肿胀激活的 Cl- 电流和细胞内的 CLC-3 参与了人肺动脉平滑肌细胞的增殖。
Swelling-activated Cl- currents and intracellular CLC-3 are involved in proliferation of human pulmonary artery smooth muscle cells.
机构信息
aDepartments of Physiology and Medicine bDivision of Cardiology, University Health Network, University of Toronto, Toronto, Canada cChina-Japan Union Hospital, Jilin University, Changchun, China dDivision of Respirology eThe Li Ka Shing Knowledge Institute, St. Michael's Hospital,University of Toronto, Toronto, Canada fCedars-Sinai Heart Institute, Los Angeles, California, USA *Wenbin Liang and Lihong Huang contributed equally to this study. **Deceased.
出版信息
J Hypertens. 2014 Feb;32(2):318-30. doi: 10.1097/HJH.0000000000000013.
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 (ICl,swell) are upregulated in PASMC proliferation and that nonspecific Cl current blockers inhibit proliferation. However, the specific role of ICl,swell in PASMC proliferation and its molecular underpinning remain unknown.
METHODS AND RESULTS
In the present study, we found that the specific ICl,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 = 2.7 μmol/l) ICl,swell and inhibited (IC50 = 6.9 μmol/l) proliferation in isolated human PASMCs (hPASMCs). To identify the Cl channel genes underlying ICl,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 ICl,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 ICl,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 ICl,swell (i.e.,CLC-2) also showed a mainly intracellular distribution.
CONCLUSION
Our findings support the conclusion that both ICl,swell and CLC-3 play a role in PASMC proliferation, but CLC-3 channels do not underlie ICl,swell in these cells.
背景
肺动脉平滑肌细胞（PASMCs）的增殖导致血管重构不良，并导致肺动脉高压，尽管进行了治疗，但这种疾病的年死亡率仍为 15%。我们之前的研究表明，肿胀激活的氯离子电流（ICl，swell）在 PASMC 增殖中上调，而非特异性氯离子电流阻滞剂可抑制增殖。然而，ICl，swell 在 PASMC 增殖中的具体作用及其分子基础仍不清楚。
方法和结果
在本研究中，我们发现，特异性 ICl，swell 阻滞剂 DCPIB（4-[(2-丁基-6,7-二氯-2-环戊基-2,3-二氢-1-氧代-1H-茚-5-基)氧基]丁酸）可剂量依赖性地阻断（IC50=2.7μmol/l）ICl，swell 并抑制（IC50=6.9μmol/l）分离的人 PASMCs（hPASMCs）的增殖。为了鉴定导致 ICl，swell 和调节 hPASMC 增殖的氯离子通道基因，我们测量了 hPASMCs 中候选氯离子通道基因（CLC-1 至 CLC-7、CLC-Ka 和 CLC-Kb 以及 BEST-1 至 BEST-4）的 mRNA 表达。CLC-2 至 CLC-7 和 BEST-1 在 hPASMCs 中表达，最丰富的基因是 CLC-3，该基因先前与 ICl，swell 相关。尽管 CLC-3 转录物的 microRNA 适应 shRNA 的稳定表达选择性地使 CLC-3 mRNA 减少超过 80%，并与对照 shRNA 相比抑制 hPASMC 增殖（超过 45%），但它并没有改变 ICl，swell。与这一观察结果一致，免疫细胞化学染色研究表明 CLC-3 蛋白主要位于培养增殖的 hPASMCs 的细胞内区域。靶向 CLC-3 的 shRNA 使细胞内 CLC-3 蛋白水平显著降低。ICl，swell 的另一个分子候选物（即 CLC-2）也表现出主要的细胞内分布。
结论
我们的研究结果支持以下结论，即 ICl，swell 和 CLC-3 均在 PASMC 增殖中发挥作用，但 CLC-3 通道不是这些细胞中 ICl，swell 的基础。
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