Project Summary Despite intensive investigation, hypertension remains a major public health problem. The pathogenesis of hypertension is incompletely characterized, and the potential role of transcriptional mechanisms is particularly unclear. Previously, we have published evidence that the E2F2 transcription factor is crucial for maintaining blood pressure (BP) homeostasis: mice carrying a deletion mutation of E2F2 are hypertensive, and their arterial vessels are hypercontractile. Historically, E2F2 is considered a cell-cycle regulator; however, in endothelial cells (ECs), E2F2 promotes the expression of endothelial converting enzyme 1b (ECE-1b), the deactivating ECE-1 isoform, thus suppressing ECE-1 activity and endothelin-1 (ET-1) biogenesis. Our results also suggest that Sam68, a Src-family?kinase (SFK) substrate, interacts with E2F2 and represses E2F2- mediated ECE-1b expression. Consistently, we found that Sam68-knockout mice are hypotensive. These findings are particularly exciting, because clinical studies have identified an E2F binding-site polymorphism in the ECE-1b promoter and another independent polymorphism in the C-terminal Src kinase (CSK, a major physiological inhibitor of SFK) gene that are strongly associated with human hypertension. Collectively, our results and observations reported by other laboratories may have identified a previously unknown mechanism of BP control that is governed by the Sam68/E2F2?ECE-1b pathway, and deregulation of this pathway may contribute to BP disorders, including hypertension in humans. However, despite strong evidence indicating that E2F2 and Sam68 regulates vessel contractility, the Sam68/E2F2?ECE-1b pathway has not been explicitly linked to BP regulation, and the mechanisms by which Sam68/E2F2 signaling regulates ECE-1b expression and vascular function remain uncharacterized. The objective of this application is to elucidate the mechanisms underlying the E2F2-mediated regulation of vascular function and BP. Our central hypothesis is that E2F2 recruits both co-activators and co-repressors (e.g., Sam68) to the ECE-1b promoter, thereby regulating ECE-1 activity and preserving a normal contractile state in arteries. Furthermore, dysregulation of E2F2 and the E2F2/Sam68 interaction contributes to BP disorders by inducing aberrations in ECE-1 activity and ET-1 biogenesis. We will accomplish our objective with a series of experiments organized under three specific aims: 1) to elucidate the molecular mechanisms by which E2F2 regulates ECE-1b expression; 2) to define the functional significance of the Sam68/E2F2 signaling in arterial ECs and intact vessels; and 3) to determine the consequences of dysfunctional Sam68/E2F2?ECE-1b pathway on BP control. We anticipate that the work proposed in this project will extensively characterize the novel SFK/Sam68?E2F2/ECE-1b pathway in the regulation of vascular function and BP control, and may potentially reveal new targets for future research into preventive and therapeutic interventions for the treatment of hypertension.