Prostaglandin E2 Production In Cells Research Articles

Sphingosine-1-phosphate induces COX-2 expression and PGE2 production in human granulosa cells through a S1P1/3-mediated YAP signaling

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid that can regulate various physiological and pathological processes. The expression of S1P has been detected in human follicular fluid. In addition, two S1P receptors, S1P1 and S1P3, are expressed at a high level in human granulosa cells. Cyclooxygenase-2 (COX-2)-derived prostaglandin E2 (PGE2) production plays a critical role in the regulation of ovulation. However, thus far, the effect of S1P on COX-2 expression and PGE2 production in human granulosa cells remains unknown. In the present study, our results demonstrated that treatment with S1P significantly induced COX-2, but not COX-1, expression and increased PGE2 production in human granulosa cells. The stimulatory effects of S1P on COX-2 expression and PGE2 production were attenuated by treatment with specific antagonist of S1P1 or S1P3 and siRNA-mediated knockdown of S1P1 or S1P3. In addition, the COX-2 expression was induced by S1P1 or S1P3 agonist treatment. Interestingly, treatment with S1P activated YAP signaling via S1P1 and S1P3. Moreover, knockdown of YAP partially attenuated S1P-induced COX-2 expression and PGE2 production. These results provide evidence that S1P induces COX-2 expression and PGE2 production in human granulosa cells through a S1P1/3-mediated YAP signaling pathway.

Picfeltarraenin IA inhibits lipopolysaccharide-induced inflammatory cytokine production by the nuclear factor-κB pathway in human pulmonary epithelial A549 cells.

The present study aimed to investigate the effect of picfeltarraenin IA (IA) on respiratory inflammation by analyzing its effect on interleukin (IL)-8 and prostaglandin E2 (PGE2) production. The expression of cyclooxygenase 2 (COX2) in human pulmonary adenocarcinoma epithelial A549 cells in culture was also examined. Human pulmonary epithelial A549 cells and the human monocytic leukemia THP-1 cell line were used in the current study. Cell viability was measured using a methylthiazol tetrazolium assay. The production of IL-8 and PGE2 was investigated using an enzyme-linked immunosorbent assay. The expression of COX2 and nuclear factor-κB (NF-κB)-p65 was examined using western blot analysis. Treatment with lipopolysaccharide (LPS; 10 µg/ml) resulted in the increased production of IL-8 and PGE2, and the increased expression of COX2 in the A549 cells. Furthermore, IA (0.1-10 µmol/l) significantly inhibited PGE2 production and COX2 expression in cells with LPS-induced IL-8, in a concentration-dependent manner. The results suggested that IA downregulates LPS-induced COX2 expression, and inhibits IL-8 and PGE2 production in pulmonary epithelial cells. Additionally, IA was observed to suppress the expression of COX2 in THP-1 cells, and also to regulate the expression of COX2 via the NF-κB pathway in the A549 cells, but not in the THP-1 cells. These results indicate that IA regulates LPS-induced cytokine release in A549 cells via the NF-κB pathway.

Effects of Camphorquinone on Cytotoxicity, Cell Cycle Regulation and Prostaglandin E2 Production of Dental Pulp Cells: Role of ROS, ATM/Chk2, MEK/ERK and Hemeoxygenase-1.

Camphorquinone (CQ) is a popularly-used photosensitizer in composite resin restoration. In this study, the effects of CQ on cytotoxicity and inflammation-related genes and proteins expression of pulp cells were investigated. The role of reactive oxygen species (ROS), ATM/Chk2/p53 and hemeoxygenase-1 (HO-1) and MEK/ERK signaling was also evaluated. We found that ROS and free radicals may play important role in CQ toxicity. CQ (1 and 2 mM) decreased the viability of pulp cells to about 70% and 50% of control, respectively. CQ also induced G2/M cell cycle arrest and apoptosis of pulp cells. The expression of type I collagen, cdc2, cyclin B, and cdc25C was inhibited, while p21, HO-1 and cyclooxygenase-2 (COX-2) were stimulated by CQ. CQ also activated ATM, Chk2, and p53 phosphorylation and GADD45α expression. Besides, exposure to CQ increased cellular ROS level and 8-isoprostane production. CQ also stimulated COX-2 expression and PGE2 production of pulp cells. The reduction of cell viability caused by CQ can be attenuated by N-acetyl-L-cysteine (NAC), catalase and superoxide dismutase (SOD), but can be promoted by Zinc protoporphyin (ZnPP). CQ stimulated ERK1/2 phosphorylation, and U0126 prevented the CQ-induced COX-2 expression and prostaglandin E2 (PGE2) production. These results indicate that CQ may cause cytotoxicity, cell cycle arrest, apoptosis, and PGE2 production of pulp cells. These events could be due to stimulation of ROS and 8-isoprostane production, ATM/Chk2/p53 signaling, HO-1, COX-2 and p21 expression, as well as the inhibition of cdc2, cdc25C and cyclin B1. These results are important for understanding the role of ROS in pathogenesis of pulp necrosis and pulpal inflammation after clinical composite resin filling.

Antagonistic effects of acetylshikonin on LPS-induced NO and PGE2 production in BV2 microglial cells via inhibition of ROS/PI3K/Akt-mediated NF-κB signaling and activation of Nrf2-dependent HO-1.

Although acetylshikonin (ACS) is known to have antioxidant and antitumor activities, whether ACS regulates the expression of proinflammatory mediators in lipopolysaccharide (LPS)-stimulated microglial cells remains unclear. In this study, it was found that ACS isolated from Lithospermum erythrorhizon inhibits LPS-induced nitric oxide (NO) and prostaglandin E2 (PGE2) release by suppressing the expression of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) in BV2 microglial cells. Furthermore, ACS reduced the LPS-induced DNA-binding activity of nuclear factor-κB (NF-κB) and subsequently suppressed iNOS and COX-2 expression. Consistent with these data, ACS attenuated the phosphorylation of PI3K and Akt and suppressed the DNA-binding activity of NF-κB by inducing the generation of reactive oxygen species (ROS) in LPS-stimulated cells. In addition, ACS enhanced heme oxygenase-1 (HO-1) expression via nuclear factor-erythroid 2-related factor 2 (Nrf2) activation. Zinc protoporphyrin, a specific HO-1 inhibitor, partially attenuated the antagonistic effects of ACS on LPS-induced NO and PGE2 production. By contrast, the presence of cobalt protoporphyrin, a specific HO-1 inducer, potently suppressed LPS-induced NO and PGE2 production. These data indicate that ACS downregulates proinflammatory mediators such as NO and PGE2 by suppressing PI3K/Akt-dependent NF-κB activity induced by ROS as well as inducing Nrf2-dependent HO-1 activity. Taken together, ACS might be a good candidate to regulate LPS-mediated inflammatory diseases.

Abstract P4-04-16: Obesity-associated systemic interleukin-6 promotes pre-adipocyte aromatase expression via increased breast cancer cell prostaglandin E2 production

Abstract Introduction: Obesity is associated with a worse breast cancer prognosis, particularly in estrogen receptor alpha (ERα) positive, postmenopausal patients. Resistance to aromatase inhibitor treatment may be one mechanism mediating this effect, as obese patients have a lower response rate to this class of drugs and higher breast tissue aromatase expression. We hypothesized that obesity-associated circulating factors promote breast cancer cell cyclooxygenase-2 (COX-2) expression and prostaglandin E2 (PGE2) production, resulting in an elevation in pre-adipocyte aromatase expression that enhances breast cancer cell estrogen receptor activity and proliferation. Methods: We utilized an in vitro model of the obese patient’s tumor microenvironment in which cultured MCF-7 breast cancer cells and pre-adipocytes were exposed to pooled serum from obese (OB; BMI≥30.0 kg/m2) or normal weight (N; BMI 18.5-24.9 kg/m2) postmenopausal women. Results: Exposure to OB versus N patient sera significantly increased MCF-7 cell COX-2 expression and PGE2 production. This was coupled with 89% greater pre-adipocyte aromatase expression following culture in conditioned media (CM) from MCF-7 cells exposed to OB versus N patient sera, a difference nullified by treatment of the MCF-7 cells with the COX-2 inhibitor celecoxib during CM generation. Previous analysis of the sera revealed significantly higher interleukin 6 (IL-6) concentrations in the OB versus N patient samples. Depletion of IL-6 from the sera resulted in neutralization of the difference between OB and N CM-stimulated aromatase expression. N CM generated with the addition of IL-6 treatment at the time of N patient sera exposure also induced pre-adipocyte aromatase expression that was statistically equivalent to the OB CM condition. Finally, CM from pre-adipocyte/MCF-7 cell co-cultures exposed to OB patient sera stimulated greater MCF-7 and T47D breast cancer cell ERα activity and proliferation in comparison to N. Conclusions: This study indicates that obesity-associated systemic IL-6 indirectly enhances pre-adipocyte aromatase expression via increased breast cancer cell PGE2 production, thereby promoting greater cancer cell ERα activity and proliferation. Investigation regarding the efficacy of a COX-2 inhibitor/aromatase inhibitor combination therapy in the obese postmenopausal patient population is warranted. Citation Format: Laura W Bowers, Andrew J Brenner, Stephen D Hursting, Rajeshwar R Tekmal, Linda A deGraffenried. Obesity-associated systemic interleukin-6 promotes pre-adipocyte aromatase expression via increased breast cancer cell prostaglandin E2 production [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-04-16.

Mechanisms for proteinase-activated receptor 1-triggered prostaglandin E2 generation in mouse osteoblastic MC3T3-E1 cells.

We analyzed signaling mechanisms for prostaglandin E2 (PGE2) production following activation of proteinase-activated receptor-1 (PAR1), a thrombin receptor, in preosteoblastic MC3T3-E1 cells. PAR1 stimulation caused PGE2 release, an effect suppressed by inhibitors of COX-1, COX-2, iPLA2, cPLA2, MAP kinases (MAPKs), Src, EGF receptor (EGFR) tyrosine kinase (EGFR-TK) and matrix metalloproteinase (MMP), but not by an intracellular Ca2+ chelator or inhibitors of PI3 kinase, protein kinase C (PKC) and NF-κB. PAR1 activation induced phosphorylation of MAPKs and upregulation of COX-2. The phosphorylation of p38 MAPK was suppressed by inhibitors of Src and EGFR-TK. The COX-2 upregulation was dependent on ERK, p38, EGFR-TK, Src, and COX-2 itself. PAR1 activation also induced MEK-dependent phosphorylation of cAMP response element binding protein (CREB). All inhibitors of EP1, EP2, EP3 and EP4 receptors suppressed the PAR1-triggered PGE2 release. Exogenously applied PGE2 facilitated PAR1-triggered COX-2 upregulation, but it alone had no effect. Together, the PAR1-mediated PGE2 production in MC3T3-E1 cells appears to involve iPLA2 and cPLA2 for arachidonic acid release, and the MEK/ERK/CREB and Src/MMP/EGFR/p38 pathways for COX-2 upregulation, which is facilitated by endogenous PGE2 formed by COX-2. These signaling mechanisms might underlie the role of the thrombin/PAR1/PGE2 system in the early stage of the bone healing.

Abstract P4-04-16: Obesity-associated systemic interleukin-6 promotes pre-adipocyte aromatase expression via increased breast cancer cell prostaglandin E2 production

Abstract P4-04-16: Obesity-associated systemic interleukin-6 promotes pre-adipocyte aromatase expression via increased breast cancer cell prostaglandin E2 production

Obesity-associated systemic interleukin-6 promotes pre-adipocyte aromatase expression via increased breast cancer cell prostaglandin E2 production.

Obesity is associated with a worse breast cancer prognosis, particularly in estrogen receptor alpha (ERα) positive, postmenopausal patients. We hypothesized that this is mediated in part by an elevation in breast cancer cell cyclooxygenase-2 (COX-2) expression and prostaglandin E2 (PGE2) production that results in greater local pre-adipocyte aromatase expression. We utilized an in vitro model of the obese patient's tumor microenvironment in which cultured MCF-7 breast cancer cells and pre-adipocytes were exposed to pooled serum from obese (OB; BMI ≥ 30.0 kg/m(2)) or normal weight (N; BMI 18.5-24.9 kg/m(2)) postmenopausal women. Exposure to OB versus N sera significantly increased MCF-7 cell COX-2 expression and PGE2 production. Pre-adipocyte aromatase expression was 89 % greater following culture in conditioned media (CM) from MCF-7 cells exposed to OB versus N sera (OB-CM and N-CM, respectively), a difference nullified by MCF-7 cell treatment with the COX-2 inhibitor celecoxib. Previous analysis of the sera revealed significantly higher interleukin-6 (IL-6) concentrations in the OB versus N samples. Depletion of IL-6 from the sera neutralized the difference in pre-adipocyte aromatase expression stimulated by OB-CM versus N-CM. Finally, CM from pre-adipocyte/MCF-7 cell co-cultures exposed to OB sera stimulated greater MCF-7 and T47D breast cancer cell ERα activity and proliferation in comparison to N sera. This study indicates that obesity-associated systemic IL-6 indirectly enhances pre-adipocyte aromatase expression via increased breast cancer cell PGE2 production. Investigation regarding the efficacy of a COX-2 inhibitor/aromatase inhibitor combination therapy in the obese postmenopausal patient population is warranted.

Sphingosine-1-phosphate mediates COX-2 expression and PGE2 /IL-6 secretion via c-Src-dependent AP-1 activation.

Sphingosine-1-phosphate (S1P) has been shown to regulate cyclooxygenase-2 (COX-2)/prostaglandin E2 (PGE2 ) expression and IL-6 secretion in various respiratory diseases. However, the mechanisms underlying S1P-induced COX-2 expression and PGE2 production in human tracheal smooth muscle cells (HTSMCs) remain unclear. Here we demonstrated that S1P markedly induced COX-2 expression. S1P also induced PGE2 and IL-6 secretion which were reduced by the inhibitors of COX-2 (NS-398 and celecoxib). Pretreatment with the inhibitor of S1PR1 (W123), S1PR3 (CAY10444), c-Src (PP1), PYK2 (PF431396), MEK1/2 (U0126), p38 MAPK (SB202190), JNK1/2 (SP600125), or AP-1 (Tanshinone IIA) and transfection with siRNA of S1PR1, S1PR3, c-Src, PYK2, p38, p42, JNK2, c-Jun, or c-Fos reduced S1P-induced COX-2 expression and PGE2 /IL-6 secretion. Moreover, S1P induced c-Src, PYK2, p42/p44 MAPK, JNK1/2, p38 MAPK, and c-Jun phosphorylation. We observed that S1P-induced p42/p44 MAPK and JNK1/2, but not p38 MAPK activation was mediated via a c-Src/PYK2-dependent pathway. S1P also enhanced c-Fos, but not c-Jun mRNA and protein expression and the AP-1 promoter activity. S1P-induced c-Fos mRNA and protein expression, c-Jun phosphorylation, and AP-1 promoter activity was reduced by W123, CAY10444, PP1, PF431396, U0126, SP600125, or SB202190. These results demonstrated that S1P-induced COX-2 expression and PGE2 /IL-6 generation was mediated through S1PR1/3/c-Src/PYK2/p42/p44 MAPK- or JNK1/2- and S1PR1/3/c-Src/p38 MAPK-dependent AP-1 activation.

Prednisone inhibits the IL-1β-induced expression of COX-2 in HEI-OC1 murine auditory cells through the inhibition of ERK-1/2, JNK-1 and AP-1 activity.

Hearing loss can be induced by multiple causes, including cochlear inflammation. Prednisone (PDN) is a well-known steroid clinically used in the treatment of hearing loss. In the present study, we investigated the inhibitory effects and the mechanisms of action of PDN on the expression of cyclooxygenase (COX)-2, an inflammatory enzyme involved in the production of prostaglandins (PGs), in House Ear Institute-Organ of Corti 1 (HEI-OC1) cells (a murine auditory cell line) treated with the inflammatory cytokine, interleukin (IL)-1β. The exposure of HEI-OC1 cells to IL-1β increased COX-2 protein and mRNA expression, COX-2 promoter-driven luciferase activity and COX-2 enzymatic activity [as indicated by the increased production of prostaglandin E2 (PGE2), a major COX-2 metabolite]. However, PDN markedly inhibited the IL-1β-induced COX-2 protein and mRNA expression, COX-2 promoter activity and PGE2 production in the HEI-OC1 cells without affecting COX-2 protein and mRNA stability. PDN further inhibited the IL-1β-induced activation of extracellular signal-regulated kinase (ERK)-1/2 and c-Jun N-terminal kinase (JNK)-1, but had no effect on the cytokine-induced activation of p38 MAPK and proteolysis of IκB-α, a nuclear factor-κB (NF-κB) inhibitory protein. PDN also partially suppressed the IL-1β‑induced activation of activator protein (AP)-1 (but not that of NF-κB) promoter-driven luciferase activity. Of note, the inhibitory effects of PDN on the IL-1β-induced expression of COX-2 and the activation of ERK-1/2 and JNK-1 in the HEI-OC1 cells were significantly diminished by RU486, a glucocorticoid receptor (GR) antagonist, suggesting that PDN exerts its inhibitory effects through GR. To the best of our knowledge, our study demonstrates for the first time that PDN inhibits the IL-1β-induced COX-2 expression and activity in HEI-OC1 cells by COX-2 transcriptional repression, which is partly associated with the inhibition of ERK-1/2, JNK-1 and AP-1 activation.

Differential regulation of c-Met signaling pathways for synovial cell function.

We previously demonstrated that blocking the hepatocyte growth factor (HGF) receptor, c-Met, using a HGF antagonist, NK4, inhibited arthritis in a rheumatoid arthritis (RA) model mice. In the present study, we investigated the role of c-Met signaling in synovial cell function. We demonstrated that synovial tissues from RA patients and MH7A cells, a human RA synovial cell line, expressed HGF and c-Met. HGF and c-Met expression in RA synovium was increased compared to osteoarthritis synovium suggesting increased c-Met signaling in RA synovial cells. The c-Met inhibitor, SU11274, inhibited ERK1/2 and AKT phosphorylation in HGF-stimulated MH7A cells. MEK and PI3K inhibitors suppressed production of matrix metalloproteinase-3 (MMP-3), vascular endothelial growth factor (VEGF) and prostaglandin E2 (PGE2) by MH7A cells, suggesting that c-Met-MEK-ERK and c-Met-PI3K-AKT pathways are involved positively regulating MH7A cell function. Although SU11274 suppressed MMP-3 and VEGF production it enhanced PGE2 production by MH7A cells suggesting that negative regulation by c-Met signaling, independent of the MEK-ERK and PI3K-AKT pathways, is involved in PGE2 production. Blocking c-Met signaling may be therapeutically useful to inhibit angiogenesis and cartilage and bone destruction by inhibiting VEGF and MMP-3 production, while enhancing PGE2 production in synovial cells in RA.

PGE2 signaling and its biosynthesis-related enzymes in cholangiocarcinoma progression.

Prostaglandin E2 (PGE2) involves in progression of various chronic inflammation-related cancers including cholangiocarcinoma (CCA). This study aimed to determine the role of PGE2 signaling, its biosynthesis-related enzymes in a clinical prognosis, and their targeted inhibition in CCA progression. The immunohistochemical staining of cyclooxygenase (COX)-1, COX-2, mPGES-1, EP1, and EP4 was examined in CCA tissues, and their expressions were compared with clinicopathological parameters. The effect of PGE2 on levels of its signaling molecules was examined in CCA cell lines using proteome profiler array. The suppression of mPGES-1 using a small-molecule inhibitor (CAY10526) and small interfering RNA (siRNA) was determined for growth and migration ability in CCA cells. The results indicated that strong expressions of COX-1, COX-2, mPGES-1, EP1, and EP4 were found in CCA tissues as 87.5, 47.5, 52.5, 55, and 80 % of frequencies, respectively. High mPGES-1 expression was significantly correlated with tumor stages III-IV (p = 0.001), lymph node metastasis (p = 0.004), shorter survival (p = 0.009), and prognostic indicator of CCA patients (HR = 2.512, p = 0.041). Expressions of COX-1, COX-2, and EP receptors did not correlate with data tested from patients. PGE2 markedly enhanced protein levels of integrinα6, VE-cadherin, Jagged1, and Notch3, and CAY10526 suppressed those protein levels as well as PGE2 production in CCA cells. CAY10526 and siRNA mPGES-1 markedly suppressed mPGES-1 protein levels, growth, and migration abilities of CCA cell lines. In conclusion, PGE2 signaling strongly promotes CCA progression. Therefore, inhibition of PGE2 synthesis by suppression of its biosynthesis-related enzymes could be useful for prevention and treatment of CCA.

Artesunate exerts an anti‐immunosuppressive effect on cervical cancer by inhibiting PGE2 production and Foxp3 expression

Artesunate (ART), derived from a common traditional Chinese medicine, has beeen used an antimalarial for several years. In this study, the effect and mechanism of ART on anti-human cervical cancer cells was examined. The level of prostaglandin E2 (PGE2 ) and the population of CD4+CD25+Foxp3 regulatory T cells (Treg) in peripheral blood were detected by flow cytometry. In vivo antitumor activity was investigated in mice with cervical cancer by the subcutaneous injection of various concentrations of ART. The concentrations of PGE2 in the supernatants of CaSki cells were measured using an ELISA kit. Cyclooxygenase-2 (COX-2) and Foxp3 expression were determined using quantitative polymerase chain reaction (qPCR) and western blot analysis. The effect of ART on the viability of CaSki and Hela cells was evaluated with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. It was identified that the level of PGE2 and the population of CD4+CD25+Foxp3 Treg cells in the peripheral blood were significantly higher in cervical cancer patients and mice with cervical cancer. ART was capable of inhibiting orthotopic tumor growth, which correlated with a decrease in the level of PGE2 and the percentage of Treg cells in mice with cervical cancer. Furthermore, ART decreased COX-2 expression and the production of PGE2 in CaSki and Hela cells. Notably, the supernatants of CaSki cells treated with ART lowered the expression of Foxp3 in Jurkat T cells, which was capable of being reversed by exogenous PGE2 . Our data revealed that ART may elicit an anti-tumor effect against cervical cancer by inhibition of PGE2 production in CaSki and Hela cells, which resulted in the decrease of Foxp3 expression in T cells. Therefore, ART may be an effective drug for immunotherapy of cervical cancer.

TGF-β1 Downregulates COX-2 Expression Leading to Decrease of PGE2 Production in Human Lung Cancer A549 Cells, Which Is Involved in Fibrotic Response to TGF-β1

Transforming growth factor-ß1 (TGF-β1) is a multifunctional cytokine that is involved in various pathophysiological processes, including cancer progression and fibrotic disorders. Here, we show that treatment with TGF-β1 (5 ng/mL) induced downregulation of cyclooxygenase-2 (COX-2), leading to reduced synthesis of prostaglandin E2 (PGE2), in human lung cancer A549 cells. Treatment of cells with specific inhibitors of COX-2 or PGE2 receptor resulted in growth inhibition, indicating that the COX-2/PGE2 pathway contributes to proliferation in an autocrine manner. TGF-β1 treatment induced growth inhibition, which was attenuated by exogenous PGE2. TGF-β1 is also a potent inducer of epithelial mesenchymal transition (EMT), a phenotype change in which epithelial cells differentiate into fibroblastoid cells. Supplementation with PGE2 or PGE2 receptor EP4 agonist PGE1-alcohol, as compared with EP1/3 agonist sulprostone, inhibited TGF-β1-induced expression of fibronectin and collagen I (extracellular matrix components). Exogenous PGE2 or PGE2 receptor agonists also suppressed actin remodeling induced by TGF-β1. These results suggest that PGE2 has an anti-fibrotic effect. We conclude that TGF-β1-induced downregulation of COX-2/PGE2 signaling is involved in facilitation of fibrotic EMT response in A549 cells.

Immunological role of prostaglandin E2 production in mouse auditory cells in response to LPS

The effect of LPS on the production of prostaglandin E2 (PGE2) in mouse HEI-OC1 auditory cells was examined. HEI-OC1 auditory cells constitutively produce a small amount of PGE2. LPS augmented the PGE2 production via enhanced cyclooxygenase 2 (COX2) expression. LPS-induced augmentation of COX2 expression was dependent on up-regulation of COX2 mRNA expression. LPS induced the production of TNF-α, but not IL-1β· An anti-TNF-α neutralizing Ab significantly inhibited PGE2 production and COX2 mRNA expression in response to LPS. LPS-induced PGE2 production was prevented by a series of pharmacological signaling inhibitors to NF-κB and MAPKs. Pam3CSK4 as a TLR2 ligand, as well as LPS as a TLR4 ligand, augmented the PGE2 production. However, poly I:C as a TLR3 ligand, imiquimod as a TLR7 ligand and CpG DNA as a TLR9 ligand did not augment it. HEI-OC1 cells expressed TLR2, TLR4 and TLR9, but not TLR3 or TLR7. The putative role of LPS-induced PGE2 production in auditory cells is discussed.

Lung inflammation caused by adenosine-5′-triphosphate is mediated via Ca2+/PKCs-dependent COX-2/PGE2 induction

Up-regulation of cyclooxygenase (COX)-2 and prostaglandin E2 (PGE2) are implicated in lung inflammation. Adenosine 5′-triphosphate (ATP) has been shown to act via activation of P2 purinoceptors, leading to COX-2 expression in various inflammatory diseases. The mechanisms of ATP-induced COX-2 expression and PGE2 release remain unclear. We showed that pretreatment with the inhibitors of P2 receptors (PPADS and Suramin), Gq protein (GPA2A), phosphatidylcholine-phospholipase C (PC-PLC; D609), phosphoinositide-phospholipase C (PI-PLC; ET-18-OCH3), Ca2+/calmodulin-dependent protein kinase II (CaMKII; KN62), protein kinase C (PKC; Gö6976, Ro-318220, GF109203X, and rottlerin), MEK1/2 (PD98059), p38 MAPK (SB202190), and nuclear factor-kappaB (NF-κB; Bay11-7082) and the intracellular calcium chelator (BAPTA/AM) or transfection with siRNAs of these molecules and cPLA2 reduced ATPγS-induced COX-2 expression or PGE2 production in A549 cells. In addition, ATPγS-induced elevation of intracellular Ca2+ concentration was attenuated by PPADS, Suramin, D609, or ET-18-OCH3. ATPγS-induced p38 MAPK, p42/p44 MAPK, and NF-κB p65 activation were inhibited by Gö6976, Ro-318220, GF109203X, or rottlerin. ATPγS also induced cPLA2 phosphorylation and activity, which were reduced via inhibition of P2 receptors, PKCs, p38 MAPK, and p42/p44 MAPK. ATPγS-induced cPLA2 expression was inhibited by SB202190, PD98059, or Bay11-7082. In the in vitro study, we established that ATPγS induced PGE2 generation via a cPLA2/COX-2-dependent pathway. In the in vivo study, we found that ATPγS induced COX-2 mRNA expression in the lungs and leukocyte (mainly eosinophils and neutrophils) count in bronchoalveolar lavage (BAL) fluid in mice via a P2 receptors-dependent signaling pathway. We concluded that ATPγS may induce lung inflammation via a cPLA2/COX-2/PGE2-dependent pathway.

Sulforaphane Inhibits Prostaglandin E2 Synthesis by Suppressing Microsomal Prostaglandin E Synthase 1

Sulforaphane (SFN) is a dietary cancer preventive with incompletely characterized mechanism(s) of cancer prevention. Since prostaglandin E2 (PGE2) promotes cancer progression, we hypothesized that SFN may block PGE2 synthesis in cancer cells. We found that SFN indeed blocked PGE2 production in human A549 cancer cells not by inhibiting COX-2, but rather by suppressing the expression of microsomal prostaglandin E synthase (mPGES-1), the enzyme that directly synthesizes PGE2. We identified the Hypoxia Inducible Factor 1 alpha (HIF-1α) as the target of SFN-mediated mPGES-1 suppression. SFN suppressed HIF-1α protein expression and the presence of HIF-1α at the mPGES-1 promoter, resulting in reduced transcription of mPGES-1. Finally, SFN also reduced expression of mPGES-1 and PGE2 production in A549 xenograft tumors in mice. Together, these results point to the HIF-1α, mPGES-1 and PGE2 axis as a potential mediator of the anti-cancer effects of SFN, and illustrate the potential of SFN for therapeutic control of cancer and inflammation. Harmful side effects in patients taking agents that target the more upstream COX-2 enzyme render the downstream target mPGES-1 a significant target for anti-inflammatory therapy. Thus, SFN could prove to be an important therapeutic approach to both cancer and inflammation.

ASC-dependent RIP2 Kinase Regulates Reduced PGE2 Production in Chronic Periodontitis

Levels of prostaglandin E2 (PGE2) and its processing enzyme, prostaglandin-endoperoxide-synthase-2/ cyclooxygenase-2 (PTGS2/COX-2), are elevated in actively progressing periodontal lesions, but suppressed in chronic disease. COX-2 expression is regulated through inflammatory signaling that converges on the mitogen-activated protein kinase (MAPK) pathway. Emerging evidence suggests a role for the inflammatory adaptor protein, ASC/Pycard, in MAPK activation. We postulated that ASC may represent a mediator of the MAPK-mediated regulatory network of PGE2 production. Using RNAi-mediated gene slicing, we demonstrated that ASC regulates COX-2 expression and PGE2 production in THP1 monocytic cells following infection with Porphyromonas gingivalis (Pg). Production of PGE2 did not require the inflammasome adaptor function of ASC, but was dependent on MAPK activation. Furthermore, the MAP kinase kinase kinase CARD domain-containing protein RIPK2 was induced by Pg in an ASC-dependent manner. Reduced ASC and RIPK2 levels were revealed by orthogonal comparison of the expression of the RIPK family in ASC-deficient THP1 cells with that in chronic periodontitis patients. We show that pharmacological inhibition of RIPK2 represses PGE2 secretion, and RNAi-mediated silencing of RIPK2 leads to diminished MAPK activation and PGE2 secretion. These findings identify a novel ASC-RIPK2 axis in the generation of PGE2 that is repressed in patients diagnosed with chronic adult periodontitis.

The Prostaglandin E2Type 4 Receptor Participates in the Response to Acute Oxidant Stress in Airway Epithelial Cells

Oxidative stress is implicated in the pathogenesis of many inflammatory pulmonary diseases, including cystic fibrosis (CF). Delineating how oxidative stress stimulates CF transmembrane conductance regulator (CFTR) in airway epithelial cells is useful, both to increase the understanding of airways host defense and suggest therapeutic approaches to reduce the oxidant stress burden in the CF lung. Using the airway epithelial cell line Calu-3, we investigated the hypothesis that hydrogen peroxide (H₂O₂), which stimulates anion efflux through CFTR, does so via the production of prostaglandin E₂ (PGE₂). Using iodide efflux as a biochemical marker of CFTR activity and short circuit current (I(sc)) recordings, we found that the H₂O₂-stimulated efflux was abolished by cyclooxygenase-1 inhibition and potentially also involves microsomal prostaglandin E synthase-1 activity, implicating a role for PGE₂ production. Furthermore, H₂O₂ application resulted in a rapid release of PGE₂ from Calu-3 cells. We additionally hypothesized that the PGE₂ subtype 4 (EP(4)) receptor was involved in mediating this response. In the presence of (4Z)-7-[(rel-1S,2S,5R)-5-((1,1'-biphenyl-4-yl)methoxy)-2-(4-morpholinyl)-3-oxocyclopentyl]-4-heptenoic acid (AH23848) (which blocks the EP₄ receptor), the H₂O₂-stimulated response was abolished. To investigate this finding in a polarized system, we measured the increase in I(sc) induced by H₂O₂ addition in the presence and absence of AH23848. H₂O₂ induced a robust increase in I(sc), which was significantly attenuated in the presence of AH23848, suggesting some role for the EP₄ receptor. In conclusion, with H₂O₂ as a model oxidant stress, stimulation of CFTR seems to involve PGE₂ production and likely EP₄ receptor activation in Calu-3 airway epithelial cells. This mechanism would be compromised in the CF airways.

Pseudolaric acid B inhibits inducible cyclooxygenase-2 expression via downregulation of the NF-κB pathway in HT-29 cells

Pseudolaric acid B (PAB) is a diterpene acid isolated from the root and trunk bark of Pseudolaric kaempferi Gordon. Previous work has found that PAB has anti-inflammatory and anti-tumor effects in xenograft models of human hepatocellular carcinoma. The aim of this study is to evaluate the correlation between anti-cancer and anti-inflammatory effects of PAB and its molecular mechanisms on HT-29 cells. Production of prostaglandin E2 (PGE2) in HT-29 cells was evaluated by ELISA. mRNA of cyclooxygenase-2 (COX-2) was analyzed by RT-PCR assay. High-content screening (HCS) method was adopted to detect the cytokine mixture (CM)-induced transcription activity of NF-κB and STAT3. Western blotting was used to evaluate the protein expression levels of inflammatory mediators induced by CM. After treatment with PAB in various concentrations, the inhibition rate of cell proliferation was measured with sulforhodamine B assays. For the in vivo studies, tumor-bearing models xenografted with HT-29 cells were developed in nude mice, and following oral administration with PAB, tumor inhibition rate was calculated. PAB inhibited the PGE2 production in HT-29 cells significantly (P < 0.05) with similar results detected at the COX-2 mRNA level. Furthermore, PAB suppressed the COX-2 protein expression and significant nuclear translocation of NF-κB and STAT3 induced by CM, which correlated with a concomitant degradation of I-κB and a decrease in constitutive STAT3 phosphorylation (P < 0.05). Moreover, various concentrations of PAB inhibited the proliferation of HT-29 cells in a dose- and time-dependent manner. In vivo, after treatment with PAB for 17 days, the tumor weight of the 50 and 100 mg/kg treated groups was 0.62 ± 0.15 and 0.54 ± 0.06 g, respectively. When compared to the control group (0.82 ± 0.16 g), the inhibition rate of tumor weight was 24.2% at 50 mg/kg (P < 0.05) and 34.7% at 100 mg/kg (P < 0.001). PAB shows potential anti-cancer activity in HT-29 cells, and its molecular mechanisms are related to the anti-inflammatory action.