Connexin 40 regulates lung endothelial permeability in acute lung injury via the ROCK1-MYPT1-MLC20 pathway
Connexin 40 regulates lung endothelial permeability in acute lung injury via the ROCK1-MYPT1-MLC20 pathway
复制标题
Connexin 40 通过 ROCK1-MYPT1-MLC20 通路调节急性肺损伤中的肺内皮通透性。
DOI：
10.1152/ajplung.00012.2018
复制
发表时间：
2019-01-01
影响因子：
4.9
通讯作者：
Kuebler, Wolfgang M.
中科院分区：
文献类型：
--
作者：
Yin, Jun;Lv, Lu;Kuebler, Wolfgang M.
肺血管通透性增加是急性肺损伤(ALI)的一个特征。间隙连接蛋白40(Cx40)是一种广泛存在于肺微血管内皮细胞的缝隙连接蛋白。然而，Cx40在肺血管通透性调节中的作用及其潜在机制尚不清楚。在这里，我们测试了Cx40通过Rho相关蛋白激酶(ROCK)依赖的肌球蛋白轻链(MLC)调节机制参与肺内皮细胞通透性调节的假设。在小鼠气管内酸性或脂多糖诱导的肺损伤模型中，Cx40的遗传缺陷减弱了ALI的关键特征，包括血管屏障失效。在人肺微血管内皮细胞(PMVECs)中，凝血酶诱导的跨内皮细胞电阻丧失可通过Cx40抑制模拟肽((40)GAP(27))、Cx40特异性shRNA或ROCK抑制剂Y27632来减弱。在隔离的灌流小鼠肺中，缝隙连接阻滞剂Carbenoxolone、(40)GAP(27)、Y27632或Cx40的遗传缺陷可消除血小板激活因子诱导的肺重量增加。在内毒素处理的PMVECs和盐酸处理的小鼠肺中，MLC20的磷酸化都显著增加。在脂多糖处理的PMVECs和盐酸处理的小鼠肺中，ROCK1的表达均增加，并且与MLC20的磷酸化平行。免疫共沉淀实验表明，ROCK1和Cx40之间存在蛋白质相互作用。脂多糖诱导的ROCK1上调和MLC20的磷酸化可被Cx40基因敲除所阻断。脂多糖引起针对亚基1的肌球蛋白磷酸酶的磷酸化，该作用可被Y27632或Cx40-shRNA所阻断。我们的发现揭示了Cx40在调节ROCK1和MLC20中的作用，而ROCK1和MLC20是导致ALI肺血管屏障衰竭的关键因素。
Increased pulmonary vascular permeability is a hallmark of acute lung injury (ALI). Connexin 40 (Cx40) is a gap junctional protein abundantly present in the lung microvascular endothelium. Yet, the role of Cx40 in the regulation of lung vascular permeability and its underlying mechanisms are unclear. Here, we tested the hypothesis that Cx40 participates in regulation of lung endothelial permeability via a mechanism involving a Rho-associated protein kinase (ROCK) dependent regulation of myosin light chain (MLC). In murine models of intratracheal acid-or LPS-induced lung injury, genetic deficiency of Cx40 attenuated key features of ALI including vascular barrier failure. In human pulmonary microvascular endothelial cells (PMVECs), thrombin-induced loss of transendothelial electrical resistance was attenuated by a Cx40-inhibiting mimetic peptide ((40)GAP(27)), Cx40-specific shRNA, or ROCK inhibitor Y27632. In isolated perfused mouse lungs, platelet-activating factor-induced lung weight gain was abrogated by gap junction blocker carbenoxolone, (40)GAP(27), Y27632, or genetic deficiency of Cx40. Phosphorylation of MLC20 increased drastically in both LPS-treated PMVECs and HCl-treated mouse lungs. Expression of ROCK1 was increased in both LPS-treated PMVECs and HCl-treated mouse lungs, and paralleled by phosphorylation of MLC20. Coimmunoprecipitation experiments revealed protein-protein interaction between ROCK1 and Cx40. LPS-induced upregulation of ROCK1 and phosphorylation of MLC20 were blocked by knockdown of Cx40. LPS caused phosphorylation of myosin phosphatase targeting subunit 1, which could be abrogated by Y27632 or Cx40-shRNA. Our findings reveal a role of Cx40 in regulation of ROCK1 and MLC20 that contributes critically to lung vascular barrier failure in ALI.