Down-regulation of p63 is required for epidermal UV-B-induced apoptosis.

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, deltaNp63alpha, 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 deltaNp63alpha in the mouse epidermis using the loricrin promoter (ML.deltaNp63alpha). The ML.deltaNp63alpha mouse epidermis developed normally, with no overt phenotype and an unaltered proliferation rate. When challenged by UV-B exposure, the ML.deltaNp63alpha mice exhibited a 40-45% decrease in the number of apoptotic cells in the epidermis as compared with nontransgenic littermates. These results suggest that aberrant expression of deltaNp63alpha altered the UV-B-induced apoptotic pathway in the transgenic epidermis, proving that down-regulation of deltaNp63alpha in response to UV-B is important to epidermal apoptosis. The forced overexpression of deltaNp63alpha may act via a dominant negative effect on the endogenous p53 transcriptional activity required for UV-B-induced apoptosis.

12-O-tetradecanoylphorbol-13-acetate promotion of transgenic mouse epidermis coexpressing transforming growth factor-alpha and v-fos: acceleration of autonomous papilloma formation and malignant conversion via c-Ha-ras activation.

To study oncoprotein cooperation in vivo, transgenic mice were established that coexpressed human transforming growth factor-alpha (TGFalpha) and v-fos exclusively in the epidermis by means of a human keratin 1 (HK1)-based vector. HK1.fos/alpha mice exhibited aberrant epidermal proliferation and differentiation and formed spontaneous papillomas that achieved tumor autonomy but did not convert to malignancy. To determine the sensitivity to a chemical promotion stimulus, HK1.fos/alpha mice were promoted with 12-O-tetradecanoylphorbol-13-acetate (TPA). Previously, after 7 mo TPA promotion of HK1.TGFalpha mice that express moderate levels of TGFalpha elicited papillomas that remained regression-prone and benign for up to 2 yr. In HK1.fos mice, 6 mo TPA elicited papillomas that required spontaneous c-Ha-ras activation and converted to malignancy after 14-16 mo. We now show that in HK1.fos/alpha transgenic genotypes, TPA promotion accelerated papillomatogenesis, with the earliest papilloma appearance at 2 mo after initiation of TPA promotion. These papillomas started to convert to malignancy by 10 mo. Analysis of HK1.fos/alpha papillomas and carcinomas revealed that the endogenous c-Ha-ras gene possessed mutations at codons 12, 13, and 61 at the papilloma stage, but no mutations of the p53 tumor suppressor gene were detected. These data indicate that coexpression of fos and TGFalpha increased epidermal sensitivity to TPA promotion, which accelerated malignant conversion. However, in this transgenic model conversion always required additional genetic events, e.g., activation of the endogenous c-Ha-ras gene.

Development of gene-switch transgenic mice that inducibly express transforming growth factor beta1 in the epidermis.

Previous attempts to establish transgenic mouse models to study the functions of transforming growth factor beta1 (TGFbeta1) in the skin revealed controversial roles for TGFbeta1 in epidermal growth (inhibition vs. stimulation) and resulted in neonatal lethality in one instance. To establish a viable transgenic model for studying functions of TGFbeta1 in the skin, we have now developed transgenic mice, which allow focal induction of the TGFbeta1 transgene in the epidermis at different expression levels and at different developmental stages. This system, termed "gene-switch," consists of two transgenic lines. The mouse loricrin vector targets the GLVPc transactivator (a fusion molecule of the truncated progesterone receptor and the GAL4 DNA binding domain), and a thymidine kinase promoter drives the TGFbeta1 target gene with GAL4 binding sites upstream of the promoter. These two transgenic lines were mated to generate bigenic mice, and TGFbeta1 transgene expression was controlled by topical application of an antiprogestin. On epidermal-specific induction of the TGFbeta1 transgene, the BrdUrd labeling index in the transgenic epidermis decreased 6-fold compared with controls. Induction of the TGFbeta1 transgene expression also caused epidermal resistance to phorbol 12-myristate 13-acetate-induced hyperplasia, with a reduction in both epidermal thickness and BrdUrd labeling compared with those in controls. In addition, TGFbeta1 transgene expression induced an increase in angiogenesis in the dermis. Given that the TGFbeta1 transgene can affect both the epidermis and dermis, this transgenic model will provide a useful tool for studying roles of TGFbeta1 in wound-healing and skin carcinogenesis in the future.

Mutations in the 1A domain of keratin 9 in patients with epidermolytic palmoplantar keratoderma.

Epidermolytic palmoplantar keratoderma is an autosomal dominant skin disorder characterized by hyperkeratosis of the palms and soles. Ultrastructurally the disease exhibits abnormal keratin filament networks and tonofilament clumping like that found in the keratin disorders of epidermolysis bullosa simplex and epidermolytic hyperkeratosis. The disease has been mapped to chromosome 17q11-q23 in the region of the type 1 keratin gene locus and more recently mutations have been found in the palmoplantar specific keratin, keratin 9. We have analyzed six unrelated incidences of epidermolytic palmoplantar keratoderma for mutations in their keratin 9 genes. In two of these, we have identified mutations that alter critical residues within the highly conserved helix initiation motif at the beginning of the rod domain of keratin 9. In a three-generation Middle Eastern kindred we found a C to T transition at codon 162 that results in an arginine to tryptophan substitution at position 10 of the 1A alpha-helical domain, thus confirming this codon as a hot spot for mutation in keratin 9. The other mutation found involves a T to C transition at codon 167 that results in the expression of a serine residue in place of the normal leucine at position 15 of the 1A segment and is the first documentation of this mutation in this gene. The identification of these substitutions extends the current catalog of disease causing mutations in keratin 9.