MECHANISMS IN THE REGULATION OF SP-A GENE EXPRESSION

Ut Southwestern Medical Center, Dallas TX

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Abstract

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