Essential Role for Endocytosis in the Growth Factor-stimulated Activation of ERK1/2 in Endothelial Cells
Essential Role for Endocytosis in the Growth Factor-stimulated Activation of ERK1/2 in Endothelial Cells
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DOI：
10.1074/jbc.m112.446401
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发表时间：
2013-03-15
影响因子：
4.8
通讯作者：
Reynolds, Andrew R.
中科院分区：
文献类型：
--
作者：
Gourlaouen, Morgane;Welti, Jonathan C.;Reynolds, Andrew R.
血管内皮生长因子（VEGF）通过与内皮细胞（EC）上的VEGF受体2（VEGFR 2）结合来刺激血管生成。细胞外相关激酶1/2（ERK 1/2）的下游激活对于血管生成的进行是重要的。受体内化与VEGFR 2信号转导有关，但其在ERK 1/2活化中的作用尚不清楚。为了探索这个问题，我们利用pitstop和dynasore，两个小分子抑制剂的内吞作用。首先，我们证实了这两种抑制剂都阻断了内皮细胞中VEGFR 2的内化。然后，我们在存在和不存在抑制剂的情况下用VEGF刺激EC，并检查VEGFR 2向ERK 1/2的信号传导。VEGFR 2和C-Raf的活化在抑制剂存在下仍然发生，而MEK 1/2和ERK 1/2的活化被废除。因此，尽管在用VEGF刺激的EC中VEGFR 2或C-Raf的活化不需要内化，但是内化对于活化级联中更远端的激酶是必需的。重要的是，当用其他促血管生成生长因子，即成纤维细胞生长因子2和肝细胞生长因子刺激EC时，抑制内化也阻止了ERK 1/2的活化。相比之下，相同的抑制剂不能阻断生长因子刺激的成纤维细胞或癌细胞中的ERK 1/2活化。最后，我们表明，这些小分子抑制剂的内吞阻断血管生成在体外和体内。因此，受体内化可能是促血管生成生长因子激活人EC中ERK 1/2信号传导的一般要求，靶向受体运输可能提供阻断肿瘤血管生成的治疗机会。
Vascular endothelial growth factor (VEGF) stimulates angiogenesis by binding to VEGF receptor 2 (VEGFR2) on endothelial cells (ECs). Downstream activation of the extracellular related kinases 1/2 (ERK1/2) is important for angiogenesis to proceed. Receptor internalization has been implicated in VEGFR2 signaling, but its role in the activation of ERK1/2 is unclear. To explore this question we utilized pitstop and dynasore, two small molecule inhibitors of endocytosis. First, we confirmed that both inhibitors block the internalization of VEGFR2 in ECs. We then stimulated ECs with VEGF in the presence and absence of the inhibitors and examined VEGFR2 signaling to ERK1/2. Activation of VEGFR2 and C-Raf still occurred in the presence of the inhibitors, whereas the activation of MEK1/2 and ERK1/2 was abrogated. Therefore, although internalization is not required for activation of either VEGFR2 or C-Raf in ECs stimulated with VEGF, internalization is necessary to activate the more distal kinases in the cascade. Importantly, inhibition of internalization also prevented activation of ERK1/2 when ECs were stimulated with other pro-angiogenic growth factors, namely fibroblast growth factor 2 and hepatocyte growth factor. In contrast, the same inhibitors did not block ERK1/2 activation in fibroblasts or cancer cells stimulated with growth factors. Finally, we show that these small molecule inhibitors of endocytosis block angiogenesis in vitro and in vivo. Therefore, receptor internalization may be a generic requirement for pro-angiogenic growth factors to activate ERK1/2 signaling in human ECs, and targeting receptor trafficking may present a therapeutic opportunity to block tumor angiogenesis.