Skip to main content
SpringerLink
Log in

Inhibitory effects of Rap1GAP overexpression on proliferation and migration of endothelial cells via ERK and Akt pathways

  • Published:
Journal of Huazhong University of Science and Technology [Medical Sciences] Aims and scope Submit manuscript

Summary

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Milkiewicz M, Ispanovic E, Doyle JL, et al. Regulators of angiogenesis and strategies for their therapeutic manipulation. Int J Biochem Cell Biol, 2006,38(3):333–357

    Article  PubMed  CAS  Google Scholar 

  2. Carmeliet P. Angiogenesis in life, disease and medicine. Nature, 2005,438(7070):932–936

    Article  PubMed  CAS  Google Scholar 

  3. Takai Y, Sasaki T, Matozaki T. Small GTP-binding proteins. Physiol Rev, 2001,81(1):153–208

    PubMed  CAS  Google Scholar 

  4. McLeod SJ, Shum AJ, Lee RL, et al. The Rap GTPases regulate integrin-mediated adhesion, cell spreading, actin polymerization, and Pyk2 tyrosine phosphorylation in B lymphocytes. J Biol Chem, 2004,279(13):12009–12019

    Article  PubMed  CAS  Google Scholar 

  5. Singh L, Gao Q, Kumar A, et al. The high-risk human papillomavirus type 16 E6 counters the GAP function of E6TP1 toward small Rap G proteins. J Virol, 2003,77(2): 1614–1620

    Article  PubMed  CAS  Google Scholar 

  6. Gao Q, Kumar A, Singh L, et al. Human papillomavirus E6-induced degradation of E6TP1 is mediated by E6AP ubiquitin ligase. Cancer Res, 2002,62(11):3315–3321

    PubMed  CAS  Google Scholar 

  7. Kurachi H, Wada Y, Tsukamoto N, et al. Human SPA-1 gene product selectively expressed in lymphoid tissues is a specific GTPase-activating protein for Rap1 and Rap2. Segregate expression profiles from a rap1GAP gene product. J Biol Chem, 1997,272(44):28081–28088

    Article  PubMed  CAS  Google Scholar 

  8. Ishida D, Kometani K, Yang H, et al. Myeloproliferative stem cell disorders by deregulated Rap1 activation in SPA-1-deficient mice. Cancer Cell, 2003,4(1):55–65

    Article  PubMed  CAS  Google Scholar 

  9. Tsygankova OM, Feshchenko E, Klein PS, et al. Thyroid-stimulating hormone/cAMP and glycogen synthase kinase 3beta elicit opposing effects on Rap1GAP stability. J Biol Chem, 2004,279(7):5501–5507

    Article  PubMed  CAS  Google Scholar 

  10. Tsygankova OM, Prendergast GV, Puttaswamy K, et al. Downregulation of Rap1GAP contributes to Ras transformation. Mol Cell Biol, 2007,27(19):6647–6658

    Article  PubMed  CAS  Google Scholar 

  11. Nellore A, Paziana K, Ma C, et al. Loss of Rap1GAP in papillary thyroid cancer. J Clin Endocrinol Metab, 2009,94(3):1026–1032

    Article  PubMed  CAS  Google Scholar 

  12. Zhang L, Chenwei L, Mahmood R, et al. Identification of a putative tumor suppressor gene Rap1GAP in pancreatic cancer. Cancer Res, 2006,66(2):898–906

    Article  PubMed  CAS  Google Scholar 

  13. Zheng H, Gao L, Feng Y, et al. Down-regulation of Rap1GAP via promoter hypermethylation promotes melanoma cell proliferation, survival, and migration. Cancer Res, 2009,69(2):449–457

    Article  PubMed  CAS  Google Scholar 

  14. Zuo H, Gandhi M, Edreira MN, et al. Downregulation of Rap1GAP through epigenetic silencing and loss of heterozygosity promotes invasion and progression of thyroid tumors. Cancer Res, 2010,70(4):1389–1397

    Article  PubMed  CAS  Google Scholar 

  15. Tsygankova OM, Ma C, Tang W, et al. Downregulation of Rap1GAP in human tumor cells alters cell/matrix and cell/cell adhesion. Mol Cell Biol, 2010,30(13):3262–3274

    Article  PubMed  CAS  Google Scholar 

  16. Rangarajan S, Enserink JM, Kuiperij HB, et al. Cyclic AMP induces integrin-mediated cell adhesion through Epac and Rap1 upon stimulation of the β2-adrenergic receptor. J Cell Biol, 2003,160(4):487–493

    Article  PubMed  CAS  Google Scholar 

  17. de Bruyn KM, Zwartkruis FJ, de Rooij J, et al. The small GTPase Rap1 is activated by turbulence and is involved in integrin αIIbβ3-mediated cell adhesion in human megakaryocytes. J Biol Chem, 2003,278(25):22412–22417

    Article  PubMed  Google Scholar 

  18. Carmona G, Göttig S, Orlandi A, et al. Role of the small GTPase Rap1 for integrin activity regulation in endothelial cells and angiogenesis. Blood, 2009,113(2):488–497

    Article  PubMed  CAS  Google Scholar 

  19. Fukuhara S, Sakurai A, Sano H, et al. Cyclic AMP potentiates vascular endothelial cadherin-mediated cell-cell contact to enhance endothelial barrier function through an Epac-Rap1 signaling pathway. Mol Cell Biol, 2005,25(1):136–146

    Article  PubMed  CAS  Google Scholar 

  20. Yan J, Li F, Ingram DA, et al. Rap1a is a key regulator of FGF2-induced angiogenesis and together with Rap1b controls human endothelial cell functions. Mol Cell Biol, 2008, 28(18):5803–5810

    Article  PubMed  CAS  Google Scholar 

  21. Chrzanowska-Wodnicka M, Kraus AE, Gale D, et al. Defective angiogenesis, endothelial migration, proliferation, and MAPK signaling in Rap1b-deficient mice. Blood, 2008,111(5):2647–2656

    Article  PubMed  CAS  Google Scholar 

  22. Nagashima K, Endo A, Ogita H, et al. Adaptor protein Crk is required for ephrin-B1-induced membrane ruffling and focal complex assembly of human aortic endothelial cells. Mol Biol Cell, 2002,13(12):4231–4242

    Article  PubMed  CAS  Google Scholar 

  23. Roberts DM, Anderson AL, Hidaka M, et al. A vascular gene trap screen defines RasGRP3 as an angiogenesis-regulated gene required for the endothelial response to phorbol esters. Mol Cell Biol, 2004,24(24):10515–10528

    Article  PubMed  CAS  Google Scholar 

  24. Fujita H, Fukuhara S, Sakurai A, et al. Local activation of Rap1 contributes to directional vascular endothelial cell migration accompanied by extension of microtubules on which RAPL, a Rap1-associating molecule, localizes. J Biol Chem, 2005,280(6):5022–5031

    Article  PubMed  CAS  Google Scholar 

  25. Wittchen ES, Worthylake RA, Kelly P, et al. Rap1 GTPase inhibits leukocyte transmigration by promoting endothelial barrier function. J Biol Chem, 2005,280(12): 11675–11682

    Article  PubMed  CAS  Google Scholar 

  26. Jaffe EA, Nachman RL, Becker CG, et al. Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria. J Clin Invest, 1973,52(11):2745–2756

    Article  PubMed  CAS  Google Scholar 

  27. Fang Y, Olah ME. Cyclic AMP-dependent, protein kinase A-independent activation of extracellular signal-regulated kinase 1/2 following adenosine receptor stimulation in human umbilical vein endothelial cells: role of exchange protein activated by cAMP 1 (Epac1). J Pharmacol Exp Ther, 2007,322(3):1189–1200

    Article  PubMed  CAS  Google Scholar 

  28. Misra UK, Kaczowka S, Pizzo SV. The cAMP-activated GTP exchange factor, Epac1 upregulates plasma membrane and nuclear Akt kinase activities in 8-CPT-2-O-Me-cAMP-stimulated macrophages: gene silencing of the cAMP-activated GTP exchange Epac1 prevents 8-CPT-2-O-Me-cAMP activation of Akt activity in macrophages. Cell Signal, 2008,20(8):1459–1470

    Article  PubMed  CAS  Google Scholar 

  29. Mitra RS, Goto M, Lee JS, et al. Rap1GAP promotes invasion via induction of matrix metalloproteinase 9 secretion, which is associated with poor survival in low N-stage squamous cell carcinoma. Cancer Res, 2008,68(10):3959–3969

    Article  PubMed  CAS  Google Scholar 

  30. Ho SM, Lau KM, Mok SC, et al. Profiling follicle stimulating hormone-induced gene expression changes in normal and malignant human ovarian surface epithelial cells. Oncogene, 2003,22(27):4243–4256

    Article  PubMed  CAS  Google Scholar 

  31. Doebele RC, Schulze-Hoepfner FT, Hong J, et al. A novel interplay between Epac/Rap1 and mitogen-activated protein kinase kinase 5/extracellular signal-regulated kinase 5 (MEK5/ERK5) regulates thrombo-spondin to control angiogenesis. Blood, 2009,114(20): 4592–4600

    Article  PubMed  CAS  Google Scholar 

  32. Hatano N, Mori Y, Oh-hora M, et al. Essential role for ERK2 mitogen-activated protein kinase in placental development. Genes Cells, 2003,8(11):847–856

    Article  PubMed  CAS  Google Scholar 

  33. Wang L, Liu F, Adamo ML. Cyclic AMP inhibits extracellular signal-regulated kinase and phosphatidylinositol 3-kinase/Akt pathways by inhibiting Rap1. J Biol Chem, 2001,276(40):37242–37249

    Article  PubMed  CAS  Google Scholar 

  34. Brader S, Eccles SA. Phosphoinositide 3-kinase signalling pathways in tumor progression, invasion and angiogenesis. Tumor, 2004,90(1):2–8

    CAS  Google Scholar 

  35. Somanath PR, Razorenova OV, Chen J, et al. Akt1 in endothelial cell and angiogenesis. Cell Cycle, 2006,5(5): 512–518

    Article  PubMed  CAS  Google Scholar 

  36. Somanath PR, Kandel ES, Hay N, et al. Akt1 signaling regulates integrin activation, matrix recognition, and fibronectin assembly. J Biol Chem, 2007,282(31):22964–22976

    Article  PubMed  CAS  Google Scholar 

  37. Favot L, Keravis T, Lugnier C. Modulation of VEGF-induced endothelial cell cycle protein expression through cyclic AMP hydrolysis by PDE2 and PDE4. Thromb Haemost, 2004,92(3):634–645

    PubMed  CAS  Google Scholar 

  38. Weber JD, Raben DM, Phillips PJ, et al. Sustained activation of extracellular-signal-regulated kinase 1 (ERK1) is required for the continued expression of cyclin D1 in G1 phase. Biochem J, 1997,326(pt 1):61–68

    PubMed  CAS  Google Scholar 

  39. Hanai J, Dhanabal M, Karumanchi SA, et al. Endostatin causes G1 arrest of endothelial cells through inhibition of Cyclin D1. J Biol Chem, 2002,277(19):16464–16469

    Article  PubMed  CAS  Google Scholar 

  40. Zhang Z, Mitra RS, Henson BS, et al. Rap1GAP inhibits tumor growth in oropharyngeal squamous cell carcinoma. Am J Pathol, 2006,168(2):585–596

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Vascular Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China

    Wenyi Li  (李文毅), Bi Jin  (金 毕), Bin Zhou  (周 斌), Xiaoyang Fu  (符晓阳), Dan Shang  (尚 丹) & Hong Zheng  (郑 鸿)

  2. Division of Dermatology, Department of Internal Medicine, Washington University School of Medicine, St. Louis, MO, 63110, USA

    Lynn A. Cornelius

Authors
  1. Wenyi Li  (李文毅)
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Bi Jin  (金 毕)
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Lynn A. Cornelius
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Bin Zhou  (周 斌)
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Xiaoyang Fu  (符晓阳)
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Dan Shang  (尚 丹)
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Hong Zheng  (郑 鸿)
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Hong Zheng  (郑 鸿).

Additional information

This project was supported by grants from the National Natural Science Foundation of China (No. 30971207) and Natural Science Foundation of Hubei Province, China (No. 2009CBD-386).

Rights and permissions

Reprints and permissions

About this article

Cite this article

Li, W., Jin, B., Cornelius, L.A. et al. Inhibitory effects of Rap1GAP overexpression on proliferation and migration of endothelial cells via ERK and Akt pathways. J. Huazhong Univ. Sci. Technol. [Med. Sci.] 31, 721–727 (2011). https://doi.org/10.1007/s11596-011-0667-x

Download citation

  • Received:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11596-011-0667-x

Key words

Search

Navigation