Mechanism study of osteoinductive LMP-1 inhibitory effect on nitric oxide production in pre-osteoclasts
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Objective To investigate the inhibitory effect of osteoinductive protein LMP-1 on synthesis of nitric oxide(NO) in pre-osteoclasts and its molecular mechanism.Methods TAT-LMP-1 was previously constructed for transduction of LMP-1 into cells.After transduction of TAT-LMP-1,pre-osteoclasts (RAW 264.7 cells) were stimulated by tumor necrosis factor-α (TNF-α) or lipopolysaccharide (LPS).Greiss assay was used to detect the synthesis of NO.Real-time RT-PCR and Western blot were used to detect themRNA and protein of inducible NO synthase (iNOS),respectively.Western blot was used to detect the phosphorylation and translocation of IκB and p65 in the activation of NF-κB pathway.Lucfferase reporter assay was used to determine p65 transcriptional activity.Results LMP-1 was observed to decrease the synthesis of NO induced by TNF-α or LPS in a concentration-dependent manner.LMP-1 also effectively inhibited the expression of iNOS.It potently suppressed the transcriptional activity and nuclear translocation of p65,and the phosphorylation of IκB.Conclusion LMP-1 can inhibit the synthesis of NO in pre-osteoclasts due to its inhibitive effects on phosphorylation of IκB,p65 translocation,and iNOS transcription partly.
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Nitric oxide; Osteoclasts; NF-κBKeywords:
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Orazipone [OR-1384; 3-[4-(methylsulfonyl)benzylidene]pentane-2,4-dione] is a novel sulfhydryl-modulating compound that has anti-inflammatory properties in experimental models of asthma and inflammatory bowel disease. In inflammation, inducible nitricoxide synthase (iNOS) generates NO, which modulates the immune response. Compounds that inhibit iNOS expression or iNOS activity possess anti-inflammatory effects. In the present study, we examined the effects of orazipone and its derivative OR-1958 [3-[3-chlorine-4-(methylsulfonyl)benzylidene]pentane-2,4-dione] on iNOS expression and NO production in J774 macrophages stimulated by bacterial lipopolysaccharide (LPS) and in human alveolar epithelial cells activated by proinflammatory cytokines. Protein expression and nuclear translocation of transcription factors were measured by Western blot. iNOS mRNA expression was determined by quantitative reverse transcription-polymerase chain reaction and iNOS mRNA stability by actinomycin D assay. iNOS promoter activity was studied in a cell line expressing luciferase under the control of iNOS promoter. Orazipone and its derivative OR-1958 but not its nonthiol-modulating analog inhibited iNOS expression and NO production in a concentration-dependent manner. Orazipone decreased LPS-induced iNOS mRNA expression, but the decay of iNOS mRNA was not affected. Orazipone extensively prevented LPS-induced activation of nuclear factor κB (NF-κB) and signal transducer and activator of transcription (STAT) 1, which are important transcription factors for iNOS. In agreement, human iNOS promoter activity was inhibited by orazipone. In conclusion, orazipone decreased activation of inflammatory transcription factors NF-κB and STAT1, and expression of iNOS in cells exposed to inflammatory stimuli. The thiolmodulating property seems to be critical in mediating the antiinflammatory effects of orazipone.
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Abstract The inhibitory effects of inotilone and methylinotilone on the induction of inducible nitric oxide synthase (iNOS) and cyclooxygenase‐2 (COX‐2) in murine RAW 264.7 cells activated with LPS were investigated. The results show that both hydroxyl groups on the benzene ring of the inotilone molecule are required for better anti‐inflammatory effect. Western blotting and RT‐PCR analyses demonstrated that inotilone blocked protein and mRNA expression of iNOS but not COX‐2. Instead, inotilone inhibited prostaglandin E 2 production through decreasing the enzyme activity of COX‐2. The repression of iNOS but not COX‐2 expression may come from the differential effect of inotilone on nuclear factor‐κB (NFκB) and CCAAT/enhancer‐binding protein beta Treatment with inotilone resulted in the reduction of LPS‐induced nuclear translocation of NFκB subunit and the NFκB‐dependent transcriptional activity by blocking phosphorylation of inhibitor κB(IκB)α and p65 and subsequent degradation of inhibitor κBα. Inotilone also inhibited LPS‐induced activation of PI3K/Akt and extracellular signal‐regulated kinase 1/2 and p38 mitogen‐activated protein kinase. Our results suggest that inotilone may have potential to be developed into an effective anti‐inflammatory agent.
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Inducible nitric oxide synthase (iNOS) critically contributes to the development of endotoxin-mediated inflammation. It can be induced by cytokines or endotoxins via distinct signaling pathways. Lipopolysaccharide (LPS) triggers iNOS expression through activation of the inhibitor of κB-α (IκB-α)-nuclear factor κB (NF-κB) cascade, whereas interferon-γ (IFN-γ) acts primarily through Janus kinase (JAK)-signal transducer and activator of transcription 1 (STAT1). Methylene blue (MB), an agent used clinically to treat numerous ailments, has been shown to reduce NO accumulation through suppression of iNOS activity. But it remains unclear whether MB affects iNOS induction. This knowledge gap is addressed in the present study using cultured cells and endotoxemic mice. With mouse macrophages, MB treatment prevented the LPS- and/or IFN-γ-stimulated iNOS protein expression. Real-time PCR experiments showed that iNOS mRNA transcription was robustly blocked by MB treatment. The inhibitory effect of MB on iNOS expression was confirmed in vivo in endotoxemic mice. Further analysis showed that MB had no significant effect on IκB-α degradation and NF-κB or STAT1 phosphorylation in LPS/IFN-γ-stimulated cells. The nuclear transport of active NF-κB or STAT1 was also not affected by MB treatment. But MB treatment markedly reduced the binding of NF-κB and STAT1 to their DNA elements. Chromatin immunoprecipitation assays confirmed that MB reduced NF-κB and STAT1 bindings to iNOS promoter inside the cell. These studies show that MB attenuates transcriptional factor binding amid iNOS mRNA transcription, providing further insight into the molecular mechanism of MB in disease therapy.
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Abstract—Nitric oxide (NO) is known to have antiatherogenic and anti-inflammatory properties, but its effects on the cytokine-induced nuclear factor-kappa B (NF-κB) activation pathway in relation to the regulation of inducible nitric oxide synthase (iNOS) gene in vascular smooth muscle cells (VSMCs) remain elusive. To elucidate the roles of NO in the regulation of cytokine-induced NF-κB activation and consequent iNOS gene expression, we studied the effects of NO donors [(±)-(E)-ethyl-2-[(E)-hydroxyamino]-5-nitro-3-hexeneamide (NOR3) and sodium nitroprusside] on interleukin (IL)-1β–induced NF-κB activation and IκB-α degradation and subsequent iNOS expression in rat VSMCs. Northern blot and Western blot analyses demonstrated that NO donors decreased IL-1β–induced iNOS mRNA and protein expression. Electrophoretic mobility shift assay using synthetic oligonucleotide corresponding to the downstream NF-κB site of rat iNOS promoter as a probe showed that NOR3 inhibited IL-1β–induced NF-κB activation and its nucl...
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