摘要
Rap1 is expressed in human umbilical vein endothelial cells(HUVECs).Rap1-GTPase ac-tivating protein(Rap1GAP),with its specific target,Rap1,has been shown to be important in the regu-lation of many physiological and certain pathological processes.In this study,we investigated the effect of Rap1GAP expression on endothelial cell function,or,more specifically,proliferation and migration of endothelial cells.HUVECs were transfected with pcDNA3.1(empty vector),pcDNA3.1 containing Flag-tagged-Rap1GAP or Myc-tagged-Rap1N17.The proliferation,migration and tube formation were examined and compared among the 3 groups.Expression of Rap1,Rap1GAP,extracellular sig-nal-regulated kinase(ERK),phospho-ERK,Akt,phosphor-Akt was detected by Western blotting.The results showed that the proliferation,migration and tube formation were significantly reduced in Rap1GAP-and Rap1N17-transfected HUVECs as compared with empty vector-transfected control.These changes were coincident with increased expression of Rap1GAP and decreased expression of ac-tivated Rap1,phospho-ERK and-Akt.After treatment of Rap1GAP-transfected HUVECs with a stimu-lator of Rap1 guanine-nucleotide-exchange factor(Rap1GEF)8CPT-2'OMe-cAMP,it was found that Rap1 activity was decreased as compared with empty vector-transfected control.Pretreatment of HUVECs with an ERK inhibitor PD98059 or a PI3K inhibitor LY294002 prior to stimulation not only blocked 8CPT-2'OMe-cAMP-induced phosphorylation of ERK and Akt,but also significantly reduced cell proliferation and migration.Finally,we examined the effect of vascular endothelial growth factor(VEGF)on HUVECs overexpressing Rap1GAP.VEGF-stimulated Rap1 activity,phosphorylation of ERK and Akt,cyclin D1 expression and cell proliferation were repressed in HUVECs overexpressing Rap1GAP as compared to empty vector-transfected control.Taken together,our findings demonstrate that Rap1GAP/Rap1 and their downstream effectors regulate proliferation and migration of HUVECs via ERK and Akt pathways.
Rapl is expressed in human umbilical vein endothelial cells （HUVECs）. Rapl-GTPase activating protein （RaplGAP）, with its specific target, Rapl, has been shown to be important in the regulation of many physiological and certain pathological processes. In this study, we investigated the effect of RaplGAP expression on endothelial cell function, or, more specifically, proliferation and migration of endothelial cells. HUVECs were transfected with pcDNA3.1 （empty vector）, pcDNA3.1 containing Flag-tagged-RaplGAP or Myc-tagged-RaplN17. The proliferation, migration and tube formation were examined and compared among the 3 groups. Expression of Rapl, RaplGAP, extracellular signal-regulated kinase （ERK）, phospho-ERK, Akt, phosphor-Akt was detected by Western blotting. The results showed that the proliferation, migration and tube formation were significantly reduced in RaplGAP- and RaplN17-transfected HUVECs as compared with empty vector-transfected control. These changes were coincident with increased expression of Rap 1GAP and decreased expression of activated Rap l, phospho-ERK and -Akt. After treatment of Rap l GAP-transfected HUVECs with a stimulator of Rapl guanine-nucleotide-exchange factor （RaplGEF） 8CPT-2＇OMe-cAMP, it was found that Rapl activity was decreased as compared with empty vector-transfected control. Pretreatment of HU- VECs with an ERK inhibitor PD98059 or a PI3K inhibitor LY294002 prior to stimulation not only blocked 8CPT-2＇OMe-cAMP-induced phosphorylation of ERK and Akt, but also significantly reduced cell proliferation and migration. Finally, we examined the effect of vascular endothelial growth factor （VEGF） on HUVECs overexpressing RaplGAP. VEGF-stimulated Rapl activity, phosphorylation of ERK and Akt, cyclin D1 expression and cell proliferation were repressed in HUVECs overexpressing RaplGAP as compared to empty vector-transfected Control. Taken together, our findings demonstrate that RaplGAP/Rapl and their downstream effectors regulate proliferation and migration of HUVECs via ERK and Akt pathways.
基金
supported by grants from the National Natural Science Foundation of China(No.30971207)
Natural Science Foundation of Hubei Province,China(No.2009CBD-386)
