TRAF6 inhibits proangiogenic signals in endothelial cells and regulates the expression of vascular endothelial growth factor.
TNF-family molecules induce the expression Vascular Endothelial Growth Factor (VEGF) in endothelial cells (EC) and elicit signaling responses that result in angiogenesis. However, the role of TNF-receptor associated factors (TRAFs) as upstream regulators of VEGF expression or as mediators of angiogenesis is not known. In this study, HUVEC were cotransfected with a full-length VEGF promoter-luciferase construct and siRNAs to TRAF 1, -2, -3, -5, -6, and promoter activity was measured. Paradoxically, rather than inhibiting VEGF expression, we found that knockdown of TRAF6 resulted in a 4-6-fold increase in basal VEGF promoter activity compared to control siRNA-transfected EC (P<0.0001). In addition, knockdown of TRAF 1, -2, -3 or -5 resulted in a slight increase or no change in VEGF promoter activation. Using [(3)H]thymidine incorporation assays as well as the in vitro wound healing assay, we also found that basal rates of EC proliferation and migration were increased following TRAF6 knockdown; and this response was inhibited by the addition of a blocking anti-VEGF antibody into cell cultures. Using a limited protein array to gain insight into TRAF6-dependent intermediary signaling responses, we observed that TRAF6 knockdown resulted in an increase in the activity of Src family kinases. In addition, we found that treatment with AZD-0530, a pharmacological Src inhibitor, reduced the regulatory effect of TRAF6 knockdown on VEGF promoter activity. Collectively, these findings define a novel pro-angiogenic signaling response in EC that is regulated by TRAF6.
Transplantation Research Center, Division of Nephrology, Department of Medicine, Children's Hospital Boston, Boston, MA 02115, United States.
Sarah
S
Bruneau
Dipak
D
Datta
Jesse A
JA
Flaxenburg
Soumitro
S
Pal
David M
DM
Briscoe
D052117:Benzodioxoles / Q000494:pharmacology; D002465:Cell Movement / Q000235:genetics; D049109:Cell Proliferation; D055785:Gene Knockdown Techniques; D061307:Human Umbilical Vein Endothelial Cells / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D008156:Luciferases / Q000235:genetics; D018919:Neovascularization, Physiologic / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011799:Quinazolines / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000235:genetics; D048029:TNF Receptor-Associated Factor 6 / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics; D019061:src-Family Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism
D052117:Benzodioxoles; D011799:Quinazolines; D034741:RNA, Small Interfering; D048029:TNF Receptor-Associated Factor 6; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; C515233:saracatinib; D008156:Luciferases; D019061:src-Family Kinases