The aquaporin 1 C-terminal tail is required for migration and growth of pulmonary arterial myocytes.

Pulmonary arterial smooth muscle cell (PASMC) proliferation and migration are important contributors to the vascular remodeling that occurs during development of pulmonary hypertension. We previously demonstrated that aquaporin (AQP)1, a member of the water channel family of proteins, was expressed in PASMCs and was necessary for hypoxia-induced migration; however, the mechanism by which AQP1 controls this response is unclear. The C-terminal tail of AQP1 contains putative calcium (EF-hand) and protein binding sites. Thus, we wanted to explore whether the C-terminal tail or the EF-hand motif of AQP1 was required for migration and proliferation. Rat PASMCs were isolated from distal pulmonary arteries, and proliferation and migration were measured using BrdU incorporation and transwell filters, respectively. To deplete AQP1, PASMCs were transfected with AQP1 small interference RNA (siRNA) or nontargeting siRNA. Knockdown of AQP1 reduced basal proliferation and hypoxia-induced migration and proliferation in PASMCs. In subsequent experiments, wild-type AQP1, AQP1 lacking the entire cytoplasmic C-terminal tail, or AQP1 with a mutation in the EF-hand motif were expressed in PASMCs using adenoviral constructs. For all AQP1 constructs, infection increased AQP1 protein levels, water permeability, and the change in cell volume induced by hypotonic challenge. Infection with wild-type and EF-hand mutated AQP1, but not C-terminal-deleted AQP1, increased PASMC migration and proliferation. Our results suggest that AQP1 controls proliferation and migration in PASMCs and that the mechanism requires the C-terminal tail of the protein but is independent of water transport or the EF-hand motif.

Hypoxia-induced migration in pulmonary arterial smooth muscle cells requires calcium-dependent upregulation of aquaporin 1.

Pulmonary arterial smooth muscle cell (PASMC) migration is a key component of the vascular remodeling that occurs during the development of hypoxic pulmonary hypertension, although the mechanisms governing this phenomenon remain poorly understood. Aquaporin-1 (AQP1), an integral membrane water channel protein, has recently been shown to aid in migration of endothelial cells. Since AQP1 is expressed in certain types of vascular smooth muscle, we hypothesized that AQP1 would be expressed in PASMCs and would be required for migration in response to hypoxia. Using PCR and immunoblot techniques, we determined the expression of AQPs in pulmonary vascular smooth muscle and the effect of hypoxia on AQP levels, and we examined the role of AQP1 in hypoxia-induced migration in rat PASMCs using Transwell filter assays. Moreover, since the cytoplasmic tail of AQP1 contains a putative calcium binding site and an increase in intracellular calcium concentration ([Ca(2+)](i)) is a hallmark of hypoxic exposure in PASMCs, we also determined whether the responses were Ca(2+) dependent. Results were compared with those obtained in aortic smooth muscle cells (AoSMCs). We found that although AQP1 was abundant in both PASMCs and AoSMCs, hypoxia selectively increased AQP1 protein levels, [Ca(2+)](i), and migration in PASMCs. Blockade of Ca(2+) entry through voltage-dependent Ca(2+) or nonselective cation channels prevented the hypoxia-induced increase in PASMC [Ca(2+)](i), AQP1 levels, and migration. Silencing AQP1 via siRNA also prevented hypoxia-induced migration of PASMCs. Our results suggest that hypoxia induces a PASMC-specific increase in [Ca(2+)](i) that results in increased AQP1 protein levels and cell migration.