Constitutive Cyclooxygenase-2 Expression Research Articles

Inactivation of TRPM7 Kinase Targets AKT Signaling and Cyclooxygenase-2 Expression in Human CML Cells.

Cyclooxygenase-2 (COX-2) is a key regulator of inflammation. High constitutive COX-2 expression enhances survival and proliferation of cancer cells, and adversely impacts antitumor immunity. The expression of COX-2 is modulated by various signaling pathways. Recently, we identified the melastatin-like transient-receptor-potential-7 (TRPM7) channel-kinase as modulator of immune homeostasis. TRPM7 protein is essential for leukocyte proliferation and differentiation, and upregulated in several cancers. It comprises of a cation channel and an atypical α-kinase, linked to inflammatory cell signals and associated with hallmarks of tumor progression. A role in leukemia has not been established, and signaling pathways are yet to be deciphered. We show that inhibiting TRPM7 channel-kinase in chronic myeloid leukemia (CML) cells results in reduced constitutive COX-2 expression. By utilizing a CML-derived cell line, HAP1, harboring CRISPR/Cas9-mediated TRPM7 knockout, or a point mutation inactivating TRPM7 kinase, we could link this to reduced activation of AKT serine/threonine kinase and mothers against decapentaplegic homolog 2 (SMAD2). We identified AKT as a direct in vitro substrate of TRPM7 kinase. Pharmacologic blockade of TRPM7 in wildtype HAP1 cells confirmed the effect on COX-2 via altered AKT signaling. Addition of an AKT activator on TRPM7 kinase-dead cells reconstituted the wildtype phenotype. Inhibition of TRPM7 resulted in reduced phosphorylation of AKT and diminished COX-2 expression in peripheral blood mononuclear cells derived from CML patients, and reduced proliferation in patient-derived CD34+ cells. These results highlight a role of TRPM7 kinase in AKT-driven COX-2 expression and suggest a beneficial potential of TRPM7 blockade in COX-2-related inflammation and malignancy.

Advances in Understanding the Role of NRF2 in Liver Pathophysiology and Its Relationship with Hepatic-Specific Cyclooxygenase-2 Expression.

Oxidative stress and inflammation play an important role in the pathophysiological changes of liver diseases. Nuclear factor erythroid 2-related factor 2 (NRF2) is a transcription factor that positively regulates the basal and inducible expression of a large battery of cytoprotective genes, thus playing a key role in protecting against oxidative damage. Cyclooxygenase-2 (COX-2) is a key enzyme in prostaglandin biosynthesis. Its expression has always been associated with the induction of inflammation, but we have shown that, in addition to possessing other benefits, the constitutive expression of COX-2 in hepatocytes is beneficial in reducing inflammation and oxidative stress in multiple liver diseases. In this review, we summarized the role of NRF2 as a main agent in the resolution of oxidative stress, the crucial role of NRF2 signaling pathways during the development of chronic liver diseases, and, finally we related its action to that of COX-2, where it appears to operate as its partner in providing a hepatoprotective effect.

COX-2 Expression in Hepatocytes Improves Mitochondrial Function after Hepatic Ischemia-Reperfusion Injury.

Cyclooxygenase 2 (COX-2) is a key enzyme in prostanoid biosynthesis. The constitutive hepatocyte expression of COX-2 has a protective role in hepatic ischemia-reperfusion (I/R) injury (IRI), decreasing necrosis, reducing reactive oxygen species (ROS) levels, and increasing autophagy and antioxidant and anti-inflammatory response. The physiopathology of IRI directly impacts mitochondrial activity, causing ATP depletion and being the main source of ROS. Using genetically modified mice expressing human COX-2 (h-COX-2 Tg) specifically in hepatocytes, and performing I/R surgery on the liver, we demonstrate that COX-2 expression has a beneficial effect at the mitochondrial level. Mitochondria derived from h-COX-2 Tg mice livers have an increased respiratory rate associated with complex I electron-feeding pathways compared to Wild-type (Wt) littermates, without affecting complex I expression or assembly. Furthermore, Wt-derived mitochondria show a loss of mitochondrial membrane potential (ΔΨm) that correlates to increased proteolysis of fusion-related OPA1 through OMA1 protease activity. All these effects are not observed in h-COX-2 Tg mitochondria, which behave similarly to the Sham condition. These results suggest that COX-2 attenuates IRI at a mitochondrial level, preserving the proteolytic processing of OPA1, in addition to the maintenance of mitochondrial respiration.

Radiosynthesis and evaluation of [18F]FMTP, a COX-2 PET ligand.

The upregulation of cyclooxygenase-2 (COX-2) is involved in neuroinflammation associated with many neurological diseases as well as cancers of the brain. Outside the brain, inflammation and COX-2 induction contribute to the pathogenesis of pain, arthritis, acute allograft rejection, and in response to infections, tumors, autoimmune disorders, and injuries. Herein, we report the radiochemical synthesis and evaluation of [18F]6-fluoro-2-(4-(methylsulfonyl)phenyl)-N-(thiophen-2-ylmethyl)pyrimidin-4-amine ([18F]FMTP), a high-affinity COX-2 inhibitor, by cell uptake and PET imaging studies. The radiochemical synthesis of [18F]FMTP was optimized using chlorine to fluorine displacement method, by reacting [18F]fluoride/K222/K2CO3 with the precursor molecule. Cellular uptake studies of [18F]FMTP was performed in COX-2 positive BxPC3 and COX-2 negative PANC-1 cell lines with unlabeled FMTP as well as celecoxib to define specific binding agents. Dynamic microPET image acquisitionwas performed in anesthetized nude mice (n = 3), lipopolysaccharide (LPS) induced neuroinflammation mice (n = 4), and phosphate-buffered saline (PBS) administered control mice (n = 4) using a Trifoil microPET/CT for a scan period of 60min. A twofold higher binding of [18F]FMTP was found in COX-2 positive BxPC3 cells compared with COX-2 negative PANC-1 cells. The radioligand did not show specific binding to COX-2 negative PANC-1 cells. MicroPET imaging in wild-type mice indicated blood-brain barrier (BBB) penetration and fast washout of [18F]FMTP in the brain, likely due to the low constitutive COX-2 expression in the normal brain. In contrast, a ~ twofold higher uptake of the radioligand was found in LPS-induced mice brain than PBS treated control mice. Specific binding to COX-2 in BxPC3 cell lines, BBB permeability, and increased brain uptake in neuroinflammation mice qualifies [18F]FMTP as a potential PET tracer for studying inflammation.

Tu1542 INCREASED PREVALENCE OF BARRETT’S ESOPHAGUS IN PATIENTS WITH DUODENAL ADENOMA

Duodenal adenomas are relatively common in patients with familial adenomatous polyposis (FAP) but sporadic duodenal adenomas are rare. Cyclooxygenase 2 (COX-2) is an important regulatory protein identified in the upper gastrointestinal adenoma-carcinoma sequence. Some studies have shown constitutive COX-2 expression in normal duodenum and esophagus and higher levels in dysplastic esophageal tissue and Barrett’s esophagus (BE). However, the prevalence of BE in patients with duodenal adenomas has not been described. A retrospective cross-sectional study was performed using our electronic endoscopy and pathology database. Study subjects included all patients with duodenal adenoma at our tertiary care academic medical center from Sept 2009 to Sept 2018. Data regarding patient demographics, comorbid conditions and medication use was noted. We also collected information regarding endoscopic and histological findings. A total of 333 patients with duodenal adenoma were included in the study. The average age was 71.6 years and 50.1% were females. A total of 13% of patients had FAP. Barrett’s esophagus was identified in 32 patients (9.6%). There was no difference in patient characteristics amongst patients with duodenal adenoma who had BE as compared to those who did not [Table 1]. As compared to the general population, 43.7% of duodenal adenoma patients who had BE were female. In patients with duodenal adenoma, patients on proton pump inhibitor (PPI) therapy have higher Barrett’s esophagus 15.2% vs 3.2% (p value 0.001). On a multivariate analysis patients with duodenal adenoma not using PPI have significantly lower Barrett’s esophagus [OR=0.14, ( 95% CI: 0.04- 0.52, p-value: 0.003] Despite our study limitations of referral bias and a single center experience, our findings demonstrate that 1 in 10 patients with duodenal adenoma have concomitant Barrett’s esophagus. The prevalence of Barrett’s esophagus in patients with duodenal adenoma is higher than in the general population. Previously some reports have shown changes in expression of some cell regularity proteins such as COX-2 in patients with duodenal adenomas, and COX-2 has also been reported to be increased in BE. Further studies are needed evaluating molecular characteristics in these patients to see if this finding has any clinical significance.

COX2 is induced in the ovarian epithelium during ovulatory wound repair and promotes cell survival†.

The ovarian surface epithelium (OSE) is a monolayer of cells surrounding the ovary that is ruptured during ovulation. After ovulation, the wound is repaired, however, this process is poorly understood. In epithelial tissues, wound repair is mediated by an epithelial-to-mesenchymal transition (EMT). Transforming Growth Factor Beta-1 (TGFβ1) is a cytokine commonly known to induce an EMT and is present throughout the ovarian microenvironment. We, therefore, hypothesized that TGFβ1 induces an EMT in OSE cells and activates signaling pathways important for wound repair. Treating primary cultures of mouse OSE cells with TGFβ1 induced an EMT mediated by TGFβRI signaling. The transcription factor Snail was the only EMT-associated transcription factor increased by TGFβ1 and, when overexpressed, was shown to increase OSE cell migration. A polymerase chain reaction array of TGFβ signaling targets determined Cyclooxygenase-2 (Cox2) to be most highly induced by TGFβ1. Constitutive Cox2 expression modestly increased migration and robustly enhanced cell survival, under stress conditions similar to those observed during wound repair. The increase in Snail and Cox2 expression with TGFβ1 was reproduced in human OSE cultures, suggesting these responses are conserved between mouse and human. Finally, the induction of Cox2 expression in OSE cells during ovulatory wound repair was shown in vivo, suggesting TGFβ1 increases Cox2 to promote wound repair by enhancing cell survival. These data support that TGFβ1 promotes ovulatory wound repair by induction of an EMT and activation of a COX2-mediated pro-survival pathway. Understanding ovulatory wound repair may give insight into why ovulation is the primary non-hereditary risk factor for ovarian cancer.

Cyclooxygenase-2 is acutely induced by CCAAT/enhancer-binding protein β to produce prostaglandin E 2 and F 2α following gonadotropin stimulation in Leydig cells.

Cyclooxygenase 2 (COX-2) is an inducible rate-limiting enzyme for prostanoid production. Because COX-2 represents one of the inducible genes in mouse mesenchymal stem cells upon differentiation into Leydig cells, we investigated COX-2 expression and production of prostaglandin (PG) in Leydig cells. Although COX-2 was undetectable in mouse testis, it was transiently induced in Leydig cells by human chorionic gonadotropin (hCG) administration. Consistent with the finding that Leydig cells expressed aldo-keto reductase 1B7 (PGF synthase) and PGE synthase 2, induction of COX-2 by hCG caused a marked increase in testicular PGF 2α and PGE 2 levels. Using mouse Leydig cell tumor-derived MA-10 cells as a model, it was indicated by reporter assays and electron mobility shift assays that transcription of the COX-2 gene was activated by CCAAT/enhancer-binding protein β (C/EBPβ) with cAMP-stimulation. C/EBPβ expression was induced by cAMP-stimulation, whereas expression of C/EBP homolog protein (CHOP) was robustly downregulated. Transfection of CHOP expression plasmid inhibited cAMP-induced COX-2 promoter activity. In addition, CHOP reduced constitutive COX-2 expression in other mouse Leydig cell tumor-derived TM3 cells. These results indicate that COX-2 is induced in Leydig cells by activation of C/EBPβ via reduction of CHOP expression upon gonadotropin-stimulation to produce PGF 2α and PGE 2 .

Cyclooxygenase-2 Selectively Controls Renal Blood Flow Through a Novel PPARβ/δ-Dependent Vasodilator Pathway.

Cyclooxygenase-2 (COX-2) is an inducible enzyme expressed in inflammation and cancer targeted by nonsteroidal anti-inflammatory drugs. COX-2 is also expressed constitutively in discreet locations where its inhibition drives gastrointestinal and cardiovascular/renal side effects. Constitutive COX-2 expression in the kidney regulates renal function and blood flow; however, the global relevance of the kidney versus other tissues to COX-2-dependent blood flow regulation is not known. Here, we used a microsphere deposition technique and pharmacological COX-2 inhibition to map the contribution of COX-2 to regional blood flow in mice and compared this to COX-2 expression patterns using luciferase reporter mice. Across all tissues studied, COX-2 inhibition altered blood flow predominantly in the kidney, with some effects also seen in the spleen, adipose, and testes. Of these sites, only the kidney displayed appreciable local COX-2 expression. As the main site where COX-2 regulates blood flow, we next analyzed the pathways involved in kidney vascular responses using a novel technique of video imaging small arteries in living tissue slices. We found that the protective effect of COX-2 on renal vascular function was associated with prostacyclin signaling through PPARβ/δ (peroxisome proliferator-activated receptor-β/δ). These data demonstrate the kidney as the principle site in the body where local COX-2 controls blood flow and identifies a previously unreported PPARβ/δ-mediated renal vasodilator pathway as the mechanism. These findings have direct relevance to the renal and cardiovascular side effects of drugs that inhibit COX-2, as well as the potential of the COX-2/prostacyclin/PPARβ/δ axis as a therapeutic target in renal disease.

Spontaneous Glutamatergic Synaptic Activity Regulates Constitutive COX-2 Expression in Neurons OPPOSING ROLES FOR THE TRANSCRIPTION FACTORS CREB (cAMP RESPONSE ELEMENT BINDING) PROTEIN AND Sp1 (STIMULATORY PROTEIN-1)

Burgeoning evidence supports a role for cyclooxygenase metabolites in regulating membrane excitability in various forms of synaptic plasticity. Two cyclooxygenases, COX-1 and COX-2, catalyze the initial step in the metabolism of arachidonic acid to prostaglandins. COX-2 is generally considered inducible, but in glutamatergic neurons in some brain regions, including the cerebral cortex, it is constitutively expressed. However, the transcriptional mechanisms by which this occurs have not been elucidated. Here, we used quantitative PCR and also analyzed reporter gene expression in a mouse line carrying a construct consisting of a portion of the proximal promoter region of the mouse COX-2 gene upstream of luciferase cDNA to characterize COX-2 basal transcriptional regulation in cortical neurons. Extracts from the whole brain and from the cerebral cortex, hippocampus, and olfactory bulbs exhibited high luciferase activity. Moreover, constitutive COX-2 expression and luciferase activity were detected in cortical neurons, but not in cortical astrocytes, cultured from wild-type and transgenic mice, respectively. Constitutive COX-2 expression depended on spontaneous but not evoked excitatory synaptic activity and was shown to be N-methyl-d-aspartate receptor-dependent. Constitutive promoter activity was reduced in neurons transfected with a dominant-negative cAMP response element binding protein (CREB) and was eliminated by mutating the CRE-binding site on the COX-2 promoter. However, mutation of the stimulatory protein-1 (Sp1)-binding site resulted in an N-methyl-d-aspartate receptor-dependent enhancement of COX-2 promoter activity. Basal binding of the transcription factors CREB and Sp1 to the native neuronal COX-2 promoter was confirmed. In toto, our data suggest that spontaneous glutamatergic synaptic activity regulates constitutive neuronal COX-2 expression via Sp1 and CREB protein-dependent transcriptional mechanisms.

Cyclooxygenase-2 expression in hepatocytes attenuates non-alcoholic steatohepatitis and liver fibrosis in mice

Cyclooxygenase-2 (COX-2) is involved in different liver diseases but little is known about the significance of COX-2 in the development and progression of non-alcoholic steatohepatitis (NASH). This study was designed to elucidate the role of COX-2 expression in hepatocytes in the pathogenesis of steatohepatitis and hepatic fibrosis. In the present work, hepatocyte-specific COX-2 transgenic mice (hCOX-2-Tg) and their wild-type (Wt) littermates were either fed methionine-and-choline deficient (MCD) diet to establish an experimental non-alcoholic steatohepatitis (NASH) model or injected with carbon tetrachloride (CCl4) to induce liver fibrosis. In our animal model, hCOX-2-Tg mice fed MCD diet showed lower grades of steatosis, ballooning and inflammation than Wt mice, in part by reduced recruitment and infiltration of hepatic macrophages, with a corresponding decrease in serum levels of pro-inflammatory cytokines. Furthermore, hCOX-2-Tg mice showed a significant attenuation of the MCD diet-induced increase in oxidative stress and hepatic apoptosis observed in Wt mice. Even more, hCOX-2-Tg mice treated with CCl4 had significantly lower stages of fibrosis and less hepatic content of collagen, hydroxyproline and pro-fibrogenic markers than Wt controls. Collectively, our data indicates that constitutive hepatocyte COX-2 expression ameliorates NASH and liver fibrosis development in mice by reducing inflammation, oxidative stress and apoptosis and by modulating activation of hepatic stellate cells, respectively, suggesting a possible protective role for COX-2 induction in NASH/NAFLD progression.

Systematic study of constitutive cyclooxygenase-2 expression: Role of NF-κB and NFAT transcriptional pathways

Cyclooxygenase-2 (COX-2) is an inducible enzyme that drives inflammation and is the therapeutic target for widely used nonsteroidal antiinflammatory drugs (NSAIDs). However, COX-2 is also constitutively expressed, in the absence of overt inflammation, with a specific tissue distribution that includes the kidney, gastrointestinal tract, brain, and thymus. Constitutive COX-2 expression is therapeutically important because NSAIDs cause cardiovascular and renal side effects in otherwise healthy individuals. These side effects are now of major concern globally. However, the pathways driving constitutive COX-2 expression remain poorly understood. Here we show that in the kidney and other sites, constitutive COX-2 expression is a sterile response, independent of commensal microorganisms and not associated with activity of the inflammatory transcription factor NF-κB. Instead, COX-2 expression in the kidney but not other regions colocalized with nuclear factor of activated T cells (NFAT) transcription factor activity and was sensitive to inhibition of calcineurin-dependent NFAT activation. However, calcineurin/NFAT regulation did not contribute to constitutive expression elsewhere or to inflammatory COX-2 induction at any site. These data address the mechanisms driving constitutive COX-2 and suggest that by targeting transcription it may be possible to develop antiinflammatory therapies that spare the constitutive expression necessary for normal homeostatic functions, including those important to the cardiovascular-renal system.

Mitochondria of a human multidrug-resistant hepatocellular carcinoma cell line constitutively express inducible nitric oxide synthase in the inner membrane.

Mitochondria play a crucial role in pathways of stress conditions. They can be transported from one cell to another, bringing their features to the cell where they are transported. It has been shown in cancer cells overexpressing multidrug resistance (MDR) that mitochondria express proteins involved in drug resistance such as P-glycoprotein (P-gp), breast cancer resistant protein and multiple resistance protein-1. The MDR phenotype is associated with the constitutive expression of COX-2 and iNOS, whereas celecoxib, a specific inhibitor of COX-2 activity, reverses drug resistance of MDR cells by releasing cytochrome c from mitochondria. It is possible that COX-2 and iNOS are also expressed in mitochondria of cancer cells overexpressing the MDR phenotype. This study involved experiments using the human HCC PLC/PRF/5 cell line with and without MDR phenotype and melanoma A375 cells that do not express the MDR1 phenotype but they do iNOS. Western blot analysis, confocal immunofluorescence and immune electron microscopy showed that iNOS is localized in mitochondria of MDR1-positive cells, whereas COX-2 is not. Low and moderate concentrations of celecoxib modulate the expression of iNOS and P-gp in mitochondria of MDR cancer cells independently from inhibition of COX-2 activity. However, A375 cells that express iNOS also in mitochondria, were not MDR1 positive. In conclusion, iNOS can be localized in mitochondria of HCC cells overexpressing MDR1 phenotype, however this phenomenon appears independent from the MDR1 phenotype occurrence. The presence of iNOS in mitochondria of human HCC cells phenotype probably concurs to a more aggressive behaviour of cancer cells.

A new target for squamous cell skin cancer?

Prostaglandins (PGs) derived from arachidonic acid of cell membrane are synthesized by PG G/H synthase (cyclooxygenase; COX-1/-2) and signal as autocrine/paracrine lipids (1, 2). Similar to other tissues, COX-1 is constitutively expressed in keratinocytes of normal epidermis, whereas COX-2 expression is more variable and regulated (3, 4). COX-2 is induced for example by cytokines and growth factors; accumulated prostaglandins from COX-2 regulate pain, inflammation, and cancers (2, 3). In the case of cancer, increased levels of PGs disrupt differentiation and thus contribute to the sensitization of cells to carcinogens and ensuing hyperplasia (5, 6). Therefore, the inhibition of COX-2 activity with aspirin like compounds has been suggested as a potent chemo-preventive therapy to suppress tumor development, not just in skin but particularly for gastrointestinal cancers (2, 7).

Hepatic cyclooxygenase-2 expression protects against diet-induced steatosis, obesity, and insulin resistance.

Accumulation evidence links obesity-induced inflammation as an important contributor to the development of insulin resistance, which plays a key role in the pathophysiology of obesity-related diseases such as type 2 diabetes and nonalcoholic fatty liver disease. Cyclooxygenase (COX)-1 and -2 catalyze the first step in prostanoid biosynthesis. Because adult hepatocytes fail to induce COX-2 expression regardless of the proinflammatory stimuli used, we have evaluated whether this lack of expression under mild proinflammatory conditions might constitute a permissive condition for the onset of insulin resistance. Our results show that constitutive expression of human COX-2 (hCOX-2) in hepatocytes protects against adiposity, inflammation, and, hence, insulin resistance induced by a high-fat diet, as demonstrated by decreased hepatic steatosis, adiposity, plasmatic and hepatic triglycerides and free fatty acids, increased adiponectin-to-leptin ratio, and decreased levels of proinflammatory cytokines, together with an enhancement of insulin sensitivity and glucose tolerance. Furthermore, hCOX-2 transgenic mice exhibited increased whole-body energy expenditure due in part by induction of thermogenesis and fatty acid oxidation. The analysis of hepatic insulin signaling revealed an increase in insulin receptor-mediated Akt phosphorylation in hCOX-2 transgenic mice. In conclusion, our results point to COX-2 as a potential therapeutic target against obesity-associated metabolic dysfunction.

Identification of the molecular pathways that drive constitutive renal COX‐2 expression: implications for novel COX‐2‐targetted therapies that spare the cardiovascular system (837.9)

Drugs such as diclofenac and celecoxib inhibit cyclo-oxygenase (COX)-2 to produce anti-inflammatory benefit, but also produce renal/cardiovascular toxicity by inhibition of constitutive COX-2 in the kidney. During inflammation COX-2 is rapidly induced by well-defined pathways including nuclear factor κB (NFκB) and nuclear factor of activated T-cells (NFAT) but it is not known if these also drive physiological renal expression of COX-2. To explore this we compared the distribution of constitutive COX-2 expression with constitutive NFκB and NFAT activity using luciferase reporter mice. COX-2 was highly expressed in the medulla of the kidney. This closely matched a ‘hotspot’ of constitutive NFAT activity but did not correlate with NFκB activity (Figure A). To determine if constitutive renal COX-2 was driven by this NFAT activity we treated mice with cyclosporine A (CsA) to inhibit calcineurin-dependent NFAT activation. CsA reduced constitutive COX-2 expression in the renal medulla but had no effect on endoto...

Cyclooxygenase‐2 over‐expression inhibits liver apoptosis induced by hyperglycemia

Increased expression of COX-2 has been linked to inflammation and carcinogenesis. Constitutive expression of COX-2 protects hepatocytes from several pro-apoptotic stimuli. Increased hepatic apoptosis has been observed in experimental models of diabetes. Our present aim was to analyze the role of COX-2 as a regulator of apoptosis in diabetic mouse liver. Mice of C57BL/6 strain wild type (Wt) and transgenic in COX-2 (hCOX-2 Tg) were separated into Control (vehicle) and SID (streptozotocin induced diabetes, 200 mg/kg body weight, i.p.). Seven days post-injection, Wt diabetic animals showed a decrease in PI3K activity and P-Akt levels, an increase of P-JNK, P-p38, pro-apoptotic Bad and Bax, release of cytochrome c and activities of caspases-3 and -9, leading to an increased apoptotic index. This situation was improved in diabetic COX-2 Tg. In addition, SID COX-2 Tg showed increased expression of anti-apoptotic Mcl-1 and XIAP. Pro-apoptotic state in the liver of diabetic animals was improved by over-expression of COX-2. We also analyzed the roles of high glucose-induced apoptosis and hCOX-2 in vitro. Non-transfected and hCOX-2-transfected cells were cultured at 5 and 25 mM of glucose by 72 h. At 25 mM there was an increase in apoptosis in non-transfected cells versus those exposed to 5 mM. This increase was partly prevented in transfected cells at 25 mM. Moreover, the protective effect observed in hCOX-2-transfected cells was suppressed by addition of DFU (COX-2 selective inhibitor), and mimicked by addition of PGE(2) in non-transfected cells. Taken together, these results demonstrate that hyperglycemia-induced hepatic apoptosis is protected by hCOX-2 expression.

Berberine ameliorates COX-2 expression in rat small intestinal mucosa partially through PPARγ pathway during acute endotoxemia

Berberine hydrochloride (BBR), a plant alkaloid, has been used to treat intestinal inflammation or infection for years. Cyclooxygenase-2 (COX-2) is pro-inflammatory mediator and involved in the induction of gut inflammation. The expression of COX-2 in small bowel mucosa was determined and the mechanism by which BBR modulated COX-2 expression was explored in a rat model of endotoxemia induced by lipopolysaccharide (LPS). The results showed that without LPS stimulation COX-2 was constitutively expressed at low levels in control rats. LPS challenge rapidly induced COX-2 gene transcription resulting in high levels of inducible COX-2 expression in endotoxemic rats. BBR pre- and post-treatment had no marked effect on constitutive COX-2 expression but inhibited inducible COX-2 overexpression. LPS challenge increased the expression and phosphorylation of peroxisome proliferator-activated receptor gamma (PPARγ), p38 and activating transcription factor 2 and 3 (ATF2, ATF3), but the effects of LPS were inhibited by BBR treatment. GW9662 did not influence constitutive COX-2 expression but enhanced inducible COX-2 overproduction. Besides, GW9662 abolished the inhibitory effect of BBR on inducible COX-2, p38, ATF2, 3 expression and phosphorylation. Collectively, these results indicated that BBR gavage could attenuate the overexpression of inducible COX-2, not constitutive COX-2, in ileal mucosa during acute endotoxemia in part via activation of PPARγ pathway, which negatively interfered with p38/ATFs cascade.

DHA induces apoptosis and differentiation in human melanoma cells in vitro : involvement of HuR-mediated COX-2 mRNA stabilization and β-catenin nuclear translocation

The pro-inflammatory phenotype accompanying melanoma progression includes an enhanced expression of cyclooxygenase-2 (COX-2), which plays an important role in the acquisition of apoptosis resistance, and is a suitable target for melanoma prevention and therapy. We observed that the WM266-4 metastatic melanoma cell line showed a constitutive COX-2 expression higher than that of the primary WM115 cells, an increased cytosolic level of the COX-2 messenger RNA (mRNA)-stabilizer human antigen R (HuR) and a lower susceptibility to basal apoptosis. The transfection of HuR siRNA induced apoptosis and reduced COX-2 protein abundance in both the cells. The same effects were observed treating the cells with the n-3 polyunsaturated fatty acid docosahexaenoic acid (DHA), which reduced the cytoplasmic location and expression of HuR and, correspondently, decreased COX-2 protein expression and induced apoptosis. DHA also decreased the expression and stability of COX-2 mRNA, increased the β-catenin expression in the nuclei and reduced it in the cytosol, where it forms a complex with HuR and COX-2 mRNA. DHA had also a pro-differentiating effect, which is compatible with the nuclear translocation of β-catenin. These findings allow us to associate for the first time the constitutive expression of COX-2 in melanoma cells to the HuR-mediated stabilization of its mRNA and suggest that also β-catenin may play a role in HuR-mediated COX-2 stabilization in these cells. The data demonstrate that the HuR-mediated stabilization of COX-2 may represent a target of DHA action in melanoma cells and suggest the application of DHA in the prevention and therapy of melanoma.

Hypoxia upregulates PGI-synthase and increases PGI2 release in human vascular cells exposed to inflammatory stimuli

Hypoxia affects vascular function and cell metabolism, survival, growth, and motility; these processes are partially regulated by prostanoids. We analyzed the effect of hypoxia and inflammation on key enzymes involved in prostanoid biosynthesis in human vascular cells. In human vascular smooth muscle cells (VSMC), hypoxia and interleukin (IL)-1β synergistically increased prostaglandin (PG)I₂ but not PGE₂ release, thereby increasing the PGI₂/PGE₂ ratio. Concomitantly, these stimuli upregulated cyclooxygenase-2 (COX-2) expression (mRNA and protein) and COX activity. Interestingly, hypoxia enhanced PGI-synthase (PGIS) expression and activity in VSMC and human endothelial cells. Hypoxia did not significantly modify the inducible microsomal-PGE-synthase (mPGES)-1. Hypoxia-inducible factor (HIF)-1α-silencing abrogated hypoxia-induced PGIS upregulation. PGIS transcriptional activity was enhanced by hypoxia; however, the minimal PGIS promoter responsive to hypoxia (-131 bp) did not contain any putative hypoxia response element (HRE), suggesting that HIF-1 does not directly drive PGIS transcription. Serial deletion and site-directed mutagenesis studies suggested several transcription factors participate cooperatively. Plasma levels of the stable metabolite of PGI₂ and PGIS expression in several tissues were also upregulated in mice exposed to hypoxia. These data suggest that PGIS upregulation is part of the adaptive response of vascular cells to hypoxic stress and could play a role in counteracting the deleterious effect of inflammatory stimuli.

Extraendothelial and constitutive COX-2 expression is involved in the contractile effect of angiotensin II in the rat aorta

1 The role of the extraendothelial and constitutive isoforms of cyclo-oxygenase-2 (COX-2) in the contractile effect of angiotensin II (Ang II) was investigated using thoracic and abdominal aortic rings without endothelium from young Wistar rats. 2 Ang II elicited similar contractions in both aortic segments, and the effect was inhibited by pretreatment with NS398 (a selective COX-2 inhibitor) but not SC-560 [selective cyclo-oxygenase-1 (COX-1) inhibitor]. 3 COX-2 mRNA was expressed under basal conditions in both aortic segments. Additionally, Ang II increased COX-2 mRNA expression in the abdominal but not the thoracic segment, while cycloheximide (a protein synthesis inhibitor) did not affect the contractile response to Ang II in either of the two segments; this suggests that the effect is not associated with de novo COX-2 synthesis. 4 In conclusion, the basal amount of COX-2 found in aortic smooth muscle cells is sufficient to explain the production of the prostanoids related to the contractile effect of Ang II. The production of these prostanoids, which are derived from constitutive COX-2, occurs independently of the endothelium vascular system.