Our long-term goal is to understand the mechanism by which chronic hypoxia (CH) causes pulmonary hypertension. Previous studies have characterized the morphologic and functional changes that occur in the pulmonary vasculature in response to CH but the cellular mechanisms underlying these changes remain poorly understood. We demonstrated that CH induces alterations in pulmonary arterial smooth muscle cell (PASMC) intracellular pH (pHi) homeostasis, due to increased Na+/H+ exchanger isoform 1 (NHE1) expression. Changes in pHi have profound influences on cell function, although the mechanism by which pHi modulates PASMC growth, migration and contraction during CH are incompletely understood. Studies have suggested that NHE1 could act as a membrane anchor independent of its ion translocation properties. NHE1 co-localizes with and binds to ezrin, a member of the ERM (ezrin/radixin/moesin) family of proteins, which contain multiple binding domains and are important mediators of protein-protein interactions. Of particular interest, the C-terminal of ezrin contains a binding site for filamentous actin, providing a possible link between NHE1 and cytoskeletal rearrangement. Ezrin also binds ezrin/radixin/ moesin-binding phosphoprotein of 50 kDa (EBP50), or Na+/H+ exchange regulatory factor 1 (NHERF1). NHERF1 acts as a scaffold linking together intracellular signaling complexes. Despite the connotation inferred by its name, NHERF1 does not bind NHE1. We found that PASMCs express NHERF1 and that during CH, NHERF1 expression decreases whereas NHE1 expression increases. We also found that PASMCs express ezrin;the effect of CH on ezrin expression is unknown. That NHE1 utilizes the same binding motif on ezrin as NHERF1 suggests competition between NHE1 and NHERF1 for ezrin binding. If true, a decrease in NHERF1 expression with increased NHE1 and/or ezrin expression during CH could create conditions favorable for membrane/cytoskeleton interactions. Thus, we hypothesize that during CH, decreased NHERF/ezrin interactions and a concomitant increase in ezrin/NHE1 binding results in enhanced NHE1-actin filament tethering, resulting in PASMC migration, hypertrophy, proliferation and contraction. To test this hypothesis, we will use a combination of techniques including confocal FRET fluorescent microscopy, knockdown and overexpression approaches, co-immunoprecipitation, migration and proliferation assays and traction microscopy in PASMCs to determine whether: 1) CH alters NHERF1 and ezrin expression, localization and/or association;2) the interaction between NHE1 and actin is altered during CH and, if so, whether this requires ezrin and 3) ezrin-mediated NHE1-actin binding is required for CH-induced changes in PASMC function.
The experiments in this proposal will explore cellular mechanisms involved in the development of hypoxic pulmonary hypertension, a devastating disease with limited treatment options. In the United States, there are an estimated 600,000 patients with chronic lung disease that are at risk for the development of pulmonary hypertension due to hypoxemia, with approximately one half of these patients likely to already have pulmonary hypertension. Understanding the cellular changes that occur in the pulmonary vasculature with development of hypoxic pulmonary hypertension is key to advancing treatment and therapeutic options and reducing the health care cost burden associated with this disease.
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