Suppression of RhoA activity by focal adhesion kinase-induced activation of p190RhoGAP - Role in regulation of endothelial permeability
Suppression of RhoA activity by focal adhesion kinase-induced activation of p190RhoGAP - Role in regulation of endothelial permeability
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DOI：
10.1074/jbc.m511248200
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发表时间：
2006-01-27
影响因子：
4.8
通讯作者：
Mehta, D
中科院分区：
文献类型：
--
作者：
Holinstat, M;Knezevic, N;Mehta, D
内皮细胞与细胞外基质蛋白在局部粘连部位的相互作用有助于血管内皮屏障的完整性。虽然黏着斑激酶（FAK）激活是需要的内皮连接通透性增加后的屏障功能的恢复，恢复的基础仍然不清楚。我们测试的假设，FAK激活p190 RhoGAP，因此，负调节RhoA的活性，促进内皮屏障恢复的通透性增加介质凝血酶。我们观察到凝血酶引起RhoA的短暂激活，但更长时间的FAK激活在时间上与屏障功能的恢复相关。凝血酶还诱导p190 RhoGAP的酪氨酸磷酸化，这与RhoA活性的降低相一致。我们进一步证明FAK与p190RhoGAP相关，重要的是，重组FAK在体外磷酸化p190RhoGAP。通过腺病毒表达FRNK（一种显性负性FAK构建体）在单层中抑制FAK阻止了p190 RhoGAP磷酸化，增加了RhoA活性，诱导了肌动蛋白应力纤维形成，并产生了对凝血酶应答的内皮通透性的不可逆增加。我们还观察到，p190 RhoGAP不能减弱RhoA激活的FAK激活的情况下，FRNK诱导。通过C3毒素（肉毒梭菌毒素）抑制RhoA恢复了FRNK表达细胞中的内皮屏障功能。内皮细胞中的这些发现在肺微循环中被重现，其中微血管内皮中的FRNK表达增加了血管通透性。我们的研究表明，FAK诱导的下调RhoA活性通过p190 RhoGAP是一个关键步骤，在信号内皮屏障恢复后，增加内皮细胞通透性。
The interaction of endothelial cells with extracellular matrix proteins at focal adhesions sites contributes to the integrity of vascular endothelial barrier. Although focal adhesion kinase (FAK) activation is required for the recovery of the barrier function after increased endothelial junctional permeability, the basis for the recovery remains unclear. We tested the hypothesis that FAK activates p190RhoGAP and, thus, negatively regulates RhoA activity and promotes endothelial barrier restoration in response to the permeability-increasing mediator thrombin. We observed that thrombin caused a transient activation of RhoA but a more prolonged FAK activation temporally coupled to the recovery of barrier function. Thrombin also induced tyrosine phosphorylation of p190RhoGAP, which coincided with decrease in RhoA activity. We further showed that FAK was associated with p190RhoGAP, and importantly, recombinant FAK phosphorylated p190RhoGAP in vitro. Inhibition of FAK by adenoviral expression of FRNK (a dominant negative FAK construct) in monolayers prevented p190RhoGAP phosphorylation, increased RhoA activity, induced actin stress fiber formation, and produced an irreversible increase in endothelial permeability in response to thrombin. We also observed that p190RhoGAP was unable to attenuate RhoA activation in the absence of FAK activation induced by FRNK. The inhibition of RhoA by the C3 toxin (Clostridium botulinum toxin) restored endothelial barrier function in the FRNK-expressing cells. These findings in endothelial cells were recapitulated in the lung microcirculation in which FRNK expression in microvessel endothelia increased vascular permeability. Our studies demonstrate that FAK-induced down-modulation of RhoA activity via p190RhoGAP is a crucial step in signaling endothelial barrier restoration after increased endothelial permeability.