Salicylate Suppresses Macrophage Nitric-oxide Synthase-2 and Cyclo-oxygenase-2 Expression by Inhibiting CCAAT/Enhancer-binding Protein-β Binding via a Common Signaling Pathway

Abstract

We determined whether salicylate at pharmacological concentrations inhibits nitric-oxide synthase-2 (NOS-2) and cyclo-oxygenase-2 (COX-2) expressions in RAW 264.7 stimulated with lipopolysaccharide (LPS) and interferon-γ (IFN-γ). Cells were treated with sodium salicylate (10−7-10−4m) or vehicle for 30 min followed by LPS+IFN-γ for up to 24 h. Salicylate suppressed NOS-2 and COX-2 protein levels and promoter activities stimulated by LPS+IFN-γ for 4 h in a concentration-dependent manner but had no effect on NOS-2 expression stimulated by the combined agonists for 24 h. Results from promoter analysis indicate that the binding of CCAAT/enhancer-binding protein β (C/EBPβ) to its cognate site at −150/−142 on the NOS-2 promoter region was essential for NOS-2 expression at 4 h but not at 24 h. Salicylate reduced C/EBPβ binding at 4 h and did not alter its binding at 24 h. NOS-2 and COX-2 protein levels and C/EBPβ binding stimulated by LPS+IFN-γ for 4 h were inhibited by a similar battery of signaling inhibitors, suggesting a common pathway for NOS-2 and COX-2 expression. Kinetic analysis indicates that NOS-2, similar to COX-2 expression, at 4 h was largely due to the action of LPS, which induced C/EBPβ binding, whereas its expression at a longer time point was contributed by IFN-γ. Our findings implicate two distinct pathways for NOS-2 expression induced by LPS+IFN-γ. Salicylate at pharmacological concentrations is capable of suppressing the early phase of NOS-2 and COX-2 expression by blocking C/EBPβ binding. We determined whether salicylate at pharmacological concentrations inhibits nitric-oxide synthase-2 (NOS-2) and cyclo-oxygenase-2 (COX-2) expressions in RAW 264.7 stimulated with lipopolysaccharide (LPS) and interferon-γ (IFN-γ). Cells were treated with sodium salicylate (10−7-10−4m) or vehicle for 30 min followed by LPS+IFN-γ for up to 24 h. Salicylate suppressed NOS-2 and COX-2 protein levels and promoter activities stimulated by LPS+IFN-γ for 4 h in a concentration-dependent manner but had no effect on NOS-2 expression stimulated by the combined agonists for 24 h. Results from promoter analysis indicate that the binding of CCAAT/enhancer-binding protein β (C/EBPβ) to its cognate site at −150/−142 on the NOS-2 promoter region was essential for NOS-2 expression at 4 h but not at 24 h. Salicylate reduced C/EBPβ binding at 4 h and did not alter its binding at 24 h. NOS-2 and COX-2 protein levels and C/EBPβ binding stimulated by LPS+IFN-γ for 4 h were inhibited by a similar battery of signaling inhibitors, suggesting a common pathway for NOS-2 and COX-2 expression. Kinetic analysis indicates that NOS-2, similar to COX-2 expression, at 4 h was largely due to the action of LPS, which induced C/EBPβ binding, whereas its expression at a longer time point was contributed by IFN-γ. Our findings implicate two distinct pathways for NOS-2 expression induced by LPS+IFN-γ. Salicylate at pharmacological concentrations is capable of suppressing the early phase of NOS-2 and COX-2 expression by blocking C/EBPβ binding. Nitric-oxide synthase-2 (NOS-2, 1The abbreviations used are: NOS-2, nitric-oxide synthase-2; COX-2, cyclooxygenase-2; LPS, lipopolysaccharide; IFN-γ, interferon γ; C/EBP, CCAAT/enhancer-binding protein; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; PI3K, phosphatidylinositol 3-kinase; PKC and PKA, protein kinases C and A, respectively; Jak, Janus activated kinase1The abbreviations used are: NOS-2, nitric-oxide synthase-2; COX-2, cyclooxygenase-2; LPS, lipopolysaccharide; IFN-γ, interferon γ; C/EBP, CCAAT/enhancer-binding protein; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; PI3K, phosphatidylinositol 3-kinase; PKC and PKA, protein kinases C and A, respectively; Jak, Janus activated kinase also known as inducible nitric-oxide synthase), a member of the NOS protein family, defends against microbial infection but may contribute to inflammation and tissue injury when overexpressed (1Nathan C. J. Clin. Invest. 1997; 100: 2417-2423Crossref PubMed Scopus (837) Google Scholar). NOS-2 is inducible by lipopolysaccharide (LPS) and a myriad pro-inflammatory cytokines (2Wu K.K. Adv. Pharmacol. 1995; 33: 179-205Crossref PubMed Scopus (194) Google Scholar). Transcriptional activation of NOS-2 by these stimuli has been extensively characterized in murine RAW 264.7 macrophages. LPS combined with interferon-γ (IFN-γ) was reported to activate NOS-2 promoter synergistically. Promoter analysis reveals that two clusters of enhancers located at −54 to −236 and −731 to −962 of NOS-2 promoter region are essential for the sustained expression of NOS-2 in response to LPS+IFN-γ stimulation (3Xie Q.-W. Kashiwabara Y. Nathan C. J. Biol. Chem. 1994; 269: 4705-4708Abstract Full Text PDF PubMed Google Scholar, 4Kamijo R. Harada H. Matsuyama T. Bosland M. Gerecitano J. Shapiro D. Le J. Koh S.I. Kimura T. Green S.J. Mak T.W. Taniguchi T. Vilcek J. Science. 1994; 263: 1612-1615Crossref PubMed Scopus (785) Google Scholar). The binding of NFκB and interferon regulatory factor-1 to the enhancer elements of these two regions are indispensable for NOS-2 transcription (5Xie Q.-W. Whisnant R. Nathan C. J. Exp. Med. 1993; 177: 1779-1784Crossref PubMed Scopus (1028) Google Scholar, 6Lowenstein C.J. Alley E.W. Raval P. Snowman A.M. Snyder S.H. Russell S.W. Murphy W.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9730-9734Crossref PubMed Scopus (1006) Google Scholar). It has been recently reported (7Gupta A.K. Kone B.C. Am. J. Physiol. 1999; 276: F599-F605PubMed Google Scholar, 8Dlaska M. Weiss G. J. Immunol. 1999; 162: 6171-6177PubMed Google Scholar) that a CAAT/enhancer-binding protein (C/EBP) element situated at −142 to −150 is also involved in NOS-2 expression, but the exact transactivators that bind to this site have not been characterized. COX-2 is also inducible in this murine macrophage by diverse pro-inflammatory mediators. Its induction by these stimuli is typical of an immediately early gene with a rapid and transient time course. Several regulatory elements on murine COX-2 promoter including a cyclic AMP response element, two C/EBP sites, and a single NFκB site have been shown to be involved in COX-2 promoter activation (9Xie W. Fletcher B.S. Andersen R.D. Herschman H.R. Mol. Cell. Biol. 1994; 14: 6531-6539Crossref PubMed Scopus (189) Google Scholar, 10Wadleigh D.J. Herschman H.R. Biochem. Biophys. Res. Commun. 1999; 264: 865-870Crossref PubMed Scopus (75) Google Scholar). The C/EBP sites have been demonstrated to be required for COX-2 expression in murine cells induced by diverse stimuli including LPS (10Wadleigh D.J. Herschman H.R. Biochem. Biophys. Res. Commun. 1999; 264: 865-870Crossref PubMed Scopus (75) Google Scholar, 11Thomas B. Berenbaum F. Humbert L. Bian H. Béréziat G. Crofford L. Olivier J.L. Eur. J. Biochem. 2000; 267: 6798-6809Crossref PubMed Scopus (109) Google Scholar, 12Inoue H. Yokoyama C. Hara S. Tone Y. Tanabe T. J. Biol. Chem. 1995; 270: 24965-24971Abstract Full Text Full Text PDF PubMed Scopus (467) Google Scholar). However, the binding of C/EBPβ to these two sites have not been fully characterized. Results from our laboratory demonstrate that C/EBPβ binding to the C/EBP site in human COX-2 promoter is required for COX-2 transcriptional activation by pro-inflammatory mediators such as phorbol 12-myristate 13-acetate and interleukin-1β (13Saunders M.A. Sansores-Garcia L. Gilroy D.W. Wu K.K. J. Biol. Chem. 2001; 276: 18897-18904Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Aspirin and sodium salicylate at pharmacological concentrations suppress COX-2 transcription by blocking the binding of C/EBPβ (13Saunders M.A. Sansores-Garcia L. Gilroy D.W. Wu K.K. J. Biol. Chem. 2001; 276: 18897-18904Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 14Xu X.-M. Sansores-Garcia L. Chen X.-M. Matijevic-Aleksic N. Du M. Wu K.K. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5292-5297Crossref PubMed Scopus (266) Google Scholar). The effect of salicylate on NOS-2 expression is controversial. Although several reports have shown that salicylate suppresses NOS-2 expression at suprapharmacological concentrations (>5 mm) by reducing NFκB binding (15Farivar R.S. Brecher P. J. Biol. Chem. 1996; 271: 31585-31592Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 16Amin A.R. Vyas P. Attur M. Leszczynska-Piziak J. Patel I.R. Weissmann G. Abramson S.B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7926-7930Crossref PubMed Scopus (288) Google Scholar), to our knowledge, there is no report on NOS-2 expression influenced by pharmacological concentrations of salicylate. Because NOS-2 and COX-2 expressions are concurrently induced by LPS+IFN-γ in RAW 264.7 cells, we used this cell line as a model to test the hypothesis that salicylate at pharmacological concentrations (10−4 to 10−6m) inhibits NOS-2 and COX-2 expressions stimulated by LPS+IFN-γ by blocking a common C/EBPβ-dependent transcriptional pathway. The results show that salicylate suppressed NOS-2 and COX-2 protein levels and promoter activities by blocking the binding of C/EBPβ to its respective C/EBP elements on NOS-2 and COX-2 promoters. Our results further show that NOS-2 promoter activity stimulated by LPS+IFN-γ for 4 h required C/EBPβ binding, and at this time period of stimulation, salicylate was effective in suppressing C/EBPβ binding, thereby reducing the extent of NOS-2 expression. Interestingly, C/EBPβ binding was no longer required for NOS-2 expression, and salicylate was incapable of inhibiting NOS-2 promoter activity stimulated by LPS+IFN-γ for 24 h. RAW 264.7 mouse macrophages (ATCC) were cultured in Dulbecco's modified Eagle's medium (Invitrogen) containing 10% fetal bovine serum (Sigma), 100 units/ml penicillin, and 100 μg/ml streptomycin (Invitrogen). For all experiments, medium was removed, and cells were washed and incubated in serum-free medium for 24 h before stimulation with 2 μg/ml LPS (Escherichia coli serotype 0111:B4, Sigma), 40 units/ml IFN-γ (Roche Molecular Biochemicals), or LPS+IFN-γ for indicated time periods. A wild-type murine 1.63-kilobase NOS-2 promoter/enhancer fragment (−1486/+145), its C/EBP site (−150/−142) mutant with the substitution of TGATGTAA for GTCGTGGC, and a 0.9-kilobase COX-2 promoter/enhancer fragment (−891/+9) were cloned into pGL3 basic luciferase expression vectors. RAW 264.7 cells at ∼60% confluence were transfected with the luciferase expression construct according to a procedure described previously (14Xu X.-M. Sansores-Garcia L. Chen X.-M. Matijevic-Aleksic N. Du M. Wu K.K. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5292-5297Crossref PubMed Scopus (266) Google Scholar). A mixture of 15 μg of Lipofectin (Invitrogen) and 4 μg of DNA was added to cells and incubated in serum-free medium for 6 h. The medium was replaced with Dulbecco's modified Eagle's medium containing 10% fetal bovine serum for 18 h. Cells were washed with serum-free medium, growth-arrested for 24 h, and stimulated with LPS and IFN-γ for an indicated period of time. Cells were harvested using Promega lysis buffer, and luciferase activity was measured on a Turner TD-20/20 luminometer. On the same cell lysate, total protein assay was performed. Western blot analysis was performed as described previously (13Saunders M.A. Sansores-Garcia L. Gilroy D.W. Wu K.K. J. Biol. Chem. 2001; 276: 18897-18904Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Cells were washed with cold phosphate-buffered saline containing 0.1 mmphenylmethylsulfonyl fluoride, harvested, and spun down at 3000 rpm for 5 min. Cell pellet was dissolved in solubilizing buffer (1% Triton X-100, 50 mm Tris-HCl, 150 mm NaCl, 10% glycerol, 1.5 mm MgCl2, 1 mm EDTA, 0.1 mm phenylmethylsulfonyl fluoride, 50 mmNaF, 1 mm Na3VO4, 0.06 mg/ml aprotinin). Cell pellet was then spun down at 10,000 rpm for 15 min. Supernatant was collected, and its protein concentration was determined. For sodium dodecylsulfate-polyacrylamide gel electrophoresis, each lane was loaded with 20 μg of protein. The gel was electrophoretically transferred, and proteins were identified by specific antibodies: anti-NOS-2 antibody (Upstate Biotechnology, Lake Placid, NY), anti-COX-2 antibody (Cayman, Ann Arbor, MI), anti-actin antibody (Oncogene, Boston, MA), and anti-C/EBPβ (Santa Cruz Biotechnology, Santa Cruz, CA). The protein bands were visualized by enhanced chemiluminescence system (Pierce, Rockford, IL). The binding of nuclear extract proteins to the DNA sequence was performed by a newly developed method using streptavidin-conjugated agarose beads to bind biotinylated DNA probe (17Zhu Y. Saunders M.A. Yeh H. Deng W.G. Wu K.K. J. Biol. Chem. 2002; 277: 6923-6928Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Nuclear extracts were prepared from RAW 264.7 cells by a method described previously (17Zhu Y. Saunders M.A. Yeh H. Deng W.G. Wu K.K. J. Biol. Chem. 2002; 277: 6923-6928Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). 400 μg of nuclear extract proteins in 400 μl of phosphate-buffered saline buffer containing multiple protease inhibitors (1 mm Na3VO4, 10 mm NaF, 25 mm β-glycerophosphate, 0.1 mm phenylmethylsulfonyl fluoride, 0.06 mg/ml aprotinin, 1 μg/ml leupeptin, 0.5 mm dithiothreitol) were incubated with a mixture of 4 μg of double-stranded biotinylated oligonucleotides containing murine NOS-2 promoter C/EBP sequence at −150 to −142 (underlined in or COX-2 C/EBP sequence at to DNA and 40 μl of with for 2 h on a at The beads were by at 3000 rpm for 1 washed with phosphate-buffered saline buffer containing multiple inhibitors, and in 40 μl of Nuclear proteins to the beads were by in at for 5 min and to Western blot an of an of kinase an of an of Janus activated kinase an of protein kinase C and an of of were from RAW 264.7 cells were with these for 30 min to the of LPS, IFN-γ, or LPS+IFN-γ. It is that RAW 264.7 cells COX-2 and NOS-2 proteins in response to LPS+IFN-γ whether sodium salicylate at pharmacological concentrations suppresses COX-2 and NOS-2 expressions we the of sodium salicylate on COX-2 and NOS-2 protein levels and promoter salicylate suppressed COX-2 protein levels in RAW 264.7 cells stimulated with LPS+IFN-γ for 4 h in a concentration-dependent manner with a of at to NOS-2 protein was stimulation of RAW 264.7 cells with LPS+IFN-γ for 4 h and was suppressed by sodium salicylate 1 salicylate at suppressed COX-2 and NOS-2 promoter activities stimulated with for 4 h to a similar extent We have in RAW 264.7 as as in human that salicylate inhibited NOS-2 and COX-2 expressions in cells were cultured in the of 10% fetal bovine salicylate did not NOS-2 or COX-2 protein levels stimulated by LPS+IFN-γ for 4 h not salicylate suppressed NOS-2 and COX-2 promoter activities stimulated by LPS+IFN-γ to a similar Promoter activity was as protein the promoter is the of response to LPS+IFN-γ stimulation, COX-2 promoter activity at 4 h and to at h and NOS-2 promoter activity up to 24 h the of C/EBP element in NOS-2 promoter we transfected cells with luciferase expression containing a NOS-2 promoter The of the C/EBP element in the of NOS-2 promoter activity at 4 h but not at 24 h suggesting pathways for 4 h stimulation by LPS+IFN-γ. We determined whether salicylate inhibited the NOS-2 expression. salicylate reduced NOS-2 promoter activity at 4 h by but had no effect on NOS-2 promoter activity stimulated with LPS+IFN-γ for 24 h of the C/EBP sequence at the NOS-2 promoter region to promoter activity stimulated by LPS+IFN-γ for 4 h but not 24 h. the of salicylate suppressed NOS-2 promoter activity stimulated by LPS+IFN-γ for 4 h but not 24 the of several C/EBPβ the C/EBPβ was in cells and its was not by LPS+IFN-γ stimulation for 4 h C/EBPβ binding to the NOS-2 promoter sequence was at which was enhanced by LPS+IFN-γ stimulation for 4 h 6 binding to COX-2 C/EBP sequence was not but the binding was at 4 h of stimulation 6 salicylate at did not alter C/EBPβ protein levels 6 but suppressed C/EBPβ binding to NOS-2 and COX-2 promoter C/EBP C/EBPβ binding to NOS-2 sequence a with which is with a of C/EBPβ protein levels salicylate had no effect on C/EBPβ binding or protein at 24 h of stimulation of C/EBP β proteins and C/EBP β binding to NOS-2 promoter sequence stimulation with LPS+IFN-γ for 24 h. lane LPS+IFN-γ stimulation for 24 and lane with sodium salicylate followed by LPS+IFN-γ stimulation for 24 LPS has been reported to COX-2 expression in RAW 264.7 cells at early time M. B. P. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar), but the of NOS-2 expression had not been We determined the of NOS-2 expression induced by LPS and IFN-γ LPS stimulation, NOS-2 proteins were at 4 h and at h NOS-2 proteins were at 4 h IFN-γ stimulation at h and at 24 h LPS+IFN-γ in stimulation of NOS-2 expression. expression was at 4 h, at h, and at 24 h. We determined whether sodium salicylate inhibited NOS-2 protein expression stimulated by LPS, IFN-γ, or LPS+IFN-γ for 4 and 24 h. Salicylate inhibited LPS and NOS-2 protein levels at 4 h to a similar extent It had no effect on NOS-2 protein levels these results that salicylate inhibited C/EBPβ binding, thereby suppressing COX-2 and NOS-2 by salicylate of NOS-2 protein levels stimulated with LPS IFN-γ, and LPS+IFN-γ for 4 h or IFN-γ for 24 protein analysis by Western and the the is of with similar into the signaling pathway which salicylate C/EBPβ binding, we the signaling pathways which NOS-2 and COX-2 expressions are stimulated by LPS+IFN-γ for 4 h. NOS-2 protein levels were reduced by and with the of PI3K, MAPK, and in NOS-2 expression. COX-2 protein levels at 4 h were also inhibited by and with an 10 and C/EBPβ binding to the NOS-2 promoter sequence the C/EBPβ protein C/EBPβ binding to COX-2 promoter sequence was by these two inhibitors results a common signaling pathway which NOS-2 and COX-2 promoter activities are stimulated by LPS+IFN-γ at 4 of C/EBP β binding to NOS-2 sequence and COX-2 sequence by and C/EBPβ protein levels in RAW 264.7 by Western show C/EBPβ binding. of A or analysis of binding from the of lane stimulation with LPS+IFN-γ for 4 lane with to LPS+IFN-γ and lane with before of of NOS-2 expression in response to IFN-γ stimulation, we were in whether the signaling pathway for NOS-2 expression stimulated by LPS+IFN-γ for 4 h is to We cells with or for 30 min followed by LPS, IFN-γ, or LPS+IFN-γ for 4 h. Cells were harvested, and NOS-2 proteins were determined. NOS-2 protein expression induced by LPS or LPS+IFN-γ was inhibited by but not by IFN-γ did not NOS-2 at 4 h, and with these inhibitors did not alter its effect on NOS-2 protein expression results that NOS-2 and COX-2 expressions a 4 h stimulation by LPS+IFN-γ were contributed by LPS a signaling pathway several signaling including Our findings indicate that salicylate at pharmacological concentrations is capable of suppressing NOS-2 and COX-2 expressions in RAW 264.7 macrophages by blocking C/EBPβ binding to specific binding sites at the NOS-2 and COX-2 promoter Our results further demonstrate that the NOS-2 and COX-2 expressions in response to LPS+IFN-γ stimulation for 4 h is to the action of LPS, IFN-γ did not NOS-2 or COX-2 expression at this early time C/EBPβ binding has been shown to be required for COX-2 transcriptional activation M. B. P. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). this our results show that C/EBPβ binding is also essential for NOS-2 transcriptional activation by LPS at 4 h. Salicylate was capable of suppressing C/EBPβ binding to the of results that salicylate suppresses NOS-2 and COX-2 transcription by a common C/EBPβ-dependent We have previously demonstrated that COX-2 stimulation by phorbol 12-myristate 13-acetate and interleukin-1β (13Saunders M.A. Sansores-Garcia L. Gilroy D.W. Wu K.K. J. Biol. Chem. 2001; 276: 18897-18904Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar) in human also C/EBPβ binding, and salicylate a similar effect on blocking C/EBPβ binding, thereby suppressing COX-2 salicylate not suppress COX-2 stimulation by stimulation not C/EBPβ binding. these results indicate that C/EBPβ binding is a of salicylate It may be that salicylate is capable of suppressing an of C/EBPβ-dependent action of salicylate the of on diverse human The by which salicylate C/EBPβ binding has not been characterized. C/EBPβ binding on is that salicylate C/EBPβ by inhibiting the kinase The exact kinase that C/EBPβ and its binding and to be Several kinases including MAPK, and have been shown to C/EBPβ at distinct or M. P. M. T. Mol. Cell. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, T. S. T. M. T. S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: PubMed Scopus Google Scholar, M. Science. PubMed Scopus Google Scholar, C. C. P. T. M. M. 1993; PubMed Scopus Google Scholar). It is whether all of the sites are required for C/EBPβ binding, is whether all the kinases are activated by each It is that each of agonists may activate several kinases that in to promoter C/EBPβ binding. Salicylate may of the kinase thereby the C/EBPβ binding. Our results that PKC is a of the of PKC or did not alter C/EBPβ binding. is in to whether or is the of salicylate Our results demonstrate of NOS-2 and COX-2 promoter activation by LPS+IFN-γ. COX-2 expression by the combined stimuli a time that is of COX-2 induction by agonists including Its promoter activity stimulated by LPS or LPS+IFN-γ at 4 h and The of IFN-γ not the effect of LPS the time of COX-2 NOS-2 expression by the combined stimuli has a time which is contributed by IFN-γ. Our time of NOS-2 expression by the combined stimuli is with the reported Murphy W.J. C.J. Snyder S.H. Russell S.W. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar, A. S. L. H. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar). However, the time of NOS-2 expression the stimulation of LPS or IFN-γ had not been extensively Our results show a time of NOS-2 expression stimulated by these two LPS induced an early and NOS-2 expression the time of COX-2 expression, whereas IFN-γ stimulated NOS-2 expression with a and a time course. is to distinct signaling pathways used by these two agonists to the binding of transactivators to respective regulatory elements on the NOS-2 The action of LPS+IFN-γ be by a time of stimulation, of multiple transactivators and to the and of binding time by these two are and The results from this for a in C/EBPβ binding that with the early NOS-2 expression. C/EBPβ binding is required for NOS-2 expression at 4 h but not at 24 h. The binding of NFκB and interferon regulatory is for the and sustained NOS-2 expression stimulated by LPS+IFN-γ. It is to that C/EBPβ proteins are by LPS+IFN-γ stimulation, which results in the of the C/EBPβ binding. It be to the time of binding of transactivators in response to stimulation by LPS, IFN-γ, or LPS+IFN-γ and the effect of salicylate on the binding of these transactivators at time NOS-2 transcriptional activation in response to stimulation by LPS+IFN-γ at 4 and 24 h may be distinct signaling Our results indicate that at 4 h of stimulation, promoter activation is a pathway PI3K, MAPK, and On the our Y. and also that NOS-2 transcriptional activation by LPS+IFN-γ for 24 h was inhibited by and and by and suggesting that NOS-2 expression at 24 h is a signaling pathway PKC and of and by PI3K, MAPK, and a of reports signaling pathways for NOS-2 expression in RAW 264.7 reports on signaling pathways for NOS-2 expression at or h but did not For B. R. A. S. J. Immunol. Google Scholar) report the of NOS-2 expression at 6 h by whereas M. R. L. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar) and Chen J. Immunol. Google Scholar) report the of PKC in NOS-2 expression at 24 h. A. P. L. J. Immunol. 1999; 162: Google Scholar) report that stimulated by LPS+IFN-γ for 24 h was by results are with our findings that NOS-2 expression at h and its expression at 24 h. these results the that NOS-2 expression stimulated by LPS+IFN-γ two distinct that are by an early phase of expression, which is PI3K, MAPK, and and a phase of expression, which is PKC and of this we have characterized the early phase of expression. The early phase NOS-2 expression the expression of an early gene such as Its signaling pathway to C/EBPβ binding and transcriptional We have demonstrated that salicylate is capable of suppressing the early phase NOS-2 expression by blocking C/EBPβ binding, and this also its of COX-2 transcriptional the phase NOS-2 expression has not been characterized. Its signaling pathways to be and signaling The of the signaling pathway in the phase NOS-2 expression is It is also which transcriptional are activated the signaling pathway and which transcription are by the pathway. Our results have the of C/EBPβ in the phase NOS-2 transcriptional It is that NFκB and interferon regulatory factor-1 are the transactivators for the phase NOS-2 expression. is in to the signaling and transcriptional pathways for the phase NOS-2 expression and the pharmacological of its expression. We Kone of of for the NOS-2 promoter expression and for