Mouse COX-2 Research Articles

Diosgenin suppresses COX-2 and mPGES-1 via GR and improves LPS-induced liver injury in mouse

Using a wild yam (Dioscorea japonica), we previously found novel anti-inflammatory and anti-carcinogenic effects via the downregulation of cyclooxygenase (COX)-2 and microsomal prostaglandin E synthase (mPGES)-1. One of the substances in wild yam is a steroidal saponin, diosgenin. We demonstrated that diosgenin suppressed COX-2 in human non–small-cell lung carcinoma A549 cells via nuclear factor-kappa B (NF-κB) translocation and the effects were reversed by a glucocorticoid receptor antagonist, RU486. In lipopolysaccharide (LPS)-induced mouse liver injury, COX-2 and mPGES-1 were induced and localized in sinusoidal macrophages and endothelial cells; however, diosgenin administration significantly suppressed Ptgs2 and Ptges expression and decreased COX-2 and mPGES-1 immunopositive cells in the sinusoids. Multiple immunohistochemical analyses showed that diosgenin had an effect on COX-2 and mPGES-1, particularly in the macrophages. Thus, we showed that diosgenin downregulated COX-2 and mPGES-1 via the glucocorticoid receptor and suppressed COX-2 and mPGES-1 in the macrophages of LPS-induced acute mouse liver injury.

The positive effect of selective prostaglandin E2 receptor EP2 and EP4 blockade on cystogenesis in vitro is counteracted by increased kidney inflammation in vivo

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a major cause of end-stage kidney disease in man. The central role of cyclic adenosine monophosphate (cAMP) in ADPKD pathogenesis has been confirmed by numerous studies including positive clinical trial data. Here, we investigated the potential role of another major regulator of renal cAMP, prostaglandin E2 (PGE2), in modifying disease progression in ADPKD models using selective receptor modulators to all four PGE2 receptor subtypes (EP1-4). In 3D-culture model systems utilizing dog (MDCK) and patient-derived (UCL93, OX161-C1) kidney cell lines, PGE2 strikingly promoted cystogenesis and inhibited tubulogenesis by stimulating proliferation while reducing apoptosis. The effect of PGE2 on tubulogenesis and cystogenesis in 3D-culture was mimicked or abolished by selective EP2 and EP4 agonists or antagonists but not those specific to EP1 or EP3. In a Pkd1 mouse model (Pkd1nl/nl), kidney PGE2 and COX-2 expression were increased by two-fold at the peak of disease (week four). However, Pkd1nl/nl mice treated with selective EP2 (PF-04418948) or EP4 (ONO-AE3-208) antagonists from birth for three weeks had more severe cystic disease and fibrosis associated with increased cell proliferation and macrophage infiltration. A similar effect was observed for the EP4 antagonist ONO-AE3-208 in a second Pkd1 model (Pax8rtTA-TetO-Cre-Pkd1f/f). Thus, despite the positive effects of slowing cyst growth invitro, the more complex effects of inhibiting EP2 or EP4 invivo resulted in a worse outcome, possibly related to unexpected pro-inflammatory effects.

MicroRNA-381 protects myocardial cell function in children and mice with viral myocarditis via targeting cyclooxygenase-2 expression.

The present study aimed to determine the expression of cyclooxygenase (COX)-2 and microRNA (miRNA/miR)-381 in the blood of children with viral myocarditis (VM), and investigate the association between COX-2 and miR-381 in the occurrence and development of the disease using a mouse model. A total of 26 children with VM (15 boys and 11 girls) were included in the present study. Peripheral blood was collected from all children. The mouse model of VM was constructed by coxsackievirus B3 (CVB3) infection. Peripheral blood and myocardial tissues were collected from all mice for analysis. Reverse transcription-quantitative polymerase chain reaction was used to determine the expression of COX-2 mRNA and miR-381 in serum and myocardial tissues. ELISA was used to measure the content of COX-2 protein in serum from humans and mice, and western blotting was employed to determine the expression of COX-2 protein in myocardial tissues from mice. Contents of creatine kinase (CK-MB) and lactate dehydrogenase (LDH) were evaluated using an automatic biochemical analyzer. A dual luciferase assay was conducted to identify interactions between COX-2 mRNA and miR-381. Children with VM had increased COX-2 levels and decreased miR-381 expression in peripheral blood, compared with those who had recovered from VM. CVB3 infection resulted in damage in the myocardium of mice, and elevated CK-MB and LDH contents. VM model mice exhibited increased COX-2 levels and decreased miR-381 expression in peripheral blood and myocardial tissues compared with normal mice. miR-381 binds to the 3'-untranslated seed regions of both human and mouse COX-2 mRNA to regulate their expression. The present study demonstrated that children with VM have decreased miR-381 expression and elevated COX-2 expression in peripheral blood. miR-381 may inhibit myocardial cell damage caused by CVB3 infection and protect myocardial cell function by targeting COX-2 expression.

Docking assisted design of novel 4-adamantanyl-2-thiazolylimino-5-arylidene-4-thiazolidinones as potent NSAIDs

Docking analysis was used to predict the effectiveness of adamantanyl insertion in improving cycloxygenase/lipoxygenase (COX/LOX) inhibitory action of previously tested 2-thiazolylimino-5-arylidene-4-thiazolidinones. The crystal structure data of human 5-LOX (3O8Y), ovine COX-1 (1EQH) and mouse COX-2 (3ln1) were used for docking analysis. All docking calculations were carried out using AutoDock 4.2 software. Following prediction results, 11 adamantanyl derivatives were synthesized and evaluated for biological action. Prediction evaluations correlated well with experimental biological results. Comparison of the novel adamantanyl derivatives with the 2-thiazolylimino-5-arylidene-4-thiazolidinones previously tested showed that insertion of the adamantanyl group led to the production of more potent COX-1 inhibitors, as well as LOX inhibitors (increased activity from 200% to 560%). Five compounds out of the 11 exhibited better activity than naproxen; while nine out of 11 showed better activity than NDGA and seven compounds possessed better anti-inflammatory activity than indomethacin.

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.

Synthesis & Characterization of 2-(substituted-phenyl)acetohydrazide Analogs, 1,3,4-oxadiazoles, and 1,2,4-triazine Ring Systems: A Novel Class of Potential Analgesic and Anti-Inflammatory Agents

The new series of 2-(substituted-phenyl)acetohydrazides analogs, S-alkylated 5-substituted-1,3,4-oxadiazoles- 2-thione derivatives and 5-arylidene-3-substituted-1,2,4-triazines have been synthesized in good yields and characterized by IR, NMR, mass spectral and elemental analyses. All the synthesized compounds 4(a-d), 5(a-d), 7(a-b), and 8(a-f) are evaluated for their in vitro DPPH scavenging, antimicrobial activity, in vivo analgesic, anti-inflammatory activities. The results of the anti-inflammatory activity are supported by molecular docking study with mouse COX-1 (PDB ID: 2CZT) and COX-2 (PDB ID: 3LN1) enzymes to predict their putative interactions. Among all the assays conducted, the compounds 5-(4-bromophenyl)-3-(naphthalen-2-ylmethyl)-1,2,4-triazine (4d) and2-{[5-(diphenylmethyl)-1,3,4-oxadiazol-2- yl]sulfanyl}-N-(pyrazin-2-yl)acetamide (8a) have emerged as the most potent molecules. Keywords: Acid hydrazide analogs, antioxidant activity, analgesic activity, anti-inflammatory activity, antimicrobial activity, molecular docking study.

A reporter mouse for optical imaging of inflammation in mdx muscles.

BackgroundDuchenne muscular dystrophy (DMD) is due to mutations in the gene coding for human DMD; DMD is characterized by progressive muscle degeneration, inflammation, fat accumulation, and fibrosis. The mdx mouse model of DMD lacks dystrophin protein and undergoes a predictable disease course. While this model has been a valuable resource for pre-clinical studies aiming to test therapeutic compounds, its utility is compromised by a lack of reliable biochemical tools to quantifiably assay muscle disease. Additionally, there are few non-invasive assays available to researchers for measuring early indicators of disease progression in mdx mice.MethodsMdx mice were crossed to knock-in mice expressing luciferase from the Cox2 promoter. These reporter mice (Cox2FLuc/+DMD−/−) were created to serve as a tool for researchers to evaluate muscle inflammation. Luciferase expression was assayed by immunohistochemistry to insure that it correlated with muscle lesions. The luciferase signal was quantified by optical imaging and luciferase assays to verify that the signal correlated with muscle damage. As proof of principle, Cox2FLuc/+DMD−/− mice were also treated with prednisolone to validate that a reduction in luciferase signal correlated with prednisone treatment.ResultsIn this investigation, a novel reporter mouse (Cox2FLuc/+DMD−/− mice) was created and validated for non-invasive quantification of muscle inflammation in vivo. In this dystrophic mouse, luciferase is expressed from cyclooxygenase 2 (Cox2) expressing cells and bioluminescence is detected by optical imaging. Bioluminescence is significantly enhanced in damaged muscle of exercised Cox2FLuc/+DMD−/− mice compared to non-exercised Cox2FLuc/+DMD+/+ mice. Moreover, the Cox2 bioluminescent signal is reduced in Cox2FLuc/+DMD−/− mice in response to a course of steroid treatment. Reduction in bioluminescence is detectable prior to measurable therapy-elicited improvements in muscle strength, as assessed by traditional means. Biochemical assay of luciferase provides a second means to quantify muscle inflammation.ConclusionsThe Cox2FLuc/+DMD−/− mouse is a novel tool to evaluate the therapeutic benefits of drugs intended to target inflammatory aspects of dystrophic pathology. This mouse model will be a useful adjunct to traditional outcome measures in assessing potential therapeutic compounds.

Species differential regulation of COX2 can be described by an NFκB-dependent logic AND gate.

Cyclooxygenase 2 (COX2), a key regulatory enzyme of the prostaglandin/eicosanoid pathway, is an important target for anti-inflammatory therapy. It is highly induced by pro-inflammatory cytokines in a Nuclear factor kappa B (NFκB)-dependent manner. However, the mechanisms determining the amplitude and dynamics of this important pro-inflammatory event are poorly understood. Furthermore, there is significant difference between human and mouse COX2 expression in response to the inflammatory stimulus tumor necrosis factor alpha (TNFα). Here, we report the presence of a molecular logic AND gate composed of two NFκB response elements (NREs) which controls the expression of human COX2 in a switch-like manner. Combining quantitative kinetic modeling and thermostatistical analysis followed by experimental validation in iterative cycles, we show that the human COX2 expression machinery regulated by NFκB displays features of a logic AND gate. We propose that this provides a digital, noise-filtering mechanism for a tighter control of expression in response to TNFα, such that a threshold level of NFκB activation is required before the promoter becomes active and initiates transcription. This NFκB-regulated AND gate is absent in the mouse COX2 promoter, most likely contributing to its differential graded response in promoter activity and protein expression to TNFα. Our data suggest that the NFκB-regulated AND gate acts as a novel mechanism for controlling the expression of human COX2 to TNFα, and its absence in the mouse COX2 provides the foundation for further studies on understanding species-specific differential gene regulation.Electronic supplementary materialThe online version of this article (doi:10.1007/s00018-015-1850-1) contains supplementary material, which is available to authorized users.

Design and synthesis of novel heterocyclic acetamide derivatives for potential analgesic, anti-inflammatory, and antimicrobial activities

A new series of heterocyclic amides were synthesized by the reaction of heterocyclic amines with substituted phenylacetic acids 1 in the presence of EDC.HCl as a coupling agent. The newly synthesized compounds were characterized by IR, NMR, mass spectral, and elemental analysis. All the synthesized compounds were evaluated for their in vivo analgesic, anti-inflammatory, and in vitro antimicrobial activities. Among the tested acetamides, the compounds 2a, 3b, 4d, 4e, and 4f showed potential anti-inflammatory and analgesic activities. The compounds which exhibited good anti-inflammatory activity were docked to mouse COX-1 (PDB ID: 2CZT) and COX-2 (PDB ID: 3LN1) enzymes to predict their putative interactions. The compound 2-(2-Bromophenyl)-N-(pyrazin-2-yl)acetamide (4f) emerged as most potent molecule with synergistic anti-inflammatory, analgesic, and antimicrobial properties.

Assessment of radioligands for PET imaging of cyclooxygenase-2 in an ischemic neuronal injury model

Cyclooxygenase-2 (COX-2) plays crucial roles in progressive neuronal death in ischemic brain injury. In the present study, we evaluated two radiolabeled COX-2 selective inhibitors, [11C]celecoxib and [11C]rofecoxib, as positron emission tomography (PET) tracers for COX-2 imaging in normal and ischemic mouse brains. We also took advantage of our newly-generated antibody highly selective for mouse COX-2 to prove accumulation of the radioligands in regions enriched with COX-2. In vitro autoradiography demonstrated specific binding of high-concentration [11C]rofecoxib but not [11C]celecoxib to the cerebellum and brain stem of normal brains wherein COX-2 immunoreactivity in neurons was most abundantly observed. Meanwhile, both of these radioligands failed to detect COX-2 expression in PET assays despite their excellent brain permeability. Hypoperfusion-induced ischemia caused marked necrotic neuron death accompanied by gliosis and enhancement of neuronal COX-2 immunoreactivity in the hippocampus. Correspondingly, in vitro autoradiographic binding of [11C]rofecoxib was increased in the injured hippocampus compared to the uninjured contralateral region, but failed in living brains of ischemia model likewise. Our work provides the rationale for monitoring COX-2 as a biomarker reflecting ischemic brain injuries and demonstrates that [11C]rofecoxib, not [11C]celecoxib, is useful for in vitro assays of COX-2, but its affinity would be insufficient for in vivo PET visualization.

Abstract 325: COX2 Expression is Increased in Human and Mouse Calcific Aortic Valve Disease and COX2 Inhibition Blocks the Induction of Osteogenic Gene Expression in Valve Interstitial Cells

Calcific aortic valve disease (CAVD) is considered a disease of aging and is characterized by calcific nodule formation, stenosis of the valve, and induction of osteogenic gene expression. The klotho deficient mouse model of premature aging exhibits CAVD evident in calcific lesions in the aortic valve annulus. Klotho deficient mice develop calcification by 6 weeks of age, with minimal immune cell infiltration and increased osteogenic gene expression. Microarray analyses performed on aortic valve tissues of wild type and klotho deficient mice demonstrate that cyclooxygenase 2 (COX2) expression is increased in klotho deficient mice. We hypothesize that increased COX2 expression contributes to aortic valve calcification and that COX2 inhibition prevents osteogenic gene expression in aortic valve interstitial cells (aVICs). In klotho deficient mice, COX2 protein expression is increased throughout regions of valve calcification relative to wild type controls. Prior to the formation of calcified valve lesions, increased COX2 expression is localized to areas at risk of developing calcification in klotho deficient mice. Thus increased COX2 expression precedes valve calcification. In human explanted calcific aortic valves, COX2 expression also is increased in mineralized regions compared to controls. In bone, COX2 deficiency inhibits osteogenic gene induction. The ability of COX2 inhibition to block osteogenic gene expression was examined using porcine aVICs. Treatment of aVICs with osteogenic media induces bone markers, including osteocalcin, in addition to increasing COX2 gene expression. Furthermore, inhibition of COX2 activity in cells cultured under osteogenic conditions results in significantly decreased expression of osteogenic genes compared to controls. In vivo studies in progress provide initial evidence that loss of COX2 activity reduces aortic valve calcification in klotho deficient mice. In conclusion, COX2 expression is increased in regions of aortic valve calcification in both human and mouse CAVD tissues, and COX2 inhibition suppresses osteogenic gene expression in cultured aVICs. Targeting COX2 enzyme activity may be a potential therapeutic option for preventing or reducing aortic valve calcification in the future.

Abstract CN04-02: A novel approach to biomolecular imaging of COX-2

Abstract Molecular imaging emerged in the early 21st century as a discipline that enables the visualization of cellular functions, molecular biology of diseases, and diagnosis of disease states in living beings. COX-2 is an attractive target for molecular imaging of cancer, because it is an inducible enzyme that is expressed in inflammation, premalignant and malignant lesions. We designed fluorescent COX-2 inhibitors by tethering bulky fluorescent functional groups onto non-steroidal anti-inflammatory drugs (NSAIDs), and COX-2 inhibitors (COXIBs). To identify the lead compounds, we synthesized a library of 200 hundred fluorescent conjugates, and evaluated them for selective inhibition of purified COX-2, COX-2 in mouse macrophages and COX-2 in human cancer cells. Although a significant number of compounds showed COX-2 inhibitory activity against purified protein, only a fraction of these compounds inhibited COX-2 activity in intact cells, and of those, most did not possess fluorescence properties suitable for in vivo imaging. Optimized lead compounds were then tested for their ability to image COX-2 in intact cells. Among the compounds that emerged from our development pathway, only 5-carboxy-X-rhodamine (5-ROX) and 6-carboxy-X-rhodamine (6-ROX) based conjugates, Fluorocoxib A and Fluorocoxib B, exhibited sufficient metabolic stability to distribute to target tissues in rodents and canines. A detailed kinetic analysis indicated that these compounds are slow, tight-binding inhibitors of COX-2 with very low rates of association and dissociation. These compounds displayed a very high degree of selectivity of uptake in inflammatory tissues or human tumor xenografts in rodents, and in naturally occurring oral or bladder cancer in dogs relative to surrounding normal tissue or muscle as determined by either optical imaging or mass spectrometry. Uptake of these compounds required the expression of COX-2 at the target site and declined as the level of COX-2 decreased. Experiments with COX-2 (-/-) animals or with animals pretreated with indomethacin or celecoxib verified that selective accumulation into inflamed tissue or tumors was due to binding to COX-2. Pharmacokinetic analysis revealed that only the intact parent compound is found in the region of interest. Site-directed mutagenesis and analysis of X-ray co-crystal structure of fluorocoxib A with COX-2 revealed that the n-butyl linker group allows the indomethacin part of fluorocoxib A to fully insert into the binding pocket of COX-2, while the bulky fluorescent 5-ROX group binds in the lobby of the enzyme. Thus, fluorocoxibs represent the first feasible optical imaging reagents for clinical detection of tissues containing high levels of COX-2 in settings amenable to fluorescent excitation and analysis by surface measurement or endoscopy (e.g., skin, esophagus, intestine, and bladder). (This work has been supported by grants from the National Cancer Institute and National Institutes of Health [CA136465 and CA128323]) Citation Format: Md. Jashim Uddin, Brenda C. Crews, Kebreab Ghebreselasie, Anna L. Blobaum, Kelsey C. Duggan, Shu Xu, Philip Kingsley, Maria Cekanova, Lawrence J. Marnett. A novel approach to biomolecular imaging of COX-2. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr CN04-02.

Inhibition of cyclooxygenase‐2 causes regression of gastric adenomas in trefoil factor 1 deficient mice

Cyclooxygenase-2 (Cox-2) expression is a marker of reduced survival in gastric cancer patients, and inhibition of Cox-2 suppresses gastrointestinal carcinogenesis in experimental animal models. To investigate the role of Cox-2 in gastric carcinogenesis in vivo, we utilized trefoil factor 1 (Tff1) deficient mice, which model the neoplastic process of the stomach by developing gastric adenomas with full penetrance. These tumors express Cox-2 protein and mRNA, and we have now investigated the effects of genetic deletion of the mouse Cox-2 gene [also known as prostaglandin-endoperoxide synthase 2 (Ptgs2)] and a Cox-2 selective drug celecoxib. Our results show that genetic deletion of Cox-2 in the Tff1 deleted background resulted in reduced adenoma size and ulceration with a chronic inflammatory reaction at the site of the adenoma. To characterize the effect of Cox-2 inhibition in more detail, mice that had already developed an adenoma were fed with celecoxib for 8-14 weeks, which resulted in disruption of the adenoma that ranged from superficial erosion to deep ulcerated destruction accompanied with chronic inflammation. Importantly, mice fed with celecoxib for 16 weeks, followed by control food for 9 weeks, redeveloped a complete adenoma with no detectable inflammatory process. Finally, we determined the identity of the Cox-2 expressing cells and found them to be fibroblasts. Our results show that inhibition of Cox-2 is sufficient to reversibly disrupt gastric adenomas in mice.

The Structural Basis of Endocannabinoid Oxygenation by Cyclooxygenase-2

The cyclooxygenases (COX-1 and COX-2) oxygenate arachidonic acid (AA) in the committed step of prostaglandin biogenesis. Substitutions of I434V, H513R, and I523V constitute the only differences in residues lining the cyclooxygenase channel between COX-1 and COX-2. These changes create a hydrophobic pocket in COX-2, with Arg-513 located at the base of the pocket, which has been exploited in the design of COX-2-selective inhibitors. Previous studies have shown that COX-2, but not COX-1, can oxygenate endocannabinoid substrates, including 2-arachidonoyl glycerol (2-AG). To investigate the isoform-specific structural basis of endocannabinoid binding to COX-2, we determined the crystal structure of the 2-AG isomer 1-arachidonoyl glycerol (1-AG) in complex with wild type and R513H murine (mu) COX-2 to 2.2 and 2.35 Å, respectively, and R513H muCOX-2 in complex with AA to 2.45 Å resolution. The 2,3-dihydroxypropyl moiety of 1-AG binds near the opening of the cyclooxygenase channel in the space vacated by the movement of the Leu-531 side chain, validating our previous hypothesis implicating the flexibility of the Leu-531 side chain as a determinant for the ability of COX-2 to oxygenate endocannabinoid substrates. Functional analyses carried out to compliment our structural findings indicated that Y355F and R513H muCOX-2 constructs had no effect on the oxygenation of 1-AG and 2-AG, whereas substitutions that resulted in a shortened side chain for Leu-531 had only modest effects. Both AA and 1-AG bind to R513H muCOX-2 in conformations similar to those observed in the co-crystal structures of these substrates with wild type enzyme.

Identification of PARP-1 as one of the transcription factors binding to the repressor element in the promoter region of COX-2

Cyclooxygenase-2 (COX-2) plays important roles in the development of many disease conditions, including pancreatic β-cell dysfunction. Although the processes involved in the transcriptional regulation of COX-2 are well documented, some key elements, especially inhibitory elements, are still unknown. In our previous study, we identified a novel repressor element located in promoter region of mouse COX-2. In this study, we isolated several DNA-binding proteins from NIT-1 cells via DNA affinity chromatography; the most prominent among these proteins was poly (ADP-ribose) polymerase-1 (PARP-1). In this study, gel-supershift assays and chromatin immunoprecipitation assays showed that PARP-1 can bind to the inhibitory element −655/−632 in the promoter region of mouse COX-2 both in vitro and in vivo. Furthermore, overexpression of PARP-1 significantly inhibited promoter activity and decreased COX-2 expression. Conversely, repression of PARP-1 by RNAi upregulated COX-2 expression. These data suggest that PARP-1 plays an important role in the regulation of COX-2 expression via binding to the inhibitory element. Collectively, our findings provide new important information on the transcriptional regulation of COX-2 in pancreatic β-cells.

Mechanisms of adiponectin‐mediated COX‐2 induction and protection against iron injury in mouse hepatocytes

Adiponectin (APN)-mediated cyclooxygenase (COX)-2 induction is known to have various protective effects on cardiovascular diseases. However, the molecular mechanisms of APN-mediated COX-2 induction and its protection against iron-mediated injury in hepatocytes are still unclear. Herein, we show that AMP-mediated peroxisome proliferator-activated receptor (PPAR)alpha activation was attributable to COX-2 induction by APN, which was further confirmed by identifying novel functional PPAR responsive elements (PPREs) in the mouse COX-2 promoter region. Prostaglandin (PG)I2 and PGE2, metabolites of COX-2, time-dependently increased in hepatocytes treated with APN. Interestingly, beraprost and misoprostol, respective agonists for PGI2 and PGE2, mimicked the protective effects of APN in iron-mediated inflammation in hepatocytes. The iron dextran-activated nuclear factor (NF)-kappaB pathway was correlated with the increased production of inflammatory cytokines including tumor necrosis factor-alpha, intercellular adhesion molecule-1, and monocyte chemotactic protein-1. This was eliminated by administration of APN, whereas blockage of PPARalpha activation, an upstream regulator of COX-2 induction by APN, and COX-2 activation reversed the anti-inflammatory effect of APN, suggesting the crucial role of COX-2 in this event. Herein, we demonstrate that APN-mediated COX-2 induction through a PPARalpha-dependent mechanism, and COX-2 exerted an anti-inflammatory effect of APN in hepatocytes subjected to iron challenge.

Embryonic Expression of Cyclooxygenase-2 Causes Malformations in Axial Skeleton

Cyclooxygenases (COXs) have important functions in various physiological and pathological processes. COX-2 expression is highly induced by a variety of stimuli and is observed during certain periods of embryonic development. In this report, the direct effect of COX-2 expression on embryonic development is examined in a novel COX-2 transgenic mouse model that ubiquitously expresses human COX-2 from the early stages of embryonic development. COX-2 transgenic fetuses exhibit severe skeletal malformations and die shortly after birth. Skeletal malformations are localized along the entire vertebral column and rib cage and are linked to defective formation of cartilage anlagen. The cartilage anlagen of axial skeleton fail to properly develop in transgenic embryos because of impaired precartilaginous sclerotomal condensation, which results from the reduction of cell number in the sclerotome. Despite the ubiquitous expression of COX-2, the number of apoptotic cells is highly increased in the sclerotome of transgenic embryos but not in other tissues, suggesting that it is a tissue-specific response. Therefore, the loss of sclerotomal cells due to an increased apoptosis is probably responsible for axial skeletal malformations in transgenic fetuses. In addition, the sclerotomal accumulation of p53 protein is observed in transgenic embryos, suggesting that COX-2 may induce apoptosis via the up-regulation of p53. Our results demonstrate that the aberrant COX-2 signaling during embryonic development is teratogenic and suggest a possible association of COX-2 with fetal malformations of unknown etiology.

Up-regulation of adipogenesis in adipocytes expressing stably cyclooxygenase-2 in the antisense direction

Adipocytes and the precursor cells express two types of cyclooxygenase (COX) isoforms that are involved in the biosynthesis of different types of prostaglandins (PGs) exerting opposite effects on adipogenesis. To evaluate the role of the inducible COX-2 isoform in the control of the differentiation and maturation of adipocytes, we employed an antisense technology to suppress specifically the expression of COX-2 in adipocytes. Cultured 3T3-L1 preadipocytes were transfected stably with a mammalian expression vector having the full-length cDNA encoding mouse COX-2 oriented in the antisense direction. The cloned transfectants with antisense COX-2 exhibited stable expression of antisense RNA for COX-2, which was accompanied by the suppressed expression of mRNA and protein levels of sense COX-2. However, almost no alteration in the expression of COX-1 was detected. The transfectants with antisense COX-2 showed significant decreases in the delayed synthesis of PGE 2 involving the inducible COX-2 in response to cell stimuli. By contrast, the immediate synthesis of PGE 2 associated with the constitutive COX-1 was not influenced appreciably. The stable expression of antisense mRNA of COX-2 resulted in significant stimulation of fat storage during the maturation phase without affecting the cell proliferation associated with the clonal expansion phase. The gene expression studies revealed higher expression levels of adipocyte-specific markers in the transfectants with antisense COX-2, indicating the mechanism that stimulates adipogenesis program. The up-regulation of fat storage was appreciably prevented by anti-adipogenic prostanoids, such as PGE 2 and PGF 2α, during the maturation phase. These results suggest that COX-2 is more preferentially involved in the generation of endogenous anti-adipogenic prostanoids during the maturation phase of adipocytes.

Genome wide analysis and comparative docking studies of new diaryl furan derivatives against human cyclooxygenase-2, lipoxygenase, thromboxane synthase and prostacyclin synthase enzymes involved in inflammatory pathway

In an effort to develop potent anti-inflammatory and antithrombotic drugs, a series of new 4-(2-phenyltetrahydrofuran-3-yl) benzene sulfonamide analogs were designed and docked against homology models of human cyclooxygenase-2 (COX-2), lipoxygenase and thromboxane synthase enzymes built using MODELLER 7v7 software and refined by molecular dynamics for 2 ns in a solvated layer. Validation of these homology models by procheck, verify-3D and ERRAT programs revealed that these models are highly reliable. Docking studies of 4-(2-phenyltetrahydrofuran-3-yl) benzene sulfonamide analogs designed by substituting different chemical groups on benzene rings replacing 1H pyrazole in celecoxib with five membered thiophene, furan, 1H pyrrole, 1H imidazole, thiazole and 1,3-oxazole showed that diaryl furan molecules showed good binding affinity towards mouse COX-2. Further, docking studies of diaryl furan derivatives are likely to have superior thromboxane synthase and COX-2 selectivity. Docking studies against site directed mutagenesis of Arg120Ala, Ser530Ala, Ser530Met and Tyr355Phe enzymes displayed the effect of inhibition of COX-2. Drug likeliness and activity decay for these inhibitors showed that these molecules act as best drugs at very low concentrations.

Sirtuin‐1‐dependent COX2 Induction is an Essential Survival Mechanism in Renal Medullary Interstitial Cells against Oxidative Stress

The present studies examined the role of NAD+‐dependant deacetylase sirtuin‐1 (SIRT1) in protecting the cells in renal medulla against oxidative stress. High levels of nitrosylated tyrosine were detected in renal medulla compared with renal cortex of C57BL6/j mice by immunohistochemistry, consistence with the presence of oxidative stress in renal medulla. Immunoblot and immunohistochemistry also showed abundant SIRT1 in renal medulla, mainly co‐localizing with COX2 in renal medullary interstitial cells (RMICs) by immunofluorescence. Selective knock‐down of SIRT1 in cultured mouse RMICs by shRNA significantly increased H2O2 (500 μM) induced cell death (from 48±5% to 78±4%, P<0.0001), accompanied with increased activated‐caspase‐3 levels. SIRT1 knock‐down also almost abolished H2O2‐induced COX2 expression (P<0.0001). ChIP showed physical association of SIRT1 with mouse COX2 gene promoter, supporting a role of SIRT1 in modulating COX2 transcription. Pre‐treatment with PGE2 (0.1, 1 μM) dose dependently protected cultured SIRT1‐deleted RMICs from H2O2 induced cell death (from 57±5% to 31±6% and 18±9% respectively, P<0.0001), suggesting COX2 derived PGE2 contributes to the protective effect of SIRT1. These data are consistent with an important role for SIRT1 in regulating COX2 expression and promoting cell adaptation to oxidative stress in renal medulla. Research supported by NIH.