Cyclooxygenase-2 Gene Transcription Research Articles

Silica Nanoparticles Shed Light on Intriguing Cellular Pathways in Human Tracheal Smooth Muscle Cells: Revealing COX-2/PGE2 Production through the EGFR/Pyk2 Signaling Axis.

The use of manufactured silica nanoparticles (SiNPs) has become widespread in everyday life, household products, and various industrial applications. While the harmful effects of crystalline silica on the lungs, known as silicosis or chronic pulmonary diseases, are well understood, the impact of SiNPs on the airway is not fully explored. This study aimed to investigate the potential effects of SiNPs on human tracheal smooth muscle cells (HTSMCs). Our findings revealed that SiNPs induced the expression of cyclooxygenase-2 (COX-2) mRNA/protein and the production of prostaglandin E2 (PGE2) without causing cytotoxicity. This induction was transcription-dependent, as confirmed by cell viability assays and COX-2 luciferase reporter assays. Further analysis, including Western blot with pharmacological inhibitors and siRNA interference, showed the involvement of receptor tyrosine kinase (RTK) EGF receptor (EGFR), non-RTK Pyk2, protein kinase Cα (PKCα), and p42/p44 MAPK in the induction process. Notably, EGFR activation initiated cellular signaling that led to NF-κB p65 phosphorylation and translocation into the cell nucleus, where it bound and stimulated COX-2 gene transcription. The resulting COX-2 protein triggered PGE2 production and secretion into the extracellular space. Our study demonstrated that SiNPs mediate COX-2 up-regulation and PGE2 secretion in HTSMCs through the sequential activation of the EGFR/Pyk2/PKCα/p42/p44MAPKs-dependent NF-κB signaling pathway. Since PGE2 can have both physiological bronchodilatory and anti-inflammatory effects, as well as pathological pro-inflammatory effects, the increased PGE2 production in the airway might act as a protective compensatory mechanism and/or a contributing factor during airway exposure to SiNPs.

Sphingosine 1-Phosphate Induces Cyclooxygenase-2/Prostaglandin E2 Expression via PKCα-dependent Mitogen-Activated Protein Kinases and NF-κB Cascade in Human Cardiac Fibroblasts.

In the regions of tissue injuries and inflammatory diseases, sphingosine 1-phosphate (S1P), a proinflammatory mediator, is increased. S1P may induce the upregulation of cyclooxygenase-2 (COX-2)/prostaglandin E2 (PGE2) system in various types of cells to exacerbate heart inflammation. However, the detailed molecular mechanisms by which S1P induces COX-2 expression in human cardiac fibroblasts (HCFs) remain unknown. HCFs were incubated with S1P and analyzed by Western blotting, real time-Polymerase chain reaction (RT-PCR), and immunofluorescent staining. Our results indicated that S1P activated S1PR1/3-dependent transcriptional activity to induce COX-2 expression and PGE2 production. S1P recruited and activated PTX-sensitive Gi or -insensitive Gq protein-coupled S1PR and then stimulated PKCα-dependent phosphorylation of p42/p44 MAPK, p38 MAPK, and JNK1/2, leading to activating transcription factor NF-κB. Moreover, S1P-activated NF-κB was translocated into the nucleus and bound to its corresponding binding sites on COX-2 promoters determined by chromatin immunoprecipitation (ChIP) and promoter-reporter assays, thereby turning on COX-2 gene transcription associated with PGE2 production in HCFs. These results concluded that in HCFs, activation of NF-κB by PKCα-mediated MAPK cascades was essential for S1P-induced up-regulation of the COX-2/PGE2 system. Understanding the mechanisms of COX-2 expression and PGE2 production regulated by the S1P/S1PRs system on cardiac fibroblasts may provide rationally therapeutic interventions for heart injury or inflammatory diseases.

Production of fever mediator PGE2 in human monocytes activated with MDP adjuvant is controlled by signaling from MAPK and p300 HAT: Key role of T cell derived factor

Fever and inflammatory responses were observed in some subjects in early clinical trials of vaccines adjuvanted with muramyl dipeptide (MDP), a NOD2 agonist. Biosynthesis of Prostaglandin E2 (PGE2) that transmits febrile signals to the brain is controlled by an inducible enzyme, Cyclooxygenase 2 (COX-2). MDP alone was not sufficient to induce expression of COX-2 and PGE2 production in vitro. Conditioned medium prepared from Peripheral Blood Mononuclear Cells (PBMCs)-derived CD3-bead purified human T cells (TCM) dramatically increased COX2 gene transcription, COX-2 protein expression, and PGE2 production in MDP-treated monocytes. We explored epigenetic changes at the COX2 promoter using Chromatin Immunoprecipitation assay (ChIP). Increase in COX2 transcription correlated with increased recruitment of RNA polymerase II (Pol II) and p300 histone acetyl transferase (HAT) to the COX2 promoter in monocytes activated with MDP and TCM. The role of p300 HAT was confirmed by using C646, an inhibitor of p300, that reduced binding of acetylated H3 and H4 histones at the COX2 promoter, COX2 transcription, and PGE2 production in monocytes. Binding of p300, Nuclear Factor Kappa B (NF-κB), and Pol II to the COX2 promoter was also sensitive to inhibitors of Mitogen-Activated Protein Kinase (MAPK) pathway and to antibodies against Macrophage-1 (Mac-1) integrin in MDP/TCM-treated monocytes. Importantly, recombinant Glycoprotein Ib alfa (GPIbα), the recently identified factor in TCM, increased binding of NF-κB, p300, and of Pol II to the COX2 promoter and COX2 transcription in MDP-treated monocytes. Our findings suggest that a second signal through Mac-1 and MAPK is triggered by a T cell derived soluble GPIbα protein leading to the assembly of the transcription machinery at the COX2 promoter and production of PGE2 in human monocytes in response to MDP/NOD2 activation.

High Glucose Induces Endothelial COX2 and iNOS Expression via Inhibition of Monomethyltransferase SETD8 Expression

Cyclooxygenase 2 (COX2) and inducible nitric oxide synthase (iNOS) overexpression results in endothelial apoptosis, thus mediating vascular endothelial injury in hyperglycaemia. E26 transformation-specific sequence transcription factor-1 (ESE-1), which belongs to the E26 transformation-specific family of transcription factors, has been demonstrated to be involved in COX2 and iNOS gene transcription. Our previous study indicated that SET domain-containing protein 8 (SETD8) downregulation is involved in high glucose-mediated endothelial inflammation in human umbilical vein endothelial cells (HUVECs). Here, we report that SETD8 plays a major role in hyperglycaemia-induced COX2 and iNOS expression. In HUVECs, upregulation of ESE-1 expression was related to high glucose-mediated apoptosis and COX2 and iNOS expression. High glucose inhibited SETD8 expression, and overexpression of SETD8 diminished the effects of high glucose treatment. Consistently, RNA silencing of SETD8 led to the opposite effect. Furthermore, SETD8 was found to interact with specificity protein 1 (SP1). Blockade of SP1 protected against high glucose-mediated endothelial injury. Mechanistically, we showed that H4K20me1, a downstream target of SETD8, and SP1 were enriched at the ESE-1 promoter region by ChIP assay. Luciferase reporter assays indicated that SETD8 overexpression attenuated ESE-1 promoter activity and augmented the inhibitory effect of siSP1 on ESE-1 promoter activity. In general, our data indicate that SETD8 interacts with SP1 to coregulate ESE-1 expression, which is involved in hyperglycaemia-mediated endothelial apoptosis in HUVECs.

Induction of COX-2 gene transcription and prostaglandin E2 (PGE2) production in primary human monocytes by MDP adjuvant: role of MAPK and p300 HAT

Abstract Muramyl dipeptide (MDP) is the minimal structure responsible for adjuvant activity of peptidoglycans. Early clinical trials of vaccines formulated with MDP revealed adjuvant-induced pyrogenic response. Here we studied signaling pathway leading to production of mediator of fever, PGE2, in human monocytes activated with MDP. MDP alone induced nuclear translocation of NF-kB but only minimal levels of COX-2 mRNA expression and PGE2 production in monocytes. T cell conditioned medium (TCM) prepared from CD3 bead-purified T cells dramatically upregulated COX-2 mRNA and PGE2 in MDP-activated monocytes but did not increase nuclear NF-kB. COX-2 mRNA expression and PGE2 production in monocytes activated with MDP and TCM were sensitive to the inhibitors of ERK1/2, p38, and JNK MAPK. Western blotting confirmed that MDP alone and TCM alone induced phosphorylation of p38 and SAPK/JNK and of ERK1/2, respectively. In addition to MAPK, the role of p300 histone acetyltransferase (HAT) was studied by ChIP assay. TCM induced binding of p300 to a COX-2 promoter. Inhibition of p300 blocked binding of p300 to COX-2 promoter, acetylation of H3 and H4 histones, upregulation of COX-2 mRNA and of PGE2 in monocytes activated with MDP/TCM. Importantly, inhibitors of either p38 or ERK1/2 prevented recruitment of p300 to COX-2 promoter. Taken together these results suggest cooperation between two signaling pathways in monocytes: nuclear translocation of NF-kB and activation of SAPK/JNK and p38 MAPK are induced by MDP, while activation of ERK1/2 and recruitment of p300 to COX-2 promoter are induced by TCM. These two pathways are equally important for p300-facilitated acetylation of H3 and H4 histones at the COX-2 promoter and for up-regulated COX-2/PGE2 production.

Activation of histone acetyltransferase p300 by MAPK pathway is required for optimal production of PGE2 in primary human monocytes activated with muramyl dipeptide adjuvant (MDP).

Abstract Prostaglandin E2 (PGE2) is a proximal mediator of fever produced by human monocytes and macrophages activated by microbial pathogens and, in some cases, by adjuvants that contain analogues of microbial products. Here we studied the signaling pathway of PGE2 production in primary human monocytes activated with MDP, the minimal structure unit responsible for the adjuvant activity of peptidoglycans. MDP alone did not induce COX-2 mRNA expression and PGE2 protein production in monocytes. However, COX-2 mRNA and PGE2 were dramatically up-regulated when MDP was combined with T cell conditioned medium (TCM) prepared from CD3 bead-purified human T cells. MDP-induced nuclear NF-kB was not further increased by TCM. However, TCM induced increase in cytosolic Ca2+ and in phosphorylated ERK1/2 in monocytes, suggesting a role for MAPK signaling. In agreement with this, inhibition of Ca2+, of Ca2+-dependent PKC, and of MAPK kinase enzymes MEK1/2 suppressed PGE2 production in MDP/TCM-treated monocytes. Chromatin immunoprecipitation analysis showed that TCM alone or combined with MDP facilitated association of histone acetyltransferase p300 to the promoter regions of COX-2, IL-1b, and of IL-6 genes. Inhibition of p300 by C646 suppressed the recruitment of p300 and the acetylation of H3 and H4 histones at the COX-2 promoter, prevented COX-2 gene transcription, and PGE2 production. Taken together these results demonstrate that MDP-induced COX-2 gene transcription and PGE2 protein production in primary monocytes requires a second signal delivered by a TCM through a Ca2+-dependent activation of PKC/MEK1/2 signaling pathway followed by phosphorylation of ERK1/2 and p300-facilitated acetylation of histones H3 and H4 at the COX-2 promoter.

Prostaglandin synthesis enzymes' gene transcription in bitches with endometritis.

Endometritis is a major cause of infertility in many domestic species. However, until now the pathogenesis of the endometritis in the bitch is unclear. The aim of this study was to evaluate the gene transcription pattern of prostaglandin (PG) synthesis enzymes (cyclooxygenase [COX2], PTGES-1 and PGFS) in the endometrium of bitches with or without endometritis. Thirty mixed breed bitches in dioestrus, aged between 1 and 5years, and weighing between 10 and 30kg were used. After ovariohysterectomy (OVX), uterine biopsy samples were collected from the middle part of both horns. Then, endometrial epithelium was collected using the cytobrush method and mRNA analysis was performed by real-time RT-PCR. Data were analysed with Kruskal-Wallis anova using the sas® software. Uterine condition was identified by endometrial biopsies (normal endometria [n=11; NE], acute endometritis [n=10; AE] and chronic endometritis [n=9; CE]). The COX2, PTGES-1 and PGFS/AKR1C3 mRNA expression in bitches with and without endometritis was similar. Except for PGFS/AKR1C3, gene transcription of COX2 and PTGES-1 was significantly increased in AE compared with CE. In addition, COX2 gene transcription was significantly increased in AE compared with NE. In contrast, no differences were found for COX2, PTGES-1 and PGFS/AKR1C3 mRNA expression in the samples of NE compared with CE.

β-naphthoflavone interferes with cyp1c1, cox2 and IL-8 gene transcription and leukotriene B4 secretion in Atlantic cod (Gadus morhua) head kidney cells during inflammation.

The objective of this study was to evaluate how β-naphthoflavone interacts with lipopolysaccharide (LPS) and polyinosinic acid: polycytidylic acid (poly I: C) induced innate immune parameters as well as phase I and phase II detoxification enzymes in head kidney cells isolated from Atlantic cod. β-naphthoflavone is a pure agonist of aryl hydrocarbon receptor (AhR) while LPS and poly I: C are not. β-naphthoflavone was added to head kidney leukocytes alone or together with LPS or poly I: C and the responses were evaluated in terms of protein and gene expression. The results showed that β-naphthoflavone (25nM), with and without LPS, significantly induced cytochrome P450 (cyp1c) transcription in cod head kidney cells. β-naphthoflavone (100nM) in the presence of the virus mimic, poly I: C, also increased cyp1c1transcription. LPS induced cyp1c1, cyclooxygenase 2 (cox2), interleukin 1β (IL-1β), interleukin 6 (IL-6) and interleukin 8 (IL-8) transcription, genes that were not affected by the tested β-naphthoflavone concentrations alone. However, β-naphthoflavone (25 and 50nM) strengthened LPS induced cox2 and IL-8 transcription. Cod head kidney cells exposed to β-naphthoflavone concentrations ranging from 25 to 100nM, with and without LPS or poly I: C, expressed AhR protein. LPS or β-naphthoflavone (5-50nM) significantly induced leukotriene B4 (LTB4) secretion compared to control. In conclusion, this study suggests that β-naphthoflavone could interfere with LPS induced immune cell signaling in cod head kidney cells.

Hepatitis C virus-mediated Aurora B kinase inhibition modulates inflammatory pathway and viral infectivity

Chronic hepatitis C is a leading cause of chronic liver disease, cirrhosis and hepatocellular carcinoma. DNA methylation and histone covalent modifications constitute crucial mechanisms of genomic instability in human disease, including liver fibrosis and hepatocellular carcinoma. The present work studies the consequences of HCV-induced histone modifications in early stages of infection. Human primary hepatocytes and HuH7.5 cells were transiently transfected with the core protein of hepatitis C virus (HCV) genotypes 1a, 1b, and 2a. Infectious genotype 2a HCV in culture was also used. We show that HCV and core protein inhibit the phosphorylation of Serine 10 in histone 3. The inhibition is due to the direct interaction between HCV core and Aurora B kinase (AURKB) that results in a decrease of AURKB activity. HCV and core significantly downregulate NF-κB and COX-2 transcription, two proteins with anti-apoptotic and proliferative effects implicated in the control of the inflammatory response. AURKB depletion reduced HCV and core repression of NF-κB and COX-2 gene transcription and AURKB overexpression reversed the viral effect. AURKB abrogation increased HCV specific infectivity which was decreased when AURKB was overexpressed. The core-mediated decrease of AURKB activity may play a role in the inflammatory pathway during the initial steps of viral infection, while ensuring HCV infectivity.

Suitable in vitro Eimeria arloingi macromeront formation in host endothelial cells and modulation of adhesion molecule, cytokine and chemokine gene transcription.

Eimeria arloingi infections can cause severe haemorrhagic enteritis in young goat kids, thereby leading to high economic losses in goat industry worldwide. We aimed to isolate a new E. arloingi strain and establish a suitable in vitro culture system for the first merogony. E. arloingi oocysts were collected from naturally infected goat kids in the province of Alentejo, Portugal. For the maintenance of E. arloingi (strain A), kids kept under strict parasite-free conditions were orally infected with 10(3) sporulated oocysts each. Further, a new excystation protocol was successfully established to obtain viable sporozoites for further in vitro development in primary bovine umbilical vein endothelial cells (BUVEC). Overall, E. arloingi first merogony was successfully accomplished in BUVEC leading to macromeront formation (up to 150 μm) and the release of fully developed merozoites I stages. Moreover, host endothelial cell-parasite interactions were investigated in order to determine the extent of modulation carried out by E. arloingi in BUVEC during the first merogony. Gene transcription of adhesion molecules (E-selectin, P-selectin, VCAM-1, ICAM-1) was enhanced in the first hours post-infection (p.i.) in E. arloingi-infected BUVEC. BUVEC activation due to invasion was also shown by increased chemokine (CXCL8, CCL2, CCL5), cytokine (GM-CSF) and COX-2 gene transcription. The new E. arloingi (strain A) will be useful for better comprehension of early host innate immune reactions against this parasite in vitro/in vivo as well as to further our investigations in the complex Eimeria-host endothelial cell interactions.

Celecoxib sensitizes Staphylococcus aureus to antibiotics in macrophages by modulating SIRT1.

We have previously shown that celecoxib in combination with an antibiotic, increase the bacterial sensitivity to antibiotics. However, the underlying molecular mechanism remained elusive. Efficacy of the combinatorial treatment of celecoxib and ampicillin in vitro was evaluated on macrophage-phagocytosed S aureus. To elucidate the mechanism, signaling pathway of infection and inflammation involving TLR2, JNK, SIRT1 and NF-κB was studied by FACS, Western blot, ELISA and activity assays. Combinatorial treatment of ampicillin and celecoxib reduced the bacterial load in the macrophages. Further studies clearly suggested the activation of the master regulator of oxidative stress and inflammation SIRT1,, by celecoxib when used alone and/or in combination with ampicillin. Also, the results indicated that celecoxib inhibited JNK phosphorylation thereby stabilizing and activating SIRT1 protein that inhibited the COX-2 gene transcription with a significant decrease in the levels of protein inflammatory cytokines like IL-6, MIP-1α and IL-1β via inhibition of NF-κB. SIRT1 activation by celecoxib also resulted in increase of catalase and peroxidase activity with a decrease in Nitric oxide levels. In conclusion, we demonstrate a novel role of celecoxib in controlling inflammation as an enhancer of antibiotic activity against bacteria by modulating SIRT1.

The role of hypoxia-inducible factor-1 and vascular endothelial growth factor in late-phase preconditioning with xenon, isoflurane and levosimendan in rat cardiomyocytes

The protective effects of late-phase preconditioning can be triggered by several stimuli. Unfortunately, the transfer from bench to bedside still represents a challenge, as concomitant medication or diseases influence the complex signalling pathways involved. In an established model of primary neonatal rat cardiomyocytes, we analysed the cardioprotective effects of three different stimulating pharmaceuticals of clinical relevance. The effect of additional β-blocker treatment was studied as these were previously shown to negatively influence preconditioning. Twenty-four hours prior to hypoxia, cells pre-treated with or without metoprolol (0.55 µg/ml) were preconditioned with isoflurane, levosimendan or xenon. The influences of these stimuli on hypoxia-inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF) as well as inducible and endothelial nitric synthase (iNOS/eNOS) and cyclooxygenase-2 (COX-2) were analysed by polymerase chain reaction and western blotting. The preconditioning was proved by trypan blue cell counts following 5 h of hypoxia and confirmed by fluorescence staining. Five hours of hypoxia reduced cell survival in unpreconditioned control cells to 44 ± 4%. Surviving cell count was significantly higher in cells preconditioned either by 2 × 15 min isoflurane (70 ± 16%; P = 0.005) or by xenon (59 ± 8%; P = 0.049). Xenon-preconditioned cells showed a significantly elevated content of VEGF (0.025 ± 0.010 IDV [integrated density values when compared with GAPDH] vs 0.003 ± 0.006 IDV in controls; P = 0.0003). The protein expression of HIF-1α was increased both by levosimendan (0.563 ± 0.175 IDV vs 0.142 ± 0.042 IDV in controls; P = 0.0289) and by xenon (0.868 ± 0.222 IDV; P < 0.0001) pretreatment. A significant elevation of mRNA expression of iNOS was measureable following preconditioning by xenon but not by the other chosen stimuli. eNOS mRNA expression was found to be suppressed by β-blocker treatment for all stimuli. In our model, independently of the chosen stimulus, β-blocker treatment had no significant effect on cell survival. We found that the stimulation of late-phase preconditioning involves several distinct pathways that are variably addressed by the different stimuli. In contrast to isoflurane treatment, xenon-induced preconditioning does not lead to an increase in COX-2 gene transcription but to a significant increase in HIF-1α and subsequently VEGF.

Upregulation of COX-2/PGE2 by ET-1 Mediated Through Ca2+-Dependent Signals in Mouse Brain Microvascular Endothelial Cells

Endothelin-1 (ET-1), a proinflammatory mediator, is elevated in the regions of several brain inflammatory disorders, implying that ET-1 may contribute to inflammatory responses. The deleterious effects of ET-1 on brain endothelial cells may aggravate brain inflammation mediated through the upregulation of cyclooxygenase-2 (COX-2)/prostaglandin E2 (PGE2) system. However, the signaling mechanisms underlying ET-1-induced COX-2 expression in mouse brain microvascular endothelial cells (bEnd.3 cells) remain unclear. Herein, we investigated the effects of Ca2+-dependent protein kinases on ET-1-induced COX-2 expression and PGE2 release in bEnd.3 cells. The data obtained with Western blotting, reverse transcription PCR, and intracellular Ca2+ analyses showed that ET-1-induced COX-2 expression was mediated through phosphatidylinositol-phospholipase C (PI-PLC) and phosphatidylcholine-phospholipase C (PC-PLC)/Ca2+-dependent activation of protein kinase C-alpha (PKC-α) and calmodulin kinase II (CaMKII) cascades. Next, we demonstrated that ET-1 stimulated intracellular Ca2+ increase, phoshorylation of PKC-α, CaMKII, and mitogen-activated protein kinases (MAPKs) (ERK1/2, p38 MAPK, and JNK1/2) and then activated the activating transcription factor 2 (ATF2)/activator protein 1 (AP-1) via Gq/i protein-coupled ETB receptors. Moreover, the data of chromatin immunoprecipitation and promoter reporter assay demonstrated that the activated ATF2/AP-1 and p300 bound to its corresponding binding sites within COX-2 promoter, thereby turning on COX-2 gene transcription. Finally, upregulation of COX-2 by ET-1 promoted PGE2 biosynthesis and release in these cells. Taken together, these results demonstrate that in bEnd.3 cells, Ca2+-dependent PKC-α and CaMKII linking to MAPKs, ATF2/AP-1, and p300 cascade is essential for ET-1-induced COX-2 upregulation. Understanding the mechanisms of COX-2/PGE2 system upregulated by ET-1 on brain microvascular endothelial cells may provide rational therapeutic interventions for brain injury and inflammatory diseases.

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

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

Telomerase reverse transcriptase inhibition stimulates cyclooxygenase 2 expression in cancer cells and synergizes with celecoxib to exert anti-cancer effects

Background:Telomerase and telomerase reverse transcriptase (hTERT) confer cancer cells sustained proliferation and survival potentials. Targeting telomerase or hTERT is a novel anti-cancer strategy. However, telomerase/hTERT inhibition alone has minimal clinical efficacy. We explored the relationship between hTERT and cyclooxygenase 2 (COX2) and evaluated synergistic anti-cancer effects of targeting both hTERT and COX2.Methods:hTERT was depleted in gastric and cervical cancer cells using small interfering RNA (siRNA) and analysed for COX2 expression using quantitative PCR and immunoblotting. Viable cells and apoptotic cells in gastric cancer cells treated with hTERT siRNA or/and the COX2 inhibitor celecoxib were measured using Trypen blue exclusion and flow cytometry. The in vivo anti-cancer effect of hTERT depletion or/and celecoxib was evaluated using mouse xenograft models.Results:Knocking down hTERT expression in cancer cells led to robust increases in mRNA and protein levels of COX2. The COX2 promoter activity increased substantially in hTERT-depleted cells. hTERT depletion led to the activation of p38 mitogen-activated protein kinase responsible for the stimulation of COX2 gene transcription. hTERT depletion or celecoxib alone did not affect cancer cell survival, whereas their combination synergistically killed them both in vitro and in vivo.Conclusion:hTERT induces COX2 expression and simultaneously targeting hTERT and COX2 synergistically kills cancer cells.

Abstract 582: hTERT inhibition stimulates cyclooxygenase2 (COX2) expression in cancer cells and synergizes with celecoxib to exert anti-cancer effects.

Abstract Telomerase and telomerase reverse transcriptase (hTERT) confer cancer cells sustained proliferation and survival potentials through telomere lengthening dependent and independent activities. Targeting telomerase or hTERT is thus a novel anti-cancer strategy. However, telomerase/hTERT inhibition alone has minimal clinical efficacy. We explored the relationship between hTERT and oncogenic cyclooxygenase 2 (COX2) and evaluated the synergistic anti-cancer effects of targeting both hTERT and COX2. hTERT depletion in gastric and cervical cancer cells led to robust increases in mRNA and protein levels of COX2. The COX2 promoter activity increased substantially in hTERT-depleted cancer cells. Mechanistically, hTERT depletion led to the activation of p38 MAPK that was responsible for the stimulation of COX2 gene transcription. hTERT depletion or the COX2 inhibitor celecoxib alone did not affect gastric cancer cell survival, while their combination synergistically killed them both in vitro and in vivo. Taken together, hTERT induces COX2 expression in cancer cells and simultaneously targeting hTERT and COX2 synergistically kills cancer cells, which may help path a new avenue in telomerase-based cancer therapeutics. Citation Format: Tiantian Liu, Magnus Björkholm, Dawei Xu. hTERT inhibition stimulates cyclooxygenase2 (COX2) expression in cancer cells and synergizes with celecoxib to exert anti-cancer effects. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 582. doi:10.1158/1538-7445.AM2013-582 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Pin1 is required for ultraviolet A-stimulated cyclooxygenase-2 induction in mouse epidermal cells

Ultraviolet A (UVA) radiation (320–400nm) is considered a major cause of human skin photoaging and skin cancer. Overexpression of cyclooxygenase-2 (COX-2) leads to prostanoid formation in skin tissue, disturbs the balance between proliferation and apoptosis, and subsequently promotes tumorigenesis. The peptidyl-prolyl isomerase Pin1 is known to be overexpressed in most cancer cell types and plays an important role in oncogenesis. Here, we studied whether exposure of JB6 Cl41 mouse epidermal cells to UVA affects COX-2 expression and the possible involvement of Pin1 activation. UVA increased COX-2 protein expression and prostaglandin E2 production in an energy-dependent manner. Pre-exposure of JB6 Cl41 cells to UVA potentiated epidermal growth factor-induced anchorage-independent growth; this effect was significantly suppressed by inhibition of COX-2. UVA-stimulated COX-2 expression was significantly decreased by inhibition of Pin1. The increased COX-2 gene transcription in response to UVA was preceded by activation of the transcription factors nuclear factor-κB (NF-κB), cAMP response element-binding protein (CREB), CCAAT/enhancer-binding proteins α and β (C/EBPα and C/EBPβ) and c-Jun/activator protein-1 (AP-1). Pin1 inhibitor treatment suppressed the activation of NF-κB, CREB, and C/EBP by UVA irradiation. Conversely, JB6 C141 cells overexpressing Pin1 showed increased basal COX-2 expression and NF-κB, CREB, C/EBP, and AP-1 activities. These results suggest that UVA-induced COX-2 expression is mediated by Pin1 activation and this is associated with malignant transformation of epidermal cells.

Up-regulation of COX-2/PGE2 by endothelin-1 via MAPK-dependent NF-κB pathway in mouse brain microvascular endothelial cells

BackgroundEndothelin-1 (ET-1) is a proinflammatory mediator and elevated in the regions of several brain injury and inflammatory diseases. The deleterious effects of ET-1 on endothelial cells may aggravate brain inflammation mediated through the regulation of cyclooxygenase-2 (COX-2)/prostaglandin E2 (PGE2) system in various cell types. However, the signaling mechanisms underlying ET-1-induced COX-2 expression in brain microvascular endothelial cells remain unclear. Herein we investigated the effects of ET-1 in COX-2 regulation in mouse brain microvascular endothelial (bEnd.3) cells.ResultsThe data obtained with Western blotting, RT-PCR, and immunofluorescent staining analyses showed that ET-1-induced COX-2 expression was mediated through an ETB-dependent transcriptional activation. Engagement of Gi- and Gq-protein-coupled ETB receptors by ET-1 led to phosphorylation of ERK1/2, p38 MAPK, and JNK1/2 and then activated transcription factor NF-κB. Moreover, the data of chromatin immunoprecipitation (ChIP) and promoter reporter assay demonstrated that the activated NF-κB was translocated into nucleus and bound to its corresponding binding sites in COX-2 promoter, thereby turning on COX-2 gene transcription. Finally, up-regulation of COX-2 by ET-1 promoted PGE2 release in these cells.ConclusionsThese results suggested that in mouse bEnd.3 cells, activation of NF-κB by ETB-dependent MAPK cascades is essential for ET-1-induced up-regulation of COX-2/PGE2 system. Understanding the mechanisms of COX-2 expression and PGE2 release regulated by ET-1/ETB system on brain microvascular endothelial cells may provide rationally therapeutic interventions for brain injury or inflammatory diseases.

C-Src-dependent EGF receptor transactivation contributes to ET-1-induced COX-2 expression in brain microvascular endothelial cells

BackgroundEndothelin-1 (ET-1) is elevated and participates in the regulation of several brain inflammatory disorders. The deleterious effects of ET-1 on endothelial cells may aggravate brain inflammation mediated through the upregulation of cyclooxygenase-2 (COX-2) gene expression. However, the signaling mechanisms underlying ET-1-induced COX-2 expression in brain microvascular endothelial cells remain unclear.ObjectiveThe goal of this study was to examine whether ET-1-induced COX-2 expression and prostaglandin E2 (PGE2) release were mediated through a c-Src-dependent transactivation of epidermal growth factor receptor (EGFR) pathway in brain microvascular endothelial cells (bEnd.3 cells).MethodsThe expression of COX-2 induced by ET-1 was evaluated by Western blotting and RT-PCR analysis. The COX-2 regulatory signaling pathways were investigated by pretreatment with pharmacological inhibitors, short hairpin RNA (shRNA) or small interfering RNA (siRNA) transfection, chromatin immunoprecipitation (ChIP), and promoter activity reporter assays. Finally, we determined the PGE2 level as a marker of functional activity of COX-2 expression.ResultsFirst, the data showed that ET-1-induced COX-2 expression was mediated through a c-Src-dependent transactivation of EGFR/PI3K/Akt cascade. Next, we demonstrated that ET-1 stimulated activation (phosphorylation) of c-Src/EGFR/Akt/MAPKs (ERK1/2, p38 MAPK, and JNK1/2) and then activated the c-Jun/activator protein 1 (AP-1) via Gq/i protein-coupled ETB receptors. The activated c-Jun/AP-1 bound to its corresponding binding sites within COX-2 promoter, thereby turning on COX-2 gene transcription. Ultimately, upregulation of COX-2 by ET-1 promoted PGE2 biosynthesis and release in bEnd.3 cells.ConclusionsThese results demonstrate that in bEnd.3 cells, c-Src-dependent transactivation of EGFR/PI3K/Akt and MAPKs linking to c-Jun/AP-1 cascade is essential for ET-1-induced COX-2 upregulation. Understanding the mechanisms of COX-2 expression and PGE2 release regulated by ET-1/ETB system on brain microvascular endothelial cells may provide rational therapeutic interventions for brain injury and inflammatory diseases.

Effect of mifepristone on COX-2 both in eutopic and ectopic endometrium in mouse endometriotic model

To study the influence of mifepristone on the expression of cyclooxygenase 2 (COX-2) protein and COX-2 mRNA and then to evaluate the mechanism. After the establishment of 30 mice endometriosis models, the mice were randomly divided into six groups with 5 mice each group and assigned to experimental and control groups of 1-, 4- and 6-week circle according to whether mifepristone (0.13 mg d(-1)) was taken or not. Small animal optical imaging system was used to detect the fluorescent intensity of the ectopic tissue. Reverse transcript-polymerase chain reaction and western blot was used to examine COX-2 protein and COX-2 mRNA expression. ELISA was used to examine concentration of PGE(2) in serum. Mifepristone could not affect the fluorescent intensity of the ectopic endometrium after it was taken 1, 4, and 6 (P > 0.05). However, it could decrease the transcription of COX-2 mRNA in the 1 and 4 week groups (P < 0.05), while the difference in the 6 week group was not significant (P > 0.05). It could decrease the expression of COX-2 protein after it was taken 4 and 6 weeks (P < 0.05). The serous PGE(2) in the trial groups was lower than that in the control groups, but the difference was not significant (P > 0.05). This study showed that mifepristone could not affect the size of the ectopic endometrium, but it could decrease the transcription of COX-2 gene and then reduce the expression of COX-2 protein and its product PGE(2) which is an important factor which mediate pain. This maybe another mechanism that mifepristone takes effect through anti-inflammatory path.