Protein kinase C-α signals Rho-guanine nucleotide dissociation inhibitor phosphorylation and Rho activation and regulates the endothelial cell barrier function
Protein kinase C-α signals Rho-guanine nucleotide dissociation inhibitor phosphorylation and Rho activation and regulates the endothelial cell barrier function
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DOI：
10.1074/jbc.m101927200
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发表时间：
2001-06-22
影响因子：
4.8
通讯作者：
Malik, AB
中科院分区：
文献类型：
--
作者：
Mehta, D;Rahman, A;Malik, AB
Rho-GDP鸟嘌呤核苷酸解离抑制剂（GDI）与GDP结合形式的Rho复合并抑制其活化。我们研究了蛋白激酶C（PKC）同工酶在Rho激活机制和内皮屏障功能丧失信号中的作用。凝血酶和佛波酯诱导人脐静脉内皮细胞GDI快速磷酸化和Rho-A激活。通过氯化白屈菜红碱抑制PKC可消除凝血酶诱导的GDI磷酸化和Rho激活。PKC的耗竭阻止凝血酶诱导的GDI磷酸化和Rho激活，从而表明这些事件发生在佛波酯敏感的PKC同工酶激活的下游。C3毒素对PKC的消耗或Rho的抑制也阻止了凝血酶诱导的跨内皮电阻的降低（增加的跨内皮渗透性的量度），因此表明PKC诱导的屏障功能障碍是通过Rho依赖性途径介导的。使用抑制剂和显性负突变体，我们发现Rho激活受PKC-α调节。此外，用凝血酶刺激人脐静脉内皮细胞诱导PKC-α与Rho的快速结合，活化的PKC-α而不是PKC-β在体外诱导显著的GDI磷酸化。总之，这些结果表明，PKC-α是至关重要的调节GDI磷酸化，Rho激活，并在信号Rho依赖性内皮屏障功能障碍。
The Rho-GDP guanine nucleotide dissociation inhibitor (GDI) complexes with the GDP-bound form of Rho and inhibits its activation. We investigated the role of protein kinase C (PKC) isozymes in the mechanism of Rho activation and in signaling the loss of endothelial barrier function. Thrombin and phorbol la-myristate 13-acetate induced rapid phosphorylation of GDI and the activation of Rho-A in human umbilical venular endothelial cells. Inhibition of PKC by chelerythrine chloride abrogated the thrombin-induced GDI phosphorylation and Rho activation. Depletion of PKC prevented the thrombin-induced GDI phosphorylation and Rho activation, thereby indicating that these events occurred downstream of phorbol ester-sensitive PKC isozyme activation. The depletion of PKC or inhibition of Rho by C3 toxin also prevented the thrombin-induced decrease in transendothelial electrical resistance (a measure of increased transendothelial permeability), thus indicating that PKC-induced barrier dysfunction was mediated through Rho-dependent pathway. Using inhibitors and dominant-negative mutants, we found that Rho activation was regulated by PKC-alpha. Moreover, the stimulation of human umbilical venular endothelial cells with thrombin induced rapid association of PKC-alpha with Rho, Activated PKC-alpha but not PKC-epsilon induced marked phosphorylation of GDI in vitro. Taken together, these results indicate that PKC-alpha is critical in regulating GDI phosphorylation, Rho activation, and in signaling Rho-dependent endothelial barrier dysfunction.