Swelling-activated Cl- currents and intracellular CLC-3 are involved in proliferation of human pulmonary artery smoothmuscle cells
Swelling-activated Cl- currents and intracellular CLC-3 are involved in proliferation of human pulmonary artery smoothmuscle cells
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DOI：
10.1097/hjh.0000000000000013
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发表时间：
2014-02-01
影响因子：
4.9
通讯作者：
Backx, Peter H.
中科院分区：
文献类型：
--
作者：
Liang, Wenbin;Huang, Lihong;Backx, Peter H.
背景：肺动脉平滑肌细胞(PASMCs)的增殖导致不利的血管重构，并导致肺动脉高压，尽管接受了治疗，这种疾病每年仍有15%的死亡率。我们先前的研究表明，肿胀激活的氯电流(I-CI，I-Swell)在PASMC增殖中上调，而非特异性的氯电流阻滞剂抑制增殖。方法与结果：在本研究中，我们发现在体外培养的人PASMC中，特异性的I-Cl、I-Swell被抑制(IC_(50)=2.7m ol/L)，I-C l、I-Swell被抑制(IC_(50)=6.9m ol/L)。方法与结果：在本研究中，我们发现特异性I-Cl、I-Swell在人PASMC增殖中的作用呈剂量依赖性地阻断(IC_(50)=2.7m ol/L)，并抑制其增殖(IC_(50)=6.9m ol/(4-[(2-butyl-6，7-dichloro-2-cyclopentyl-2，3-dihydro-1-oxo-1H-inden-5-yl)oxy])。为了确定可能的氯通道基因(ClC-1到ClC-7，ClC-KA和ClC-KB，以及BEST-1到BEST-4)在hPASMCs中的表达，我们研究了氯通道基因在hPASMC中的表达。ClC-2到ClC-7和BEST-1在hPASMCs中均有表达，其中最丰富的基因是ClC-3，这是一种与I-Cl、I-Swell连锁的通道基因。在hPASMCs中稳定表达针对ClC-3转录本的适合microRNA的shRNA，与对照shRNA相比，选择性地使ClC-3mRNA降低80%以上，抑制hPASMC增殖(~gt；45%)，但不改变I-Cl、I-Swell。与此一致的是，免疫细胞染色显示ClC-3蛋白主要位于培养的增殖期hPASMCs的胞内区。靶向ClC-3的shRNA显著降低细胞内ClC-3蛋白水平。I-C l的另一个候选分子I-Swell(即CLC-2)也主要分布在细胞内。结论：我们的研究结果支持I-C l、I-Swell和CLC-3在PASMC增殖中起作用的结论，但CLC-3通道不是这些细胞I-Cl、I-Swell的基础。
Background:Proliferation of pulmonary artery smooth muscle cells (PASMCs) leads to adverse vascular remodeling and contributes to pulmonary arterial hypertension, a condition associated with a 15% annual mortality despite treatment. We previously showed that swelling-activated Cl- currents (I-Cl,I-swell) are upregulated in PASMC proliferation and that nonspecific Cl- current blockers inhibit proliferation. However, the specific role of I-Cl,I-swell in PASMC proliferation and its molecular underpinning remain unknown.Methods and results:In the present study, we found that the specific I-Cl,I-swell blocker, DCPIB (4-[(2-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy] butanoic acid), dose-dependently blocked (IC50=2.7mol/l) I-Cl,I-swell and inhibited (IC50=6.9mol/l) proliferation in isolated human PASMCs (hPASMCs). To identify the Cl- channel genes underlying I-Cl,I-swell and regulating hPASMC proliferation, we measured the mRNA expression of candidate Cl- channel genes (CLC-1 to CLC-7, CLC-Ka and CLC-Kb, and BEST-1 to BEST-4) in hPASMCs. CLC-2 to CLC-7 and BEST-1 are expressed in hPASMCs, with the most abundant gene being CLC-3, a channel gene previously linked to I-Cl,I-swell. Although stable expression of a microRNA-adapted shRNA targeting CLC-3 transcripts in hPASMCs selectively reduced CLC-3 mRNA by more than 80% and inhibited hPASMC proliferation (by >45%) compared with control-shRNA, it did not alter I-Cl,I-swell. Consistent with this observation, immunocytostaining studies revealed that CLC-3 protein is primarily located in intracellular areas of cultured proliferative hPASMCs. The intracellular CLC-3 protein levels were profoundly reduced by shRNA targeting CLC-3. The other molecular candidate for I-Cl,I-swell (i.e.,CLC-2) also showed a mainly intracellular distribution.Conclusion:Our findings support the conclusion that both I-Cl,I-swell and CLC-3 play a role in PASMC proliferation, but CLC-3 channels do not underlie I-Cl,I-swell in these cells.