Increased Prostaglandin E2 Synthesis Research Articles

Paradoxical Induction of ALOX15/15B by Cortisol in Human Amnion Fibroblasts: Implications for Inflammatory Responses of the Fetal Membranes at Parturition

Inflammation of the fetal membranes is an indispensable event of parturition, with increasing prostaglandin E2 (PGE2) synthesis as one of the ultimate products that prime labor onset. In addition to PGE2, the fetal membranes also boast a large capacity for cortisol regeneration. It is intriguing how increased PGE2 synthesis is achieved in the presence of increasing amounts of classical anti-inflammatory glucocorticoids in the fetal membranes at parturition. 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE) synthesized by lipoxygenase 15/15B (ALOX15/15B) has been shown to enhance inflammation-induced PGE2 synthesis in amnion fibroblasts. Here, we examined whether glucocorticoids could induce ALOX15/15B expression and 15(S)-HETE production to promote PGE2 synthesis in amnion fibroblasts at parturition. We found that cortisol and 15(S)-HETE abundance increased parallelly in the amnion at parturition. Cortisol induced ALOX15/15B expression and 15(S)-HETE production paradoxically in amnion fibroblasts. Mechanism study revealed that this paradoxical induction was mediated by p300-mediated histone acetylation and interaction of glucocorticoid receptor with transcription factors CREB and STAT3. Conclusively, cortisol regenerated in the fetal membranes can paradoxically induce ALOX15/15B expression and 15(S)-HETE production in human amnion fibroblasts, which may further assist in the induction of PGE2 synthesis in the inflammatory responses of the fetal membranes for parturition.

Low Salt Delivery Triggers Autocrine Release of Prostaglandin E2 From the Aldosterone-Sensitive Distal Nephron in Familial Hyperkalemic Hypertension Mice.

Aberrant activation of with-no-lysine kinase (WNK)-STE20/SPS1-related proline-alanine-rich protein kinase (SPAK) kinase signaling in the distal convoluted tubule (DCT) causes unbridled activation of the thiazide-sensitive sodium chloride cotransporter (NCC), leading to familial hyperkalemic hypertension (FHHt) in humans. Studies in FHHt mice engineered to constitutively activate SPAK specifically in the DCT (CA-SPAK mice) revealed maladaptive remodeling of the aldosterone sensitive distal nephron (ASDN), characterized by decrease in the potassium excretory channel, renal outer medullary potassium (ROMK), and epithelial sodium channel (ENaC), that contributes to the hyperkalemia. The mechanisms by which NCC activation in DCT promotes remodeling of connecting tubule (CNT) are unknown, but paracrine communication and reduced salt delivery to the ASDN have been suspected. Here, we explore the involvement of prostaglandin E2 (PGE2). We found that PGE2 and the terminal PGE2 synthase, mPGES1, are increased in kidney cortex of CA-SPAK mice, compared to control or SPAK KO mice. Hydrochlorothiazide (HCTZ) reduced PGE2 to control levels, indicating increased PGE2 synthesis is dependent on increased NCC activity. Immunolocalization studies revealed mPGES1 is selectively increased in the CNT of CA-SPAK mice, implicating low salt-delivery to ASDN as the trigger. Salt titration studies in an in vitro ASDN cell model, mouse CCD cell (mCCD-CL1), confirmed PGE2 synthesis is activated by low salt, and revealed that response is paralleled by induction of mPGES1 gene expression. Finally, inhibition of the PGE2 receptor, EP1, in CA-SPAK mice partially restored potassium homeostasis as it partially rescued ROMK protein abundance, but not ENaC. Together, these data indicate low sodium delivery to the ASDN activates PGE2 synthesis and this inhibits ROMK through autocrine activation of the EP1 receptor. These findings provide new insights into the mechanism by which activation of sodium transport in the DCT causes remodeling of the ASDN.

Increased inflammatory lipid metabolism and anaplerotic mitochondrial activation follow acquired resistance to vemurafenib in BRAF-mutant melanoma cells

BackgroundBRAF inhibitors, such as vemurafenib, have shown efficacy in BRAF-mutant melanoma treatment but acquired-resistance invariably develops. Unveiling the potential vulnerabilities associated with vemurafenib resistance could provide rational strategies for combinatorial treatment.MethodsThis work investigates the metabolic characteristics and vulnerabilities of acquired resistance to vemurafenib in three generated BRAF-mutant human melanoma cell clones, analysing metabolic profiles, gene and protein expression in baseline and nutrient withdrawal conditions. Preclinical findings are correlated with gene expression analysis from publicly available clinical datasets.ResultsTwo vemurafenib-resistant clones showed dependency on lipid metabolism and increased prostaglandin E2 synthesis and were more responsive to vemurafenib under EGFR inhibition, potentially implicating inflammatory lipid and EGFR signalling in ERK reactivation and vemurafenib resistance. The third resistant clone showed higher pyruvate-carboxylase (PC) activity indicating increased anaplerotic mitochondrial metabolism, concomitant with reduced GLUT-1, increased PC protein expression and survival advantage under nutrient-depleted conditions. Prostaglandin synthase (PTGES) expression was inversely correlated with melanoma patient survival. Increases in PC and PTGES gene expression were observed in some patients following progression on BRAF inhibitors.ConclusionsAltogether, our data highlight heterogeneity in metabolic adaptations during acquired resistance to vemurafenib in BRAF-mutant melanoma, potentially uncovering key clinically-relevant mechanisms for combinatorial therapeutic targeting.

Prognostic association of PTGS2 (COX-2) over-expression according to BRAF mutation status in colorectal cancer: Results from two prospective cohorts and CALGB 89803 (Alliance) trial

BackgroundProstaglandin-endoperoxide synthase 2 (PTGS2, cyclooxygenase-2, COX-2)-prostaglandin E2 (PGE2) pathway promotes tumour progression. Considering evidence suggesting increased PGE2 synthesis by BRAF mutation in tumour cells, we hypothesised that the association of tumour PTGS2 (COX-2) expression with colorectal cancer mortality might be stronger in BRAF-mutated tumours than in BRAF-wild-type tumours. MethodsUsing 1708 patients, including 1200 stage I-IV colorectal carcinoma cases in the Nurses’ Health Study (NHS) and the Health Professionals Follow-up Study (HPFS) and 508 stage III colon cancer cases in a National Cancer Institute–sponsored randomised controlled trial of adjuvant therapy (CALGB/Alliance 89803), we evaluated tumour PTGS2 (COX-2) expression status using immunohistochemistry. We examined the prognostic association of PTGS2 (COX-2) expression in strata of BRAF mutation status by multivariable Cox proportional hazards regression models to adjust for potential confounders, including disease stage, tumour differentiation, microsatellite instability status and KRAS and PIK3CA mutations. ResultsIn NHS and HPFS, the association of PTGS2 (COX-2) expression with colorectal cancer-specific survival differed by BRAF mutation status (Pinteraction = 0.0005); compared with PTGS2 (COX-2)-negative/low carcinomas, the multivariable-adjusted hazard ratios for PTGS2 (COX-2)-high carcinomas were 2.44 (95% confidence interval, 1.39–4.28) in BRAF-mutated cases and 0.82 (95% confidence interval, 0.65–1.04) in BRAF-wild-type cases. Differential prognostic associations of PTGS2 (COX-2) expression in strata of BRAF mutation status were similarly observed in CALGB/Alliance 89803 trial (Pinteraction = 0.03). ConclusionsThe association of tumour PTGS2 (COX-2) expression with colorectal cancer mortality is stronger in BRAF-mutated tumours than in BRAF-wild-type tumours, supporting interactive roles of PTGS2 (COX-2) expression and BRAF mutation statuses in prognostication of patients with colorectal cancer; ClinicalTrials.gov Identifier, NCT00003835.

Effects of ethanol on human periodontal ligament fibroblasts subjected to static compressive force.

Consumption of toxic substances such as alcohol is widespread in the general population and thus also in patients receiving orthodontic treatment. Since human periodontal ligament (hPDL) fibroblasts play a key role in orthodontic tooth movement (OTM) by expressing cytokines and chemokines, we wanted to clarify whether ethanol modulates the physiological activity and expression pattern of hPDL fibroblasts during static compressive force application. We pre-incubated hPDL fibroblasts for 24h with different ethanol concentrations, corresponding to casual (0.041% blood alcohol concentration [BAC], % by volume) and excessive (0.179%) alcohol consumption. At each ethanol concentration, we incubated the cells for another 48h with and without an additional physiological compressive force of 2g/cm2 occurring during orthodontic tooth movement in compression areas of the periodontal ligament. Thereafter, we analyzed expression and secretion of genes and proteins involved in OTM regulation by RT-qPCR and ELISA. We also performed co-culture experiments to observe hPDL-fibroblast-mediated osteoclastogenesis. We observed no effects of ethanol on cytotoxicity or cell viability of hPDL fibroblasts in the applied concentrations. Ethanol showed an enhancing effect on angiogenesis and activity of alkaline phosphatase. Simultaneously, ethanol reduced the induction of IL-6 and increased prostaglandin E2 synthesis as well as hPDL-fibroblast-mediated osteoclastogenesis without affecting the RANK-L/OPG-system. hPDL fibroblasts thus seem to be a cell type quite resistant to ethanol, as no cytotoxic effects or influence on cell viability were detected. High ethanol concentrations, however, seem to promote bone formation and angiogenesis. Ethanol at 0.179% also enhanced hPDL-induced osteoclastogenesis, indicating increased bone resorption and thus tooth movement velocity to be expected during orthodontic therapy.

The role of bile acids in cellular invasiveness of gastric cancer

BackgroundBile acids have been implicated in the development of digestive tract malignancy by epidemiological, clinical and animal studies. The growth and transformation signaling by most of the bile acids is thought to be related to the induced cyclooxygenase-2 (COX-2) expression and increased production of prostaglandin E2 (PGE2). The highly hydrophobic bile acids such as chenodeoxycholic acid (CD) and deoxycholic acid can promote carcinogenesis and stimulate the invasion of colon cancer cells. On the contrary, ursodeoxycholic acid (UDCA), a less hydrophobic stereoisomer of CD, inhibits proliferation and induces apoptosis in colon cancer cells. We examined the effects of bile acid on human gastric cancer cells MKN-74.MethodsEarly-passage human gastric cancer MKN-74 cells were used for drug treatment, preparation of whole cell lysates, subcellular extracts and Western blot analysis. The levels of PGE2 released by the cells were measured by enzyme inummoassay to indicate COX-2 enzymatic activity. Cellular invasion assay was performed in Boyden chamber.ResultsExposure of CD led to activation of protein kinase C (PKC) alpha, increased COX-2 expression and increased PGE2 synthesis. The induced COX-2 protein expression could be detected within 4 h exposure of 200 μM CD, and it was dose- and time-dependent. PGE2 is the product of COX-2, and has been reported to cause tumor invasion and angiogenesis in animal study. Safingol (SAF), a PKC inhibitor, suppressed the COX-2 protein expression and PGE2 production by CD in MKN-74. Furthermore, UDCA suppressed PGE2 production by CD but did not affect COX-2 protein expression induced by CD. Using a Boyden chamber invasion assay, both SAF and UDCA impeded CD induced tumor invasiveness of MKN-74 by 30–50%.ConclusionsOur results indicate that signaling of hydrophobic bile acid such as CD in gastric cancer cells is through PKC activation and COX-2 induction, which leads to increased cellular invasion. By perturbing the bile acid pool, UDCA attenuates CD-induced PGE2 synthesis and tumor invasiveness.

EHD4 Regulates Prostaglandin E2 Synthesis in Renal Collecting Duct Principal Cells: Potential Implications for AQP2

Eps15 homology domain‐containing protein 4 (EHD4) regulates endocytic recycling. We previously reported that global EHD4 knockout (EHD4‐KO) mice have a higher urine flow and lower urine osmolality than wild‐type (WT) mice. Further, the diuretic phenotype in EHD4‐KO mice is associated with a reduced accumulation of aquaporin 2 (AQP2) in the apical membrane of principal cells of EHD4‐KO mice. However, the mechanism by which EHD4 regulates AQP2 localization is not known. EHD4 and AQP2 share common protein interacting partners, therefore we hypothesized that EHD4 interacts with AQP2, thereby regulating its trafficking. To test this hypothesis, we transfected mouse cortical collecting duct principal (mpkCCD) cells with a GFP‐tagged EHD4 plasmid and visualized the localization of EHD4 and AQP2. EHD4 was found to co‐localize with AQP2 in mpkCCD cells. To assess the interaction between EHD4 and AQP2, co‐immunoprecipitation of AQP2 was performed and the presence of EHD4 in the immunecomplex was measured by Western blotting. EHD4 was not detected in the immune‐complex containing AQP2, suggesting that these two proteins may not interact directly. Pointing to a potential indirect mechanism of regulation, evidence from the literature suggests that the paralog EHD1 binds to cytosolic phospholipase A2α, a key enzyme for eicosanoid synthesis. Prostaglandin E2 (PGE2) is a known negative regulator of AQP2 trafficking. Therefore, we hypothesize that EHD4 deletion increases PGE2 synthesis in principal cells, thereby contributing to the reduced membrane accumulation of AQP2. To test our hypothesis we examined urinary PGE2 excretion by female mice and found it to be significantly higher in EHD4‐KO mice (3595 ± 581 pg/day, n = 6) compared to WT mice (1042 ± 131 pg/day, n = 4; P = 0.008). To exclude a role for flow‐mediated stimulation of PGE2 synthesis, we examined the role of EHD4 in PGE2 synthesis in the mpkCCD cell line. PGE2 concentrations in the surrounding media of EHD4‐siRNA treated mpkCCD cells was significantly higher than of scrambled siRNA‐treated cells (84 ± 7 versus 53 ± 5 pg/ml respectively; n = 6; P = 0.0002). Together, these data suggest that EHD4 regulates collecting duct PGE2 production, providing a possible mechanism by which EHD4 regulates AQP2 localization and urine excretion in vivo.Support or Funding InformationAmerican Heart Association Grant‐In‐Aid 17GRNT33661008This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Rocuronium Bromide Inhibits Inflammation and Pain by Suppressing Nitric Oxide Production and Enhancing Prostaglandin E2 Synthesis in Endothelial Cells.

PurposeRocuronium bromide is a nondepolarizing neuromuscular blocking drug and has been used as an adjunct for relaxation or paralysis of the skeletal muscles, facilitation of endotracheal intubation, and improving surgical conditions during general anesthesia. However, intravenous injection of rocuronium bromide induces injection pain or withdrawal movement. The exact mechanism of rocuronium bromide-induced injection pain or withdrawal movement is not yet understood. We investigated whether rocuronium bromide treatment is involved in the induction of inflammation and pain in vascular endothelial cells.MethodsFor this study, calf pulmonary artery endothelial (CPAE) cells were used, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, Western blot, nitric oxide detection, and prostaglandin E2 immunoassay were conducted.ResultsRocuronium bromide treatment inhibited endothelial nitric oxide synthase and suppressed nitric oxide production in CPAE cells. Rocuronium bromide activated cyclooxygenase-2, inducible nitric oxide synthase and increased prostaglandin E2 synthesis in CPAE cells.ConclusionsRocuronium bromide induced inflammation and pain in CPAE cells. Suppressing nitric oxide production and enhancing prostaglandin E2 synthesis might be associated with rocuronium bromide-induced injection pain or withdrawal movement.

Vitamin D Induces Cyclooxygenase 2 Dependent Prostaglandin E2 Synthesis in HaCaT Keratinocytes.

The active metabolite of vitamin D calcitriol and its analogs are well-known for their anti-inflammatory action in the skin, while their main side effect associated with topical treatment of inflammatory disorders is irritant contact dermatitis. Prostaglandin E2 (PGE2 ) is pro-inflammatory at the onset of inflammation and anti-inflammatory at its resolution. We hypothesized that induction of PGE2 synthesis by calcitriol in epidermal keratinocytes may contribute both to its pro-inflammatory and anti-inflammatory effects on the skin. Treatment of human immortalized HaCaT keratinocytes with calcitriol (3-100 nM, 2-24 h) increased PGE2 production due to increased mRNA and protein expression of COX-2, but not to increase of COX-1 or release of arachidonic acid. The effect of calcitriol on COX-2 mRNA was observed also in primary human keratinocytes. The increase in COX-2 mRNA is associated with COX-2 transcript stabilization. Calcitriol exerts this effect by a rapid (2 h) and protein synthesis independent mode of action that is dependent on PKC and Src kinase activities. Treatment with a COX-2 inhibitor partially prevented the attenuation of the keratinocyte inflammatory response by calcitriol. We conclude that upregulation of COX-2 expression with the consequent increase in PGE2 synthesis may be one of the mechanisms explaining the Janus face of calcitriol as both a promoter and attenuator of cutaneous inflammation. J. Cell. Physiol. 231: 837-843, 2016. © 2015 Wiley Periodicals, Inc.

Sustained adrenergic signaling leads to increased metastasis in ovarian cancer via increased PGE2 synthesis.

Adrenergic stimulation adversely affects tumor growth and metastasis, but the underlying mechanisms are not well understood. Here, we uncovered a novel mechanism by which catecholamines induce inflammation by increasing prostaglandin E2 (PGE2) levels in ovarian cancer cells. Metabolic changes in tumors isolated from patients with depression and mice subjected to restraint stress showed elevated PGE2 levels. Increased metabolites and PTGS2 and PTGES protein levels were found in Skov3-ip1 and HeyA8 cells treated with norepinephrine, and these changes were shown to be mediated by ADRB2 receptor signaling. Silencing PTGS2 resulted in significantly decreased migration and invasion in ovarian cancer cells in the presence of norepinephrine and decreased tumor burden and metastasis in restraint stress orthotopic models. In human ovarian cancer samples, concurrent increased ADRB2, PTGS2 and PTGES expression was associated with reduced overall and progression-free patient survival. In conclusion, increased adrenergic stimulation results in increased PGE2 synthesis via ADRB2-Nf-kB-PTGS2 axis, which drives tumor growth and metastasis.

Prostaglandin E2 EP3 receptor regulates the diuretic response to hypertonic stress (892.38)

We recently showed that activation of prostaglandin E2 (PGE2) EP3 receptors during normal sodium intake is part of a negative feedback mechanism that attenuates cyclooxygenase‐2 (COX‐2) expression in the thick ascending limb (TAL). However, the functional effects of this mechanism have not been determined. Since PGE2 exhibits natriuretic and diuretic functions, we determined the consequences of EP3 receptor manipulation and its relationship to COX‐2 expression and activity in response to hypertonic saline stress. EP3 receptor expression in the outer medulla increased about 2‐fold in Sprague‐Dawley rats given free access to 1% NaCl in the drinking water for 7 days compared with tap water; p<0.05. Ingestion of 1% NaCl also increased COX‐2 mRNA accumulation and protein expression in freshly isolated mTAL tubules, an effect that was augmented when EP3 was blocked. Urine volume increased approximately 2.5‐fold during ingestion of 1% NaCl but was reduced by ~40% on days 1‐7 in rats pretreated with a EP3 receptor agonist (sulprostone: 20 μg·kg−1·day−1) for 3 days. The decrease in urine volume was associated with a decline in COX‐2‐dependent PGE2 synthesis. In contrast, the increase in urine volume induced by 1% NaCl intake was significantly augmented after pretreatment with a selective EP3 receptor antagonist (L‐798106: 100 μg·kg−1·day−1) and was associated with an increase in PGE2 synthesis. Collectively, the data suggest that the COX‐2/PGE2 signaling pathway involving EP3 underlies the diuretic response to increased NaCl intake. The mechanism that contributes to these effects is currently under investigation.Grant Funding Source: NIH HL34300, American Heart Association 12GRNT12060350, Fondecyt 1130741, PGB12‐2007 CARE UC‐SQM

Paricalcitol Attenuates Ischemia-Reperfusion Injury in Mice Kidney

Ischemia-reperfusion injury (IRI) is unavoidable event in renal transplantation, causing the delayed graft function and increased immunogenicity. We investigated whether paricalcitol is renoprotective in a mouse model of IRI, and whether potential mechanism is related with modulation of renal inflammation and prostaglandin E2 (PGE2) synthesis. Paricalcitol (0.3 μ/Kg) was administered to male C57BL/6 mice 24 hours before IRI, and mice were killed at 72 hours after IRI. Treatment with paricalcitol decreased blood urea nitrogen levels, serum creatinine levels, tubular necrosis score. Paricalcitol decreased the number of TUNEL-positive cells and reduced the expression of apoptotic markers. The production of RANTES and tumor necrosis factor-α were reduced by paricalcitol and the infiltration of CD40-positive antigen-presenting cells were decreased in the paricalcitol-treated kidneys. Paricalcitol inhibited the production of Th1 cytokines interleukin (IL)-2 and interferon-γ, which was accompanied with decreased infiltration of T-cells and macrophages. The immunomodulatory effects of paricalcitol also affected the decreased production of Th2 cytokines IL-4 and IL-10. Paricalcitol induced the increased cyclooxygenase (COX)-2 expression and decreased the 15-hydroxyprostaglandin dehydrogenase (15-PGDH) production, which lead to increased endogenous PGE2 synthesis. Of the PGE2 receptors, the expression of EP1 and EP4 were increased in the paricalcitol-treated kidneys and the activation of EP4 receptors increased the expression of survival kinase Akt. In conclusion, our study demonstrated that paricalcitol has a protective effect on renal IRI and that this effect is associated with inhibition of renal inflammatory infiltration, increased PGE2 synthesis and EP4 receptor activation.

Anti-ulcerogenic mechanisms of a lyophilized aqueous extract of Dalbergia monetaria L. in rats, mice and guinea-pigs.

The decoction of Dalbergia monetaria L. is popularly used in Brazil for the treatment of gastric ulcer and the lyophilized aqueous extract (LAE) of D. monetaria has significant anti-ulcerogenic activity and inhibits gastric ulcer lesions induced by pylorus-ligature, ethanol and hypothermic-restraint stress. This work was conducted to identify the antiulcerogenic mechanisms of action of the LAE of D. monetaria. We analysed the effect of the LAE on prostaglandin E2 (PGE2) synthesis and on the characteristics (pH, volume and total acid content) of gastric juice. The antagonism between the LAE and histamine or carbachol was also analysed. The LAE increased gastric mucosal PGE2 synthesis compared with control (89.7+/-21.5 and 52.6+/-11.8 pg mg(-1), respectively) as assayed by enzyme immunoassay in the rat stomach. The LAE reduced the total acid content of gastric juice, but did not modify pH or gastric volume significantly, in Shay rats. Dose-response curves to histamine were shifted to the right in guinea-pig isolated right atria (pD2 values were 5.77+/-0.2 and 5.42+/-0.3, respectively, in the absence and presence of the LAE), with a significant reduction in maximum response (140+/-15.1 and 98+/-13.0, respectively). The same effect was observed when the agonist was isoprenaline. The LAE had no effect on the dose-response curve to carbachol in rat fundus strips. Thus, the protective effect of the LAE on induced gastric lesions might be because of synergistic effects, e.g. increased PGE2 synthesis and antagonism of H2 histamine and beta-adrenergic receptors, reducing gastric acid secretion. Increased PGE2 synthesis results in increased protection, and antagonism of H2 histamine and beta-adrenergic receptors reduces aggressive factors against the gastric mucosa.

Soluble iron modulates iron oxide particle-induced inflammatory responses via prostaglandin E2 synthesis: In vitro and in vivo studies

BackgroundAmbient particulate matter (PM)-associated metals have been shown to play an important role in cardiopulmonary health outcomes. To study the modulation of PM-induced inflammation by leached off metals, we investigated intracellular solubility of radio-labeled iron oxide (59Fe2O3) particles of 0.5 and 1.5 μm geometric mean diameter. Fe2O3 particles were examined for the induction of the release of interleukin 6 (IL-6) as pro-inflammatory and prostaglandin E2 (PGE2) as anti-inflammatory markers in cultured alveolar macrophages (AM) from Wistar Kyoto (WKY) rats. In addition, we exposed male WKY rats to monodispersed Fe2O3 particles by intratracheal instillation (1.3 or 4.0 mg/kg body weight) to examine in vivo inflammation.ResultsParticles of both sizes are insoluble extracellularly in the media but moderately soluble in AM with an intracellular dissolution rate of 0.0037 ± 0.0014 d-1 for 0.5 μm and 0.0016 ± 0.0012 d-1 for 1.5 μm 59Fe2O3 particles. AM exposed in vitro to 1.5 μm particles (10 μg/mL) for 24 h increased IL-6 release (1.8-fold; p < 0.05) and also PGE2 synthesis (1.9-fold; p < 0.01). By contrast, 0.5 μm particles did not enhance IL-6 release but strongly increased PGE2 synthesis (2.5-fold, p < 0.005). Inhibition of PGE2 synthesis by indomethacin caused a pro-inflammatory phenotype as noted by increased IL-6 release from AM exposed to 0.5 μm particles (up to 3-fold; p < 0.005). In the rat lungs, 1.5 but not 0.5 μm particles (4.0 mg/kg) induced neutrophil influx and increased vascular permeability.ConclusionsFe2O3 particle-induced neutrophilic inflammatory response in vivo and pro-inflammatory cytokine release in vitro might be modulated by intracellular soluble iron via PGE2 synthesis. The suppressive effect of intracellular released soluble iron on particle-induced inflammation has implications on how ambient PM-associated but soluble metals influence pulmonary toxicity of ambient PM.

Prostaglandin E2 receptors EP1 and EP4 are up-regulated in rabbit chondrocytes by IL-1β, but not by TNFα

Prostaglandin E2 (PGE2) exerts its actions through the binding of the high affinity EP receptors. We wanted to evaluate the regulation of EP1 and EP4, and the expression of cyclooxygenase (COX)-2, main enzyme responsible for PGE2 synthesis in inflammatory situations, in healthy rabbit chondrocytes stimulated with inflammatory mediators locally increased during osteoarthritis. Articular chondrocytes obtained from healthy rabbits were stimulated with interleukin (IL)-1beta (0.1-100 u/ml) or tumour necrosis factor (TNF)alpha (100 ng/ml). Where indicated, cells were preincubated with non-steroidal antiinflammatory drugs (NSAIDs) (10(-6) M) to inhibit PGE2 synthesis. IL-1beta induced a dose and time-dependent increase in EP1, EP4 and COX-2 expression. However, TNFalpha presence did not induce a significant modification in EP1, EP4 or COX-2 gene expression at any time of study. NSAID presence significantly inhibited PGE2 release but did not modify the EP receptors or COX-2 expression induced by IL-1beta. Our results indicate that EP1 and EP4 receptors, and COX-2 are up-regulated in IL-1beta-stimulated chondrocytes, while no significant modifications are observed in TNFalpha-stimulated cells. NSAIDs were unable to modify the expression of these mediators induced by IL-1beta. Therefore, the increase in PGE2 synthesis, induced by IL-1beta, does not seem to mediate the increase in EP receptor expression, in rabbit chondrocytes.

Protein Kinase Cα-dependent Phosphorylation of the mRNA-stabilizing Factor HuR: Implications for Posttranscriptional Regulation of Cyclooxygenase-2

In this study, we investigated the molecular mechanisms underlying the ATP analogue adenosine-5'-O-(3-thio)triphosphate-induced nucleocytoplasmic shuttling of the mRNA stabilizing factor HuR in human (h) mesangial cells (MC). Using synthetic protein kinase C (PKC) inhibitors and small interfering RNA approaches, we demonstrated that knockdown of PKC alpha efficiently blocked the ATP-dependent nuclear HuR export to the cytoplasm. The functional importance of PKC alpha in HuR shuttling is highlighted by the high cytosolic HuR content detected in hMC stably overexpressing PKC alpha compared with mock-transfected cells. The ATP-induced recruitment of HuR to the cytoplasm is preceded by a direct interaction of PKC alpha with nuclear HuR and accompanied by increased Ser phosphorylation as demonstrated by coimmunoprecipitation experiments. Mapping of putative PKC target sites identified serines 158 and 221 as being indispensable for HuR phosphorylation by PKC alpha. RNA pull-down assay and RNA electrophoretic mobility shift assay demonstrated that the HuR shuttling by ATP is accompanied by an increased HuR binding to cyclooxygenase (COX)-2 mRNA. Physiologically, the ATP-dependent increase in RNA binding is linked with an augmentation in COX-2 mRNA stability and subsequent increase in prostaglandin E(2) synthesis. Regulation of HuR via PKC alpha-dependent phosphorylation emphasizes the importance of posttranslational modification for stimulus-dependent HuR shuttling.

Feed Forward Cycle of Hypotonic Stress-induced ATP Release, Purinergic Receptor Activation, and Growth Stimulation of Prostate Cancer Cells

ATP is released in many cell types upon mechanical strain, the physiological function of extracellular ATP is largely unknown, however. Here we report that ATP released upon hypotonic stress stimulated prostate cancer cell proliferation, activated purinergic receptors, increased intracellular [Ca(2+)](i), and initiated downstream signaling cascades that involved MAPKs ERK1/2 and p38 as well as phosphatidylinositol 3-kinase (PI3K). MAPK activation, the calcium response as well as induction of cell proliferation upon hypotonic stress were inhibited by preincubation with the ATP scavenger apyrase, indicating that hypotonic stress-induced signaling pathways are elicited by released ATP. Hypotonic stress increased prostaglandin E(2) (PGE(2)) synthesis. Consequently, ATP release was inhibited by antagonists of PI3K (LY294002 and wortmannin), phospholipase A(2) (methyl arachidonyl fluorophosphonate (MAFP)), cyclooxygenase-2 (COX-2) (indomethacin, etodolac, NS398) and 5,8,11,14-eicosatetraynoic acid (ETYA), which are involved in arachidonic acid metabolism. Furthermore, ATP release was abolished in the presence of the adenylate cyclase (AC) inhibitor MDL-12,330A, indicating regulation of ATP-release by cAMP. The hypotonic stress-induced ATP release was significantly blunted when the ATP-mediated signal transduction cascade was inhibited on different levels, i.e. purinergic receptors were blocked by suramin and pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), the Ca(2+) response was inhibited upon chelation of intracellular Ca(2+) by 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), and ERK1,2 as well as p38 were inhibited by UO126 and SB203580, respectively. In summary our data demonstrate that hypotonic stress initiates a feed forward cycle of ATP release and purinergic receptor signaling resulting in proliferation of prostate cancer cells.

Effects of Cyclic Mechanical Stress on the Production of Inflammatory Agents by Nucleus Pulposus and Anulus Fibrosus Derived Cells In Vitro

Cyclic mechanical stress (CMS) was applied to cultured nucleus pulposus and anulus fibrosus cells, and the production of inflammatory agents by these cells was evaluated. To investigate the involvement of CMS in the production of inflammatory agents by disc cells. It has been reported that CMS affects degeneration of the disc. However, little is known about the effect of CMS on the production of inflammatory agents by both cell types in vitro. Cells derived from nucleus pulposus and anulus fibrosus of Sprague-Dawley rat tails were cultured with or without CMS applied by the Flexercell Strain Unit (Flexcell International Corp., Hillsborough, NC) in the presence or absence of inflammatory stimulus. Doses of prostaglandin-E2 (PGE2) were measured in the culture supernatants. Semiquantitative evaluations of the expressions of cyclooxygenase (COX)-2 and phospholipase-A2 IIA messenger ribonucleic acids (mRNAs) were also examined. Sole application of CMS on nucleus pulposus and anulus fibrosus cells increased PGE2 synthesis. Coincidence of CMS and inflammatory stimulus synergistically enhanced PGE2 synthesis of both cell types. Anulus fibrosus cells showed a stronger reactivity to these stimuli than nucleus pulposus cells. The expression of COX-2 mRNA of anulus fibrosus cells tended to correlate to the amount of PGE2, whereas COX-2 mRNA was constitutively expressed in nucleus pulposus cells, suggesting that the roles of COX-2 might be different between nucleus pulposus and anulus fibrosus. Phospholipase-A2 IIA mRNA was constitutively expressed in both cell types. The results of this study suggested that CMS might be involved in the pathomechanism of pain induction of lumbar disc diseases.

Molecular mechanism of adaptive cytoprotection induced by ethanol in human gastric cells.

Adaptive cytoprotection is the process by which the pretreatment of cells with low concentrations of a noxious agent prevents the damage caused by a subsequent exposure of those cells to higher concentrations of that same agent. In this study, a human gastric carcinoma cell line was used to examine the molecular mechanism of adaptive cytoprotection induced by ethanol. Pretreatment of cells with 1%-4% ethanol made cells resistant to a subsequent exposure to 8% ethanol. This adaptive cytoprotection was accompanied by an increase in prostaglandin E2 synthesis and was partially inhibited by inhibitors of cyclooxygenase-2, but not by an inhibitor of cyclooxygenase-1. Furthermore, the adaptive cytoprotection was not dependent on newly synthesized proteins and was inhibited by a protein tyrosine kinase inhibitor. Based on these results, it is proposed that the stimulation of cyclooxygenase-2-dependent prostaglandin E2 synthesis, which is regulated post-translationally by protein tyrosine phosphorylation, plays an important role in adaptive cytoprotection induced by ethanol in gastric cells.

Ionizing radiation up-regulates cyclooxygenase-2 in I407 cells through p38 mitogen-activated protein kinase.

The epithelial cell line I407 up-regulates cyclooxygenase-2 (COX-2) mRNA and protein expression following ionizing radiation exposure. Prostaglandin E2 (PGE2) production is concomitantly up-regulated. Irradiation of I407 cells also results in phosphorylation of the p38 mitogen-activated protein kinase and the p38 inhibitor SB203580 abrogates radiation-induced PGE2 synthesis. Wild-type p38alpha (p38alphaWT) and dominant-negative p38alpha (p38alphaDN) stable-transfectant clones of I407 cells were used to examine the role of the p38 mitogen-activated protein kinase pathway in the events controlling PGE2 synthesis after ionizing radiation. Treatment of p38alphaWT clones with gamma-radiation resulted in increased COX-2 protein levels and PGE2 synthesis similar to treated control-transfected cells. In contrast, the p38alphaDN clones failed to up-regulate COX-2 protein or increase PGE2 synthesis when irradiated. Exogenous arachidonate did not restore PGE2 synthesis by p38alphaDN cells. Radiation increased COX-2 mRNA stability and the p38 inhibitor SB203580 attenuated COX-2 mRNA stability in irradiated I407 cells. In contrast, irradiation had no effect on transcription from a COX-2 promoter/luciferase reporter plasmid in the presence or absence of SB203580. The data demonstrate a crucial role for p38alpha in COX-2 expression and PGE2 synthesis in an irradiated transformed epithelial cell line. Furthermore, they indicate that p38 activity is required at a step distal to arachidonate release, most probably COX-2 up-regulation, since exogenous arachidonate did not restore PGE2 synthesis.