Octamer motif is required for the NF-kappaB-mediated induction of the inducible nitric oxide synthase gene expression in RAW 264.7 macrophages

The promoter of the mouse inducible nitric oxide synthase (iNOS) has a putative octamer motif (ATGCAAAA) which exists 24 bp upstream from the TATA box and is mismatched at a single residue from the consensus octamer motif. To examine whether this site is involved in iNOS expression, we constructed various deletions and site-directed mutants of the iNOS promoter linked to the chloramphenicol acetyltransferase (CAT) reporter gene, transfected the constructs into RAW 264.7 macrophages, and stimulated the cells with interferon-gamma (IFN-gamma) and/or lipopolysaccharide (LPS). CAT activity was not induced by LPS in constructs containing only the octamer motif (-71 to +82), but was induced with constructs containing the octamer motif and the upstream sequences of the NF-kappaB site (-91 to +82). However, a site-directed mutation of the octamer motif in the context of the -91 to +82 promoter construct or an extended promoter construct (-1542 to +82) abolished IFN-gamma and/or LPS-induced CAT activity. Similar results were obtained from site-directed mutants at either the NF-kappaB site or both the NF-kappaB site and octamer motif in these two constructs. In addition, we demonstrated that the conversion of the iNOS octamer motif into a consensus sequence increased CAT activity. Electrophoretic mobility shift assay (EMSA) performed with the NF-kappaB site or the octamer motif-containing oligonucleotide probe revealed that NF-kappaB binding was induced by LPS treatment, while the Oct-1 binding was constitutive. Competition assays performed with octamer-related oligonucleotide competitors derived from the immunoglobulin-kappaB or SV40 promoter confirmed the identity of the iNOS promoter sequence as being a Oct-1 binding site. EMSA carried out using a probe containing both the NF-kappaB site and the octamer motif identified two LPS-induced complexes. Competition assays with each NF-kappaB site or octamer motif competitor revealed that NF-kappaB and Oct-1 were present in these two complexes. These data suggest that, besides the NF-kappaB site, the octamer motif is essential for the maximal expression of the iNOS gene in murine macrophages, and the direct interaction of Oct-1 and NF-kappaB is important for the regulation of this gene.     

8-Hydroxyquinoline inhibits iNOS expression and nitric oxide production by down-regulating LPS-induced activity of NF-kappaB and C/EBPbeta in Raw 264.7 cells

In activated macrophage, large amounts of nitric oxide (NO) are generated by inducible nitric oxide synthase (iNOS), resulting in acute or chronic inflammatory disorders. In Raw 264.7 cells stimulated with lipopolysaccharide (LPS) to mimic inflammation, 8-hydroxyquinoline (8HQ) inhibited the LPS-induced expression of both iNOS protein and mRNA in a parallel dose-dependent manner. 8HQ did not enhance the degradation of iNOS mRNA. To investigate the mechanism by which 8HQ inhibits iNOS gene expression, we examined the activation of MAP kinases in Raw 264.7 cells. We did not observe any significant change in the phosphorylation of MAPKs between LPS alone and LPS plus 8HQ-treated cells. Moreover, 8HQ significantly inhibited the DNA-binding activity of nuclear factor-kappaB (NF-kappaB) and CCAAT/enhancer-binding protein beta (C/EBPbeta), but not activator protein-1 and cAMP response element-binding protein. Taken together, these results suggest that 8HQ acts to inhibit inflammation through inhibition of NO production and iNOS expression through blockade of C/EBPbeta DNA-binding activity and NF-kappaB activation.     

Inhibition of lipopolysaccharide-induced expression of inducible nitric oxide synthase by butein in RAW 264.7 cells

Butein has been reported to exert anti-inflammatory effect but the possible mechanism involved is still unclear. Here, we report the inhibitory effect of butein on nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) gene expression. Butein also inhibited the induction of tumor necrosis factor-alpha and cyclooxygenase 2 by LPS. To further investigate the mechanism responsible for the inhibition of iNOS gene expression by butein, we examined the effect of butein on LPS-induced nuclear factor-kappaB (NF-kappaB) activation. The LPS-induced DNA binding activity of NF-kappaB was significantly inhibited by butein, and this effect was mediated through inhibition of the degradation of inhibitory factor-kappaB and phosphorylation of Erk1/2 MAP kinase. Furthermore, increased binding of the osteopontin alphavbeta3 integrin receptor by butein may explain its inhibitory effect on LPS-mediated NO production. Taken together, these results suggest that butein inhibits iNOS gene expression, providing possible mechanisms for its anti-inflammatory action.     

Inhibition of nitric oxide synthase inhibitors and lipopolysaccharide induced inducible NOS and cyclooxygenase-2 gene expressions by rutin, quercetin, and quercetin pentaacetate in RAW 264.7 macrophages

Several natural flavonoids have been demonstrated to perform some beneficial biological activities, however, higher-effective concentrations and poor-absorptive efficacy in body of flavonoids blocked their practical applications. In the present study, we provided evidences to demonstrate that flavonoids rutin, quercetin, and its acetylated product quercetin pentaacetate were able to be used with nitric oxide synthase (NOS) inhibitors (N-nitro-L-arginine (NLA) or N-nitro-L-arginine methyl ester (L-NAME)) in treatment of lipopolysaccharide (LPS) induced nitric oxide (NO) and prostaglandin E2 (PGE2) productions, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) gene expressions in a mouse macrophage cell line (RAW 264.7). The results showed that rutin, quercetin, and quercetin pentaacetate-inhibited LPS-induced NO production in a concentration-dependent manner without obvious cytotoxic effect on cells by MTT assay using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide as an indicator. Decrease of NO production by flavonoids was consistent with the inhibition on LPS-induced iNOS gene expression by western blotting. However, these compounds were unable to block iNOS enzyme activity by direct and indirect measurement on iNOS enzyme activity. Quercetin pentaacetate showed the obvious inhibition on LPS-induced PGE2 production and COX-2 gene expression and the inhibition was not result of suppression on COX-2 enzyme activity. Previous study demonstrated that decrease of NO production by L-arginine analogs effectively stimulated LPS-induced iNOS gene expression, and proposed that stimulatory effects on iNOS protein by NOS inhibitors might be harmful in treating sepsis. In this study, NLA or L-NAME treatment stimulated significantly on LPS-induced iNOS (but not COX-2) protein in RAW 264.7 cells which was inhibited by these three compounds. Quercetin pentaacetate, but not quercetin and rutin, showed the strong inhibitory activity on PGE2 production and COX-2 protein expression in NLA/LPS or L-NAME/LPS co-treated RAW 264.7 cells. These results indicated that combinatorial treatment of L-arginine analogs and flavonoid derivates, such as quercetin pentaacetate, effectively inhibited LPS-induced NO and PGE2 productions, at the same time, inhibited enhanced expressions of iNOS and COX-2 genes.     

Activation of phosphoinositide 3-kinase in response to inflammation and nitric oxide leads to the up-regulation of cyclooxygenase-2 expression and subsequent cell proliferation in mesangial cells

In this study, we showed that nitric oxide (NO) donors induced the mesangial cell proliferation and cyclooxygenase-2 (COX-2) protein expression in murine mesangial cells. An inflammatory condition [lipopolysaccharide (LPS) plus interferon-gamma (IFN-gamma)] could also induce cell proliferation and significantly enhance inducible nitric oxide synthase (iNOS) and COX-2 expression. Phosphoinositide 3-kinase (PI3K) inhibitor, LY294002, inhibited these responses. LPS/IFN-gamma-induced COX-2 expression in mesangial cells could be inhibited by iNOS inhibitor, aminoguanidine. Selective COX-2 inhibitor, NS398, was capable of inhibiting NO donor- or LPS/IFN-gamma-induced mesangial cell proliferation. Both NO donor and LPS/IFN-gamma markedly activated the PI3K activity and the phosphorylation of Akt and nuclear factor (NF)-kappaB DNA binding activity in mesangial cells, which could be inhibited by LY294002 and transfection of dominant-negative vectors of PI3K/p85 and Akt. These results indicate that a PI3K/Akt-dependent pathway involved in the NO-regulated COX-2 expression and cell proliferation in mesangial cells under inflammatory condition.     

Phospholipase D2 acts as an important regulator in LPS-induced nitric oxide synthesis in Raw 264.7 cells

The purpose of this study was to identify the role of phospholipase D2 (PLD2) in lipopolysaccharide (LPS)-induced nitric oxide (NO) synthesis. LPS enhanced NO synthesis and inducible nitric oxide synthase (iNOS) expression in macrophage cell line, Raw 264.7 cells. When Raw 264.7 cells were stimulated with LPS, the expressions of PLDs were increased. Thus, to investigate the role of PLD in NO synthesis, we transfected PLD1, PLD2, and their dominant negative forms to Raw 264.7 cells, respectively. Interestingly, only PLD2 overexpression, but not that of PLD1, increased NO synthesis and iNOS expression. Moreover, LPS-induced NO synthesis and iNOS expression were blocked by PLD2 siRNA, suggesting that LPS upregulates NO synthesis through PLD2. Next, we investigated the S6K1-p42/44 MAPK-STAT3 signaling pathway in LPS-induced NO synthesis mechanism. Knockdown of PLD2 with siRNA also decreased phosphorylation of S6K1, p42/44 MAPK and STAT3 induced by LPS. Furthermore, we found that STAT3 bound with the iNOS promoter, and their binding was mediated by PLD2. Taken together, our results demonstrate the importance of PLD2 for LPS-induced NO synthesis in Raw 264.7 cells with involvement of the S6K1-p42/44 MAPK-STAT3 pathway.     

Osteopontin is induced by nitric oxide in RAW 264.7 cells

Nitric oxide (NO) produced by macrophages is thought to contribute to various pathological conditions. Osteopontin (OPN) is a phosphorylated glycoprotein produced principally by macrophages. OPN inhibits inducible nitric oxide synthase (iNOS), which generates large amounts of NO production. However, the relationship between NO and endogenous OPN in activated macrophages has not yet been elucidated. We therefore examined expression of endogenous iNOS and OPN in a murine macrophage cell line, RAW 264.7 cells, by treating the cells with lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma). Treatment of cells with LPS and IFN-gamma resulted in an increase of iNOS mRNA to maximum at 12 h after stimulation. In contrast, OPN mRNA was induced more slowly than iNOS mRNA. Induction of both iNOS and OPN mRNA in RAW 264.7 cells was markedly suppressed by addition of the specific iNOS inhibitor S-2-aminoethyl isothiourea dihydrobromide. The NOS inhibitor NG-methyl-L-arginine also suppressed induction of OPN mRNA but hardly affected iNOS mRNA expression. The NO-releasing agent spermine-NONOate but not peroxynitrite enhanced induction of OPN mRNA. These results suggest that NO directly up-regulates the endogenous OPN in macrophages stimulated with LPS and IFN-gamma. This up-regulation of endogenous OPN may represent a negative feedback system acting to reduce iNOS expression.     

Interleukin 4 induces the expression of inducible nitric oxide synthase in eosinophils

We investigated the effects of the Th2-like cytokines IL-4 and IL-13 and of IL-10 on the induction of iNOS and NO production in rat eosinophils. Addition of mIL-4 to the eosinophil culture increased iNOS activity and nitrite production but did not improve the stimulatory effect of IFN-gamma and LPS. In contrast to eosinophils, addition of mIL-4 to macrophage cultures inhibited the iNOS expression and nitrite production induced by IFN-gamma plus LPS. Addition of mIL-13 to the eosinophil cultures did not significantly change iNOS activity and nitrite production in cells stimulated or not with IFN-gamma plus LPS. On the other hand, IL-13 inhibited iNOS activity in IFN-gamma plus LPS-stimulated macrophages. In the presence of IL-10, iNOS activity in non-stimulated eosinophil or macrophage cultures was not significantly altered, but the enzyme expression was inhibited in IFN-gamma plus LPS-stimulated eosinophils or macrophages. The production of nitrite by eosinophils stimulated by IFN-gamma plus LPS was inhibited by the presence of IL-10 in the medium. In conclusion, eosinophils might exhibit differential modulation of the L-arginine/iNOS pathway depending on the profile of Th2 cytokines produced during allergic diseases. IL-4 appears to be an important Th2 cytokine involved in the induction of the L-arginine/iNOS pathway in eosinophils.