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. 2001 Aug;10(4):209–215. doi: 10.1080/09629350120080401

Diphenyleneiodonium inhibits NF-kappaB activation and iNOS expression induced by IL-1beta: involvement of reactive oxygen species.

A F Mendes 1, A P Carvalho 1, M M Caramona 1, M C Lopes 1
PMCID: PMC1781711  PMID: 11577997

Abstract

AIMS: In this work, we studied the mechanisms by which diphenyleneiodonium chloride (DPI) inhibits nitric oxide (NO) synthesis induced by the proinflammatory cytokine interleukin-1beta (IL-1) in bovine articular chondrocytes. To achieve this, we evaluated the ability of DPI to inhibit the expression and activity of the inducible isoform of the NO synthase (iNOS) induced by IL-1. We also studied the ability of DPI to prevent IL-1-induced NF-kappaB activation and reactive oxygen species (ROS) production. RESULTS: Northern and Western blot analysis, respectively, showed that DPI dose-dependently inhibited IL-1-induced iNOS mRNA and protein synthesis in primary cultures of bovine articular chondrocytes. DPI effectively inhibited NO production (IC50=0.03+/-0.004 microM), as evaluated by the method of Griess. Nuclear factor-kappa B (NF-kappaB) activation, as evaluated by electrophoretic mobility shift assay, was inhibited by DPI (1-10 microM) in a dose-dependent manner. IL-1-induced ROS production, as evaluated by measurement of dichlorofluorescein fluorescence, was inhibited by DPI at concentrations that also prevented NF-kappaB activation and iNOS expression. CONCLUSIONS: DPI inhibits IL-1-induced NO production in chondrocytes by two distinct mechanisms: (i) by inhibiting NOS activity, and (ii) by preventing iNOS expression through the blockade of NF-kappaB activation. These results also support the involvement of reactive oxygen species in IL-1-induced NF-kappaB activation and expression of NF-kappaB-dependent genes, such as iNOS.

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Selected References

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  1. Akamatsu Y., Ohno T., Hirota K., Kagoshima H., Yodoi J., Shigesada K. Redox regulation of the DNA binding activity in transcription factor PEBP2. The roles of two conserved cysteine residues. J Biol Chem. 1997 Jun 6;272(23):14497–14500. doi: 10.1074/jbc.272.23.14497. [DOI] [PubMed] [Google Scholar]
  2. Bass D. A., Parce J. W., Dechatelet L. R., Szejda P., Seeds M. C., Thomas M. Flow cytometric studies of oxidative product formation by neutrophils: a graded response to membrane stimulation. J Immunol. 1983 Apr;130(4):1910–1917. [PubMed] [Google Scholar]
  3. Beiqing L., Chen M., Whisler R. L. Sublethal levels of oxidative stress stimulate transcriptional activation of c-jun and suppress IL-2 promoter activation in Jurkat T cells. J Immunol. 1996 Jul 1;157(1):160–169. [PubMed] [Google Scholar]
  4. Blanco F. J., Lotz M. IL-1-induced nitric oxide inhibits chondrocyte proliferation via PGE2. Exp Cell Res. 1995 May;218(1):319–325. doi: 10.1006/excr.1995.1161. [DOI] [PubMed] [Google Scholar]
  5. Blanco F. J., Ochs R. L., Schwarz H., Lotz M. Chondrocyte apoptosis induced by nitric oxide. Am J Pathol. 1995 Jan;146(1):75–85. [PMC free article] [PubMed] [Google Scholar]
  6. Cruz T. F., Mills G., Pritzker K. P., Kandel R. A. Inverse correlation between tyrosine phosphorylation and collagenase production in chondrocytes. Biochem J. 1990 Aug 1;269(3):717–721. doi: 10.1042/bj2690717. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dorsam G., Taher M. M., Valerie K. C., Kuemmerle N. B., Chan J. C., Franson R. C. Diphenyleneiodium chloride blocks inflammatory cytokine-induced up-regulation of group IIA phospholipase A(2) in rat mesangial cells. J Pharmacol Exp Ther. 2000 Jan;292(1):271–279. [PubMed] [Google Scholar]
  8. Du X., Stocklauser-Färber K., Rösen P. Generation of reactive oxygen intermediates, activation of NF-kappaB, and induction of apoptosis in human endothelial cells by glucose: role of nitric oxide synthase? Free Radic Biol Med. 1999 Oct;27(7-8):752–763. doi: 10.1016/s0891-5849(99)00079-9. [DOI] [PubMed] [Google Scholar]
  9. Eberhardt W., Kunz D., Pfeilschifter J. Pyrrolidine dithiocarbamate differentially affects interleukin 1 beta- and cAMP-induced nitric oxide synthase expression in rat renal mesangial cells. Biochem Biophys Res Commun. 1994 Apr 15;200(1):163–170. doi: 10.1006/bbrc.1994.1429. [DOI] [PubMed] [Google Scholar]
  10. Eberhardt W., Plüss C., Hummel R., Pfeilschifter J. Molecular mechanisms of inducible nitric oxide synthase gene expression by IL-1beta and cAMP in rat mesangial cells. J Immunol. 1998 May 15;160(10):4961–4969. [PubMed] [Google Scholar]
  11. Farrell A. J., Blake D. R., Palmer R. M., Moncada S. Increased concentrations of nitrite in synovial fluid and serum samples suggest increased nitric oxide synthesis in rheumatic diseases. Ann Rheum Dis. 1992 Nov;51(11):1219–1222. doi: 10.1136/ard.51.11.1219. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Goldring M. B., Fukuo K., Birkhead J. R., Dudek E., Sandell L. J. Transcriptional suppression by interleukin-1 and interferon-gamma of type II collagen gene expression in human chondrocytes. J Cell Biochem. 1994 Jan;54(1):85–99. doi: 10.1002/jcb.240540110. [DOI] [PubMed] [Google Scholar]
  13. Green L. C., Wagner D. A., Glogowski J., Skipper P. L., Wishnok J. S., Tannenbaum S. R. Analysis of nitrate, nitrite, and [15N]nitrate in biological fluids. Anal Biochem. 1982 Oct;126(1):131–138. doi: 10.1016/0003-2697(82)90118-x. [DOI] [PubMed] [Google Scholar]
  14. Janssen-Heininger Y. M., Macara I., Mossman B. T. Cooperativity between oxidants and tumor necrosis factor in the activation of nuclear factor (NF)-kappaB: requirement of Ras/mitogen-activated protein kinases in the activation of NF-kappaB by oxidants. Am J Respir Cell Mol Biol. 1999 May;20(5):942–952. doi: 10.1165/ajrcmb.20.5.3452. [DOI] [PubMed] [Google Scholar]
  15. Kinugawa K., Shimizu T., Yao A., Kohmoto O., Serizawa T., Takahashi T. Transcriptional regulation of inducible nitric oxide synthase in cultured neonatal rat cardiac myocytes. Circ Res. 1997 Dec;81(6):911–921. doi: 10.1161/01.res.81.6.911. [DOI] [PubMed] [Google Scholar]
  16. Knott I., Dieu M., Burton M., Houbion A., Remacle J., Raes M. Induction of cyclooxygenase by interleukin 1: comparative study between human synovial cells and chondrocytes. J Rheumatol. 1994 Mar;21(3):462–466. [PubMed] [Google Scholar]
  17. Kusuhara H., Komatsu H., Sumichika H., Sugahara K. Reactive oxygen species are involved in the apoptosis induced by nonsteroidal anti-inflammatory drugs in cultured gastric cells. Eur J Pharmacol. 1999 Nov 3;383(3):331–337. doi: 10.1016/s0014-2999(99)00599-3. [DOI] [PubMed] [Google Scholar]
  18. LeBel C. P., Ali S. F., McKee M., Bondy S. C. Organometal-induced increases in oxygen reactive species: the potential of 2',7'-dichlorofluorescin diacetate as an index of neurotoxic damage. Toxicol Appl Pharmacol. 1990 Jun 1;104(1):17–24. doi: 10.1016/0041-008x(90)90278-3. [DOI] [PubMed] [Google Scholar]
  19. Lee Y. J., Galoforo S. S., Berns C. M., Chen J. C., Davis B. H., Sim J. E., Corry P. M., Spitz D. R. Glucose deprivation-induced cytotoxicity and alterations in mitogen-activated protein kinase activation are mediated by oxidative stress in multidrug-resistant human breast carcinoma cells. J Biol Chem. 1998 Feb 27;273(9):5294–5299. doi: 10.1074/jbc.273.9.5294. [DOI] [PubMed] [Google Scholar]
  20. Li Y., Trush M. A. Diphenyleneiodonium, an NAD(P)H oxidase inhibitor, also potently inhibits mitochondrial reactive oxygen species production. Biochem Biophys Res Commun. 1998 Dec 18;253(2):295–299. doi: 10.1006/bbrc.1998.9729. [DOI] [PubMed] [Google Scholar]
  21. Lo Y. Y., Conquer J. A., Grinstein S., Cruz T. F. Interleukin-1 beta induction of c-fos and collagenase expression in articular chondrocytes: involvement of reactive oxygen species. J Cell Biochem. 1998 Apr 1;69(1):19–29. doi: 10.1002/(sici)1097-4644(19980401)69:1<19::aid-jcb3>3.0.co;2-y. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Lowenstein C. J., Alley E. W., Raval P., Snowman A. M., Snyder S. H., Russell S. W., Murphy W. J. Macrophage nitric oxide synthase gene: two upstream regions mediate induction by interferon gamma and lipopolysaccharide. Proc Natl Acad Sci U S A. 1993 Oct 15;90(20):9730–9734. doi: 10.1073/pnas.90.20.9730. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Loyau G., Pujol J. P. The role of cytokines in the development of osteoarthritis. Scand J Rheumatol Suppl. 1990;81:8–12. doi: 10.3109/03009749009096939. [DOI] [PubMed] [Google Scholar]
  25. McCartney-Francis N., Allen J. B., Mizel D. E., Albina J. E., Xie Q. W., Nathan C. F., Wahl S. M. Suppression of arthritis by an inhibitor of nitric oxide synthase. J Exp Med. 1993 Aug 1;178(2):749–754. doi: 10.1084/jem.178.2.749. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. McGuire J. J., Anderson D. J., McDonald B. J., Narayanasami R., Bennett B. M. Inhibition of NADPH-cytochrome P450 reductase and glyceryl trinitrate biotransformation by diphenyleneiodonium sulfate. Biochem Pharmacol. 1998 Oct 1;56(7):881–893. doi: 10.1016/s0006-2952(98)00216-0. [DOI] [PubMed] [Google Scholar]
  27. Miesel R., Kurpisz M., Kroger H. Suppression of inflammatory arthritis by simultaneous inhibition of nitric oxide synthase and NADPH oxidase. Free Radic Biol Med. 1996;20(1):75–81. doi: 10.1016/0891-5849(95)02026-8. [DOI] [PubMed] [Google Scholar]
  28. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983 Dec 16;65(1-2):55–63. doi: 10.1016/0022-1759(83)90303-4. [DOI] [PubMed] [Google Scholar]
  29. O'Donnell B. V., Tew D. G., Jones O. T., England P. J. Studies on the inhibitory mechanism of iodonium compounds with special reference to neutrophil NADPH oxidase. Biochem J. 1993 Feb 15;290(Pt 1):41–49. doi: 10.1042/bj2900041. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Pagano P. J., Ito Y., Tornheim K., Gallop P. M., Tauber A. I., Cohen R. A. An NADPH oxidase superoxide-generating system in the rabbit aorta. Am J Physiol. 1995 Jun;268(6 Pt 2):H2274–H2280. doi: 10.1152/ajpheart.1995.268.6.H2274. [DOI] [PubMed] [Google Scholar]
  31. Palmer R. M., Hickery M. S., Charles I. G., Moncada S., Bayliss M. T. Induction of nitric oxide synthase in human chondrocytes. Biochem Biophys Res Commun. 1993 May 28;193(1):398–405. doi: 10.1006/bbrc.1993.1637. [DOI] [PubMed] [Google Scholar]
  32. Pelletier J. P., Jovanovic D., Fernandes J. C., Manning P., Connor J. R., Currie M. G., Di Battista J. A., Martel-Pelletier J. Reduced progression of experimental osteoarthritis in vivo by selective inhibition of inducible nitric oxide synthase. Arthritis Rheum. 1998 Jul;41(7):1275–1286. doi: 10.1002/1529-0131(199807)41:7<1275::AID-ART19>3.0.CO;2-T. [DOI] [PubMed] [Google Scholar]
  33. Pelletier J. P., Martel-Pelletier J., Howell D. S., Ghandur-Mnaymneh L., Enis J. E., Woessner J. F., Jr Collagenase and collagenolytic activity in human osteoarthritic cartilage. Arthritis Rheum. 1983 Jan;26(1):63–68. doi: 10.1002/art.1780260110. [DOI] [PubMed] [Google Scholar]
  34. Potoka D. A., Takao S., Owaki T., Bulkley G. B., Klein A. S. Endothelial cells potentiate oxidant-mediated Kupffer cell phagocytic killing. Free Radic Biol Med. 1998 May;24(7-8):1217–1227. doi: 10.1016/s0891-5849(97)00453-x. [DOI] [PubMed] [Google Scholar]
  35. Rusyn I., Bradham C. A., Cohn L., Schoonhoven R., Swenberg J. A., Brenner D. A., Thurman R. G. Corn oil rapidly activates nuclear factor-kappaB in hepatic Kupffer cells by oxidant-dependent mechanisms. Carcinogenesis. 1999 Nov;20(11):2095–2100. doi: 10.1093/carcin/20.11.2095. [DOI] [PubMed] [Google Scholar]
  36. Santos L. L., Morand E. F., Yang Y., Hutchinson P., Holdsworth S. R. Suppression of adjuvant arthritis and synovial macrophage inducible nitric oxide by N-iminoethyl-L-ornithine, a nitric oxide synthase inhibitor. Inflammation. 1997 Jun;21(3):299–311. doi: 10.1023/a:1027397816209. [DOI] [PubMed] [Google Scholar]
  37. Schmidt K. N., Amstad P., Cerutti P., Baeuerle P. A. The roles of hydrogen peroxide and superoxide as messengers in the activation of transcription factor NF-kappa B. Chem Biol. 1995 Jan;2(1):13–22. doi: 10.1016/1074-5521(95)90076-4. [DOI] [PubMed] [Google Scholar]
  38. Sen C. K. Redox signaling and the emerging therapeutic potential of thiol antioxidants. Biochem Pharmacol. 1998 Jun 1;55(11):1747–1758. doi: 10.1016/s0006-2952(97)00672-2. [DOI] [PubMed] [Google Scholar]
  39. Stadler J., Stefanovic-Racic M., Billiar T. R., Curran R. D., McIntyre L. A., Georgescu H. I., Simmons R. L., Evans C. H. Articular chondrocytes synthesize nitric oxide in response to cytokines and lipopolysaccharide. J Immunol. 1991 Dec 1;147(11):3915–3920. [PubMed] [Google Scholar]
  40. Stefanovic-Racic M., Meyers K., Meschter C., Coffey J. W., Hoffman R. A., Evans C. H. N-monomethyl arginine, an inhibitor of nitric oxide synthase, suppresses the development of adjuvant arthritis in rats. Arthritis Rheum. 1994 Jul;37(7):1062–1069. doi: 10.1002/art.1780370712. [DOI] [PubMed] [Google Scholar]
  41. Stuehr D. J., Fasehun O. A., Kwon N. S., Gross S. S., Gonzalez J. A., Levi R., Nathan C. F. Inhibition of macrophage and endothelial cell nitric oxide synthase by diphenyleneiodonium and its analogs. FASEB J. 1991 Jan;5(1):98–103. doi: 10.1096/fasebj.5.1.1703974. [DOI] [PubMed] [Google Scholar]
  42. Taskiran D., Stefanovic-Racic M., Georgescu H., Evans C. Nitric oxide mediates suppression of cartilage proteoglycan synthesis by interleukin-1. Biochem Biophys Res Commun. 1994 Apr 15;200(1):142–148. doi: 10.1006/bbrc.1994.1426. [DOI] [PubMed] [Google Scholar]
  43. Weinberg J. B., Granger D. L., Pisetsky D. S., Seldin M. F., Misukonis M. A., Mason S. N., Pippen A. M., Ruiz P., Wood E. R., Gilkeson G. S. The role of nitric oxide in the pathogenesis of spontaneous murine autoimmune disease: increased nitric oxide production and nitric oxide synthase expression in MRL-lpr/lpr mice, and reduction of spontaneous glomerulonephritis and arthritis by orally administered NG-monomethyl-L-arginine. J Exp Med. 1994 Feb 1;179(2):651–660. doi: 10.1084/jem.179.2.651. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Westacott C. I., Whicher J. T., Barnes I. C., Thompson D., Swan A. J., Dieppe P. A. Synovial fluid concentration of five different cytokines in rheumatic diseases. Ann Rheum Dis. 1990 Sep;49(9):676–681. doi: 10.1136/ard.49.9.676. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Xie Q. W., Kashiwabara Y., Nathan C. Role of transcription factor NF-kappa B/Rel in induction of nitric oxide synthase. J Biol Chem. 1994 Feb 18;269(7):4705–4708. [PubMed] [Google Scholar]
  46. Yea C. M., Cross A. R., Jones O. T. Purification and some properties of the 45 kDa diphenylene iodonium-binding flavoprotein of neutrophil NADPH oxidase. Biochem J. 1990 Jan 1;265(1):95–100. doi: 10.1042/bj2650095. [DOI] [PMC free article] [PubMed] [Google Scholar]

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