VEGF activation of protein kinase C stimulates occludin phosphorylation and contributes to endothelial permeability
VEGF activation of protein kinase C stimulates occludin phosphorylation and contributes to endothelial permeability
复制标题
DOI：
10.1167/iovs.06-0322
复制
发表时间：
2006-11-01
影响因子：
4.4
通讯作者：
Antonetti, David A.
中科院分区：
文献类型：
--
作者：
Harhaj, Nicole S.;Felinski, Edward A.;Antonetti, David A.
目的. VEGF是一种有效的透化因子，有助于糖尿病视网膜病变和脑肿瘤的发病机制。VEGF诱导的血管通透性在体内和细胞培养中需要PKC活性，但PKC调节屏障特性的机制仍不清楚。本研究旨在检测VEGF和糖尿病如何改变occludin磷酸化和内皮细胞通透性。化学PKC抑制剂和激活剂用于处理培养的原代视网膜内皮细胞。体外激酶试验和蛋白质印迹分析的二维（2D）和一维（1D）凝胶阻滞试验用于分析闭合蛋白磷酸化。通过测量70-kDa葡聚糖通过培养物中细胞单层的通量来确定内皮细胞渗透性。显性负性PKC β II突变体（S217 A）的外源性表达被用来评估VEGF诱导的occludin磷酸化和内皮通透性的PKC依赖性。在链脲佐菌素诱导的糖尿病大鼠视网膜中也测定了封闭蛋白磷酸化。VEGF刺激原代视网膜内皮细胞中occludin的磷酸化。PKC活性的化学抑制剂阻断VEGF诱导的occludin磷酸化的增加，如通过Western印迹分析中的2D凝胶和凝胶阻滞所评估的，并且阻断VEGF诱导的70 kDa葡聚糖单层渗透性的一部分。显性负性PKC β II突变体的表达阻断VEGF诱导的occludin磷酸化和内皮通透性。最后，在糖尿病动物的视网膜中观察到occludin磷酸化水平升高。这些结果强烈表明VEGF诱导的内皮通透性需要PKC依赖的occludin磷酸化。PKC活性的调节和紧密连接蛋白的修饰可能对糖尿病视网膜病变和脑肿瘤的治疗具有治疗意义。
PURPOSE. VEGF is a potent permeabilizing factor that contributes to the pathogenesis of diabetic retinopathy and brain tumors. VEGF-induced vascular permeability in vivo and in cell culture requires PKC activity, but the mechanism by which PKC regulates barrier properties remains unknown. This study was conducted to examine how VEGF and diabetes alter occludin phosphorylation and endothelial cell permeability.METHODS. Chemical PKC inhibitors and activators were used to treat primary retinal endothelial cells in culture. In vitro kinase assays and Western blot analysis of two-dimensional (2D) and one-dimensional (1D) gel retardation assays were used to analyze occludin phosphorylation. Endothelial cell permeability was determined by measuring the flux of 70-kDa dextran through a cell monolayer in culture. Exogenous expression of a dominant negative PKC beta II mutant (S217A) was used to assess the PKC dependence of VEGF-induced occludin phosphorylation and endothelial permeability. Occludin phosphorylation was also determined in retinas of streptozotocin-induced diabetic rats.RESULTS. VEGF stimulated the phosphorylation of occludin in primary retinal endothelial cells. Chemical inhibitors of PKC activity blocked the VEGF-induced increase in occludin phosphorylation, as assessed by 2D gel and gel retardation in Western blot analysis, and blocked part of the VEGF-induced monolayer permeability to 70-kDa dextran. Expression of a dominant negative PKC beta II mutant blocked VEGF-induced occludin phosphorylation and endothelial permeability. Finally, elevated occludin phosphorylation was observed in the retina of diabetic animals.CONCLUSIONS. These results strongly suggest that VEGF-induced endothelial permeability requires PKC-dependent phosphorylation of occludin. Regulation of PKC activity and tight junction protein modifications may have therapeutic implications for treatment of diabetic retinopathy and brain tumors.