1 Guangzhou Institute of Respiratory Diseases, State Key Laboratory of Respiratory Diseases, The First Affiliated Hospital, Guangzhou Medical University, Guangzhou, People's Republic of China; and.
Am J Respir Cell Mol Biol. 2014 Jun;50(6):1010-20. doi: 10.1165/rcmb.2013-0374OC.
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.
肺血管平滑肌细胞（PASMC）增殖和迁移是肺动脉高压发展过程中血管重构的重要因素。我们之前的研究表明，水通道蛋白家族的成员水通道蛋白 1（AQP1）在 PASMC 中表达，并对低氧诱导的迁移是必需的；然而，AQP1 控制这种反应的机制尚不清楚。AQP1 的 C 末端尾部包含假定的钙（EF 手）和蛋白质结合位点。因此，我们想探讨 AQP1 的 C 末端尾部或 EF 手基序是否是迁移和增殖所必需的。从远端肺动脉分离大鼠 PASMC，分别用 BrdU 掺入和 Transwell 过滤器测量增殖和迁移。为了耗尽 AQP1，用 AQP1 小干扰 RNA（siRNA）或非靶向 siRNA 转染 PASMC。AQP1 的敲低减少了 PASMC 的基础增殖和低氧诱导的迁移和增殖。在随后的实验中，使用腺病毒构建体在 PASMC 中表达野生型 AQP1、缺失整个细胞质 C 末端尾部的 AQP1 或具有 EF 手基序突变的 AQP1。对于所有 AQP1 构建体，感染均增加了 AQP1 蛋白水平、水通透性以及低渗刺激引起的细胞体积变化。野生型和 EF 手突变型 AQP1 的感染，但不是 C 末端缺失型 AQP1 的感染，增加了 PASMC 的迁移和增殖。我们的结果表明，AQP1 控制 PASMC 的增殖和迁移，其机制需要该蛋白的 C 末端尾部，但不依赖于水转运或 EF 手基序。
