Ceramide inhibits lipopolysaccharide-mediated nitric oxide synthase and cyclooxygenase-2 induction in macrophages: effects on protein kinases and transcription factors

The goal of this study was to elucidate whether triggering the sphingomyelin pathway modulates LPS-initiated responses. For this purpose we investigated the effects of N-acetylsphingosine (C(2)-ceramide) on LPS-induced production of NO and PGE(2) in murine RAW 264.7 macrophages and explored the signaling pathways involved. We found that within a range of 10-50 microM, C(2)-ceramide inhibited LPS-elicited NO synthase and cyclooxygenase-2 induction accompanied by a reduction in NO and PGE(2) formation. By contrast, a structural analog of C(2)-ceramide that does not elicit functional activity, C(2)-dihydroceramide, did not affect the LPS response. The nuclear translocation and DNA binding study revealed that ceramide can inhibit LPS-induced NF-kappaB and AP-1 activation. The immunocomplex kinase assay indicated that IkappaB kinase activity stimulated by LPS was inhibited by ceramide, which concomitantly reduced the IkappaBalpha degradation caused by LPS within 1-6 h. In concert with the decreased cytosolic p65 protein level, LPS treatment resulted in rapid nuclear accumulation of NF-kappaB subunit p65 and its association with the cAMP-responsive element binding protein. Ceramide coaddition inhibited all the LPS responses. In addition, LPS-induced PKC and p38 mitogen-activated protein kinase activation were overcome by ceramide. In conclusion, we suggest that ceramide inhibition of LPS-mediated induction of inducible NO synthase and cyclooxygenase-2 is due to reduction of the activation of NF-kappaB and AP-1, which might result from ceramide's inhibition of LPS-stimulated IkappaB kinase, p38 mitogen-activated protein kinase, and protein kinase C.     

Suppression of nuclear factor-kappa B activity by nitric oxide and hyperoxia in oxygen-resistant cells

Inhaled nitric oxide (iNO) is used clinically to treat pulmonary hypertension in newborns, often in conjunction with hyperoxia (NO/O2). Prolonged exposure to NO/O2 causes synergistic lung injury and death of lung epithelial cells. To explore the mechanisms involved, oxygen-resistant HeLa-80 cells were exposed to NO +/- O2. Exposure to NO and O2 induced a synergistic cytotoxicity, accompanied with apoptotic characteristics, including elevated caspase-3-like activity, Annexin V incorporation, and nuclear condensation. This apoptosis was associated with a synergistic suppression of NF-kappaB activity. Cells lacking functional NF-kappaB p65 subunit were more sensitive to NO/O2 than their wild type counterparts. This injury was partially rescued by transfection with a p65 expression construct, suggesting an inverse relationship between NF-kappaB and susceptibility to the cytotoxicity of NO/O2. Despite the reduced NF-kappaB activity in cells exposed to NO +/- O2, IkappaBalpha was degraded, suggesting that pathways regulating the steady-state levels of IkappaB were not involved. However, exposure to NO/O2 caused a marked reduction in nuclear localization and an increase in protein carbonyl formation of NF-kappaB p65 subunit. These results suggest that NO/O2-induced apoptosis occurs by suppressing NF-kappaB activity.     

Induction of glutathione peroxidase in response to inactivation by nitric oxide.

To determine effect of nitric oxide (NO) on cellular glutathione peroxidase (GPX) level in living cells, we measured the activity, protein and mRNA of GPX in rat kidney (KNRK) cells under a high NO condition. Combined treatment of lipopolysaccharide (LPS, 1 microgram/ml) and tumor necrosis factor-alpha (TNF-alpha, 50 ng/ml) synergistically enhanced (23-folds) nitrite production from KNRK cells. This was suppressed by an inducible NO synthase (iNOS) inhibitor (aminoguanidine, N-nitro-L-arginine methylester hydrochloride) and arginase. iNOS expression was detected by RT-PCR in the treated cells. GPX was inactivated irreversibly when the cells had been homogenized before exposure to a NO donor, S-nitroso-N-acetylpenicillamine (SNAP). In living KNRK cells, SNAP and LPS + TNF-alpha exerted a transient effect on the GPX activity. The treatment with SNAP (200 microM) or sodium nitroprusside (200 microM) enhanced GPX gene expression, which was blocked by a NO scavenger, 2-phenyl-4,4,5,5,-tetramethylimidazoline-1-oxyl-3-oxide. GPX mRNA was markedly increased by the treatment with LPS + TNF-alpha, and aminoguanidine blocked the effect. In cells metabolically labeled with 75Se, LPS + TNF-alpha accelerated the incorporation of radioactivity into GPX molecule by 2.1-fold. These results suggest that inactivation of GPX by NO triggers a signal for inducing GPX gene expression in KNRK cells, thereby restoring the intracellular level of this indispensable enzyme.     

Inhibitory effect of curcumin on nitric oxide production from lipopolysaccharide-activated primary microglia

Curcumin has been shown to exhibit anti-inflammatory, antimutagenic, and anticarcinogenic activities. However, the modulatory effect of curcumin on the functional activation of primary microglial cells, brain mononuclear phagocytes causing the neuronal damage, largely remains unknown. The current study examined whether curcumin influenced NO production in rat primary microglia and investigated its underlying signaling pathways. Curcumin decreased NO production in LPS-stimulated microglial cells in a dose-dependent manner, with an IC(50) value of 3.7 microM. It also suppressed both mRNA and protein levels of inducible nitric oxide synthase (iNOS), indicating that this drug may affect iNOS gene expression process. Indeed, curcumin altered biochemical patterns induced by LPS such as phosphorylation of all mitogen-activated protein kinases (MAPKs), and DNA binding activities of nuclear factor-kappaB (NF-kappaB) and activator protein (AP)-1, assessed by reporter gene assay. By analysis of inhibitory features of specific MAPK inhibitors, a series of signaling cascades including c-Jun N-terminal kinase (JNK), p38 and NF-kappaB was found to play a critical role in curcumin-mediated NO inhibition in microglial cells. The current results suggest that curcumin is a promising agent for the prevention and treatment of both NO and microglial cell-mediated neurodegenerative disorders.     

Inhibition of interleukin-12 p40 transcription and NF-kappaB activation by nitric oxide in murine macrophages and dendritic cells

Nitric oxide (NO), an important effector molecule of the innate immune system, can also regulate adaptive immunity. In this study, the molecular effects of NO on the toll-like receptor signaling pathway were determined using interleukin-12 (IL-12) as an immunologically relevant target gene. The principal conclusion of these experiments is that NO inhibits IL-1 receptor-associated kinase (IRAK) activity and attenuates the molecular interaction between tumor necrosis factor receptor-associated factor-6 and IRAK. As a consequence, the NO donor S-nitroso-N-acetylpenicillamine (SNAP) inhibits lipopolysaccharide (LPS)-induced IL-12 p40 mRNA expression, protein production, and promoter activity in murine macrophages, dendritic cells, and the murine macrophage cell line RAW 264.7. Splenocytes from inducible nitric-oxide synthase-deficient mice demonstrate markedly increased IL-12 p40 protein and mRNA expression compared with wild type splenocytes. The inhibitory action of NO on IL-12 p40 is independent of the cytokine IL-10. The effects of NO can be directly attributed to inhibition of NF-kappaB activation through IRAK-dependent pathways. Accordingly, SNAP strongly reduces LPS-induced NF-kappaB DNA binding to the p40 promoter and inhibits LPS-induced IkappaB phosphorylation. Similarly, NO attenuates IL-1beta-induced NF-kappaB activation. These experiments provide another example of how an innate immune molecule may have a profound effect on adaptive immunity.     

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.