Zalfa A. Abdel-Malek

Cellular senescence in naevi and immortalisation in melanoma: a role for p16?

Cellular senescence, the irreversible proliferative arrest seen in somatic cells after a limited number of divisions, is considered a crucial barrier to cancer, but direct evidence for this in vivo was lacking until recently. The best-known form of human cell senescence is attributed to telomere shortening and a DNA-damage response through p53 and p21. There is also a more rapid form of senescence, dependent on the p16-retinoblastoma pathway. p16 (CDKN2A) is a known melanoma susceptibility gene. Here, we use retrovirally mediated gene transfer to confirm that the normal form of senescence in cultured human melanocytes involves p16, since disruption of the p16/retinoblastoma pathway is required as well as telomerase activation for immortalisation. Expression (immunostaining) patterns of senescence mediators and markers in melanocytic lesions provide strong evidence that cell senescence occurs in benign melanocytic naevi (moles) in vivo and does not involve p53 or p21 upregulation, although p16 is widely expressed. In comparison, dysplastic naevi and early (radial growth-phase, RGP) melanomas show less p16 and some p53 and p21 immunostaining. All RGP melanomas expressed p21, suggesting areas of p53-mediated senescence, while most areas of advanced (vertical growth-phase) melanomas lacked both p16 and p21, implying escape from both forms of senescence (immortalisation). Moreover, nuclear p16 but not p21 expression can be induced in human melanocytes by oncogenic BRAF, as found in around 80% of naevi. We conclude that cell senescence can form a barrier to melanoma development. This also provides a potential explanation of why p16 is a melanoma suppressor gene.

A-Melanocortin and Endothelin-1 Activate Antiapoptotic Pathways and Reduce DNA Damage in Human Melanocytes

UV radiation is an important etiologic factor for skin cancer, including melanoma. Constitutive pigmentation and the ability to tan are considered the main photoprotective mechanism against sun-induced carcinogenesis. Pigmentation in the skin is conferred by epidermal melanocytes that synthesize and transfer melanin to keratinocytes. Therefore, insuring the survival and genomic stability of epidermal melanocytes is critical for inhibiting photocarcinogenesis, particularly melanoma, the most deadly form of skin cancer. The paracrine factors alpha-melanocortin and endothelin-1 are critical for the melanogenic response of cultured human melanocytes to UV radiation. We report that alpha-melanocortin and endothelin-1 rescued human melanocytes from UV radiation-induced apoptosis and reduced DNA photoproducts and oxidative stress. The survival effects of alpha-melanocortin and endothelin-1 were mediated by activation of the melanocortin 1 and endothelin receptors, respectively. Treatment of melanocytes with alpha-melanocortin and/or endothelin-1 before exposure to UV radiation activated the inositol triphosphate kinase-Akt pathway and increased the phosphorylation and expression of the microphthalmia-related transcription factor. Treatment with alpha-melanocortin and/or endothelin-1 enhanced the repair of cyclobutane pyrimidine dimers and reduced the levels of hydrogen peroxide induced by UV radiation. These effects are expected to reduce genomic instability and mutagenesis.

Mesenchymal–epithelial interactions in the skin: increased expression of dickkopf1 by palmoplantar fibroblasts inhibits melanocyte growth and differentiation

We investigated whether or not the topographic regulation of melanocyte differentiation is determined by mesenchymal–epithelial interactions via fibroblast-derived factors. The melanocyte density in palmoplantar human skin (i.e., skin on the palms and the soles) is five times lower than that found in nonpalmoplantar sites. Palmoplantar fibroblasts significantly suppressed the growth and pigmentation of melanocytes compared with nonpalmoplantar fibroblasts. Using cDNA microarray analysis, fibroblasts derived from palmoplantar skin expressed high levels of dickkopf 1 (DKK1; an inhibitor of the canonical Wnt signaling pathway), whereas nonpalmoplantar fibroblasts expressed higher levels of DKK3. Transfection studies revealed that DKK1 decreased melanocyte function, probably through β-catenin–mediated regulation of microphthalmia-associated transcription factor activity, which in turn modulates the growth and differentiation of melanocytes. Thus, our results provide a basis to explain why skin on the palms and the soles is generally hypopigmented compared with other areas of the body, and might explain why melanocytes stop migrating in the palmoplantar area during human embryogenesis.

Melanocortin receptors: their functions and regulation by physiological agonists and antagonists.

Abstract: The melanocortins are a family of bioactive peptides derived from proopiomelanocortin, and share significant structural similarity. Those peptides are best known for their stimulatory effects on pigmentation and steroidogenesis. Melanocortins are synthesized in various sites in the central nervous system and in peripheral tissues, and participate in regulating multiple physiological functions. Research during the past decade has provided evidence that melanocortins elicit their diverse biological effects by binding to a distinct family of G protein-coupled receptors with seven transmembrane domains. To date, five melanocortin receptor genes have been cloned and characterized. Those receptors differ in their tissue distribution and in their ability to recognize the various melanocortins and the physiological antagonists, agouti signaling protein and agouti-related protein. These advances have opened new horizons for exploring the significance of melanocortins, their antagonists, and their receptors in a variety of important physiological functions.

What are melanocytes really doing all day long...

Abstract:  Everyone knows and seems to agree that melanocytes are there to generate melanin – an intriguing, but underestimated multipurpose molecule that is capable of doing far more than providing pigment and UV protection to skin ( 1 ). What about the cell that generates melanin, then? Is this dendritic, neural crest‐derived cell still serving useful (or even important) functions when no‐one looks at the pigmentation of our skin and its appendages and when there is essentially no UV exposure? In other words, what do epidermal and hair follicle melanocytes do in their spare time – at night, under your bedcover? How much of the full portfolio of physiological melanocyte functions in mammalian skin has really been elucidated already? Does the presence or absence of melanoctyes matter for normal epidermal and/or hair follicle functions (beyond pigmentation and UV protection), and for skin immune responses? Do melanocytes even deserve as much credit for UV protection as conventional wisdom attributes to them? In which interactions do these promiscuous cells engage with their immediate epithelial environment and who is controlling whom? What lessons might be distilled from looking at lower vertebrate melanophores and at extracutaneous melanocytes in the endeavour to reveal the ‘secret identity’ of melanocytes? The current Controversies feature explores these far too infrequently posed, biologically and clinically important questions. Complementing a companion viewpoint essay on malignant melanocytes ( 2 ), this critical re‐examination of melanocyte biology provides a cornucopia of old, but under‐appreciated concepts and novel ideas on the slowly emerging complexity of physiological melanocyte functions, and delineates important, thought‐provoking questions that remain to be definitively answered by future research.

Influence of α-melanocyte-stimulating hormone and ultraviolet radiation on the transfer of melanosomes to keratinocytes

The epidermal melanin unit in human skin is composed of melanocytes and keratinocytes. Melanocytes, located in the basal layer of the epidermis, manufacture melanin‐loaded organelles called melanosomes. Through their dendritic processes, melanocytes distribute melanosomes to neighboring keratinocytes, where their presence confers to the skin its characteristic color and photoprotective properties. In this study, we used murine melanocytes and keratinocytes alone and in coculture to characterize the processes involved in melanosome transfer. Ultraviolet (UV) radiation induced an accumulation of melanosomes in melanocytes, whereas treatment with α‐melanocyte‐stimulating hormone (MSH) induced exocytosis of melanosomes accompanied by ruffling of the melanocyte membrane. We found that keratinocytes phagocytose melanosomes and latex beads equally well and that this phagocytic process was increased by exposure of keratinocytes to UV radiation or to MSH. Coculture of melanocytes and keratinocytes resulted in an increase in MSH released to the medium. Gene array analysis of MSH‐treated melanocytes showed up‐regulation of many genes associated with exocytosis. In our studies, we never observed cytophagocytosis of melanosome‐filled processes. This result, together with the other findings, suggests that a combination of signals that increase melanosome production and release by melanocytes and that stimulate phagocytosis by keratinocytes are the most relevant mechanisms involved in skin tanning.

Significance of the melanocortin 1 receptor in regulating human melanocyte pigmentation, proliferation, and survival.

Abstract: The characterization of the melanocortin 1 receptor (MC1R) expressed on human melanocytes and the findings that certain mutations in the POMC gene or the MC1R gene result in red hair phenotype underscore the significance of melanocortins and MC1R in regulating human pigmentation. We demonstrated that human melanocytes respond to α‐melanocortin (α‐MSH) or ACTH with increased proliferation and melanogenesis, and to agouti signaling protein by abrogation of these effects. α‐MSH and ACTH were equipotent and more potent than β‐MSH, and γ‐MSH was the least potent in activating the MC1R and stimulating melanogenesis and proliferation of human melanocytes. We characterized the MC1R genotype in a panel of human melanocyte cultures and identified three cultures that were homozygous for Arg160Trp, heterozygous for Arg151Cys and Asp294His, and heterozygous for Arg160Trp and Asp294His substitutions, respectively. Those cultures failed to respond to α‐MSH with increase in cAMP levels, tyrosinase activity, or proliferation and had an exaggerated response to the cytotoxic effect of ultraviolet (UV) radiation. These loss‐of‐function mutations have been associated with red hair phenotype and increased risk for skin cancer. Melanocytes homozygous for Val29Met substitution in MC1R responded normally to α‐MSH and UVB, suggesting that this variant is a polymorphism. We observed that α‐MSH promotes human melanocyte survival by inhibiting the UV‐induced apoptosis independently of melanin synthesis. This effect was absent in human melanocytes with loss of function MC1R mutations. We predict that the survival effect of α‐MSH is caused by reduction of UV‐induced DNA damage and contributes to the prevention of melanoma.

α‐MSH activates immediate defense responses to UV‐induced oxidative stress in human melanocytes

Exposure of cultured human melanocytes to ultraviolet radiation (UV) results in DNA damage. In melanoma, UV‐signature mutations resulting from unrepaired photoproducts are rare, suggesting the possible involvement of oxidative DNA damage in melanocyte malignant transformation. Here we present data demonstrating immediate dose‐dependent generation of hydrogen peroxide in UV‐irradiated melanocytes, which correlated directly with a decrease in catalase activity. Pretreatment of melanocytes with α‐melanocortin (α‐MSH) reduced the UV‐induced generation of 7,8‐dihydro‐8‐oxyguanine (8‐oxodG), a major form of oxidative DNA damage. Pretreatment with α‐MSH also increased the protein levels of catalase and ferritin. The effect of α‐MSH on 8‐oxodG induction was mediated by activation of the melanocortin 1 receptor (MC1R), as it was absent in melanocytes expressing loss‐of‐function MC1R, and blocked by concomitant treatment with an analog of agouti signaling protein (ASIP), ASIP‐YY. This study provides unequivocal evidence for induction of oxidative DNA damage by UV in human melanocytes and reduction of this damage by α‐MSH. Our data unravel some mechanisms by which α‐MSH protects melanocytes from oxidative DNA damage, which partially explain the strong association of loss‐of‐function MC1R with melanoma.

Modulation of murine melanocyte function in vitro by agouti signal protein

Molecular and biochemical mechanisms that switch melanocytes between the production of eumelanin or pheomelanin involve the opposing action of two intercellular signaling molecules, α‐melanocyte‐stimulating hormone (MSH) and agouti signal protein (ASP). In this study, we have characterized the physiological effects of ASP on eumelanogenic melanocytes in culture. Following exposure of black melan‐a murine melanocytes to purified recombinant ASP in vitro, pigmentation was markedly inhibited and the production of eumelanosomes was decreased significantly. Melanosomes that were produced became pheomelanosome‐like in structure, and chemical analysis showed that eumelanin production was significantly decreased. Melanocytes treated with ASP also exhibited time‐ and dose‐dependent decreases in melanogenic gene expression, including those encoding tyrosinase and tyrosinase‐related proteins 1 and 2. Conversely, melanocytes exposed to MSH exhibited an increase in tyrosinase gene expression and function. Simultaneous addition of ASP and MSH at approximately equimolar concentrations produced responses similar to those elicited by the hormone alone. These results demonstrate that eumelanogenic melanocytes can be induced in culture by ASP to exhibit features characteristic of pheomelanogenesis in vivo. Our data are consistent with the hypothesis that the effects of ASP on melanocytes are not mediated solely by inhibition of MSH binding to its receptor, and provide a cell culture model to identify novel factors whose presence is required for pheomelanogenesis.

Melanocytes as instigators and victims of oxidative stress

Epidermal melanocytes are particularly vulnerable to oxidative stress due to the pro-oxidant state generated during melanin synthesis, and to intrinsic antioxidant defences that are compromised in pathologic conditions. Melanoma is thought to be oxidative stress-driven, and melanocyte death in vitiligo is thought to be instigated by a highly pro-oxidant state in the epidermis. We review the current knowledge about melanin and the redox state of melanocytes, how paracrine factors help counteract oxidative stress, the role of oxidative stress in melanoma initiation and progression and in melanocyte death in vitiligo, and how this knowledge can be harnessed for melanoma and vitiligo treatment.