Mucociliary functions of respiratory tract epithelium play a very important role in pulmonary defense. Vitamin A or its analogs (retinoids) is required for the expression of these differentiated functions. In vitamin A deficiency, the epithelium changes to a squamous keratinizing one (squamous metaplasia). Both in vivo and in vitro organ culture studies show that the addition of vitamin A or other retinoids can convert the squamous epithelium to a normal mucociliary one. Excess vitamin A can convert even the stratified skin epithelium to a one containing mucus-secreting granules (mucous cell metaplasia). However, the nature of mucociliary differentiation and the mechanism by which retinoid controls the expression of these differentiated functions are still unknown. An interdisciplinary approach is proposed to investigate the nature of retinoid-responsive mucous cell differentiation in cultured hamster tracheal epithelial (HTE) cells. We have developed a serum-free medium that permits the growth and differentiation of protease-dissociated respiratory tract epithelial cells, including HTE cells, on collagen gel substrata. Vitamin A or other retinoids is required in HTE culture to induce mucous cell differentiation and ciliogenesis. Based on the preliminary studies, it is proposed that retinoids regulate mucous cell differentiation, especially the synthesis of mucin core protein, apomucin, at the genetic level in the squamous basal cells. To test this hypothesis and examine the mechanisms of vitamin A regulation at the genetic and biochemical levels, it will be necessary to develop genetic and immunological probes for mucin. Development of these probes will be facilitated by the well-defined culture system for HTE cells. Monoclonal antibodies specific for hamster tracheal mucin and apomucin will be developed for monitoring the stages of mucin biosynthesis during the differentiation. cDNA probes to retinoid-responsive genes, in particular cDNA complementary to apomucin, will be isolated. The cDNA library of HTE cells cultured in the presence of retinoid will be developed and screened with oligonucleotide probes corresponding to apomucin sequence or vitamin A-responsive genes. Mabs will be used to immunoprecipitate polysome fractions enriched with apomucin mRNA. Furthermore, a recently developed biphasic culture chamber (the Whitcutt chamber) which enhances the polarity of differentiation of cultured HTE cells similar to in vivo, will be used to identify the precursor cell type involved in retinoid-reponsiove mucous cell differentiation in vitro.
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