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.

Light or Dark Pigmentation of Engineered Skin Substitutes Containing Melanocytes Protects Against Ultraviolet Light-Induced DNA Damage In Vivo.

Engineered skin substitutes (ESS) containing autologous fibroblasts and keratinocytes provide stable wound closure in patients with large, full-thickness burns, but are limited by hypopigmentation due to absence of added melanocytes. DNA damage caused by ultraviolet radiation (UV) increases risk for skin cancer development. In human skin, melanocytes provide pigmentation that protects skin from UV-induced DNA damage. This study investigated whether inclusion of human melanocytes (hM) affects the response of ESS to UV in vivo. Specifically, pigmentation and formation of cyclobutane pyrimidine dimers (CPDs), the most prevalent UV-induced DNA photoproduct, were analyzed. Three groups of ESS were prepared with fibroblasts and keratinocytes, ± melanocytes, and grafted orthotopically to immunodeficient mice: ESS without melanocytes (ESS-hM), ESS with light skin-derived (Caucasian) melanocytes (ESS+hM-L), and ESS with dark skin-derived (African-American) melanocytes (ESS+hM-D). Pigmentation of ESS+hM-L and ESS+hM-D increased significantly after grafting; pigmentation levels were significantly different among groups. Mean melanocyte densities in ESS+hM-L and ESS+hM-D were similar to each other and to densities in normal human skin. After 8 weeks in vivo, grafts were irradiated with 135 mJ/cm2 UV; non-UV-treated mice served as controls. UV modestly increased pigmentation in the ESS+hM groups. UV significantly increased CPD levels in ESS-hM, and levels in ESS-hM were significantly greater than in ESS+hM-L or ESS+hM-D. The results demonstrate that light or dark melanocytes in ESS decreased UV-induced DNA damage. Therefore, melanocytes in ESS play a photoprotective role. Protection against UV-induced DNA damage is expected to reduce skin cancer risk in patients grafted with ESS containing autologous melanocytes.

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.

MC1R: Front and Center in the Bright Side of Dark Eumelanin and DNA Repair.

Melanin, the pigment produced by specialized cells, melanocytes, is responsible for skin and hair color. Skin pigmentation is an important protective mechanism against the DNA damaging and mutagenic effects of solar ultraviolet radiation (UV). It is acknowledged that exposure to UV is the main etiological environmental factor for all forms of skin cancer, including melanoma. DNA repair capacity is another major factor that determines the risk for skin cancer. Human melanocytes synthesize eumelanin, the dark brown form of melanin, as well as pheomelanin, which is reddish-yellow in color. The relative rates of eumelanin and pheomelanin synthesis by melanocytes determine skin color and the sensitivity of skin to the drastic effects of solar UV. Understanding the complex regulation of melanocyte function and how it responds to solar UV has a huge impact on developing novel photoprotective strategies to prevent skin cancer, particularly melanoma, the most fatal form, which originates from melanocytes. This review provides an overview of the known differences in the photoprotective effects of eumelanin versus pheomelanin, how these two forms of melanin are regulated genetically and biochemically, and their impact on the DNA damaging effects of UV exposure. Additionally, this review briefly discusses the role of paracrine factors, focusing on α-melanocortin (α-melanocyte stimulating hormone; α-MSH), in regulating melanogenesis and the response of melanocytes to UV, and describes a chemoprevention strategy based on targeting the melanocortin 1 receptor (MC1R) by analogs of its physiological agonist α-MSH.

Restoration of cutaneous pigmentation by transplantation to mice of isogeneic human melanocytes in dermal-epidermal engineered skin substitutes.

Autologous engineered skin substitutes (ESS) containing melanocytes (hM) may restore pigmentation and photoprotection after grafting to full-thickness skin wounds. In this study, normal hM were isolated from discard skin, propagated with or without tyrosinase inhibitors, cryopreserved, recovered into culture, and added to ESS (ESS-P) before transplantation. ESS-P were incubated in either UCMC160/161 or UCDM1 medium, scored for hM densities, and grafted to mice. The results showed that sufficient hM can be propagated to expand donor tissue by 100-fold; incubation of hM in tyrosinase inhibitors reduced pigment levels but did not change hM recovery after cryopreservation; hM densities in ESS-P were greater after incubation in UCDM1 than UCMC160 medium; hM were localized to the dermal-epidermal junction of ESS-P; and UCDM1 medium promoted earlier pigment distribution and density. These results indicate that hM can be incorporated into ESS-P efficiently to restore cutaneous pigmentation and UV photoprotection after full-thickness skin loss conditions.

Significance of the Melanocortin 1 and Endothelin B Receptors in Melanocyte Homeostasis and Prevention of Sun-Induced Genotoxicity.

The membrane bound melanocortin 1 receptor (MC1R), and the endothelin B receptor (ENDBR) are two G-protein coupled receptors that play important roles in constitutive regulation of melanocytes and their response to ultraviolet radiation (UVR), the main etiological factor for melanoma. The human MC1R is a Gs protein-coupled receptor, which is activated by its agonists α-melanocyte stimulating hormone (α-melanocortin; α-MSH) and adrenocorticotropic hormone (ACTH). The ENDBR is a Gq coupled-receptor, which is activated by Endothelin (ET)-3 during embryonic development, and ET-1 postnatally. Pigmentation and the DNA repair capacity are two major factors that determine the risk for melanoma. Activation of the MC1R by its agonists stimulates the synthesis of eumelanin, the dark brown photoprotective pigment. In vitro studies showed that α-MSH and ET-1 interact synergistically in the presence of basic fibroblast growth factor to stimulate human melanocyte proliferation and melanogenesis, and to inhibit UVR-induced apoptosis. An important function of the MC1R is reduction of oxidative stress and activation of DNA repair pathways. The human MC1R is highly polymorphic, and MC1R variants, particularly those that cause loss of function of the expressed receptor, are associated with increased melanoma risk independently of pigmentation. These variants compromise the DNA repair and antioxidant capacities of human melanocytes. Recently, activation of ENDBR by ET-1 was reported to reduce the induction and enhance the repair of UVR-induced DNA photoproducts. We conclude that α-MSH and ET-1 and their cognate receptors MC1R and ENDBR reduce the risk for melanoma by maintaining genomic stability of melanocytes via modulating the DNA damage response to solar UVR. Elucidating the response of melanocytes to UVR should improve our understanding of the process of melanomagenesis, and lead to effective melanoma chemoprevention, as well as therapeutic strategies.

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.

Assessment of replication rates of human keratinocytes in engineered skin substitutes grafted to athymic mice.

Stable closure of skin wounds with engineered skin substitutes (ESS) requires indefinite mitotic capacity to generate the epidermis. To evaluate whether keratinocytes in ESS exhibit the stem cell phenotype of label retention, ESS (n = 6-9/group) were pulsed with 5-bromo-2'-deoxyuridine (BrdU) in vitro, and after grafting to athymic mice (n = 3-6/group). Pulse and immediate chase in vitro labeled virtually all basal keratinocytes at day 8, with label uptake decreasing until day 22. Label retention in serial chase decreased more rapidly from day 8 to day 22, with a reorganization of BrdU-positive cells into clusters. Similarly, serial chase of labeled basal keratinocytes in vivo decreased sharply from day 20 to day 48 after grafting. Label uptake was assessed by immediate chases of basal keratinocytes, and decreased gradually to day 126, while total labeled cells remained relatively unchanged. These results demonstrate differential rates of label uptake and retention in basal keratinocytes of ESS in vitro and in vivo, and a proliferative phenotype with potential for long-term replication in the absence of hair follicles. Regulation of a proliferative phenotype in keratinocytes of ESS may improve the biological homology of tissue-engineered skin to natural skin, and contribute to more rapid and stable wound healing.

Alpha-melanocyte-stimulating hormone suppresses oxidative stress through a p53-mediated signaling pathway in human melanocytes.

Epidermal melanocytes are skin cells specialized in melanin production. Activation of the melanocortin 1 receptor (MC1R) on melanocytes by α-melanocyte-stimulating hormone (α-MSH) induces synthesis of the brown/black pigment eumelanin that confers photoprotection from solar UV radiation (UVR). Contrary to keratinocytes, melanocytes are slow proliferating cells that persist in the skin for decades, in an environment with high levels of UVR-induced reactive oxygen species (ROS). We previously reported that in addition to its role in pigmentation, α-MSH also reduces oxidative stress and enhances the repair of DNA photoproducts in melanocytes, independent of melanin synthesis. Given the significance of ROS in carcinogenesis, here we investigated the mechanisms by which α-MSH exerts antioxidant effects in melanocytes. We show that activation of the MC1R by α-MSH contributes to phosphorylation of p53 on serine 15, a known requirement for stabilization and activation of p53, a major sensor of DNA damage. This effect is mediated by the cAMP/PKA pathway and by the activation of phosphoinositide 3-kinase (PI3K) ATR and DNA protein kinase (DNA-PK). α-MSH increases the levels of 8-oxoguanine DNA glycosylase (OGG1) and apurinic apyrimidinic endonuclease 1 (APE-1/Ref-1), enzymes essential for base excision repair. Nutlin-3, an HDM2 inhibitor, mimicked the effects of α-MSH resulting in reduced phosphorylation of H2AX (γ-H2AX), a marker of DNA damage. Conversely, the p53 inhibitor pifithrin-α or silencing of p53 abolished the effects of α-MSH and augmented oxidative stress. These results show that p53 is an important target of the downstream MC1R signaling that reduces oxidative stress and possibly malignant transformation of melanocytes.

Stepping up melanocytes to the challenge of UV exposure.

Exposure to solar ultraviolet radiation (UV) is the main etiological factor for skin cancer, including melanoma. Cutaneous pigmentation, particularly eumelanin, afforded by melanocytes is the main photoprotective mechanism, as it prevents UV-induced DNA damage in the epidermis. Therefore, maintaining genomic stability of melanocytes is crucial for prevention of melanoma, as well as keratinocyte-derived basal and squamous cell carcinoma. A critical independent factor for preventing melanoma is DNA repair capacity. The response of melanocytes to UV is mediated mainly by a network of paracrine factors that not only activate melanogenesis, but also DNA repair, anti-oxidant, and survival pathways that are pivotal for maintenance of genomic stability and prevention of malignant transformation or apoptosis. However, little is known about the stress response of melanocytes to UV and the regulation of DNA repair pathways in melanocytes. Unraveling these mechanisms might lead to strategies to prevent melanoma, as well as non-melanoma skin cancer.

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.