[CANCER RESEARCH 60, 4016 – 4020, August 1, 2000] Advances in Brief Down-Regulation of p63 Is Required for Epidermal UV-B-induced Apoptosis Kristin M. Liefer, Maranke I. Koster, Xiao-Jing Wang, Annie Yang, Frank McKeon, and Dennis R. Roop 1 Departments of Molecular and Cellular Biology [K. M. L., X-J. W., D. R. R.] and Dermatology [X-J. W., D. R. R.] and Program in Developmental Biology [M. I. K., D. R. R], Baylor College of Medicine, Houston, Texas 77030, and Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115 [A. Y., F. M.] Abstract In the epidermis, p53 plays an important role in UV-B protection that led us to examine the role, if any, that p63, a p53 homologue highly expressed in the basal layer of the epidermis, might play in the epidermal UV-B response. One p63 isoform, DNp63a, decreased dramatically in normal keratinocytes or newborn epidermis at both the protein and RNA levels after UV-B irradiation. In an attempt to further investigate the significance of the UV-B-induced decrease of this p63 isoform as well as further delineate the function of p63 in the epidermis, we generated transgenic mice that constitutively express DNp63a in the mouse epider- mis using the loricrin promoter (ML.DNp63a). The ML.DNp63a mouse epidermis developed normally, with no overt phenotype and an unaltered proliferation rate. When challenged by UV-B exposure, the ML.DNp63a mice exhibited a 40 – 45% decrease in the number of apoptotic cells in the epidermis as compared with nontransgenic littermates. These results sug- gest that aberrant expression of DNp63a altered the UV-B-induced ap- optotic pathway in the transgenic epidermis, proving that down-regula- tion of DNp63a in response to UV-B is important to epidermal apoptosis. The forced overexpression of DNp63a may act via a dominant negative effect on the endogenous p53 transcriptional activity required for UV-B- induced apoptosis. Introduction For many years, p53 has been considered the prototypical tumor suppressor and remains the subject of intense research. The protein product of the p53 gene responds to cellular stresses such as DNA damage and hypoxia and plays important roles in regulating cell cycle progression, genomic stability, and apoptosis (1, 2). Mutations in the p53 gene have been detected in ;50% of all human cancers (3), demonstrating the universality of this tumor suppressor. DNA damage can be caused by a number of genotoxic agents, and UV-B radiation is one of the most biologically relevant inducers of DNA damage. UV-B radiation in sunlight is the carcinogen respon- sible for most human skin cancers (4, 5). In response to the DNA damage induced by UV-B, the p53 protein is stabilized and translo- cated to the nucleus, where it triggers an arrest of the cell cycle or induces apoptosis (1, 4, 6). In the epidermis, the response to UV-B irradiation is frequently characterized by the induction of apoptosis, primarily mediated by p53 (7). In the absence of p53, fewer apoptotic cells are observed in the epidermis in response to UV-B (7). Also, mice null for p53 are more susceptible to UV-B-induced tumorigen- esis than wild-type mice, implying a critical function for p53 in combating the detrimental effects on the epidermis of this carcinogen (8, 9). Recently, two homologues of p53, p63 and p73, have been discov- ered and cloned, generating a new family of p53-like genes. The p63 gene shares extensive homology to p53 and produces multiple tran- scripts with varying functions (10 –13). Unlike the fairly ubiquitous nature of the p53 protein, p63 exhibits a rather tissue-specific distri- bution in that it is most detectable in the basal layer of stratified epithelia, including the epidermis (13). It is reported that the most highly expressed p63 isoform in the epidermis is DNp63a (13). This isoform lacks the 59 region that exhibits extensive homology to the transactivation domain of p53. In contrast to p53, DNp63a fails to induce apoptosis when overexpressed in cultured cells. DNp63a has also been shown to inhibit p53 transcriptional activity (13). The p63 gene was disrupted by homologous recombination, resulting in severe limb, craniofacial, and epithelial defects and leading to death shortly after birth (14, 15). The newborns lacking functional p63 did not have a recognizable epidermis or hair follicles but possessed a thin, single cell layer covering the body (14, 15). This cell layer did not exhibit epidermal characteristics such as keratin expression, but in one model, this cell layer showed a few isolated patches of highly differentiated clusters of epidermal-like cells (14, 15). Because p63 is essential to normal epidermal development and is a homologue of the p53 tumor suppressor, we investigated the role of p63 in the epidermis in response to UV-B irradiation. We found that in contrast to p53, DNp63a is down-regulated in response to UV-B. To further characterize the importance of this down-regulation as well as the functional importance of p63 in the epidermis, we generated transgenic mice that overexpress DNp63a in the epidermis. The epidermis of these transgenic mice developed normally but exhibited a reduction in UV-B-induced apoptosis, indicating that the down- regulation of this p63 isoform is critical for normal epidermal UV-B- induced apoptosis. Materials and Methods Primary Keratinocyte Culture. Primary keratinocytes were obtained from newborn ICR mouse epidermis as described previously (16). Cells (1.5 3 10 5 ) were plated on 60-mm dishes and cultured in 50% fibroblast- conditioned medium supplemented with 0.05 mM calcium and 4 ng/ml epider- mal growth factor. Media were refreshed 1 day after plating, and cells were exposed to UV-B 2 days after plating. UV-B Treatment of Keratinocytes and Mice. Before exposure to UV radiation, the media were removed, and cells were rinsed twice with Dulbec- co’s PBS (Life Technologies, Inc., Gaithersburg, MD) and covered with 1.5 ml of PBS during the UV treatment. Cells were exposed to 50 mJ/cm 2 UV-B from FS40T12 bulbs (National Biological/ETA Systems, Twinsburg, OH) as mon- itored by a radiometer/photometer (International Light, Inc, Newburyport, MA). PBS was removed, and media were added to cells as soon as UV treatment was complete. Samples were taken at 0 (no UV radiation), 6, 12, and 24 h after UV treatment. Culture dishes were rinsed three times with PBS, and PBS was removed before freezing the dish on dry ice and storing it at 280°C until protein or RNA extraction was performed. Newborn pups were immo- bilized, and back skin was exposed to 100 mJ/cm 2 UV-B from FS40T12 lamps (National Biological/ETA Systems) as monitored by a photometer/radiometer (International Light, Inc.). Samples of back skin were taken at 0 (no UV radiation), 6, 12, and 24 h after UV exposure for normal ICR pups or at 24 h after UV exposure for transgenic litters. Portions of the skin were frozen in OCT, fixed in 10% neutral buffered formalin, and embedded in paraffin or frozen in liquid nitrogen for storage. Adult mice were exposed to 100 mJ/cm 2 UV-B, monitored at the height of the ears. Ear samples were processed as described here for newborn back skin. Received 4/18/00; accepted 6/15/00. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 To whom requests for reprints should be addressed, at Department of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030. 4016 Research. on February 23, 2016. © 2000 American Association for Cancer cancerres.aacrjournals.org Downloaded from