Department of Cell and Molecular Biology, Cardiovascular Research Center and the John A Burns School of Medicine, University of Hawaii at Manoa, Honolulu, Hawaii 96813, USA.
J Cell Physiol. 2011 Sep;226(9):2287-96. doi: 10.1002/jcp.22565.
The molecular mechanisms regulating vascular barrier integrity remain incompletely elucidated. We have previously reported an association between the GTPase R-Ras and repeat 3 of Filamin A (FLNa). Loss of FLNa has been linked to increased vascular permeability. We sought to determine whether FLNa's association with R-Ras affects endothelial barrier function. We report that in endothelial cells endogenous R-Ras interacts with endogenous FLNa as determined by co-immunoprecipitations and pulldowns with the FLNa-GST fusion protein repeats 1-10. Deletion of FLNa repeat 3 (FLNaΔ3) abrogated this interaction. In these cells FLNa and R-Ras co-localize at the plasma membrane. Knockdown of R-Ras and/or FLNa by siRNA promotes vascular permeability, as determined by TransEndothelial Electrical Resistance and FITC-dextran transwell assays. Re-expression of FLNa restored endothelial barrier function in cells lacking FLNa whereas re-expression of FLNaΔ3 did not. Immunostaining for VE-Cadherin in cells with knocked down R-Ras and FLNa demonstrated a disorganization of VE-Cadherin at adherens junctions. Loss of R-Ras and FLNa or blocking R-Ras function via GGTI-2133, a selective R-Ras inhibitor, induced vascular permeability and increased phosphorylation of VE-Cadherin (Y731) and Src (Y416). Expression of dominant negative R-Ras promoted vascular permeability that was blocked by the Src inhibitor PP2. These findings demonstrate that maintaining endothelial barrier function is dependent upon active R-Ras and association between R-Ras and FLNa and that loss of this interaction promotes VE-Cadherin phosphorylation and changes in downstream effectors that lead to endothelial leakiness.
调控血管屏障完整性的分子机制仍不完全清楚。我们之前曾报道过 GTPase R-Ras 与细丝蛋白 A（FLNa）重复 3 之间的关联。FLNa 的缺失与血管通透性增加有关。我们试图确定 FLNa 与 R-Ras 的关联是否会影响内皮屏障功能。我们报告称，在内皮细胞中，内源性 R-Ras 与内源性 FLNa 相互作用，这可通过与 FLNa-GST 融合蛋白重复 1-10 的共免疫沉淀和下拉实验来确定。FLNa 重复 3 缺失（FLNaΔ3）消除了这种相互作用。在这些细胞中，FLNa 和 R-Ras 在质膜处共定位。通过 TransEndothelial Electrical Resistance 和 FITC-葡聚糖 Transwell 测定，用 siRNA 敲低 R-Ras 和/或 FLNa 可促进血管通透性。在缺乏 FLNa 的细胞中重新表达 FLNa 可恢复内皮屏障功能，而重新表达 FLNaΔ3 则不能。用 siRNA 敲低 R-Ras 和 FLNa 的细胞中的 VE-Cadherin 免疫染色显示，在黏着连接处 VE-Cadherin 的排列紊乱。R-Ras 和 FLNa 的缺失或通过 GGTI-2133（一种选择性的 R-Ras 抑制剂）阻断 R-Ras 功能，会诱导血管通透性增加，并增加 VE-Cadherin（Y731）和Src（Y416）的磷酸化。表达显性失活的 R-Ras 可促进血管通透性，而 Src 抑制剂 PP2 可阻断这种通透性。这些发现表明，维持内皮屏障功能依赖于活性 R-Ras 以及 R-Ras 和 FLNa 之间的相互作用，而这种相互作用的缺失会促进 VE-Cadherin 的磷酸化以及下游效应物的改变，从而导致内皮通透性增加。
