A putative GSK3β binding site in the aquaporin 1 (AQP1) C‐terminal tail controls pulmonary arterial smooth muscle cell (PASMC) function.

Abstract

Pulmonary hypertension (PH) is a devastating disease, characterized by functional and structural pulmonary vascular changes. In distal, small diameter pulmonary arteries, PASMC migration and proliferation are important contributors to the development of PH. In past studies, we showed that hypoxia, a stimulus for PH, elevated the expression of the water channel, aquaporin 1 (AQP1). We also showed that AQP1 induced PASMC migration and proliferation via a mechanism involving β‐catenin protein accumulation, which required the AQP1 C‐terminal tail but was independent of water transport. In this study, we wanted to elucidate the mechanism underlying AQP1‐mediated regulation of β‐catenin in PASMCs. β‐catenin can play various roles in the cell, and its protein levels are tightly regulated by the serine/threonine kinase, glycogen synthase kinase 3 β (GSK3β). Under control conditions, GSK3β is an important factor regulating the turnover of β‐catenin, binding to β‐catenin and targeting it for degradation. Recently, we performed in silico analysis of the AQP1 C‐terminal tail, and found in the proximal portion of the tail a potential binding site for GSK3β. Thus, we explored whether mutating the putative GSK3β binding site on the C‐terminal tail of AQP1 alters PASMC migration and proliferation and, if so, whether β‐catenin was involved. Primary cultured rat distal PASMCs were harvested and used for experiments. Cells were infected with adenoviral constructs expressing wild‐type AQP1 (AdAQP1), AQP1 with the putative GSK3β binding site mutated (AdAQP1M), or green fluorescent protein (AdGFP), as a control for infection. Infection with AdAQP1 and AdAQP1M increased total and surface AQP1 protein levels compared with AdGFP. The rate of change in water flux into PASMCs, tested by a water permeability assay using calcein self‐quenching, indicates that both AQP1 constructs function similarly as a water channel. While infection with AdAQP1 increased PASMC migration and proliferation, measured by BrdU incorporation and transwell assays, respectively, infection with AdAQP1M had no effect. Our results suggest that AQP1 controls PASMCs migration and proliferation via a mechanism that requires specific residues within the tail region that may form a putative GSK3β binding site. With increased AQP1 levels, as during PH, we think GSK3β is sequestered, β‐catenin escapes degradation, translocates to the nucleus and initiates transcription of the target genes involved in cell proliferation and survival.Support or Funding InformationThis work was funded by HL12651, 18POST34030262