Tubedown associates with cortactin and controls permeability of retinal endothelial cells to albumin
Tubedown associates with cortactin and controls permeability of retinal endothelial cells to albumin
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DOI：
10.1242/jcs.028597
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发表时间：
2008-06-15
影响因子：
4
通讯作者：
Gendron, Robert L.
中科院分区：
文献类型：
--
作者：
Paradis, Helene;Islam, Thasin;Gendron, Robert L.
Tubedown（Narg 1，Tbdn）是Nat 1蛋白家族的成员，与乙酰转移酶Ard 1结合，在成人视网膜血管稳态中发挥血管生成抑制作用。本研究的目的是更好地了解Tbdn蛋白复合物的性质以及它如何对血管产生稳态影响。Tbdn从内皮细胞的免疫沉淀，然后凝胶电泳和液相色谱串联质谱鉴定肌动蛋白细胞骨架结合蛋白corprin作为一个共同的免疫纯化物种。Western blotting证实了Tbdn与coronin的相关性。免疫荧光共聚焦显微镜显示，Tbdn共定位与coronin和F-肌动蛋白在细胞质区域和培养的内皮细胞的皮质。因为已知皮质激素通过其与肌动蛋白细胞骨架的相互作用来调节细胞渗透性，这是一个对内皮细胞稳态至关重要的过程，所以检查了Tbdn对内皮细胞渗透性的作用。内皮细胞中Tbdn表达的敲低导致Ard 1蛋白表达的共同抑制，并通过FITC-白蛋白穿过细胞单层的转运来测量细胞通透性的显著增加。此外，在转基因小鼠中诱导内皮中Tbdn敲低导致的增殖性视网膜新血管形成和增厚与体内异常视网膜血管中白蛋白外渗或渗漏的显著增加相关。这些结果提供了证据表明，Tbdn和coronin之间发生关联，Tbdn参与调节视网膜内皮细胞对白蛋白的渗透性。这项工作暗示了Tbdn在血管通透性动力学中的功能作用，这对血管稳态至关重要。
Tubedown (Narg1, Tbdn), a member of the Nat1 family of proteins, associates with the acetyltransferase Ard1 and exerts an angiostatic function in adult retinal-blood-vessel homeostasis. The purpose of the present study was to gain a better understanding of the nature of the Tbdn protein complex and how it might exert a homeostatic influence on blood vessels. Immunoprecipitation of Tbdn from endothelial cells followed by gel electrophoresis and liquid-chromatography-tandem-mass-spectrometry identified the actin-cytoskeleton-binding protein cortactin as a co-immunopurifying species. Western blotting confirmed the association between Tbdn and cortactin. Immunofluorescence confocal microscopy revealed that Tbdn colocalizes with cortactin and F-actin in cytoplasmic regions and at the cortex of cultured endothelial cells. Because cortactin is known to regulate cellular permeability through its interaction with the actin cytoskeleton, a process that is crucial for endothelial cell homeostasis, the role of Tbdn on endothelial cell permeability was examined. Knockdown of Tbdn expression in endothelial cells led to the co-suppression of Ard1 protein expression and to a significant increase in cellular permeability measured by the transit of FITC-albumin across the cellular monolayer. Furthermore, the proliferative retinal neovascularization and thickening resulting from induction of Tbdn knockdown in endothelium in transgenic mice was associated with a significant increase in extravasation or leakage of albumin from abnormal retinal blood vessels in vivo. These results provide evidence that an association occurs between Tbdn and cortactin, and that Tbdn is involved in the regulation of retinal-endothelial-cell permeability to albumin. This work implicates a functional role for Tbdn in blood-vessel permeability dynamics that are crucial for vascular homeostasis.