Exploring the Role of NHE1 in Pulmonary Arterial Smooth Muscle Cell (PASMC) Migration and Proliferation

Abstract

Pulmonary hypertension (PH) is a severe condition defined by mean pulmonary artery pressure >20 mmHg. PH pathogenesis involves vascular contraction and remodeling characterized by increased PASMC migration and proliferation. Na+/H+ exchanger isoform 1 (NHE1) is a membrane protein that regulates intracellular pH (pHi) and cell volume via export of H+ and import of Na+. This protein also has a cytosolic tail region shown to be involved in non-canonical functions such as cytoskeletal interactions via protein-protein interactions. Of particular interest, the tail region contains a binding site for ERM (ezrin/radixin/moesin) proteins. In other cell types, this binding site regulates actin filament organization, cell shape and migration by interacting with ezrin.We previously found NHE activity, and in some cases NHE1 expression, is increased in PASMCs from PH patients and animal models and was associated with enhanced proliferation and migration. Additionally, NHE inhibition with ethyl isopropyl amiloride reduced migration and proliferation in PASMCs from PH models; however, it is unclear if dysregulation of pHi or other functions of NHE1 are driving these abnormalities. In this study, we wanted to elucidate the mechanism by which NHE1 promotes increased PASMC migration and proliferation. We hypothesized dysregulation of pHi by NHE1 increases proliferation, whereas the interaction between NHE1 and ezrin mediates increased migration.PASMCs were isolated from distal pulmonary arteries of male Wistar rats. Cells were infected with adenovirus containing green fluorescent protein (AdGFP), wild-type NHE1 (AdNHE1-WT) or NHE1 mutants. To analyze the role of pHi regulation, a mutant containing a single amino acid substitution at position 262 was used to prevent ion translocation activity (AdNHE1-I). To analyze the role of protein-protein interactions, we generated a mutant in which a cluster of lysines and arginines in the ezrin binding region was replaced by alanines (AdNHE1-E). To confirm correct protein localization and efficacy of Na+/H+ translocation of the overexpressed proteins, NHE activity was assessed via Na+-dependent recovery following an ammonium pulse. Protein overexpression was confirmed via immunoblotting.Finally, cells underwent experiments to analyze migration (transwell assay) and proliferation (BrdU incorporation). All measurements were made in a blinded fashion.Enhanced expression of wildtype NHE1 increased PASMC migration and proliferation. Expression of the translocation deficient mutant (AdNHE1-I) also increased migration; however, proliferation was unchanged compared to AdGFP. Interestingly, AdNHE1-E (ezrin binding mutant) was unable to increase migration or proliferation. Our results suggest the protein-protein interaction between NHE1 and ezrin is essential for NHE1 to increase PASMC migration and proliferation. In contrast, pH regulation by NHE1 contributes to increased PASMC proliferation, but increased migration is pH-independent. R01HL073859 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.