title: Inhibitory effects of Rap1GAP overexpression on proliferation and migration of endothelial cells via ERK and Akt pathways.
abstract: Rap1 is expressed in human umbilical vein endothelial cells (HUVECs). Rap1-GTPase activating protein (Rap1GAP), with its specific target, Rap1, has been shown to be important in the regulation 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 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 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 activated Rap1, phospho-ERK and -Akt. After treatment of Rap1GAP-transfected HUVECs with a stimulator 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.
authors.affiliation: Department of Vascular Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China.
authors.forename: Wenyi
authors.initials: W
authors.lastname: Li
authors.affiliation: Department of Vascular Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China.
authors.forename: Bi
authors.initials: B
authors.lastname: Jin
authors.affiliation: Division of Dermatology, Department of Internal Medicine, Washington University School of Medicine, St. Louis, MO, 63110, USA.
authors.forename: Lynn A
authors.initials: LA
authors.lastname: Cornelius
authors.affiliation: Department of Vascular Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China.
authors.forename: Bin
authors.initials: B
authors.lastname: Zhou
authors.affiliation: Department of Vascular Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China.
authors.forename: Xiaoyang
authors.initials: X
authors.lastname: Fu
authors.affiliation: Department of Vascular Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China.
authors.forename: Dan
authors.initials: D
authors.lastname: Shang
authors.affiliation: Department of Vascular Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China. zhenghong559@gmail.com.
authors.forename: Hong
authors.initials: H
authors.lastname: Zheng
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism; D002465:Cell Movement; D049109:Cell Proliferation; D002478:Cells, Cultured; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D020690:GTPase-Activating Proteins / Q000096:biosynthesis* / Q000235:genetics; D061307:Human Umbilical Vein Endothelial Cells / Q000166:cytology* / Q000378:metabolism; D006801:Humans; D015398:Signal Transduction; D014162:Transfection
chemical_list: C486377:AKTIP protein, human; D048868:Adaptor Proteins, Signal Transducing; D051017:Apoptosis Regulatory Proteins; D020690:GTPase-Activating Proteins; C484658:RAP1GAP protein, human; D048049:Extracellular Signal-Regulated MAP Kinases