Our objective is to elucidate the mechanisms underlying growth and differentiation in human epithelial tissues. The human epidermis has been chosen as a model system, since we can cultivate these cells under conditions where differentiative properties, including stratification and keratinization, are retained. The major epidermal proteins are keratins. Basal keratinocytes express K14 (50 kd) and K5 (58 kd). As cells undergo terminal differentiation, they downregulate K5 and K14 mRNAs and induce expression of K1 and K10. This switch may enhance filament bundling, an early indication of differentiation. In squamous cell carcinomas, K1 and K10 is reduced, and K6 and K16 appear in suprabasal cells. These keratins are not normally expressed, but are induced transiently during wound- healing. We have shown that both normal and abnormal differentiation are controlled negatively by vitamin A. When elevated to ~10X higher than physiological, synthesis of all suprabasal keratins are downregulated. SQCC cells are more sensitive to retinoic acid (RA) than normal cells, and differentiation can be completely blocked with RA treatment of SQCC lines. Prerequisite to understanding how retinoids control differentiation and why they are useful therapeutically is to elucidate the biochemical behavior of basal cells, i.e. the precursors of the differentiating cells. Central to this study is investigating the sequences and factors controlling basal keratinocyte genes. We have isolated the functional K5 and K14 genes. Using a reporter or tagged K14 gene, coupled with (1) transfection of cultured keratinocytes, and (2) transgenic mouse technology, we have begun to elucidate mechanisms underlying K14/K5 gene expression. Parallel to these studies, we will explore events leading to Ra-mediated inhibition of differentiation. We will examine the kinetics and level of regulation of each Ra-induced change. Of particular interest will be whether K5 and K14 and their keratinocyte transcription factors are regulated by retinoids. Finally, we will (1) examine the role of the steroid hormone-like retinoic acid receptors (RARs) in controlling the RA response, and (2) investigate whether these receptors are involved in the induction of expression of autocrine regulators, including TGF-alpha and TGF-betas, which may act in conjunction with vitamin A to control growth and differentiation. A knowledge of the factors controlling this balance is a prerequisite to understanding how this equilibrium goes awry in the course of human diseases, e.g. psoriasis and basal- and squamous- cell carcinomas. The global aim of this proposal is to coordinate the molecular biology of human differentiation with the application of this research to medicine.
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