Aquaporin 1 (AQP1)/ GSK3β interactions control pulmonary arterial smooth muscle cell (PASMC) function

Pulmonary hypertension (PH) is a complex, incurable, devastating condition, due to pathology of the pulmonary circulation. PH can be induced by hypoxia and is notable for severe wall thickening in the distal vasculature, characterized by enhanced PASMC migration and proliferation. Previously our lab discovered AQP1, a water channel, was highly expressed in PASMCs and mediated hypoxia-induced migration and proliferation. Moreover, increasing AQP1 protein was sufficient to induce PASMC migration and proliferation without hypoxia. We showed the C-terminal tail was required for regulating cell function via accumulation of β-catenin independent of water transport.β-catenin protein is tightly controlled by glycogen synthase kinase 3 β (GSK3β). Binding to GSK3β results in β-catenin degradation; when stabilized, β-catenin mediates transcription of pro-growth genes. Through in silico analysis, we identified a putative binding site for GSK3β in the AQP1 C-terminal tail, allowing us to hypothesize a potential mechanism whereby AQP1 binds GSK3β, releasing β-catenin from degradation, and thereby increases PASMC migration and proliferation.First, we used BioID, a proximity-based assay, to identify GSK3β and AQP1 interactions. We designed BioID constructs with wild-type AQP1 (BioID-AQP1) and AQP1 with mutations in the putative GSK3β binding site (BioID-AQP1M) fused to biotin ligase and compared results to wild-type AQP1 (AQP1), as a control for transfection. We used HEK293 cells which do not express endogenous AQP1. In cells transfected with AQP1 and incubated with cell permeable biotin, following biotin-affinity capture of streptavidin, no biotinylated GSK3b was measured. In cells transfected with BioID-AQP1, a significant amount of GSK3β protein was biotinylated, suggesting AQP1/GSK3β protein-protein interactions. When the putative GSK3b binding site was mutated, very low levels of GSK3b were biotinylated.Next, primary cultured rat distal PASMCs were infected with adenoviral constructs expressing wild-type AQP1 (AdAQP1), AQP1 with the putative GSK3β binding site mutated (AdAQP1M), or green fluorescent protein (AdGFP; control for infection). Compared to AdGFP, infection with AdAQP1 and AdAQP1M increased PASMC total and surface AQP1 protein levels. Using calcein self-quenching, the rate change of water influx was similar for both AQP1 constructs, indicating the mutation did not impair function as a water channel. Infection with AdAQP1 significantly increased both PASMC migration and proliferation, measured by BrdU incorporation and transwell assays, respectively, whereas infection with AdAQP1M had no effect.Our results suggest AQP1 controls PASMC migration and proliferation via a mechanism that requires GSK3β interactions. We speculate high levels of AQP1, as occur during PH, act as a sink for GSK3β, allowing β-catenin to escape from the destruction complex and facilitate cell migration and initiate gene transcription involved in cell proliferation. HL126514 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.