Induced Cyclooxygenase-2 mRNA Research Articles

Protein Never in Mitosis Gene A Interacting-1 regulates calpain activity and the degradation of cyclooxygenase-2 in endothelial cells

BackgroundThe peptidyl-proline isomerase, Protein Never in Mitosis Gene A Interacting-1 (PIN1), regulates turnover of inducible nitric oxide synthase (iNOS) in murine aortic endothelial cells (MAEC) stimulated with E. coli endotoxin (LPS) and interferon-γ (IFN). Degradation of iNOS was reduced by a calpain inhibitor, suggesting that PIN1 may affect induction of other calpain-sensitive inflammatory proteins, such as cyclooxygenase (COX)-2, in MAEC.MethodsMAEC, transduced with lentivirus encoding an inactive control short hairpin (sh) RNA or one targeting PIN1 that reduced PIN1 by 85%, were used. Cells were treated with LPS/IFN, calpain inhibitors (carbobenzoxy-valinyl-phenylalaninal (zVF), PD150606), cycloheximide and COX inhibitors to determine the effect of PIN1 depletion on COX-2 and calpain.ResultsLPS or IFN alone did not induce COX-2. However, treatment with 10 μg LPS plus 20 ng IFN per ml induced COX-2 protein 10-fold in Control shRNA MAEC. Induction was significantly greater (47-fold) in PIN1 shRNA cells. COX-2-dependent prostaglandin E2 production increased 3-fold in KD MAEC, but did not increase in Control cells. The additional increase in COX-2 protein due to PIN1 depletion was post-transcriptional, as induction of COX-2 mRNA by LPS/IFN was the same in cells containing or lacking PIN1. Instead, the loss of COX-2 protein, after treatment with cycloheximide to block protein synthesis, was reduced in cells lacking PIN1 in comparison with Control cells, indicating that degradation of the enzyme was reduced. zVF and PD150606 each enhanced the induction of COX-2 by LPS/IFN. zVF also slowed the loss of COX-2 after treatment with cycloheximide, and COX-2 was degraded by exogenous μ-calpain in vitro. In contrast to iNOS, physical interaction between COX-2 and PIN1 was not detected, suggesting that effects of PIN1 on calpain, rather than COX-2 itself, affect COX-2 degradation. While cathepsin activity was unaltered, depletion of PIN1 reduced calpain activity by 55% in comparison with Control shRNA cells.ConclusionPIN1 reduced calpain activity and slowed the degradation of COX-2 in MAEC, an effect recapitulated by an inhibitor of calpain. Given the sensitivity of COX-2 and iNOS to calpain, PIN1 may normally limit induction of these and other calpain substrates by maintaining calpain activity in endothelial cells.

Biomarkers of inflammation in cattle determining the effectiveness of anti-inflammatory drugs

The impact of nonsteroidal anti-inflammatory drugs (NSAID) on prostaglandin E(2) (PGE(2)) production and cyclooxygenase 2 (COX-2) mRNA expression in bovine whole blood (WB) cultures stimulated by lipopolysaccharide (LPS) was determined, using the blood from six Holstein dairy cattle in various stages of lactation. Peak production of PGE(2) occurred 24 h after LPS stimulation but did not result in detectable concentrations of thromboxane B(2) (TXB(2)). The NSAID indomethacin, aspirin, flunixin meglumine, and 4-[5-phenyl-3-(trifluoromethyl)-1H-pyrazol-1-yl] benzene sulfonamide (PTPBS; celecoxib analogue), along with dexamethasone, were all equally effective in reducing the concentration of PGE(2) in the bovine WB culture supernatants. Bradykinin exhibited peak supernatant concentrations 1 h after LPS stimulation. Dexamethasone and the NSAID used in this study were equally effective at inhibiting bradykinin production. Peak induction of COX-2 mRNA occurred 3 h post-LPS stimulation. However, neither dexamethasone nor any of the NSAID used in this study altered COX-2 mRNA concentrations. In contrast, aspirin, flunixin meglumine, and PTPBS reduced tumor necrosis factor-alpha (TNFalpha) mRNA concentration. These results demonstrate that bovine blood cells respond to NSAID therapy like other mammalian cells with respect to inhibition of PGE(2) production and suppression of TNF mRNA induction, but do not inhibit induction of COX-2 mRNA.

PIN1 regulates calpain‐mediated degradation of cyclooxygenase‐2 in endothelial cells

Although the peptidyl‐proline isomerase, PIN1, regulates pro‐inflammatory factors, its role in cyclooxygenase‐2 (COX‐2) expression is unknown. Here, PIN1 was depleted in murine aortic endothelial cells (MAEC) by stable expression short hairpin RNA targeting PIN1 or a control construct. E. coli endotoxin (LPS) plus interferon‐gamma (IFN) induced COX‐2 4.7 times more in PIN1 knockdown (KD) than in control MAEC. KD did not affect induction of COX‐2 mRNA. Instead, KD increased stability of COX‐2 in cycloheximide‐treated cells. Previously, it was found that PIN1 KD reduced calpain activity and increased expression of the endogenous inhibitor, calpastatin. Here, induction COX‐2 was enhanced by the calpain inhibitor, MDL 28170, and loss of COX‐2 after cycloheximide was similar in KD cells and in controls treated with MDL 28170. COX‐2 in extracts of control MAEC was more sensitive to calpain‐1 digestion in vitro than COX‐2 from knockdown cells. Immunodepletion of PIN1 from control cell extracts did not affect calpain sensitivity, and no direct interaction between COX‐2 and PIN1 was seen. These results suggest that PIN1 regulates COX‐2 degradation mainly by its indirect effect on calpain via calpastatin. Thus, PIN1 may normally restrain expression of COX‐2 and other calpain substrates in endothelial cells. Modulation of PIN1 may significantly affect calpain‐dependent vascular activity and inflammatory diseases.

The concentrations of visfatin in the follicular fluids of women undergoing controlled ovarian stimulation are correlated to the number of oocytes retrieved

To compare the concentrations of visfatin in the plasma with those in follicular fluid of women undergoing controlled ovarian stimulation and to discover their correlation to the number of oocytes retrieved. Further, to examine whether FSH or hCG affects the expression of visfatin and whether visfatin affects COX-2 expression in cultured granulosa luteal (GL) cells. A clinical and in vitro study. University hospital. Women subjected to IVF procedures were enrolled in the study. Plasma and follicular fluid visfatin levels were analyzed using ELISA. HCG, FSH, PGE2 and visfatin were added to cultured GL cells. Enzyme immunoassay and RT-PCR were performed. There was no correlation between follicular fluid and plasma visfatin levels (r = 0.443). The number of oocytes retrieved was significantly correlated to follicular visfatin levels in multiple linear regression analysis (r = 0.891, r(2) = 0.794). In vitro experiments on GL cells revealed that hCG and PGE(2) considerably increased visfatin mRNA expression. FSH did not affect visfatin mRNA expression. Treatment with visfatin caused an induction of COX-2 mRNA. The follicular fluid visfatin concentrations are correlated to the number of oocytes retrieved. Human GL cells produce visfatin, and visfatin synthesis is increased by hCG and PGE2 treatment. Visfatin can induce expression of COX-2 mRNA in GL cells.

The role of macrophage mediators in respirable quartz-elicited inflammation

The instigation and persistence of an inflammatory response is widely considered to be critically important in quartz-induced lung cancer and fibrosis. Macrophages have been long recognised as a crucial player in pulmonary inflammation, but evidence for the role of type II epithelial cells is accumulating. Investigations were performed in the rat lung type II cell line RLE and the rat alveolar macrophage cell line NR8383 using Western blotting, NF-κB immunohistochemistry and qRT-PCR of the pro-inflammatory genes iNOS and COX-2, as well as the cellular stress gene HO-1. The direct effect of quartz on pro-inflammatory signalling cascades and gene expression in RLE cells was compared to the effect of conditioned media derived from quartz-treated NR8383 cells. Conditioned media activated the NF-κB signalling pathway and induced a far stronger upregulation of iNOS mRNA than quartz itself. Quartz elicited a stronger, progressive induction of COX-2 and HO-1 mRNA. Our results suggest a differentially mediated inflammatory response, in which reactive particles themselves induce oxidative stress and activation of COX-2, while mediators released from particle-activated macrophages trigger NF-κB activation and iNOS expression in type II cells.

S1P/S1P 2 Signaling Induces Cyclooxygenase-2 Expression in Wilms Tumor

Cyclooxygenase-2 has been reported to be ubiquitously expressed in Wilms tumor, the most common malignant renal tumor in children. However, to our knowledge the regulation mechanism of cyclooxygenase-2 expression remains unexplored. Quantitative real-time polymerase chain reaction and Western blot were performed to detect cyclooxygenase-2 mRNA and protein expression in WiT49 cells upon stimulation by S1P (Biomol(R)), and S1P(2) and cyclooxygenase-2 mRNA expression in 10 freshly frozen Wilms tumor tissues and matched normal tissues. Over expression, blockade and down-regulation of S1P(2) were determined using adenoviral transduction, the S1P(2) antagonist JTE-013 (Tocris Bioscience, Ellisville, Missouri) and small interfering RNA (Dharmacon, Lafayette, Colorado) transfection, respectively. The prostaglandin E(2) level in WiT49 cells was determined by gas chromatography/mass spectrometry. S1P induced cyclooxygenase-2 mRNA and protein expression in WiT49 cells in a concentration dependent manner. Over expression of S1P(2) in WiT49 cells led to a significant increase in cyclooxygenase-2 mRNA and protein expression as well as subsequent prostaglandin E(2) synthesis. In addition, pretreatment of those cells that over expressed S1P(2) with the S1P(2) selective antagonist JTE-013 completely blocked S1P induced cyclooxygenase-2 protein expression. In accordance with these results silencing S1P(2) in WiT49 cells down-regulated S1P induced cyclooxygenase-2 expression. Further research in 10 Wilms tumor specimens showed that S1P(2) mRNA is greatly increased in Wilms tumor. S1P induced cyclooxygenase-2 expression in Wilms tumor and this effect was mediated by S1P(2). This finding extends the biological function of S1P(2) and provides the biochemical basis for developing inhibitors targeting the S1P/cyclooxygenase-2 signaling pathway.

Hypertonic induction of COX2 expression requires TonEBP/NFAT5 in renal epithelial cells

TonEBP/NFAT5 transcription factor is a master regulator of genes involved in osmoprotection. Cyclooxygenase 2 (COX2) has been reported to be a cytoprotective molecule in the inner renal medulla, where cells are physiologically exposed to the highest osmolality of the body. Our aim was to study whether COX2 expression requires TonEBP/NFAT5. Incubation of MDCK cells in hypertonic NaCl medium (500 mOsm/kg H 2O) caused fully translocation of TonEBP/NFAT5 from cytoplasm to nucleoplasm and significantly increased COX2 mRNA, protein and activity levels. TonEBP/NFAT5- siRNA prevented hypertonic induction of COX2 mRNA and protein, leading to a depressed-prostaglandin synthesis and to a decreased cell survival. By using COX2- siRNA and COX2 specific inhibitor NS398, we found that cell survival does not depend on endogenous COX2-induced prostaglandin synthesis, but that cytoprotection strongly correlates with COX2 protein levels. These results demonstrate a new function for TonEBP/NFAT5, i.e., to mediate hypertonic-induced COX2 expression, and suggest that osmoprotection strongly depends on COX2 protein levels.

Targeting of Aryl Hydrocarbon Receptor-Mediated Activation of Cyclooxygenase-2 Expression by the Indole-3-Carbinol Metabolite 3,3′-Diindolylmethane in Breast Cancer Cells

Ligands of the aryl hydrocarbon receptor (AhR) include the environmental xenobiotic 2,3,7,8 tetrachlorodibenzo(p)dioxin (TCDD), polycyclic aryl hydrocarbons, and the dietary compounds 3, 3′-diindolylmethane (DIM), a condensation product of indol-3-carbinol found in Brassica vegetables, and the phytoalexin resveratrol (RES). The AhR and its cofactors regulate the expression of target genes at pentameric (GCGTG) xenobiotic responsive elements (XRE). Because the activation of cyclooxygenase-2 (COX-2) expression by AhR ligands may contribute to inflammation and tumorigenesis, we investigated the epigenetic regulation of the COX-2 gene by TCDD and the reversal effects of DIM in MCF-7 breast cancer cells. Results of DNA binding and chromatin immunoprecipitation (ChIP) studies documented that the treatment with TCDD induced the association of the AhR to XRE harbored in the COX-2 promoter and control CYP1A1 promoter oligonucleotides. The TCDD-induced binding of the AhR was reduced by small-interfering RNA for the AhR or the cotreatment with synthetic (3-methoxy-4-naphthoflavone) or dietary AhR antagonists (DIM, RES). In time course ChIP studies, TCDD induced the rapid (15 min) occupancy by the AhR, the histone acetyl transferase p300, and acetylated histone H4 (AcH4) at the COX-2 promoter. Conversely, the cotreatment of MCF-7 cells with DIM (10 μmol/L) abrogated the TCDD-induced recruitment of the AhR and AcH4 to the COX-2 promoter and the induction of COX-2 mRNA and protein levels. Taken together, these data suggest that naturally occurring modulators of the AhR such as DIM may be effective agents for dietary strategies against epigenetic activation of COX-2 expression by AhR agonists.

COX-2 Inhibition Suppresses the Interferon-γ-Induced Expression of Indoleamine 2,3-Dioxygenase (IDO) in Human Leukemia Cell Lines.

Avoidance of immunosurveillance is presently considered as a hallmark of cancer. Indoleamine 2,3-dioxygenase (IDO) is a cytosolic enzyme which metabolizes L-tryptophan to kynurenines and induces T cell suppression either directly on T cells, or by altering antigen presenting cell function and generating regulatory T cells (Treg). Recently, IDO expression by cancer cells and/or cancer-associated stromal cells has been correlated with the induction of immune dysfunction by human solid tumors, such as ovarian and colon cancer. It has been previously shown that IDO may be expressed by human acute myeloid leukemia, where it promotes the differentiation of Treg cells (Curti A et al. Blood 2007; 109:2871–7). Cyclooxygenase (COX) is the rate-limiting enzyme in the synthesis of prostaglandins and exists in two isoforms, COX-1 and COX-2. COX-2 is over-expressed by several tumor types and reportedly affects multiple pathways involved in tumorigenesis, including angiogenesis, invasion, and tumor-induced immune suppression. The present study aimed to determine whether COX-2 inhibitors may interfere with the IFN-γ-induced expression of IDO-1 in leukemia cells. The IDO-negative HL-60 acute myeloid leukemia cells, characterized by a CD34− CD33+CD119+ phenotype, were challenged with 100 ng/ml IFN-γ, a well-known inducer of IDO. Cytokine-treated HL-60 cells markedly up-regulated IDO-1 mRNA (~ 66-fold after 96 hours of culture) and protein and released a significant amount of kynurenines (12.58±2.66 μM after 72 hours and 19.21±9.8 μM after 96 hours of culture compared with 1.35±0.36 μM and 1.31±0.42 μM from untreated HL-60 cells; p =0.0004). This was associated with an average 35% depletion of tryptophan from culture supernatants. Not unexpectedly, IFN-γ-treated HL-60 cells promoted the in vitro conversion of naïve allogeneic CD4+CD25− T cells into bona fide CD4+CD25+FoxP3+ Treg cells. Phosphorylated signal transducer and activator of transcription (STAT)-1 could be detected in HL-60 cells starting from 48 hours of IFN-γ treatment, but not in untreated HL-60 cells, and its expression kinetics and relative amount closely paralleled those of IDO-1. IFN-γ strongly induced COX-2 mRNA (average fold-induction equal to 17.7±1.37 compared with untreated HL-60 cells) and protein. Conversely, IFN-γ exerted no appreciable effects on the expression levels of the tryptophan transporter molecule (LAT-1/CD98 receptor complex) on HL-60 leukemia cells. The addition of 100 μM nimesulide, a preferential COX-2 inhibitor, to HL-60 cells that were pre-treated with IFN-γ as above detailed translated into an average 54% reduction of kynurenines in culture supernatants (18.78±4.02 μM in the absence of nimesulide compared with 8.75±3.26 μM in cultures established with the drug; p = 0.0082). Superimposable results in terms of kynurenine production and tryptophan depletion were recorded in cultures of HL-60 cells that were simultaneously established with IFN-γ and nimesulide. Exposure to nimesulide attenuated mRNA signals for IDO-1, suggesting that the overall inhibition of IDO activity leading to suppressed kynurenine synthesis might also be attributed to the reduction of IDO-1 gene transcription. Interestingly, nimesulide diminished the expression of phosphorylated STAT-1 in HL-60 cells, indicating the involvement of this signaling pathway in the regulation of IDO-1 expression. In a further set of experiments, aspirin was added for 24 hours at 1 mM (final concentration) to IFN-γ-treated HL-60 cells in order to investigate its effects on IDO expression and function. In good agreement with the above results with nimesulide, aspirin promoted an average 65% inhibition of kynurenine release from IFN-γ-treated HL-60 cells and abrogated tryptophan consumption from culture supernatants. Collectively, these data suggest that COX-2 inhibition might be exploited as a valuable strategy to interfere with IDO-mediated tryptophan catabolism and hopefully to interfere with the tumor-induced immune dysfunction.

Nickel and the Microbial Toxin, MALP-2, Stimulate Proangiogenic Mediators from Human Lung Fibroblasts via a HIF-1α and COX-2–Mediated Pathway

Hypoxia-inducible factor (HIF-1alpha) and cyclooxygenase-2 (COX-2) have been implicated in the regulation of inflammatory-like processes that lead to angiogenesis and fibrotic disorders. Here we demonstrate that in human lung fibroblasts (HLFs) treated with mixed exposures to chemical and microbial stimuli, HIF-1alpha stabilization plays a pivotal role in the induction of COX-2 mRNA and protein, driving the release of vascular endothelial growth factor (VEGF) and proangiogenic and profibrotic chemokines. Upon costimulation with Ni and the mycoplasma-derived lipopeptide macrophage-activating lipopeptide-2 (MALP-2), there was a synergistic induction of CXCL1 and CXCL5 mRNA and protein release from HLF, as well as an enhanced response in VEGF compared to either stimulus alone. Consistent with our previous findings that Ni and MALP-2 stimulates the induction of CXCL8 via a COX-2-mediated pathway, CXCL1, CXCL5, and VEGF release were also regulated by COX-2. Ni induced the stabilization of HIF-1alpha protein in HLF, which was further enhanced in the presence of MALP-2. Depletion of HIF-1alpha using siRNA blocked COX-2 induction by Ni and MALP-2 along with the release of VEGF, CXCL1, CXCL5, and CXCL8. Our results indicate that Ni and MALP-2 interact to promote an angiogenic profibrotic phenotype in HLF. Moreover, these findings reveal a potential role for HIF-1alpha in mediating chemical-induced alterations in cellular response to microbial stimuli, modulating pulmonary inflammation and its consequences such as fibrosis and angiogenesis.

17-Beta Estradiol and Hydroxyestradiols Interact via the NF-Kappa B Pathway to Elevate Cyclooxygenase 2 Expression and Prostaglandin E2 Secretion in Human Bronchial Epithelial Cells

Some epidemiological studies suggest women may be at greater risk for lung cancer than men. Hydroxyestradiols (OHE(2)) are genotoxic and considered as carcinogenic metabolites of estrogens. In this study, we demonstrate that treatment with 0.1 or 1 nM 2/4 OHE(2) significantly increased intracellular oxidative stress, nuclear factor kappa B (NF-kappaB) activity, and cyclooxygenase-2 (COX-2) expression within 24 h in human bronchial epithelial cells BEAS-2B. Cotreatment with the NF-kappaB inhibitor, Bay 117085, prevented OHE(2)-induced COX-2 mRNA accumulation, suggesting that OHE(2) induced COX-2 expression via the NF-kappaB dependent pathway. Furthermore, cotreatment with 10nM 17-beta estradiol (E(2)) significantly enhanced OHE(2)-increased intracellular oxidative stress and significantly increased not only NF-kappaB activity but also COX-2 levels. As COX-2 participates in biosynthesis of prostaglandin E2 (PGE2), PGE2 secretion was enhanced by the cotreatment of 1 nM OHE(2) and 10nM E(2). To understand the enhancement mechanism between OHE(2) and E(2), cells were cotreated with an antioxidant, N-acetylcysteine (NAC), or NF-kB inhibitor, Bay 117085. Both NAC and Bay 117085 prevented the enhancement in COX-2 expression and PGE2 secretion by the cotreatment of E(2) and OHE(2) in BEAS-2B cells. Similarly, Bay 117085 prevented PGE2 secretion induced by the cotreatment of E(2) and OHE(2) in rat lung slice cultures. These results suggest that E(2) enhanced OHE(2)-increased intracellular oxidative stress which increased NF-kappaB activity, COX-2 expression, and PGE2 secretion. Elevated COX-2 expression and PGE2 secretion has been shown to increase the risk of cancer development. Our present data suggest a pathway that contributes an epigenetic mechanism to the overall mechanism of carcinogenesis.

Down‐regulation of CNS cyclooxygenase‐2 message by inhibitors of soluble epoxide hydrolase lead to anti‐hyperalgesia in inflammatory pain

Epoxyeicosatrienoic acids (EETs), metabolites of arachidonic acid produced by cytochrome P450s, have recently been shown to possess anti‐inflammatory and anti‐hyperalgesic activities. Through inhibition of soluble epoxide hydrolase (sEH), the major enzyme that inactivates these metabolites, we have shown that plasma concentrations and residence times of EETs can be increased dramatically. Interestingly, in models of inflammation, inhibition of sEH leads to decreases in circulating pro‐inflammatory lipid mediator concentrations, most prominently prostaglandin E2. We previously showed that sEH inhibitors transcriptionally down‐regulate cyclooxygenase (COX2) message in mouse liver. Indeed co‐administration of sEH inhibitors and selective COX‐2 inhibitors lead to a synergistic reduction in circulating prostaglandin concentrations. Here we show that the analgesic potency of inhibitors of sEH are comparable to that of morphine and that sEH inhibitors penetrate the blood brain barrier and down‐regulate the spinal induction of COX2 mRNA in response to peripheral local inflammation.

Nontypeable Haemophilus influenzae induces COX-2 and PGE2 expression in lung epithelial cells via activation of p38 MAPK and NF-kappa B

BackgroundNontypeable Haemophilus influenzae (NTHi) is an important respiratory pathogen implicated as an infectious trigger in chronic obstructive pulmonary disease, but its molecular interaction with human lung epithelial cells remains unclear. Herein, we tested that the hypothesis that NTHi induces the expression of cyclooxygenase (COX)-2 and prostaglandin E2 (PGE2) via activation of p38 mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-kappa B in pulmonary alveolar epithelial cells.MethodsHuman alveolar epithelial A549 cells were infected with different concentrations of NTHi. The phosphorylation of p38 MAPK was detected by Western blot analysis, the DNA binding activity of NF-kappa B was assessed by electrophoretic mobility shift assay (EMSA), and the expressions of COX-1 and 2 mRNA and PGE2 protein were measured by reverse transcription-polymerase chain reaction (RT-PCR) and enzyme linked immunosorbent assay (ELISA), respectively. The roles of Toll-like receptor (TLR) 2 and TLR4, well known NTHi recognizing receptor in lung epithelial cell and gram-negative bacteria receptor, respectively, on the NTHi-induced COX-2 expression were investigated in the HEK293 cells overexpressing TLR2 and TLR4 in vitro and in the mouse model of NTHi-induced pneumonia by using TLR2 and TLR4 knock-out mice in vivo. In addition, the role of p38 MAPK and NF-kappa B on the NTHi-induced COX-2 and PGE2 expression was investigated by using their specific chemical inhibitors.ResultsNTHi induced COX-2 mRNA expression in a dose-dependent manner, but not COX-1 mRNA expression in A549 cells. The enhanced expression of PGE2 by NTHi infection was significantly decreased by pre-treatment of COX-2 specific inhibitor, but not by COX-1 inhibitor. NTHi induced COX-2 expression was mediated by TLR2 in the epithelial cell in vitro and in the lungs of mice in vivo. NTHi induced phosphorylation of p38 MAPK and up-regulated DNA binding activity of NF-kappa B. Moreover, the expressions of COX-2 and PGE2 were significantly inhibited by specific inhibitors of p38 MAPK and NF-kappa B. However, NTHi-induced DNA binding activity of NF-kappa B was not affected by the inhibition of p38 MAPK.ConclusionNTHi induces COX-2 and PGE2 expression in a p38 MAPK and NF-kappa B-dependent manner through TLR2 in lung epithelial cells in vitro and lung tissues in vivo. The full understanding of the role of endogenous anti-inflammatory PGE2 and its regulation will bring new insight to the resolution of inflammation in pulmonary bacterial infections.

Nickel Alterations of TLR2-Dependent Chemokine Profiles in Lung Fibroblasts Are Mediated by COX-2

Particulate matter air pollution (PM) has been linked with chronic respiratory diseases. Real-life exposures are likely to involve a mixture of chemical and microbial stimuli, yet little attention has been paid to the potential interactions between PM components (e.g., Ni) and microbial agents on the development of inflammatory-like conditions in the lung. Using the Toll-like receptor (TLR)-2 agonist MALP-2 as a lipopeptide relevant to microbial colonization, we hypothesized that nickel sensitizes human lung fibroblasts (HLF) for microbial-driven chemokine release through modulation of TLR signaling pathways. NiSO(4) (200 muM) synergistically enhanced CXCL8, yet antagonized CXCL10 mRNA expression and protein release from HLF in response to MALP-2. RT(2)-PCR pathway-focused array results indicated that NiSO(4) exposure did not alter the expression of TLRs or their downstream signaling mediators, yet significantly increased the expression of cyclooxygenase 2 (COX-2). Moreover, when NiSO(4) was given in combination with MALP-2, there was an amplified induction of COX-2 mRNA and protein along with its metabolic product, PGE2, in HLF. The COX-2 inhibitor, NS-398, attenuated NiSO(4) and MALP-2-induced PGE2 and CXCL8 release and partially reversed the NiSO(4)-dependent inhibition of MALP-2-induced CXCL10 release from HLF. These data indicate that NiSO(4) alters the pattern of TLR-2-dependent chemokine release from HLF via a COX-2-mediated pathway. The quantitative and qualitative effects of NiSO(4) on microbial-driven chemokine release from HLF shed new light on how PM-derived metals can exacerbate respiratory diseases.

Extracellular HIV-Tat Induces Cyclooxygenase-2 in Glial Cells through Activation of Nuclear Factor of Activated T Cells

Both the HIV-1 protein Tat and cyclooxygenase-2 (COX-2) have been involved in the neuropathogenesis associated with HIV-1 infection. However, the relationship among them has not been addressed. Here, we found that extracellular Tat was able to induce COX-2 mRNA and protein expression and PGE2 synthesis in astrocytoma cell lines and primary human astrocytes. Moreover, Tat induced COX-2 promoter transcription. Deletion of NF-kappaB sites of the promoter did not diminish Tat-dependent transcription. Interestingly, Tat did not induce NF-kappaB activity, suggesting that NF-kappaB was not necessary to control COX-2 transcription induced by Tat. In contrast, deletion or mutation of the NFAT and/or AP-1 site abrogated COX-2 induction by Tat. Moreover, Tat induced transcription of NFAT- and AP-1-dependent reporter genes. Transfection of a dominant negative c-Jun mutant protein, TAM-67, or of a dominant negative version of NFAT, efficiently blocked the induction of COX-2 promoter by Tat, confirming the requirement of both transcription factors. Moreover, Tat induced NFAT translocation to the nucleus and binding to the distal site of the COX-2 promoter. The importance of NFAT and AP-1 in COX-2 induction and PGE2 synthesis by Tat was corroborated by using pharmacological inhibitors of the NFAlphaTau, ERK, and JNK pathways. In summary, our results indicate that HIV-1 Tat was able to induce COX-2 and PGE2 synthesis in astrocytic cells through an NFAT/AP-1-dependent mechanism.

Nuclear transcription factors and lipid homeostasis in liver

Nuclear transcription factors and lipid homeostasis in liver

Role of cyclooxygenase-2 in tetrahydrobiopterin-induced dopamine oxidation

Dopamine is considered one of the main contributing factors in the induction of oxidative stress and selective dopaminergic neurodegeneration in Parkinson’s disease. We have previously reported that tetrahydrobiopterin (BH4) leads to dopamine oxidation and renders dopamine-producing cells vulnerable. In the present study, we found that BH4 selectively upregulates cyclooxygenase-2 (COX-2) expression in dopaminergic cells. BH4 caused an induction of COX-2 mRNA, and a critical regulatory motif for BH4-induced transcriptional activation of COX-2 is CRE/AP-1. COX-2 can oxidize dopamine and cause oxidative stress, which is evidenced by the findings that significant increase in dopamine-chrome formation and protein carbonyl contents by BH4-induced COX-2 up-regulation, and the increases are abolished by COX-2 selective inhibitor meloxicam. Increased COX-2 promotes dopaminergic neurodegeneration in both SH-SY5Y cells and rat mesencephalic neurons. These data suggest that BH4-induced COX-2 expression is responsible for dopamine oxidation, leading to the preferential vulnerability of dopaminergic cells in Parkinson’s disease.

An upstream CRE-E-box element is essential for gastrin-dependent activation of the cyclooxygenase-2 gene in human colon cancer cells

Cyclooxygenase-2, the inducible enzyme of arachidonic acid metabolism and prostaglandin synthesis, is over expressed in colorectal cancer. Inhibition of COX-1/-2 by non-steroidal anti-inflammatory drugs is associated with a decreased risk for these malignancies, whereas high serum gastrin levels elevate this risk. As gastrin exhibits trophical effects on colonic epithelium we sought to explore whether it is capable to induce COX-2 expression in a human colon cancer cell line. The aim of this study is the description of the gastrin evoked effects on the transcriptional activity of the COX-2 gene in colorectal cancer cells and the identification of regulatory promoter elements. Reporter gene assays were performed with the gastrin-stimulated human colorectal cancer cell-line Colo-320, which was stable transfected with the human cholecystokinin-B/gastrin receptor cDNA and COX-2-promoter-luciferase constructs containing different segments of the 5′-region of the COX-2 gene or with mutated promoter constructs. Transcription factors were characterized with electrophoretic mobility shift assays. Gastrin-dependent induction of COX-2 mRNA was shown using “real-time” PCR. Resulting elevated Prostaglandin E2-levels were measured using ELISA. Gastrin stimulated the PGE2-generation and COX-2-mRNA expression in human Colo-320-B cells potently, obviously by transactivating the COX-2-promoter using a region between − 68 bp and + 70 bp. Further examinations identified a CRE-E-box element between − 56 bp and − 48 bp mediating the gastrin-effects on the COX-2 gene. Transcription factors binding to this promoter element were USF-1 und -2. These results show the necessity to perform succeeding studies, which could describe possible mechanisms in which gastrin and COX-2 contribute to the induction of colorectal carcinomas.

2,2′,4,4′-Tetrachlorobiphenyl upregulates cyclooxygenase-2 in HL-60 cells via p38 mitogen-activated protein kinase and NF-κB

Polychlorinated biphenyls (PCBs) are ubiquitous, persistent environmental contaminants that affect a number of cellular systems, including neutrophils. Among the effects caused by the noncoplanar PCB 2,2′,4,4′-tetrachlorobiphenyl (2244-TCB) in granulocytic HL-60 cells are increases in superoxide anion production, activation of phospholipase A 2 with subsequent release of arachidonic acid (AA) and upregulation of the inflammatory gene cyclooxygenase-2 (COX-2). The objective of this study was to determine the signal transduction pathways involved in the upregulation of COX-2 by 2244-TCB. Treatment of HL-60 cells with 2244-TCB led to increased expression of COX-2 mRNA. This increase was prevented by the transcriptional inhibitor actinomycin D in cells pretreated with 2244-TCB for 10 min. The increase in COX-2 mRNA was associated with release of 3H-AA, phosphorylation of p38 and extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinases, increased levels of nuclear NF-κB and increased superoxide anion production. Bromoenol lactone, an inhibitor of the calcium-independent phospholipase A 2, reduced 3H-AA release but had no effect on COX-2 mRNA, protein or activity. Pretreatment with SB-202190 or SB-203580, inhibitors of the p38 MAP kinase pathway, prevented the 2244-TCB-mediated induction of COX-2 and phosphorylation of p38 and ERK MAP kinases. These inhibitors did not alter 3H-AA release. Treatment with PD 98059 or U 0126, inhibitors of the MAP/ERK (MEK) pathway, prevented the 2244-TCB-mediated activation of ERK but had no effect on COX-2 induction or p38 phosphorylation. 2244-TCB treatment did not affect c-Jun N-terminal kinase (JNK) phosphorylation. 2244-TCB exposure increased the amount of nuclear NF-κB. This increase was prevented by pretreatment with p38 MAP kinase inhibitors, but not by pretreatment with MEK inhibitors. Pretreatment with inhibitors of NF-κB prevented the 2244-TCB-mediated induction of COX-2 mRNA. 2244-TCB-mediated increases in superoxide anion were prevented by the NADPH oxidase inhibitor apocynin or the free radical scavenger 4-hydroxy TEMPO, but neither of these inhibitors affected the 2244-TCB-induced changes in COX-2 mRNA levels or 3H-AA release. Taken together these data suggest that p38 MAP kinase-dependent activation of NF-κB is critical for the 2244-TCB-mediated upregulation of COX-2 mRNA.

Trifunctional inhibition of COX-2 by extracts of Lonicera japonica: Direct inhibition, transcriptional and post-transcriptional down regulation

The anti-inflammatory properties of aqueous extracts from Lonicera japonica (LJ) flower, an anti-inflammatory treatment in traditional Chinese medicine, were tested by radioimmunoassay of cyclooxygenase isoenzyme-generated prostaglandin E 2 (PGE 2) synthesis as well as by Western and Northern blot analysis of COX-2 protein and mRNA expression, respectively. Boiled LJ aqueous extracts directly inhibited both COX-1 and COX-2 activity, while non-boiled extracts stimulated COX-1. Boiled LJ extracts also inhibited expression of IL-1β-induced COX-2 protein expression and suppressed its mRNA induction by IL-1β in A549 cells. Suppression of COX-2 mRNA induction required a significantly higher dose of aqueous extract than did suppression of protein expression, indicating that compounds in the extract act translationally or post-translationally at lower doses and transcriptionally or post-transcriptionally at higher doses. Direct inhibition of COX isoenzymes as well as down-regulation of COX-2 mRNA and protein may represent the mechanism by which this ancient herbal treatment decreases inflammation.