Skip to main content
PMC home page
The Journal of Experimental Medicine logoLink to The Journal of Experimental Medicine
. 1994 Sep 1;180(3):969–976. doi: 10.1084/jem.180.3.969

Iron regulates nitric oxide synthase activity by controlling nuclear transcription

PMCID: PMC2191642  PMID: 7520477

Abstract

Recently, it was reported that nitric oxide (NO) directly controls intracellular iron metabolism by activating iron regulatory protein (IRP), a cytoplasmic protein that regulates ferritin translation. To determine whether intracellular iron levels themselves affect NO synthase (NOS), we studied the effect of iron on cytokine-inducible NOS activity and mRNA expression in the murine macrophage cell line J774A.1. We show here that NOS activity is decreased by about 50% in homogenates obtained from cells treated with interferon gamma plus lipopolysaccharide (IFN-gamma/LPS) in the presence of 50 microM ferric iron [Fe(3+)] as compared with extracts from cells treated with IFN- gamma/LPS alone. Conversely, addition of the iron chelator desferrioxamine (100 microM) at the time of stimulation with IFN- gamma/LPS increases NOS activity up to 2.5-fold in J774 cells. These effects of changing the cellular iron state cannot be attributed to a general alteration of the IFN-gamma/LPS signal, since IFN-gamma/LPS- mediated major histocompatibility complex class II antigen expression is unaffected. Furthermore, neither was the intracellular availability of the NOS cofactor tetrahydrobiopterin altered by treatment with Fe(3+) or desferrioxamine, nor do these compounds interfere with the activity of the hemoprotein NOS in vitro. We demonstrate that the mRNA levels for NOS are profoundly increased by treatment with desferrioxamine and reduced by Fe(3+). The half-life of NOS mRNA appeared not to be significantly altered by administration of ferric ion, and NOS mRNA stability was only slightly prolonged by desferrioxamine treatment. Nuclear run-off experiments demonstrate that nuclear transcription of cytokine-inducible NOS mRNA is strongly increased by desferrioxamine whereas it is decreased by Fe(3+). Thus, this transcriptional response appears to account quantitatively for the changes in enzyme activity. Our results suggest the existence of a regulatory loop between iron metabolism and the NO/NOS pathway.

Full Text

The Full Text of this article is available as a PDF (1.0 MB).

Selected References

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

  1. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  2. Bredt D. S., Snyder S. H. Nitric oxide mediates glutamate-linked enhancement of cGMP levels in the cerebellum. Proc Natl Acad Sci U S A. 1989 Nov;86(22):9030–9033. doi: 10.1073/pnas.86.22.9030. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  4. Cleveland D. W., Lopata M. A., MacDonald R. J., Cowan N. J., Rutter W. J., Kirschner M. W. Number and evolutionary conservation of alpha- and beta-tubulin and cytoplasmic beta- and gamma-actin genes using specific cloned cDNA probes. Cell. 1980 May;20(1):95–105. doi: 10.1016/0092-8674(80)90238-x. [DOI] [PubMed] [Google Scholar]
  5. Drapier J. C., Hibbs J. B., Jr Differentiation of murine macrophages to express nonspecific cytotoxicity for tumor cells results in L-arginine-dependent inhibition of mitochondrial iron-sulfur enzymes in the macrophage effector cells. J Immunol. 1988 Apr 15;140(8):2829–2838. [PubMed] [Google Scholar]
  6. Drapier J. C., Hirling H., Wietzerbin J., Kaldy P., Kühn L. C. Biosynthesis of nitric oxide activates iron regulatory factor in macrophages. EMBO J. 1993 Sep;12(9):3643–3649. doi: 10.1002/j.1460-2075.1993.tb06038.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Drapier J. C., Pellat C., Henry Y. Generation of EPR-detectable nitrosyl-iron complexes in tumor target cells cocultured with activated macrophages. J Biol Chem. 1991 Jun 5;266(16):10162–10167. [PubMed] [Google Scholar]
  8. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  9. Fuchs D., Hausen A., Reibnegger G., Werner E. R., Werner-Felmayer G., Dierich M. P., Wachter H. Immune activation and the anaemia associated with chronic inflammatory disorders. Eur J Haematol. 1991 Feb;46(2):65–70. doi: 10.1111/j.1600-0609.1991.tb00524.x. [DOI] [PubMed] [Google Scholar]
  10. Fuchs D., Zangerle R., Artner-Dworzak E., Weiss G., Fritsch P., Tilz G. P., Dierich M. P., Wachter H. Association between immune activation, changes of iron metabolism and anaemia in patients with HIV infection. Eur J Haematol. 1993 Feb;50(2):90–94. doi: 10.1111/j.1600-0609.1993.tb00147.x. [DOI] [PubMed] [Google Scholar]
  11. Fukushima T., Nixon J. C. Analysis of reduced forms of biopterin in biological tissues and fluids. Anal Biochem. 1980 Feb;102(1):176–188. doi: 10.1016/0003-2697(80)90336-x. [DOI] [PubMed] [Google Scholar]
  12. Gay D. A., Yen T. J., Lau J. T., Cleveland D. W. Sequences that confer beta-tubulin autoregulation through modulated mRNA stability reside within exon 1 of a beta-tubulin mRNA. Cell. 1987 Aug 28;50(5):671–679. doi: 10.1016/0092-8674(87)90325-4. [DOI] [PubMed] [Google Scholar]
  13. Gordeuk V. R., Thuma P. E., Brittenham G. M., Zulu S., Simwanza G., Mhangu A., Flesch G., Parry D. Iron chelation with desferrioxamine B in adults with asymptomatic Plasmodium falciparum parasitemia. Blood. 1992 Jan 15;79(2):308–312. [PubMed] [Google Scholar]
  14. Gordeuk V., Thuma P., Brittenham G., McLaren C., Parry D., Backenstose A., Biemba G., Msiska R., Holmes L., McKinley E. Effect of iron chelation therapy on recovery from deep coma in children with cerebral malaria. N Engl J Med. 1992 Nov 19;327(21):1473–1477. doi: 10.1056/NEJM199211193272101. [DOI] [PubMed] [Google Scholar]
  15. Granger D. L., Hibbs J. B., Jr, Perfect J. R., Durack D. T. Specific amino acid (L-arginine) requirement for the microbiostatic activity of murine macrophages. J Clin Invest. 1988 Apr;81(4):1129–1136. doi: 10.1172/JCI113427. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. Gross S. S., Levi R. Tetrahydrobiopterin synthesis. An absolute requirement for cytokine-induced nitric oxide generation by vascular smooth muscle. J Biol Chem. 1992 Dec 25;267(36):25722–25729. [PubMed] [Google Scholar]
  18. Hibbs J. B., Jr, Taintor R. R., Vavrin Z., Rachlin E. M. Nitric oxide: a cytotoxic activated macrophage effector molecule. Biochem Biophys Res Commun. 1988 Nov 30;157(1):87–94. doi: 10.1016/s0006-291x(88)80015-9. [DOI] [PubMed] [Google Scholar]
  19. Ignarro L. J. Biosynthesis and metabolism of endothelium-derived nitric oxide. Annu Rev Pharmacol Toxicol. 1990;30:535–560. doi: 10.1146/annurev.pa.30.040190.002535. [DOI] [PubMed] [Google Scholar]
  20. Lancaster J. R., Jr, Hibbs J. B., Jr EPR demonstration of iron-nitrosyl complex formation by cytotoxic activated macrophages. Proc Natl Acad Sci U S A. 1990 Feb;87(3):1223–1227. doi: 10.1073/pnas.87.3.1223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Liew F. Y., Millott S., Parkinson C., Palmer R. M., Moncada S. Macrophage killing of Leishmania parasite in vivo is mediated by nitric oxide from L-arginine. J Immunol. 1990 Jun 15;144(12):4794–4797. [PubMed] [Google Scholar]
  22. Mayer B., John M., Böhme E. Purification of a Ca2+/calmodulin-dependent nitric oxide synthase from porcine cerebellum. Cofactor-role of tetrahydrobiopterin. FEBS Lett. 1990 Dec 17;277(1-2):215–219. doi: 10.1016/0014-5793(90)80848-d. [DOI] [PubMed] [Google Scholar]
  23. McMillan K., Bredt D. S., Hirsch D. J., Snyder S. H., Clark J. E., Masters B. S. Cloned, expressed rat cerebellar nitric oxide synthase contains stoichiometric amounts of heme, which binds carbon monoxide. Proc Natl Acad Sci U S A. 1992 Dec 1;89(23):11141–11145. doi: 10.1073/pnas.89.23.11141. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Means R. T., Jr, Krantz S. B. Progress in understanding the pathogenesis of the anemia of chronic disease. Blood. 1992 Oct 1;80(7):1639–1647. [PubMed] [Google Scholar]
  25. Melefors O., Hentze M. W. Iron regulatory factor--the conductor of cellular iron regulation. Blood Rev. 1993 Dec;7(4):251–258. doi: 10.1016/0268-960x(93)90012-s. [DOI] [PubMed] [Google Scholar]
  26. Mellouk S., Green S. J., Nacy C. A., Hoffman S. L. IFN-gamma inhibits development of Plasmodium berghei exoerythrocytic stages in hepatocytes by an L-arginine-dependent effector mechanism. J Immunol. 1991 Jun 1;146(11):3971–3976. [PubMed] [Google Scholar]
  27. Moncada S., Higgs A. The L-arginine-nitric oxide pathway. N Engl J Med. 1993 Dec 30;329(27):2002–2012. doi: 10.1056/NEJM199312303292706. [DOI] [PubMed] [Google Scholar]
  28. Nathan C. Nitric oxide as a secretory product of mammalian cells. FASEB J. 1992 Sep;6(12):3051–3064. [PubMed] [Google Scholar]
  29. Nüssler A., Drapier J. C., Rénia L., Pied S., Miltgen F., Gentilini M., Mazier D. L-arginine-dependent destruction of intrahepatic malaria parasites in response to tumor necrosis factor and/or interleukin 6 stimulation. Eur J Immunol. 1991 Jan;21(1):227–230. doi: 10.1002/eji.1830210134. [DOI] [PubMed] [Google Scholar]
  30. Pantopoulos K., Weiss G., Hentze M. W. Nitric oxide and the post-transcriptional control of cellular iron traffic. Trends Cell Biol. 1994 Mar;4(3):82–86. doi: 10.1016/0962-8924(94)90179-1. [DOI] [PubMed] [Google Scholar]
  31. Reif D. W., Simmons R. D. Nitric oxide mediates iron release from ferritin. Arch Biochem Biophys. 1990 Dec;283(2):537–541. doi: 10.1016/0003-9861(90)90680-w. [DOI] [PubMed] [Google Scholar]
  32. Sanceau J., Merlin G., Wietzerbin J. Tumor necrosis factor-alpha and IL-6 up-regulate IFN-gamma receptor gene expression in human monocytic THP-1 cells by transcriptional and post-transcriptional mechanisms. J Immunol. 1992 Sep 1;149(5):1671–1675. [PubMed] [Google Scholar]
  33. Snyder S. H. Nitric oxide: first in a new class of neurotransmitters. Science. 1992 Jul 24;257(5069):494–496. doi: 10.1126/science.1353273. [DOI] [PubMed] [Google Scholar]
  34. Stuehr D. J., Cho H. J., Kwon N. S., Weise M. F., Nathan C. F. Purification and characterization of the cytokine-induced macrophage nitric oxide synthase: an FAD- and FMN-containing flavoprotein. Proc Natl Acad Sci U S A. 1991 Sep 1;88(17):7773–7777. doi: 10.1073/pnas.88.17.7773. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Stuehr D. J., Marletta M. A. Synthesis of nitrite and nitrate in murine macrophage cell lines. Cancer Res. 1987 Nov 1;47(21):5590–5594. [PubMed] [Google Scholar]
  36. Stuehr D. J., Nathan C. F. Nitric oxide. A macrophage product responsible for cytostasis and respiratory inhibition in tumor target cells. J Exp Med. 1989 May 1;169(5):1543–1555. doi: 10.1084/jem.169.5.1543. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Weinberg E. D. Iron depletion: a defense against intracellular infection and neoplasia. Life Sci. 1992;50(18):1289–1297. doi: 10.1016/0024-3205(92)90279-x. [DOI] [PubMed] [Google Scholar]
  38. Weiss G., Fuchs D., Hausen A., Reibnegger G., Werner E. R., Werner-Felmayer G., Wachter H. Iron modulates interferon-gamma effects in the human myelomonocytic cell line THP-1. Exp Hematol. 1992 Jun;20(5):605–610. [PubMed] [Google Scholar]
  39. Weiss G., Goossen B., Doppler W., Fuchs D., Pantopoulos K., Werner-Felmayer G., Wachter H., Hentze M. W. Translational regulation via iron-responsive elements by the nitric oxide/NO-synthase pathway. EMBO J. 1993 Sep;12(9):3651–3657. doi: 10.1002/j.1460-2075.1993.tb06039.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Weiss G., Lutton J. D., Fuchs D., Werner-Felmayer G., Bock G., Abraham N. G., Kappas A., Levere R. D., Wachter H. Comparative effects of heme and metalloporphyrins on interferon-gamma-mediated pathways in monocytic cells (THP-1). Proc Soc Exp Biol Med. 1993 Apr;202(4):470–475. doi: 10.3181/00379727-202-43561. [DOI] [PubMed] [Google Scholar]
  41. Werner-Felmayer G., Werner E. R., Fuchs D., Hausen A., Reibnegger G., Schmidt K., Weiss G., Wachter H. Pteridine biosynthesis in human endothelial cells. Impact on nitric oxide-mediated formation of cyclic GMP. J Biol Chem. 1993 Jan 25;268(3):1842–1846. [PubMed] [Google Scholar]
  42. Werner-Felmayer G., Werner E. R., Fuchs D., Hausen A., Reibnegger G., Wachter H. Tetrahydrobiopterin-dependent formation of nitrite and nitrate in murine fibroblasts. J Exp Med. 1990 Dec 1;172(6):1599–1607. doi: 10.1084/jem.172.6.1599. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Werner E. R., Fuchs D., Hausen A., Reibnegger G., Wachter H. Simultaneous determination of neopterin and creatinine in serum with solid-phase extraction and on-line elution liquid chromatography. Clin Chem. 1987 Nov;33(11):2028–2033. [PubMed] [Google Scholar]
  44. Werner E. R., Werner-Felmayer G., Fuchs D., Hausen A., Reibnegger G., Yim J. J., Wachter H. Impact of tumour necrosis factor-alpha and interferon-gamma on tetrahydrobiopterin synthesis in murine fibroblasts and macrophages. Biochem J. 1991 Dec 15;280(Pt 3):709–714. doi: 10.1042/bj2800709. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Werner E. R., Werner-Felmayer G., Wachter H. Tetrahydrobiopterin and cytokines. Proc Soc Exp Biol Med. 1993 May;203(1):1–12. doi: 10.3181/00379727-203-43566a. [DOI] [PubMed] [Google Scholar]
  46. White K. A., Marletta M. A. Nitric oxide synthase is a cytochrome P-450 type hemoprotein. Biochemistry. 1992 Jul 28;31(29):6627–6631. doi: 10.1021/bi00144a001. [DOI] [PubMed] [Google Scholar]
  47. Xie Q. W., Cho H. J., Calaycay J., Mumford R. A., Swiderek K. M., Lee T. D., Ding A., Troso T., Nathan C. Cloning and characterization of inducible nitric oxide synthase from mouse macrophages. Science. 1992 Apr 10;256(5054):225–228. doi: 10.1126/science.1373522. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Experimental Medicine are provided here courtesy of The Rockefeller University Press

RESOURCES