Elastin is a critical component of the lung extracellular matrix and provides elastic recoil to the alveolar wall. Because most lung elastin is produced during fetal and neonatal development, and there is essentially no turnover of deposited elastin in the healthy lung, this elastin is critical to lung function for the life of the organism. We have identified glucocorticoids as transcriptional activators of fetal lung tropoelastin expression. Importantly, maternal glucocorticoid treatment results in both increased fetal tropoelastin expression and accelerated, but architecturally normal, lung morphogenesis. However, glucocorticoid upregulation of lung tropoelastin production is tissue-specific and is confined to fetal periods; late in the neonatal period and in the adult, glucocorticoids actually repress the production of tropoelastin. Together, these findings indicate that glucocorticoids are potential specific regulators of lung elastin expression in vivo. The goals of this project are to determine the molecular mechanisms of glucocorticoid-mediated induction of fetal lung tropoelastin, the effect of this regulation on elastic fiber assembly and lung morphogenesis, and the basis for the tissue-specific and age-specific regulation of tropoelastin by glucocorticoids. We will determine the glucocorticoid-responsive elements in the tropoelastin promoter by transfecting fetal, neonatal, and adult lung interstitial fibroblasts with deletion and mutated tropoelastin promoter expression constructs. We will use gel-shift and supershift assays to determine specific nuclear proteins binding to those sequences. Transacting factors will be overexpressed in elastogenic cells to assess if they influence endogenous tropoelastin expression. To determine if glucocorticoids coordinately regulate elastin-associated genes in vivo, we will use Northern and in situ hybridization to define the patterns of expression of mRNAs encoding microfibrillar proteins and lysyl oxidase during fetal and neonatal lung development. Using a lung mince organ culture system which supports continued morphological development, we will compare the effects of glucocorticoids and other stimulators of tropoelastin expression on lung development, and on the expression of tropoelastin and elastin-associated microfibrillar proteins. These studies should provide important information regarding the molecular mechanisms of glucocorticoid-mediated tropoelastin expression in the lung, and will likely define tissue-specific and age-specific cis-acting elements of the tropoelastin promoter. We may also develop an understanding of the specific trans-acting factors which regulate tropoelastin expression in normal and accelerated lung development. These proposed studies will also, for the first time, coordinately assess the patterns of expression of several genes important in lung elastic fiber assembly and deposition. Our findings should provide important information regarding the mechanisms regulating the assembly of an elastic matrix during lung development. This, in turn, will provide a sound basis for future studies dissecting abnormal elastic matrix production and assembly in diseases such as pulmonary fibrosis.
