Differential Induction of Reactive Oxygen Species and Expression of Antioxidant Enzymes in Human Melanocytes Correlate with Melanin Content: Implications on the Response to Solar UV and Melanoma Susceptibility.

Constitutive pigmentation determines the response to sun exposure and the risk for melanoma, an oxidative stress-driven tumor. Using primary cultures of human melanocytes, we compared the effects of constitutive pigmentation on their antioxidant response to solar UV. The quantitation of eumelanin and pheomelanin showed that the eumelanin content and eumelanin to pheomelanin ratio correlated inversely with the basal levels of reactive oxygen species (ROS). Irradiation with 7 J/cm2 solar UV increased ROS generation without compromising melanocyte viability. Among the antioxidant enzymes tested, the basal levels of heme oxygenase-1 (HO-1) and the glutamate cysteine ligase catalytic subunit and modifier subunit (GCLC and GCLM) correlated directly with the eumelanin and total melanin contents. The levels of HO-1 and GCLM decreased at 6 h but increased at 24 h post-solar UV. Consistent with the GCLC and GCLM levels, the basal glutathione (GSH) content was significantly lower in light than in dark melanocytes. The expression of HMOX1, GCLC, GCLM, and CAT did not correlate with the melanin content and was reduced 3 h after solar UV irradiation, particularly in lightly pigmented melanocytes. Solar UV increased p53 and lipid peroxidation, which correlated inversely with the eumelanin and total melanin contents. These intrinsic differences between light and dark melanocytes should determine their antioxidant response and melanoma risk.

Development of hMC1R Selective Small Agonists for Sunless Tanning and Prevention of Genotoxicity of UV in Melanocytes.

Activation of the human melanocortin 1 receptor (hMC1R) expressed on melanocytes by α-melanocortin plays a central role in regulating human pigmentation and reducing the genotoxicity of UV by activating DNA repair and antioxidant defenses. For the development of a hMC1R-targeted photoprotection strategy, we designed tetra- and tripeptide agonists with modifications that provide the necessary lipophilicity and hMC1R selectivity to be effective drugs. These peptides proved to be superior to most of the existing analogs of the physiological tridecapeptide α-melanocortin because of their small size and high hMC1R selectivity. Testing on primary cultures of human melanocytes showed that these peptides are highly potent with prolonged stimulation of melanogenesis, enhanced repair of UV-induced DNA photoproducts, and reduced apoptosis. One of the tripeptides, designated as LK-514 (5), with a molecular weight of 660 Da, has unprecedented (>100,000) hMC1R selectivity when compared with the other melanocortin receptors hMC3R, hMC4R, and hMC5R, and increases pigmentation (sunless tanning) in a cultured, three-dimensional skin model. These new analogs should be efficacious in preventing skin cancer, including melanoma, and treatment of skin disorders, such as vitiligo and polymorphic light eruptions.

Endothelin-1 and α-melanocortin have redundant effects on global genome repair in UV-irradiated human melanocytes despite distinct signaling pathways.

Human melanocyte homeostasis is sustained by paracrine factors that reduce the genotoxic effects of ultraviolet radiation (UV), the major etiological factor for melanoma. The keratinocyte-derived endothelin-1 (End-1) and α-melanocyte-stimulating hormone (α-MSH) regulate human melanocyte function, proliferation and survival, and enhance repair of UV-induced DNA photoproducts by binding to the Gq - and Gi -protein-coupled endothelin B receptor (EDNRB), and the Gs -protein-coupled melanocortin 1 receptor (MC1R), respectively. We hereby report that End-1 and α-MSH regulate common effectors of the DNA damage response to UV, despite distinct signaling pathways. Both factors activate the two DNA damage sensors ataxia telangiectasia and Rad3-related and ataxia telangiectasia mutated, enhance DNA damage recognition by reducing soluble nuclear and chromatin-bound DNA damage binding protein 2, and increase total and chromatin-bound xeroderma pigmentosum (XP) C. Additionally, α-MSH and End-1 increase total levels and chromatin localization of the damage verification protein XPA, and the levels of γH2AX, which facilitates recruitment of DNA repair proteins to DNA lesions. Activation of EDNRB compensates for MC1R loss of function, thereby reducing the risk of malignant transformation of these vulnerable melanocytes. Therefore, MC1R and EDNRB signaling pathways represent redundant mechanisms that inhibit the genotoxic effects of UV and melanomagenesis.

In vitro behavior and UV response of melanocytes derived from carriers of CDKN2A mutations and MC1R variants.

Coinheritance of germline mutation in cyclin-dependent kinase inhibitor 2A (CDKN2A) and loss-of-function (LOF) melanocortin 1 receptor (MC1R) variants is clinically associated with exaggerated risk for melanoma. To understand the combined impact of these mutations, we established and tested primary human melanocyte cultures from different CDKN2A mutation carriers, expressing either wild-type MC1R or MC1RLOF variant(s). These cultures expressed the CDKN2A product p16 (INK4A) and functional MC1R. Except for 32ins24 mutant melanocytes, the remaining cultures showed no detectable aberrations in proliferation or capacity for replicative senescence. Additionally, the latter cultures responded normally to ultraviolet radiation (UV) by cell cycle arrest, JNK, p38, and p53 activation, hydrogen peroxide generation, and repair of DNA photoproducts. We propose that malignant transformation of melanocytes expressing CDKN2A mutation and MC1RLOF allele(s) requires acquisition of somatic mutations facilitated by MC1R genotype or aberrant microenvironment due to CDKN2A mutation in keratinocytes and fibroblasts.

Endothelin-1 protects human melanocytes from UV-induced DNA damage by activating JNK and p38 signalling pathways.

Endothelin-1 is a paracrine factor with mitogenic, melanogenic and survival effects on cultured human melanocytes. We report that endothelin-1 signalling reduced the generation and enhanced the repair of ultraviolet radiation (UV)-induced DNA photoproducts, and inhibited apoptosis of human melanocytes, without increasing cAMP levels, melanin content or proliferation. Treatment with endothelin-1 activated the MAP kinases JNK and p38, as evidenced by phosphorylation of their target, activating transcription factor-2 (ATF-2). Endothelin-1 also enhanced the phosphorylation of JNK, p38 and ATF-2 by UV. The effects of endothelin-1 were dependent on increasing intracellular calcium mobilization by endothelin B receptor signalling. Activation of both JNK and p38 was required for reducing DNA photoproducts, but only JNK partially contributed to the survival effect of endothelin-1. ATF-2 activation depended mainly on JNK, yet was not sufficient for the effect of endothelin-1 on UV-induced DNA damage, suggesting the requirement for other JNK and p38 targets for this effect. Our results underscore the significance of endothelin-1 and endothelin B receptor signalling in reducing the genotoxic effects of UV via activating JNK and p38, hence restoring genomic stability of melanocytes.

Melanocortins and the melanocortin 1 receptor, moving translationally towards melanoma prevention.

Beginning in the last decade of the twentieth century, the fields of pigment cell research and melanoma have witnessed major breakthroughs in the understanding of the role of melanocortins in human pigmentation and the DNA damage response of human melanocytes to solar ultraviolet radiation (UV). This began with the cloning of the melanocortin 1 receptor (MC1R) gene from human melanocytes and the demonstration that the encoded receptor is functional. Subsequently, population studies found that the MC1R gene is highly polymorphic, and that some of its variants are associated with red hair phenotype, fair skin and poor tanning ability. Using human melanocytes cultured from donors with different MC1R genotypes revealed that the alleles associated with red hair color encode for a non-functional receptor. Epidemiological studies linked the MC1R red hair color variants to increased melanoma risk. Investigating the impact of different MC1R variants on the response of human melanocytes to UV led to the important discovery that the MC1R signaling activates antioxidant, DNA repair and survival pathways, in addition to stimulation of eumelanin synthesis. These effects of MC1R were absent in melanocytes expressing 2 MC1R red hair color variants that result in loss of function of the receptor. The importance of the MC1R in reducing UV-induced genotoxicity in melanocytes led us to design small peptide analogs of the physiological MC1R agonist α-melanocortin (α-melanocyte stimulating hormone; α-MSH) for the goal of utilizing them for melanoma chemoprevention.

PGE(2) is a UVR-inducible autocrine factor for human melanocytes that stimulates tyrosinase activation.

Prostaglandins activate signalling pathways involved in growth, differentiation and apoptosis. Prostaglandin E(2) (PGE(2)) is released by keratinocytes following ultraviolet irradiation (UVR) and stimulates the formation of dendrites in melanocytes. We show that multiple irradiations of human melanocytes with UVR-activated cPLA(2), the rate-limiting enzyme in eicosanoid synthesis and stimulated PGE(2) secretion. PGE(2) increased cAMP production, tyrosinase activity and proliferation in melanocytes. PGE(2) binds to four distinct G-protein coupled receptors (EP(1-4)). We show that PGE(2) stimulates EP(4) receptor signalling in melanocytes, resulting in cAMP production. Conversely, PGE(2) also stimulated the EP(3) receptor in melanocytes, resulting in lowered basal cAMP levels. These data suggest that relative levels or activity of these receptors controls effects of PGE(2) on cAMP in melanocytes. The data are the first to identify PGE(2) as an UVR-inducible autocrine factor for melanocytes. These data also show that PGE(2) activates EP(3) and EP(4) receptor signalling, resulting in opposing effects on cAMP production, a critical signalling pathway that regulates proliferation and melanogenesis in melanocytes.