The melanocortin-1 receptor (MC1R) is highly polymorphic in human populations, with individuals carrying the variant alleles R151C, R160W and D294H shown to be strongly associated with red hair, fair skin, inability to tan, and increased risk of developing skin cancers (RHC phenotype). Carriers of other low penetrant variant alleles such as V60L, V92M, and R163Q (designated ‘r’) show intermediate responses to UV when compared to wild type MC1R (WT) and the high penetrant RHC variants. The purpose of this study was to understand the involvement of MC1R in the mechanism of UV-sensitivity in the skin and also the protective role that the melanogenic enzyme Dopachrome Tautomerase (DCT) may play in it. Examination of MC1R polymorphism function in model systems may allow a molecular distinction between people at risk of melanoma development on sun exposed skin, and those who may develop melanoma through nevus transformation on covered skin areas. Furthermore, the assessment of DCT as a protective agent and possible therapeutic target in the UV radiation response of skin is a major goal of this research.
A series of neonatal foreskin samples have been collected and primary cells isolated from the epidermis to establish strains of various cell types. Keratinocytes were grown in selective media, and multiple cultures pooled before freezing. Melanocytes (MC) or melanoblasts (MB) of specific MC1R genotype were retained as individual strains by growth in selective medias. MB are immature MC lacking melanin pigment with reduced melanogenic protein levels. From these stocks, particular strains of defined MC1R variant genotype could be used for experimental purposes. Co-cultures of MB or MC with keratinocytes were grown to establish a more physiologically relevant system similar to that in the skin. Our studies have revealed that a potent MC1R agonist NDP-MSH and the cAMP activator drug forskolin increased expression of DCT and the dendricity of WT MC1R MC strains in co-culture with keratinocytes. MC homozygous for the R151C variant showed reduced dendricity increases and no changes in expression of DCT in response to NDP-MSH. The ‘r’ homozygotes V60L, V92M and the R/WT heterozygote MC1R MC R151C/WT, R160W/WT, and D294H/WT were examined in the same fashion. V60L and R151C/WT cells showed intermediate responses between RHC variant and WT MC1R MC. V92M MC showed responses that were similar or greater to that seen in WT MC1R cells. R160W/WT and D294H/WT exhibited a dominant negative effect of reduced responses over the WT allele similar to RHC homozygous variants. As the propagation of mature MC was time consuming, MB were co-cultured and compared with MC in the same conditions. Dendricity, melanogenic protein and mRNA responses were comparable between the two cell types.
To begin an assessment of the role DCT induction by MC1R ligand treatment plays in MC cell function, siRNA knockdown experiments using melanoma cells and MB cell monocultures were carried out. DCT knockdown in the melanoma cell lines A2058, MM96L, and WM266-4 reduced cell survival when assayed by MTT assay, which was further reduced after treatment with 25 mJ/cm2 UVB when assayed 3 or 24 hours later. Expression of p53 and pp53 increased after DCT siRNA treatment, UVB irradiation, or both. A similar pattern of survival and protein expression was observed in WT MC1R MB cell strains. siRNA knockdown of DCT appeared to negatively affect DNA damage responses via p53 in melanoma cell lines and primary WT MB cell strains in monoculture. When RHC MC1R variant MB strains were examined in the same manner, responses including reduction in DCT with subsequent increases in p53 and pp53 were not seen or were very weak. The effect of knockdown of DCT expression using lentivirus particles containing hairpin shRNA against human DCT in co-cultures of WT MC1R MB cells and keratinocytes showed the reduction in DCT protein levels was not as great as that in monocultured cells using siRNA against DCT. Changes in cell survival, levels of p53, p38 and other proteins were also not as great as that seen in the monoculture experiments. It is possible that keratinocytes conferred a protective function to the MB in co-culture, which allowed increased survival post-UVB, and reduced DNA damage, thus requiring less increase in expression of proteins such as p53 and p38. Assay of hydrogen peroxide found that this was increased in WT MB cells after DCT siRNA and UVB treatment in monoculture. Without functional DCT protein, the cells were more sensitive to oxidative as well as DNA damage.
The experimental studies presented here have left little doubt that DCT is involved in protection of skin from UV damage. These experiments have shown its contribution by the reduction in survival of melanogenic cells in monoculture after treatment with DCT siRNA and UVB radiation, the increases seen in pp53 and p53 occurring after knockdown of DCT by both siRNA in monocultures and co-cultures with keratinocytes. That these observations were not as great in RHC MC1R variant cells treated with DCT siRNA and UVB suggests that one of the mechanisms whereby MC1R variants result in increased sun sensitivity of the skin, in that the loss of the DCT response results in reduced increases in DNA damage response proteins, indirectly contributing to the accumulation of mutations that may eventually lead to melanoma.