Subtilase Cytotoxin Enhances Escherichia coli Survival in Macrophages by Suppression of Nitric Oxide Production through the Inhibition of NF-κB Activation

ABSTRACTSubtilase cytotoxin (SubAB), which is produced by certain strains of Shiga-toxigenicEscherichia coli(STEC), cleaves an endoplasmic reticulum (ER) chaperone, BiP/Grp78, leading to induction of ER stress and caspase-dependent apoptosis. SubAB alters the innate immune response. SubAB pretreatment of macrophages inhibited lipopolysaccharide (LPS)-induced production of both monocyte chemoattractant protein 1 (MCP-1) and tumor necrosis factor α (TNF-α). We investigated here the mechanism by which SubAB inhibits nitric oxide (NO) production by mouse macrophages. SubAB suppressed LPS-induced NO production through inhibition of inducible NO synthase (iNOS) mRNA and protein expression. Further, SubAB inhibited LPS-induced IκB-α phosphorylation and nuclear localization of the nuclear factor-κB (NF-κB) p65/p50 heterodimer. Reporter gene and chromatin immunoprecipitation (ChIP) assays revealed that SubAB reduced LPS-induced NF-κB p65/p50 heterodimer binding to an NF-κB binding site on the iNOS promoter. In contrast to the native toxin, a catalytically inactivated SubAB mutant slightly enhanced LPS-induced iNOS expression and binding of NF-κB subunits to the iNOS promoter. The SubAB effect on LPS-induced iNOS expression was significantly reduced in macrophages from NF-κB1 (p50)-deficient mice, which lacked a DNA-binding subunit of the p65/p50 heterodimer, suggesting that p50 was involved in SubAB-mediated inhibition of iNOS expression. Treatment of macrophages with an NOS inhibitor or expression of SubAB byE. coliincreasedE. colisurvival in macrophages, suggesting that NO generated by macrophages resulted in efficient killing of the bacteria and SubAB contributed toE. colisurvival in macrophages. Thus, we hypothesize that SubAB might represent a novel bacterial strategy to circumvent host defense during STEC infection.

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Subtilase Cytotoxin-Coding Genes in Verotoxin-Producing Escherichia coli Strains from Sheep and Goats Differ from Those from Cattle

Applied and Environmental Microbiology ◽

10.1128/aem.05604-11 ◽

2011 ◽

Vol 77 (23) ◽

pp. 8259-8264 ◽

Cited By ~ 11

Author(s):

José A. Orden ◽

Pilar Horcajo ◽

Ricardo de la Fuente ◽

José A. Ruiz-Santa-Quiteria ◽

Gustavo Domínguez-Bernal ◽

...

Keyword(s):

Escherichia Coli ◽

Human Disease ◽

Virulence Genes ◽

Small Ruminants ◽

Human Beings ◽

Content Type ◽

Sheep And Goats ◽

E Coli ◽

Healthy Cattle ◽

Subtilase Cytotoxin

ABSTRACTSubtilase cytotoxin (SubAB) from verotoxin (VT)-producingEscherichia coli(VTEC) strains was first described in the 98NK2 strain and has been associated with human disease. However, SubAB has recently been found in two VT-negativeE. colistrains (ED 591 and ED 32). SubAB is encoded by two closely linked, cotranscribed genes (subAandsubB). In this study, we investigated the presence ofsubABgenes in 52 VTEC strains isolated from cattle and 209 strains from small ruminants, using PCR. Most (91.9%) VTEC strains from sheep and goats and 25% of the strains from healthy cattle possessedsubABgenes. The presence ofsubABin a high percentage of the VTEC strains from small ruminants might increase the pathogenicity of these strains for human beings. Some differences in the results of PCRs and in the association with some virulence genes suggested the existence of different variants ofsubAB. We therefore sequenced thesubAgene in 12 strains and showed that thesubAgene in most of thesubAB-positive VTEC strains from cattle was almost identical (about 99%) to that in the 98NK2 strain, while thesubAgene in most of thesubAB-positive VTEC strains from small ruminants was almost identical to that in the ED 591 strain. We propose the termssubAB1to describe the SubAB-coding genes resembling that in the 98NK2 strain andsubAB2to describe those resembling that in the ED 591 strain.

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Transcription of the Subtilase Cytotoxin Gene subAB1 in Shiga Toxin-Producing Escherichia coli Is Dependent on hfq and hns

Applied and Environmental Microbiology ◽

10.1128/aem.01281-19 ◽

2019 ◽

Vol 85 (20) ◽

Cited By ~ 2

Author(s):

Laura Heinisch ◽

Katharina Zoric ◽

Maike Krause ◽

Herbert Schmidt

Keyword(s):

Gene Expression ◽

Escherichia Coli ◽

Shiga Toxin ◽

Promoter Activity ◽

Deletion Mutants ◽

Content Type ◽

E Coli ◽

Intensive Investigation ◽

Subtilase Cytotoxin

ABSTRACT Certain foodborne Shiga toxin-producing Escherichia coli (STEC) strains carry genes encoding the subtilase cytotoxin (SubAB). Although the mode of action of SubAB is under intensive investigation, information about the regulation of subAB gene expression is currently not available. In this study, we investigated the regulation of the chromosomal subAB1 gene in laboratory E. coli strain DH5α and STEC O113:H21 strain TS18/08 using a luciferase reporter gene assay. Special emphasis was given to the role of the global regulatory protein genes hfq and hns in subAB1 promoter activity. Subsequently, quantitative real-time PCR was performed to analyze the expression of Shiga toxin 2a (Stx2a), SubAB1, and cytolethal distending toxin V (Cdt-V) genes in STEC strain TS18/08 and its isogenic hfq and hns deletion mutants. The deletion of hfq led to a significant increase of up to 2-fold in subAB1 expression, especially in the late growth phase, in both strains. However, deletion of hns showed different effects on the promoter activity during the early and late exponential growth phases in both strains. Furthermore, upregulation of stx2a and cdt-V was demonstrated in hfq and hns deletion mutants in TS18/08. These data showed that the expression of subAB1, stx2a, and cdt-V is integrated in the regulatory network of global regulators Hfq and H-NS in Escherichia coli. IMPORTANCE Shiga toxin-producing Escherichia coli (STEC) strains are responsible for outbreaks of foodborne diseases, such as hemorrhagic colitis and the hemolytic uremic syndrome. The pathogenicity of those strains can be attributed to, among other factors, the production of toxins. Recently, the subtilase cytotoxin was detected in locus of enterocyte effacement (LEE)-negative STEC, and it was confirmed that it contributes to the cytotoxicity of those STEC strains. Although the mode of action of SubAB1 is under intensive investigation, the regulation of gene expression is currently not known. The global regulatory proteins H-NS and Hfq have impact on many cellular processes and have been described to regulate virulence factors as well. Here, we investigate the role of hns and hfq in expression of subAB1 as well as stx2a and cdt-V in an E. coli laboratory strain as well as in wild-type STEC strain TS18/08.

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Augmented inducible nitric oxide synthase expression and increased NO production reduce sepsis-induced lung injury and mortality in myeloperoxidase-null mice

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00450.2007 ◽

2008 ◽

Vol 295 (1) ◽

pp. L96-L103 ◽

Cited By ~ 50

Author(s):

Viktor Brovkovych ◽

Xiao-Pei Gao ◽

Evan Ong ◽

Svitlana Brovkovych ◽

Marie-Luise Brennan ◽

...

Keyword(s):

Nitric Oxide ◽

Nitric Oxide Synthase ◽

Inducible Nitric Oxide Synthase ◽

Bacterial Colonization ◽

Inos Expression ◽

No Production ◽

Bacterial Killing ◽

E Coli ◽

Oxide Synthase ◽

Inducible Nitric Oxide

The myeloperoxidase (MPO)-hydrogen peroxide-halide system is an efficient oxygen-dependent antimicrobial component of polymorphonuclear leukocyte (PMN)-mediated host defense. However, MPO deficiency results in few clinical consequences indicating the activation of compensatory mechanisms. Here, we determined possible mechanisms protecting the host using MPO−/−mice challenged with live gram-negative bacterium Escherichia coli. We observed that MPO−/−mice unexpectedly had improved survival compared with wild-type (WT) mice within 5–12 h after intraperitoneal E. coli challenge. Lungs of MPO−/−mice also demonstrated lower bacterial colonization and markedly attenuated increases in microvascular permeability and edema formation after E. coli challenge compared with WT. However, PMN sequestration in lungs of both groups was similar. Basal inducible nitric oxide synthase (iNOS) expression was significantly elevated in lungs and PMNs of MPO−/−mice, and NO production was increased two- to sixfold compared with WT. Nitrotyrosine levels doubled in lungs of WT mice within 1 h after E. coli challenge but did not change in MPO−/−mice. Inhibition of iNOS in MPO−/−mice significantly increased lung edema and reduced their survival after E. coli challenge, but iNOS inhibitor had the opposite effect in WT mice. Thus augmented iNOS expression and NO production in MPO−/−mice compensate for the lack of HOCl-mediated bacterial killing, and the absence of MPO-derived oxidants mitigates E. coli sepsis-induced lung inflammation and injury.

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Single oral inoculation with Escherichia coli (ATCC 25922) stimulates generalised production of nitric oxide in mice

Acta Veterinaria Hungarica ◽

10.1556/avet.57.2009.1.13 ◽

2009 ◽

Vol 57 (1) ◽

pp. 127-138 ◽

Cited By ~ 3

Author(s):

Ana Nemec ◽

Zlatko Pavlica ◽

David Crossley ◽

Irena Zdovc ◽

Damijan Eržen ◽

...

Keyword(s):

Nitric Oxide ◽

Escherichia Coli ◽

Spin Trap ◽

Oral Route ◽

No Production ◽

Paramagnetic Resonance ◽

E Coli ◽

Oral Inoculation ◽

Nos Inhibitors ◽

Oxide Synthase

Nitric oxide (NO) production was investigated in the lungs, thoracic aorta, heart, liver, spleen, kidneys and brain of mice inoculated orally withEscherichia coliATCC 25922. Detection of NO was performed by electron paramagnetic resonance (EPR) using diethyldithiocarbamate (DETC) spin trap. Nitric oxide synthase (NOS) inhibitors [nonselective: L-NAME and inducible NOS (iNOS) selective: 1400W] were used to determine the source of NO. Spin-trap only and untreated mice were included as controls. Within 2.5 hours (h) of a single oral inoculation withE. colihalf of the animals had increased NO levels in all investigated organs. Thereafter the signals dropped before increasing again to reach maximal median values by 25 h in all organs of all inoculated mice. The most intense response occurred in livers, followed by aorta and lungs. Early (2.5 h) inhibition of the signal was achieved using both NOS inhibitors. L-NAME was also effective at 25 h, while 1400W-treated mice had increased NO levels beyond 7 h. The generalised increase in NO production in the short and longer term indicates a host response toE. coliadministered by the oral route of infection.

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Epithelial Invasion by Escherichia coli Bearing Dr Fimbriae Is Controlled by Nitric Oxide-Regulated Expression of CD55

Infection and Immunity ◽

10.1128/iai.72.5.2907-2914.2004 ◽

2004 ◽

Vol 72 (5) ◽

pp. 2907-2914 ◽

Cited By ~ 14

Author(s):

Li Fang ◽

Bogdan J. Nowicki ◽

Petri Urvil ◽

Pawel Goluszko ◽

Stella Nowicki ◽

...

Keyword(s):

Nitric Oxide ◽

Escherichia Coli ◽

Epithelial Cell Line ◽

No Production ◽

Dependent Manner ◽

E Coli ◽

Regulated Expression ◽

Ishikawa Cells ◽

Epithelial Invasion

ABSTRACT We previously reported that inhibition of nitric oxide (NO) increases the rate of bacteremia and maternal mortality in pregnant rats with uterine infection by Escherichia coli expressing the Dr fimbria (Dr+). Epithelial binding and invasion by Dr+ E. coli has also been shown to be dependent upon the expression level of the cellular receptor decay-accelerating factor (DAF; CD55). Here, we hypothesize that NO-related severity of infection could be mediated by changes in DAF expression and in the rate of epithelial invasion. The cellular basis of NO effects on epithelial invasion with Dr+ E. coli was studied using Ishikawa endometrial carcinoma cells as an in vitro model of the human endometrial epithelium. Initially, we show that Ishikawa cells produce NO and express both NO synthase enzymes, NOS II and NOS III, and DAF protein. We next tested the abilities of both Dr+ E. coli and a Dr− E. coli mutant to invade Ishikawa cells, and invasion was seen only with Dr+ E. coli. Invasion by Dr+ E. coli was decreased by elevated NO production and increased by NO inhibition. Elevated NO production significantly decreased DAF protein and mRNA expression in Ishikawa cells in a time- and dose-dependent manner. Here, we propose that in vitro invasion of an epithelial cell line is directly related to NO-regulated expression of DAF. The significance of NO-regulated receptor-ligand invasion is that it may represent a novel unrecognized phenomenon of epithelial defense against infection.

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Monocyte Chemoattractant Protein 1 Regulates Pulmonary Host Defense via Neutrophil Recruitment during Escherichia coli Infection

Infection and Immunity ◽

10.1128/iai.00067-11 ◽

2011 ◽

Vol 79 (7) ◽

pp. 2567-2577 ◽

Cited By ~ 59

Author(s):

Gayathriy Balamayooran ◽

Sanjay Batra ◽

Theivanthiran Balamayooran ◽

Shanshan Cai ◽

Samithamby Jeyaseelan

Keyword(s):

Escherichia Coli ◽

Host Defense ◽

Neutrophil Recruitment ◽

Bacterial Clearance ◽

Content Type ◽

Monocyte Chemoattractant Protein 1 ◽

Neutrophil Influx ◽

E Coli ◽

Monocyte Chemoattractant ◽

Monocyte Chemoattractant Protein

ABSTRACTNeutrophil accumulation is a critical event to clear bacteria. Since uncontrolled neutrophil recruitment can cause severe lung damage, understanding neutrophil trafficking mechanisms is important to attenuate neutrophil-mediated damage. While monocyte chemoattractant protein 1 (MCP-1) is known to be a monocyte chemoattractant, its role in pulmonary neutrophil-mediated host defense against Gram-negative bacterial infection is not understood. We hypothesized that MCP-1/chemokine (C-C motif) ligand 2 is important for neutrophil-mediated host defense. Reduced bacterial clearance in the lungs was observed in MCP-1−/−mice followingEscherichia coliinfection. Neutrophil influx, along with cytokines/chemokines, leukotriene B4(LTB4), and vascular cell adhesion molecule 1 levels in the lungs, was reduced in MCP-1−/−mice after infection.E. coli-induced activation of NF-κB and mitogen-activated protein kinases in the lung was also reduced in MCP-1−/−mice. Administration of intratracheal recombinant MCP-1 (rMCP-1) to MCP-1−/−mice induced pulmonary neutrophil influx and cytokine/chemokine responses in the presence or absence ofE. coliinfection. Ourin vitromigration experiment demonstrates MCP-1-mediated neutrophil chemotaxis. Notably, chemokine receptor 2 is expressed on lung and blood neutrophils, which are increased uponE. coliinfection. Furthermore, our findings show that neutrophil depletion impairsE. coliclearance and that exogenous rMCP-1 after infection improves bacterial clearance in the lungs. Overall, these new findings demonstrate thatE. coli-induced MCP-1 causes neutrophil recruitment directly via chemotaxis as well as indirectly via modulation of keratinocyte cell-derived chemokine, macrophage inflammatory protein 2, and LTB4.

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The Nitrite Transporter Facilitates Biofilm Formation via Suppression of Nitrite Reductase and Is a New Antibiofilm Target in Pseudomonas aeruginosa

mBio ◽

10.1128/mbio.00878-20 ◽

2020 ◽

Vol 11 (4) ◽

Author(s):

Ji-Su Park ◽

Ha-Young Choi ◽

Won-Gon Kim

Keyword(s):

Nitric Oxide ◽

Escherichia Coli ◽

Pseudomonas Aeruginosa ◽

Nitrite Reductase ◽

Biofilm Formation ◽

No Production ◽

Content Type ◽

A Genome ◽

Biofilm Dispersal ◽

Gmp Production

ABSTRACT Biofilm-forming bacteria, including the Gram-negative Pseudomonas aeruginosa, cause multiple types of chronic infections and are responsible for serious health burdens in humans, animals, and plants. Nitric oxide (NO) has been shown to induce biofilm dispersal via triggering a reduction in cyclic-di-GMP levels in a variety of bacteria. However, how NO, at homeostatic levels, also facilitates biofilm formation is unknown. Here, we found that complestatin, a structural analog of vancomycin isolated from Streptomyces, inhibits P. aeruginosa biofilm formation by upregulating NO production via nitrite reductase (NIR) induction and c-di-GMP degradation via phosphodiesterase (PDE) stimulation. The complestatin protein target was identified as a nitrite transporter from a genome-wide screen using the Keio Escherichia coli knockout library and confirmed using nitrite transporter knockout and overexpression strains. We demonstrated that the nitrite transporter stimulated biofilm formation by controlled NO production via appropriate NIR suppression and subsequent diguanylate cyclase (DGC) activation, not PDE activity, and c-di-GMP production in E. coli and P. aeruginosa. Thus, this study provides a mechanism for NO-mediated biofilm formation, which was previously not understood. IMPORTANCE Bacterial biofilms play roles in infections and avoidance of host defense mechanisms of medically important pathogens and increase the antibiotic resistance of the bacteria. Nitric oxide (NO) is reported to be involved in both biofilm formation and dispersal, which are conflicting processes. The mechanism by which NO regulates biofilm dispersal is relatively understood, but there are no reports about how NO is involved in biofilm formation. Here, by investigating the mechanism by which complestatin inhibits biofilm formation, we describe a novel mechanism for governing biofilm formation in Escherichia coli and Pseudomonas aeruginosa. Nitrite transporter is required for biofilm formation via regulation of NO levels and subsequent c-di-GMP production. Additionally, the nitrite transporter contributes more to P. aeruginosa virulence than quorum sensing. Thus, this study identifies nitrite transporters as new antibiofilm targets for future practical and therapeutic agent development.

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Distinct Nitrite and Nitric Oxide Physiologies inEscherichia coliandShewanella oneidensis

Applied and Environmental Microbiology ◽

10.1128/aem.00559-18 ◽

2018 ◽

Vol 84 (12) ◽

pp. e00559-18 ◽

Cited By ~ 6

Author(s):

Qiu Meng ◽

Jianhua Yin ◽

Miao Jin ◽

Haichun Gao

Keyword(s):

Nitric Oxide ◽

Escherichia Coli ◽

Growth Inhibition ◽

Shewanella Oneidensis ◽

Food Preservation ◽

Content Type ◽

E Coli ◽

Cell Extracts ◽

Copper Oxidase

ABSTRACTNitrite has been used as a bacteriostatic agent for centuries in food preservation. It is widely accepted that this biologically inert molecule functions indirectly, serving as a stable reservoir of bioactive nitric oxide (NO) and other reactive nitrogen species to impact physiology. As a result, to date, we know surprisingly little aboutin vivotargets of nitrite. Here, we carry out comparative analyses of nitrite and NO physiology inEscherichia coliand inShewanella oneidensis, a Gram-negative environmental bacterium renowned for respiratory versatility. These two bacteria differ from each other in many aspects of nitrite and NO physiology, including NO generation, NO degradation, and unexpectedly, their contrary susceptibility to nitrite and NO. In cell extracts of both bacteria, most of the NO targets are also susceptible to nitrite, and vice versa. However, with respect to growth inhibition caused by NO, the targets are impacted distinctly; NO targets are responsible for the inhibition of growth ofE. colibut not ofS. oneidensis. More surprisingly, all proteins identified to be implicated in NO tolerance in other bacteria appear to play a dispensable role in protectingS. oneidensisagainst NO. These data suggest thatS. oneidensisis equipped with a robust but yet unknown NO protecting system. In the case of nitrite, it is clear that the target of physiological significance in both bacteria is cytochrome heme-copper oxidase.IMPORTANCENitrite is toxic to living organisms at high levels, but such antibacterial effects of nitrite are attributable to the formation of nitric oxide (NO), a highly reactive radical gas molecule. Here, we report thatShewanella oneidensisis highly resistant to NO but sensitive to nitrite compared toEscherichia coliby approximately 4-fold. In both bacteria, nitrite inhibits bacterial growth by targeting cytochrome heme-copper oxidase. In contrast, the targets of NO are diverse. Although these targets are similar inE. coliandS. oneidensis, they are responsible for growth inhibition caused by NO in the former but not in the latter. Overall, the presented data, along with the previous data, solidify a proposal that thein vivotargets of NO and nitrite in bacteria are largely different.

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JAK Inhibitors AG-490 and WHI-P154 Decrease IFN-γ-Induced iNOS Expression and NO Production in Macrophages

Mediators of Inflammation ◽

10.1155/mi/2006/16161 ◽

2006 ◽

Vol 2006 ◽

pp. 1-7 ◽

Cited By ~ 13

Author(s):

Outi Sareila ◽

Riku Korhonen ◽

Outi Kärpänniemi ◽

Riina Nieminen ◽

Hannu Kankaanranta ◽

...

Keyword(s):

Nitric Oxide ◽

Inos Expression ◽

Janus Kinase ◽

Inos Mrna ◽

No Production ◽

Dependent Manner ◽

Jak Inhibitors ◽

Concentration Dependent Manner ◽

Inflammatory Processes ◽

Nuclear Levels

In inflammation, inducible nitric oxide synthase (iNOS) produces nitric oxide (NO), which modulates inflammatory processes. We investigated the effects of Janus kinase (JAK) inhibitors, AG-490 and WHI-P154, on iNOS expression and NO production in J774 murine macrophages stimulated with interferon-γ(IFN-γ). JAK inhibitors AG-490 and WHI-P154 decreased IFN-γ-induced nuclear levels of signal transducer and activator of transcription 1α(STAT1α). JAK inhibitors AG-490 and WHI-P154 decreased also iNOS protein and mRNA expression and NO production in a concentration-dependent manner. Neither of the JAK inhibitors affected the decay of iNOS mRNA when determined by actinomycin D assay. Our results suggest that the inhibition of JAK-STAT1-pathway by AG-490 or WHI-P154 leads to the attenuation of iNOS expression and NO production in IFN-γ-stimulated macrophages.

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Exendin-4 inhibits interleukin-1β-induced iNOS expression at the protein level, but not at the transcriptional and posttranscriptional levels, in RINm5F β-cells

Journal of Endocrinology ◽

10.1677/joe-08-0507 ◽

2009 ◽

Vol 202 (1) ◽

pp. 65-75 ◽

Cited By ~ 17

Author(s):

Jung-Hoon Kang ◽

Seo-Yoon Chang ◽

Hyun-Jong Jang ◽

Dong-Bin Kim ◽

Gyeong Ryul Ryu ◽

...

Keyword(s):

Nitric Oxide ◽

Inos Expression ◽

Interleukin 1Β ◽

Inos Mrna ◽

Β Cells ◽

Β Cell ◽

Inos Protein ◽

Inos Gene ◽

Nitrite Production ◽

Inos Promoter

Cytokines such as interleukin-1β (IL-1β) stimulate inducible nitric oxide synthase (iNOS) expression and nitric oxide overproduction leading to β-cell damage. Meanwhile, glucagon-like peptide-1 (GLP-1) and its potent analog exendin-4 (EX-4) were well known for β-cell proliferation. However, the protective mechanisms of GLP-1 in β-cells exposed to cytokines were not fully elucidated. Therefore, the effects of EX-4 on the IL-1β-induced iNOS gene expression were investigated employing RINm5F β-cells. EX-4 inhibited IL-1β-induced iNOS protein expression and nitrite production. However, northern blot and promoter analyses showed that EX-4 failed to inhibit IL-1β-induced iNOS mRNA expression and iNOS promoter activity. By electrophoretic mobility shift assay (EMSA), EX-4 did not alter the binding activity of NF-κB to the iNOS promoter. Consistent with the EMSA result, EX-4 did not inhibit nuclear translocation of p65. We also tested the effect of EX-4 on iNOS mRNA stability. Actinomycin D chase experiments showed that EX-4 did not affect the decay rate of iNOS mRNA and the promoter assay using the construct containing 3′-untranslated region of iNOS showed that EX-4 did not alter the stability of iNOS mRNA. Meanwhile, forskolin significantly inhibited IL-1β-induced iNOS protein, which was reversed by H-89, a protein kinase A (PKA) inhibitor. Moreover, EX-4 pretreatment restored IL-1β-induced decrease in cAMP toward control level. Additionally, the cycloheximide chase study demonstrated that EX-4 significantly accelerated iNOS protein degradation. We therefore concluded that EX-4 inhibited IL-1β-induced iNOS protein and nitrite production via cAMP/PKA system irrespective of both transcriptional and posttranscriptional mechanisms of iNOS gene, and this inhibitory effect of EX-4 appears to be regulated at posttranslational level.

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