Harrington Elizabeth O, Shannon Christopher J, Morin Nicole, Rowlett Heather, Murphy Christopher, Lu Qing
Pulmonary Vascular Research Laboratory, Providence VA Medical Center, Department of Medicine, Brown Medical School, 151 Research Services, 830 Chalkstone Avenue, Providence, RI 02908, USA.
Exp Cell Res. 2005 Aug 15;308(2):407-21. doi: 10.1016/j.yexcr.2005.05.005.
We have shown that PKCdelta enhanced microvascular endothelial basal barrier function, correlating with elevated RhoA GTPase activity and increased focal contact formation. In the current study, we investigated signaling pathways important in PKCdelta modulation of barrier function in unstimulated endothelial cell monolayers by assessing the effects of PKCdelta inhibition in endothelial cells (EC) derived from rat pulmonary artery (PAEC) and epididymus (FPEC). Rottlerin exposure or Ad PKCdeltadn infection significantly enhanced monolayer permeability in both EC. Immunofluorescence analyses demonstrated fewer stress fibers and focal contacts in rottlerin-treated or Ad PKCdeltadn-infected EC; yet, PKCdelta inhibition caused no significant changes in microtubule structures. These changes correlated with a reduction in both focal adhesion kinase (FAK) and RhoA GTPase activities. Microfilament stabilization significantly attenuated the focal contact and barrier disruptive effects of rottlerin. FAK overexpression did not blunt the effects of rottlerin-induced barrier dysfunction or stress fiber and focal contact disruption. Conversely, GFP-linked dominant active RhoA overexpression protected EC from stress fiber and focal contact disruption induced by both rottlerin exposure and overexpression of PKCdelta dominant negative protein. Additionally, PKCdelta immunoprecipitated with p190RhoGAP and p120RasGAP, modulators of RhoA activity. Thus, PKCdelta may regulate basal endothelial barrier function by stabilizing microfilaments and focal contacts by regulating RhoA GTPase activity through upstream modulators, p190RhoGAP and p120RasGAP.
我们已经表明，蛋白激酶Cδ（PKCδ）增强了微血管内皮细胞的基础屏障功能，这与RhoA GTP酶活性升高和黏着斑形成增加相关。在本研究中，我们通过评估PKCδ抑制对源自大鼠肺动脉的内皮细胞（PAEC）和附睾的内皮细胞（FPEC）的影响，研究了在未刺激的内皮细胞单层中PKCδ调节屏障功能的重要信号通路。暴露于rottlerin或感染Ad PKCdeltadn均显著增强了两种内皮细胞单层的通透性。免疫荧光分析显示，在经rottlerin处理或Ad PKCdeltadn感染的内皮细胞中，应力纤维和黏着斑较少；然而，PKCδ抑制并未引起微管结构的显著变化。这些变化与黏着斑激酶（FAK）和RhoA GTP酶活性的降低相关。微丝稳定显著减弱了rottlerin对黏着斑和屏障的破坏作用。FAK过表达并未减弱rottlerin诱导的屏障功能障碍或应力纤维及黏着斑破坏的作用。相反，绿色荧光蛋白（GFP）连接的显性活性RhoA过表达可保护内皮细胞免受rottlerin暴露和PKCδ显性负性蛋白过表达所诱导的应力纤维和黏着斑破坏。此外，PKCδ与RhoA活性调节剂p190RhoGAP和p120RasGAP进行免疫共沉淀。因此，PKCδ可能通过上游调节剂p190RhoGAP和p120RasGAP调节RhoA GTP酶活性，从而稳定微丝和黏着斑，进而调节内皮细胞的基础屏障功能。
