Department of Ion Channel Pharmacology, School of Pharmacy, China Medical University, Shenyang 110122, China.
J Adv Res. 2020 Sep 15;29:23-32. doi: 10.1016/j.jare.2020.09.003. eCollection 2021 Mar.
Ca-activated Cl channel TMEM16A is expressed in endothelial cells, and contributes to many diseases such as hypertension, blood-brain barrier dysfunction, and pulmonary hypertension. It remains unclear whether TMEM16A regulates endothelial angiogenesis, which participates in many physiological and pathological processes. Cholesterol regulates many ion channels including TMEM16A, and high cholesterol levels contribute to endothelial dysfunction. It remains to be determined whether cholesterol regulates TMEM16A expression and function in endothelial cells.
This study aimed to investigate whether cholesterol regulated TMEM16A expression and function in endothelial angiogenesis.
Whole-cell patch clamp techniques were used to record Ca-activated Cl currents in human aortic endothelial cells (HAECs) and HEK293 cells transfected with TMEM16A-overexpressing plasmids. Western blot was used to examine the expression of TMEM16A and DNA methyltransferase 1 (DNMT1) in HAECs. CCK-8 assay, would healing assay, and tube formation assay were used to test endothelial cell proliferation, migration and angiogenesis, respectively.
TMEM16A mediates the Ca-activated Cl channel in HAECs. Cholesterol treatment inhibited TMEM16A expression via upregulation of DNMT1 in HAECs, and the inhibitory effect of cholesterol on TMEM16A expression was blocked by 5-aza, the DNMT1 inhibitor. In addition, direct application of cholesterol inhibited TMEM16A currents in heterologous HEK293 cells with an IC of 0.1209 μM. Similarly, cholesterol directly inhibited TMEM16A currents in HAECs. Furthermore, TMEM16A knockdown increased tube formation, cell migration and proliferation of HAECs, and TMEM16A overexpression produced the opposite effect.
This study reveals a novel mechanism of cholesterol-mediated TMEM16A inhibition, by which cholesterol reduces TMEM16A expression via DNMT1-mediated methylation and directly inhibits channel activities. TMEM16A channel inhibition promotes endothelial cell angiogenesis.
钙激活氯离子通道 TMEM16A 在内皮细胞中表达，与高血压、血脑屏障功能障碍和肺动脉高压等多种疾病有关。TMEM16A 是否调节参与许多生理和病理过程的内皮血管生成尚不清楚。胆固醇调节包括 TMEM16A 在内的许多离子通道，高胆固醇水平导致内皮功能障碍。胆固醇是否调节内皮细胞中 TMEM16A 的表达和功能仍有待确定。
本研究旨在探讨胆固醇是否调节内皮血管生成中的 TMEM16A 表达和功能。
使用全细胞膜片钳技术记录人主动脉内皮细胞（HAECs）和转染 TMEM16A 过表达质粒的 HEK293 细胞中的 Ca 激活 Cl 电流。Western blot 用于检测 HAECs 中 TMEM16A 和 DNA 甲基转移酶 1（DNMT1）的表达。CCK-8 测定、伤口愈合测定和管形成测定分别用于测试内皮细胞增殖、迁移和血管生成。
TMEM16A 介导 HAECs 中的 Ca 激活 Cl 通道。胆固醇处理通过上调 HAECs 中的 DNMT1 抑制 TMEM16A 表达，DNMT1 抑制剂 5-氮杂胞苷可阻断胆固醇对 TMEM16A 表达的抑制作用。此外，胆固醇直接在异源 HEK293 细胞中以 0.1209 μM 的 IC 抑制 TMEM16A 电流。同样，胆固醇直接抑制 HAECs 中的 TMEM16A 电流。此外，TMEM16A 敲低增加了 HAECs 的管形成、细胞迁移和增殖，而 TMEM16A 过表达则产生相反的效果。
本研究揭示了胆固醇介导的 TMEM16A 抑制的一种新机制，即胆固醇通过 DNMT1 介导的甲基化降低 TMEM16A 表达，并直接抑制通道活性。TMEM16A 通道抑制促进内皮细胞血管生成。
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