Differential actions of PAR2 and PAR1 in stimulating human endothelial cell exocytosis and permeability -: The role of Rho-GTPases
Differential actions of PAR2 and PAR1 in stimulating human endothelial cell exocytosis and permeability -: The role of Rho-GTPases
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DOI：
10.1161/01.res.0000057386.15390.a3
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发表时间：
2003-02-21
影响因子：
20.1
通讯作者：
Murray, AG
中科院分区：
文献类型：
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作者：
Klarenbach, SW;Chipiuk, A;Murray, AG
内皮细胞蛋白酶激活受体(PARs)属于异源三聚体G蛋白偶联受体家族，参与白细胞聚集和加强再灌注损伤。我们表征了内皮PAR(2)刺激后的作用和招募的信号转导通路。我们用von Willebrand因子(VWF)释放和过氧化物酶单层通透性分别报告了Webel-Palade小体(WPB)的胞吐和孔道形成。用选择性PAR(2)激动肽SLIGRL-NH2或PAR(1)激动肽TFLLR-NH2刺激人脐静脉内皮细胞(HUVEC)。PAR(2)刺激可引起类似PAR(1)刺激的WPB胞吐，但与PAR(1)不同的是，PAR(1)不能增加单层通透性。BAPTA-AM抑制PAR(2)诱导的胞吐，提示内皮细胞存在PAR(2)钙依赖信号。此外，PAR(2)类似PAR(1)刺激的胞吐需要肌动蛋白细胞骨架重塑，因为如果用茉莉酸激动素处理细胞，vWF的释放被抑制。Rho-GTPase活性是PAR刺激的胞吐作用所必需的，因为艰难梭菌毒素B使这一家族肌动蛋白调节蛋白失活阻止了胞吐作用。显性负性突变体CDC42(17N)的表达抑制了胞吐作用，而显性负性Rac(17N)的表达和C3外毒素处理都不影响vWF的释放。与PAR(1)激动剂相比，PAR(2)对RhoA-GTP的刺激作用较弱。我们得出结论，PAR(2)和PAR(1)均以钙和CDC42 GTP酶依赖的方式诱导WP小体胞吐。相反，PAR(1)和PAR(2)激活增加单层通透性的不同作用与PAR(2)激动剂对RhoA的微弱激活有关。
Endothelial cell proteinase activated receptors (PARs) belong to a family of heterotrimeric G protein-coupled receptors that are implicated in leukocyte accumulation and potentiation of reperfusion injury. We characterized the effect and the signal transduction pathways recruited after stimulation of endothelial PAR(2). We used von Willebrand Factor (vWF) release and monolayer permeability to peroxidase to report Weibel-Palade body (WPB) exocytosis and pore formation, respectively. Human umbilical vein endothelial cells (HUVECs) were stimulated with the selective PAR(2) agonist peptide SLIGRL-NH2 or PAR(1) agonist peptide TFLLR-NH2. PAR(2) stimulation resulted in WPB exocytosis like PAR(1) stimulation but, unlike PAR(1), failed to increase monolayer permeability. BAPTA-AM inhibited PAR(2)-induced exocytosis, indicating a PAR(2) calcium-dependent signal in ECs. Moreover, PAR(2)-like PAR(1)-stimulated exocytosis requires actin cytoskeleton remodeling, because vWF release is inhibited if the cells were pretreated with Jasplakinolide. Rho-GTPase activity is required for PAR-stimulated exocytosis, because inactivation of this family of actin-regulatory proteins with Clostridium difficile toxin B blocked exocytosis. Expression of dominant-negative mutant Cdc42(17N) inhibited exocytosis whereas neither dominant-negative Rac(17N) expression nor C3 exotoxin treatment affected vWF release. PAR(2) stimulated RhoA-GTP weakly compared with the PAR(1) agonist. We conclude that both PAR(2) and PAR(1) elicit WP body exocytosis in a calcium and Cdc42 GTPase-dependent manner. In contrast, the differential effect of PAR(1) versus PAR(2) activation to increase monolayer permeability correlates with weak RhoA activation by the PAR(2) agonist.