Faculty Bibliography

TGFbeta is an important regulator of epidermal keratinocyte function because it suppresses cell proliferation, while it induces synthesis of extracellular matrix proteins and their cells surface receptors. To examine whether TGFbeta affects synthesis of intracellular proteins as well, specifically the transcription of keratin genes, we transfected a series of DNA constructs that contain keratin gene promoters into human epidermal keratinocytes. The transfected cells were grown in the presence and absence of TGFbeta. We found that TGFbeta specifically induces transcription controlled by the promoters of K:5 and K:14 keratin genes, markers of basal cells. No other keratin gene promoters were induced. The effect of TGFbeta is concentration-dependent, can be demonstrated in HeLa cells, does not depend on keratinocyte growth conditions and can be elicited by both TGFbeta1 and TGFbeta2. We conclude that TGFbeta promotes the basal cell phenotype in stratified epithelia such as the epidermis

In 1987, a tremendous boost in our understanding of the action of dietary vitamin A occurred with the discovery and characterization of nuclear receptors for retinoic acid, the active form of the vitamin, in the laboratories of P. Chambon and R. Evans. They have shown that the nuclear receptors are ligand-activated transcription factors capable of specific gene regulation. Since that discovery, it has been determined that there are at least six retinoic acid receptors belonging to two families, RARs and RXRs, that they are differentially expressed in various mammalian tissues, and that they act as homo- and heterodimers interacting with other ligand-activated nuclear receptors. The domain structure of the receptors has been described, and their DNA-binding, ligand-binding, dimerization, and transcriptional activation regions characterized. Among the most important retinoid-regulated genes are the homeobox proteins, regulatory transcription factors which are responsible for body axis formation, patterning, limb formation, and other crucial processes during development. Retinoic acid and its receptors also regulate many differentiation markers which are particularly important in stratified epithelia, such as skin and oral epithelia. Our increased understanding led to improved therapy of a large number of skin disorders, ranging from acne to wrinkles and including epidermal and oral carcinomas

The promoter of human K14 keratin gene, specific for the basal layer of stratified epithelia, is regulated by nuclear receptors for retinoic acid and thyroid hormone. However, the DNA sequences responsible for this regulation have not yet been identified. To identify the retinoic acid-responsive site, we have devised a simple site-specific mutagenesis method and introduced mutations into the K14 keratin gene promoter. These mutations identify the retinoic acid-responsive site. The site consists of a cluster of consensus palindrome half-sites in various orientations. As shown previously, retinoic acid and thyroid hormone receptors can recognize and bind common sequences in regulated genes. Here, we describe mutations that abolish regulation by both receptors. Interestingly, the hormone-dependent and -independent regulatory sites of the thyroid hormone nuclear receptor can be separated. Clusters of half-sites that share structural organization with the K14 regulatory site were found in the K5 and K10 keratin gene promoters. Similar clusters may be responsible for retinoic acid-mediated transcription regulation in epidermis

In the epidermis, retinoic acid (RA) is known to regulate the gene expression of keratins, the intermediate filament proteins of epithelial cells. We have cloned the upstream regulatory regions of three human epidermal keratin genes, K5, K10, and K14, and engineered DNA constructs in which these regions drive expression of the CAT reporter gene. By co-transfecting the constructs into various epithelial cell types along with the vectors expressing the nuclear receptors for RA and thyroid hormone (T3), we have shown that RA and T3 directly regulate expression of these three keratin genes through the action of their nuclear receptors. In this paper, we review our previous results to stress that RA has a dual effect on keratin expression in epidermis: both direct and indirect. We also analyze the DNA sequences upstream from those three RA-regulated keratin genes and identify the clusters of degenerate consensus half-site motifs, which may comprise the putative retinoic acid recognition elements (RAREs). Furthermore, our recent results concerning the regulation of K5 and K14 expression by the RA receptor are also shown; these confirm our predictions regarding the location of the RAREs in epidermal keratin genes

Keratin K5 is expressed in the basal layer of stratified epithelia in mammals and its synthesis is regulated by hormones and vitamins such as retinoic acid. The molecular mechanisms that regulate K5 expression are not known. To initiate analysis of the protein factors that interact with the human K5 keratin gene upstream region, we have used gel-retardation and DNA-mediated cell-transfection assays. We found five DNA sites that specifically bind nuclear proteins. DNA-protein interactions at two of the sites apparently increase transcription levels, at one decrease it. The importance of the remaining two sites is, at present, unclear. In addition, the location of the retinoic acid and thyroid hormone nuclear receptor action site has been determined, and we suggest that it involves a cluster of five sites similar to the consensus recognition elements. The complex constellation of protein binding sites upstream from the K5 gene probably reflects the complex regulatory circuits that govern the expression of the K5 keratin in mammalian tissues

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