Role of cGMP-dependent protein kinase in regulation of pulmonary vascular smooth muscle cell adhesion and migration: effect of hypoxia
Role of cGMP-dependent protein kinase in regulation of pulmonary vascular smooth muscle cell adhesion and migration: effect of hypoxia
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DOI：
10.1152/ajpheart.00077.2008
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发表时间：
2009-07-01
影响因子：
4.8
通讯作者：
Raj, J. Usha
中科院分区：
文献类型：
--
作者：
Negash, S.;Narasimhan, S. R.;Raj, J. Usha
[10] Negash S，Narasimhan SR，Zhou W，Liu J，Wei FL，Tian J，Raj JU. cGMP依赖性蛋白激酶在调节肺血管平滑肌细胞粘附和迁移中的作用：缺氧的影响。Am J Physiol Heart Circ Physiol 297：H304-H312，2009.首次发表于2009年5月1日; doi：10.1152/ajpheart.00077.2008。长期暴露于缺氧可导致肺血管重构和肺动脉高压。缺氧通过影响细胞粘附、迁移和细胞外基质蛋白的分泌，诱导肺血管平滑肌细胞（PVSMC）增殖和血管重塑。我们以前表明，急性缺氧降低cGMP依赖性蛋白激酶（PKG）在PVSMC的活性和PKG在维持常氧分化的收缩表型中发挥作用。在这项研究中，我们研究了缺氧对PVSMC粘附和迁移的影响以及PKG在这些功能中的作用。将绵羊胎肺动脉平滑肌细胞在常氧（Po（2）= 100 Torr）或缺氧（Po（2）= 30-40 Torr）条件下孵育，或在常氧条件下用PKG抑制剂DT-3处理24 h。为了进一步研究PKG在调节粘附和迁移中的作用，用全长PKG 1 α [PKG-绿色荧光蛋白（GFP）]或显性阴性构建体（G1 α R-GFP）瞬时转染PVSMC。测定细胞与细胞外基质蛋白的粘附，并通过α/β-整合素介导的细胞粘附阵列评估整合素介导的粘附。暴露于缺氧（24 h）和DT-3对PKG 1的药理学抑制显著促进了α（4）-、β（1）-和α（5）β（1）-整合素介导的与纤连蛋白、层粘连蛋白和腱生蛋白的粘附，并导致细胞迁移增加。同样地，通过显性负性PKG 1 α构建体的表达抑制PKG增加了细胞粘附和迁移，与缺氧诱导的细胞粘附和迁移相当。与整合素介导的细胞粘附相关的动态肌动蛋白重组部分受肌动蛋白结合蛋白cofilin调节，其（Ser 3）磷酸化抑制其肌动蛋白切断活性。我们发现PKG表达和活性的增加与丝切蛋白（Ser 3）磷酸化的减少有关，这意味着PKG在调节丝切蛋白活性和肌动蛋白动力学中发挥作用。总之，这些发现确定cGMP/PKG 1信号传导作为暴露于常氧与缺氧的PVSMC之间的功能差异的中心。
Negash S, Narasimhan SR, Zhou W, Liu J, Wei FL, Tian J, Raj JU. Role of cGMP-dependent protein kinase in regulation of pulmonary vascular smooth muscle cell adhesion and migration: effect of hypoxia. Am J Physiol Heart Circ Physiol 297: H304-H312, 2009. First published May 1, 2009; doi: 10.1152/ajpheart.00077.2008.-Exposure to prolonged hypoxia can result in pulmonary vascular remodeling and pulmonary hypertension. Hypoxia induces pulmonary vascular smooth muscle cell (PVSMC) proliferation and vascular remodeling by affecting cell adhesion and migration and secretion of extracellular matrix proteins. We previously showed that acute hypoxia decreases cGMP-dependent protein kinase (PKG) activity in PVSMC and that PKG plays a role in maintaining the differentiated contractile phenotype in normoxia. In this study, we investigated the effect of hypoxia on PVSMC adhesion and migration and the role of PKG in these functions. Ovine fetal pulmonary artery SMC were incubated in normoxia (Po(2) similar to 100 Torr) or hypoxia (Po(2) similar to 30-40 Torr) or treated with the PKG inhibitor DT-3 for 24 h in normoxia. To further study the role of PKG in the modulation of adhesion and migration, PVSMC were transiently transfected with a full-length PKG1 alpha [PKG-green fluorescent protein (GFP)] or a dominant-negative construct (G1 alpha R-GFP). Cell adhesion to extracellular matrix proteins was determined, and integrin-mediated adhesion was assessed by alpha/beta-integrin-mediated cell adhesion array. Exposure to hypoxia (24 h) and pharmacological inhibition of PKG1 by DT-3 significantly promoted adhesion mediated by alpha(4)-, beta(1)-, and alpha(5)beta(1)-integrins to fibronectin, laminin, and tenacin and also resulted in increased cell migration. Likewise, inhibition of PKG by expression of a dominant-negative PKG1 alpha construct increased cell adhesion and migration, comparable to that induced by hypoxia. Dynamic actin reorganization associated with integrin-mediated cell adhesion is partly regulated by the actin-binding protein cofilin, the (Ser3) phosphorylation of which inhibits its actin-severing activity. We found that increased PKG expression and activity is associated with decreased cofilin (Ser3) phosphorylation, implying a role for PKG in the modulation of cofilin activity and actin dynamics. Together, these findings identify cGMP/PKG1 signaling as central to the functional differences between PVSMC exposed to normoxia versus hypoxia.