Infants born prematurely are at risk for developing respiratory distress syndrome (RDS). In these infants, RDS results from insufficient levels of pulmonary surfactant and has an associated high mortality. Surfactant protein C (SP-C) is a highly hydrophobic protein that contributes to the surface tension lowering properties of pulmonary surfactant. This grant will test the central hypothesis that SP-C is essential for pulmonary surfactant function. This hypothesis is supported by the identification of term infants that succumb to RDS and have a deficiency in SP-C with apparently normal levels of other surfactant proteins. This application seeks to determine the molecular mechanisms controlling SP-C gene transcription in the surfactant producing Type II cells and determine the role of SP-C in surfactant function. The experiments proposed in this research application will test specific hypothesis that pulmonary Type II cell selective expression of SP-C results from the combinatorial assembly of transcription factors on a proximal cis-active region of the SP-C promoter (Aim 1) and on a cell selective enhancer (CSE) element (Aim 2). The experiments will focus to two distinct factors that potentially modify SP-C activation by the transcription factor TTF-1. In vitro gene expression assays will be used to determine how a Type II cell isoform of SSBP (a single stranded binding protein) and the factor GATA6 alter TTF-1 dependent transactivation of SP-C. Mutational analysis of the SSBP and GATA6 binding sites will be used to define sequences required for maximal SP-C activation. Factors critical to cell selective enhancer function will be identified in Aim 2. Core binding site probes will be used to clone transactivating proteins that bind the CSE by screening of a mouse lung cDNA expression library. Cloned factors will then be assayed for their ability to stimulate transcription of CSE-promoter constructs. Transgenic mice will be generated to determine the extent to which the SSBP/GATA6 activation elements and CSE elements, direct Type II cell specific expression in vivo.
In Aim 3, SP-C deficient mice will be generated by gene targeting techniques to discern the role of SP-C in surfactant function in vivo and provide a model of the SP-C deficient infant. The results of these studies will provide insight into Type II cell specific gene transcription and greatly facilitate the design of an appropriate therapy for surfactant deficiencies.
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