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Proc Natl Acad Sci U S A. Oct 15, 1993; 90(20): 9730–9734.

Macrophage nitric oxide synthase gene: two upstream regions mediate induction by interferon gamma and lipopolysaccharide.


The promoter region of the mouse gene for macrophage-inducible nitric oxide synthase (mac-NOS; EC has been characterized. A putative TATA box is 30 base pairs upstream of the transcription start site. Computer analysis reveals numerous potential binding sites for transcription factors, many of them associated with stimuli that induce mac-NOS expression. To localize functionally important portions of the regulatory region, we constructed deletion mutants of the mac-NOS 5' flanking region and placed them upstream of a luciferase reporter gene. The macrophage cell line RAW 264.7, when transfected with a minimal promoter construct, expresses little luciferase activity when stimulated by lipopolysaccharide (LPS), interferon gamma (IFN-gamma), or both. Maximal expression depends on two discrete regulatory regions upstream of the putative TATA box. Region I (position -48 to -209) increases luciferase activity approximately 75-fold over the minimal promoter construct. Region I contains LPS-related responsive elements, including a binding site for nuclear factor interleukin 6 (NF-IL6) and the kappa B binding site for NF-kappa B, suggesting that this region regulates LPS-induced expression of the mac-NOS gene. Region II (position -913 to -1029) alone does not increase luciferase expression, but together with region I it causes an additional 10-fold increase in expression. Together the two regions increase expression 750-fold over activity obtained from a minimal promoter construct. Region II contains motifs for binding IFN-related transcription factors and thus probably is responsible for IFN-mediated regulation of LPS-induced mac-NOS. Delineation of these two cooperative regions explains at the level of transcription how IFN-gamma and LPS act in concert to induce maximally the mac-NOS gene and, furthermore, how IFN-gamma augments the inflammatory response to LPS.

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  • Lowenstein CJ, Snyder SH. Nitric oxide, a novel biologic messenger. Cell. 1992 Sep 4;70(5):705–707. [PubMed]
  • Moncada S, Palmer RM, Higgs EA. Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol Rev. 1991 Jun;43(2):109–142. [PubMed]
  • Nathan C. Nitric oxide as a secretory product of mammalian cells. FASEB J. 1992 Sep;6(12):3051–3064. [PubMed]
  • Stuehr DJ, Marletta MA. Mammalian nitrate biosynthesis: mouse macrophages produce nitrite and nitrate in response to Escherichia coli lipopolysaccharide. Proc Natl Acad Sci U S A. 1985 Nov;82(22):7738–7742. [PMC free article] [PubMed]
  • Lorsbach RB, Murphy WJ, Lowenstein CJ, Snyder SH, Russell SW. Expression of the nitric oxide synthase gene in mouse macrophages activated for tumor cell killing. Molecular basis for the synergy between interferon-gamma and lipopolysaccharide. J Biol Chem. 1993 Jan 25;268(3):1908–1913. [PubMed]
  • Hibbs JB, Jr, Taintor RR, Vavrin Z, Rachlin EM. Nitric oxide: a cytotoxic activated macrophage effector molecule. Biochem Biophys Res Commun. 1988 Nov 30;157(1):87–94. [PubMed]
  • Liew FY, Li Y, Millott S. Tumour necrosis factor (TNF-alpha) in leishmaniasis. II. TNF-alpha-induced macrophage leishmanicidal activity is mediated by nitric oxide from L-arginine. Immunology. 1990 Dec;71(4):556–559. [PMC free article] [PubMed]
  • Schini VB, Junquero DC, Scott-Burden T, Vanhoutte PM. Interleukin-1 beta induces the production of an L-arginine-derived relaxing factor from cultured smooth muscle cells from rat aorta. Biochem Biophys Res Commun. 1991 Apr 15;176(1):114–121. [PubMed]
  • Radomski MW, Palmer RM, Moncada S. Glucocorticoids inhibit the expression of an inducible, but not the constitutive, nitric oxide synthase in vascular endothelial cells. Proc Natl Acad Sci U S A. 1990 Dec;87(24):10043–10047. [PMC free article] [PubMed]
  • Busse R, Mülsch A. Induction of nitric oxide synthase by cytokines in vascular smooth muscle cells. FEBS Lett. 1990 Nov 26;275(1-2):87–90. [PubMed]
  • Ding A, Nathan CF, Graycar J, Derynck R, Stuehr DJ, 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]
  • al-Ramadi BK, Meissler JJ, Jr, Huang D, Eisenstein TK. Immunosuppression induced by nitric oxide and its inhibition by interleukin-4. Eur J Immunol. 1992 Sep;22(9):2249–2254. [PubMed]
  • Marotta P, Sautebin L, Di Rosa M. Modulation of the induction of nitric oxide synthase by eicosanoids in the murine macrophage cell line J774. Br J Pharmacol. 1992 Nov;107(3):640–641. [PMC free article] [PubMed]
  • Lowenstein CJ, Glatt CS, Bredt DS, Snyder SH. 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. [PMC free article] [PubMed]
  • Lyons CR, Orloff GJ, Cunningham JM. 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]
  • Xie QW, Cho HJ, Calaycay J, Mumford RA, Swiderek KM, Lee TD, 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. [PubMed]
  • Morrison DC, Leive L. Fractions of lipopolysaccharide from Escherichia coli O111:B4 prepared by two extraction procedures. J Biol Chem. 1975 Apr 25;250(8):2911–2919. [PubMed]
  • Ghosh D. A relational database of transcription factors. Nucleic Acids Res. 1990 Apr 11;18(7):1749–1756. [PMC free article] [PubMed]
  • Cassady AI, Stacey KJ, Nimmo KA, Murphy KM, von der Ahe D, Pearson D, Botteri FM, Nagamine Y, Hume DA. Constitutive expression of the urokinase plasminogen activator gene in murine RAW264 macrophages involves distal and 5' non-coding sequences that are conserved between mouse and pig. Nucleic Acids Res. 1991 Dec 25;19(24):6839–6847. [PMC free article] [PubMed]
  • Baeuerle PA. The inducible transcription activator NF-kappa B: regulation by distinct protein subunits. Biochim Biophys Acta. 1991 Apr 16;1072(1):63–80. [PubMed]
  • Akira S, Kishimoto T. IL-6 and NF-IL6 in acute-phase response and viral infection. Immunol Rev. 1992 Jun;127:25–50. [PubMed]
  • Reid LE, Brasnett AH, Gilbert CS, Porter AC, Gewert DR, Stark GR, Kerr IM. A single DNA response element can confer inducibility by both alpha- and gamma-interferons. Proc Natl Acad Sci U S A. 1989 Feb;86(3):840–844. [PMC free article] [PubMed]
  • Benech PD, Sastry K, Iyer RR, Eichbaum QG, Raveh DP, Ezekowitz RA. 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. [PMC free article] [PubMed]
  • Leitman DC, Ribeiro RC, Mackow ER, Baxter JD, West BL. 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]
  • Klemsz MJ, McKercher SR, Celada A, Van Beveren C, Maki RA. The macrophage and B cell-specific transcription factor PU.1 is related to the ets oncogene. Cell. 1990 Apr 6;61(1):113–124. [PubMed]
  • Goyal N, Knox J, Gronostajski RM. Analysis of multiple forms of nuclear factor I in human and murine cell lines. Mol Cell Biol. 1990 Mar;10(3):1041–1048. [PMC free article] [PubMed]
  • Nishizawa M, Nagata S. Regulatory elements responsible for inducible expression of the granulocyte colony-stimulating factor gene in macrophages. Mol Cell Biol. 1990 May;10(5):2002–2011. [PMC free article] [PubMed]
  • Boulukos KE, Pognonec P, Sariban E, Bailly M, Lagrou C, Ghysdael J. Rapid and transient expression of Ets2 in mature macrophages following stimulation with cMGF, LPS, and PKC activators. Genes Dev. 1990 Mar;4(3):401–409. [PubMed]
  • Vincenti MP, Burrell TA, Taffet SM. Regulation of NF-kappa B activity in murine macrophages: effect of bacterial lipopolysaccharide and phorbol ester. J Cell Physiol. 1992 Jan;150(1):204–213. [PubMed]
  • Tebo JM, Hamilton TA. Lipopolysaccharide induces DNA binding activity specific for the IFN-stimulated response element in murine peritoneal macrophages. J Immunol. 1992 Oct 1;149(7):2352–2357. [PubMed]
  • Driggers PH, Ennist DL, Gleason SL, Mak WH, Marks MS, Levi BZ, Flanagan JR, Appella E, Ozato K. An interferon gamma-regulated protein that binds the interferon-inducible enhancer element of major histocompatibility complex class I genes. Proc Natl Acad Sci U S A. 1990 May;87(10):3743–3747. [PMC free article] [PubMed]
  • Farber JM. A collection of mRNA species that are inducible in the RAW 264.7 mouse macrophage cell line by gamma interferon and other agents. Mol Cell Biol. 1992 Apr;12(4):1535–1545. [PMC free article] [PubMed]
  • Improta T, Pine R, Pfeffer LM. Interferon-gamma potentiates the antiviral activity and the expression of interferon-stimulated genes induced by interferon-alpha in U937 cells. J Interferon Res. 1992 Apr;12(2):87–94. [PubMed]
  • Eilers A, Seegert D, Schindler C, Baccarini M, Decker T. The response of gamma interferon activation factor is under developmental control in cells of the macrophage lineage. Mol Cell Biol. 1993 Jun;13(6):3245–3254. [PMC free article] [PubMed]
  • Finn PW, Kara CJ, Douhan J, 3rd, Van TT, Folsom V, Glimcher LH. Interferon gamma regulates binding of two nuclear protein complexes in a macrophage cell line. Proc Natl Acad Sci U S A. 1990 Feb;87(3):914–918. [PMC free article] [PubMed]
  • Shannon MF, Pell LM, Lenardo MJ, Kuczek ES, Occhiodoro FS, Dunn SM, Vadas MA. A novel tumor necrosis factor-responsive transcription factor which recognizes a regulatory element in hemopoietic growth factor genes. Mol Cell Biol. 1990 Jun;10(6):2950–2959. [PMC free article] [PubMed]
  • LeClair KP, Blanar MA, Sharp PA. The p50 subunit of NF-kappa B associates with the NF-IL6 transcription factor. Proc Natl Acad Sci U S A. 1992 Sep 1;89(17):8145–8149. [PMC free article] [PubMed]
  • Mukaida N, Mahe Y, Matsushima K. Cooperative interaction of nuclear factor-kappa B- and cis-regulatory enhancer binding protein-like factor binding elements in activating the interleukin-8 gene by pro-inflammatory cytokines. J Biol Chem. 1990 Dec 5;265(34):21128–21133. [PubMed]
  • Baeuerle PA, Baltimore D. Activation of DNA-binding activity in an apparently cytoplasmic precursor of the NF-kappa B transcription factor. Cell. 1988 Apr 22;53(2):211–217. [PubMed]
  • Baeuerle PA, Baltimore D. I kappa B: a specific inhibitor of the NF-kappa B transcription factor. Science. 1988 Oct 28;242(4878):540–546. [PubMed]
  • Xie QW, Whisnant R, Nathan C. Promoter of the mouse gene encoding calcium-independent nitric oxide synthase confers inducibility by interferon gamma and bacterial lipopolysaccharide. J Exp Med. 1993 Jun 1;177(6):1779–1784. [PMC free article] [PubMed]

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