title: Essential role for endocytosis in the growth factor-stimulated activation of ERK1/2 in endothelial cells.
abstract: 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.
authors: Tumour Biology Team, Breakthrough Breast Cancer Research Centre, The Institute of Cancer Research, London SW3 6JB, United Kingdom.
authors: Morgane
authors: M
authors: Gourlaouen
authors: Tumour Biology Team, Breakthrough Breast Cancer Research Centre, The Institute of Cancer Research, London SW3 6JB, United Kingdom.
authors: Jonathan C
authors: JC
authors: Welti
authors: Tumour Biology Team, Breakthrough Breast Cancer Research Centre, The Institute of Cancer Research, London SW3 6JB, United Kingdom.
authors: Naveen S
authors: NS
authors: Vasudev
authors: Tumour Biology Team, Breakthrough Breast Cancer Research Centre, The Institute of Cancer Research, London SW3 6JB, United Kingdom. Electronic address: andrew.reynolds@icr.ac.uk.
authors: Andrew R
authors: AR
authors: Reynolds
mesh_terms: D020533:Angiogenesis Inhibitors / Q000494:pharmacology; D000818:Animals; D002460:Cell Line; D045744:Cell Line, Tumor; D004705:Endocytosis / Q000502:physiology*; D042783:Endothelial Cells / Q000201:enzymology; D005260:Female; D016220:Fibroblast Growth Factor 1 / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D015972:Gene Expression Regulation, Neoplastic; D017228:Hepatocyte Growth Factor / Q000378:metabolism; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism*; D009369:Neoplasms / Q000188:drug therapy* / Q000378:metabolism; D017978:Receptors, Growth Factor / Q000378:metabolism; D015398:Signal Transduction; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000378:metabolism
chemical_list: D020533:Angiogenesis Inhibitors; D036341:Intercellular Signaling Peptides and Proteins; D017978:Receptors, Growth Factor; D042461:Vascular Endothelial Growth Factor A; D016220:Fibroblast Growth Factor 1; D017228:Hepatocyte Growth Factor; D040301:Vascular Endothelial Growth Factor Receptor-2; C535150:MAPK1 protein, human; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3