentation enhancers.

The highest incidences of cancer are found in the skin, but endogenous pigmentation is associated with markedly reduced risk. Agents that enhance skin pigmentation have the potential to reduce both photodamage and skin cancer incidence. The purpose of this review is to evaluate agents that have the potential to increase skin pigmentation. These include topically applied substances that simulate natural pigmentation: dihydroxyacetone and melanins; and substances that stimulate the natural pigmentation process: psoralens with UVA (PUVA), dimethylsulfoxide (DMSO), L-tyrosine, L-Dopa, lysosomotropic agents, diacylglycerols, thymidine dinucleotides, DNA fragments, melanocyte stimulating hormone (MSH) analogs, 3-isobutyl-1-methylxanthine (IBMX), nitric oxide donors, and bicyclic monoterpene (BMT) diols. These agents are compared with regards to efficacy when administered to melanoma cells, normal human epidermal melanocytes, animal skin, and human skin. In addition, mechanisms of action are reviewed since these may reveal issues related to both efficacy and safety. Both dihydroxyacetone and topically applied melanins are presently available to the consumer, and both of these have been shown to provide some photoprotection. Of the pigmentation stimulators, only PUVA and MSH analogs have been tested extensively on humans, but there are concerns about the safety and side effects of both. At least some of the remaining pigmentation stimulators under development have the potential to safely induce a photoprotective tan.     

Gene mapping study for constitutive skin color in an isolated Mongolian population

To elucidate the genes responsible for constitutive human skin color, we measured the extent of skin pigmentation in the buttock, representative of lifelong non-sun-exposed skin, and conducted a gene mapping study on skin color in an isolated Mongolian population composed of 344 individuals from 59 families who lived in Dashbalbar, Mongolia. The heritability of constitutive skin color was 0.82, indicating significant genetic association on this trait. Through the linkage analysis using 1,039 short tandem repeat (STR) microsatellite markers, we identified a novel genomic region regulating constitutive skin color on 11q24.2 with an logarithm of odds (LOD) score of 3.39. In addition, we also found other candidate regions on 17q23.2, 6q25.1, and 13q33.2 (LOD ≥ 2). Family-based association tests on these regions with suggestive linkage peaks revealed ten and two significant single nucleotide polymorphisms (SNPs) on the linkage regions of chromosome 11 and 17, respectively. We were able to discover four possible candidate genes that would be implicated to regulate human skin color: ETS1, UBASH3B, ASAM, and CLTC.     

A Case–Sibling Assessment of the Association Between Skin Pigmentation and Other Vitamin D-related Factors and Type 1 Diabetes Mellitus

Fair skin pigmentation has been associated with a higher risk of type 1 diabetes mellitus (T1DM). The aim is to compare children with T1DM directly to a sibling in relation to their skin pigmentation in sun-exposed and unexposed sites, past sun exposure and methylation of the VDR gene promoter. The sample consisted of children with T1DM attending a diabetes outpatient clinic and siblings (total n  = 42). Cutaneous melanin density was estimated using a spectrophotometer. Parental report on past sun exposure was obtained. DNA methylation analysis of the VDR gene promoter was conducted. Matched data analysis was performed comparing each case directly to their sibling. Cases were significantly more likely to have lighter skin pigmentation at the upper arm (AOR 0.69 [95% CI: 0.52, 0.90]; P  = 0.01). Low infant sun exposure was imprecisely associated with a two-fold increase in T1DM risk (AOR 2.43 [95% CI: 0.91, 6.51]; P  = 0.08 for under 1 h of winter sun exposure per leisure day). The VDR gene promoter was completely unmethylated in both cases and siblings. The previously demonstrated association between light skin pigmentation and T1DM risk was evident even in this comparison across sibling pairs. Further work on past UVR exposure and related factors such as skin pigmentation is required.     

A solid-phase approach to mouse melanocortin receptor agonists derived from a novel thioether cyclized peptidomimetic scaffold

The solid-phase synthesis of a novel thioether cyclized peptidomimetic scaffold, displaying functionality at the i to i + 3 positions, is reported. The thioether bridge is formed on-bead by an intramolecular reaction between a chloroacetylated reduced peptide bond and the free thiol from a cysteine. The crude products were obtained in moderate to very high purity. A series of 19 compounds were prepared and tested for agonist activity at the mouse melanocortin receptors 1, 3, 4, and 5 (mMC1-5R). From these results, several compounds were identified as having low micromolar agonist activity at the mMC1R and mMC4R. The former is involved in skin pigmentation and animal coat coloration. The latter is involved in the regulation of appetite and food intake and is currently a drug target for potential treatment of obesity. The most potent compound 1n with the pharmacophore motif "His-DPhe-Arg-Trp" was identified as having an EC(50) value of 165 nM at mMC1R, 7600 nM at mMC3R, 650 nM at mMC4R, and 335 nM at mMC5R. In addition, some of the compounds showed moderate selectivity for the mMC1R.     

Spectrophotometric methods for quantifying pigmentation in human hair-influence of MC1R genotype and environment.

Eumelanin (brown/black melanin) and pheomelanin (red/yellow melanin) in human hair can be quantified using chemical methods or approximated using spectrophotometric methods. Chemical methods consume greater resources, making them less attractive for epidemiological studies. This investigation sought to identify the spectrophotometric measures that best explain the light-dark continuum of hair color and the measure that is best able to distinguish red hair from nonred hair. Genetic analysis was performed on these two measures to determine the proportion of genetic and environmental influences on variation in these traits. Reflectance curves along the visible spectrum and subjective ratings of hair color were collected from 1730 adolescent twin individuals. Discriminant class analyses were performed to determine the spectrophotometric measure that could best proxy for eumelanin and pheomelanin quantities. The ratio of light reflected in the green portion of the spectrum to that reflected in the red portion of the spectrum was best able to distinguish red hair from nonred hair. Melanocortin 1 receptor (MC1R) genotype explained some, but not all, variation in this measure. Light absorbed in the red portion of the spectrum was best able to explain the light-dark continuum of hair color. Variance components analysis showed that there were qualitatively different genetic influences between males and females for the light-dark continuum of hair. Our results show that spectrophotometric measures approximating variation in eumelanin and pheomelanin may be considered as an alternative to chemical methods in larger epidemiological studies.     

Structural and molecular evolutionary analysis of Agouti and Agouti-related proteins

Agouti (ASIP) and Agouti-related protein (AgRP) are endogenous antagonists of melanocortin receptors that play critical roles in the regulation of pigmentation and energy balance, respectively, and which arose from a common ancestral gene early in vertebrate evolution. The N-terminal domain of ASIP facilitates antagonism by binding to an accessory receptor, but here we show that the N-terminal domain of AgRP has the opposite effect and acts as a prodomain that negatively regulates antagonist function. Computational analysis reveals similar patterns of evolutionary constraint in the ASIP and AgRP C-terminal domains, but fundamental differences between the N-terminal domains. These studies shed light on the relationships between regulation of pigmentation and body weight, and they illustrate how evolutionary structure function analysis can reveal both unique and common mechanisms of action for paralogous gene products.     

The protective role of melanin against UV damage in human skin

Human skin is repeatedly exposed to UVR that influences the function and survival of many cell types and is regarded as the main causative factor in the induction of skin cancer. It has been traditionally believed that skin pigmentation is the most important photoprotective factor, as melanin, besides functioning as a broadband UV absorbent, has antioxidant and radical scavenging properties. Besides, many epidemiological studies have shown a lower incidence for skin cancer in individuals with darker skin compared to those with fair skin. Skin pigmentation is of great cultural and cosmetic importance, yet the role of melanin in photoprotection is still controversial. This article outlines the major acute and chronic effects of UVR on human skin, the properties of melanin, the regulation of pigmentation and its effect on skin cancer prevention.     

MOLECULAR MECHANISMS OF ULTRAVIOLET RADIATION CARCINOGENESIS

UV radiation is a potent DNA damaging agent and a known inducer of skin cancer in experimental animals. There is excellent scientific evidence to indicate that most non-melanoma human skin cancers are induced by repeated exposure to sunlight. UV radiation is unique in that it induces DNA damage that differs from the lesions induced by any other carcinogen. The prevalence of skin cancer on sun-exposed body sites in individuals with the inherited disorder XP suggests that defective repair of UV-induced DNA damage can lead to cancer induction. Carcinogenesis in the skin, as elsewhere, is a multistep process in which a series of genetic and epigenetic events leads to the emergence of a clone of cells that have escaped normal growth control mechanisms. The principal candidates that are involved in these events are oncogenes and tumor suppressor genes. Oncogenes display a positive effect on transformation, whereas tumor suppressor genes have an essentially negative effect, blocking transformation. Activated ras oncogenes have been identified in human skin cancers. In most cases, the mutations in the ras oncogenes have been localized to pyrimidine-rich sequences, which indicates that these sites are probably the targets for UV-induced DNA damage and subsequent mutation and transformation. The finding that activation of ras oncogenes in benign and self-regressing keratoacanthomas in both humans and in animals indicates that they play a role in the early stages of carcinogenesis (Corominas et al., 1989; Kumar et al., 1990). Since cancers do not arise immediately after exposure to physical or chemical carcinogens, ras oncogenes must remain latent for long periods of time. Tumor growth and progression into the more malignant stages may require additional events involving activation of other oncogenes or deletion of growth suppressor genes. In addition, amplification of proto-oncogenes or other genes may also be involved in tumor induction or progression. In contrast to the few studies that implicate the involvement of oncogenes in UV carcinogenesis, the role of tumor suppressor genes in UV carcinogenesis is unknown. Since cancer-prone individuals, particularly XP patients, lack one or more repair pathways, one can speculate that DNA repair enzymes would confer susceptibility to both spontaneous and environmentally induced cancers. Another potential candidate that can function as a tumor suppressor gene is the normal c-Ha-ras gene. Spandidos and Wilkie (1988) have shown that the normal c-Ha-ras gene can suppress transformation induced by the mutated ras gene.(ABSTRACT TRUNCATED AT 400 WORDS)     

Comparable Photoreactivity of Hair Melanosomes,Eu‐ and Pheomelanins at Low Concentrations: Low Melanin a Risk Factor for UVA Damage and Melanoma?†

Melanin is known to be photoreactive and photoprotective, but its function in skin in vivo is still debated. Data is lacking of the effects of UVA irradiation on human skin melanosomes of different pigmentation, which have not been extensively degraded by isolation procedures. We have shown previously that melanosomes isolated from human oriental and black cat hair, and synthetic eumelanins, are photoreactive producing superoxide at low concentrations when exposed to UVA irradiation comparable to UK levels of sunlight. Here we investigated the UVA-irradiation of melanosomes, isolated from different colored human hair samples, using electron spin resonance spectroscopy and spin trapping. Comparable irradiation of synthetic pheomelanins synthesized from L-dopa and L-cysteine was also studied. An alkali method (5 min NaOH at 90 degrees C) could be used to isolate oriental hair melanosomes but was not suitable for auburn and blonde hair. Dithiothreitol and proteinase K resulted in melanin release from possible over-digestion of melanosomes; however, dithiothreitol and papain resulted in no melanin release and good melanosome yields with separation from residual keratin for brown, auburn and blonde hair. Melanosomes isolated by the latter method and synthetic pheomelanins were similar in UVA-photoreactivity at low concentrations, independent of hair color, and broadly comparable to synthetic melanins. Melanosome concentration at constant fluence may be more significant with respect to photodamage and UVA photocarcinogenesis (melanoma) via superoxide radical production than pigment type.     

Molecular characterization and analysis of the biosynthetic gene cluster for the antitumor antibiotic mitomycin C from Streptomyces lavendulae NRRL 2564

BACKGROUND: The mitomycins are natural products that contain a variety of functional groups, including aminobenzoquinone- and aziridine-ring systems. Mitomycin C (MC) was the first recognized bioreductive alkylating agent, and has been widely used clinically for antitumor therapy. Precursor-feeding studies showed that MC is derived from 3-amino-5-hydroxybenzoic acid (AHBA), D-glucosamine, L-methionine and carbamoyl phosphate. A genetically linked AHBA biosynthetic gene and MC resistance genes were identified previously in the MC producer Streptomyces lavendulae NRRL 2564. We set out to identify other genes involved in MC biosynthesis. RESULTS: A cluster of 47 genes spanning 55 kilobases of S. lavendulae DNA governs MC biosynthesis. Fourteen of 22 disruption mutants did not express or overexpressed MC. Seven gene products probably assemble the AHBA intermediate through a variant of the shikimate pathway. The gene encoding the first presumed enzyme in AHBA biosynthesis is not, however, linked within the MC cluster. Candidate genes for mitosane nucleus formation and functionalization were identified. A putative MC translocase was identified that comprises a novel drug-binding and export system, which confers cellular self-protection on S. lavendulae. Two regulatory genes were also identified. CONCLUSIONS: The overall architecture of the MC biosynthetic gene cluster in S. lavendulae has been determined. Targeted manipulation of a putative MC pathway regulator led to a substantial increase in drug production. The cloned genes should help elucidate the molecular basis for creation of the mitosane ring system, as well efforts to engineer the biosynthesis of novel natural products.     

Parallel synthesis of a lipopeptide library by hydrazone-based chemical ligation

alpha-Melanocyte-stimulating hormone (alpha-MSH) is an endogeneous linear tridecapeptide with potential application for the modulation of skin tanning. To evaluate the interest of introducing a lipid moiety onto this peptide, we developed an efficient chemoselective parallel method to prepare a large series of analogues of alpha-melanocortin with high purity, varying the nature or the relative position of the lipid moiety. Two sets of building blocks containing lipidic alpha-oxo-aldehydes or alpha-hydrazinoacetyl peptides were combined to obtain a 102-membered library of amphiphilic alpha-MSH analogues. This library was pharmacologically tested at 1 x 10(-7) M for the ability to induce AMPc production in M4Be melanoma cell line after stimulation of the human melanocortin MC1 receptor. Among theses lipopeptides, 84 compounds exhibited an AMPc induction higher than Melitane, a patented alpha-MSH agonist. These results provide strong evidence of the interest of introduction of a lipid tail for the pharmacomodulation of bioactive peptides.     

Constitutive and Relative Facultative Skin Pigmentation among Victorian Children Including Comparison of Two Visual Skin Charts for Determining Constitutive Melanin Density

Our aim was to examine the association between ethnicity, phenotype, sun behavior and other characteristics, and constitutive and relative facultative skin pigmentation. A total of 191 participants were recruited, with a mean age of 7.6 years (SD 3.4), during 2009–2011 from Maternal and Child Health Centres (MCHC) and schools in Melbourne, Australia. Parental questionnaire data were obtained on sun behavior and examination consisted of noting the child's natural skin, hair and eye color, ethnicity, nevi count and spectrophotometric melanin density (MD). Constitutive skin pigmentation was estimated from buttock MD. Relative facultative skin pigmentation was estimated by hand compared with buttock absorption. Ethnicity, hair color and skin color were associated with constitutive and facultative skin pigmentation on univariate analysis. Higher ambient ultraviolet radiation (UVR) in the past month, greater freckling, greater nevi and increased sun exposure over the past year were related to darker facultative skin pigmentation. Sun exposure over the life course was not. The two skin charts accounted for 39.7% and 21.4% of buttock MD, respectively. Relative facultative skin pigmentation is associated with recent UVR levels, not life‐course sun exposure. Relative facultative skin pigmentation may not be a useful measure of sun exposure over the early life course. Skin color charts can be used to assess constitutive skin pigmentation if spectrophotometry is not available.     

Acute response of human skin to solar radiation: regulation and function of the p53 protein.

p53 is a tumor suppressor gene and mutation of p53 is a frequent event in skin cancer. The wild-type p53 encodes for a 53-kD phosphoprotein that plays a pivotal role in regulating cell growth and cell death. The wt-p53 gene is also called "guardian of the genome", for its role in preventing the accumulation of genetic alterations, observed in cancer cells. The wild-type p53 protein plays a central role in the response of the cell to DNA damage. UV, present in sunlight, is one of the most ubiquitously present DNA damage inducing stress conditions to which skin cells are exposed. The wt-p53 protein accumulates in human skin cells in vitro and in human skin in vivo upon UV irradiation. This upregulation mounts a protective response against permanent DNA damage through transactivation of either cell cycle arrest genes and DNA repair genes or genes that mediate the apoptotic response. The molecular events which regulate the activity of the wt-p53 protein activity are only beginning to be described.