This application is a request to continue work to discern mechanisms regulating expression of surfactant protein SP-B gene. SP-B is a hydrophobic polypeptide that enhances the spreading and stability of pulmonary surfactant phospholipids. SP-B synthesis in pulmonary epithelial cells increases prior to birth and is a critical component of the surfactant complex required for adaptation to air breathing. Lack of surfactant at birth is a major cause of morbidity and mortality of premature infants. This application will test the hypothesis that cell-specific, ontogenic and humoral controls of SP-B gene expression are mediated, at least in part, by the cis-active elements of the SP-B gene and their interaction with nuclear transcription proteins. Regions of the SP-B gene conferring cell-specific, ontogenic and the humorally regulated pattern of SP-B expression will be delineated using DNase hypersensitivity, gel retardation analysis, DNA footprinting and by transcriptional assays using chimeric SP-B-reporter gene constructs in transient assays and in transgenic mice. Expression of the SP-B-CAT transgenes will be studied both in vitro and in vivo. Transgenic mice bearing SP-B-CAT constructs will be generated for comparison of CAT expression with that of the endogenous SP-B gene and other cellular markers of epithelial cell maturation including CC10, SP-A and SP-C. Temporal-spatial and humoral regulation of SP-B and SP-B chimeric genes will be assessed. Transgenic mice will be generated expressing human proSP-B mRNA (sense and antisense SP-B mRNAs) using enhancer elements from the human SP-C gene to discern the effects of increased and decreased expression of SP-B on surfactant structure, function and on perinatal adaptation to air breathing. Alternatively, homologous recombination will be used to disrupt the SP-B locus and determine the role of SP-B in lung function in SP-B ablated animals in vivo. Further understanding of the role of SP-B in lung function and the mechanisms regulating SP-B may provide insights critical to developing new therapeutic strategies for prevention of lung disease in premature infants.
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