Our research is directed at epithelial differentiation in normal and pathologic states. This grant focuses on the roles of vitamins and hormones, as exemplified by retinoids and thyroid hormones, in the control of keratin synthesis and in the process of keratinization. For decades, clinicians have recognized that thyroid hormones and vitamin A had profound effects upon normal and abnormal skin and within recent years the interest in the use of vitamin A and its derivatives on diseases of keratinization has become widespread. At the same time studies of other systems have defined on cellular and molecular levels the mechanism by which these substances effect regulation of gene expression; they bind to specific receptors which in turn react with recognition elements in the non-coding regions of DNA to profoundly affect transcription. The experiments will focus upon several genes for epidermal keratins whose expression we have shown to be negatively regulated by both retinoic acid and its receptor as well as thyroid hormone and its receptor. The assays used will be based upon transfection involving regulatory regions of keratin genes linked to a reporter gene, either chloramphenicol acetyl transferase for quantitative studies or luciferase for localization studies. The recognition elements in the keratin gene for both receptor complexes will be identified through deletion studies. Specific point mutations will define their exact positions. Throughout these studies, the polymerase chain reaction (PCR) will be used to amplify appropriate areas of DNA. Parallel studies will be undertaken in cell lines and primary cultures of epithelial cells. Finally, the effects of retinoids and thyroid hormones on differentiating epidermis in tissue culture will be determined. Thus, our aims in the 5 years for which we are requesting support will be: 1. to identify and characterize the recognition elements (REs) for the nuclear receptors of retinoic acid and thyroid hormone in human keratin genes - basal cell-specific K#5 and K#14, stratum corneum-specific K#10, hyperproliferation-associated K#6 and K#16, and the simple-epithelia- specific K#8, K#17, K#18 and K#19. 2. to define the molecular mechanisms by which retinoids and thyroid hormones regulate keratin gene expression and to delineate the interactions among the two classes of mediators and their receptors; 3. to analyze the effects of retinoids and thyroid hormones on epidermal differentiation in vitro. Our proposal, which will define the mechanisms of action of retinoids and thyroid hormones in epidermis, has important implications for health care for it will ultimately lead to understanding of the normal and pathologic processes in skin and should indicate the potential of new therapeutic agents.
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