Lade Julie M, Andrade Manuella R, Undem Clark, Walker Jasmine, Jiang Haiyang, Yun Xin, Shimoda Larissa A
Division of Pulmonary and Critical Care Medicine, Department of Medicine, Baltimore, MD, United States.
Department of Physiology, Johns Hopkins School of Medicine, Baltimore, MD, United States.
Front Physiol. 2023 Feb 28;14:1108304. doi: 10.3389/fphys.2023.1108304. eCollection 2023.
Exposure to hypoxia, due to high altitude or chronic lung disease, leads to structural changes in the pulmonary vascular wall, including hyperplasia and migration of pulmonary arterial smooth muscle cells (PASMCs). Previous studies showed that hypoxia upregulates the expression of Na/H exchanger isoform 1 (NHE1) and that inhibition or loss of NHE1 prevents hypoxia-induced PASMC migration and proliferation. The exact mechanism by which NHE1 controls PASMC function has not been fully delineated. In fibroblasts, NHE1 has been shown to act as a membrane anchor for actin filaments, binding of the adaptor protein, ezrin. Thus, in this study, we tested the role of ezrin and NHE1/actin interactions in controlling PASMC function. Using rat PASMCs exposed to hypoxia (4% O, 24 h) we found that hypoxic exposure increased phosphorylation (activation) of ezrin, and promoted interactions between NHE1, phosphorylated ezrin and smooth muscle specific -actin (SMA) as measured immunoprecipitation and co-localization. Overexpression of wild-type human NHE1 in the absence of hypoxia was sufficient to induce PASMC migration and proliferation, whereas inhibiting ezrin phosphorylation with NSC668394 suppressed NHE1/SMA co-localization and migration in hypoxic PASMCs. Finally, overexpressing a version of human NHE1 in which amino acids were mutated to prevent NHE1/ezrin/SMA interactions was unable to increase PASMC migration and proliferation despite exhibiting normal Na/H exchange activity. From these results, we conclude that hypoxic exposure increases ezrin phosphorylation in PASMCs, leading to enhanced ezrin/NHE1/SMA interaction. We further speculate that these interactions promote anchoring of the actin cytoskeleton to the membrane to facilitate the changes in cell movement and shape required for migration and proliferation.
由于高海拔或慢性肺部疾病导致的缺氧，会引起肺血管壁的结构变化，包括肺动脉平滑肌细胞（PASMCs）的增生和迁移。先前的研究表明，缺氧会上调钠/氢交换体1（NHE1）的表达，而抑制或缺失NHE1可阻止缺氧诱导的PASMC迁移和增殖。NHE1控制PASMC功能的确切机制尚未完全阐明。在成纤维细胞中，NHE1已被证明可作为肌动蛋白丝的膜锚，与衔接蛋白埃兹蛋白结合。因此，在本研究中，我们测试了埃兹蛋白以及NHE1/肌动蛋白相互作用在控制PASMC功能中的作用。使用暴露于缺氧环境（4%氧气，24小时）的大鼠PASMC，我们发现缺氧暴露增加了埃兹蛋白的磷酸化（激活），并促进了NHE1、磷酸化埃兹蛋白和平滑肌特异性肌动蛋白（SMA）之间的相互作用，这通过免疫沉淀和共定位来测量。在无缺氧情况下过表达野生型人NHE1足以诱导PASMC迁移和增殖，而用NSC668394抑制埃兹蛋白磷酸化可抑制缺氧PASMC中NHE1/SMA共定位和迁移。最后，过表达一种人NHE1变体，其中氨基酸发生突变以阻止NHE1/埃兹蛋白/SMA相互作用，尽管其钠/氢交换活性正常，但仍无法增加PASMC迁移和增殖。从这些结果中，我们得出结论，缺氧暴露会增加PASMC中埃兹蛋白的磷酸化，导致埃兹蛋白/NHE1/SMA相互作用增强。我们进一步推测，这些相互作用促进肌动蛋白细胞骨架与膜的锚定，以促进迁移和增殖所需的细胞运动和形状变化。
