Thioredoxin-interacting protein mediates sustained VEGFR2 signaling in endothelial cells required for angiogenesis.
Thioredoxin-interacting protein mediates sustained VEGFR2 signaling in endothelial cells required for angiogenesis.
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DOI：
10.1161/atvbaha.112.300386
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发表时间：
2013-04
期刊：
影响因子：
--
通讯作者：
Berk BC
中科院分区：
文献类型：
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作者：
Park SY;Shi X;Pang J;Yan C;Berk BC
硫氧还蛋白相互作用蛋白（Thioredoxin-interacting protein，TXNIP）是一种α-arrestin蛋白，其功能对调节血管内皮生长因子受体2（VEGFR 2）信号传导和内皮细胞（EC）存活具有重要作用。由于VEGFR 2是血管生成的关键，我们探讨了TXNIP在VEGF诱导的血管生成中的作用。TXNIP敲低抑制VEGF诱导的人脐静脉EC（HUVEC）管形成和增殖。为了阐明TXNIP改变HUVEC中VEGFR 2信号的机制，我们研究了VEGFR 2、PLCγ1、eNOS和Akt的磷酸化。TXNIP敲除在> 5 min时显著降低VEGFR 2和PLCγ1的磷酸化，但在2 min时磷酸化没有变化; Akt和eNOS磷酸化也是如此。细胞表面生物素化检测显示TXNIP敲低显著减弱了VEGFR 2的内化。这些结果表明，TXNIP是持续的VEGFR 2信号传导所必需的，其主要由内化的VEGFR 2介导。Rab 5敲除抑制早期核内体的运输和融合，显着阻断了VEGF诱导的VEGFR 2内化以及VEGFR 2和PLCγ1的磷酸化。免疫荧光和免疫共沉淀显示TXNIP是Rab 5和VEGFR 2复合物的一部分。最后，TXNIP敲低阻止了VEGFR 2和Rab 5的结合。我们的研究结果表明，TXNIP是必不可少的Rab 5阳性内体，这是EC生长和血管生成所需的VEGFR 2内化。
Thioredoxin-interacting protein (TXNIP) is an α-arrestin protein whose function is important for the regulation of vascular endothelial growth factor receptor 2 (VEGFR2) signaling and endothelial cell (EC) survival. Since VEGFR2 is critical for angiogenesis, we explored the role of TXNIP in VEGF-induced angiogenesis. TXNIP knockdown inhibited VEGF-induced EC tube formation and proliferation in cultured human umbilical vein EC (HUVEC). To elucidate the mechanism by which TXNIP altered VEGFR2 signaling in HUVEC, we studied phosphorylation of VEGFR2, PLCγ1, eNOS, and Akt. TXNIP knockdown significantly decreased phosphorylation of VEGFR2 and PLCγ1 at times > 5min, but phosphorylation was unchanged at 2min; as was Akt and eNOS phosphorylation. Cell surface biotinylation assay showed that TXNIP knockdown significantly attenuated VEGFR2 internalization. These results suggested that TXNIP was required for sustained VEGFR2 signaling, which is mediated largely by internalized VEGFR2. Rab5 knockdown to inhibit the trafficking and fusion of early endosomes significantly blocked VEGF-induced VEGFR2 internalization and phosphorylation of VEGFR2 and PLCγ1. Immunofluorescence and co-immunoprecipitation showed that TXNIP was part of a complex that included Rab5 and VEGFR2. Finally, TXNIP knockdown prevented the association of VEGFR2 and Rab5. Our results show that TXNIP is essential for VEGFR2 internalization in Rab5 positive endosomes, which is required for EC growth and angiogenesis.