Journey Through the Spectacular Landscape of Melanocortin 1 Receptor

Abstract

The physiological role of α-melanocyte stimulating hormone in regulating integumental pigmentation of many vertebrate species has been recognized since the 1960’s. However, its physiological significance for human pigmentation remained enigmatic until the 1990’s. α-Melanocyte stimulating hormone and related melanocortins are synthesized locally in the skin, primarily by keratinocytes, in addition to the pituitary gland, and therefore act as paracrine factors for melanocytes. Human melanocytes express the melanocortin 1 receptor, which recognizes α-melanocyte stimulating hormone and the related adrenocorticotropic hormone as agonists. This review summarizes the current knowledge of the pleotropic effects of the activated melanocortin 1 receptor that maintain human melanocyte homeostasis by regulating melanogenesis and the response to environmental stressors, mainly solar radiation. Certain allelic variants of the melanocortin 1 receptor gene are associated with specific pigmentary phenotypes in various human populations. Variants associated with red hair phenotype compromise the function of the encoded receptor. Activation of the human melanocortin 1 receptor regulates eumelanin synthesis and enhances DNA damage response of melanocytes to solar radiation and oxidative stressors. We describe how synthetic selective melanocortin 1 receptor agonists can be efficacious as sunless tanning agents, for treatment of vitiligo and photosensitivity disorders, and for prevention of skin cancer, including melanoma.

Regulation of vertebrate pigmentation by α-melanocyte stimulating hormone

The bioactive peptide α-melanocyte stimulating hormone (α-MSH) has been known since the 1960’s as the physiological regulator of integumental pigmentation of many vertebrate species ^1–4. In poikilotherms, such as fish, reptiles, and amphibians, α-MSH regulates rapid color change by dispersion of melanosomes within melanophores for camouflage to deter predators, and for thermoregulation. Mice have served as a useful experimental model for mammalian pigmentation. Studies on mouse coat color revealed that it is mainly determined by the synthesis of the dark brown/black pigment eumelanin and the yellow pigment pheomelanin, and that α-MSH stimulates the synthesis of eumelanin ^5,6. Although the function of α-MSH is evolutionarily conserved across vertebrate species, its role in regulating human pigmentation remained enigmatic until the early 1990’s. The melanocortins, α, β, and γ-MSH are structurally-related bioactive peptides derived from the large precursor peptide pro-opiomelanocortin (POMC), which is classically known to be processed in the intermediate lobe of the pituitary gland in many vertebrate species ^{7 8}. Differential processing of POMC in the anterior lobe of the pituitary gland results in the production of adrenocorticotropic hormone (ACTH), known best for regulating steroidogenesis in the adrenal glands. This has led to the assumption that melanocortins act as endocrine factors that are transported by the blood circulation from the pituitary to their targets, including the melanocytes in skin. Since in humans the intermediate lobe of the pituitary is vestigial and plasma levels of α-MSH are hardly detectable, the role of α-MSH in regulating human pigmentation was dismissed for many years ⁹.

Role of α-MSH and the melanocortin 1 receptor in regulating human pigmentation

In the 1960’s, Lerner and McGuire reported that injection of human subjects with dark complexion with α-MSH increased skin pigmentation ¹⁰. These results demonstrated that exogenous α-MSH darkens human skin, but the mechanism by which melanocytes respond to α-MSH directly or indirectly was not determined. Evidence for the physiological significance of α-MSH and its direct effects on human melanocytes began to accumulate beginning in the mid-1980’s. It started with the discovery that POMC is synthesized and processed into its active metabolites in human skin, primarily by epidermal keratinocytes ^11,12 (Fig. 1). Expression of POMC was found to be up regulated in the epidermis upon exposure of skin to ultraviolet radiation (UV) ^13,14. This up regulation was reported to be mediated by transactivation of p53, which is activated by DNA damage ¹⁵ (Fig. 1), suggesting that increased POMC expression is part of the cutaneous DNA damage response. Human scalp hair follicle keratinocytes and melanocytes were also found to express POMC and its processing enzymes proconvertases 1 and 2 ¹⁶. This revoked the concept that melanocortins act exclusively as endocrine factors and suggested that they can function as paracrine regulators of human melanocytes.

Figure 1.

Anatomical sites for POMC synthesis and processing into its active melanocortin peptides. (A) POMC is synthesized and differentially processed in the pituitary gland into its bioactive peptides the melanocortins α-MSH, β-MSH and γ-MSH, or ACTH. (B, C) POMC is synthesized in the skin, primarily by epidermal keratinocytes, and its synthesis is upregulated upon exposure to UV, and in the hair follicles, and processed into α-MSH- and ACTH-related peptides, which act as paracrine factors for epidermal and follicular melanocytes, respectively.

The cloning of the gene of the α-MSH receptor, termed the melanocortin 1 receptor (MC1R), from human epidermal melanocytes, and the demonstration that human melanocytes express functional MC1R that can be activated by melanocortins, primarily α-MSH and ACTH, provided conclusive evidence for the direct role of these agonists in regulating human pigmentation ^17–21 (Fig. 2). Similar to epidermal melanocytes, follicular melanocytes express the MC1R and respond to α-MSH by increased pigmentation, which supports the role of the activated MC1R in regulating hair color, in addition to skin pigmentation ¹⁶. Since the cloning of human MC1R, about 600 research articles and over 100 review articles on different aspects of this gene and the function and significance of the MC1R receptor have been published.

Figure 2.

Keratinocyte-derived α-MSH and ACTH stimulate eumelanin synthesis by melanocytes by activating the human MC1R expressed on melanocytes. POMC is synthesized and processed into α-MSH and ACTH by keratinocytes. α-MSH and ACTH act as agonists for the human MC1R expressed on melanocytes, and increase eumelanin synthesis by, and the transfer of melanosomes from, melanocytes to keratinocytes. These effects require expression of functional MC1R and are absent or attenuated in melanocytes expressing loss-of-function MC1R.

The best evidence that In human skin cells, functional MC1R expression seems to be mainly restricted to melanocytes and is not expressed in keratinocytes or fibroblasts was provided by Roberts et al. ^22,23. Unpublished data from our laboratory support these findings, demonstrating that unlike cultured human melanocytes, primary cultures of human keratinocytes did not respond to treatment with α-MSH with increase in intracellular levels of the second messenger cAMP ^19,24. In these experiments, treatment with cholera toxin, which activates adenylate cyclase, resulted in a robust increase in cAMP levels in keratinocytes. Other studies reported on the expression of MC1R in keratinocytes, but the results were based on using HaCaT immortalized keratinocytes stably transfected with MC1R, rather than primary human keratinocyte cultures ^25,26. A third study demonstrated low expression of MC1R in keratinocytes using quantitative real-time PCR, but no functional assays were performed ²⁷. Others reported expression of MC1R in fibroblasts, however, the pharmacological properties of the identified receptor differed markedly from that expressed in human melanocytes in that it had a remarkably lower affinity for α-MSH ^19,28. It is important to note that the physiological relevance of the MC1R in keratinocytes, fibroblasts or any other cell type can best be supported by functional assays to prove activity of the receptor, in addition to evidence for its expression at the RNA or protein levels.

Currently, the MC1R, which codes for a G protein-coupled receptor, is considered a major determinant of the diversity of human pigmentation. Population studies from different countries and across continents led to the identification of many allelic variants of the MC1R, with a few, namely R151C, R160W and D294H, strongly associated with light skin and red hair color (Fig. 3) ^29–31. These epidemiological studies revealed that the human MC1R is highly polymorphic, with about 200 allelic variants expressed in different human populations, with some variants associated with specific pigmentary phenotypes ³². The consensus protein sequence of the receptor is predominantly expressed in African populations where dark skin pigmentation is thought to be needed for photoprotection from the equatorial sun ³³. The R163Q is very highly expressed in Eastern and Southeastern Asians ³³. The R163Q and V92M variants are considered “pseudo alleles” since they do not significantly alter the activity of the encoded MC1R ^34,35. The V60L variant is thought to be associated with blonde hair, and is considered a “mild” MC1R allele ³⁰. A reported true null MC1R individual was compound heterozygote for two rare insertions c.86_87insA and c.537_538insC ³⁶. The former insertion occurs in the N-terminal region of MC1R and causes a frameshift that results in a stop codon36 base pairs downstream, and the latter frameshift occurs in the fourth transmembrane region of MC1R and results in a premature stop codon 174 base pairs downstream.

Figure 3.

Schematic representation and amino acid sequence of the MC1R, a Gs protein-coupled receptor, and the localization of the well-characterized variants resulting from single amino acid substitution. In red are the so-called red-hair allelic variants, in orange is the mild allelic V60L, and in blue are the pseudoalleles V92M and R163Q.

Regulation of eumelanin synthesis by MC1R and α-MSH

Based on studies on mouse coat color, it became recognized that α-MSH stimulates eumelanin synthesis ⁵. Treatment of C57BL/6A^y mice with the potent α-MSH analog NDP-α-MSH resulted in the growth of dark hair follicles due to increased eumelanin synthesis ³⁷. It has been known for many decades that stimulation of eumelanin synthesis by α-MSH requires the expression of functional α-MSH receptor by melanocytes ⁶. In mice, the Extension (E) locus codes for the receptor for α-MSH. The loss-of-function recessive yellow mutation in Extension (e/e) results in yellow coat color due to absence of eumelanin and exclusive synthesis of pheomelanin ^6,38. Treatment of cultured human melanocytes with the potent full length α-MSH analog NDP-α-MSH stimulated eumelanin synthesis ³⁹. As in mice, the response of human melanocytes to α-MSH requires expression of functional MC1R. The three human MC1R variant alleles, R151C, R160W, and D294H, the so-called red-hair-color variants, result in loss of function of MC1R by impairing signaling of the receptor following α-MSH binding, thereby inhibiting eumelanin synthesis ^34,35 (Fig. 3, 4). In vitro expression of some MC1R variants in heterologous cells, including R151C and R160W, reduced the cell surface expression of the encoded MC1R, which could contribute to reduced melanogenesis in melanocytes in response to α-MSH ^40,41.

Figure 4.

Mechanism of action of the human MC1R physiological agonists α-MSH and ACTH and physiological antagonists ASIP and HBD3. Activation of the MC1R by α-MSH and ACTH increases eumelanin synthesis by transactivating MITF. These effects are absent in melanocytes expressing loss-of-function MC1R. Inhibition of MC1R and its signaling pathway by ASIP and HBD3 inhibit eumelanin synthesis.

Compared to other G protein-coupled receptors, the human MC1R is unique in that it has 2 physiological agonists, α-MSH and ACTH, as well as 2 known physiological antagonists, agouti signaling protein (ASIP) and human β-defensin 3 (HBD3) ^42,43 (Fig. 4). The agouti phenotype in mice is the outcome of the switch from eumelanin to pheomelanin synthesis within hair follicles in response to the temporal expression of ASIP, which acts as a competitive inhibitor of α-MSH binding to MC1R ^44,45. Similarly, in human melanocytes, ASIP inhibits the binding of α-MSH to MC1R, thereby blocking eumelanin synthesis ⁴². β-defensin 3 was first discovered as a regulator of pigmentation in dogs, as mutations in its gene resulted in black coat color ⁴⁶. In human melanocytes, HBD3 was shown to inhibit α-MSH-induced cAMP formation, thereby antagonizing the melanogenic effect of α-MSH ⁴³. The central role of MC1R in regulating human pigmentation is further substantiated by the findings that its expression is up regulated by its agonist α-MSH and by the paracrine factor endothelin-1. The latter is synthesized by epidermal keratinocytes and has potent effects on promoting the proliferation, survival, and nucleotide excision repair of human melanocytes ^47–50.

Signaling pathways of the activated MC1R

The human MC1R gene is located on chromosome 16p24.3 and codes for a G_s protein-coupled receptor with 7 transmembrane domains, consisting of 317 amino acids ^17,51. The mechanism of action of α-MSH was discovered using mouse melanoma cell lines, and shown to involve activation of the membrane bound adenylate cyclase, resulting in increased levels of the intracellular second messenger cyclic AMP (cAMP) ²⁴. This is also true for the human MC1R ¹⁹. However, unlike its mouse counterpart, the human MC1R recognizes ACTH, in addition to α-MSH, as agonist ^19,52 (Fig. 2,4). The MC1R expressed on cultured human melanocytes recognizes α-MSH and ACTH with the same affinity but has a lower affinity to β- or γ-MSH ¹⁹ (Fig. 4).

The cAMP pathway is the central and best recognized signaling pathway for the activated MC1R and for stimulating melanogenesis. The significance of this pathway is supported by reports demonstrating increased melanogenesis upon treatment of melanocytes with the adenylate cyclase activator forskolin, the phosphodiesterase inhibitor isobutyl methylxanthine, or with dibutyryl cAMP ^34,53,54. In vivo, treatment of recessive yellow (e/e) mice with dibutyryl cAMP or forskolin rescued the pigmentary phenotype and stimulated the synthesis of eumelanin, thereby restoring black coat color ^6,53. Conversely, inhibition of cAMP intracellular levels or downstream targets of cAMP reduces melanogenesis. The phosphodiesterase 4D3 (PDE4D3) was reported to be a direct target of MITF, thereby acting in a negative feedback loop to render melanocytes refractory to cAMP stimulation, e.g. by forskolin, thereby limiting the stimulation of melanogenesis ⁵⁵. Rolipram, the phosphodiesterase inhibitor of PDE4 prolonged forskolin-induced MITF expression, thereby sustained stimulation of melanogenesis. Salt-inducible kinases (SIK), which inhibit the cAMP-dependent CREB-regulated transcriptional co-activator (CRTC) family of proteins, result in inhibition of CREB activation, upstream of MITF in melanocytes, which is expected to inhibit melanogenesis ⁵⁶. Based on the effects of SIK, small molecule SIK inhibitors were developed and shown to stimulate melanogenesis by upregulating MITF expression in melanocytes in vitro, increasing pigmentation of human skin ex vivo, and stimulating eumelanin synthesis in the skin of e/e mice in vivo ⁵⁷. Targeting SIK has been proposed as a strategy for modulating skin pigmentation, with their activation causing hypopigmentation, and their inhibition resulting in increased pigmentation without UV exposure ⁵⁸. The above strategies (inhibition of phosphodiesterase and targeting SIK, downstream of MC1R) underscore the significance of the cAMP pathway as a central regulator of pigmentation. However, given that phosphodiesterases and SIK are expressed on multiple cell types raises concerns about the effects of these strategies on cellular targets other than melanocytes.

In addition to the cAMP pathway, activation of MC1R was reported to activate ERK1/2 ⁵⁹. It should be noted that although pERK1/2 was observed in human melanocytes expressing wild-type or two loss-of-function variants of MC1R after brief treatment with 1 nM α-MSH, this increase in phosphorylation was markedly less than that induced by 1 nM endothelin 1, which binds the endothelin B G_q-bound receptor and activates signaling pathways that include protein kinase C and intracellular calcium mobilization ⁵⁹. In this same study, activation of ERK1/2 was detected using a human melanoma cell line expressing wild-type MC1R after treatment with the potent α-MSH analog NDP-α-MSH at the high concentration of 0.1 μM. The requirement for such a high concentration of NDP-α-MSH suggests that MC1R signaling does not potently activate ERK1/2. The biological significance of the observed phosphorylation of ERK1/2 by MC1R agonists in human melanocytes deserves to be further investigated.

Additionally, α-MSH was shown to activate the IP3K/Akt pathway in B16-F10 melanoma cells and human melanocytes ⁶⁰. In cultured human melanocytes, we found that α-MSH, as well as endothelin-1, activate Akt, thereby promoting survival by inhibiting UV-induced apoptosis ⁴⁸. Whether or not the effect of α-MSH on Akt is mediated by cAMP or another putative signaling pathway downstream of MC1R requires investigation. One study showed that activation of MC1R reduced oxidative DNA damage by a cAMP pathway downstream of wild-type MC1R expressed in melanoma cells, and by activation of Akt in melanoma cells expressing various MC1R variants ⁶¹. Paradoxically, stimulation of cAMP production in wild-type melanocytic cells blocked Akt activation ⁶¹. Given the complexity, and at times the inconsistency of the published results, and in order to define with certainty the signaling pathway(s) downstream of human MC1R and their physiological significance for human pigmentation, further studies are warranted comparing human melanocytes that constitutively express wild type versus various MC1R variants.

Activation of the MAP kinase p38 by α-MSH was reported in mouse B16 melanoma cells and found to be accompanied by stimulation of melanogenesis and inhibition of proliferation ⁶². We published that human melanocytes respond to UV by marked phosphorylation of p38 within one hour, and that treatment with α-MSH immediately after UV exposure further augments this effect ⁶³. This effect of α-MSH is required for augmenting the UV-induced activation of p53, which proved to be pivotal for the antioxidant effect of α-MSH. We found that forskolin had a similar effect as α-MSH on UV-induced p38 phosphorylation (unpublished data), suggesting that it is mediated by activation of the cAMP pathway. The possible involvement of other MC1R-activated signaling pathway in mediating the phosphorylation of p38 remains to be determined.

Photoprotective role of eumelanin

Reduction of solar UV-induced DNA photoproducts

Stimulation of eumelanin synthesis by the α-MSH/MC1R axis is essential for photoprotection of human skin. Human melanocytes regardless of genotype or pigmentary phenotype synthesize both eumelanin and pheomelanin, with the ratio of eumelanin to pheomelanin correlating directly with visual skin pigmentation ^64–67. The extent of skin pigmentation correlates directly with eumelanin content in melanocytes ^65,66. Clinical studies have unequivocally shown that individuals with dark complexion have better photoprotection and a markedly lower risk for sun-induced skin cancers, including melanoma ^68–70. The penetration of solar ultraviolet rays (UV) through the layers of dark skin, which has high eumelanin content, was found to be remarkably less than through light skin, which is expected to reduce the extent of cutaneous DNA damage ⁷¹. Eumelanin is known to be more stable and more resistant to photodegradation than pheomelanin ^72,73. Melanosomes enriched with eumelanin in dark skin persist in the skin epidermal layers and form supranuclear caps in keratinocytes, thereby shielding cellular DNA from impinging UV rays and preventing UV-induced mutations ⁷⁴. In contrast, pheomelanosomes are photolabile, leaving only “melanin dust” in the epidermis upon degradation, which results in poor photoprotection of lightly pigmented skin ^73,75,76.

The photoprotective role of eumelanin was supported by an in vivo study on human subjects from different ethnicities and pigmentary phenotypes, which showed that in response to the same dose of UV, less DNA photoproducts were induced in dark skin than in light skin ⁷⁷. A study from our laboratory comparing the response of human melanocytes cultured from different skin phenotypes and expressing different total melanin and eumelanin and pheomelanin contents confirmed that induction of cyclobutane pyrimidine dimers (CPD), the major form of DNA photoproducts, in response to the same doses of UV correlated inversely with eumelanin content ⁷⁸. It is established that the ability to tan determines the risk for sun-induced skin cancers, with individuals who burn rather than tan upon sun exposure having a high risk ^79,80. Activation of the cAMP pathway, the main signaling pathway for MC1R, is critical for UV-induced increase in pigmentation (i.e., tanning response) ⁸¹. The tanning capacity is determined by constitutive pigmentation and is mainly regulated by the MC1R genotype and the activity of the encoded receptor ^34,76. Individuals with red hair and fair skin, a phenotype strongly associated with loss-of-function variants of MC1R, have a poor tanning ability, which reduces their photoprotection and increases their risk for photocarcinogenesis ^29,76.

Antioxidant effects of eumelanin versus the oxidative effects of pheomelanin

Eumelanin is also known to be an efficient scavenger of reactive oxygen species, unlike pheomelanin that is considered to be a pro-oxidant ^73,82–85. It is established that pheomelanin chromophore is excited to the singlet state and decays to the triplet state, thereby generating superoxide anion, and that it induces oxidative base lesions and DNA stand breaks through the generation of hydrogen peroxide, hydroxy radicals and possibly singlet oxygen (reviewed in ⁸⁶). Purified pheomelanin from red hair was found to directly accelerate the autoxidation of the antioxidants glutathione (GSH) and NADPH, which is expected to increase the vulnerability of cells to oxidative stress ⁸⁷. It was reported that generation of CPD in UV-irradiated melanin-containing cells (melanocytes and keratinocytes) persisted for hours after cessation of UV exposure ⁸⁵. The generation of the so-called “dark CPD” was mostly evident in cells enriched with pheomelanin and was attributed to increased generation of oxidative radicals ⁸⁵. An in vivo study on recessive yellow (e/e) mice that co-express the common somatic activating BRAF^V600E mutation in melanoma concluded that pheomelanin can be oncogenic, due to its induction of extensive oxidative damage, which led to spontaneous melanoma formation ⁸⁴.

We recently published that cultured human melanocytes from light or dark skin, expressing low or high ratio of eumelanin to pheomelanin, respectively, differ markedly in the response to the oxidative effect of solar UV ⁶⁶. Light melanocytes with a low eumelanin to pheomelanin ratio had significantly higher basal levels of reactive oxygen species (ROS) and exhibited greater induction of ROS upon irradiation with solar UV, as compared to their dark counterparts with high eumelanin to pheomelanin ratio. Moreover, light melanocytes had lower levels of the antioxidant enzymes heme-oxygenase 1, glutamate-cysteine ligase catalytic subunit (GCLC) and glutamate-cysteine ligase modifying subunit (GCLM) that regulate the synthesis of the intracellular antioxidant GSH and had lower basal levels of GSH. Lightly pigmented melanocytes also had higher solar UV-induced p53 levels than their dark counterparts, indicative of greater induction of DNA damage that further augments p53 accumulation, as we had previously reported ⁸⁸, and greater extent of lipid peroxidation. These findings further confirm that light melanocytes, as compared to dark melanocytes, are more susceptible to solar UV-induced oxidative stress due to increased levels of ROS, which can be attributed to lower eumelanin content, and to diminished expression of antioxidant enzymes that function for reducing oxidative stress and oxidative damage. Cutaneous melanoma is thought to be an oxidative stress-driven tumor ^89,90. The higher induction of oxidative stress and the lower antioxidant response of light, as compared to dark melanocytes, can have important implications on the risk for cutaneous melanoma in individuals with light pigmentary phenotype, which is determined to a large extent by MC1R genotype.

Augmentation of DNA repair capacity in melanocytes by the activated MC1R

In addition to the classical role of α-MSH on cutaneous pigmentation and the demonstration of its melanogenic effect on human melanocytes, a seminal discovery from our laboratory and that of Markus Bohm was the participation of α-MSH in reducing the extent of UV-induced DNA damage in human melanocytes ^34,48,91 (Fig. 5). Both research groups reported that α-MSH reduces the apoptotic effect of UV on cultured human melanocytes by enhancing the repair of DNA photoproducts. We demonstrated that α-MSH treatment activates the Akt survival pathway in UV-irradiated human melanocytes, and increases the levels of the antiapoptotic protein Bcl2, a known target of the transcription factor Mitf, which in turn is a downstream target of the activated MC1R ^48,92. These effects confer resistance of melanocytes to UV-induced apoptosis and maintain their long-term survival. We attribute the survival effects of α-MSH to the reduction of the burden of UV-induced DNA damage. We further demonstrated that treatment of human melanocytes with α-MSH activates the global genome sub-pathway of nucleotide excision repair (GGR) following UV exposure ^50,93. We reported that in UV-irradiated human melanocytes, α-MSH activates the DNA damage sensors Ataxia Telangiectasia and Rad3-Related (ATR) and Ataxia Telangiectasia-Mutated (ATM) and enhances the translocation of the DNA damage recognition protein XPC and of the DNA damage verification protein XPA from the cytoplasm to chromatin, the site of DNA photoproducts. Activation of this pathway results in reduction of CPD, due to enhancement of their repair. The effects of α-MSH on GGR and survival of melanocytes require activation of MC1R and are compromised in melanocytes expressing two loss-of-function MC1R variants ^34,50,93,94.

Figure 5.

Activation of the MC1R and its signaling pathway by α-MSH or ACTH augment the DNA damage response and survival in melanocytes. Activation of MC1R transactivates the transcription factors p53, Nrf2 and MITF, resulting in increased survival, which is attributed to enhanced DNA repair and antioxidant defenses, thereby conferring photoprotection and inhibiting oxidative stress.

The antioxidant effects of α-MSH and the activated MC1R

Exposure of human melanocytes to UV results in oxidative stress, which can damage multiple cellular components, including DNA, lipids and proteins ^{63,66,95 84}. There is accumulating evidence that melanoma is an oxidative stress-driven tumor ^89,90. Also, in vitiligo, extensive oxidative stress due to high concentrations of hydrogen peroxide in the skin is a causative factor for melanocyte death and skin depigmentation ⁹⁶. We reported that treatment of human melanocytes with α-MSH reduces the levels of hydrogen peroxide generated immediately after UV exposure, and the levels of 8-oxoguanine, the major form of oxidative DNA damage ⁹⁵. These effects are mediated by activation of MC1R, as they were blocked by its physiological antagonist ASIP ⁹⁵, and were absent in melanocytes expressing two loss-of-function MC1R variants ⁶³.

The antioxidant effects of α-MSH were found to be mediated by activation of the transcription factor nuclear factor erythroid 2–related factor 2 (Nrf2), the master regulator of phase II detoxification genes with ARE elements in their promoters ⁹⁷ (Fig. 5). Microarray data from our laboratory provided further support for the role of Nrf2 in the antioxidant effect of α-MSH, as treatment with α-MSH upregulated the expression of a number of known Nrf2 target genes, such as peroxiredoxin 1 and thioredoxin-like 1 ³⁴. The antioxidant effects of α-MSH on human melanocytes were also found to be mediated by the transcription factor p53, a major regulator of the DNA damage response, including DNA repair and antioxidant response ^63,98 (Fig. 5). Treatment of melanocytes with α-MSH augmented the UV-induced accumulation of p53 and phosphorylation of p53 on Ser15, and this was observed in melanocytes expressing functional MC1R, but not in melanocytes expressing loss-of-function MC1R. Silencing of p53 markedly inhibited the antioxidant effects of α-MSH, preventing the reduction in the burden of oxidative DNA damage, thereby maintaining a high oxidative stress state in melanocytes.

Development of α-MSH analogs as MC1R agonists

The first described pigmentary effect of α-MSH on human skin sparked interest in developing α-MSH analogs as “sunless tanning agents”. The collaborative team that spear-headed this endeavor was led by the Medicinal Chemist Victor Hruby and the late Comparative Endocrinologist Mac Hadley at the University of Arizona. They were the ones to develop potent α-MSH analogs, the best known of which is the full-length tridecapeptide [Nle⁴, D-Phe⁷]- α-MSH (NDP-α-MSH) ^99,100. This analog proved to be markedly more potent than the physiological α-MSH in stimulation mouse and human MC1R ^19,92,101 (Fig. 6). In a clinical trial, systemic administration of NDP-α-MSH to human subjects resulted in increased pigmentation in the absence of any sun exposure, thereby validating the efficacy of the peptide as a sunless tanning agent ¹⁰². Several clinical studies have supported the efficacy of NDP-α-MSH (also known as Melanotan I and afamelanotide) for the treatment of the rare photosensitivity disorder erythropoietic porphyria (EPP) and led to its approval by the European Commission in 2014, and by the FDA in late 2019 ^103,104. Other multicenter clinical trials showed that systemic administration of afamelanotide in combination with narrow band UVB was efficacious in inducing rapid and persistent repigmentation of vitiligo skin ^105,106. However, NDP-α-MSH is not selective for the MC1R, and therefore has off-target effects due to its binding and activation of other melanocortin receptors expressed in other skin and extracutaneous cells ^92,102. Being a full-length analog (13 amino acids in length), NDP-α-MSH can only be administered systemically, resulting in extra-cutaneous side effects such as loss of appetite and nausea due to binding and activation of other melanocortin receptors, such as the neuronal MC3 and MC4R.

Figure 6.

Comparison of the potency and prolonged effect of one of our tetrapeptide MC1R agonists and NDP-α-MSH. (A) comparison of the dose-dependent effects of the tetrapeptide 1 (LK 184) analog of α-MSH and the full-length analog NDP-α-MSH on stimulation of tyrosinase activity, indicative of increased melanogenesis, after 6 days of treatment of primary cultures of human melanocytes. * =statistically different from NDP-α-MSH at p<0.5. (B) Comparison of the residual effect of the tetrapeptide 1, NDP-α-MSH and α-MSH, after 4 days of treatment. *= statistically different from NDP-α-MSH and α-MSH at p<0.5. The results in A and B were reproduced in 5 and 3 different melanocyte cultures, respectively.

The safety of using melanocortin agonists as tanning agents or clinically, especially for long term use, is a concern that is being seriously addressed. There have been a number of case reports on darkening and eruption of nevi upon systemic self-administration of synthetic α-MSH analogs, e.g., Melanotan I, which raised concerns about the potential for melanoma development. However, in all cases, the analogs were purchased from unreliable sources online, with no information about their purity or dosage ^107–109. Importantly, their effects on nevi were reversed shortly after cessation of treatment. Data from multicenter clinical trials with afamelanotide used by EPP patients for 8 years point to the safety of using MC1R agonists, with minor and well-tolerated side effects, including nausea, headache and fatigue, and no reported incidence of melanoma ^103,110,111. There are reports about the possible association of over expression of MC1R with colon adenocarcinoma and breast cancer ^112–114. The latter reported that high levels of MC1R are associated with breast cancer progression and worse patient outcome ¹¹⁴. However, the drawback of the in vitro data in this study is that they were obtained after treatment of breast cancer cell lines with a very high dose of 0.2 μM NDP-α- MSH, an analog that is at least 10-fold more potent than α-MSH ¹⁹. Further studies are needed to determine the safety of melanocortins in a variety of clinical settings.

Our discoveries of the pleiotropic effects of α-MSH, namely enhanced nucleotide excision repair, antioxidant effects, and increased survival of human melanocytes, in addition to stimulation of melanogenesis, led us to develop small peptide analogs consisting only of the 6–9 (His-Phe-Arg-Trp) or the 6–8 (His-Phe-Arg) amino acid sequence of α-MSH, with various N-capping groups, which are highly selective for MC1R ^{48,50,63,92,93,95}. Previous structure-activity studies revealed that the 6–9 amino acid residues of α-MSH are required for its pigmentary effects ^115,116. Given the unprecedented small size (tetra- and tripeptides) of our MC1R agonists should allow for their skin permeation, and our goal is to develop them as topical agents, for practical application and prevention of extracutaneous systemic effects.

Two of our tetrapeptides with high selectivity for MC1R are equipotent, and 10-fold more potent than α-MSH in activating the MC1R and stimulating melanogenesis in primary cultures of human melanocytes ^19,92. When comparing one of these tetrapeptides to the full-length tridecapeptide analog NDP-α-MSH, the former proved to be more potent in stimulating tyrosinase activity of normal human melanocytes, having a significantly greater effect at 1 pm concentration than NDP- α-MSH (Fig. 6A). Our tetrapeptide also had a greater and more prolonged residual effect than NDP-α-MSH (Fig. 6B). Three of our tripeptides with high MC1R selectivity have equal potency and are only 10-fold less potent than α-MSH, despite their unprecedented small size. Remarkably, one representative tripeptide had a more prolonged melanogenic effect than either α-MSH or NDP-α-MSH. All five peptides mimicked the effects of α-MSH on repairing of CPD and enhancing survival of human melanocytes irradiated with UV. When one representative tetrapeptide and one representative tripeptide were tested in 3D-engineered human skin substitute, they resulted in significant and obvious increase in pigmentation, further validating their melanogenic effects on skin.

These unique MC1R agonists represent a platform technology, including sunless tanning, cutaneous melanoma prevention and treatment of vitiligo and photosensitivity disorders. A main etiological factor for cutaneous melanoma is exposure to solar UV ^79,117. These analogs are expected to prevent melanomagenesis and maintain genomic stability of melanocytes, since similar to α-MSH, they activate multiple innate photoprotective mechanisms, namely DNA repair and melanogenesis, which reduce the burden of solar UV-induced DNA damage. Vitiligo, the most common depigmentary disease, is characterized by death of melanocytes, caused by high levels of oxidative stress in vitiligo skin, loss of adhesion of, and autoimmune attack on, melanocytes ¹¹⁸. Given that α-MSH promotes the survival, increases the adhesion and activates antioxidant defenses of melanocytes ^{34,48,63,95,119}, our analogs are expected to be efficacious in repigmenting vitiligo skin and inhibiting the progression of the disease. The potent full length α-MSH analog NDP-α-MSH has been approved by the FDA and European Commission for the treatment of the rare severe photosensitivity disorder EPP. We expect our analogs to be highly effective in treating EPP, without the off-target side effects of the non-selective NDP-α-MSH. The efficacy of our small MC1R selective analogs as “sunless tanning agents” will fulfill the desire of millions for a cosmetically desirable and safe tan and will be a safe alternative to sunbathing and tanning beds. The acquired natural tan without the damaging effects of the sun or tanning beds should reduce the risk of skin cancers, including melanoma.

Final Remarks

α-Melanocyte stimulating hormone was originally named after the discovery of its first and most obvious effect on integumental pigmentation. Many additional physiological functions have been discovered for α-MSH, including immunosuppression, regulation of food intake, and enhancement of sexual potency ^120,121. These effects are elicited mostly by binding melanocortin receptors, other than MC1R (MC3, 4, and 5), and have stirred interest in the development of α-MSH analogs as drugs for specifically targeting these receptors. The challenge that still exists is the lack of selectivity of these analogs to their putative receptors, which invariably results in off-target effects on MC1R, evident as skin darkening. In our laboratory, we were among the first to report on the various effects of α-MSH via activation of MC1R, on the DNA damage response of human melanocytes, which enable melanocytes to reduce the extent of solar UV-induced DNA photoproducts and oxidative damage and to survive with genomic stability. From a drug development standpoint, our success in developing analogs of α-MSH with unprecedented small size and high potency, with high selectivity for MC1R, makes these analogs superior to previously developed α-MSH analogs ^92,122–124. Our goal to develop our small MC1R agonists as topical agents should mitigate the systemic effects and restrict their influence on melanocytes, the main cellular target in skin, thereby reducing potential toxicity. The seemingly restricted expression of MC1R in human skin to melanocytes and the selectivity of our unique tri- and tetrapeptide α-MSH analogs for MC1R should further contribute to minimizing off-target effects of our agonists. The journey through the landscape of melanocortin and MC1R promises to advance us from the long bench side to the translational path, which includes rigorous toxicological testing, in the hope that this will lead to multiple and safe clinical applications for the benefits of millions worldwide.

Significance:

The significance of the melanocortin 1 receptor and its physiological agonist α-melanocyte stimulating hormone for human pigmentation became evident in the 1990’s. Based on extensive epidemiological and experimental evidence, it is unequivocal that the human melanocortin 1 receptor is a central regulator of melanocyte homeostasis and constitutive pigmentation, and a bona fide melanoma susceptibility gene. Activation of the encoded receptor by α-melanocyte stimulating hormone stimulates eumelanin synthesis, activates DNA repair, antioxidant, and survival pathways in melanocytes. These pleotropic effects sparked the interest in developing analogs of this physiological agonist that can serve as sunless tanning agents, for photoprotection and re-pigmentation of vitiligo skin.