Cyclooxygenase-2 Transcriptional Activity Research Articles

Withdrawal: Histone deacetylase inhibitors suppress the induction of c-Jun and its target genes including COX-2.

Cyclooxygenase-2 (COX-2) is considered to be a target for anticancer therapy. Histone deacetylase (HDAC) inhibitors exhibit antitumor activity, but the mechanisms of action are incompletely understood. We investigated whether HDAC inhibitors blocked AP-1-mediated activation of COX-2 transcription. Trichostatin A and suberoylanilide hydroxamic acid, two structurally related inhibitors of HDAC activity, blocked AP-1-mediated induction of COX-2 expression and prostaglandin E2 biosynthesis. Chromatin immunoprecipitation assays indicated that HDAC inhibitors suppressed c-Jun binding to the COX-2 promoter and thereby blocked transcription. The observed reduction in binding reflected reduced levels of c-Jun. HDAC inhibitors suppressed the induction of c-jun transcription by blocking the recruitment of the preinitiation complex (RNA polymerase II and TFIIB) to the c-jun promoter. HDAC3 but not HDAC1 or HDAC2 was required for AP-1-mediated stimulation of c-jun expression. Because HDAC inhibitors suppressed the induction of c-jun gene expression, resulting in reduced COX-2 transcription, it was important to determine whether other known AP-1 target genes were also modulated. Cyclin D1 and collagenase-1 are AP-1-dependent genes that have been implicated in carcinogenesis. HDAC inhibitors suppressed the induction of both cyclin D1 and collagenase-1 transcription by inhibiting the binding of c-Jun to the respective promoters. Taken together, these results suggest that HDAC inhibitors block the induction of c-jun transcription by inhibiting the recruitment of the preinitiation complex to the c-jun promoter. This led, in turn, to reduced expression of several activator protein-1-dependent genes (COX-2, cyclin D1, collagenase-1). These findings provide new insights into the mechanisms underlying the antitumor activity of HDAC inhibitors.

Effect of quercetin on doxorubicin-induced expression of MDR1 gene in HL-60 cells

Leukemia cells can acquire a multidrug resistant (MDR) phenotype in response to a wide variety of chemotherapeutic agents including doxorubicin (Dox). In addition to the constitutive expression in the leukemia prior to chemotherapy, a complex phenotype of pleiotropic resistance is presented in the residual or recurrent leukemia. Recent studies showed Dox-induced coexpression of COX2 and MDR1 genes in human leukaemia cells, and whether Dox-induced MDR1 up-regulation in acute leukaemia cells is dependent on COX2-transcriptional activity and thus might be overcome or prevented with COX2-promotor inhibitor quercetin interfering with COX2 expression and activity. This study was purposed to investigate the impacts of quercetin on Dox-induced mRNA expression of MDR1 and COX2 genes in HL-60 leukemia cells. The MDR1 and COX2 mRNA expression in HL-60 cells was detected by RT-PCR; the prostaglandin E2 (PGE2) release was measured by ELISA; the cytotoxicity of Dox was determined by MTT test. The incubation of HL-60 cells with Dox not only up-regulated MDR1 mRNA, but also COX2 mRNA expression, and after co-incubation with quercetin or celecoxib, Dox-induced overexpression of MDR1 and COX2 mRNA were reduced by quercetin, not by celecoxib, whereas PGE2 release was significantly decreased with subsequent enhancement of Dox cytotoxic efficacy by both of them. Dox-induced MDR1 up-regulation may be dependent on COX2-transcriptional activity, not PGE2, suggesting that the existence of causal link between COX2 and MDR1 expression induced by Dox, and modulation of COX2 transcriptional expression by quercetin would not only sensitize leukemia cells to Dox, but also prevent the acquisition of MDR during chemotherapy.

Involvement of NADPH oxidases in suppression of cyclooxygenase-2 promoter-dependent transcriptional activities by sesamol.

Cyclooxygenase-2 (COX-2) has been shown to play an important role in colon carcinogenesis. Moreover, one of the components of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, NADPH oxidase 1 (NOX1), dominantly expressed in the colon, is implicated in the pathogenesis of colon cancer. We have reported that sesamol, one of the lignans in sesame seeds, suppressed COX-2 gene transcriptional activity in human colon cancer cells, and also suppressed intestinal polyp formation in Apc-mutant mice. In the present study, we investigated the involvement of NADPH oxidase in the inhibition of COX-2 transcriptional activity by sesamol. We found that several NADPH oxidase inhibitors, such as apocynin, showed suppressive effects on COX-2 transcriptional activity. Moreover, sesamol significantly suppressed NOX1 mRNA levels in a dose-dependent manner. In addition, we demonstrated that knockdown of NOX1 successfully suppressed COX-2 transcriptional activity. These results suggest that inhibition of NADPH oxidase, especially NOX1, may be involved in the mechanism of the suppression of COX-2 transcriptional activity by sesamol.

Proliferating fibroblasts suppress cancer cell epithelial‐to‐mesenchymal transition (EMT) and migration via control inhibition of p300 HAT activation by 5‐methoxytryptophan (LB492)

We recently identified 5‐methoxytryptophan (5‐MTP), a novel tryptophan metabolite, as an endogenous inhibitor of cyclooxygenase‐2 (COX‐2) expression. Our reported data show that cancer cells are defective in 5‐MTP production and exogenous 5‐MTP suppresses cancer cell COX‐2 overexpression. Furthermore, 5‐MTP inhibits cancer cell migration and invasion. However, it is unclear whether endogenous 5‐MTP controls cancer cell COX‐2 and COX‐2 mediated cancer growth and metastasis. Since human proliferative fibroblasts (pFb) produce robust 5‐MTP, we determined whether pFb controls cancer cell COX‐2 expression and behavior. pFb co‐cultured with A549 lung cancer cells in a two‐chamber system inhibited PMA‐induced COX‐2 expression and migration in a time‐ and cell number‐dependent manner. pFb blocked A549 epithelial mesenchymal transition (EMT) induced by transforming growth factor‐β (TGFβ). Hydroxyindole O‐methyltransferase (HIOMT) catalyzes the final step of 5‐MTP synthesis. pFb expressed abundant HIOMT proteins. Knockdown of HIOMT in pFb resulted in abrogation of control of A549 COX‐2 expression, migration and EMT in the two‐chamber co‐culture system. These results are consistent with control of A549 COX‐2 expression and EMT/migration by 5‐MTP. We next determined whether 5‐MTP targets p300 histone acetyltransferase (HAT) which is essential for COX‐2 transcriptional activation. Basal p300 HAT activity in A549 was stimulated by proinflammatory cytokines and phorbol esters, which was inhibited by 5‐MTP. 5‐MTP inhibited TGFβ. Co‐cultured with pFb suppressed A549 p300 HAT, TGFβ and COX‐2 which were abrogated by treating pFb with HIOMT siRNA. These findings suggest that fibroblasts control cancer cell COX‐2 expression via inhibition of p300 HAT and TGFβ activation which results in suppression of cancer cell EMT and migration. Fibroblast produced 5‐MTP may confer endogenous resistance to cancer growth and metastasis.

Highlights of This Issue

EGFR-mutations and ALK rearrangements could co-exist in non-small cell lung cancer (NSCLC). To explore the predictive biomarkers in EGFR/ALK co-altered tumors, Yang and colleagues, investigated molecular alterations of EGFR and ALK in a 977-case Chinese cohort and correlated phospho-EGFR and phospho-ALK with responses to EGFR-TKIs and crizotinib. Tumors with such co-alterations could have diverse responses to first-line EGFR TKIs, which were associated with phospho-EGFR levels. Elevated phospho-ALK levels correlated the efficacy of subsequent crizotinib treatment. This study showed the potential utility of receptors phosphorylation status to predict the treatment by EGFR-TKI or crizotinib in EGFR/ALK co-altered NSCLC.Glioblastomas are highly fatal brain tumors that are extremely radioresistant. This study identifies NVP-BEZ235 (a dual PI3K/mTOR inhibitor) as a potent inhibitor of key DNA repair enzymes, DNA-PKcs and ATM, in vivo. Treatment with this drug blocks the repair of radiation-induced DNA damage in tumors, resulting in remarkable radiosensitization and prolonging survival of brain-tumor bearing mice. Based on the results presented, blocking DNA repair with potent and bioavailable inhibitors of DNA-PKcs and ATM appears to be a feasible option for improving GBM therapy in the future.Gong and colleagues demonstrate an important activity of the natural compound, Nexrutine on inflammation-associated carcinogenesis and inhibition of fibrosis. The study elaborates on the noteworthy signaling axis among STAT3, NFκB, COX-2 and EP4. The authors show that STAT3 negatively regulates NFκB activation. Suppression of this crosstalk inhibits COX-2 transcriptional activity and subdues prostaglandins. The immediate outcome of this study can influence current modalities for pancreatic cancer treatment through the combinatorial use of Nexrutine as a dual inhibitor of NFκB and STAT3. It also has clinical implications for other inflammation and fibrosis-associated cancers.Relapse of myeloma after autologous stem cell transplantation (ASCT) is frequent. Posttransplant immunotherapy using adoptive T-cell transfers and tumor antigen vaccines may improve clinical responses if a high frequency of tumor antigen-directed immune responses can be generated. Rapoport and colleagues show that adoptive transfer of costimulated autologous T cells which are primed and boosted by a MAGE-A3 Trojan peptide vaccine combined with the TLR-3 agonist and novel vaccine adjuvant Poly-ICLC induces vaccine-directed cellular immune responses in 76% of patients after ASCT. A high frequency of vaccine-directed antibody responses also occurred but only in the presence of montanide.

Sesamol suppresses cyclooxygenase-2 transcriptional activity in colon cancer cells and modifies intestinal polyp development in ApcMin/+ mice

Excessive prostaglandin production by cyclooxygenase-2 in stromal and epithelial cells is a causative factor of colorectal carcinogenesis. Thus, compounds which inhibit cyclooxygenase-2 transcriptional activity in colon epithelial cells could be candidates for anti-carcinogenic agents. A cyclooxygenase-2 transcriptional activity in the human colon cancer cell line DLD-1 has been measured using a β-galactosidase reporter gene system. Using this system, we demonstrated that the decrease in basal cyclooxygenase-2 transcriptional activities at 100 µM sesamol, one of the lignans in sesame seeds, was 50%. Other compounds in sesame seeds such as sesamin, sesamolin, ferulic acid, and syringic acid did not exhibit significant suppression of cyclooxygenase-2 transcriptional activity at up to 100 µM. In a following experiment, 6-week-old male Min mice, Apc-deficient mice, were divided into a non-treated and 500 ppm sesamol groups. At the age of 15 weeks, it was found that treatment with sesamol decreased the number of polyps in the middle part of small intestine to 66.1% of the untreated value. Moreover, sesamol suppressed cyclooxygenase-2 and cytosolic prostaglandin E2 synthase mRNA in the polyp parts. The present findings may demonstrate the novel anti-carcinogenetic property of sesamol, and imply that agents that can suppress cyclooxygenase-2 expression may be useful cancer chemopreventive agents.

5-methoxyindole metabolites of L-tryptophan: control of COX-2 expression, inflammation and tumorigenesis

Cyclooxygenase-2(COX-2) overexpression promotes inflammation and tumorigenesis. COX-2 expression in response to diverse stimuli is tightly controlled to avoid persistent overexpression. 5-methoxyindole metabolites of L-tryptophan represent a new class of compounds that control COX-2 expression at the transcriptional level. Two of the metabolites, the newly discovered 5-methoxytryptophan (5-MTP, also known as cytoguardin) and N-acetyl 5-methoxytryptamine (melatonin) are the focus of this review. 5-MTP is produced by mesenchymal cells such as fibroblasts via 5-hydroxytryptophan (5-HTP). It inhibits COX-2 transcriptional activation induced by diverse proinflammatory and mitogenic factors. Cancer cells are deficient in cytoguardin production which contributes to COX-2 overexpression. Fibroblast-generated 5-MTP is capable of restoring the control of COX-2 overexpression in cancer cells. 5-MTP blocks cancer cell migration and invasion in vitro and inhibits tumor growth and cancer metastasis in a xenograft model. Melatonin possesses similar COX-2 suppressing and anti-cancer properties albeit at supra-pharmacological concentrations. By contrast, 5-hydroxyindole metabolites of L-tryptophan such as 5-hydroxytryptamine (serotonin), 5-hydroxytryptophol and other serotonin catabolites do not control COX-2 expression. 5-hydroxytryptophan inhibits COX-2 expression through conversion to 5-MTP. The physiological relevance of 5-MTP as an endogenous regulator of inflammation and cancer metastasis remains to be investigated. On the other hand, 5-methoxyindole metabolites of tryptophan are valuable lead compounds for development of new anti-inflammatory drugs and cancer chemoprevention.

Involvement of PGE2 and the cAMP signalling pathway in the up-regulation of COX-2 and mPGES-1 expression in LPS-activated macrophages

PG (prostaglandin) E2 plays an important role in the modulation of the immune response and the inflammatory process. In the present study, we describe a PGE2 positive feedback for COX (cyclo-oxygenase)-2 and mPGES-1 [microsomal PGES (PGE synthase)-1] expression in the macrophage cell line RAW 264.7. Our results show that PGE2 induces COX-2 and mPGES-1 expression, an effect mimicked by dbcAMP (dibutyryl-cAMP) or forskolin. Furthermore, the cAMP signalling pathway co-operates with LPS (lipopolysaccharide) in the induction of COX-2 and mPGES-1 transcriptional activation. Analysis of the involvement of PGE receptors [EPs (E-prostanoids)] showed that incubation with EP2 agonists up-regulated both COX2 and mPGES-1 mRNA levels. Moreover, EP2 receptor overexpression enhanced the transcriptional activation of COX2 and mPGES-1 promoters. This induction was repressed by the PKA (protein kinase A) inhibitor H89. Activation of the PGE2/EP2/PKA signalling pathway induced the phosphorylation of CREB [CRE (cAMP-response element)-binding protein] in macrophages and stimulated the specific binding of this transcription factor to COX2 and mPGES-1 promoters. Deletion or mutation of potential CRE sites in both promoters diminished their transcriptional activity. In summary, the results of the present study demonstrate that activation of PKA/CREB signalling through the EP2 receptor by PGE2 plays a key role in the expression of COX-2 and mPGES-1 in activated macrophages.

Transient receptor potential vanilloid-1 participates in the inhibitory effect of ginsenoside Rg1 on capsaicin-induced interleukin-8 and prostaglandin E2 production in HaCaT cells

Ginsenoside Rg1 (GRg1), one of the major active constituents of Panax notoginseng, has shown anti-inflammatory and antinocioceptic activity, but its role in keratinocytes needs further study. We have examined the inhibitory effect of GRg1 on transient receptor potential vanilloid-1 (TRPV1) activation in keratinocyte HaCaT cells and explored its involved mechanism. HEK 293T cells over-expressing exogenous TRPV1 were constructed and named HEK 293T-TRPV1 cells. The effects of GRg1 on production of interleukin-8 (IL-8) and prostaglandin E(2) (PGE(2) ), calcium influx, the expression of cyclooxygenase-2 (COX-2) and nuclear factor-κB (NF-κB) transcriptional activity in HEK 293T-TRPV1 and HaCaT cells were examined by ELISA, Fluo 3-AM fluorescence probe, Western blot and Dual-Luciferase Reporter Assay, respectively. The results showed that GRg1 blocked intracellular calcium by both capsaicin and proton activation in a TRPV1-dependent manner. Furthermore, GRg1 inhibited the expression of COX-2 and NF-κB transcriptional activity induced by capsaicin in keratinocytes. The inhibitory effect of GRg1 was similar to capsazepine, an antagonist of TRPV1. More importantly, GRg1 dose-dependently inhibited capsaicin-induced PGE(2) and IL-8 secretion in HaCaT cells and HEK 293T-TRPV1 cells. These data showed that GRg1 could inhibit TRPV1 mediated responses in HaCaT cells, indicating that GRg1 acted as a TRPV1 antagonist.

Angiotensin II differentially modulates cyclooxygenase-2, microsomal prostaglandin E2 synthase-1 and prostaglandin I2 synthase expression in adventitial fibroblasts exposed to inflammatory stimuli

To assess whether angiotensin II (Ang II) modulates key enzymes of the cyclooxygenase (COX)-2/prostanoid pathway, including prostaglandin E synthase-1 (mPGES-1) and prostacyclin synthase (PGIS) in rat aortic adventitial fibroblasts in the presence or absence of an inflammatory stimulus [interleukin (IL)-1β]. Fibroblasts stimulated with IL-1β (10 ng/ml, 24 h) and/or Ang II (0.1 μmol/l, 24 h) were used. IL-1β up-regulated COX-2 and mPGES-1 (protein and mRNA) and increased PGI2 and PGE2 release, without altering PGIS protein expression. Ang II did modify neither COX-2 and mPGES-1 expression nor prostanoid levels, but it induced PGIS expression. Interestingly, Ang II further enhanced IL-1β-induced COX-2 expression and PGI2 release and concomitantly reduced IL-1β-induced mPGES-1 expression. The AT1 receptor antagonist losartan prevented the effects of Ang II on IL-1β-induced COX-2 or mPGES-1 expression. IL-1β activated p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK)1/2 pathways, and coincubation with Ang II resulted in a higher and more sustained phosphorylation of both MAPK. Inhibition of either p38 MAPK (SB203580) or ERK1/2 (PD98059) reduced COX-2 and mPGES-1 expression in cells treated with IL-1β or the combination of IL-1β and Ang II. Ang II did not modify COX-2 transcriptional activity but increased COX-2 mRNA stability in IL-1β-treated cells; by contrast, it increased PGIS mRNA levels through a transcriptional mechanism. Ang II differentially modulates key enzymes involved in prostanoid biosynthesis thereby altering the balance between PGI2/PGE2 in vascular cells exposed to inflammatory stimuli.

Tumor necrosis factor-alpha - mediated 2-hydroxyethyl methacrylate cytotoxic and inflammatory effect on human gingival fibroblasts

2-Hydroxyethyl methacrylate (HEMA), deriving from polymerized dental resinous biomaterials, can diffuse throughout the dentin organic matrix, preventing collagen collapse, but also at gingival and tooth pulp level [1]. HEMA could induce toxic effects, such as tissue inflammation, also at relatively low concentrations. Our study aimed to investigate the cytotoxic and inflammatory effect exerted on human gingival fibroblasts (HGFs) by a low HEMA concentration evaluating cell viability by Trypan blue dye exclusion test, early apoptosis and reactive oxygen species (ROS) production by flow cytometry and gene expression of specific proteins involved in the inflammatory process, such as tumor necrosis factor-alpha (TNF-α) and cyclooxygenase-2 (COX-2), by real-time reverse transcription polymerase chain reaction (real-time RT PCR). Cultured HGFs, obtained from fragments of gingival tissue, were exposed to 3 mM HEMA in Dulbecco’s modified Eagle’s medium for 0, 24 or 96 hours. In our experimental model, both 24- and 96-hour HEMA treatment decreased cell viability of about 20%. In parallel Annexin-V/PI assay, which detects apoptosis, indicated a 18% of Annexin-V positive cells after 24- and 96-hour HEMA incubation. After 24-hour HEMA treatment we observed an increase of ROS persisting up to 96 hours. Interestingly, 24-hour HEMA treatment increased TNF-α gene expression of about 80% and COX-2 mRNA levels of about 70% compared to control. After 96-hour HEMA incubation, TNF-α gene expression was about sixfold and COX-2 mRNA levels were about fivefold compared to control. Increase of TNF-α and COX-2 gene expression was hence HEMA exposure time-dependent. Since TNF-α - induced inflammation has been shown to be mediated by the activation of COX-2 transcription in HGFs [2], we can hypothesize that, in our experimental model, 24- or 96-hour HEMA treatment in HGFs induces a ROS-mediated cytotoxicity and an inflammatory process modulated by increase of TNF-α gene expression, which could rapidly produce the observed up-regulation of COX-2 transcription. Thus, the knowledge of molecular mechanisms underlying cellular response to dental resinous biomaterials, identifying threshold over which these compounds become toxic, could allow to set up protocols for a more effective clinical practice and for a better performance of tested materials. [1] Schweikl H, Spagnuolo G, Schmalz G. J Dent Res 2006; 85: 870-7. [2] Nakao S, Ogata Y, Shimizu E, Yamazaki M, Furuyama S, Sugiya H. Mol Cell Biochem 2002; 238: 11-8.

Butein downregulates phorbol 12-myristate 13-acetate-induced COX-2 transcriptional activity in cancerous and non-cancerous breast cells

Butein is a flavonoid isolated from the bark of Rhus verniciflua Stokes and the flowers of Butea monosperma, and is known to be a potential therapeutic drug for treating inflammation and cancer. Cyclooxygenase (COX) converts arachidonic acid to prostanoids, and increased expression of its isoform COX-2 has been observed in breast cancer tissues. It has been suggested that COX inhibitors can be used as chemopreventive agents against breast carcinogenesis. This study examined the potential suppressive effect of the flavonoid on phorbol 12-myristate 13-acetate (PMA)-induced COX-2 expression in the non-tumorigenic MCF-10A and cancerous MCF-7 breast cells. Immunoblot and mRNA analyses revealed that butein at or below 10 μM significantly inhibited PMA-induced COX-2 expression in these breast cells. The blocking of the PKC signaling pathway appeared to be the underlying mechanism. Butein treatment reduced the amount of phospho-mitogen activated protein kinase (MAPK) ERK-1/2, and the total activity of PKC. Activated ERKs might trigger the transcriptional activation of COX-2. Reporter gene assays as well as electrophoretic mobility shift assays (EMSA) illustrated that butein inhibited transcription of this gene. This study showed that butein down-regulated PMA-induced COX-2 expression in both cancerous and non-cancerous breast cells, and such findings could provide the basis for pharmaceutical development of butein.

The dietary flavonoid apigenin blocks phorbol 12-myristate 13-acetate-induced COX-2 transcriptional activity in breast cell lines

Cyclooxygenase (COX)-2 is the inducible isozyme catalyzing the conversion of arachidonic acid to prostanoids. Its expression has been linked to the process of carcinogenesis, including tumorigenesis of the breast. Apigenin (APG) is a flavone commonly found in fruit and vegetables, and is shown to be a potential modulator in inflammatory diseases. This study examined the potential suppressive effect of the flavone on phorbol ester-induced COX-2 expression in the breast cell lines MCF-10A and MCF-7. Real-time PCR and/or Western blotting indicated that APG in micromolar range significantly inhibited phorbol 12-myristate 13-acetate (PMA)-induced COX-2 expression in these breast cells. APG treatment reduced the amount of phospho-mitogen-activated protein kinase (MAPK) ERK-1/2, but it did not alter the activity of PKC. Activated ERKs might trigger the transactivation of AP-1 or CRE, which can be located at COX-2 promoter region (−72/−53). Reporter gene assay as well as electrophoretic mobility shift assays (EMSA) illustrated that APG inhibited transcription factor binding at this region in a dose-dependent manner. This study showed that APG down-regulated PMA-induced COX-2 expression in breast cells without affecting PKC activity. These findings could provide a scientific basis for developing APG nutraceutical against breast carcinogenesis.

Cancer Chemopreventive Activity of Maslinic Acid: Suppression of COX-2 Expression and Inhibition of NF-κB and AP-1 Activation in Raji Cells

Chronic inflammation is one of the predisposing factors for neoplastic transformation. Targeting inflammation through suppression of the pro-inflammatory pathway by dietary phytochemicals provides an important strategy for cancer prevention. Maslinic acid is a novel natural triterpenoid known to inhibit proliferation and induce apoptosis in some tumor cell lines. Although maslinic acid has cytotoxic and pro-apoptotic effects on cancer cells, the underlying mechanisms of its effects on the inflammatory pathway have yet to be elucidated. It has been reported that abnormal expression of pro-inflammatory enzyme cyclooxygenase-2 (COX-2) causes promotion of cellular proliferation, suppression of apoptosis, enhancement of angiogenesis and invasiveness. In the present study, the suppressive effect of maslinic acid on COX-2 expression and the binding activity of upstream transcription factors NF- κB and AP-1, which are known to regulate COX-2 transcriptional activation, were assessed using Raji cells. The anti-inflammatory action of maslinic acid was benchmarked against oleanolic acid and other standard drugs. Western blot analysis and electrophoretic mobility shift assay (EMSA) were employed to analyze COX-2 expression as well as NF- κB and AP-1 binding activity. Our results showed that maslinic acid suppresses COX-2 expression in a concentration-dependent manner. Likewise, the constitutive nuclear NF- κB (p65) activity as well as phorbol 12-myristate 13-acetate (PMA)- and sodium N-butyrate (SnB)-induced AP-1 binding activity in Raji cells were significantly reduced following treatment with maslinic acid. Since maslinic acid suppresses COX-2 expression in Raji cells at concentrations that also lowered the NF- κB (p65) and AP-1 binding activity, it is possible that the suppression of COX-2 by this natural triterpenoid might be achieved, at least in part, via the NF- κB and AP-1 signaling pathways.

P38 MAPK contributes to angiotensin II-induced COX-2 expression in aortic fibroblasts from normotensive and hypertensive rats

To investigate the effect of angiotensin II on cyclooxygenase-2 (COX-2) expression in aortic adventitial fibroblasts from normotensive [Wistar-Kyoto (WKY)] rats and spontaneously hypertensive rats (SHRs). Protein expression was determined by western blot, mRNA levels by real-time PCR, transcriptional activity by luciferase assays, superoxide anion (O2*-) production by dihydroethidine fluorescence and prostaglandin E2 by enzyme immunoassay. Angiotensin II (0.1 micromol/l, 0.5-6 h) time dependently induced COX-2 protein expression, this effect being transient in fibroblasts from WKY rats and maintained over time in SHRs. Angiotensin II effect was abolished by valsartan (1 micromol/l), an angiotensin II type 1 receptor antagonist. Angiotensin II-induced prostaglandin E2 production was reduced by valsartan and the COX-2 inhibitor NS398 (1 micromol/l). Angiotensin II increased O2*- production more in SHR than WKY rats. This increase was reduced by apocynin (30 micromol/l) and allopurinol (10 micromol/l), respective nicotinamide adenine dinucleotide phosphate (NADPH) and xanthine oxidase inhibitors. However, angiotensin II-induced COX-2 expression was unaffected by apocynin, allopurinol, tempol (1 mmol/l) or catalase (1000 U/ml). Angiotensin II (2-30 min) induced p38 mitogen-activated protein kinase (MAPK) phosphorylation, transiently in WKY rats but sustained in SHRs. The p38 inhibitor SB203580 (10 micromol/l) reduced angiotensin II-induced COX-2 protein and mRNA levels. The angiotensin II effect was not prevented by inhibition of mRNA synthesis, and angiotensin II was unable to modulate COX-2 transcriptional activity. Angiotensin II increases COX-2 expression in aortic fibroblasts through mechanisms including p38 MAPK pathway, independent of reactive oxygen species production and nonmediated by COX-2 transcriptional activity modulation. The sustained angiotensin-induced p38 MAPK activation in SHR cells might be related to the maintained COX-2 expression in this strain.

Feedback regulation of cyclooxygenase-2 transcription ex vivo and in vivo

Cyclooxygenase-2 (COX-2) is a rate-limiting enzyme for prostaglandin biosynthesis. Its inducible expression is regulated by complex pathways. To monitor Cox-2 transcriptional activity in vivo, we generated a knock-in mouse expressing a firefly luciferase reporter. In this study we examined, by comparing luciferase activity of Cox-2 luc/+ and Cox-2 luc/− cells and mice, effects of prostanoid products on Cox-2 promoter transcriptional activation. In peritoneal macrophages, luciferase induction by LPS in Cox-2 luc/− cells was less than that of Cox-2 luc/+ cells. However, in the presence of PGE 2, induction was comparable, suggesting positive Cox-2 feedback regulation by PGE 2 occurs for macrophages. In contrast, feedback modulation was not observed in TPA-induced Cox-2 luc/+ and Cox-2 luc/− mouse embryonic fibroblasts (MEFs). Using non-invasive in vivo imaging, we observed negative feedback regulation of Cox-2 expression during paw inflammation in living mice. Our results suggest Cox-2 expression is regulated by cell type specific feedback mechanisms, both in cultured cells and in living animals.

Induction of COX-2 in human airway cells by manganese: Role of PI3K/PKB, p38 MAPK, PKCs, Src, and glutathione depletion

The exposure of manganese is believed to be the risk of respiratory diseases. COX-2 is a protein involved in biosynthesis of inflammatory prostaglandins. Evidence suggests that COX-2 involves in the pathogenesis of lung inflammation. In this study, the effect of manganese-chloride (manganese) on COX-2 expression in A549 human lung epithelial cells was investigated. Treatment with manganese induced COX-2 at both protein and mRNA levels that were due to COX-2 transcriptional activation. Interestingly, manganese treatment led to activation of ERKs, p38 MAPK, JNKs, ATF-2, and PKB, but not NF-kappaB, and also cellular GSH depletion in A549 cells. Importantly, the manganese-induced COX-2 expression was suppressed by treatment with the inhibitor of p38 MAPK (SB203580), PI3K/PKB (LY294002), PKCs (GO6983, GF109203X, Rottlerin), Src (PP1), or the thiol-containing compound (NAC). There was crosstalk between p38 MAPK and GSH depletion or Src in response to manganese signal. Induction of COX-2 by manganese was also seen in different human airway cells, including H292 (bronchial) or Hep2 (laryngeal). These results collectively suggest that manganese induces COX-2 by transcriptional up-regulation in human airway cells and the induction appears to be cooperatively mediated via multiple signaling pathways and GSH depletion.

HIV-2 induces NF-κB activation and cyclooxygenase-2 expression in human astroglial cells

HIV-2 invades CNS and causes neurological disease as well as HIV-1 does. Induction of COX-2 in CNS of HIV-1 infected people has been proposed as a cause of cognitive impairment, so we tested whether HIV-2 may cause damage by a similar mechanism. COX-2 mRNA and protein expression were induced in human astrocytes upon interaction with HIV-2, being this induction abrogated by CXCR4 antagonists. HIV-2 induced COX-2 promoter transcription and deletion of the two NF-κB binding sites of the promoter abrogated this induction. Neither AP-1 nor NFAT seem to be involved in COX-2 transcriptional activation. Overexpression of IκBα completely abrogated COX-2 induction, and transfection of p65/relA NF-κB induced COX-2 transcription. Interestingly, HIV-2 activated NF-κB by inducing p65/relA transactivating activity through Ser536 phosphorylation. Moreover, the astrocyte activation induced by HIV-2 was abrogated by different COX inhibitors. In summary, HIV-2 induces COX-2 in human astrocytes depending on CXCR4 coreceptor.

ERM, an ETS Transcription Factor, Is Crucial for Folliculogenesis and Ovulation in Mice.

PEA3, ER81, and ERM are members of PEA3 subfamily of ETS transcription factors which are known to influence a host of biological processes. We previously showed that ERM, expressed in Sertoli cells, plays a crucial role in maintaining spermatogonial niche in the mouse testis. However, it is not yet known whether PEA3 family members are expressed in the ovary or play any role in ovarian functions. Here we show that ERM and PEA3 are expressed in mouse ovaries in specific spatiotemporal manners. While PEA3 is expressed in nuclei of granulosa cells prior to ovulation, ERM is expressed during follicular growth at steady-state levels. Both ERM and PEA3 mRNAs are also detected in cumulus-oocyte-complexes (COCs) and in denuded oocytes. Notably, PEA3 is highly expressed in the cumulus cells of ovulated COCs at 16 hr post-hCG. Cyclooxygenase-2 (COX-2), a rate-limiting enzyme for prostaglandin synthesis, is critical to oocyte maturation and ovulation. Since several putative ETS-binding sites (EBS) are present in the COX-2 promoter, we examined whether ERM influences COX-2 transcriptional activity. Indeed, we found that addition of ERM increases the transcriptional activity of the 3.2-kb mCOX-2 promoter by 2.5 fold in luciferase reporter assays. The results show that PEA3 and ERM are expressed in granulosa and cumulus cells during folliculogenesis and ovulation, suggesting they influence cellular events in the ovary by regulating downstream genes such as COX-2. In ERM deficient female mice, ovulation is severely impaired even after gonadotropin treatments to induce superovulation. Histological observations revealed that ovaries from wildtype female mice have follicles at various developmental stages with well-formed granulosa cell (GC) layers, while ERM-/- ovaries have smaller follicles with thin GC layers. Even at 16 hr post-hCG which is normally after ovulation, development of follicles in the ERM-/- ovary were halted at preovulatory stages. The results show that ERM, expressed in granulosa and cumulus cells, plays important roles in folliculogenesis and ovulation. We will further perform physiological and molecular evaluations of ERM-/- ovaries and identify ERM target genes in the ovary using gene expression profiling analysis. This research was supported by MOEHRD KRF-2006-312-C00642.

Proteinase-activated Receptor-2 Induces Cyclooxygenase-2 Expression through β-Catenin and Cyclic AMP-response Element-binding Protein

Chronic inflammation of mucosae is associated with an increased cancer risk. Tumorigenesis in these tissues is associated with the activity of some proteinases, cyclooxygenase-2 (COX-2), and beta-catenin. Serine proteinases participate in both inflammation and tumorigenesis through the activation of proteinase-activated receptor-2 (PAR(2)), which up-regulates COX-2 by an unknown mechanism. We sought to determine whether beta-catenin participated in PAR(2)-induced COX-2 expression and through what cellular mechanism. In A549 epithelial cells, we showed that PAR(2) activation increased COX-2 expression through the beta-catenin/T cell factor transcription pathway. This effect was dependent upon ERK1/2 MAPK, which inhibited the beta-catenin-regulating protein, glycogen synthase kinase-3beta, and induced the activity of the cAMP-response element-binding protein (CREB). Knockdown of CREB by small interfering RNA revealed that PAR(2)-induced beta-catenin transcriptional activity and COX-2 expression were CREB-dependent. A co-immunoprecipitation assay revealed a physical interaction between CREB and beta-catenin. Thus, PAR(2) up-regulated COX-2 expression via an ERK1/2-mediated activation of the beta-catenin/Tcf-4 and CREB pathways. These findings reveal new cellular mechanisms by which serine proteinases may participate in tumor development and are particularly relevant to cancers associated with chronic mucosal inflammation, where serine proteinases are abundant and COX-2 overexpression is a common feature.