Kininostatin Associates With Membrane Rafts and Inhibits αvβ3 Integrin Activation in Human Umbilical Vein Endothelial Cells
Kininostatin Associates With Membrane Rafts and Inhibits αvβ3 Integrin Activation in Human Umbilical Vein Endothelial Cells
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DOI：
10.1161/atvbaha.107.148759
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发表时间：
2007-09
期刊：
影响因子：
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通讯作者：
Yi Wu;V. Rizzo;Yuchuan Liu;Irma M. Sainz;N. G. Schmuckler;R. Colman
中科院分区：
文献类型：
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作者：
Yi Wu;V. Rizzo;Yuchuan Liu;Irma M. Sainz;N. G. Schmuckler;R. Colman
目的：高分子量激肽原（HKa）的裂解形式是体内血管生成和肿瘤生长的有效抑制剂；功能域已被确定为结构域5 （D5，命名为kininostatatin）。我们现在确定了内皮细胞（ECs）上D5的亚细胞靶向位点，并研究了D5对整合素功能的抑制。方法与结果：采用蔗糖密度梯度离心法分离内皮膜筏。与ECs结合的D5主要与膜筏相关，其中也定位了HKa受体uPAR。相比之下，其他HKa受体，细胞角蛋白1和gC1q受体在膜筏中未检测到。共聚焦显微镜显示D5与caveolin-1在ECs上共定位。去除胆固醇对膜筏的破坏降低了D5与ECs的结合。在血管内皮生长因子的刺激下，αvβ3整合素与uPAR和caveolin-1形成复合物，αvβ3整合素的配体结合亲和力增加。这些事件被D5抑制。同样，D5抑制αvβ3整合素介导的EC特异性粘附和扩散以及小鸟苷三磷酸酶Rac1的激活。结论- d5通过膜筏与ECs结合，下调αvβ3整合素双向信号通路及下游Rac1激活途径。
Objective—The cleaved form of high molecular weight kininogen (HKa) is a potent inhibitor of angiogenesis and tumor growth in vivo; the functional domain has been identified as domain 5 (D5, named as kininostatin). We now identify the subcellular targeting site for D5 on endothelial cells (ECs), and investigate D5 inhibition of integrin functions. Methods and Results—Endothelial membrane rafts were isolated using sucrose density gradient centrifugation. D5, bound to ECs, was predominantly associated with membrane rafts, in which uPAR, a HKa receptor, was also localized. In contrast, other HKa receptors, cytokeratin-1 and gC1q receptor, were not detected in membrane rafts. Colocalization of D5 with caveolin-1 was demonstrated on ECs by confocal microscopy. Disruption of membrane rafts by cholesterol removal decreased D5 binding to ECs. On stimulation with vascular endothelial growth factor, αvβ3 integrin formed a complex with uPAR and caveolin-1, which was accompanied by an increase in ligand binding affinity of αvβ3 integrin. These events were inhibited by D5. Consistently, D5 suppressed specific αvβ3 integrin-mediated EC adhesion and spreading as well as small guanosine triphosphatase Rac1 activation. Conclusions—D5 binds to ECs via membrane rafts and downregulates αvβ3 integrin bidirectional signaling and the downstream Rac1 activation pathway.