생명과학회지 (Journal of Life Science)

  • 제18권12호
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  • Pages.1651-1656
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  • 2008
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  • 1225-9918(pISSN)
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  • 2287-3406(eISSN)
한국생명과학회 (Korean Society of Life Science)
권호 표지
DOI QR코드

DOI QR Code

마우스 내피세포주 MAE의 NO 생성과정에서 과발현된 Ref-1의 AKT 활성 조절에 대한 연구

Effect of Overexpressed Ref-1 on AKT Phosphorylation for NO Production in Mouse Aortic Endothelial Cell Line

  • 송주동 (부산대학교 의학전문대학원 미생물학 및 면역학교실) ;
  • 이상권 (부산대학교 의학전문대학원 흉부외과학교실) ;
  • 박영철 (부산대학교 의학전문대학원 미생물학 및 면역학교실)
  • Song, Ju-Dong (Department of Cardiovascular Surgery, Pusan National University School of Medicine) ;
  • Lee, Sang-Kwon (Department of Microbiology & Immunology, Pusan National University School of Medicine) ;
  • Park, Young-Chul (Department of Cardiovascular Surgery, Pusan National University School of Medicine)
  • 발행 : 2008.12.30
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초록

Redox factor-1 (Ref-1)은 산화적으로 손상된 DNA의 복구와 세포내 산화환원에 민감한 전사인자들의 활성화에 필수적인 역할을 수행한다. 본 연구에서는 마우스 유래 혈관내피세포주 (MAE)에서 nitric oxide (NO) 생성과정에 관여하는 AKT 활성화의 측면에서 adenoviral vector를 사용하여 과발현된 Ref-1의 역할을 살펴보았다. NO 측정을 위하여 fluorophore DAF-2를 사용하였다. 과발현된 Ref-1은 bradykinin으로 자극한 세포뿐만 아니라 자극되지 않은 세포의 NO 생성도 증가시켰다. 놀랍게도 이 과발현된 Ref-1은 AKT의 직접적인 인산화를 유도하였으며, AKT 저해제로 널리 사용되는 wortmannin에 의해 반응이 억제되었다. 또한, Ref-1에 의한 직접적인 AKT 활성화를 증명하기 위하여 HA-tagged activation-deficient AKT를 과발현시키는 adenoviral vector를 사용하였다. 이 방법을 이용한 AKT 활성의 저해는 과발현된 Ref-1에 의한 NO 생성 및 bradykinin 자극에 의한 NO 생성을 억제하였다. 이들 결과는 Ref-1이 마우스 혈관내피세포에서 직접적인 AKT 인산화를 통하여 eNOS 활성화를 유도한다는 것을 의미한다.

Redox factor-1 (Ref-1) is essential for repair of oxidatively damaged DNA and also govern the reductive activation of many transcription factors. In this study, we examined the effect of overexpressed Ref-1 on AKT activation for nitric oxide (NO) production in mouse aortic endothelial (MAE) cells. Adenoviral-mediated overexpression of Ref-1 enhanced NO production in unstimulated- as well as bradykinin-stimulated MAE cells. Importantly, forced overexpression of Ref-1 induced direct phosphorylation of AKT in cells. And, a PI3K inhibitor wortmannin completely abolished the increase in AKT phosphorylation by stimulation of bradykinin and/or overexpressed Ref-1. In addition, inhibition of AKT activity with HA-tagged activation-deficient AKT suppressed Ref-1-induced endothelial NO synthase (eNOS) phosphorylation and resulted in a corresponding inhibition of unstimulated- and bradykinin-stimulated NO production. These results suggest that Ref-1 stimulates direct phosphorylation of AKT for eNOS enzyme activity in murine endothelial cells.

키워드

참고문헌

  1. Furchgott, R. F. and J. V. Zawadzki. 1980. The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature 288, 373-376 https://doi.org/10.1038/288373a0
  2. Alheid, U., J. C. Frolich and U. Forstermann. 1987. Endothelium-derived relaxing factor from cultured human endothelial cells inhibits aggregation of human platelets. Thromb. Res. 47, 561-571 https://doi.org/10.1016/0049-3848(87)90361-6
  3. Westendorp, R. G., R Draijer, A. E. Meinders and V. W. van Hinsbergh. 1994. Cyclic-GMP-mediated decrease in permeability of human umbilical and pulmonary artery endothelial cell monolayers. J. Vasco Res. 31, 42-51 https://doi.org/10.1159/000159030
  4. Busse, R. and A. Mulsch. 1990. Calcium-dependent nitric oxide synthesis in endothelial cytosol is mediated by calmodulin. FEBS Lett. 265, 133-136 https://doi.org/10.1016/0014-5793(90)80902-U
  5. Bult, H. 1996. Nitric oxide and atherosclerosis: possible implications for therapy. Mol. Med. Today 2, 510-518 https://doi.org/10.1016/S1357-4310(97)81455-4
  6. Kawashima, S. and M. Yokoyama. 2004. Dysfunction of endothelial nitric oxide synthase and atherosclerosis. Arterioscler. Thromb. Vasco BioI. 24, 998-1005 https://doi.org/10.1161/01.ATV.0000125114.88079.96
  7. Cooke, J. P. and V. J. Dzau. 1997. Nitric oxide synthase: role in the genesis of vascular disease. Annu. Rev. Med. 48, 489-509 https://doi.org/10.1146/annurev.med.48.1.489
  8. Nakamura, H, K. Nakamura and J. Yodoi. 1997. Redox regulation of cellular activation. Annu. Rev. Immunol. 15, 351-369 https://doi.org/10.1146/annurev.immunol.15.1.351
  9. Huang, L. E., Z. Arany, D. M. Livingston and H. F. Bunn. 1996. Activation of hypoxia-inducible transcription factor depends primarily upon redox-sensitive stabilization of its alpha subunit. J. BioI. Chem. 271, 32253-32259 https://doi.org/10.1074/jbc.271.50.32253
  10. Jayaraman, L., K G. Murthy, C. Zhu, T. Curran, S. Xanthoudakis and C. Prives. 1997. Identification of redox/repair protein Ref-1 as a potent activator of p53. Genes Dev. 11, 558-570 https://doi.org/10.1101/gad.11.5.558
  11. Mitomo, K, K. Nakayama, K. Fujimoto, X. Sun, S. Seki and K. Yamamoto. 1994. Two different cellular redox systems regulate the DNA-binding activity of the p50 subunit of NF-kappa B in vitro. Gene 145, 197-203 https://doi.org/10.1016/0378-1119(94)90005-1
  12. Demple, B., T. Herman and D. S. Chen. 1991. Cloning and expression of APE, the cDNA encoding the major human apurinic endonuclease: definition of a family of DNA repair enzymes. Proc. Natl. A cad. Sci. USA 88, 11450-11454 https://doi.org/10.1073/pnas.88.24.11450
  13. Song, J. D., K. M. Kim, S. K. Lee, J. M. Kim and Y. C. Park. 2007. Adenoviral mediated Ref-1 overexpression potentiates NO production in bradykinin-stimulated endothelial cells. J. Life Sci. 17, 905-909 https://doi.org/10.5352/JLS.2007.17.7.905
  14. Xing, Z., Y. Ohkawara, M. Jordana, F. L. Graham and J. Gauldie. 1996. Transfer of granulocyte-macrophage colony-stimulating factor gene to rat lung induces eosinophilia, monocytosis, and fibrotic reactions. J. Clin. Invest. 97, 1102-1110 https://doi.org/10.1172/JCI118503
  15. Jeon, B. H., G. Gupta, Y. C. Park, B. Qi, A. Haile, F. A. Khanday, Y. X. Liu, J. M. Kim, M. Ozaki, A. R White, D. E. Berkowitz and K Irani. 2004. Apurinic/ apyrimidic endonuclease 1 regulates endothelial NO production and vascular tone. Circ. Res. 95, 902-910 https://doi.org/10.1161/01.RES.0000146947.84294.4c
  16. Kojima, H., H. Kojima, K. Sakurai, K. Kikuchi, S. Kawahara, Y. Kirino, H. Nagoshi, Y. Hirata and T. Nagano. 1998. Development of a fluorescent indicator for nitric oxide based on the fluorescein chromophore. Chem. Pharm. Bull. 46, 373-375 https://doi.org/10.1248/cpb.46.373
  17. Dai, J., W. Li, L. Chang, Z. Zhang, C. Tang, N. Wang, Y. Zhu and X. Wang. 2006. Role of redox factor-1 in hyperhomocysteinemia-accelerated atherosclerosis. Free Radic. BioI. Med. 41, 1566-1577 https://doi.org/10.1016/j.freeradbiomed.2006.08.020
  18. Martinet, W., M. W. Knaapen, G. R. De Meyer, A. G. Herman and M. M. Kockx. 2002. Elevated levels of oxidative DNA damage and DNA repair enzymes in human atherosclerotic plaques. Circulation 106, 927-932 https://doi.org/10.1161/01.CIR.0000026393.47805.21
  19. Kuchan, M. J. and J. A. Frangos. 1994. Role of calcium and calmodulin in flow-induced nitric oxide production in endothelial cells. Am. J. Physiol. 266, C628-C636 https://doi.org/10.1152/ajpcell.1994.266.3.C628
  20. Ayajiki, K, M. Kindermann, M. Hecker, I. Fleming and R. Busse. 1996. Intracellular pH and tyrosine phosphorylation but not calcium determine shear stress-induced nitric oxide production in native endothelial cells. Circ. Res. 78, 750-758 https://doi.org/10.1161/01.RES.78.5.750
  21. Dimmeler, S., B. Assmus, C. Hermann, J. Haendeler and A. M. Zeiher. 1998. Fluid shear stress stimulates phosphorylation of Akt in human endothelial cells: involvement in suppression of apoptosis. Circ. Res. 83, 334-342 https://doi.org/10.1161/01.RES.83.3.334
  22. Fulton, D., J. P. Gratton, T. J. McCabe, J. Fontana, Y. Fujio, K Walsh, T. F. Franke, A. Papapetropoulos and W. C. Sessa. 1999. Regulation of endothelium-derived nitric oxide production by the protein kinase Akt. Nature 399, 597-601 https://doi.org/10.1038/21218
  23. Dimmeler, S., I. Fleming, B. Fisslthaler, C. Hermann, R. Busse and A. M. Zeiher. 1999. Activation of nitric oxide synthase in endothelial cells by Akt-dependent phosphorylation. Nature 399, 601-605 https://doi.org/10.1038/21224
  24. Dugid, J. R., J. N. Eble, T. M. Wilson and M. R. Kelley. 1995. Differential cellular and subcellular expression of the human multifunctional apurinic/ apyrimidinic endonuclease (APE/ref-1) DNA repair enzyme. Cancer Res. 55, 6097-6102
  25. Kakolyris, S., L. Kaklamanis, K. Engels, S. B. Fox, M. Taylor, I. D. Hickson, K. C. Gatter and A. L. Harris. 1998. Expression and subcellular localization of human AP endo-nuclease 1 (HAP1/Ref-1) protein: a basis for its role in human disease. Histopathology 33, 561-569 https://doi.org/10.1046/j.1365-2559.1998.00541.x
  26. Rivkees, S. A. and M. R. Kelley. 1994. Expression of multifunctional DNA repair enzyme gene, apurinic/ apyrimidic endonuclease (APE; Ref-1)in the suprachiasmatic, supraoptic and paraventricular nuclei. Brain Res. 666, 137-142 https://doi.org/10.1016/0006-8993(94)90296-8