The goal of our research is to define the molecular mechanisms involved in lung type II cell-specific, developmental and hormonal regulation of surfactant synthesis in fetal lung. To achieve this goal, we have focused on the gene encoding surfactant protein-A (SP-A), a major surfactant protein expressed primarily in type II pneumonocytes and is developmentally regulated in concert with surfactant glycerophospholipid synthesis. SP-A gene transcription in midgestation rabbit and human fetal lung in culture is induced by hormones and factors that increase cyclic AMP (cAMP). In deletion mapping and mutagenesis studies, we have found that basal and cAMP induction of rabbit and human SP-A promoter activity is critically dependent upon cooperative interactions of at least four identified DNA response elements which lie within 400 bp upstream of the SP-A transcription initiation site. Mutagenesis of any one of these elements has a marked effect to reduce basal and cAMP induction of SP-A promoter activity. In studies using transgenic mice carrying SP-A:human growth hormone (hGH) reporter genes, we have found that as little as 400 bp of SP-A 5'-flanking sequence mediates lung- specific and appropriate developmental regulation of hGH expression. In the proposed research, we will use transgenic mice for deletion mapping and mutagenesis to further define critical response elements, and for a promoter building strategy, whereby tissue/cell-specific and developmental regulation of transgene expression will be analyzed in mice carrying SP-A:hGH fusion genes containing the SP-A basal promoter fused downstream of the identified response elements, either individually or in various combinations. The functional role of these elements within the endogenous mouse SP-A gene will be analyzed by gene targeting. Transcription factors (TFs) that bind to these and other identified response elements will be isolated by expression screening or yeast one-hybrid system. Upon identification of TFs crucial for SP-A gene regulation, we will explore effects of cAMP on their DNA binding and transcriptional activities, their interactions with each other and with co-activators, and their functional role in mice by gene targeting or dominant-negative inactivation. Finally, in light of the important role of cAMP in type II cell differentiation and the regulation of SP-A gene expression, we will analyze developmental changes in expression and subcellular localization of protein kinase A (PKA) regulatory (R) and catalytic (c) subunits and of anchor proteins important for PKA nuclear localization, during lung development and type II cell differentiation. To assess the role of PKA in lung development and surfactant synthesis, we will create transgenic mice carrying a dominant negative R subunit under control of SP-A and SP-C promoters to inhibit PKA activity in a type II cell-specific, developmentally timed manner.
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