Skip to main content
PMC home page
The Journal of Experimental Medicine logoLink to The Journal of Experimental Medicine
. 1993 Jun 1;177(6):1779–1784. doi: 10.1084/jem.177.6.1779

Promoter of the mouse gene encoding calcium-independent nitric oxide synthase confers inducibility by interferon gamma and bacterial lipopolysaccharide

PMCID: PMC2191051  PMID: 7684434

Abstract

Inducible nitric oxide synthase (iNOS) can be expressed by many types of mammalian cells in response to diverse signals acting synergistically, including cytokines and microbial products. We previously showed that induction of iNOS in mouse macrophages by interferon gamma (IFN-gamma) and lipopolysaccharide (LPS) was at the transcriptional level. From a mouse genomic library, we now cloned a 1,749-bp fragment from the 5'-flanking region of the iNOS gene, and used S1 nuclease mapping and primer extension to identify the mRNA transcription start site within it. The mRNA initiation site is preceded by a TATA box and at least 22 oligonucleotide elements homologous to consensus sequences for the binding of transcription factors involved in the inducibility of other genes by cytokines or bacterial products. These include 10 copies of IFN-gamma response element; 3 copies of gamma-activated site; 2 copies each of nuclear factor-kappa B, IFN-alpha-stimulated response element, activating protein 1, and tumor necrosis factor response element; and one X box. Plasmids in which all or the downstream one half or one third of this region of iNOS were linked to a reporter gene encoding chloramphenicol acetyltransferase were transfected into cells of the RAW264.7 macrophage-like line. All these constructs conferred inducibility of the iNOS promoter by LPS, but only the construct containing all 1,749 bp conferred synergistic inducibility by IFN-gamma plus LPS.

Full Text

The Full Text of this article is available as a PDF (712.9 KB).

Selected References

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

  1. Benech P. D., Sastry K., Iyer R. R., Eichbaum Q. G., Raveh D. P., Ezekowitz R. A. Definition of interferon gamma-response elements in a novel human Fc gamma receptor gene (Fc gamma RIb) and characterization of the gene structure. J Exp Med. 1992 Oct 1;176(4):1115–1123. doi: 10.1084/jem.176.4.1115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Benoist C., Mathis D. Regulation of major histocompatibility complex class-II genes: X, Y and other letters of the alphabet. Annu Rev Immunol. 1990;8:681–715. doi: 10.1146/annurev.iy.08.040190.003341. [DOI] [PubMed] [Google Scholar]
  3. Beutler B., Brown T. A CAT reporter construct allows ultrasensitive estimation of TNF synthesis, and suggests that the TNF gene has been silenced in non-macrophage cell lines. J Clin Invest. 1991 Apr;87(4):1336–1344. doi: 10.1172/JCI115137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. Cho H. J., Xie Q. W., Calaycay J., Mumford R. A., Swiderek K. M., Lee T. D., Nathan C. Calmodulin is a subunit of nitric oxide synthase from macrophages. J Exp Med. 1992 Aug 1;176(2):599–604. doi: 10.1084/jem.176.2.599. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Decker T., Lew D. J., Mirkovitch J., Darnell J. E., Jr Cytoplasmic activation of GAF, an IFN-gamma-regulated DNA-binding factor. EMBO J. 1991 Apr;10(4):927–932. doi: 10.1002/j.1460-2075.1991.tb08026.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Ding A. H., Nathan C. F., Stuehr D. J. Release of reactive nitrogen intermediates and reactive oxygen intermediates from mouse peritoneal macrophages. Comparison of activating cytokines and evidence for independent production. J Immunol. 1988 Oct 1;141(7):2407–2412. [PubMed] [Google Scholar]
  8. Ding A., Nathan C. F., Graycar J., Derynck R., Stuehr D. J., Srimal S. Macrophage deactivating factor and transforming growth factors-beta 1 -beta 2 and -beta 3 inhibit induction of macrophage nitrogen oxide synthesis by IFN-gamma. J Immunol. 1990 Aug 1;145(3):940–944. [PubMed] [Google Scholar]
  9. Drapier J. C., Wietzerbin J., Hibbs J. B., Jr Interferon-gamma and tumor necrosis factor induce the L-arginine-dependent cytotoxic effector mechanism in murine macrophages. Eur J Immunol. 1988 Oct;18(10):1587–1592. doi: 10.1002/eji.1830181018. [DOI] [PubMed] [Google Scholar]
  10. Golub E. I., Kim H., Volsky D. J. Transfection of DNA into adherent cells by DEAE-dextran/DMSO method increases drastically if the cells are removed from surface and treated in suspension. Nucleic Acids Res. 1989 Jun 26;17(12):4902–4902. doi: 10.1093/nar/17.12.4902. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gorman C. M., Moffat L. F., Howard B. H. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol Cell Biol. 1982 Sep;2(9):1044–1051. doi: 10.1128/mcb.2.9.1044. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hamada H., Kakunaga T. Potential Z-DNA forming sequences are highly dispersed in the human genome. Nature. 1982 Jul 22;298(5872):396–398. doi: 10.1038/298396a0. [DOI] [PubMed] [Google Scholar]
  13. Heck D. E., Laskin D. L., Gardner C. R., Laskin J. D. Epidermal growth factor suppresses nitric oxide and hydrogen peroxide production by keratinocytes. Potential role for nitric oxide in the regulation of wound healing. J Biol Chem. 1992 Oct 25;267(30):21277–21280. [PubMed] [Google Scholar]
  14. Hevel J. M., White K. A., Marletta M. A. Purification of the inducible murine macrophage nitric oxide synthase. Identification as a flavoprotein. J Biol Chem. 1991 Dec 5;266(34):22789–22791. [PubMed] [Google Scholar]
  15. Iyengar R., Stuehr D. J., Marletta M. A. Macrophage synthesis of nitrite, nitrate, and N-nitrosamines: precursors and role of the respiratory burst. Proc Natl Acad Sci U S A. 1987 Sep;84(18):6369–6373. doi: 10.1073/pnas.84.18.6369. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kilbourn R. G., Gross S. S., Jubran A., Adams J., Griffith O. W., Levi R., Lodato R. F. NG-methyl-L-arginine inhibits tumor necrosis factor-induced hypotension: implications for the involvement of nitric oxide. Proc Natl Acad Sci U S A. 1990 May;87(9):3629–3632. doi: 10.1073/pnas.87.9.3629. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Leitman D. C., Ribeiro R. C., Mackow E. R., Baxter J. D., West B. L. Identification of a tumor necrosis factor-responsive element in the tumor necrosis factor alpha gene. J Biol Chem. 1991 May 25;266(15):9343–9346. [PubMed] [Google Scholar]
  18. Lenardo M. J., Baltimore D. NF-kappa B: a pleiotropic mediator of inducible and tissue-specific gene control. Cell. 1989 Jul 28;58(2):227–229. doi: 10.1016/0092-8674(89)90833-7. [DOI] [PubMed] [Google Scholar]
  19. Lew D. J., Decker T., Strehlow I., Darnell J. E. Overlapping elements in the guanylate-binding protein gene promoter mediate transcriptional induction by alpha and gamma interferons. Mol Cell Biol. 1991 Jan;11(1):182–191. doi: 10.1128/mcb.11.1.182. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Liew F. Y., Li Y., Severn A., Millott S., Schmidt J., Salter M., Moncada S. A possible novel pathway of regulation by murine T helper type-2 (Th2) cells of a Th1 cell activity via the modulation of the induction of nitric oxide synthase on macrophages. Eur J Immunol. 1991 Oct;21(10):2489–2494. doi: 10.1002/eji.1830211027. [DOI] [PubMed] [Google Scholar]
  21. Lowenstein C. J., Glatt C. S., Bredt D. S., Snyder S. H. Cloned and expressed macrophage nitric oxide synthase contrasts with the brain enzyme. Proc Natl Acad Sci U S A. 1992 Aug 1;89(15):6711–6715. doi: 10.1073/pnas.89.15.6711. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lyons C. R., Orloff G. J., Cunningham J. M. Molecular cloning and functional expression of an inducible nitric oxide synthase from a murine macrophage cell line. J Biol Chem. 1992 Mar 25;267(9):6370–6374. [PubMed] [Google Scholar]
  23. Mackman N., Brand K., Edgington T. S. Lipopolysaccharide-mediated transcriptional activation of the human tissue factor gene in THP-1 monocytic cells requires both activator protein 1 and nuclear factor kappa B binding sites. J Exp Med. 1991 Dec 1;174(6):1517–1526. doi: 10.1084/jem.174.6.1517. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Nathan C. Nitric oxide as a secretory product of mammalian cells. FASEB J. 1992 Sep;6(12):3051–3064. [PubMed] [Google Scholar]
  25. Nussler A. K., Di Silvio M., Billiar T. R., Hoffman R. A., Geller D. A., Selby R., Madariaga J., Simmons R. L. Stimulation of the nitric oxide synthase pathway in human hepatocytes by cytokines and endotoxin. J Exp Med. 1992 Jul 1;176(1):261–264. doi: 10.1084/jem.176.1.261. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Pearse R. N., Feinman R., Ravetch J. V. Characterization of the promoter of the human gene encoding the high-affinity IgG receptor: transcriptional induction by gamma-interferon is mediated through common DNA response elements. Proc Natl Acad Sci U S A. 1991 Dec 15;88(24):11305–11309. doi: 10.1073/pnas.88.24.11305. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Pfeilschifter J., Vosbeck K. Transforming growth factor beta 2 inhibits interleukin 1 beta- and tumour necrosis factor alpha-induction of nitric oxide synthase in rat renal mesangial cells. Biochem Biophys Res Commun. 1991 Mar 15;175(2):372–379. doi: 10.1016/0006-291x(91)91574-v. [DOI] [PubMed] [Google Scholar]
  28. Roberts A. B., Vodovotz Y., Roche N. S., Sporn M. B., Nathan C. F. Role of nitric oxide in antagonistic effects of transforming growth factor-beta and interleukin-1 beta on the beating rate of cultured cardiac myocytes. Mol Endocrinol. 1992 Nov;6(11):1921–1930. doi: 10.1210/mend.6.11.1282674. [DOI] [PubMed] [Google Scholar]
  29. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Schindler C., Fu X. Y., Improta T., Aebersold R., Darnell J. E., Jr Proteins of transcription factor ISGF-3: one gene encodes the 91-and 84-kDa ISGF-3 proteins that are activated by interferon alpha. Proc Natl Acad Sci U S A. 1992 Aug 15;89(16):7836–7839. doi: 10.1073/pnas.89.16.7836. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Schindler C., Shuai K., Prezioso V. R., Darnell J. E., Jr Interferon-dependent tyrosine phosphorylation of a latent cytoplasmic transcription factor. Science. 1992 Aug 7;257(5071):809–813. doi: 10.1126/science.1496401. [DOI] [PubMed] [Google Scholar]
  32. Shuai K., Schindler C., Prezioso V. R., Darnell J. E., Jr Activation of transcription by IFN-gamma: tyrosine phosphorylation of a 91-kD DNA binding protein. Science. 1992 Dec 11;258(5089):1808–1812. doi: 10.1126/science.1281555. [DOI] [PubMed] [Google Scholar]
  33. 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]
  34. 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]
  35. 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]
  36. Yang Z., Sugawara M., Ponath P. D., Wessendorf L., Banerji J., Li Y., Strominger J. L. Interferon gamma response region in the promoter of the human DPA gene. Proc Natl Acad Sci U S A. 1990 Dec;87(23):9226–9230. doi: 10.1073/pnas.87.23.9226. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Yui Y., Hattori R., Kosuga K., Eizawa H., Hiki K., Kawai C. Purification of nitric oxide synthase from rat macrophages. J Biol Chem. 1991 Jul 5;266(19):12544–12547. [PubMed] [Google Scholar]

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

RESOURCES