Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1996 Sep 1;318(Pt 2):379–382. doi: 10.1042/bj3180379

Regulation of reactive-oxygen-species generation in fibroblasts by Rac1.

M Sundaresan 1, Z X Yu 1, V J Ferrans 1, D J Sulciner 1, J S Gutkind 1, K Irani 1, P J Goldschmidt-Clermont 1, T Finkel 1
PMCID: PMC1217632  PMID: 8809022

Abstract

In a variety of non-phagocytic cell types, there is a marked increase in intracellular levels of reactive oxygen species (ROS), including superoxide and H2O2, after ligand stimulation. We demonstrate that in NIH 3T3 cells transient expression of constitutively activated forms of the small GTP-binding proteins Ras or Rac1 leads to a significant increase in intracellular ROS. An increase in intracellular ROS is also demonstrated after growth factor [platelet-derived growth factor (PDGF) or epidermal growth factor (EGF)] or cytokine [tumour necrosis factor-alpha (TNF-alpha) or interleukin (IL)-1 beta] stimulation of NIH 3T3 cells. Expression of a dominant negative allele of Rac1 inhibits the rise in ROS seen after Ras expression or after stimulation by either growth factors or cytokines. These results provide the first demonstration of the pathway by which ligand stimulation of ROS occurs in non-phagocytic cells and suggest that the family of Ras-related small GTP-binding proteins may function as regulators of the intracellular redox state.

Full Text

The Full Text of this article is available as a PDF (259.4 KB).

Selected References

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

  1. Abate C., Patel L., Rauscher F. J., 3rd, Curran T. Redox regulation of fos and jun DNA-binding activity in vitro. Science. 1990 Sep 7;249(4973):1157–1161. doi: 10.1126/science.2118682. [DOI] [PubMed] [Google Scholar]
  2. Abo A., Pick E., Hall A., Totty N., Teahan C. G., Segal A. W. Activation of the NADPH oxidase involves the small GTP-binding protein p21rac1. Nature. 1991 Oct 17;353(6345):668–670. doi: 10.1038/353668a0. [DOI] [PubMed] [Google Scholar]
  3. Baas A. S., Berk B. C. Differential activation of mitogen-activated protein kinases by H2O2 and O2- in vascular smooth muscle cells. Circ Res. 1995 Jul;77(1):29–36. doi: 10.1161/01.res.77.1.29. [DOI] [PubMed] [Google Scholar]
  4. Bokoch G. M., Der C. J. Emerging concepts in the Ras superfamily of GTP-binding proteins. FASEB J. 1993 Jun;7(9):750–759. doi: 10.1096/fasebj.7.9.8330683. [DOI] [PubMed] [Google Scholar]
  5. Bokoch G. M. Regulation of the human neutrophil NADPH oxidase by the Rac GTP-binding proteins. Curr Opin Cell Biol. 1994 Apr;6(2):212–218. doi: 10.1016/0955-0674(94)90138-4. [DOI] [PubMed] [Google Scholar]
  6. Burdon R. H. Superoxide and hydrogen peroxide in relation to mammalian cell proliferation. Free Radic Biol Med. 1995 Apr;18(4):775–794. doi: 10.1016/0891-5849(94)00198-s. [DOI] [PubMed] [Google Scholar]
  7. Chen Z., Silva H., Klessig D. F. Active oxygen species in the induction of plant systemic acquired resistance by salicylic acid. Science. 1993 Dec 17;262(5141):1883–1886. doi: 10.1126/science.8266079. [DOI] [PubMed] [Google Scholar]
  8. Coso O. A., Chiariello M., Yu J. C., Teramoto H., Crespo P., Xu N., Miki T., Gutkind J. S. The small GTP-binding proteins Rac1 and Cdc42 regulate the activity of the JNK/SAPK signaling pathway. Cell. 1995 Jun 30;81(7):1137–1146. doi: 10.1016/s0092-8674(05)80018-2. [DOI] [PubMed] [Google Scholar]
  9. Griendling K. K., Minieri C. A., Ollerenshaw J. D., Alexander R. W. Angiotensin II stimulates NADH and NADPH oxidase activity in cultured vascular smooth muscle cells. Circ Res. 1994 Jun;74(6):1141–1148. doi: 10.1161/01.res.74.6.1141. [DOI] [PubMed] [Google Scholar]
  10. Guehmann S., Vorbrueggen G., Kalkbrenner F., Moelling K. Reduction of a conserved Cys is essential for Myb DNA-binding. Nucleic Acids Res. 1992 May 11;20(9):2279–2286. doi: 10.1093/nar/20.9.2279. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Guyton K. Z., Liu Y., Gorospe M., Xu Q., Holbrook N. J. Activation of mitogen-activated protein kinase by H2O2. Role in cell survival following oxidant injury. J Biol Chem. 1996 Feb 23;271(8):4138–4142. doi: 10.1074/jbc.271.8.4138. [DOI] [PubMed] [Google Scholar]
  12. Hall A. Ras-related proteins. Curr Opin Cell Biol. 1993 Apr;5(2):265–268. doi: 10.1016/0955-0674(93)90114-6. [DOI] [PubMed] [Google Scholar]
  13. Hawkins P. T., Eguinoa A., Qiu R. G., Stokoe D., Cooke F. T., Walters R., Wennström S., Claesson-Welsh L., Evans T., Symons M. PDGF stimulates an increase in GTP-Rac via activation of phosphoinositide 3-kinase. Curr Biol. 1995 Apr 1;5(4):393–403. doi: 10.1016/s0960-9822(95)00080-7. [DOI] [PubMed] [Google Scholar]
  14. Hecht D., Zick Y. Selective inhibition of protein tyrosine phosphatase activities by H2O2 and vanadate in vitro. Biochem Biophys Res Commun. 1992 Oct 30;188(2):773–779. doi: 10.1016/0006-291x(92)91123-8. [DOI] [PubMed] [Google Scholar]
  15. Jones O. T. The regulation of superoxide production by the NADPH oxidase of neutrophils and other mammalian cells. Bioessays. 1994 Dec;16(12):919–923. doi: 10.1002/bies.950161211. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. Knaus U. G., Heyworth P. G., Evans T., Curnutte J. T., Bokoch G. M. Regulation of phagocyte oxygen radical production by the GTP-binding protein Rac 2. Science. 1991 Dec 6;254(5037):1512–1515. doi: 10.1126/science.1660188. [DOI] [PubMed] [Google Scholar]
  18. Krieger-Brauer H. I., Kather H. Antagonistic effects of different members of the fibroblast and platelet-derived growth factor families on adipose conversion and NADPH-dependent H2O2 generation in 3T3 L1-cells. Biochem J. 1995 Apr 15;307(Pt 2):549–556. doi: 10.1042/bj3070549. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kumar S., Rabson A. B., Gélinas C. The RxxRxRxxC motif conserved in all Rel/kappa B proteins is essential for the DNA-binding activity and redox regulation of the v-Rel oncoprotein. Mol Cell Biol. 1992 Jul;12(7):3094–3106. doi: 10.1128/mcb.12.7.3094-3106.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Levine A., Tenhaken R., Dixon R., Lamb C. H2O2 from the oxidative burst orchestrates the plant hypersensitive disease resistance response. Cell. 1994 Nov 18;79(4):583–593. doi: 10.1016/0092-8674(94)90544-4. [DOI] [PubMed] [Google Scholar]
  21. Lo Y. Y., Cruz T. F. Involvement of reactive oxygen species in cytokine and growth factor induction of c-fos expression in chondrocytes. J Biol Chem. 1995 May 19;270(20):11727–11730. doi: 10.1074/jbc.270.20.11727. [DOI] [PubMed] [Google Scholar]
  22. Marui N., Offermann M. K., Swerlick R., Kunsch C., Rosen C. A., Ahmad M., Alexander R. W., Medford R. M. Vascular cell adhesion molecule-1 (VCAM-1) gene transcription and expression are regulated through an antioxidant-sensitive mechanism in human vascular endothelial cells. J Clin Invest. 1993 Oct;92(4):1866–1874. doi: 10.1172/JCI116778. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. Meier B., Radeke H. H., Selle S., Younes M., Sies H., Resch K., Habermehl G. G. Human fibroblasts release reactive oxygen species in response to interleukin-1 or tumour necrosis factor-alpha. Biochem J. 1989 Oct 15;263(2):539–545. doi: 10.1042/bj2630539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Minden A., Lin A., Claret F. X., Abo A., Karin M. Selective activation of the JNK signaling cascade and c-Jun transcriptional activity by the small GTPases Rac and Cdc42Hs. Cell. 1995 Jun 30;81(7):1147–1157. doi: 10.1016/s0092-8674(05)80019-4. [DOI] [PubMed] [Google Scholar]
  26. Ohba M., Shibanuma M., Kuroki T., Nose K. Production of hydrogen peroxide by transforming growth factor-beta 1 and its involvement in induction of egr-1 in mouse osteoblastic cells. J Cell Biol. 1994 Aug;126(4):1079–1088. doi: 10.1083/jcb.126.4.1079. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Olson M. F., Ashworth A., Hall A. An essential role for Rho, Rac, and Cdc42 GTPases in cell cycle progression through G1. Science. 1995 Sep 1;269(5228):1270–1272. doi: 10.1126/science.7652575. [DOI] [PubMed] [Google Scholar]
  28. Qiu R. G., Chen J., Kirn D., McCormick F., Symons M. An essential role for Rac in Ras transformation. Nature. 1995 Mar 30;374(6521):457–459. doi: 10.1038/374457a0. [DOI] [PubMed] [Google Scholar]
  29. Ridley A. J., Paterson H. F., Johnston C. L., Diekmann D., Hall A. The small GTP-binding protein rac regulates growth factor-induced membrane ruffling. Cell. 1992 Aug 7;70(3):401–410. doi: 10.1016/0092-8674(92)90164-8. [DOI] [PubMed] [Google Scholar]
  30. Rosenkranz A. R., Schmaldienst S., Stuhlmeier K. M., Chen W., Knapp W., Zlabinger G. J. A microplate assay for the detection of oxidative products using 2',7'-dichlorofluorescin-diacetate. J Immunol Methods. 1992 Nov 25;156(1):39–45. doi: 10.1016/0022-1759(92)90008-h. [DOI] [PubMed] [Google Scholar]
  31. Schreck R., Meier B., Männel D. N., Dröge W., Baeuerle P. A. Dithiocarbamates as potent inhibitors of nuclear factor kappa B activation in intact cells. J Exp Med. 1992 May 1;175(5):1181–1194. doi: 10.1084/jem.175.5.1181. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Schreck R., Rieber P., Baeuerle P. A. Reactive oxygen intermediates as apparently widely used messengers in the activation of the NF-kappa B transcription factor and HIV-1. EMBO J. 1991 Aug;10(8):2247–2258. doi: 10.1002/j.1460-2075.1991.tb07761.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Shibanuma M., Kuroki T., Nose K. Stimulation by hydrogen peroxide of DNA synthesis, competence family gene expression and phosphorylation of a specific protein in quiescent Balb/3T3 cells. Oncogene. 1990 Jul;5(7):1025–1032. [PubMed] [Google Scholar]
  34. Sundaresan M., Yu Z. X., Ferrans V. J., Irani K., Finkel T. Requirement for generation of H2O2 for platelet-derived growth factor signal transduction. Science. 1995 Oct 13;270(5234):296–299. doi: 10.1126/science.270.5234.296. [DOI] [PubMed] [Google Scholar]
  35. Verheij M., Bose R., Lin X. H., Yao B., Jarvis W. D., Grant S., Birrer M. J., Szabo E., Zon L. I., Kyriakis J. M. Requirement for ceramide-initiated SAPK/JNK signalling in stress-induced apoptosis. Nature. 1996 Mar 7;380(6569):75–79. doi: 10.1038/380075a0. [DOI] [PubMed] [Google Scholar]
  36. Xanthoudakis S., Miao G., Wang F., Pan Y. C., Curran T. Redox activation of Fos-Jun DNA binding activity is mediated by a DNA repair enzyme. EMBO J. 1992 Sep;11(9):3323–3335. doi: 10.1002/j.1460-2075.1992.tb05411.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES