The synthesis of pulmonary surfactant, a lipoprotein complex that acts to reduce lung alveolar surface tension, is developmentally and hormonally regulated in fetal lung tissue. Surfactant protein B (SP-B) is critical in the function of surfactant;the lung is susceptible to injury and failure when SP-B levels decrease below 25% of normal. Prematurely-born infants that lack adequate surfactant can develop Respiratory Distress Syndrome, a leading cause of neonatal morbidity and mortality. Antenatal administration of glucocorticoids accelerates fetal lung maturity and enhances SP-B expression by increasing human SP-B mRNA stability by unknown mechanisms. Since glucocorticoids are used clinically in the treatment of premature infants against RDS, it is important to understand the molecular mechanism(s) by which glucocorticoids act to regulate surfactant protein gene expression in the lung. We hypothesize that regulation of SP-B mRNA stability is mediated by specific mRNA:protein interactions localized to the SP-B mRNA 3'-untranslated (UTR) regions. We have identified regions necessary and sufficient for mediating glucocorticoid-induced stabilization of SP-B mRNA which are restricted to the 3'-UTR. Cytosolic proteins specifically and independently bind to a 126 nt long region of the SP-B 3'-UTR. Recently, we have identified a small 30 nt mRNA element in this region predicted to form a stem-loop structure that is sufficient for mediating glucocorticoid-induced stabilization of mRNA and reduces intrinsic stability of SP-B mRNA. This element may provide a target to increase SP-B mRNA levels without the use of glucocorticoids. The objective of this application is to more completely define the molecular mechanisms whereby glucocorticoids enhance SP-B mRNA stability through elements in the 3'- UTR and the molecular mechanisms by which these elements mediate intrinsic SP-B mRNA stability. The following specific aims are proposed in this application: (1) to identify elements of the human SP-B mRNA 3'-UTR that are sufficient for mediating in vivo glucocorticoid stabilization and/or intrinsic stability of SP-B mRNA, (2) to identify proteins that may mediate glucocorticoid regulation or intrinsic regulation of SP-B mRNA stability through interaction with elements of the SP-B mRNA 3'-UTR, and (3) to identify microRNAs (miRNAs) that may mediate glucocorticoid regulation and intrinsic regulation of SP-B mRNA stability through interaction with elements of the SP-B mRNA 3'-UTR. The proposed research will define molecular mechanisms by which SP-B mRNA stability is regulated by glucocorticoids through identification of specific mRNA sequences, proteins and miRNAs that may be involved in hormonal and intrinsic regulation of mRNA stability. Identification and characterization of these components will delineate the complex mechanisms whereby hormones regulate gene expression in lung. The understanding of these mechanisms may lead to development of improved therapeutic strategies that enhance lung maturation and prevent RDS and its consequences. PROJECT NARRATIVE: The widespread use of glucocorticoids for treatment of prematurely-born infants makes it necessary to understand the mechanisms of regulation of surfactant protein mRNA by hormones. Ultimately, the information may allow the design of treatment regimes where the advantages of glucocorticoid treatment are retained and the deleterious effects of glucocorticoids are avoided.
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