Integrin-linked kinase regulates inducible nitric oxide synthase and cyclooxygenase-2 expression in an NF-kappa B-dependent manner.

Nitric oxide (NO) and prostaglandins are produced as a result of the stimulation of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2, respectively, in response to cytokines or lipopolysaccharide (LPS). We demonstrate that the activity of integrin-linked kinase (ILK) is stimulated by LPS activation in J774 macrophages. Inhibition of ILK activity by dominant-negative ILK or a highly selective small molecule ILK inhibitor, in epithelial cells or LPS-stimulated J774 cells and murine macrophages, resulted in inhibition of iNOS expression and NO synthesis. LPS stimulates the phosphorylation of IkappaB on Ser-32 and promotes its degradation. Inhibition of ILK suppressed this LPS-stimulated IkappaB phosphorylation and degradation. Similarly, ILK inhibition suppressed the LPS-stimulated iNOS promoter activity. Mutation of the NF-kappaB sites in the iNOS promoter abolished LPS- and ILK-mediated regulation of iNOS promoter activity. Overexpression of ILK-stimulated NF-kappaB activity and inhibition of ILK or protein kinase B (PKB/Akt) suppressed this activation. We conclude that ILK can regulate NO production in macrophages by regulating iNOS expression through a pathway involving PKB/Akt and NF-kappaB. Furthermore, we also demonstrate that ILK activity is required for LPS stimulated cyclooxygenase-2 expression in murine and human macrophages. These findings implicate ILK as a potential target for anti-inflammatory applications.     

Penehyclidine hydrochloride attenuates LPS-induced iNOS production by inhibiting p38 MAPK activation in endothelial cells

The aim of this study was to investigate the inhibitory effect of penehyclidine hydrochloride (PHC) on lipopolysaccharide (LPS)-induced nitric oxide (NO) and inducible nitric oxide synthase (iNOS) production in human endothelial cell. Cultured endothelial cells were pretreated with PHC, followed by LPS treatment. NO activity were determined. iNOS expression and p38 mitogen-activated protein kinase (p38 MAPK) protein expression were measured by Western blot analysis. LPS treatment significantly induced p38 MAPK activation, iNOS expression, and NO production, which could be attenuated by 2 μg/ml PHC pretreatment. Furthermore, our study showed LPS-induced NO production and iNOS expression were suppressed by p38 MAPK inhibitor SB203580 pretreatment. We concluded that PHC attenuates NO production and iNOS expression by suppressing the activation of p38 MAPK pathway, thereby implicating a mechanism by which PHC may exert its protective effects against LPS-induced endothelial cell injury.     

Role of the cyclic AMP-protein kinase A pathway in lipopolysaccharide-induced nitric oxide synthase expression in RAW 264.7 macrophages. Involvement of cyclooxygenase-2.

The signaling pathway for lipopolysaccharide (LPS)-induced nitric oxide (NO) release in RAW 264.7 macrophages involves the protein kinase C and p38 activation pathways (Chen, C. C., Wang, J. K., and Lin, S. B. (1998) J. Immunol. 161, 6206-6214; Chen, C. C., and Wang, J. K. (1999) Mol. Pharmacol. 55, 481-488). In this study, the role of the cAMP-dependent protein kinase A (PKA) pathway was investigated. The PKA inhibitors, KT-5720 and H8, reduced LPS-induced NO release and inducible nitric oxide synthase (iNOS) expression. The direct PKA activator, Bt(2)cAMP, caused concentration-dependent NO release and iNOS expression, as confirmed by immunofluorescence studies. The intracellular cAMP concentration did not increase until after 6 h of LPS treatment. Two cAMP-elevating agents, forskolin and cholera toxin, potentiated the LPS-induced NO release and iNOS expression. Stimulation of cells with LPS or Bt(2)cAMP for periods of 10 min to 24 h caused nuclear factor-kappaB (NF-kappaB) activation in the nuclei, as shown by detection of NF-kappaB-specific DNA-protein binding. The PKA inhibitor, H8, inhibited the NF-kappaB activation induced by 6- or 12-h treatment with LPS but not that induced after 1, 3, or 24 h. The cyclooxygenase-2 (COX-2) inhibitors, NS-398 and indomethacin, attenuated LPS-induced NO release, iNOS expression, and NF-kappaB DNA-protein complex formation. LPS induced COX-2 expression in a time-dependent manner, and prostaglandin E(2) production was induced in parallel. These results suggest that 6 h of treatment with LPS increases intracellular cAMP levels via COX-2 induction and prostaglandin E(2) production, resulting in PKA activation, NF-kappaB activation, iNOS expression, and NO production.     

Anti-Inflammatory Effects of Progesterone in Lipopolysaccharide-Stimulated BV-2 Microglia

Female sex is associated with improved outcome in experimental brain injury models, such as traumatic brain injury, ischemic stroke, and intracerebral hemorrhage. This implies female gonadal steroids may be neuroprotective. A mechanism for this may involve modulation of post-injury neuroinflammation. As the resident immunomodulatory cells in central nervous system, microglia are activated during acute brain injury and produce inflammatory mediators which contribute to secondary injury including proinflammatory cytokines, and nitric oxide (NO) and prostaglandin E₂ (PGE₂), mediated by inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2), respectively. We hypothesized that female gonadal steroids reduce microglia mediated neuroinflammation. In this study, the progesterone’s effects on tumor necrosis factor alpha (TNF-α), iNOS, and COX-2 expression were investigated in lipopolysaccharide (LPS)-stimulated BV-2 microglia. Further, investigation included nuclear factor kappa B (NF-κB) and mitogen activated protein kinase (MAPK) pathways. LPS (30 ng/ml) upregulated TNF-α, iNOS, and COX-2 protein expression in BV-2 cells. Progesterone pretreatment attenuated LPS-stimulated TNF-α, iNOS, and COX-2 expression in a dose-dependent fashion. Progesterone suppressed LPS-induced NF-κB activation by decreasing inhibitory κBα and NF-κB p65 phosphorylation and p65 nuclear translocation. Progesterone decreased LPS-mediated phosphorylation of p38, c-Jun N-terminal kinase and extracellular regulated kinase MAPKs. These progesterone effects were inhibited by its antagonist mifepristone. In conclusion, progesterone exhibits pleiotropic anti-inflammatory effects in LPS-stimulated BV-2 microglia by down-regulating proinflammatory mediators corresponding to suppression of NF-κB and MAPK activation. This suggests progesterone may be used as a potential neurotherapeutic to treat inflammatory components of acute brain injury.     

B1 cells produce nitric oxide in response to a series of toll-like receptor ligands

The effect of a series of toll-like receptor (TLR) ligands on the production of nitric oxide (NO) in mouse B1 cells was examined by using CD5⁺ IgM⁺ WEHI 231 cells. The stimulation with a series of TLR ligands, which were Pam3Csk4 for TLR1/2, poly I:C for TLR3, lipopolysaccharide (LPS) for TLR4, imiquimod for TLR7 and CpG DNA for TLR9, resulted in enhanced NO production via augmented expression of an inducible type of NO synthase (iNOS). LPS was most potent for the enhancement of NO production, followed by poly I:C and Pam3Csk4. Imiquimod and CpG DNA led to slight NO production. The LPS-induced NO production was dependent on MyD88-dependent pathway consisting of nuclear factor (NF)-κB and a series of mitogen-activated protein kinases (MAPKs). Further, it was also dependent on the MyD88-independent pathway consisting of toll-IL-1R domain-containing adaptor-inducing IFN-β (TRIF) and interferon regulatory factor (IRF)-3. Physiologic peritoneal B1 cells also produced NO via the iNOS expression in response to LPS. The immunological significance of TLR ligands-induced NO production in B1 cells is discussed.     

IFN-gamma + LPS induction of iNOS is modulated by ERK, JNK/SAPK, and p38(mapk) in a mouse macrophage cell line

Nitric oxide (NO.) produced by inducible nitric oxide synthase (iNOS) mediates a number of important physiological and pathophysiological processes. The objective of this investigation was to examine the role of mitogen-activated protein kinases (MAPKs) in the regulation of iNOS and NO. by interferon-gamma (IFN-gamma) + lipopolysaccharide (LPS) in macrophages using specific inhibitors and dominant inhibitory mutant proteins of the MAPK pathways. The signaling pathway utilized by IFN-gamma in iNOS induction is well elucidated. To study signaling pathways that are restricted to the LPS-signaling arm, we used a subclone of the parental RAW 264.7 cell line that is unresponsive to IFN-gamma alone with respect to iNOS induction. In this RAW 264.7gammaNO(-) subclone, IFN-gamma and LPS are nevertheless required for synergistic activation of the iNOS promoter. We found that extracellular signal-regulated kinase (ERK) augmented and p38(mapk) inhibited IFN-gamma + LPS induction of iNOS. Dominant-negative MAPK kinase-4 inhibited iNOS promoter activation by IFN-gamma + LPS, also implicating the c-Jun NH(2)-terminal kinase (JNK) pathway in mediating iNOS induction. Inhibition of the ERK pathway markedly reduced IFN-gamma + LPS-induced tumor necrosis factor-alpha protein expression, providing a possible mechanism by which ERK augments iNOS expression. The inhibitory effect of p38(mapk) appears more complex and may be due to the ability of p38(mapk) to inhibit LPS-induced JNK activation. These results indicate that the MAPKs are important regulators of iNOS-NO. expression by IFN-gamma + LPS.     

Lipopolysaccharide induces relaxation in lung pericytes by an iNOS-independent mechanism

Lipopolysaccharide (LPS)-regulated contractility in pericytes may play an important role in mediating pulmonary microvascular fluid hemodynamics during inflammation and sepsis. LPS has been shown to regulate inducible nitric oxide (NO) synthase (iNOS) in various cell types, leading to NO generation, which is associated with vasodilatation. The purpose of this study was to test the hypothesis that LPS can regulate relaxation in lung pericytes and to determine whether this relaxation is mediated through the iNOS pathway. As predicted, LPS stimulated NO synthesis and reduced basal tension by 49% (P < 0.001). However, the NO synthase inhibitors N (omega)-nitro-L-arginine methyl ester, aminoguanidine, and N (omega)-monomethyl-L-arginine did not block the relaxation produced by LPS. In fact, aminoguanidine and N (omega)-monomethyl-L-arginine potentiated the LPS response. The possibility that NO might mediate either contraction or relaxation of the pericyte was further investigated through the use of NO donor compounds; however, neither sodium nitroprusside nor S-nitroso-N-acetylpenicillamine had any significant effect on pericyte contraction. The inhibitory effect of aminoguanidine on LPS-stimulated NO production was confirmed. This ability of LPS to inhibit contractility independent of iNOS was also demonstrated in lung pericytes derived from iNOS-deficient mice. This suggests the presence of an iNOS-independent but as yet undetermined pathway by which lung pericyte contractility is regulated.     

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.     

Toll-like receptor 9-mediated cytosolic phospholipase A2 activation regulates expression of inducible nitric oxide synthase

Although CpG containing DNA is an important regulator of innate immune responses via toll-like receptor 9 (TLR9), excessive activation of this receptor is detrimental to the host. Here, we show that cytosolic phospholipase A₂ (cPLA₂) activation is important for TLR9-mediated inducible nitric oxide synthase (iNOS) expression. Activation of TLR9 signaling by CpG induces iNOS expression and NO production. Inhibition of TLR9 blocked the iNOS expression and NO production. The CpG also stimulates cPLA₂-hydrolyzed arachidonic acid (AA) release. Inhibition of cPLA₂ activity by inhibitor attenuated the iNOS expression by CpG response. Additionally, knockdown of cPLA₂ protein by miRNA also suppressed the CpG-induced iNOS expression. Furthermore, the CpG rapidly phosphorylates three MAPKs and Akt. A potent inhibitor for p38 MAPK or Akt blocked the CpG-induced AA release and iNOS expression. These results suggest that TLR9 activation stimulates cPLA₂ activity via p38 or Akt pathways and mediates iNOS expression.     

Dioscorealide B suppresses LPS‐induced nitric oxide production and inflammatory cytokine expression in RAW 264.7 macrophages: The inhibition of NF‐κB and ERK1/2 activation

Dioscorealide B (DB), a naphthofuranoxepin has been purified from an ethanolic extract of the rhizome of Dioscorea membranacea Pierre ex Prain & Burkill which has been used to treat inflammation and cancer in Thai Traditional Medicine. Previously, DB has been reported to have anti‐inflammatory activities through reducing nitric oxide (NO) and tumor necrosis factor‐α (TNF‐α) production in lipopolysaccharides (LPS)‐induced RAW 264.7 macrophage cells. In this study, the mechanisms of DB on LPS‐induced NO production and cytokine expression through the activation of nuclear factor‐κB (NF‐κB) and ERK1/2 are demonstrated in RAW 264.7 cells. Through measurement with Griess's reagent, DB reduced NO level with an IC₅₀ value of 2.85 ± 0.62 µM that was due to the significant suppression of LPS‐induced iNOS mRNA expression as well as IL‐1β, IL‐6, and IL‐10 mRNA at a concentration of 6 µM. At the signal transduction level, DB significantly inhibited NF‐κB binding activity, as determined using pNFκB‐Luciferase reporter system, which action resulted from the prevention of IκBα degradation. In addition, DB in the range of 1.5–6 µM significantly suppressed the activation of the ERK1/2 protein. In conclusion, the molecular mechanisms of DB on the inhibition of NO production and mRNA expression of iNOS, IL‐1β, IL‐6, and IL‐10 were due to the inhibition of the upstream kinases activation, which further alleviated the NF‐κB and MAPK/ERK signaling pathway in LPS‐induced RAW264.7 macrophage cells. J. Cell. Biochem. 109: 1057–1063, 2010. © 2010 Wiley‐Liss, Inc.     

Expression of Inducible Nitric Oxide Synthase (iNOS) in Microglia of the Developing Quail Retina

Inducible nitric oxide synthase (iNOS), which produce large amounts of nitric oxide (NO), is induced in macrophages and microglia in response to inflammatory mediators such as LPS and cytokines. Although iNOS is mainly expressed by microglia that become activated in different pathological and experimental situations, it was recently reported that undifferentiated amoeboid microglia can also express iNOS during normal development. The aim of this study was to investigate the pattern of iNOS expression in microglial cells during normal development and after their activation with LPS by using the quail retina as model. iNOS expression was analyzed by iNOS immunolabeling, western-blot, and RT-PCR. NO production was determined by using DAR-4M AM, a reliable fluorescent indicator of subcellular NO production by iNOS. Embryonic, postnatal, and adult in situ quail retinas were used to analyze the pattern of iNOS expression in microglial cells during normal development. iNOS expression and NO production in LPS-treated microglial cells were investigated by an in vitro approach based on organotypic cultures of E8 retinas, in which microglial cell behavior is similar to that of the in situ retina, as previously demonstrated in our laboratory. We show here that amoeboid microglia in the quail retina express iNOS during normal development. This expression is stronger in microglial cells migrating tangentially in the vitreal part of the retina and is downregulated, albeit maintained, when microglia differentiate and become ramified. LPS treatment of retina explants also induces changes in the morphology of amoeboid microglia compatible with their activation, increasing their lysosomal compartment and upregulating iNOS expression with a concomitant production of NO. Taken together, our findings demonstrate that immature microglial cells express iNOS during normal development, suggesting a certain degree of activation. Furthermore, LPS treatment induces overactivation of amoeboid microglia, resulting in a significant iNOS upregulation.     

p38MAPK介导的胶质细胞iNOS的转录激活机制

丝裂原激活蛋白激酶(MAPK)酶级联反应系统参与胶质细胞中iNOS的合成.通过瞬时转染p38MAPK途径中上游激酶,MAPK激酶3(MKK3)和MAPK激酶6 (MKK6 )表达质粒,进一步了解p38MAPK级联传导信号系统调节iNOS基因在胶质细胞中的转录激活机制.MKK3或MKK6表达质粒与接有荧光素酶(luciferase ,Luc)的大鼠iNOS启动基因质粒(iNOS Luc)联合转染C6星形胶质细胞株引起iNOS Luc的激活,并且使细胞因子诱导的iNOSmRNA的表达增强.这两种效应都能够被p38MAPK抑制剂SB2 0 35 80所抑制.MKK3 6也可以诱导核因子κB(NFκB Luc)依赖的转录活性.这些分子水平的研究结果为p38MAPK信号级联传导途径在调节大鼠胶质细胞中iNOS基因转录激活中的重要作用,包括转录因子NFκB的作用提供了证据.通过阻断iNOS表达或NO的生成,抑制细胞炎症发生,为防治神经细胞炎症反应性疾病提供实验依据.