Kristin Ostrom , and Barbara

Although the ability of UV irradiation to induce pigmentation in vivo and in vitro is well documented, the intracellular signals that trigger this response are poorly understood. We have recently shown that increasing DNA repair after irradiation enhances UV-induced melanization. Moreover, addition of small DNA fragments, particularly thymine dinucleotides (pTpT), selected to mimic sequences excised during the repair of UV-induced DNA photoproducts, to unirradiated pigment cells in vitro or to guinea pig skin in vivo induces a pigment response indistinguishable from UVinduced tanning. Here we present further evidence that DNA damage and/or the repair of this damage increases melanization. (i) Treatment with the restriction enzyme Pvu II or the DNA-damaging chemical agents methyl methanesulfonate (MMS) or 4-nitroquinoline 1-oxide (4-NQO) produces a 4to 10-fold increase in melanin content in Cloudman S91 murine melanoma cells and an up to 70% increase in normal human melanocytes. (ii) UV irradiation, MMS, and pTpT all upregulate the mRNA level for tyrosinase, the rate-limiting enzyme in melanin biosynthesis. (iii) Treatment with pTpT or MMS increases the response of S91 cells to melanocytestimulating hormone (MSH) and increases the binding of MSH to its cell surface receptor, as has been reported for UV irradiation. Together, these data suggest that UV-induced DNA damage and/or the repair of this damage is an important signal in the pigmentation response to UV irradiation. Because Pvu II acts exclusively on DNA and because MMS and 4-NQO, at the concentrations used, primarily interact with DNA, such a stimulus alone appears sufficient to induce melanogenesis. Of possible practical importance, the dinucleotide pTpT mimics most, if not all, of the effects of UV irradiation on pigmentation, tyrosinase mRNA regulation, and response to MSH without the requirement for antecedent DNA damage. The prokaryotic response to UV irradiation, the so-called SOS response, is well documented and is now known to include the induction of a set of >20 genes involved in DNA repair and cell survival (reviewed in ref. 1). In this case, the single-stranded DNA generated after UV irradiation interacts with and activates a protease, the Rec A protein (2). Activated Rec A protein then cleaves and inactivates the repressors of specific genes, leading to their induction (2). In eukaryotic cells, the existence of a UV-induced DNA damage-responsive SOS-like system mediated by one common transcription regulator has been the subject of considerable controversy. Although a variety of genes are known to be induced by DNA damage (3-6), many of these genes are also induced by agents such as phorbol esters (3, 7) and by metabolic or oxidative stress (8-10). Because UV irradiation is reported, like phorbol esters, to activate protein kinase C directly (11, 12) and to produce oxidative damage through generation of free radicals from membrane lipids and other The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. extranuclear cellular constituents, it cannot be determined whether the effects ofUV are due directly to DNA damage or instead to other impacts on the cell. Indeed, two of the major UV-induced transcription factors, AP-1 and NF-KB, are now thought to initiate their responses at or near the plasma membrane (13, 14). Perhaps the best-characterized example of DNA damagespecific gene induction involves a DNA repair enzyme, photolyase, encoded by the PHR1 gene in Saccharomyces cerevisiae (6). This gene is induced by a variety of DNA-damaging agents including UV light, methyl methanesulfonate (MMS) and 4-nitroquinoline 1-oxide (4-NQO). Up-regulation of PHR1 gene transcription is, at least in part, accomplished by the removal of a damage-responsive repressor which binds to a specific site in the 5' region of the gene (15). Another well-studied UV and DNA damage-inducible gene is the mammalian GADD45 gene. This gene is transcriptionally activated not only by UV irradiation but also by ionizing radiation and chemical agents that specifically cause base damage (10, 16). The induction of GADD45 by ionizing radiation is mediated by the p53 tumor suppressor protein and the ataxia telangiectasia gene product (17), but the UVand base-damage responses are less well understood. Recently, GADD45 protein was shown to stimulate DNA excision repair as well as to inhibit DNA replication by blocking the cell cycle at the GI checkpoint (18). Thus GADD45 plays a major role in maintaining the integrity of the genome after damage by coordinating repair with replication. As would be expected teleologically, the majority of the prokaryotic genes responsive to DNA damage are, like PHRI and GADD45, involved in the repair or prevention of such damage. As well, UV irradiation and DNA-damaging carcinogenic chemical agents appear to induce DNA repair systems in mammalian cells (19), consistent with the existence of an SOS-like response analogous to that in yeast. Because melanin is photoprotective in human skin and is generally acknowledged as the body's major defense against photocarcinogenesis (20), acting to inhibit the formation of UV-induced DNA photoproducts (21, 22) that give rise to "signature mutations" responsible for skin cancer development (23), enhanced melanogenesis might logically be part of the SOS response in skin. The clinical and histological manifestations of tanning have been extensively studied, but the mechanisms by which UV light stimulates melanogenesis are poorly understood. Several lines of evidence now suggest that DNA damage and/or the repair of this damage is an important signal in the melanogenic response to UV irradiation. (i) The action spectrum for the formation of the major UV-induced DNA photoproducts in human skin is essentially the same as that for UV-induced tanning (24, 25). (ii) Increasing DNA repair after UV irradiation by addition of T4 endonuclease V (T4N5), the prokaryotic DNA repair enzyme that catalyzes the rate-limiting step in Abbreviations: MSH, melanocyte-stimulating hormone; MMS, methyl methanesulfonate; 4-NQO, 4-nitroquinoline 1-oxide. *To whom reprint requests should be addressed at: Department of Dermatology, Boston University School of Medicine, 80 East Concord Street, Boston, MA 02118-2394.