Skip to main content
Biochemical Journal logoLink to Biochemical Journal
. 1998 May 15;332(Pt 1):5–8. doi: 10.1042/bj3320005

Rac1, and not Rac2, is involved in the regulation of the intracellular hydrogen peroxide level in HepG2 cells.

R H Cool 1, E Merten 1, C Theiss 1, H Acker 1
PMCID: PMC1219445  PMID: 9576845

Abstract

In order to elucidate the components of the oxygen sensory complex in HepG2 cells which regulates the production of erythropoietin, we have microinjected recombinant variants of the human small GTP-binding protein hRac1 and measured their effects on the production of reactive oxygen species (ROS) by the dihydrorhodamine-123 technique. The dominant-negative mutant hRac1(T17N) inhibits the NADH-stimulated production of ROS in HepG2 cells, whereas the constitutively activated hRac1(G12V) leads to an increase in intracellular ROS concentration. Reverse transcriptase PCR analysis showed that the hRac1, but not the hRac2, gene is expressed in HepG2 cells. These results demonstrate that hRac1, and not hRac2, is involved in the regulation of ROS production in HepG2 cells and suggest that hRac1 specifically functions in the non-phagocytic NAD(P)H oxidase complex.

Full Text

The Full Text of this article is available as a PDF (302.2 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Acker H. Mechanisms and meaning of cellular oxygen sensing in the organism. Respir Physiol. 1994 Jan;95(1):1–10. doi: 10.1016/0034-5687(94)90043-4. [DOI] [PubMed] [Google Scholar]
  2. Bastian N. R., Hibbs J. B., Jr Assembly and regulation of NADPH oxidase and nitric oxide synthase. Curr Opin Immunol. 1994 Feb;6(1):131–139. doi: 10.1016/0952-7915(94)90044-2. [DOI] [PubMed] [Google Scholar]
  3. Bunn H. F., Poyton R. O. Oxygen sensing and molecular adaptation to hypoxia. Physiol Rev. 1996 Jul;76(3):839–885. doi: 10.1152/physrev.1996.76.3.839. [DOI] [PubMed] [Google Scholar]
  4. Cross A. R., Henderson L., Jones O. T., Delpiano M. A., Hentschel J., Acker H. Involvement of an NAD(P)H oxidase as a pO2 sensor protein in the rat carotid body. Biochem J. 1990 Dec 15;272(3):743–747. doi: 10.1042/bj2720743. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Didsbury J., Weber R. F., Bokoch G. M., Evans T., Snyderman R. rac, a novel ras-related family of proteins that are botulinum toxin substrates. J Biol Chem. 1989 Oct 5;264(28):16378–16382. [PubMed] [Google Scholar]
  6. Dorseuil O., Reibel L., Bokoch G. M., Camonis J., Gacon G. The Rac target NADPH oxidase p67phox interacts preferentially with Rac2 rather than Rac1. J Biol Chem. 1996 Jan 5;271(1):83–88. doi: 10.1074/jbc.271.1.83. [DOI] [PubMed] [Google Scholar]
  7. Ehleben W., Porwol T., Fandrey J., Kummer W., Acker H. Cobalt and desferrioxamine reveal crucial members of the oxygen sensing pathway in HepG2 cells. Kidney Int. 1997 Feb;51(2):483–491. doi: 10.1038/ki.1997.67. [DOI] [PubMed] [Google Scholar]
  8. Fandrey J., Frede S., Jelkmann W. Role of hydrogen peroxide in hypoxia-induced erythropoietin production. Biochem J. 1994 Oct 15;303(Pt 2):507–510. doi: 10.1042/bj3030507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gabig T. G., Crean C. D., Mantel P. L., Rosli R. Function of wild-type or mutant Rac2 and Rap1a GTPases in differentiated HL60 cell NADPH oxidase activation. Blood. 1995 Feb 1;85(3):804–811. [PubMed] [Google Scholar]
  10. Gleadle J. M., Ebert B. L., Ratcliffe P. J. Diphenylene iodonium inhibits the induction of erythropoietin and other mammalian genes by hypoxia. Implications for the mechanism of oxygen sensing. Eur J Biochem. 1995 Nov 15;234(1):92–99. doi: 10.1111/j.1432-1033.1995.092_c.x. [DOI] [PubMed] [Google Scholar]
  11. Goldberg M. A., Dunning S. P., Bunn H. F. Regulation of the erythropoietin gene: evidence that the oxygen sensor is a heme protein. Science. 1988 Dec 9;242(4884):1412–1415. doi: 10.1126/science.2849206. [DOI] [PubMed] [Google Scholar]
  12. Goldwasser E., Alibali P., Gardner A. Differential inhibition by iodonium compounds of induced erythropoietin expression. J Biol Chem. 1995 Feb 10;270(6):2628–2629. doi: 10.1074/jbc.270.6.2628. [DOI] [PubMed] [Google Scholar]
  13. Görlach A., Holtermann G., Jelkmann W., Hancock J. T., Jones S. A., Jones O. T., Acker H. Photometric characteristics of haem proteins in erythropoietin-producing hepatoma cells (HepG2). Biochem J. 1993 Mar 15;290(Pt 3):771–776. doi: 10.1042/bj2900771. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Haataja L., Groffen J., Heisterkamp N. Characterization of RAC3, a novel member of the Rho family. J Biol Chem. 1997 Aug 15;272(33):20384–20388. doi: 10.1074/jbc.272.33.20384. [DOI] [PubMed] [Google Scholar]
  15. Heyworth P. G., Bohl B. P., Bokoch G. M., Curnutte J. T. Rac translocates independently of the neutrophil NADPH oxidase components p47phox and p67phox. Evidence for its interaction with flavocytochrome b558. J Biol Chem. 1994 Dec 9;269(49):30749–30752. [PubMed] [Google Scholar]
  16. Heyworth P. G., Knaus U. G., Xu X., Uhlinger D. J., Conroy L., Bokoch G. M., Curnutte J. T. Requirement for posttranslational processing of Rac GTP-binding proteins for activation of human neutrophil NADPH oxidase. Mol Biol Cell. 1993 Mar;4(3):261–269. doi: 10.1091/mbc.4.3.261. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hirshberg M., Stockley R. W., Dodson G., Webb M. R. The crystal structure of human rac1, a member of the rho-family complexed with a GTP analogue. Nat Struct Biol. 1997 Feb;4(2):147–152. doi: 10.1038/nsb0297-147. [DOI] [PubMed] [Google Scholar]
  18. Huang L. E., Arany Z., Livingston D. M., Bunn H. F. Activation of hypoxia-inducible transcription factor depends primarily upon redox-sensitive stabilization of its alpha subunit. J Biol Chem. 1996 Dec 13;271(50):32253–32259. doi: 10.1074/jbc.271.50.32253. [DOI] [PubMed] [Google Scholar]
  19. Inoue N., Kawashima S., Kanazawa K., Yamada S., Akita H., Yokoyama M. Polymorphism of the NADH/NADPH oxidase p22 phox gene in patients with coronary artery disease. Circulation. 1998 Jan 20;97(2):135–137. doi: 10.1161/01.cir.97.2.135. [DOI] [PubMed] [Google Scholar]
  20. Jelkmann W. Erythropoietin: structure, control of production, and function. Physiol Rev. 1992 Apr;72(2):449–489. doi: 10.1152/physrev.1992.72.2.449. [DOI] [PubMed] [Google Scholar]
  21. Jones S. A., Wood J. D., Coffey M. J., Jones O. T. The functional expression of p47-phox and p67-phox may contribute to the generation of superoxide by an NADPH oxidase-like system in human fibroblasts. FEBS Lett. 1994 Nov 28;355(2):178–182. doi: 10.1016/0014-5793(94)01201-6. [DOI] [PubMed] [Google Scholar]
  22. Kummer W., Acker H. Immunohistochemical demonstration of four subunits of neutrophil NAD(P)H oxidase in type I cells of carotid body. J Appl Physiol (1985) 1995 May;78(5):1904–1909. doi: 10.1152/jappl.1995.78.5.1904. [DOI] [PubMed] [Google Scholar]
  23. Lander H. M. An essential role for free radicals and derived species in signal transduction. FASEB J. 1997 Feb;11(2):118–124. [PubMed] [Google Scholar]
  24. Liu Y., Cox S. R., Morita T., Kourembanas S. Hypoxia regulates vascular endothelial growth factor gene expression in endothelial cells. Identification of a 5' enhancer. Circ Res. 1995 Sep;77(3):638–643. doi: 10.1161/01.res.77.3.638. [DOI] [PubMed] [Google Scholar]
  25. Marshall C., Mamary A. J., Verhoeven A. J., Marshall B. E. Pulmonary artery NADPH-oxidase is activated in hypoxic pulmonary vasoconstriction. Am J Respir Cell Mol Biol. 1996 Nov;15(5):633–644. doi: 10.1165/ajrcmb.15.5.8918370. [DOI] [PubMed] [Google Scholar]
  26. Marti H. H., Gassmann M., Wenger R. H., Kvietikova I., Morganti-Kossmann M. C., Kossmann T., Trentz O., Bauer C. Detection of erythropoietin in human liquor: intrinsic erythropoietin production in the brain. Kidney Int. 1997 Feb;51(2):416–418. doi: 10.1038/ki.1997.55. [DOI] [PubMed] [Google Scholar]
  27. Meier B., Jesaitis A. J., Emmendörffer A., Roesler J., Quinn M. T. The cytochrome b-558 molecules involved in the fibroblast and polymorphonuclear leucocyte superoxide-generating NADPH oxidase systems are structurally and genetically distinct. Biochem J. 1993 Jan 15;289(Pt 2):481–486. doi: 10.1042/bj2890481. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Moulton P. J., Goldring M. B., Hancock J. T. NADPH oxidase of chondrocytes contains an isoform of the gp91phox subunit. Biochem J. 1998 Feb 1;329(Pt 3):449–451. doi: 10.1042/bj3290449. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Nakano H., Yamazaki T., Ikeda M., Masai H., Miyatake S., Saito T. Purification of glutathione S-transferase fusion proteins as a non-degraded form by using a protease-negative E. coli strain, AD202. Nucleic Acids Res. 1994 Feb 11;22(3):543–544. doi: 10.1093/nar/22.3.543. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Royall J. A., Ischiropoulos H. Evaluation of 2',7'-dichlorofluorescin and dihydrorhodamine 123 as fluorescent probes for intracellular H2O2 in cultured endothelial cells. Arch Biochem Biophys. 1993 May;302(2):348–355. doi: 10.1006/abbi.1993.1222. [DOI] [PubMed] [Google Scholar]
  31. Shweiki D., Itin A., Soffer D., Keshet E. Vascular endothelial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesis. Nature. 1992 Oct 29;359(6398):843–845. doi: 10.1038/359843a0. [DOI] [PubMed] [Google Scholar]
  32. Sundaresan M., Yu Z. X., Ferrans V. J., Sulciner D. J., Gutkind J. S., Irani K., Goldschmidt-Clermont P. J., Finkel T. Regulation of reactive-oxygen-species generation in fibroblasts by Rac1. Biochem J. 1996 Sep 1;318(Pt 2):379–382. doi: 10.1042/bj3180379. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Van Aelst L., D'Souza-Schorey C. Rho GTPases and signaling networks. Genes Dev. 1997 Sep 15;11(18):2295–2322. doi: 10.1101/gad.11.18.2295. [DOI] [PubMed] [Google Scholar]
  34. Wang D., Youngson C., Wong V., Yeger H., Dinauer M. C., Vega-Saenz Miera E., Rudy B., Cutz E. NADPH-oxidase and a hydrogen peroxide-sensitive K+ channel may function as an oxygen sensor complex in airway chemoreceptors and small cell lung carcinoma cell lines. Proc Natl Acad Sci U S A. 1996 Nov 12;93(23):13182–13187. doi: 10.1073/pnas.93.23.13182. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Wenger R. H., Marti H. H., Schuerer-Maly C. C., Kvietikova I., Bauer C., Gassmann M., Maly F. E. Hypoxic induction of gene expression in chronic granulomatous disease-derived B-cell lines: oxygen sensing is independent of the cytochrome b558-containing nicotinamide adenine dinucleotide phosphate oxidase. Blood. 1996 Jan 15;87(2):756–761. [PubMed] [Google Scholar]
  36. Wientjes F. B., Hsuan J. J., Totty N. F., Segal A. W. p40phox, a third cytosolic component of the activation complex of the NADPH oxidase to contain src homology 3 domains. Biochem J. 1993 Dec 15;296(Pt 3):557–561. doi: 10.1042/bj2960557. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Youngson C., Nurse C., Yeger H., Curnutte J. T., Vollmer C., Wong V., Cutz E. Immunocytochemical localization on O2-sensing protein (NADPH oxidase) in chemoreceptor cells. Microsc Res Tech. 1997 Apr 1;37(1):101–106. doi: 10.1002/(SICI)1097-0029(19970401)37:1<101::AID-JEMT10>3.0.CO;2-V. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES