Modulation of gastric mucosal inflammatory responses to Helicobacter pylori by ghrelin: Role of cNOS-dependent IKK- β S-nitrosylation in the regulation of COX-2 activation

Disturbances in nitric oxide synthase (NOS) and cyclooxygenase (COX) isozyme systems, manifested by the excessive NO and prostaglandin (PGE2) generation, are well-recognized features of gastric mucosal inflammatory responses to H. pylori infection. In this study, we report that H. pylori LPS-induced enhancement in gastric mucosal inducible (i) iNOS expression and COX-2 activation was accompanied by the impairment in constitutive (c) cNOS phosphorylation, up-regulation in the inhibitory κB kinase-β (IKKβ) activation and the increase in the transcriptional factor, NF-κB, nuclear translocation. Further, we show that abrogation of cNOS control over NF-κB activation has lead to induction of iNOS expression and COX-2 activation through S-nitrosylation. Moreover, we demonstrate that the modulatory effect of peptide hormone, ghrelin, on the LPS-induced changes was reflected in the increase in Src/Akt-dependent cNOS activation through phosphorylation and the suppression of IKK-β activity through cNOS-mediated IKK-β protein S-nitrosylation. As a result, ghrelin exerted the inhibitory effect on NF-κB nuclear translocation, thus causing the repression of iNOS gene induction and the inhibition in COX-2 activation through iNOS-dependent S-nitrosylation. Our findings point to cNOS activation as a pivotal element in the signaling cascade by which ghrelin exerts modulatory control over proinflammatory events triggered in gastric mucosa by H. pylori infection.

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title: Helicobacter pylori Up-regulates Cyclooxygenase-2 mRNA Expression and Prostaglandin E2 Synthesis in MKN 28 Gastric Mucosal Cells in Vitro *
abstract: Helicobacter pylori has been suggested to play a role in the development of gastric carcinoma in humans. Also, mounting evidence indicates that cyclooxygenase-2 overexpression is associated with gastrointestinal carcinogenesis. We studied the effect of H. pylori on the expression and activity of cyclooxygenase-1 and cyclooxygenase-2 in MKN 28 gastric mucosal cells. H. pylori did not affect cyclooxygenase-1 expression, whereas cyclooxygenase-2 mRNA levels increased by 5-fold at 24 h after incubation of MKN 28 cells with broth culture filtrates or bacterial suspensions from wild-type H. pyloristrain. Also, H. pylori caused a 3-fold increase in the release of prostaglandin E2, the main product of cyclooxygenase activity. This effect was specifically related toH. pylori because it was not observed withEscherichia coli and was independent of VacA, CagA, or ammonia. H. pylori isogenic mutants specifically lackingpicA or picB, which are responsible for cytokine production by gastric cells, were less effective in the up-regulation of cyclooxygenase-2 mRNA expression and in the stimulation of prostaglandin E2 release compared with the parental wild-type strain. This study suggests that development of gastric carcinoma associated with H. pylori infection may depend on the activation of cyclooxygenase-2-related events.

title: Increased expression and cellular localization of inducible nitric oxide synthase and cyclooxygenase 2 in Helicobacter pylori gastritis.
abstract: Inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 are important regulators of mucosal inflammation and epithelial cell growth. To determine the role of iNOS and COX-2 in Helicobacter pylori-induced tissue injury, we compared their gene expression in H. pylori-induced gastritis with that in normal gastric mucosa and in non-H. pylori gastritis.



In 43 patients, we assessed H. pylori infection status, histopathology, messenger RNA (mRNA) and protein expression, and cellular localization of iNOS and COX-2.



By reverse-transcription polymerase chain reaction (RT-PCR), antral iNOS and COX-2 mRNA expression was absent to low in normal mucosa (n = 10), significantly increased in H. pylori-negative gastritis (n = 13), and even more markedly increased in H. pylori-positive gastritis (n = 20). Increased iNOS and COX-2 levels were confirmed by Northern and Western blot analysis and were both greater in the gastric antrum than in the gastric body of infected patients. Immunohistochemistry also showed increased expression of both genes in H. pylori gastritis: iNOS protein was detected in epithelium, endothelium, and lamina propria inflammatory cells, and COX-2 protein localized to mononuclear and fibroblast cells in the lamina propria.



iNOS and COX-2 are induced in H. pylori-positive gastritis and thus may modulate the inflammation and alterations in epithelial cell growth that occur in this disease. Higher levels of iNOS and COX-2 in H. pylori-positive vs. -negative gastritis and in gastric antrum, where bacterial density is greatest, suggest that expression of these genes is a direct response to H. pylori infection.

title: Suppression of Gastric Mucosal Inflammatory Responses to Helicobacter pylori Lipopolysaccharide by Peroxisome Proliferator‐Activated Receptor γ Activation
abstract: Peroxisome proliferator‐activated receptor‐ γ(PPAR γ) is a nuclear transcription factor that regulates the expression of genes associated with inflammation. We applied the animal model of H. pylori lipopolysaccharide(LPS)‐induced gastritis to assess the effect of a specific PPAR γligand, ciglitazone, on the apoptotic processes and the mucosal activity of inducible nitric oxide synthase (NOS‐2), and the expression of COX‐1 and ‐2 cyclooxygenases. In the absence of ciglitazone, the LPS‐elicited mucosal inflammatory responses were accompanied by a massive epithelial cell apoptosis, upregulation of NOS‐2 and COX‐2 protein expression, and a marked increase in the mucosal PGE 2 generation and NOS‐2 activity. The expression of COX‐1 protein, however, remained unchanged. Administration of ciglitazone led to dose‐dependent reduction (up to 48%) in the severity of mucosal inflammatory involvement elicited by the LPS and this effect of the agent was reflected in a 72.5% reduction in apoptosis, a 58.7% decline in the mucosal PGE 2 generation and a 75.6% drop in NOS‐2 activity, and produced a marked decrease inCOX‐2 and NOS‐2 protein expression. Our findings demonstrate that PPAR γactivation suppresses gastric mucosal inflammatory responses to H. pylori LPS, and suggest that pharmacological manipulation of PPAR γactivation may provide therapeutic benefits in the resolution of inflammation associated with H. pylori infection.

title: Increased expression of inducible nitric oxide synthase and cyclooxygenase-2 in Barrett's esophagus and associated adenocarcinomas.
abstract: Barrett's esophagus is a premalignant condition arising in response to chronic reflux esophagitis. Inducible nitric oxide synthase (iNOS; NOS-2) and cyclooxygenase-2 (COX-2) are mediators of inflammation and regulators of epithelial cell growth. Expression levels of iNOS and COX-2 are high in colorectal adenomas and carcinomas, and COX-2 expression is elevated in gastric cancers. To determine the involvement of iNOS and COX-2 in Barrett's-associated neoplasia, we measured expression of these genes in metaplastic Barrett's and esophageal adenocarcinomas. We detected elevated iNOS and COX-2 mRNA levels in Barrett's mucosa compared with paired gastric control tissues in 16 of 21 (76%) and 17 of 21 (80%) patients, respectively (P < 0.001 for both genes). In esophageal adenocarcinomas, iNOS and COX-2 mRNA levels were increased in four of five and five of five cases, respectively. Furthermore, in 10 of 10 Barrett's patients, immunohistochemical staining for iNOS and COX-2 expression was strongly positive and higher than in matched gastric controls. Increased COX-2 expression was confirmed by Western blotting. These findings support the hypothesis that iNOS and COX-2 are involved early and often in Barrett's-associated neoplastic progression.

title: Helicobacter pylori and Gastric Cancer: Factors That Modulate Disease Risk
abstract: Helicobacter pylori is a gastric pathogen that colonizes approximately 50% of the world's population. Infection with H. pylori causes chronic inflammation and significantly increases the risk of developing duodenal and gastric ulcer disease and gastric cancer. Infection with H. pylori is the strongest known risk factor for gastric cancer, which is the second leading cause of cancer-related deaths worldwide. Once H. pylori colonizes the gastric environment, it persists for the lifetime of the host, suggesting that the host immune response is ineffective in clearing this bacterium. In this review, we discuss the host immune response and examine other host factors that increase the pathogenic potential of this bacterium, including host polymorphisms, alterations to the apical-junctional complex, and the effects of environmental factors. In addition to host effects and responses, H. pylori strains are genetically diverse. We discuss the main virulence determinants in H. pylori strains and the correlation between these and the diverse clinical outcomes following H. pylori infection. Since H. pylori inhibits the gastric epithelium of half of the world, it is crucial that we continue to gain understanding of host and microbial factors that increase the risk of developing more severe clinical outcomes.

title: Nitric Oxide Synthase/COX Cross-Talk: Nitric Oxide Activates COX-1 But Inhibits COX-2-Derived Prostaglandin Production1
abstract: It is recognized that there is molecular cross-talk between the inflammatory mediators NO and PGs that may regulate tissue homeostasis and contribute to pathophysiological processes. However, the literature is divided with respect to whether NO activates or inhibits PG production. In this study, we sought to determine whether conflicting observations could be accounted for by divergent effects of NO on the two cyclooxygenase (COX) isoforms. Exposure of resting macrophages to NO (30 μM) enhanced PGE2 release by 4.5-fold. This enhancement was inhibited by indomethacin but not by the COX-2 selective inhibitor NS398. To separate the activation of phospholipase A2 and COX, we performed experiments using fibroblasts derived from COX-1-deficient or COX-2-deficient mice. These cells exhibit increased basal PG production, which is due to a constitutively stimulated cytosolic phospholipase A2 and enhanced basal expression of the remaining COX isozyme. The exposure of COX- 2-deficient cells to exogenous NO (10 μM) resulted in a 2.4-fold increase of PGE2 release above controls. Further studies indicated that NO stimulated PGE2 release in COX-2-deficient cells, without altering COX-1 mRNA or protein expression. In contrast, NO inhibited COX-2-derived PGE2 production in both LPS-stimulated macrophages and COX-1 knockout cells. This inhibition was associated with both decreased expression and nitration of COX-2. Thus, these studies demonstrate divergent effects of NO on the COX isoforms. The regulation of PGE production by NO is therefore complex and will depend on the local environment in which these pleiotropic mediators are produced.

title: Molecular mechanisms involved in the reciprocal regulation of cyclooxygenase and nitric oxide synthase enzymes.
abstract: The nitric oxide (NO) and cyclooxygenase (COX) pathways share a number of similarities. NO is the mediator generated from the NO synthase (NOS) pathway and COX converts arachidonic acid to prostaglandins (PGs), prostacyclin, and thromboxane A2. Two major forms of NOS and COX have been identified to date. The constitutive isoforms of these enzymes play an important role in the regulation of several physiological states. On the other hand, in an inflammatory setting, the inducible isoforms of these enzymes are induced in a variety of cells resulting in the production of large amounts NO and PGs, which play pathological roles in several disease states. An important link between the NOS and COX pathways was made by our group when we demonstrated that NO activates the COX enzymes, an event leading to overt production of PGs, suggesting that COX enzymes represent important endogenous 'receptor' targets for modulating the multifaceted roles of NO. More importantly, mechanistic studies of how NO activates the COX enzymes have been undertaken and additional pathways through which NO modulates PG production unraveled. The purpose of this article is to cover the advances, which have occurred over the years and in particular to summarize experimental data that outline how the discovery that NO modulates PG production has impacted and extended our understanding of these two systems in physiopathological events.

title: Cyclooxygenase-2 Inducible Nitric Oxide Synthase Binds , S-Nitrosylates , and Activates
abstract: clicking here. colleagues, clients, or customers by , you can order high-quality copies for your If you wish to distribute this article to others here. following the guidelines can be obtained by Permission to republish or repurpose articles or portions of articles ):  February 23, 2013  www.sciencemag.org (this information is current as of The following resources related to this article are available online at

title: The role of eNOS, iNOS, and NF-kappaB in upregulation and activation of cyclooxygenase-2 and infarct size reduction by atorvastatin.
abstract: Pretreatment with atorvastatin (ATV) reduces infarct size (IS) and increases myocardial expression of phosphorylated endothelial nitric oxide synthase (p-eNOS), inducible NOS (iNOS), and cyclooxygenase-2 (COX2) in the rat. Inhibiting COX2 abolished the ATV-induced IS limitation without affecting p-eNOS and iNOS expression. We investigated 1) whether 3-day ATV pretreatment limits IS in eNOS(-/-) and iNOS(-/-) mice and 2) whether COX2 expression and/or activation by ATV is eNOS, iNOS, and/or NF-kappaB dependent. Male C57BL/6 wild-type (WT), University of North Carolina eNOS(-/-) and iNOS(-/-) mice received ATV (10 mg.kg(-1).day(-1); ATV(+)) or water alone (ATV(-)) for 3 days. Mice underwent 30 min of coronary artery occlusion and 4 h of reperfusion, or hearts were harvested and subjected to ELISA, immunoblotting, biotin switch, and electrophoretic mobility shift assay. As a result, ATV reduced IS only in the WT mice. ATV increased eNOS, p-eNOS, iNOS, and COX2 levels and activated NF-kappaB in WT mice. It also increased myocardial COX2 activity. In eNOS(-/-) mice, ATV increased COX2 expression but not COX2 activity or iNOS expression. NF-kappaB was not activated by ATV in the eNOS(-/-) mice. In the iNOS(-/-) mice, eNOS and p-eNOS levels were increased but not iNOS and COX2 levels; however, NF-kappaB was activated. In conclusion, both eNOS and iNOS are essential for the IS-limiting effect of ATV. The expression of COX2 by ATV is iNOS, but not eNOS or NF-kappaB, dependent. Activation of COX2 is dependent on iNOS.

title: Regulation of Prostaglandin Biosynthesis by Nitric Oxide Is Revealed by Targeted Deletion of Inducible Nitric-oxide Synthase*
abstract: We investigated the effects of targeted deletion of the inducible NO synthase (iNOS) gene on the formation of prostaglandins in vivo and ex vivo. Peritoneal macrophages were obtained from control and iNOS-deficient mice, and prostaglandin E2 (PGE2) was quantified after stimulation with γ-interferon and lipopolysaccharide to induce COX-2. Total nitrate and nitrite production was completely abolished in cells from iNOS-deficient animals compared with control cells. PGE2 formation by cells from iNOS-deficient animals was decreased compared with cells from control animals 80% at 12 h (0.85 ± 0.90 ng/106 cells versus15.4 ± 2.1 ng/106 cells, p < 0.01) and 74% at 24 h (9.4 ± 4.3 ng/106 cellsversus 36.8 ± 4.1 ng/106 cells,p < 0.01). COX-2 protein expression was not significantly different in cells from control or knockout animals. Levels of PGE2 in the urine of iNOS-deficient mice were decreased 78% (0.24 ± 0.14 ng/mg of creatinineversus 1.09 ± 0.66 ng/mg of creatinine,p < 0.01) compared with control animals. In addition, the levels of urinary F2-isoprostanes, an index of endogenous oxidant stress, were significantly decreased in iNOS-deficient animals. In contrast, the levels of thromboxane B2 derived from platelets allowed to aggregate ex vivo were significantly increased in iNOS-deficient mice compared with wild-type mice. These studies support the hypothesis that NO and/or NO-derived species modulate cyclooxygenase activity and eicosanoid production in vivo.

title: Inducible nitric oxide synthase gene deletion exaggerates MAPK-mediated cyclooxygenase-2 induction by inflammatory stimuli.
abstract: Cyclooxygenase (COX)-2 and inducible nitric oxide (NO) synthase (iNOS) are responsive to a wide array of inflammatory stimuli, have been localized to vascular smooth muscle cells (SMCs), and are intimately linked to the progression of vascular disease, including atherosclerotic lesion formation. We and others have shown that the production and subsequent impact of COX products appear to be correlative with the status of NO synthesis. This study examined the impact of inflammation-driven NO production on COX-2 expression in SMCs. Concurrent stimulation of quiescent rat aortic SMCs with lipopolysaccharide (LPS) and interferon (IFN)-gamma increased COX-2, iNOS, and nitrite production. Pharmacological inhibition of NO synthase (N(G)-monomethyl-l-arginine) concentration- and time-dependently magnified LPS + IFN-gamma-mediated COX-2 mRNA and protein induction in a cGMP-independent manner. COX-2 induction was associated with activation of the ERK, p38, and JNK mitogen-activated protein kinase (MAPK) pathways. Interestingly, NO synthase inhibition enhanced ERK, p38, and to a lesser extent JNK phosphorylation but suppressed MAPK phosphatase (MKP)-1 induction in response to LPS + IFN-gamma. Similarly, the exposure of SMCs from iNOS(-/-) mice to LPS + IFN-gamma produced an enhancement of COX-2 induction, p38, and JNK phosphorylation and an attenuated upregulation of MKP-1 versus their wild-type counterparts. Taken together, our data indicate that NO, in part derived from iNOS, negatively regulates the immediate early induction of COX-2 in response to inflammatory stimuli.

title: Helicobacter pylori Induces Disturbances in Gastric Mucosal Akt Activation through Inducible Nitric Oxide Synthase-Dependent S-Nitrosylation: Effect of Ghrelin
abstract: Gastric mucosal inflammatory response to H. pylori and its key virulence factor, lipopolysaccharide (LPS), are characterized by a massive rise in apoptosis and the disturbances in NO signaling pathways. Here, we report that H. pylori LPS-induced enhancement in the mucosal inducible nitric oxide synthase (iNOS) was associated with the suppression in Akt kinase activity and the impairment in constitutive nitric oxide synthase (cNOS) phosphorylation. Further, we demonstrate that the LPS effect on Akt inactivation, manifested in the kinase protein S-nitrosylation and a decrease in its phosphorylation at Ser473, was susceptible to suppression by iNOS inhibition. Moreover, the countering effect of hormone, ghrelin, on the LPS-induced changes in Akt activity was reflected in the loss in Akt S-nitrosylation and the increase in its phosphorylation at Ser473, as well as cNOS activation through phosphorylation. Our findings demonstrate that up-regulation in iNOS with H. pylori infection leads to Akt inactivation through S-nitrosylation that exerts the detrimental effect on the processes of cNOS activation through phosphorylation. We also report that ghrelin protection against H. pylori-induced disturbances is manifested in a marked increase in Akt activity and evoked by a decrease in the kinase S-nitrosylation and the increase in its phosphorylation at Ser473.

title: Role of ghrelin-induced cSrc activation in modulation of gastric mucosal inflammatory responses to Helicobacter pylori
abstract: A peptide hormone, ghrelin, is recognized as an important modulator of gastric mucosal inflammatory responses to H. pylori through the regulation of nitric oxide synthase (NOS) system. As cSrc kinase plays a major role in transduction of signals that regulate the activity of NOS isozyme system, we investigated the influence of H. pylori LPS on the processes associated with Src activation in gastric mucosal cells. The LPS-induced drop in constitutive (c) cNOS activity and up-regulation in inducible (i) iNOS was associated with the suppression in cSrc kinase activity that was reflected in a decrease in its phosphorylation at Tyr416. Further, the countering effect of ghrelin on the LPS-induced changes in cSrc activity and the extent of its phosphorylation was accompanied by a marked reduction in the activity of iNOS and an increase in cNOS activation through phosphorylation at Ser1179. Moreover, the effect of ghrelin on cSrc activation and its Tyr416 phosphorylation was associated with the kinase S-nitrosylation that was susceptible to the blockage by cNOS inhibition. Our findings suggest that up-regulation in iNOS with H. pylori infection leads to disturbances in cNOS phosphorylation that exerts the detrimental effect on the processes of cSrc activation through cNOS-mediated S-nitrosylation. We also show that ghrelin attenuation of H. pylori-induced gastric mucosal inflammatory responses involves the enhancement in cSrc activation, elicited by the kinase S-nitrosylation and the increase in its phosphorylation at Tyr416.

title: Role of constitutive nitric oxide synthase in regulation of Helicobacter pylori-induced gastric mucosal cyclooxygenase-2 ac-tivation through S-nitrosylation: mechanism of ghrelin action
abstract: Gastric mucosal inflammatory responses to H. pylori lipopolysaccharide (LPS), are characterized by the excessive NO and prostaglandin (PGE2) generation due to the disturbances in nitric oxide synthase (NOS) and cyclooxygenase (COX) systems. Here, we report that the LPS-induced enhancement in gastric mucosal inducible (i) iNOS) activity and up-regulation in PGE2 production was associated with the suppression in Akt kinase activity and the impairment in constitutive (c) cNOS activation. The stimulatory effect of the LPS on PGE2 production, furthermore, was susceptible to suppression by COX-2 inhibitor, NS-398, and iNOS inhibitor, 1400 W. Further, we show that the countering effect of peptide hormone, ghrelin, on the LPS-induced changes was reflected in up-regu- lation in Akt activity and the increase in cNOS activation through phosphorylation, and accompanied by the suppression in iNOS expression and the reduction in COX-2 activity associated with the loss in COX-2 protein S-nitrosylation. Moreover, the effect of ghre-lin on the LPS-induced COX-2 S-nitrosylation was subject to repression by Akt inhibition. Our findings demonstrate that induction in iNOS with H. pylori in- fection leads to COX-2 activation through S-nitro- sylation and up-regulation in PGE2 generation, and that ghrelin counters these untoward consequences of the LPS through Akt-mediated up-regulation in cNO- S activation required for the iNOS gene repression.

title: Yin Yang 1 enhances cyclooxygenase-2 gene expression in macrophages.
abstract: Expression of cyclooxygenase-2 (COX-2) is associated with the pathogenesis of inflammation and various cancers, including lung cancer. Yin Yang 1 (YY1) is a zinc-finger transcription factor that interacts with histone acetyltransferases and deacetylases for its transcriptional activity and also is involved in inflammation and tumorigenesis. We investigated whether YY1 regulates COX-2 expression. We located a possible YY1 binding site proximal to the transcription initiation site of the COX-2 promoter. Electrophoretic mobility shift assays show that YY1 bound to the putative YY1 site in vitro. To show biological relevance, we performed chromatin immunoprecipitation assays showing that lipopolysaccharide (LPS) treatment induced YY1 binding to the cognate site in the endogenous COX-2 promoter. Overexpression of YY1 in macrophages treated with either LPS or live Pseudomonas aeruginosa increased COX-2 transcriptional activity. Furthermore, YY1 enhanced COX-2 protein expression and prostaglandin D(2) production elicited by LPS treatment. Mechanistically, we observed that LPS treatment resulted in disruption of an interaction between YY1 and p300, a histone acetyltransferase, but did not affect the interaction between YY1 and histone deacetylase 1/2. These data suggest that in response to LPS, YY1 dissociates from p300 and binds to the COX-2 promoter, contributing to COX-2 expression in an inflammatory milieu.

title: Lipopolysaccharide Induces Cyclooxygenase-2 in Intestinal Epithelium via a Noncanonical p38 MAPK Pathway1
abstract: Necrotizing enterocolitis (NEC), a severe intestinal inflammation in neonates, occurs following bacterial colonization of the gut. LPS-induced production of inflammatory factors in immature enterocytes may be a factor in NEC. Previously, we described LPS-induced p38 MAPK-dependent expression of cyclooxygenase-2 (COX-2) in rat IEC-6 cells. In this study, we examine COX-2 expression in newborn rat intestinal epithelium and further characterize the mechanisms of COX-2 regulation in enterocytes. Induction of NEC by formula feeding/hypoxia increased phospho-p38 and COX-2 levels in the intestinal mucosa. Celecoxib, a selective COX-2 inhibitor, exacerbated the disease, suggesting a protective role for COX-2. COX-2 was induced in the intestinal epithelium by LPS in vivo and ex vivo. The latter response was attenuated by the p38 inhibitor SB202190, but not by inhibitors of ERK, JNK, or NF-κB. In IEC-6 enterocytes, COX-2 was induced by the expression of MAPK kinase 3 EE (MKK3EE), a constitutive activator of p38, but not of activators of ERK or JNK pathways. However, neither MKK3/6 nor MKK4, the known p38 upstream kinases, were activated by LPS. Dominant-negative MKK3 or MKK4 or SB202190 failed to prevent LPS-induced, p38-activating phosphorylation, ruling out important roles of these kinases or p38 autophosphorylation. LPS increased COX-2 and activating phosphorylation of p38 with similar dose-response. Blockade of LPS-induced expression of COX-2-luciferase reporter and destabilization of COX-2 message by SB202190 indicate that p38 regulates COX-2 at transcription and mRNA stability levels. Our data indicate that p38-mediated expression of COX-2 proceeds through a novel upstream pathway and support the role of the neonate’s enterocytes as bacterial sensors.

title: Nitric oxide production and signaling in inflammation.
abstract: Nitric oxide (NO) is recognized as a mediator and regulator of inflammatory responses. It possesses cytotoxic properties that are aimed against pathogenic microbes, but it can also have damaging effects on host tissues. NO reacts with soluble guanylate cyclase to form cyclic guanosine monophosphate (cGMP), which mediates many of the effects of NO. NO can also interact with molecular oxygen and superoxide anion to produce reactive nitrogen species that can modify various cellular functions. These indirect effects of NO have a significant role in inflammation, where NO is produced in high amounts by inducible nitric oxide synthase (iNOS) and reactive oxygen species are synthesized by activated inflammatory cells. The present review deals with NO production and signaling in inflammation, especially in relation to human neutrophils and eosinophils.

title: A novel mitogen-activated protein kinase phosphatase-1 and glucocorticoid receptor (GR) interacting protein-1-dependent combinatorial mechanism of gene transrepression by GR.
abstract: Glucocorticoids have major antiinflammatory effects. Because COX-2 is the rate-limiting enzyme for proinflammatory prostaglandins, this study investigated the combinatorial inhibitory role of glucocorticoid receptor (GR) in COX-2 gene induction in macrophages and sought to identify the molecular mechanisms for that inhibition. Glucocorticoid-activated GR repressed COX-2 gene induction by lipopolysaccharide (LPS). Activated GR inhibited LPS-induced activator protein 1 activity, which in turn decreased activating transcription factor 2/c-Jun phosphorylation. The inhibition of MAPK-dependent activating transcription factor 2/c-Jun phosphorylation by GR in COX-2 repression was a result of MAPK phosphatase-1 (MKP-1) induction. Although GR did not inhibit LPS-induced p65 phosphorylation or nuclear factor-kappaB DNA binding activity, deletion of the nuclear factor-kappaB binding site in the COX-2 gene suppressed the ability of glucocorticoid to attenuate COX-2 induction. Chromatin immunoprecipitation and transfection assays revealed that a p65 DNA complex involving GR-bound GR-interacting protein 1 (GRIP1) also contributed to COX-2 repression. Additional knockdown and transfection assays identified other inflammatory genes coordinately regulated by MKP-1 and GRIP1. In summary, activated GR was found to antagonize the LPS-dependent induction of the COX-2 gene via a novel combinatorial mechanism involving MKP-1-mediated activator protein 1 inhibition and GR/GRIP1 recruitment to the p65 DNA complex; moreover, this work facilitated the identification of other GR-responding MKP-1/GRIP1-regulated genes.

title: Cyclooxygenase-2 Gene Transcription in a Macrophage Model of Inflammation1
abstract: Infections involving LPS-bearing, Gram-negative bacteria can lead to acute inflammation and septic shock. Cyclooxygenase-2 (COX-2), the target of nonsteroidal anti-inflammatory drugs and selective COX-2 inhibitors, is importantly involved in these responses. We examined the dynamics of COX-2 gene expression in RAW264.7 murine macrophages treated with LPS as a model for COX-2 gene expression during inflammation. We established, using Northern blotting, nuclear run-on assays, and RT-PCR, that COX-2 transcriptional activation continues for at least 12 h after LPS treatment and involves at least three phases. Previous studies with murine macrophages identified an NF-κB site, a C/EBP site, and a cAMP response element-1 (CRE-1) as cis-acting elements in the COX-2 promoter. We identified three additional functional elements including a second CRE (CRE-2), an AP-1 site, and an E-box that overlaps CRE-1. The E-box mediates transcriptional repression whereas the other cis-elements are activating. Using electrophoretic mobility supershift and chromatin immunoprecipitation assays, we cataloged binding to each functional cis element and found them occupied to varying extents and by different transcription factors during the 12 h following LPS treatment. This suggests that the cis elements and their cognate transcription factors participate in a sequential, coordinated regulation of COX-2 gene expression during an inflammatory response. In support of this concept, we found, using inhibitors of Jun kinase and NF-κB p50 nuclear localization, that COX-2 gene transcription was completely dependent on phospho-c-Jun plus p50 at 6 h after LPS treatment but was only partially dependent on the combination of these factors at later treatment times.

title: Ghrelin is a growth-hormone-releasing acylated peptide from stomach
abstract: Small synthetic molecules called growth-hormone secretagogues (GHSs) stimulate the release of growth hormone (GH) from the pituitary. They act through GHS-R, a G-protein-coupled receptor for which the ligand is unknown. Recent cloning of GHS-R strongly suggests that an endogenous ligand for the receptor does exist and that there is a mechanism for regulating GH release that is distinct from its regulation by hypothalamic growth-hormone-releasing hormone (GHRH). We now report the purification and identification in rat stomach of an endogenous ligand specific for GHS-R. The purified ligand is a peptide of 28 amino acids, in which the serine 3 residue is n-octanoylated. The acylated peptide specifically releases GH both in vivo and in vitro, and O-n-octanoylation at serine 3 is essential for the activity. We designate the GH-releasing peptide ‘ghrelin’ (ghre is the Proto-Indo-European root of the word ‘grow’). Human ghrelin is homologous to rat ghrelin apart from two amino acids. The occurrence of ghrelin in both rat and human indicates that GH release from the pituitary may be regulated not only by hypothalamic GHRH, but also by ghrelin.

title: Central ghrelin gastroprotection involves nitric oxide/prostaglandin cross‐talk
abstract: Ghrelin, a gut‐brain peptide, is considered a gastroprotective factor in gastric mucosa. We investigated the role of prostaglandins (PG) and the possible interplay between PGs and nitric oxide (NO) in ghrelin gastroprotection against ethanol (EtOH)‐induced gastric lesions.

title: Molecular mechanisms of ghrelin-mediated endothelial nitric oxide synthase activation.
abstract: Metabolic syndrome accelerates the atherosclerotic process, and the earliest event of which is endothelial dysfunction. Ghrelin, a newly discovered gastric peptide, improves endothelial function and inhibits proatherogenic changes. In particular, low ghrelin concentration has been associated with several features of metabolic syndrome, including obesity, insulin resistance, and high blood pressure. However, the molecular mechanisms underlying ghrelin vascular actions remain largely unclear. Here, we showed that ghrelin activated endothelial nitric oxide (NO) synthase (eNOS) in cultured endothelial cells (ECs) and in intact vessels. Specifically, ghrelin rapidly induced phosphorylation of eNOS on an activation site and production of NO in human umbilical vein ECs and bovine aortic ECs. The eNOS phosphorylation was also observed in mouse aortas ex vivo perfused with ghrelin and in aortic tissues isolated from mice injected with ghrelin. Mechanistically, ghrelin stimulated AMP-activated protein kinase (AMPK) and Akt activation in cultured ECs and intact vessels. Inhibiting AMPK and Akt with their pharmacological inhibitors, small interference RNA and adenoviruses carried dominant-negative mutants, markedly attenuated ghrelin-induced eNOS activation, and NO production. Furthermore, ghrelin receptor/Gq protein/calcium-dependent pathway mediates activation of AMPK, Akt, and eNOS, and calmodulin-dependent kinase kinase is a potential convergent point to regulate Akt and AMPK activation in ghrelin signaling. Importantly, eNOS activation is critical for ghrelin inhibition of vascular inflammation. Together, both in vitro and in vivo data demonstrate a new role of ghrelin signaling for eNOS activation, and highlight the therapeutic potential for ghrelin to correct endothelial dysfunction associated with atherosclerotic vascular diseases and metabolic syndrome.

title: Discovery of a novel IKK-β inhibitor by ligand-based virtual screening techniques

title: Inhibition of SRC Tyrosine Kinases Suppresses Activation of Nuclear Factor-κB, and Serine and Tyrosine Phosphorylation of IκB-α in Lipopolysaccharide-Stimulated Raw 264.7 Macrophages
abstract: Involvement of protein tyrosine kinases (PTK) in lipopolysaccharide (LPS)-induced nuclear factor-kappa B (NF-κB) activation has been demonstrated. Studies investigated the role of PTK and the underlying mechanisms by which PTK play a role in LPS induction of pathways leading to NF-κB activation in macrophages. Inhibitors of PTK—genistein, herbimycin A, or AG126—blocked LPS-induced NF-κB activation. Genistein also blocked pervanadate-induced NF-κB activation. Furthermore, Src TK selective inhibitors—damnacanthal or PP1—blocked LPS-induced NF-κB activation over a range of nanomolar concentrations. Genistein, damnacanthal, or PP1 blocked the LPS-induced serine phosphorylation, the degradation of IκB-α, and the consequent translocation of the p65 subunit of NF-κB to the nucleus. In addition to serine phosphorylation of IκB-α, LPS-induced NF-κB activation also required tyrosine phosphorylation of IκB-α. These TK inhibitors blocked substantially LPS induction of tyrosine phosphorylation of IκB-α. Furthermore, cSrc and Lck were physically associated with IκB-α. These results suggest that the LPS-induced NF-κB pathways are dependent on both serine and tyrosine phosphorylation of IκB-α, and that Src TK, such as cSrc and Lck, are key components of the LPS signaling pathway through at least two different mechanisms associated with NF-κB activation. This work was supported by grant R04-2002-000-00023-0 from the Basic Research Program of the Korea Science and Engineering Foundation.

title: Assessment and Application of the Biotin Switch Technique for Examining Protein S-Nitrosylation under Conditions of Pharmacologically Induced Oxidative Stress*
abstract: Protein S-nitrosylation has emerged as a principal mechanism by which nitric oxide exerts biological effects. Among methods for studying protein S-nitrosylation, the biotin switch technique (BST) has rapidly gained popularity because of the ease with which it can detect individual S-nitrosylated (SNO) proteins in biological samples. The identification of SNO sites by the BST relies on the ability of ascorbate to generate a thiol from an S-nitrosothiol, but not from alternatively S-oxidized thiols (e.g. disulfides, sulfenic acids). However, the specificity of this reaction has recently been challenged, prompting several claims that the BST may produce false-positive results and raising concerns about the application of the BST under oxidizing conditions. Here we perform a comparative analysis of the BST using differentially S-oxidized and S-nitrosylated forms of protein tyrosine phosphatase 1B, as well as intact and lysed human embryonic kidney 293 cells treated with S-oxidizing and S-nitrosylating agents, and verify that the assay is highly specific for SNO. Strikingly, exposure of samples to indirect sunlight from a laboratory window resulted in artifactual ascorbate-dependent signals that are likely promoted by the semidehydroascorbate radical; protection from sunlight eliminated the artifact. In contrast, exposure of SNO proteins to a strong ultraviolet light source (SNO photolysis) prior to the BST provided independent verification of assay specificity. By combining BST with photolysis, we have shown that anti-cancer drug-induced oxidative stress facilitates the S-nitrosylation of the major apoptotic effector glyceraldehyde-3-phosphate dehydrogenase. Collectively, these experiments demonstrate that SNO-dependent signaling pathways can be modulated by oxidative conditions and suggest a potential role for S-nitrosylation in antineoplastic drug action.

title: What a disorder: proinflammatory signaling pathways induced by Helicobacter pylori.
abstract: Infection of gastric epithelial cells with Helicobacter pylori induces strong proinflammatory responses by activating nuclear transcription factors NF-κB and AP-1. Several reports indicate that multiple bacterial factors and cellular molecules are involved in this signaling. Injected peptidoglycan, CagA or OipA and urease, and at least 16 different signaling cascades have been implicated in H. pylori-induced proinflammatory signaling. Many of these reports are contradictory, thus generating a highly puzzling scenario. Here we discuss the pros and cons of the multiple signaling activities in the induction of proinflammatory responses and associated problems, and give suggestions for finding ways out of this dilemma.

title: c-Src Regulates Akt Signaling in Response to Ghrelin via β-Arrestin Signaling-Independent and -Dependent Mechanisms
abstract: The aim of the present study was to identify the signaling mechanisms to ghrelin-stimulated activation of the serine/threonine kinase Akt. In human embryonic kidney 293 (HEK293) cells transfected with GHS-R1a, ghrelin leads to the activation of Akt through the interplay of distinct signaling mechanisms: an early Gi/o protein-dependent pathway and a late pathway mediated by β-arrestins. The starting point is the Gi/o-protein dependent PI3K activation that leads to the membrane recruitment of Akt, which is phosphorylated at Y by c-Src with the subsequent phosphorylation at A-loop (T308) and HM (S473) by PDK1 and mTORC2, respectively. Once the receptor is activated, a second signaling pathway is mediated by β-arrestins 1 and 2, involving the recruitment of at least β-arrestins, c-Src and Akt. This β-arrestin-scaffolded complex leads to full activation of Akt through PDK1 and mTORC2, which are not associated to the complex. In agreement with these results, assays performed in 3T3-L1 preadipocyte cells indicate that β-arrestins and c-Src are implicated in the activation of Akt in response to ghrelin through the GHS-R1a. In summary this work reveals that c-Src is crucially involved in the ghrelin-mediated Akt activation. Furthermore, the results support the view that β-arrestins act as both scaffolding proteins and signal transducers on Akt activation.

title: Inhibition of nitric oxide synthase expression by PPM-18, a novel anti-inflammatory agent, in vitro and in vivo.
abstract: We studied the effect of PPM-18, a chemically synthesized naphthoquinone derivative and also an anti-inflammatory agent, on the lipopolysaccharide (LPS)-activated inducible NO synthase (iNOS) expression in rat alveolar macrophages. Pretreatment of macrophages with PPM-18 (0.1-10 microM) significantly inhibited nitrite production, iNOS protein expression and iNOS mRNA accumulation. PPM-18 did not directly affect the enzymic activities of iNOS and other constitutive NOS forms. The LPS-induced increase in nuclear transcription factor kappaB (NF-kappaB) p65 and p50 in nucleus was suppressed by PPM-18 (10 microM). Moreover electrophoretic mobility-shift assays demonstrated that PPM-18 inhibited DNA binding to NF-kappaB induced by LPS in whole cells but not when added in the nuclear extract, suggesting that PPM-18 did not interfere directly with the binding of NF-kappaB to DNA and that some events had to be processed before NF-kappaB could bind DNA. Examination of NF-kappaB showed that PPM-18 stabilized the NF-kappaB inhibitor, IkappaBalpha, by preventing its degradation from NF-kappaB. Therefore the stabilization of IkappaBalpha might have contributed to the inhibition of NF-kappaB activation. These results also indicate strongly that NF-kappaB is involved in the production of NO on stimulation by LPS. PPM-18 significantly decreased the production of tumour necrosis factor alpha in response to LPS. PPM-18 protects mice against LPS-induced lethal toxicity. These results also indicate that PPM-18 is a potent inhibitor of iNOS expression by blocking the binding of NF-kappaB to promoter and exerts a beneficial effect in the mouse model of sepsis.

title: Bay 11-7082 inhibits transcription factor NF-kappaB and induces apoptosis of HTLV-I-infected T-cell lines and primary adult T-cell leukemia cells.
abstract: Human T-cell leukemia virus type I (HTLV-I) is the causative agent of an aggressive form of leukemia designated adult T-cell leukemia (ATL). We have previously demonstrated that all T-cell lines infected with HTLV-I and primary leukemic cells from ATL patients display constitutively high activity of transcription factor NF-kappaB. In this study we showed that Bay 11-7082, an inhibitor of NF-kappaB, induced apoptosis of HTLV-I-infected T-cell lines but only negligible apoptosis of HTLV-I-negative T cells. Bay 11-7082 rapidly and efficiently reduced the DNA binding of NF-kappaB in HTLV-I-infected T-cell lines and down-regulated the expression of the antiapoptotic gene, Bcl-x(L), regulated by NF-kappaB, whereas it had little effect on the DNA binding of another transcription factor, AP-1. Although the viral protein Tax is an activator of NF-kappaB, Bay 11-7082-induced apoptosis of HTLV-I-infected cells was not associated with reduced expression of Tax. Furthermore, Bay 11-7082- induced apoptosis of primary ATL cells was more prominent than that of normal peripheral blood mononuclear cells, and apoptosis of these cells was also associated with down-regulation of NF-kappaB activity. Our results indicate that NF-kappaB plays a crucial role in the pathogenesis and survival of HTLV-I-infected leukemic cells and that it is a suitable target for the prevention and treatment of ATL.

title: Ghrelin Suppression of Helicobacter pylori-Induced Gastric Mucosal Expression of iNOS is Mediated through the Inhibition of IKK-β Activation by cNOS-Dependent S-Nitrosylation
abstract: Excessive nitric oxide generation, caused by the disturbances in nitric oxide synthase (NOS) isozyme system, plays a key role in defining the extent of gastric mucosal inflammatory response to H. pylori infection. Here, we report that H. pylori LPS-induced enhancement in gastric mucosal inducible (i) iNOS expression and the impairment in constitutive (c) cNOS activity was associated with up-regulation in the inhibitory kB kinase-β (IKK?β) activation through phosphorylation, rise in IκB-α degradation, and the increase in the transcriptional factor, NF-κB, nuclear translocation. Further, we show that the countering effect of peptide hormone, ghrelin, on the LPS-induced disturbances in NOS isozyme system was reflected in the increase in Src/Akt-dependent cNOS activation through phosphorylation and the suppression of IKK-β activity through cNOSmediated IKK-β protein S-nitrosylation. As a consequence, ghrelin exerted the inhibitory effect on the LPS-induced rise in IκB-α degradation and NF-κB nuclear translocation, thus leading to iNOS gene suppression and the repression of iNOS induction. These results point to a central role of cNOS activation in controlling the signaling pathways of the LPS-triggered iNOS gene induction. Moreover, our findings suggest a molecular mechanism by which ghrelin suppresses the gastric mucosal proinflammatory consequences of H. pylori infection.

title: Nitric oxide represses inhibitory κB kinase through S-nitrosylation
abstract: Nitric oxide (NO) possesses antiinflammatory effects, which may be exerted via its ability to inhibit the transcription factor, NF-κB. A commonly proposed mode of action for inhibition of NF-κBbyNO involves interference with NF-κB binding to DNA. Because activation of inhibitory κB kinase (IKK), the prerequisite enzyme complex necessary to induce NF-κB, is subject to redox regulation, we assessed whether IKK could present a more proximal target for NO to inhibit NF-κB activation. We demonstrate here that S-nitrosothiols (SNO) caused a dose-dependent inhibition of the enzymatic activity of IKK, in lung epithelial cells and in Jurkat T cells, which was associated with S-nitrosylation of the IKK complex. Using biotin derivatization of SNO, we revealed that IKKβ, the catalytic subunit required for NF-κB activation, was a direct target for S-nitrosylation. A mutant version of IKKβ containing a Cys-179-to-Ala mutation was refractory to inhibition by SNO or to increases in S-nitrosylation, in contrast to wild-type IKKβ, demonstrating that Cys-179 is the main target for attack by SNO. Importantly, inhibition of NO synthase activity in Jurkat T cells resulted in activation of IKK, in association with its denitrosylation. Moreover, NO synthase inhibition enhanced the ability of tumor necrosis factor α to activate IKK, illustrating the importance of endogenous NO in regulating the extent of NF-κB activation by cytokines. Collectively, our findings demonstrate that IKKβ is an important target for the redox regulation of NF-κB by endogenous or exogenous NO, providing an additional mechanism for its antiinflammatory properties.

title: Integrating cell-signalling pathways with NF-κB and IKK function
abstract: Nuclear factor (NF)-κB and inhibitor of NF-κB kinase (IKK) proteins regulate many physiological processes, including the innate- and adaptive-immune responses, cell death and inflammation. Disruption of NF-κB or IKK function contributes to many human diseases, including cancer. However, the NF-κB and IKK pathways do not exist in isolation and there are many mechanisms that integrate their activity with other cell-signalling networks. This crosstalk constitutes a decision-making process that determines the consequences of NF-κB and IKK activation and, ultimately, cell fate.