Analysis of the UV-Induced Melanogenesis and Growth Arrest of Human Melanocytes

Cultured human melanocytes derived from different skin types responded to frequent treatment with ultraviolet (UV) light with increased melanin synthesis, decreased proliferation, and morphologic signs of aging. These effects were augmented by increased frequency of irradiation with 15.5 mJ/cm² UV light. Stimulation of melanogenesis by UV light involved an increase in tyrosinase activity, without any change in the amounts of either tyrosinase or tyrosinase-related protein (TRP)-1, and a decrease in the amount of TRP-2, as determined by Western blot analysis. These results are different from the mechanisms by which other melanogenic agents, such as cholera toxin and isobutyl methylxanthine, stimulated melanogenesis, whereby the amounts of tyrosinase, TRP-1 and TRP-2 were increased. The decrease in the amount of TRP-2 might be significant in that it might alter the properties of the newly synthesized melanin. The UV irradiation protocol that was followed blocked melanocytes in G₂-M phase of the cell cycle without compromising cellular viability. Following three rounds of UV irradiation, melanocytes could recover from the growth arrest and resume proliferation. Treatment with 0.1 μM α-melanocyte stimulating hormone (α-MSH) postirradiation enhanced the melanogenic effect of UV light and stimulated the melanocytes to proliferate. The effects of α-MSH on the UV induced responses and their implications on photocarcinogenesis are being further investigated. Analyzing the mechanisms by which UV light exposure affects normal melanocytes might lead to a better understanding of how these cells undergo malignant transformation, and why individuals with different skin types differ in their susceptibility to skin cancers.     

Role of the Melanocyte in Epidermal Inflammatory and Immune Responses

The epidermis is composed of four major cell types in the mouse. In addition to keratinocytes and Langerhans cells, there are also melanocytes and Thyl⁺ lymphocytes. We propose that for the epidermis to maintain homeostasis, all four cell types must interact together in an integrated fashion. Vitiligo is a form of depigmentation that affects human subjects. Depigmentation is common in the animal kingdom. In at least several animal species, depigmentation is accompanied by loss of responsiveness of the epidermis to potent contact allergens like dinitrofluorobenzene. The C57Bl/Ler vit/vit mouse spontaneously depigments and is phenotypically similar to humans with vitiligo. We have studied this species extensively and have found that it has an isolated immune defect to contact allergens. In this report, we document that there is loss of epidermal melanocytes. Although there is a loss of epidermal contact sensitivity in this animal, other immune parameters such as dermal delayed-type hypersensitivity are normal. We attribute this loss of immune sensitivity in the epidermis to be associated with loss of the melanocyte. We report and review in detail the many peptides and other types of inflammatory mediators that affect both melanocyte function as well as immune responsiveness.     

The Interaction of Agouti Signal Protein and Melanocyte Stimulating Hormone to Regulate Melanin Formation in Mammals

Important regulatory controls of melanogenesis that operate at the subcellular level to modulate the structural and/or the functional nature of the melanins and melanin granules produced in melanocytes are reviewed. Melanocyte stimulating hormone and agouti signal protein have antagonistic roles and possibly opposing mechanisms of action in the melanocyte. In the mouse, melanocyte stimulating hormone promotes melanogenic enzyme function and elicits increases in the amount of eumelanins produced, while agouti signal protein reduces total melanin production and elicits the synthesis of pheomelanin rather than eumelanin. We are now beginning to understand the complex controls involved in regulating this switch at the molecular and biochemical levels. The quality and quantity of melanins produced by melanocytes have important physiological consequences for melanocyte function and undoubtedly play important roles in the various functions of the melanins per se, including hair and skin coloration and photoprotection.     

Proliferation and Propagation of Human Melanocytes in Vitro Are Affected by Donor Age and Anatomical Site

We report the effects of two factors, donor age and anatomical site, on the proliferation and melanization of human melanocytes (MC) derived from (1) neonatal foreskins, (2) adult foreskins, or (3) adult breast or arm skin. Two different growth media have been used for this purpose. Medium I supports the long-term proliferation of neonatal MC, and medium II supports the growth of both neonatal and adult MC. We found that neonatal foreskin MC proliferated equally well in both media for more than 12 passages. However, adult MC behaved differently in the two growth media. In very early passages (passages 1-5), all three types of MC proliferated well and reached comparable final cell densities in medium I, but adult foreskin MC proliferated at a higher rate than neonatal or adult non-foreskin MC in medium II. The non-foreskin adult MC had the least proliferative rate in medium II. Unlike neonatal MC, both adult foreskin and non-foreskin MC underwent a drastic reduction in their proliferative rate after only a few (9-10) passages in culture. While the three types of MC differed in their proliferative rates, they responded similarly to melanogenic stimulation by cholera toxin (CT) and isobutyl methylxanthine (IBMX). These results demonstrate that the proliferation of human MC in standard culture media is affected by the donor age and the anatomical site from which these cells are derived. We conclude that human MC are heterogeneous, and caution must be used in extrapolating the results of studies on MC derived from different anatomical sites or age groups.