Distinct cAMP Signaling Microdomains Differentially Regulate Melanosomal pH and Pigmentation.

cAMP signaling is a well-established regulator of melanin synthesis. Two distinct cAMP signaling pathways-the transmembrane adenylyl cyclase pathway, activated primarily by the MC1R, and the soluble adenylyl cyclase (sAC) pathway-affect melanin synthesis. The sAC pathway affects melanin synthesis by regulating melanosomal pH, and the MC1R pathway affects melanin synthesis by regulating gene expression and post-translational modifications. However, whether MC1R genotype affects melanosomal pH is poorly understood. We now report that loss of function MC1R does not affect melanosomal pH. Thus, sAC signaling appears to be the only cAMP signaling pathway that regulates melanosomal pH. We also addressed whether MC1R genotype affects sAC-dependent regulation of melanin synthesis. Although sAC loss of function in wild-type human melanocytes stimulates melanin synthesis, sAC loss of function has no effect on melanin synthesis in MC1R nonfunctional human and mouse melanocytes or skin and hair melanin in e/e mice. Interestingly, activation of transmembrane adenylyl cyclases, which increases epidermal eumelanin synthesis in e/e mice, leads to enhanced production of eumelanin in sAC-knockout mice relative to that in sAC wild-type mice. Thus, MC1R- and sAC-dependent cAMP signaling pathways define distinct mechanisms that regulate melanosomal pH and pigmentation.

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.

Participation of keratinocyte- and fibroblast-derived factors in melanocyte homeostasis, the response to UV, and pigmentary disorders.

Human epidermal melanocytes play a central role in sensing the environment and protecting the skin from the drastic effects of solar ultraviolet radiation and other environmental toxins or inflammatory agents. Melanocytes survive in the epidermis for decades, which subjects them to chronic environmental insults. Melanocytes have a poor self-renewal capacity; therefore, it is critical to ensure their survival with genomic stability. The function and survival of melanocytes is regulated by an elaborate network of paracrine factors synthesized mainly by epidermal keratinocytes and dermal fibroblasts. A symbiotic relationship exists between epidermal melanocytes and keratinocytes on the one hand, and between melanocytes and dermal fibroblasts on the other hand. Melanocytes protect epidermal keratinocytes and dermal fibroblasts from the damaging effects of solar radiation, and the latter cells synthesize biochemical mediators that maintain the homeostasis, and regulate the stress response of melanocytes. Disruption of the paracrine network results in pigmentary disorders, due to abnormal regulation of melanin synthesis, and compromise of melanocyte survival or genomic stability. This review provides an update of the current knowledge of keratinocyte- and fibroblast-derived paracrine factors and their contribution to melanocyte physiology, and how their abnormal production is involved in the pathogenesis of common pigmentary disorders.

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.

Measurement of Melanin Metabolism in Live Cells by [U-13C]-L-Tyrosine Fate Tracing Using Liquid Chromatography-Mass Spectrometry.

Melanin synthesis occurs within a specialized organelle called the melanosome. Traditional methods for measuring melanin levels rely on the detection of chemical degradation products of melanin by high-performance liquid chromatography. Although these methods are robust, they are unable to distinguish between melanin synthesis and degradation and are best suited to measure melanin changes over long periods of time. We developed a method that actively measures both eumelanin and pheomelanin synthesis by fate tracing [U-13C] L-tyrosine using liquid chromatography-mass spectrometry. Using this method, we confirmed the previous reports of the differences in melanin synthesis between melanocytes derived from individuals with different skin colors and MC1R genotype and uncovered new information regarding the differential de novo synthesis of eumelanin and pheomelanin, also called mixed melanogenesis. We also revealed that distinct mechanisms that alter melanosomal pH differentially induce new eumelanin and pheomelanin synthesis. Finally, we revealed that the synthesis of L-3,4-dihydroxyphenylalanine, an important metabolite of L-tyrosine, is differentially controlled by multiple factors. Because L-tyrosine fate tracing is compatible with untargeted liquid chromatography-mass spectrometry‒based metabolomics, this approach enables the broad measurement of cellular metabolism in combination with melanin metabolism, and we anticipate that this approach will shed new light on multiple mechanisms of melanogenesis.

The enigma and challenges of vitiligo pathophysiology and treatment.

Vitiligo is the most common acquired pigmentary disorder, which afflicts 0.5%-1% of the world population, and is characterized by depigmented skin patches resulting from melanocyte loss. Vitiligo has a complex etiology and varies in its manifestations, progression, and response to treatment. It presents as an autoimmune disease, evidenced by circulating melanocyte-specific antibodies, and association with other autoimmune diseases. However, autoimmunity may be secondary to the high oxidative stress in vitiligo skin and to intrinsic defects in melanocytes and their microenvironment, which contribute to aberrant stress response, neo-antigenicity, and susceptibility of melanocytes to immune attack and apoptosis. There is also a genetic predisposition to vitiligo, which sensitizes melanocytes to environmental agents, such as phenolic compounds. Currently, there are different treatment modalities for re-pigmenting vitiligo skin. However, when repigmentation is achieved, the major challenge is maintaining the pigmentation, which is lost in 40% of cases. In this review, we present an overview of the clinical aspects of vitiligo, its pathophysiology, the intrinsic defects in melanocytes and their microenvironment, and treatment strategies. Based on lessons from the biology of human melanocytes, we present our perspective of how repigmentation of vitiligo skin can be achieved and sustained.

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.

Fueling Melanocytes with ATP from Keratinocytes Accelerates Melanin Synthesis.

Melanocyte homoeostasis and their response to ultraviolet radiation (UVR) are mediated to a large extent by keratinocyte-derived factors, many of which have been well-characterized. Lee et al. describe novel effects of adenosine 5'-triphosphate (ATP), which is secreted by keratinocytes and can stimulate melanogenesis by melanocytes following UVA exposure. The investigators attribute the melanogenic effect of ATP to binding purinergic receptors type 2 X7 (P2X7), which are expressed on human melanocytes, leading to activation of the protein kinase C pathway. This report is the first to identify expression of specific purinergic receptors on human melanocytes, and it suggests ATP as a signaling molecule that stimulates pigmentation. Follow up on these results should clarify the physiological role of ATP in mediating the tanning response to solar UVR.

The potential role of antioxidants in mitigating skin hyperpigmentation resulting from ultraviolet and visible light-induced oxidative stress.

Oxidative stress is an integral element that influences a variety of biochemical reactions throughout the body and is known to play a notable role in melanogenesis. Exogenous triggers of oxidative stress, such as ultraviolet radiation (UVR) and visible light (VL), lead to pigment formation through somewhat different pathways, but both share a common endpoint-the potential to generate cosmetically undesirable hyperpigmentation. Though organic and inorganic sunscreens are available to protect against the UVR portion of the electromagnetic spectrum, coverage is lacking to protect against the VL spectrum. In this manuscript, we review the phases of tanning, pathways of melanogenesis triggered by UVR and VL, and the associated impact of oxidative stress. We also discuss the known intrinsic mechanisms and paracrine regulation of melanocytes that influence their response to UVR. Understanding these mechanisms and their role in UVR-induced hyperpigmentation should potentially lead to identification of useful targets that can be coupled with antioxidant therapy to alleviate this effect.

Chemoprevention agents for melanoma: A path forward into phase 3 clinical trials.

Recent progress in the treatment of advanced melanoma has led to unprecedented improvements in overall survival and, as these new melanoma treatments have been developed and deployed in the clinic, much has been learned about the natural history of the disease. Now is the time to apply that knowledge toward the design and clinical evaluation of new chemoprevention agents. Melanoma chemoprevention has the potential to reduce dramatically both the morbidity and the high costs associated with treating patients who have metastatic disease. In this work, scientific and clinical melanoma experts from the national Melanoma Prevention Working Group, composed of National Cancer Trials Network investigators, discuss research aimed at discovering and developing (or repurposing) drugs and natural products for the prevention of melanoma and propose an updated pipeline for translating the most promising agents into the clinic. The mechanism of action, preclinical data, epidemiological evidence, and results from available clinical trials are discussed for each class of compounds. Selected keratinocyte carcinoma chemoprevention studies also are considered, and a rationale for their inclusion is presented. These data are summarized in a table that lists the type and level of evidence available for each class of agents. Also included in the discussion is an assessment of additional research necessary and the likelihood that a given compound may be a suitable candidate for a phase 3 clinical trial within the next 5 years.

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.

Suppression of MAPK signaling in BRAF-activated PTEN-deficient melanoma by blocking ß-catenin signaling in cancer-associated fibroblasts.

Cancer-associated fibroblasts (CAFs) in the tumor microenvironment have been associated with formation of a dynamic and optimized niche for tumor cells to grow and evade cell death induced by therapeutic agents. We recently reported that ablation of ß-catenin expression in stromal fibroblasts and CAFs disrupted their biological activities in in vitro studies and in an in vivo B16F10 mouse melanoma model. Here, we show that the development of a BRAF-activated PTEN-deficient mouse melanoma was significantly suppressed in vivo after blocking ß-catenin signaling in CAFs. Further analysis revealed that expression of phospho-Erk1/2 and phospho-Akt was greatly reduced, effectively abrogating the activating effects and abnormal cell cycle progression induced by Braf and Pten mutations. In addition, the epithelial-mesenchymal transition (EMT)-like process was also suppressed in melanoma cells. Taken together, our data highlight an important crosstalk between CAFs and the RAF-MEK-ERK signaling cascade in BRAF-activated melanoma and may offer a new approach to abrogate host-dependent drug resistance in targeted therapy.

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.