Faculty Bibliography

Keratinization, the orderly process of differentiation of epidermal keratinocytes from stratum basale to stratum corneum, is influenced by hormones and vitamins. We have used expression of epidermal keratins as a paradigm of keratinization processes and analyzed the effects of retinoic acid, thyroid hormone, and vitamin D3 on keratin gene expression. DNA constructs in which keratin gene promoters drive expression of reporter genes were co-transfected with vectors expressing nuclear receptors for the above molecules into various cell types. The keratin promoters studied included K3, K5, K10, K14, and K16. The recipient cell types were HeLa and primary cultures of rabbit corneal and esophageal epithelial cells and of human epidermal keratinocytes. We found that retinoic acid, via its nuclear receptor, suppresses expression of all the above-listed keratin genes. Thyroid hormone and its receptor similarly suppressed those genes. The site of interaction between these two receptors and the promoter sequences of K10 and K14 genes has been identified. Surprisingly, vitamin D3 and its receptor had no direct effect on keratin promoters. Our results suggest that a retinoic acid has a twofold effect on keratin gene expression: by regulating keratinocyte differentiation it determines which keratins are expressed, basal cell specific or differentiation specific; by direct interaction between its receptor and keratin genes, retinoic acid determines the total amount of keratin protein within the cell. Vitamin D3, on the other hand, also regulates keratinocyte differentiation, but does not directly interact with the keratin genes

Among extrinsic modulators of keratinization are certain hormones and vitamins, which makes them potentially important pharmacological tools for treatment of keratinization disorders. Vitamin D3 and vitamin A, and their metabolites, promote and inhibit keratinization, respectively. We have shown that retinoic acid, via its nuclear receptor, directly suppresses the expression of the keratin genes which are markers of keratinocyte differentiation. Here we present evidence that 1,25(OH)2 vitamin D3 and its nuclear receptor do not directly regulate keratin gene expression. Co-transfection of a vector expressing the nuclear receptor for vitamin D3 with responder DNA constructs containing keratin gene promoters had no effect on the level of activity of keratin gene promoters either in the presence or in the absence of vitamin D3. We conclude that vitamin D3, unlike retinoic acid, modifies keratin synthesis indirectly, by changing the differentiation phenotype of the keratinocyte

Several methods for DNA-mediated cell transfection were tested to determine the optimal conditions for transfection of human epidermal keratinocytes. The following methods were compared: electroporation, lipofection, Ca3(PO4)2 co-precipitation, DEAE-dextran, and polybrene-mediated transfection. The transfected DNA included human keratinocyte-specific promoter for keratin K14 as well as SV40 and RSV viral promoters. Enzyme assays and in situ staining were used to evaluate both quantitative and qualitative aspects of transfection, and both subconfluent and post-confluent, stratifying keratinocytes were examined. Lipofection, Ca3(PO4)2 co-precipitation, and polybrene methods transfect very efficiently, but lipofection is expensive and Ca++ in the co-precipitation procedure induces keratinocytes to differentiate. We have found that polybrene-mediated transfection followed by a 27% DMSO shock is optimal for introducing DNA into human epidermal keratinocytes