A pyrrolidinone derivative inhibits cytokine-induced iNOS expression and NF-kappaB activation by preventing phosphorylation and degradation of IkappaB-alpha

We previously showed that 1-[3-(3-pyridyl)-acryloyl]-2-pyrrolidinone hydrochloride (N2733) inhibits lipopolysaccharide (LPS)-induced tumour necrosis factor (TNF)-alpha secretion and improves the survival of endotoxemic mice. Since overproduction of nitric oxide (NO) by inducible NO synthase (iNOS) in vascular smooth muscle cells (VSMCs) is largely responsible for the development of endotoxemic shock, and iNOS gene expression is mainly regulated by LPS and inflammatory cytokines, we studied whether or not N2733 affects interleukin (IL)-1beta-induced iNOS gene expression, NF-kappaB activation, and NF-kappaB inhibitor (IkappaB)-alpha degradation in cultured rat VSMCs. N2733 dose-dependently (10-100 microM) inhibited IL-1beta-stimulated NO production, and decreased IL-1beta-induced iNOS mRNA and protein expression, as found on Northern and Western blot analyses, respectively. Gel shift assay and an immunocytochemical study showed that N2733 inhibited IL-1beta-induced NF-kappaB activation and its nuclear translocation. Western blot analyses involving anti-IkappaB-alpha and anti-phospho IkappaB-alpha antibodies showed that IL-1beta induced transient degradation of IkappaB-alpha preceded by the rapid appearance of phosphorylated IkappaB-alpha, both of which were markedly blocked by N2733. N2733 blocked IL-1beta-induced phosphorylated IkappaB-alpha even in the presence of a proteasome inhibitor (MG115). Immunoblot analysis involving anti-IkappaB kinase (IKK)-alpha and anti-phosphoserine antibodies revealed that N2733 inhibited IL-1beta-induced IKK-alpha phosphorylation, whereas N2733 had no inhibitory effect on IL-1beta-stimulated p42/p44 MAP kinase or p38 MAP kinase activity. Our results suggest that the inhibitory action of N2733 toward IL-1beta-induced NF-kappaB activation and iNOS expression is due to its blockade of the upstream signal(s) leading to IKK-alpha activation, and subsequent phosphorylation and degradation of IkappaB-alpha in rat VSMCs.     

Alachlor and carbaryl suppress lipopolysaccharide-induced iNOS expression by differentially inhibiting NF-kappaB activation

Nitric oxide (NO) produced by macrophages plays an important role in host defense and inflammation. We found that two agrochemicals, alachlor and carbaryl, inhibit lipopolysaccharide (LPS)-induced NO production by macrophages. In the present study, we investigated this inhibitory mechanism in RAW 264 cells. Both chemicals inhibited LPS-induced iNOS protein and mRNA expression as well as murine iNOS promoter activity. When treating these chemicals with reducing agents, the inhibition by carbaryl was reversed, but not the inhibition by alachlor. These chemicals also inhibited LPS-induced interferon-beta (IFN-beta) expression, an indispensable factor for LPS-induced iNOS expression. The inhibited iNOS expression, however, was not restored by exogenous IFN-beta supplementation. LPS-induced nuclear translocation of NF-kappaB, which is necessary for the expression of IFN-beta and iNOS, was inhibited by these chemicals: however, the LPS-induced degradation of IkappaB-alpha and IkappaB-beta was inhibited only by alachlor. These results indicate that alachlor and carbaryl differentially impair the LPS-induced NF-kappaB activation, leading to the inhibition of NO production.     

Insulin-like growth factor-II, phosphatidylinositol 3-kinase, nuclear factor-kappaB and inducible nitric-oxide synthase define a common myogenic signaling pathway.

Insulin-like growth factors (IGFs) are potent inducers of skeletal muscle differentiation and phosphatidylinositol (PI) 3-kinase activity is essential for this process. Here we show that IGF-II induces nuclear factor-kappaB (NF-kappaB) and nitric-oxide synthase (NOS) activities downstream from PI 3-kinase and that these events are critical for myogenesis. Differentiation of rat L6E9 myoblasts with IGF-II transiently induced NF-kappaB DNA binding activity, inducible nitric-oxide synthase (iNOS) expression, and nitric oxide (NO) production. IGF-II-induced iNOS expression and NO production were blocked by NF-kappaB inhibition. Both NF-kappaB and NOS activities were essential for IGF-II-induced terminal differentiation (myotube formation and expression of skeletal muscle proteins: myosin heavy chain, GLUT 4, and caveolin 3), which was totally blocked by NF-kappaB or NOS inhibitors in rat and human myoblasts. Moreover, the NOS substrate L-Arg induced myogenesis in the absence of IGFs in both rat and human myoblasts, and this effect was blocked by NOS inhibition. Regarding the mechanisms involved in IGF-II activation of NF-kappaB, PI 3-kinase inhibition prevented NF-kappaB activation, iNOS expression, and NO production. Moreover, IGF-II induced, through a PI 3-kinase-dependent pathway, a decrease in IkappaB-alpha protein content that correlated with a decrease in the amount of IkappaB-alpha associated with p65 NF-kappaB.     

Diphenyleneiodonium inhibits NF-kappaB activation and iNOS expression induced by IL-1beta: involvement of reactive oxygen species

AIMS: In this work, we studied the mechanisms by which diphenyleneiodonium chloride (DPI) inhibits nitric oxide (NO) synthesis induced by the proinflammatory cytokine interleukin-1beta (IL-1) in bovine articular chondrocytes. To achieve this, we evaluated the ability of DPI to inhibit the expression and activity of the inducible isoform of the NO synthase (iNOS) induced by IL-1. We also studied the ability of DPI to prevent IL-1-induced NF-kappaB activation and reactive oxygen species (ROS) production. RESULTS: Northern and Western blot analysis, respectively, showed that DPI dose-dependently inhibited IL-1-induced iNOS mRNA and protein synthesis in primary cultures of bovine articular chondrocytes. DPI effectively inhibited NO production (IC50=0.03+/-0.004 microM), as evaluated by the method of Griess. Nuclear factor-kappa B (NF-kappaB) activation, as evaluated by electrophoretic mobility shift assay, was inhibited by DPI (1-10 microM) in a dose-dependent manner. IL-1-induced ROS production, as evaluated by measurement of dichlorofluorescein fluorescence, was inhibited by DPI at concentrations that also prevented NF-kappaB activation and iNOS expression. CONCLUSIONS: DPI inhibits IL-1-induced NO production in chondrocytes by two distinct mechanisms: (i) by inhibiting NOS activity, and (ii) by preventing iNOS expression through the blockade of NF-kappaB activation. These results also support the involvement of reactive oxygen species in IL-1-induced NF-kappaB activation and expression of NF-kappaB-dependent genes, such as iNOS.     

解脲脲原体脂质相关膜蛋白通过激活核因子κB调控诱导性一氧化氮合酶在小鼠巨噬细胞中的表达

为探讨解脲脲原体(Uu)的脂质相关膜蛋白(LAMPs)诱导小鼠巨噬细胞表达诱导性一氧化氮合酶(iNOS)的分子机制,从解脲脲原体提取的脂质相关膜蛋白,刺激小鼠巨噬细胞,以RT_PCR、Western blot等方法分析iNOS的表达及NO的产生;用细胞免疫化学、间接免疫荧光及Western blot等方法检测核因子κB(NF_κB)的激活,另外检测了NF_κB的特异性抑制剂二硫代氨基甲酸吡咯烷(PDTC)和蛋白酶抑制剂放线菌酮(CHX)对iNOS的表达及NF_κB激活的影响。结果表明,解脲脲原体的LAMPs通过激活NF_κB诱导小鼠巨噬细胞表达iNOS的mRNA和蛋白,且能以时间和剂量依赖方式刺激小鼠巨噬细胞产生NO,NF_κB的抑制剂PDTC或蛋白酶抑制剂放线菌酮(CHX),可抑制NF_κB的激活及iNOS的表达。由于解脲脲原体的脂质相关膜蛋白通过激活NF_κB诱导小鼠巨噬细胞表达iNOS和产生NO,因而可能是一个重要的致病因素。     

Methanol extracts of Stewartia koreana inhibit cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) gene expression by blocking NF-kappaB transactivation in LPS-activated RAW 264.7 cells

Cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) are involved in various pathophysiological processes such as inflammation and carcinogenesis. In a search for inhibitors of COX-2 and iNOS production we found that extracts of Stewartia koreana strongly inhibited NO and PGE2 production in LPS-treated macrophage RAW 264.7 cells. We have now shown that the mRNA and protein levels of iNOS and COX-2 are reduced by the Stewartia koreana extract (SKE). SKE inhibited expression of an NF-kappaB reporter gene in response to LPS, and gel mobility shift assays revealed that SKE reduced NF-kappaB DNA-binding activity. The extract also inhibited LPS-induced phosphorylation of IkappaB-alpha and nuclear translocation of p65. Administration of the extract reduced the symptoms of arthritis in a collagen-induced arthritic mouse model. These results indicate that Stewartia extracts contain potentially useful agents for preventing and treating inflammatory diseases.     

Inhibitory effect of DCDC on lipopolysaccharide-induced nitric oxide synthesis in RAW 264.7 cells

In the present study we have examined the effect of DCDC (2',5'-dihydroxy-4-chloro-dihydrochalcone) on lipopolysaccharide (LPS)-induced responses in murine macrophage cell line RAW 264.7. Exposure of LPS-stimulated cells to DCDC inhibited the nitrite accumulation in culture medium. DCDC also concentration-dependently inhibited LPS-stimulated increase of iNOS expression; however, it had little effect on iNOS enzyme activity, suggesting that the inhibitory action to DCDC is mainly due to the inhibition on iNOS expression rather than iNOS enzyme activity. DCDC significantly inhibited LPS-evoked degradation of IkappaB-alpha and the nuclear translocation of NF-kappaB; it also exhibited the activity of scavenging the stable free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH). DCDC also inhibited cyclooxygenase-2 activity in RAW 264.7 cells with an IC50 of 3.0 microM; furthermore, it also significantly decreased LPS-induced mortality rate in mice. Taken together, we demonstrate that DCDC exhibits inhibitory effects on nitric oxide production through the inhibition of IkappaB-alpha degradation and NF-kappaB activation, and therefore the suppression of iNOS expression. DCDC also shows the antioxidant activity and COX-2 inhibitory action. Moreover, it improves survival in a murine model of endotoxaemia suggesting that DCDC may be potential in the therapy of septic shock.     

Differential effects of cyclic AMP on induction of nitric oxide synthase in 3T3-L1 cells and brown adipocytes

The aim of this study was to determine whether cyclic AMP (cAMP) pathways alter the nitric oxide (NO) production mediated by inducible NO synthase (iNOS) in adipocytes. The treatment of 3T3-L1 cells, a model of white adipocytes, with the combination of lipopolysaccharide (L), tumor necrosis factor-alpha (T), and interferon-gamma (I) synergistically induced iNOS, leading to the production of NO. Enhancers of intracellular cAMP (dibutyryl cAMP, forskolin, and IBMX) inhibited the NO production elicited by LTI, whereas H89, a specific inhibitor of PKA, stimulated the NO production in 3T3-L1 cells. In rat brown adipocyte cell line, the combined treatment with LT synergistically elicited the NO production, and the cAMP analogues further enhanced it. Forskolin inhibited the NO production in 3T3-L1 cells, but enhanced it in brown adipocytes, in a dose-dependent manner. The changes in NO production paralleled the change in iNOS mRNA and protein level in both cell types. The activation of NF-kappaB by LTI/LT was blocked in 3T3-L1 cells, but enhanced in brown adipocytes, by the co-treatment with cAMP analogues. The protein level of 1-kappaBalpha, a NF-kappaB stabilizer, changed reciprocally to that of NF-kappaB activity in each cell type. These results suggest that cAMP regulates iNOS expression in adipocytes through modulating NF-kappaB activity. The differential regulation of iNOS in 3T3-L1 cells from that in the brown adipocytes indicates that intracellular signal pathways activated by cAMP are different between the cell types.