Role of p115RhoGEF in lipopolysaccharide-induced mouse brain microvascular endothelial barrier dysfunction
Role of p115RhoGEF in lipopolysaccharide-induced mouse brain microvascular endothelial barrier dysfunction
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p115RhoGEF在脂多糖诱导的小鼠脑微血管内皮屏障功能障碍中的作用
DOI：
10.1016/j.brainres.2011.02.059
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发表时间：
2011-04-28
期刊：
影响因子：
2.9
通讯作者：
Yin Fei
中科院分区：
文献类型：
--
作者：
Deng Xiaolu;Peng Jing;Yin Fei
背景：在内皮细胞中，接触脂多糖（LPS）会通过复杂的信号传导机制导致屏障功能障碍。 RhoA/Rho 激酶途径在内皮细胞通透性中发挥重要作用。 p115RhoGEF 是一种特定的鸟嘌呤核苷酸交换因子 (GEF)，可激活 RhoA，引发 RhoA 依赖性细胞骨架重塑。然而，人们对 p115RhoGEF 在 LPS 诱导的脑内皮屏障破坏中的作用知之甚少。我们假设抑制 p115RhoGEF 可能会抑制 RhoA 的激活并防止 LPS 诱导的脑微血管内皮细胞通透性过高。方法：本研究使用培养的单层 bEnd.3 细胞（一种永生化小鼠脑内皮细胞系）。 bEnd.3细胞用特异性siRNA预处理以敲低p115RhoGEF或C3转移酶以抑制RhoA活性，然后与LPS(5μg/ml)一起孵育。通过基于Rhotekin的pull-down测定法测定RhoA激活程度，并通过蛋白质印迹分析检测p115RhoGEF、zonula occlusionns-1 (ZO-1)、occludin和claudin-5蛋白的表达。通过跨内皮电阻（TEER）测量屏障功能。 F-肌动蛋白细胞骨架通过罗丹明-鬼笔环肽染色可视化。结果：LPS处理的bEnd.3细胞中p115RhoGEF蛋白的表达水平显着增加。如 Pull-down 测定所示，LPS 刺激和 p115RhoGEF siRNA 或外切酶 C3 转移酶预处理后，RhoA 活性增强，显着降低了 RhoA 活化。此外，p115RhoGEF 的消耗部分阻止了 LPS 诱导的 TEER 降低、应力纤维形成和紧密连接蛋白降解。结论：这些结果表明 p115RhoGEF 对于 RhoA 的 LPS 信号传导和 LPS 诱导的内皮屏障功能障碍很重要，为了解 RhoGEF 在炎症中的功能提供了新的见解。 (C) 2011 Elsevier B.V. 保留所有权利。
Background: In endothelial cells, exposure to lipopolysaccharide (LPS) results in barrier dysfunction through a complex signaling mechanism. The RhoA/Rho-kinase pathway plays a significant role in endothelial cell permeability. p115RhoGEF, a specific guanine nucleotide exchange factors (GEFs) activates RhoA, triggering RhoA-dependent cytoskeletal remodeling. However, little is known about the role of p115RhoGEF in LPS-induced brain endothelial barrier breakdown. We hypothesized that suppression of p115RhoGEF may inhibit activation of RhoA and prevent LPS-induced brain microvascular endothelial cell hyperpermeability. Methods: The cultured monolayer of bEnd.3 cells, an immortalized mouse brain endothelial cell line, was used in this study. bEnd.3 cells were pretreated with specific siRNA to knockdown p115RhoGEF or C3 transferase to inhibit RhoA activity, and then incubated with LPS (5 mu g/ml). The degree of RhoA activation was determined by a Rhotekin-based pull-down assay, and expression of p115RhoGEF, zonula occludens-1 (ZO-1), occludin and claudin-5 proteins were detected by Western blot analysis. The barrier function was measured by transendothelial electrical resistance (TEER). F-actin cytoskeleton was visualized by Rhodamine-phalloidin staining. Results: The expression level of p115RhoGEF protein was significantly increased in LPS-treated bEnd.3 cells. The activity of RhoA was enhanced after LPS stimulation and pretreatment with p115RhoGEF siRNA or exoenzyme C3 transferase reduced RhoA activation significantly as shown by the pull-down assay. Furthermore, depletion of p115RhoGEF partially prevented the LPS-induced decrease in TEER, stress fiber formation and tight junction proteins degradation. Conclusions: These results suggest that p115RhoGEF is important for LPS signaling to RhoA and LPS-induced endothelial barrier dysfunction, providing new insight into the function of RhoGEFs in inflammation. (C) 2011 Elsevier B.V. All rights reserved.