The objective is an understanding of the biochemical mechanisms that regulate the expression of human genes during differentiation. The human epidermis has been chosen as a model system, since we are able to serially cultivate these cells in vitro under conditions where many of the differentiative properties, including stratification, are retained. A simple squamous epithelium, the peritoneal mesothelium, has been chosen for purposes of comparison. These cells can also be cultured, using a similar system. The major differentiation-specific proteins of the epidermis are the keratins, a group of related proteins (M.W. 40-70kd) that form 8 nm cytoskeletal filaments in all epithelial cells. We have identified two distinct multigene families encoding the keratins. Pairs of these genes are differentially expressed in different epithelial cells and at different stages of keratinization. Commitment to terminal differentiation results in a shift in the synthesis of mRNAs encoding two pairs of smaller keratins to the synthesis of a new pair of larger keratins (MW 56.5 and 67K). We have found that epidermal cells in culture will terminally differentiate if the vitamin A in the medium is removed. In addition to causing increased stratification and s. corneum formation, removal of the vitamin regulates the synthesis of the mRNAs encoding the large keratins. In contrast, increasing the vitamin A level leads to the expression of two keratins (53kd and 40kd) not normally seen in epidermal cells, but made by some cancerous cell lines and also mesothelial cells. Recently, we constructed a cDNA library to human mesothelial mRNA. We have identified cDNAs encoding the keratins of the 53kd and 40kd keratins that are positively regulated by vitamin A. We now propose to isolate and sequence the genes for these proteins in order to define the regulatory sequences responsible for the tissue-specific and vitamin A-specific expression of these genes. We will utilize mammalian gene transfer systems to study the transcriptional and translational regulation of these genes. Finally, using purified cellular receptors for the retinoids, we will investigate whether retinoic acid and retinol act directly or indirectly to influence the expression of these specific keratin genes. It is our aim to elucidate the significance of the expression of this pair of vitamin A-regulated keratins in human epidermis and to determine the precise mechanism by which vitamin A differentially regulates their expression.
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