Transcriptional Regulation of a Surfactant Enzyme

University Of Iowa, Iowa City IA

Investigators

Abstract

DESCRIPTION (provided by applicant): The developing fetal lung critically relies on the availability of maternal lipoproteins to maintain adequate synthesis of surfactant by alveolar type II epithelial cells. Lipoproteins are a rich source of fatty acids, which serve as substrates for synthesis of phosphatidylcholine (PC), the major 1ipid of surfactant. Lipoproteins also serve as potent post-transnational activators of the rate-limiting enzyme required for surfactant PC synthesis, cytidylyltransferase (CT). Although much is known about post-transnational control of CT, transcriptional regulation of this enzyme has not been evaluated. In addition, feedback control mechanisms that exist in the lung to maintain surfactant PC homeostasis under conditions of lipoprotein deficiency remain largely unknown. The revised proposal expands from recent advances made in our laboratory showing that chronic lipoprotein deficiency increases CT activity and surfactant synthesis in fetal type II cells by increasing transcription of the CT gene. These in vitro results suggest that lipoprotein deprivation stimulates CT gene transcription as a novel compensatory mechanism. These preliminary results led us to hypothesize that CT is regulated, in vivo, at the transcriptional level. We propose to test this hypothesis by generating a CT promoter-reporter mouse (AIM 1). This transgenetic mouse will be used to study constitutive and induced CT transcription after lipoprotein deprivation during various phases of fetal lung development (AIM 2). In addition, transgenic mice harboring specific CT promoter segments linked to the beta-galactosidase (beta gal) reporter gene will be used to determine the functional relevance of regulatory elements within the 5' flanking region of the CT promoter in vivo. To achieve our Aims, maternal and fetal lungs will be analyzed for reporter expression by tissue immunohistochemical staining, immunoblotting, beta gal activity, and Northern analysis for beta gal. Results will be correlated with endogenous CT expression and surfactant production. These in vivo studies will be supplemented by use of a lipoprotein-sensitive type II line. The unique contributions of this proposal impacting the field of surfactant metabolism include providing a springboard by which investigators can understand for the first time how the CT gene is regulated transcriptionally, in vitro and in vivo. Ultimately, the results from these studies could lead to therapeutic strategies directed at modulating expression of this key surfactant enzyme at the molecular level.