Pulmonary surfactant is a mixture of phospholipids and three major proteins --SP-A, SP-B and SP-C. Trnscripts for the three proteins appear at distinct developmental stages in fetal alveolar type II epithelial cells. Multiple regulators have been identified, including glucocorticoids, cAMP TGF betta, insulin, EGF, and gamma-interferon. Each of the three genes is induced by glucocorticoids, but to different extents and at different stages of development; moreover, SP-A expresion is subsequently repressed by glucocorticoids. We will investigate the mechanisms by which glucocorticoids control the transcription of these genes, alone and in combination with other regulators. Three general approaches will be developed. First, we will determine whether glucocorticoid regulation of the surfactant protein genes corresponds to primary or secondary effects of the glucocorticoid receptor; glococorticoid response elements (GREs) will be localized by deletion mutagenesis and cell transfection of cloned promoter and upstream regions for SP-A, -B and -C, by binding of purified glucocorticoid receptor, and by point mutagenesis. Other regulatory loci that interact functionally with GREs will also be identified. Second glucocorticoid receptor gene expression will be monitored during development, testing in particular whether distinct receptor promoters are differentially utilized in developing lung. We will also define specific regions within the receptor responsible for each aspec of the developmental regulation of SP-A, -B and -C. Finally, we will characterize combinatorial interactions with other regulators,, including some of those noted above. As part of these studies, we will test the potential involvement of C/EBP, an enhancer activating protein that is expressed in a restricted subset of tissues, including lung; C/EBP expression may itself be stimulated by glucocorticoids. Together, these experiments will yield new information about multiple regulators, receptors and transcription factors that control gene expression in the developing lung.
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