The long term goal of this project is to elucidate the developmental and hormonal regulation of the genes for surfactant lipid biosynthesis. Glucocorticoids stimulate phosphatidylcholine (PC) synthesis in late gestation fetal lung. Of the enzymes involved in PC synthesis, fatty- acid synthase (FAS) is the only one whose gene expression is increased by the hormone. Fatty acids are integral components of lipids and hence of surfactant and there is evidence that they also have a role in the activation of the rate limiting enzyme in PC biosynthesis. Enhanced expression of the FAS gene is, therefore, a critical factor in glucocorticoid stimulated surfactant production in fetal lung. The objective of this proposal is to determine how glucocorticoids enhance expression of the FAS gene and to elucidate the importance of FAS in overall surfactant production in fetal lung.
The Specific Aims are to: (1) Identify the sequences in the FAS gene and flanking regions that are responsible for the glucocorticoid effect. A glucocorticoid responsive region of the FAS gene was identified by deletion analysis and reporter gene expression. DNase footprinting, expression of mutated sequences in reporter gene assays, gel shift and supershift assays and Southwestern blotting will be used to further characterize the precise DNA sequences involved in the glucocorticoid effect and to identify transcription factors binding to them. (2) Determine the effect of inhibiting FAS expression and/or activity on glucocorticoid stimulated surfactant phospholipid production. An antisense strategy will be used to inhibit FAS expression and a metabolic inhibitor to suppress its activity in fetal rat lung explants. (3). Determine if glucocorticoids increase FAS expression in fetal type II cells. A deficiency in surfactant leads to the Respiratory Distress Syndrome (RDS) which remains a serious problem in premature infants. The incidence of RDS can be diminished by maternal glucocorticoid administration. To design better treatment strategies for the prevention of RDS, it is imperative to elucidate how the hormone stimulates surfactant production. Because of its pivotal position in surfactant synthesis, it is therefore crucial to elucidate the mechanism by which glucocorticoids regulate FAS expression.
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