Klotho is associated with VEGF receptor-2 and the transient receptor potential canonical-1 Ca2+ channel to maintain endothelial integrity
Klotho is associated with VEGF receptor-2 and the transient receptor potential canonical-1 Ca2+ channel to maintain endothelial integrity
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DOI：
10.1073/pnas.1008544107
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发表时间：
2010-11-09
影响因子：
11.1
通讯作者：
Matsubara, Hiroaki
中科院分区：
文献类型：
--
作者：
Kusaba, Tetsuro;Okigaki, Mitsuhiko;Matsubara, Hiroaki
Klotho是一种循环蛋白，Klotho缺乏会干扰内皮完整性，但其分子机制尚未完全阐明。我们报告说，Klotho缺陷小鼠的血管内皮细胞表现出高通透性，细胞凋亡增加，血管内皮细胞（VE）-钙粘蛋白的下调，因为VEGF介导的内部钙浓度（[Ca 2 +] i）内流和Ca 2+依赖性蛋白酶的超活化增加。免疫组织化学分析，使用Klotho固定的琼脂糖的下拉测定和FRET共聚焦成像证实Klotho蛋白直接结合VEGF受体2（VEGFR-2）和内皮，瞬时受体电位经典Ca 2+通道1（TRPC-1），并加强该关联以促进其共内化。体外诱变研究显示Klotho的第二水解酶结构域与VEGFR-2的第六和第七IG结构域以及TRPC-1的第三胞外环相互作用。在Klotho缺陷型内皮细胞中，VEGF介导的VEGFR-2/TRPC-1复合物的内化受损，表面TRPC-1表达增加2.2倍;这些作用可通过补充Klotho蛋白逆转。VEGF介导的[Ca 2 +] i升高以细胞外Ca 2+依赖的方式维持在较高水平，TRCP-1表达的正常化恢复了异常的[Ca 2 +] i处理。这些发现提供证据表明Klotho蛋白与VEGFR-2/TRPC-1相关，引起共内化，从而调节TRPC-1介导的Ca 2+内流以维持内皮完整性。
Klotho is a circulating protein, and Klotho deficiency disturbs endothelial integrity, but the molecular mechanism is not fully clarified. We report that vascular endothelium in Klotho-deficient mice showed hyperpermeability with increased apoptosis and down-regulation of vascular endothelial (VE)-cadherin because of an increase in VEGF-mediated internal calcium concentration ([Ca2+] i) influx and hyperactivation of Ca2+-dependent proteases. Immunohistochemical analysis, the pull-down assay using Klotho-fixed agarose, and FRET confocal imaging confirmed that Klotho protein binds directly to VEGF receptor 2 (VEGFR-2) and endothelial, transient-receptor potential canonical Ca2+ channel 1 (TRPC-1) and strengthens the association to promote their cointernalization. An in vitro mutagenesis study revealed that the second hydrolase domain of Klotho interacts with sixth and seventh Ig domains of VEGFR-2 and the third extracellular loop of TRPC-1. In Klotho-deficient endothelial cells, VEGF-mediated internalization of the VEGFR-2/TRPC-1 complex was impaired, and surface TRPC-1 expression increased 2.2-fold; these effects were reversed by supplementation of Klotho protein. VEGF-mediated elevation of [Ca2+] i was sustained at higher levels in an extracellular Ca2+-dependent manner, and normalization of TRCP-1 expression restored the abnormal [Ca2+] i handling. These findings provide evidence that Klotho protein is associated with VEGFR-2/TRPC-1 in causing cointernalization, thus regulating TRPC-1-mediated Ca2+ entry to maintain endothelial integrity.