Molecular aspects of tanning.

Tanning is commonly understood as increased melanization of the epidermis observed in skin following UV exposure. It is further understood to represent a host response that protects against future UV-induced damage. Although best studied in humans, tanning can also be observed in other mammals and even in more primitive animals such as sharks. The molecular basis of tanning was poorly understood well into the 1990s, although UV irradiation of murine melanoma cells had been shown to increase the number of cell surface receptors to a-melanocyte–stimulating hormone (a-MSH) and to increase the melanogenic response to a-MSH via increased protein levels and activity of tyrosinase, the rate-limiting enzyme in melanin synthesis (Bolognia et al., 1989; Chakraborty et al., 1991). However, the initiating molecular events were unknown. Identification of the responsible molecules ultimately evolved from studies of the UV action spectrum for critical events in human skin, as well as from a philosophical appreciation of tanning as a genome protective response. In the 1980s, investigators at Harvard’s Wellman Laboratories exposed normal skin of volunteers to a wide range of light doses using narrow band sources across the UV spectrum and observed them for several days to determine the lowest dose at each wavelength capable of producing a delayed tan (Parrish et al., 1982). In other experiments, they used the same approach to define the action spectrum for induction of cyclobutane pyrimidine dimers (CPDs), the most common form of DNA damage following UV exposure (Freeman et al., 1989). The spectra were virtually superimposable, peaking at B300 nm, suggesting a cause-and-effect relationship between the immediate DNA damage and subsequent increased production and dispersion of epidermal melanin. Eller et al. (1996) first documented that agents acting exclusively on DNA, such as DNA restriction enzymes, stimulate melanin production in cultured pigment cells, at least in part by increasing a-MSH binding and the melanogenic response to a-MSH. Although this did not exclude a role for UV-mediated membrane effects, it did directly implicate UV-mediated DNA damage in the tanning response. Similarly, accelerating DNA damage repair by providing UV-irradiated pigment cells with T4 endonuclease V, the bacterial enzyme that catalyzes the first step in CPD resolution, also enhanced the melanization response (Gilchrest et al., 1993). By the 1990s, DNA damage responses were recognized to be largely mediated by the transcription factor and tumor suppressor protein p53, also termed the ‘‘guardian of the genome’’ (Lane, 1992), and two groups independently asked whether p53 was involved in mediating the tanning response. Using p53 null osteosarcoma cells transfected with wild-type p53, Nylander et al. (2000) showed that p53 activation increased read-out from a transfected tyrosinase promoter linked to a reporter gene, implying that p53 could directly or indirectly stimulate tyrosinase transcription. This link between p53 and tanning was expanded and refined using both human melanoma cells and a mouse model. Compared with a p53-null parental melanoma line and to the same line transfected with an empty vector, melanoma cells transfected with wildtype p53 increased their tyrosinase mRNA levels progressively over 72 hours following p53 activation (Khlgatian et al., 1999; Khlgatian et al., 2002). Confirming an earlier report (Kichina et al., 1996), these studies also showed an inverse relationship between total p53 protein (inactive) and tyrosinase levels. The requirement for p53 activation to increase tyrosinase protein level and epidermal melanin content in vivo was confirmed by documenting the tanning ability of wild type versus p53 knockout mice (Khlgatian et al., 2002). The question remained whether p53 directly increased tyrosinase transcription, as no p53 consensus sequence had been identified in its promoter region. The key observation that the UV action spectrum for tanning is virtually identical to that for CPD production, had suggested that thymidine dinucleotide (abbreviated ‘‘TT’’), the obligate substrate for the majority of CPDs, might itself serve as a molecular signal for increased melanogenesis (tanning). Using various models, the responses to UV irradiation versus this DNA fragment were therefore compared. Indeed, TT increased mRNA and protein expression of tyrosinase as well as melanin content of cultured human and murine pigment cells and of intact guinea pig skin-containing melanocytes in the interfollicular epidermis, in a time frame similar to that observed after UV irradiation (Eller et al., 1994). As well, in guinea pig skin, the histological features were virtually identical following either treatment: increased total melanin with prominent