Activation of Anoctamin-1 Limits Pulmonary Endothelial Cell Proliferation via p38-Mitogen-activated Protein Kinase-Dependent Apoptosis
Activation of Anoctamin-1 Limits Pulmonary Endothelial Cell Proliferation via p38-Mitogen-activated Protein Kinase-Dependent Apoptosis
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DOI：
10.1165/rcmb.2016-0344oc
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发表时间：
2018-05-01
影响因子：
6.4
通讯作者：
Choudhary, Gaurav
中科院分区：
文献类型：
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作者：
Allawzi, Ayed M.;Vang, Alexander;Choudhary, Gaurav
过度增殖的内皮细胞（EC）在肺动脉高压（PAH）的发病机制中起重要作用。Anoctamin（Ano）-1是一种钙激活的氯离子通道，可调节多种细胞类型的细胞增殖和细胞周期。然而，Ano 1在肺内皮中的表达和功能尚不清楚。我们研究了Ano 1是否在肺EC中表达，以及改变Ano 1活性是否会影响EC存活。采用免疫印迹、免疫荧光和亚细胞分级技术检测Ano 1在大鼠肺微血管内皮细胞（RLMVECs）中的表达和定位。使用细胞计数、流式细胞术和半胱天冬酶-3活性来评估响应于小分子Ano 1激活剂Eact的细胞数量和凋亡的变化。分别用5，5 '，6，6'-四氯-1，1 '，3，3'-四乙基苯并咪唑羰花青、碘化物（线粒体膜电位染料）和线粒体活性氧（ROS）染料检测线粒体膜电位和线粒体活性氧（mtROS）的变化。Ano 1在RLMVEC中表达，并在线粒体中富集。用Eact激活Ano 1通过增加细胞凋亡减少RLMVEC计数。Ano 1敲低阻断了Eact的作用。Ano 1激活增加线粒体ROS，降低线粒体膜电位，增加p38磷酸化，并诱导凋亡诱导因子的释放。mtROS抑制减弱Eact介导的p38磷酸化。与对照组相比，从特发性PAH（IPAH）患者分离的肺动脉EC具有较高的Ano 1表达和细胞计数增加。Eact处理减少IPAH细胞中的细胞计数，这与细胞凋亡增加相关。总之，Ano 1在肺EC线粒体中表达。Ano 1的激活促进肺EC和人IPAH-肺动脉EC的凋亡，可能通过增加mtROS和p38磷酸化，导致凋亡。
Hyperproliferative endothelial cells (ECs) play an important role in the pathogenesis of pulmonary arterial hypertension (PAH). Anoctamin (Ano)-1, a calcium-activated chloride channel, can regulate cell proliferation and cell cycle in multiple cell types. However, the expression and function of Ano1 in the pulmonary endothelium is unknown. We examined whether Ano1 was expressed in pulmonary ECs and if altering Ano1 activity would affect EC survival. Expression and localization of Ano1 in rat lung microvascular ECs (RLMVECs) was assessed using immunoblot, immunofluorescence, and subcellular fractionation. Cell counts, flow cytometry, and caspase-3 activity were used to assess changes in cell number and apoptosis in response to the small molecule Ano1 activator, Eact. Changes in mitochondrial membrane potential and mitochondrial reactive oxygen species (mtROS) were assessed using 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine, iodide (mitochondrial membrane potential dye) and mitochondrial ROS dye, respectively. Ano1 is expressed in RLMVECs and is enriched in the mitochondria. Activation of Ano1 with Eact reduced RLMVEC counts through increased apoptosis. Ano1 knockdown blocked the effects of Eact. Ano1 activation increased mtROS, reduced mitochondrial membrane potential, increased p38 phosphorylation, and induced release of apoptosis-inducing factor. mtROS inhibition attenuated Eact-mediated p38 phosphorylation. Pulmonary artery ECs isolated from patients with idiopathic PAH (IPAH) had higher expression of Ano1 and increased cell counts compared with control subjects. Eact treatment reduced cell counts in IPAH cells, which was associated with increased apoptosis. In summary, Ano1 is expressed in lung EC mitochondria. Activation of Ano1 promotes apoptosis of pulmonary ECs and human IPAH-pulmonary artery ECs, likely via increased mtROS and p38 phosphorylation, leading to apoptosis.