Cyclooxygenase-2 Expression In Macrophages Research Articles

Cyclooxygenase-2/prostaglandin E2 pathway regulates infectious bronchitis virus replication in avian macrophages.

Infectious bronchitis virus (IBV) is a significant respiratory pathogen that affects chickens worldwide. As an avian coronavirus, IBV leads to productive infection in chicken macrophages. However, the effects of IBV infection in macrophages on cyclooxygenase-2 (COX-2) expression are still to be elucidated. Therefore, we investigated the role of IBV infection on the production of COX-2, an enzyme involved in the synthesis of prostaglandin E2 (PGE2) in chicken macrophages. The chicken macrophage cells were infected with two IBV strains, and the cells and culture supernatants were harvested at predetermined time points to measure intracellular and extracellular IBV infection. IBV infection was quantified as has been the COX-2 and PGE2 productions. We found that IBV infection enhances COX-2 production at both mRNA and protein levels in chicken macrophages. When a selective COX-2 antagonist was used to reduce the COX-2 expression in macrophages, we observed that IBV replication decreased. When IBV-infected macrophages were treated with PGE2 receptor (EP2 and EP4) inhibitors, IBV replication was reduced. Upon utilizing a selective COX-2 antagonist to diminish PGE2 expression in macrophages, a discernible decrease in IBV replication was observed. Treatment of IBV-infected macrophages with a PGE2 receptor (EP2) inhibitor resulted in a reduction in IBV replication, whereas the introduction of exogenous PGE2 heightened viral replication. Additionally, pretreatment with a Janus-kinase two antagonist attenuated the inhibitory effect of recombinant chicken interferon (IFN)-γ on viral replication. The evaluation of immune mediators, such as inducible nitric oxide (NO) synthase (iNOS), NO, and interleukin (IL)-6, revealed enhanced expression following IBV infection of macrophages. In response to the inhibition of COX-2 and PGE2 receptors, we observed a reduction in the expressions of iNOS and IL-6 in macrophages, correlating with reduced IBV infection. Overall, IBV infection increased COX-2 and PGE2 production in addition to iNOS, NO, and IL-6 expression in chicken macrophages in a time-dependent manner. Inhibition of the COX-2/PGE2 pathway may lead to increased macrophage defence mechanisms against IBV infection, resulting in a reduction in viral replication and iNOS and IL-6 expressions. Understanding the molecular mechanisms underlying these processes may shed light on potential antiviral targets for controlling IBV infection.

Salmonella fimbrial protein StcD induces cyclooxygenase-2 expression via Toll-like receptor 4.

The genome of Salmonella enterica serovar Typhimurium contains 13 operons with homology to fimbrial genes. To investigate the involvement of these fimbrial gene clusters in the expression of cyclooxygenase-2 (COX-2), which is an inducible enzyme involved in the synthesis of prostanoids, in J774 macrophages infected with S. enterica serovar Typhimurium, we constructed strains carrying a mutation in genes encoding the putative subunit proteins in 12 fimbrial operons. The level of COX-2 expression was lower in macrophages infected with fimA or stcA mutant Salmonella than in those infected with wild-type Salmonella. Therefore, we focused on putative subunit protein StcA and adhesive like protein StcD encoded in the stc operon. Treatment of macrophages with purified recombinant StcD protein, but not StcA, resulted in the activation of the mitogen-activated protein kinase and nuclear factor kappa B signaling pathways, leading to the expression of not only COX-2 but also of pro-inflammatory cytokines such as interleukin (IL)-1β, IL-6, and tumor necrosis factor alpha. The expression of StcD-induced COX-2 was inhibited by treatment with the Toll-like receptor 4 (TLR4) inhibitor TAK-242, but not by treatment with the lipopolysaccharide (LPS) antagonist polymyxin B. Furthermore, StcD treatment stimulated HEK293cells expressing TLR4 in the presence of CD14 and MD-2. StcD is a pathogen-associated molecular pattern of S. enterica serovar Typhimurium that is recognized by TLR4 and plays a significant role in the induction of COX-2 expression in macrophages.

Chemical Constituents of Ulmus minor subsp. minor Fruits Used in the Italian Phytoalimurgic Tradition and Their Anti-inflammatory Activity Evaluation.

The phytochemical investigation of Ulmus minor subsp. minor samaras EtOAc and n-BuOH extracts is reported in this work for the first time, resulting in the isolation and characterization of twenty compounds (1: - 20: ) including one new flavan-3-ol (1: ), one new trihydroxy fatty acid (2: ), and two glycosylated flavonoids (6: - 7: ) whose NMR data are not available in the literature. Structure elucidation of the isolated compounds was obtained by 1D and 2D NMR and HRESIMS data. Prior to further pharmacological investigations, the extracts (100 - 6.25 µg/mL) and compounds 1: - 12: (50 - 5 µM) were tested for their influence on viability of a murine macrophage cell line (J774A.1). Subsequently, extracts and compounds that did not impede viability, were studied for their inhibitory effect on some mediators of inflammation in J774A.1 cells stimulated with lipopolysaccharide of Escherichia coli (LPS). The NO release and the expression of iNOS and COX-2 were then evaluated and both extracts (50 - 6.25 µg/mL) and compounds (20 - 5 µM) significantly inhibited NO release as well as iNOS and COX-2 expression in macrophages. These data highlight the anti-inflammatory properties of several isolated compounds from U. minor samaras supporting their possible alimentary use.

Nicotinamide Mononucleotide Alleviates LPS-Induced Inflammation and Oxidative Stress via Decreasing COX-2 Expression in Macrophages.

Macrophage activation is an important process in controlling infection, but persistent macrophage activation leads to chronic inflammation and diseases, such as tumor progression, insulin resistance and atherosclerosis. Characterizing metabolic signatures of macrophage activation is important for developing new approaches for macrophage inactivation. Herein, we performed metabolomic analysis on lipopolysaccharide (LPS)-activated macrophages and identified the associated changes in metabolites. Notably, the cellular Nicotinamide adenine dinucleotide+ levels were decreased while NADPH was increased, proposing that NAD+ restoration can inhibit macrophage activation. Indeed, supplementation of nicotinamide mononucleotide (NMN) increased cellular NAD+ levels and decreased cytokine productions in LPS-activated cells. Quantitative proteomics identified that nicotinamide mononucleotide downregulated the expressions of LPS-responsive proteins, in which cyclooxygenase-2 (COX-2) expression was significantly decreased in NMN-treated cells. Consequently, the cellular levels of prostaglandin E2 (PGE2) was also decreased, indicating that NMN inactivated macrophages via COX-2-PGE2 pathway, which was validated in activated THP-1 cells and mouse peritoneal macrophages. In conclusion, the present study identified the metabolic characteristics of activated macrophages and revealed that NMN replenishment is an efficient approach for controlling macrophage activation.

LncRNA-MAP3K4 regulates vascular inflammation through the p38 MAPK signaling pathway and cis-modulation of MAP3K4.

Chronic vascular inflammation plays a key role in the pathogenesis of atherosclerosis. Long non-coding RNAs (lncRNAs) have emerged as essential inflammation regulators. We identify a novel lncRNA termed lncRNA-MAP3K4 that is enriched in the vessel wall and regulates vascular inflammation. In the aortic intima, lncRNA-MAP3K4 expression was reduced by 50% during the progression of atherosclerosis (chronic inflammation) and 70% during endotoxemia (acute inflammation). lncRNA-MAP3K4 knockdown reduced the expression of key inflammatory factors (eg, ICAM-1, E-selectin, MCP-1) in endothelial cells or vascular smooth muscle cells and decreased monocytes adhesion to endothelium, as well as reducing TNF-α, IL-1β, COX2 expression in macrophages. Mechanistically, lncRNA-MAP3K4 regulates inflammation through the p38 MAPK signaling pathway. lncRNA-MAP3K4 shares a bidirectional promoter with MAP3K4, an upstream regulator of the MAPK signaling pathway, and regulates its transcription in cis. lncRNA-MAP3K4 and MAP3K4 show coordinated expression in response to inflammation in vivo and in vitro. Similar to lncRNA-MAP3K4, MAP3K4 knockdown reduced the expression of inflammatory factors in several different vascular cells. Furthermore, lncRNA-MAP3K4 and MAP3K4 knockdown showed cooperativity in reducing inflammation in endothelial cells. Collectively, these findings unveil the role of a novel lncRNA in vascular inflammation by cis-regulating MAP3K4 via a p38 MAPK pathway.

12-HETE is a regulator of PGE2 production via COX-2 expression induced by a snake venom group IIA phospholipase A2 in isolated peritoneal macrophages

The snake venom miotoxin (MT)-III is a group IIA secreted phospholipase A2 (sPLA2) with pro-inflammatory activities. Previous studies have demonstrated that MT-III has the ability to stimulate macrophages to release inflammatory lipid mediators derived from arachidonic acid metabolism. Among them, we highlight prostaglandin (PG)E2 produced by the cyclooxygenase (COX)-2 pathway, through activation of nuclear factor (NF)-κB. However, the mechanisms coordinating this process are not fully understood. This study investigates the regulatory mechanisms exerted by other groups of bioactive eicosanoids derived from 12-lipoxygenase (12-LO), in particular 12-hydroxyeicosatetraenoic (12-HETE), on group IIA sPLA2-induced (i) PGE2 release, (ii) COX-2 expression, and (iii) activation of signaling pathways p38 mitogen-activated protein kinases(p38MAPK), protein C kinase (PKC), extracellular signal-regulated kinase 1/2 (ERK1/2), and NF-κB. Stimulation of macrophages with group IIA sPLA2 resulted in release of 12-HETE without modification of 12-LO protein levels. Pre-treatment of these cells with baicalein, a 12-LO inhibitor, decreased the sPLA2-induced PGE2 production, significantly reduced COX-2 expression, and inhibited sPLA2-induced ERK; however, it did not affect p38MAPK or PKC phosphorylation. In turn, sPLA2-induced PGE2 release and COX-2 expression, but not NF-κB activation, was attenuated by pre-treating macrophages with PD98059 an inhibitor of ERK1/2. These results suggest that, in macrophages, group IIA sPLA2-induced PGE2 release and COX-2 protein expression are distinctly mediated through 12-HETE followed by ERK1/2 pathway activation, independently of NF-κB activation. These findings highlight an as yet undescribed mechanism by which 12-HETE regulates one of the distinct signaling pathways for snake venom group IIA sPLA2-induced PGE2 release and COX-2 expression in macrophages.

Regulatory T cells protected against abdominal aortic aneurysm by suppression of the COX-2 expression.

CD4+CD25+ regulatory T cells (Tregs) have been shown to protect against the development of abdominal aortic aneurysm (AAA). Cyclooxygenase‐2 (COX‐2), a pro‐inflammatory protein, can convert arachidonic acid into prostaglandins (PGs). The present study was aimed to investigate the effect of Tregs on COX‐2 expression in angiotension II (Ang II)‐induced AAA in ApoE−/− mice. Tregs were injected via tail vein in every 2 weeks. Ang II was continuously infused by a micropump for 28 days to induce AAA. In vivo, compared with the control group, adoptive transfer of Tregs significantly reduced the incidence of AAA, maximal diameter, and the mRNA and protein expression of COX‐2 in mice. Immunofluorescence showed that Tregs treatment reduced COX‐2 expression both in smooth muscle cells (SMCs) and macrophages in AAA. In vitro, the Western blot analysis showed that Tregs reduced Ang II‐induced COX‐2 expression in macrophages and SMCs. Meanwhile, ELISA showed that Tregs reduced Ang II‐induced prostaglandin E2 (PGE2) secretion. Moreover, Tregs increased SMC viability and induced transition of macrophages phenotype from M1 to M2. In conclusion, Tregs treatment dramatically decreased the expression of COX‐2 in vivo and in vitro, suggesting that Tregs could protect against AAA through inhibition of COX‐2. The study may shed light on the immune treatment of AAA.

Paracrine cyclooxygenase-2 activity by macrophages drives colorectal adenoma progression in the ApcMin/+ mouse model of intestinal tumorigenesis

Genetic deletion or pharmacological inhibition of cyclooxygenase (COX)-2 abrogates intestinal adenoma development at early stages of colorectal carcinogenesis. COX-2 is localised to stromal cells (predominantly macrophages) in human and mouse intestinal adenomas. Therefore, we tested the hypothesis that paracrine Cox-2-mediated signalling from macrophages drives adenoma growth and progression in vivo in the ApcMin/+ mouse model of intestinal tumorigenesis. Using a transgenic C57Bl/6 mouse model of Cox-2 over-expression driven by the chicken lysozyme locus (cLys-Cox-2), which directs integration site-independent, copy number-dependent transgene expression restricted to macrophages, we demonstrated that stromal macrophage Cox-2 in colorectal (but not small intestinal) adenomas from cLys-Cox-2 x ApcMin/+ mice was associated with significantly increased tumour size (P = 0.025) and multiplicity (P = 0.025), compared with control ApcMin/+ mice. Transgenic macrophage Cox-2 expression was associated with increased dysplasia, epithelial cell Cox-2 expression and submucosal tumour invasion, as well as increased nuclear β-catenin translocation in dysplastic epithelial cells. In vitro studies confirmed that paracrine macrophage Cox-2 signalling drives catenin-related transcription in intestinal epithelial cells. Paracrine macrophage Cox-2 activity drives growth and progression of ApcMin/+ mouse colonic adenomas, linked to increased epithelial cell β-catenin dysregulation. Stromal cell (macrophage) gene regulation and signalling represent valid targets for chemoprevention of colorectal cancer.

Butyrate attenuates lipolysis in adipocytes co-cultured with macrophages through non-prostaglandin E2-mediated and prostaglandin E2-mediated pathways.

BackgroundInteractions between adipocytes and macrophages are associated with metabolic disorders. Production of pro-inflammatory mediators and the release of free fatty acids (FFAs) increase when these cells are co-cultured; butyrate significantly diminishes these effects by suppressing both the macrophage inflammatory and adipocyte lipolysis pathways. Butyrate is known to up-regulate the expression of prostaglandin E2 (PGE2). Therefore, we hypothesized that PGE2 is associated with the suppression of lipolysis by butyrate in co-culture.MethodsUsing contact or transwell co-culture methods with differentiated 3T3-L1 adipocytes and RAW264.7 macrophages, we investigated the effects of butyrate on the release of PGE2 into the medium and on lipolysis in adipocytes. To elucidate the underlying mechanism, we examined the effects of butyrate on cyclooxygenase-2 (COX2) and phospholipase A2 (PLA2) in co-cultured cells, and cyclic adenine monophosphate (cAMP) and protein kinase A type 1-α regulatory subunit (PRKAR1A) in co-cultured adipocytes. Silent interfering (si)RNA targeting of G-protein–coupled receptor (GPR)41 and 109A was employed to examine the effect on lipolysis in TNF-α–stimulated adipocytes.ResultsCo-culture increased PGE2 release into the medium, compared with cells cultured separately. Butyrate significantly increased PGE2 production. Co-culture elevated COX2 expression in macrophages and adipocytes, and butyrate further enhanced this effect. Co-culture enhanced cytosolic PLA2 activity in macrophages, which was further enhanced by butyrate. As for lipolysis, co-culture increased the release of FFAs and free glycerol into the medium, whereas butyrate (and to a lesser extent, PGE2) suppressed FFAs and free glycerol release. An inhibition study using a prostaglandin E receptor 3–selective antagonist suggested that approximately 40% of the suppressive effect of butyrate depends on the PGE2-mediated pathway, whereas 60% depends on a non-PGE2–mediated pathway. Co-culture increased cAMP and PRKAR1A levels in adipocytes, whereas butyrate restored the levels to those of the control. Similarly, in TNF-α–stimulated adipocytes, butyrate reduced FFAs and free glycerol release. siRNA inhibition of GPR41 and GPR109A suggested that the GPR109A-mediated pathway predominates, but the GPR41-mediated pathway also regulates the effect of butyrate on lipolysis in TNF-α–stimulated 3T3-L1 cells.ConclusionsButyrate attenuates lipolysis in adipocytes co-cultured with macrophages via non-PGE2–mediated and PGE2-mediated pathways.

Perinatal Endotoxemia Induces Sustained Hepatic COX-2 Expression through an NFκB-Dependent Mechanism

Background: Exposure to perinatal infection is associated with the multiple morbidities complicating preterm birth. How a relatively immature innate immune response contributes to this is unknown. Objective: We sought to determine if the perinatal innate immune response to endotoxemia induces a unique pattern of cyclooxygenase-2 (COX-2) expression via an NFκB-dependent mechanism. Methods: Hepatic and pulmonary COX-2 mRNA expression was assessed following perinatal (at embryonic days 15 and 19 and after birth) or adult endotoxemia. Hepatic NFκB activity was assessed by cytosolic inhibitory protein degradation and subunit nuclear translocation. Immunohistochemistry and isolated cell preparations determined hepatic macrophage COX-2 expression, and the effect of pharmacologic and genetic inhibition of NFκB activity was tested. Results: Perinatal endotoxemia induced sustained hepatic macrophage COX-2 expression and NFκB activity compared to in exposed adults. Isolated hepatic macrophages and immunohistochemistry demonstrated enriched LPS-induced COX-2 expression that was sensitive to pharmacologic and genetic approaches to attenuate NFκB activity. Finally, pharmacologic inhibition of endotoxemia-induced NFκB activity in neonatal mice prevented hepatic NFκB activity and attenuated COX-2 expression. Conclusion: Our findings of sustained neonatal hepatic NFκB activity and COX-2 expression in response to endotoxemia support a robust perinatal innate immune response. This may represent a link between the innate immune response and the pathogenesis of diseases associated with preterm birth.

5-methoxytryptophan is a circulating anti-inflammatory molecule that controls lipopolysaccharide-induced systemic inflammation and sepsis (IRM11P.622)

Abstract We identified in the conditioned medium of human fibroblasts a tryptophan metabolite, 5-methoxytryptophan (5-MTP) which controls cyclooxgenase-2 expression and inhibits NF-κB binding and p300 activity. We postulated that as with fibroblasts, human endothelial cells produce 5-MTP which plays an important role in controlling systemic inflammatory responses. Human endothelial cells produced and released 5-MTP into the conditioned medium. Lipopolysaccharide (LPS) suppressed 5-MTP production in endothelial cells and induced 5-MTP deficiency in the murine sepsis model. Intraperitoneal infusion of 5-MTP restored serum 5-MTP accompanied by inhibition of systemic pro-inflammatory cytokines, chemokines, and pro-inflammatory mediators. 5-MTP administration rescued organ failure and prevented mortality. The beneficial effect of 5-MTP was correlated with inhibition of cytokine production and COX-2 expression in macrophages. 5-MTP inhibited LPS-induced p38 MAPK, p300 HAT and NF-κB activation in macrophages and endotoxemic lung tissues in vivo. Importantly, a considerable amount of 5-MTP was detected in 30 healthy subjects (1.05 ± 0.39 μM) which was significantly reduced in 50 sepsis patients (0.37 ± 0.15 μM, p<0.0001). 5-MTP is a novel circulating anti-inflammatory molecule which protects against excessive systemic inflammatory responses. It ameliorates LPS-induced sepsis and is a valuable lead compound for developing new drugs to treat sepsis and systemic inflammatory disorders.

NSAID use reduces breast cancer recurrence in overweight and obese women: role of prostaglandin-aromatase interactions.

Obesity is associated with a worse breast cancer prognosis and elevated levels of inflammation, including greater cyclooxygenase-2 (COX-2) expression and activity in adipose-infiltrating macrophages. The product of this enzyme, the proinflammatory eicosanoid prostaglandin E2 (PGE2), stimulates adipose tissue aromatase expression and subsequent estrogen production, which could promote breast cancer progression. This study demonstrates that daily use of a nonsteroidal anti-inflammatory drug (NSAID), which inhibits COX-2 activity, is associated with reduced estrogen receptor α (ERα)-positive breast cancer recurrence in obese and overweight women. Retrospective review of data from ERα-positive patients with an average body mass index of >30 revealed that NSAID users had a 52% lower recurrence rate and a 28-month delay in time to recurrence. To examine the mechanisms that may be mediating this effect, we conducted in vitro studies that utilized sera from obese and normal-weight patients with breast cancer. Exposure to sera from obese patients stimulated greater macrophage COX-2 expression and PGE2 production. This was correlated with enhanced preadipocyte aromatase expression following incubation in conditioned media (CM) collected from the obese-patient, sera-exposed macrophages, an effect neutralized by COX-2 inhibition with celecoxib. In addition, CM from macrophage/preadipocyte cocultures exposed to sera from obese patients stimulated greater breast cancer cell ERα activity, proliferation, and migration compared with sera from normal-weight patients, and these differences were eliminated or reduced by the addition of an aromatase inhibitor during CM generation. Prospective studies designed to examine the clinical benefit of NSAID use in obese patients with breast cancer are warranted.

Vitamin D Inhibits COX-2 Expression and Inflammatory Response by Targeting Thioesterase Superfamily Member 4

Inadequate vitamin D status has been linked to increased risk of type 2 diabetes and cardiovascular disease. Inducible cyclooxygenase (COX) isoform COX-2 has been involved in the pathogenesis of such chronic inflammatory diseases. We found that the active form of vitamin D, 1,25(OH)2D produces dose-dependent inhibition of COX-2 expression in murine macrophages under both basal and LPS-stimulated conditions and suppresses proinflammatory mediators induced by LPS. Administration of 1,25(OH)2D significantly alleviated local inflammation in a carrageenan-induced paw edema mouse model. Strikingly, the phosphorylation of both Akt and its downstream target IκBα in macrophages were markedly suppressed by 1,25(OH)2D in the presence and absence of LPS stimulation through up-regulation of THEM4 (thioesterase superfamily member 4), an Akt modulator protein. Knockdown of both vitamin D receptor and THEM4 attenuated the inhibitory effect of 1,25(OH)2D on COX-2 expression in macrophages. A functional vitamin D-responsive element in the THEM4 promoter was identified by chromatin immunoprecipitation and luciferase reporter assay. Our results indicate that vitamin D restrains macrophage-mediated inflammatory processes by suppressing the Akt/NF-κB/COX-2 pathway, suggesting that vitamin D supplementation might be utilized for adjunctive therapy for inflammatory disease.

Lack of RNase L attenuates macrophage functions.

BackgroundMacrophages are one of the major cell types in innate immunity against microbial infection. It is believed that the expression of proinflammatory genes such as tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL–6, and cyclooxygenase-2 (Cox-2) by macrophages is also crucial for activation of both innate and adaptive immunities. RNase L is an interferon (IFN) inducible enzyme which is highly expressed in macrophages. It has been demonstrated that RNase L regulates the expression of certain inflammatory genes. However, its role in macrophage function is largely unknown.MethodologyBone marrow-derived macrophages (BMMs) were generated from RNase L+/+and −/− mice. The migration of BMMs was analyzed by using Transwell migration assays. Endocytosis and phagocytosis of macrophages were assessed by using fluorescein isothiocyanate (FITC)-Dextran 40,000 and FITC-E. coli bacteria, respectively. The expression of inflammatory genes was determined by Western Blot and ELISA. The promoter activity of Cox-2 was measured by luciferase reporter assays.Conclusions/FindingsLack of RNase L significantly decreased the migration of BMMs induced by M-CSF, but at a less extent by GM-CSF and chemokine C-C motif ligand-2 (CCL2). Interestingly, RNase L deficient BMMs showed a significant reduction of endocytic activity to FITC-Dextran 40,000, but no any obvious effect on their phagocytic activity to FITC-bacteria under the same condition. RNase L impacts the expression of certain genes related to cell migration and inflammation such as transforming growth factor (TGF)-β, IL-1β, IL-10, CCL2 and Cox-2. Furthermore, the functional analysis of the Cox-2 promoter revealed that RNase L regulated the expression of Cox-2 in macrophages at its transcriptional level. Taken together, our findings provide direct evidence showing that RNase L contributes to innate immunity through regulating macrophage functions.

Muramyl dipeptide potentiates staphylococcal lipoteichoic acid induction of cyclooxygenase-2 expression in macrophages

Gram-positive bacteria contain lipoteichoic acid (LTA) and peptidoglycan (PGN) layers, both of which are considered as major virulence factors associated with inflammation. Cyclooxygenase-2 (COX-2) plays an important role in the inflammation by generating prostaglandins at infections. Since LTA and PGN are thought to cooperate in the establishment of inflammation, we examined the ability of staphylococcal LTA (Sa.LTA) to induce COX-2 expression in the presence of muramyl dipeptide (MDP), which is the minimal structural unit of PGN required for inflammation, in macrophages. While MDP failed to induce COX-2 expression, Sa.LTA alone was sufficient to induce COX-2 production. Treatment with MDP enhanced Sa.LTA-induced COX-2 and prostaglandin E2 production. The cooperative effect between Sa.LTA and MDP was not observed in COX-2 expression by macrophages derived from Toll-like receptor 2 (TLR2)- or nucleotide-binding oligomerization domain 2 (NOD2)-deficient mice. In addition, MDP enhanced Sa.LTA-induced activation of the transcription factors NF-κB and CRE, which are known to modulate COX-2 gene transcription. Conclusively, these results suggest that MDP and Sa.LTA cooperatively induce inflammatory response by overproducing COX-2 through NOD2 and TLR2.

NOD2-Mediated Innate Immune Signaling Regulates the Eicosanoids in Atherosclerosis

The activity of eicosanoid pathways is critical to the inflammatory and immune responses that are associated with the progression of atherosclerosis. Yet, the signals that regulate these pathways are poorly understood. Here, we address whether the innate immune signals of nucleotide-binding oligomerization domain-containing protein (NOD) 2 affect eicosanoids metabolism in atherosclerosis. Analysis of human carotid plaques revealed that NOD2 was abundantly expressed at both mRNA and protein levels by endothelial cells and macrophages. Stimulation of NOD2 in ex vivo-cultured carotid plaques by muramyl dipeptide, an extrinsic ligand of NOD2, led to release of prostaglandin E2, upregulation of cyclooxygenase-2 and microsomal prostaglandin E synthase-1, and to downregulation of cyclooxygenase-1. NOD2 was coexpressed with cyclooxygenase-2 in lesional macrophages. NOD2-induced cyclooxygenase-2 expression in macrophages was dependent on p38 mitogen-activated protein kinase activation and was mediated by interleukin-1β and tumor necrosis factor-α. Selective lipidomic analysis of the eicosanoids released by the carotid plaques characterized the metabolites of 12-, 5-, and 15-lipoxygenase as the predominant eicosanoids that were produced by the atherosclerotic lesion in the absence of additional stimuli. Unlike the prostaglandin E2 pathway, metabolic activity of the lipoxygenase pathways was not altered on the short-term activation of NOD2 in carotid plaques. These results suggest that atherosclerosis may involve enhanced NOD2-mediated innate immunity. Activation of NOD2 preferentially upregulates the prostaglandin E2 pathway. Nevertheless, lipoxygenase pathways, such as 12-lipoxygenase, predominate the basal synthesis and metabolism of eicosanoids in atherosclerotic plaques. These findings provide new insights into the regulation of eicosanoids in atherosclerosis.

Osteopontin signaling upregulates cyclooxygenase-2 expression in tumor-associated macrophages leading to enhanced angiogenesis and melanoma growth via α9β1 integrin

Tumor-associated macrophages (TAMs) have multifaceted roles in tumor development, particularly linked with tumor angiogenesis and invasion, but the molecular mechanism underlying this association remains unclear. In this study, we report that lack of osteopontin (OPN) suppresses melanoma growth in opn(-/-) mice and macrophages are the crucial component responsible for OPN-regulated melanoma growth. In tumor microenvironment, OPN activates macrophages and influences angiogenesis by enhancing cyclooxygenase-2 (COX-2)-dependent prostaglandin E2 (PGE2) production in an autocrine manner. Furthermore, we identify α9β1 integrin as a functional receptor for OPN that mediates its effect and activates ERK and p38 signaling, which ultimately leads to COX-2 expression in macrophages. The major role played by OPN and PGE2 in angiogenesis are further amplified by upregulation of MMP-9. OPN-activated macrophages promote the migration of endothelial and cancer cells via PGE2. These findings provide evidence that TAMs serve as source of key components such as OPN and COX-2-derived PGE2 and MMP-9 in melanoma microenvironment. Clinical specimens analyses revealed that increased infiltration of OPN-positive TAMs correlate with melanoma growth and angiogenesis. These data provide compelling evidence that OPN and COX-2 expressing macrophages are obligatory factors in melanoma growth. We conclude that OPN signaling is involved in macrophage recruitment into tumor, and our results emphasize the potential role of macrophage in modulation of tumor microenvironment via secretion of OPN, PGE2 and MMP-9, which trigger angiogenesis and melanoma growth. Thus, blockade of OPN and its regulated signaling network provides unique strategy to eradicate melanoma by manipulating TAMs.

Homocysteine induces COX-2 expression in macrophages through ROS generated by NMDA receptor-calcium signaling pathways

Homocysteine (Hcy) at elevated levels is a putative risk factor for many cardiovascular disorders including atherosclerosis. In the present study, we investigated the effect of Hcy on the expression of cyclooxygenase (COX)-2 in murine macrophages and the mechanisms involved. Hcy increased the expression of COX-2 mRNA and protein in dose- and time-dependent manners, but did not affect COX-1 expression. Hcy-induced COX-2 expression was attenuated not only by the calcium chelators, EGTA and BAPTA-AM, but also by an antioxidant, N-acetylcysteine. Calcium chelators also attenuated Hcy-induced reactive oxygen species (ROS) production in macrophages, indicating that Hcy-induced COX-2 expression might be mediated through ROS generated by calcium-dependent signaling pathways. In another series of experiments, Hcy increased the intracellular concentration of calcium in a dose-dependent manner, which was attenuated by MK-801, an N-methyl-D-aspartate (NMDA) receptor inhibitor, but not by bicuculline, a gamma-aminobutyric acid receptor inhibitor. Molecular inhibition of NMDA receptor using small interfering RNA also attenuated Hcy-induced increases in intracellular calcium. Furthermore, both ROS production and Hcy-induced COX-2 expression were also inhibited by MK-801 as well as by molecular inhibition of NMDA receptor. Taken together, these findings suggest that Hcy enhances COX-2 expression in murine macrophages by ROS generated via NMDA receptor-mediated calcium signaling pathways.

Inhibitory effect of dihydroartemisinin against phorbol ester-induced cyclooxygenase-2 expression in macrophages

Dihydroartemisinin (DHA), a semi-synthetic derivative of artemisinin isolated from the traditional Chinese herb Artemisia annua L., has recently been shown to possess antitumor activity in various cancer cells. However, the effect of anti-inflammatory potentials of DHA in murine macrophage RAW 264.7 cells has not been studied. The present study investigated the effect of COX-2 and molecular mechanisms by DHA in PMA stimulated RAW 264.7 cells. DHA dose-dependently decreased PMA-induced COX-2 expression and PGE2 production, as well as COX-2 promoter-driven luciferase activity. Additionally, DHA decreased luciferase activity of COX-2 regulation-related transcription factors including NF-κB, AP-1, C/EBP and CREB. DHA also remarkably reduced PMA-induced p65, C/EBPβ, c-jun and CREB nuclear translocation. Furthermore, DHA evidently inhibited PMA-induced phosphorylation of AKT and the MAP Kinases, such as ERK, JNK and p38. Taken together, our data indicated that DHA effectively attenuates COX-2 production via down-regulation of AKT and MAPK pathway, revealing partial molecular basis for the anti-inflammatory properties of DHA.

A catalytically-inactive snake venom Lys49 phospholipase A2 homolog induces expression of cyclooxygenase-2 and production of prostaglandins through selected signaling pathways in macrophages

The effects of a snake venom Lys-49 phospholipase A2 (PLA2) homolog named MT-II, devoid of enzymatic activity, on the biosynthesis of prostaglandins and protein expression of cyclooxygenase-2 (COX-2) and signaling pathways involved were evaluated in mouse macrophages in culture and in peritoneal cells ex vivo. Stimulation of macrophages with MT-II leads to production of prostaglandin D2 (PGD2) and prostaglandin E2 (PGE2) and protein expression of COX-2 and microsomal prostaglandin E synthase-1 (mPGES-1). Inhibition of cytosolic PLA2 (cPLA2), but not Ca2+ independent PLA2 (iPLA2) reduced release of PGD2 and PGE2 and expression of COX-2 induced by MT-II. Inhibition of nuclear factor κB (NF-κB) significantly reduced MT-II-induced PGE2, but not PGD2 production and COX-2 expression. Inhibitors of either protein kinase C (PKC), protein tyrosine kinase (PTK), or extracellular signal-regulated kinase (ERK) pathways abrogated MT-II-induced NF-κB activation and reduced COX-2 expression and PGE2 release, whereas the p38 mitogen-activated protein kinase (MAPK) inhibitor reduced MT-II-induced COX-2 expression and PGD2 production. Inhibition of phosphatidylinositol-3-kinase (PI3K) pathway abrogated MT-II-induced NF-κB activation, but affected neither prostaglandins production nor COX-2 expression. MT-II-induced production of PGD2 and PGE2 and COX-2 expression were also observed in vivo after intraperitoneal injection into mice. Collectively, our data demonstrate that a catalytically-inactive PLA2 homolog is capable of inducing prostaglandins biosynthesis and COX-2 expression in macrophages in both in vitro and in vivo models, indicating that the enzymatic activity of PLA2 is not necessary to trigger these effects. MT-II-activated NF-κB, cPLA2 and distinct protein kinases are the principal steps involved in these cellular events.