Stimulated Prostaglandin E2 Production Research Articles

Abstract 3698: Epigenetic modification induced reprogramming of PUFAs-associated metabolism during trastuzumab resistance formation of HER2-positive breast cancer

Abstract Background: Secondary trastuzumab resistance seriously affects the treatment of HER2-positive breast cancer. Although studies have demonstrated several potential reasons that cause trastuzumab resistance, we still have poor knowledge about the changes in cell itself as well as its interaction with tumor microenvironment (TME) components during resistance formation. Here we suggested that altered histone modification reprograms polyunsaturated fatty acids (PUFAs) metabolism, which reduces cellular PUFAs and stimulates prostaglandin E2 (PGE2) production. Excessive PGE2 may further affect both cancer cells as well as immune cells in TME. Materials and Methods: Secondary trastuzumab-resistant cell line SKBR3_HR was generated from sensitive SKBR3 cells by raising trastuzumab concentration gently from 1ug/ml to 20ug/ml in 30 weeks. Cell viabilities were measured by CCK8 test. CUT&Tag with anti-H3K4me3 and anti-H3K27me3 antibodies were applied to make sequencing libraries. Total RNA and protein were collected for transcriptome and proteome analysis. Activity scores of metabolism processes were defined and calculated as the relative gene expression value averaged over all genes in this pathway in certain cell types. Oxidized PUFAs and PGE2 were assessed by flow cytometry with BODIPY-C11 and ELISA, respectively. Results: SKBR3_HR cells showed similar viability as primary resistant JIMT1 cells and were significantly higher than original SKBR3 cells. Pathway analysis indicated reduced PUFAs synthesis and activated arachidonic acid metabolism. As a consequence, oxidized PUFAs decreased while PGE2 accumulated heavily in SKBR3_HR cells, which may promote tumor and vascular endothelial cell proliferation but interrupt immune cell activity in TME. Transcriptional activity of two critical PUFAs synthesis-related genes, FASN and SCD, were reduced in resistant cells, while two key genes in arachidonic acid metabolism, PTGS1 and PTGES, were activated. H3K4me3 and H3K27me3 play opposite but critical rules in transcriptional regulation. During resistance formation, changes happened on 3256 H3K27me3 peaks as well as 316 H3K4me3 peaks. In detail, although little H3K27me3 changes were found at the promoter regions of four genes (FASN, SCD, PTGS1 and PTGES), reduced H3K4me3 levels may inhibit FASN and SCD expression while raised levels could stimulate PTGS1 and PTGES transcription. Conclusions: Combining the transcriptome and histone modification data, we have a deeper understanding of the cellular adaptions to trastuzumab. Altered trimethylation of H3K4 but not H3K27 remodels the expressions of FASN, SCD, PTGS1 and PTGES, drives the reprogramming of PUFAs-associated metabolism, and finally reduces cellular PUFAs while stimulates PGE2 production. High PGE2 level may affect the activities of tumor and TME-related cells. Citation Format: Yongmei Yin, Ningjun Duan, Yijia Hua, Shuang Hu. Epigenetic modification induced reprogramming of PUFAs-associated metabolism during trastuzumab resistance formation of HER2-positive breast cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3698.

Gastroprotective Effect of Myricetin on Ethanol-Induced Acute Gastric Injury in Rats.

The flavonoid myricetin is abundant in vegetables and has various bioactive properties, including anti-inflammatory and antioxidative activities. In the present study, we explored the effects of myricetin on alcohol-induced gastric ulcer in a rat model. To induce gastric ulcer, absolute ethanol (5 mL/kg body weight) was orally administrated to each rat. The positive control and myricetin-treated groups were given oral doses of omeprazole (20 mg/kg) or myricetin (12 mg/kg), respectively, 1 hour prior to the administration of absolute alcohol. We found that pretreatment with myricetin significantly decreased alcohol-induced gastric ulcer, hemorrhage, hyperemia, and epithelial cell loss in the gastric mucosa. Myricetin pretreatment reduced the level of malondialdehyde (MDA) and increased that of total glutathione (GSSG/GSH) and superoxide dismutase (SOD) in gastric tissues. In addition, it elevated the expression levels of cyclooxygenase-1 (COX-1) and prostaglandin E2 (PGE2) and decreased the phosphorylation of nuclear factor kappa B (NF-κB). Together, these results indicate that myricetin effectively inhibits ethanol-induced acute gastric injury by preventing oxidative damage, stimulating PGE2 production, and inhibiting NF-κB activation. We suggest that myricetin may be an alternative treatment for gastric injury caused by alcohol intake.

Intratumoral 18F-FLT infusion in metabolic targeted radiotherapy

BackgroundThe goal of targeted radiotherapy (TRT) is to administer radionuclides to tumor cells, while limiting radiation exposure to normal tissues. 3′-Deoxy-3′-[18F]-fluorothymidine (18F-FLT) is able to target tumor cells and emits a positron with energy appropriate for local (~ 1 mm range) radiotherapy. In the present work, we investigated the potential of TRT with a local administration of 18F-FLT alone or in combination with 5-fluorouracil (5FU), which acts as a chemotherapeutic agent and radiosensitizer. Treatment efficiency of 18F-FLT combined or not with 5FU was evaluated by intratumoral (i.t.) infusion into subcutaneous HCT116 colorectal tumors implanted in nu/nu mice. The tumor uptake and kinetics of 18F-FLT were determined and compared to 2-deoxy-2-[18F]-fluoro-D-glucose (18F-FDG) by dynamic positron emission tomography (PET) imaging following i.t. injection. The therapeutic responses of 18F-FLT alone and with 5FU were evaluated and compared with 18F-FDG and external beam radiotherapy (EBRT). The level of prostaglandin E2 (PGE2) biosynthesis was measured by liquid chromatography/tandem mass spectrometry (LC/MS/MS) in order to determine the level of inflammation to healthy tissues surrounding the tumor, after i.t. injection of 18F-FLT, and compared to EBRT.ResultsWe found that i.t. administration of 18F-FLT offers (1) the highest tumor-to-muscle uptake ratio not only in the injected tumor, but also in distant tumors, suggesting potential for concurrent metastases treatment and (2) a sixfold gain in radiotherapeutic efficacy in the primary tumor relative to EBRT, which can be further enhanced with concurrent i.t. administration of the radiosensitizer 5FU. While EBRT stimulated PGE2 production in peritumoral tissues, no significant increase of PGE2 was measured in this area following i.t. administration of 18F-FLT.ConclusionConsidering the biochemical stability of 18F-FLT and the physical properties of localized 18F, this study shows that TRT via intratumoral infusion of 18F-FLT and 5FU could provide a new effective treatment option for solid tumors. Using this approach in a colorectal tumor model, the tumor and its metastases could be efficiently irradiated locally with much lower doses absorbed by healthy tissues than with i.t. administration of 18F-FDG or conventional EBRT.

Reactive Oxygen Species Mediated Prostaglandin E2 Contributes to Acute Response of Epithelial Injury.

Reactive oxygen species (ROS) generated after tissue injury play a crucial role during wound healing through initiating acute inflammation, clarifying infection and dead tissue, and mediating various intracellular signal transduction. Prostaglandin E2 (PGE2) has been identified as one of the major factors responsible for inflammation and tissue repair. In this study, we tested our hypothesis that ROS produced by damaged human keratinocytes induces the synthesis of PGE2. In vitro epithelial wounding model was used to observe the production of ROS and secretion of PGE2 as well as the involved signal pathway. The mechanical injury caused the rapid production of ROS in in vitro cultured keratinocytes, which was significantly blocked by an inhibitor of nicotinamide adenine dinucleotide phosphate oxidase. The increased intracellular ROS caused by mechanical injury stimulates PGE2 production in a time-dependent manner via the activation of cyclooxygenase-2 (COX-2), which was stimulated by phosphorylation of extracellular signal-regulated protein kinase (ERK). These results indicate ROS-induced ERK activation leading to the activation of COX-2 and the synthesis of PGE2 in human keratinocytes responding to mechanical injury in the acute phase.

Evaluation of inhibitory effect of Benjakul on lipopolysaccharid- stimulated prostaglandin E2 production during accelerated storage condition

Inflammation of muscles is a common symptom of all ages caused by various reasons, which could occur in specific areas or spread extensively. Muscle relaxants and non-steroid anti-inflammatory drugs (NSAID) are commonly used to relieve pain and symptom. However, both have several side effects. Therefore, drugs for external use are often used together with oral drugs to relieve symptoms. Benjakul, a Thai herbal remedy, is commonly used in Thai traditional medicine. It consist of five hot herbs, namely Piper chaba Hunt., Piper sarmentosum Roxb., Piper interuptum Opiz., Plumbago indica Linn., (Plumbago rosea Linn), and Zingiber officinale Roscoe. The anti-inflammatory effects of Benjakul have been recorded since ancient times. A recent study has found that Benjakul has effect to inhibit PGE2 production but the stability of the activity has not been reported. However, in view of developing an anti-inflammatory product, the stability of this activity has to be evaluated. The objective of this study was to evaluate the inhibitory effect of Benjakul on lipopolysaccharid (LPS)-stimulated prostaglandin E2 (PGE2) [1] production during accelerated storage conditions. The 95% ethanol extract of Benjakul was used. It was kept at 40 degree Celsius with 75% humidity condition, which was called accelerated condition [2], then collected every 30 day for 6 months and tested for inhibition of PGE2 production. The study found that there was no statistically significant difference in inhibition of PGE2 production activity among samples collected from different periods of time. From the result, it can refer to the abilities that the inhibitory effect of Benjakul extract is stable to inhibit PGE2 production activity for 2 years. These results suggest that Benjakul extract may potentially be developed as phytopharmaceutical product to treat muscle inflammation.

Alginate micro-encapsulation of mesenchymal stromal cells enhances modulation of the neuro-inflammatory response

Background aimsModulation of inflammation after brain trauma is a key therapeutic goal aimed at limiting the consequences of the subsequent injury cascade. Mesenchymal stromal cells (MSCs) have been demonstrated to dynamically regulate the inflammatory environment in several tissue systems, including the central nervous system. There has been limited success, however, with the use of direct implantation of cells in the brain caused by low viability and engraftment at the injury site. To circumvent this, we encapsulated MSCs in alginate microspheres and evaluated the ability of these encapsulated MSCs to attenuate inflammation in rat organotypic hippocampal slice cultures (OHSC). MethodsOHSC were administered lipopolysaccharide to induce inflammation and immediately co-cultured with encapsulated or monolayer human MSCs. After 24 h, culture media was assayed for the pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-α) produced by OHSC, as well as MSC-produced trophic mediators. ResultsEncapsulated MSCs reduced TNF-α more effectively than did monolayer MSCs. Additionally, there was a strong correlation between increased prostaglandin E2 (PGE2) and reduction of TNF-α. In contrast to monolayer MSCs, inflammatory signals were not required to stimulate PGE2 production by encapsulated MSCs. Further encapsulation-stimulated changes were revealed in a multiplex panel analyzing 27 MSC-produced cytokines and growth factors, from which additional mediators with strong correlations to TNF-α levels were identified. ConclusionsThese results suggest that alginate encapsulation of MSCs may not only provide an improved delivery vehicle for transplantation but may also enhance MSC therapeutic benefit for treating neuro-inflammation.

Mimicking the Diabetic State in the Non‐Diabetic β‐cell to Elucidate Critical Pathways in β‐cell Dysfunction

EP3, a receptor for prostaglandin E2 (PGE2), plays a key role in the β‐cell dysfunction of diabetes. We demonstrated EP3 expression and PGE2 production are increased in islets isolated from diabetic mice and humans, and targeting this pathway in vitro restores proper β‐cell function. Yet, the mechanisms involved in the pathogenesis of β‐cell dysfunction upstream and downstream of EP3 remain elusive. The INS‐1 832/3 cell line has high EP3 expression, just as in diabetic islets. Interestingly, even though EP3 expression is high, PGE2 production is not; thus, an EP3 antagonist has no effect on β‐cell function. We hypothesized one or more of the multitude of insults β‐cells encounter in the diabetic state are necessary to stimulate PGE2 production and resulting β‐cell dysfunction. We mimicked diet by enriching INS‐1 cells with arachidonic acid (AA), an ω‐6 polyunsaturated fatty acid and PGE2 precursor, and inflammation by IL‐1β treatment. This combination significantly increased PGE2 production, blunting insulin secretion. Further, β‐cell function could be restored by an EP3 antagonist. In islets isolated from non‐diabetic mice and humans, AA and IL‐1β had a strong effect on PGE2 synthetic enzyme expression, but EP3 itself was relatively unchanged, and EP3‐mediated β‐cell dysfunction minimal. We hypothesized that high glucose or free fatty acids are responsible for the dysfunctional up‐regulation of β‐cell EP3 in vivo. Finally, we demonstrate the relevance of targeting this pathway in vivo, as mice lacking the EP3 effector, Gαz, specifically in the β‐cell are protected from the β‐cell dysfunction induced by inflammation.

Salt restriction leads to activation of adult renal mesenchymal stromal cell-like cells via prostaglandin E2 and E-prostanoid receptor 4.

Despite the importance of juxtaglomerular cell recruitment in the pathophysiology of cardiovascular diseases, the mechanisms that underlie renin production under conditions of chronic stimulation remain elusive. We have previously shown that CD44+ mesenchymal-like cells (CD44+ cells) exist in the adult kidney. Under chronic sodium deprivation, these cells are recruited to the juxtaglomerular area and differentiate to new renin-expressing cells. Given the proximity of macula densa to the juxtaglomerular area and the importance of macula densa released prostanoids in renin synthesis and release, we hypothesized that chronic sodium deprivation induces macula densa release of prostanoids, stimulating renal CD44+ cell activation and differentiation. CD44+ cells were isolated from adult kidneys and cocultured with the macula densa cell line, MMDD1, in normal or low-sodium medium. Low sodium stimulated prostaglandin E2 production by MMDD1 and induced migration of CD44+ cells. These effects were inhibited by addition of a cyclooxygenase 2 inhibitor (NS398) or an E-prostanoid receptor 4 antagonist (AH23848) to MMDD1 or CD44+ cells, respectively. Addition of prostaglandin E2 to CD44+ cells increased cell migration and induced renin expression. In vivo activation of renal CD44+ cells during juxtaglomerular recruitment was attenuated in wild-type mice subjected to salt restriction in the presence of cyclooxygenase 2 inhibitor rofecoxib. Similar results were observed in E-prostanoid receptor 4 knockout mice subjected to salt restriction. These results show that the prostaglandin E2/E-prostanoid receptor 4 pathway plays a key role in the activation of renal CD44+ mesenchymal stromal cell-like cells during conditions of juxtaglomerular recruitment; highlighting the importance of this pathway as a key regulatory mechanism of juxtaglomerular recruitment.

Stimulation of prostanoids and IL-8 production in human gingival fibroblasts by Porphyromonas gingivalis LPS is associated with MEK/ERK signaling

Background/purposeVarious toxic products are generated by periodontal pathogens. Porphyromonas gingivalis has been found to generate lipopolysaccharide (LPS) that may potentially affect periodontal health. However, the precise effects of P. gingivalis LPS on human gingival fibroblasts (GFs) await further investigation. Materials and methodsHuman GFs were cultured and exposed to different concentrations of P. gingivalis LPS (0.1–10 μg/mL) for 24 hours. Cytotoxicity was analyzed by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. Total ribonucleic acid (RNA) was isolated and subjected to reverse transcriptase-polymerase chain reaction (RT-PCR) using specific primer sets. Culture medium was collected for determination of prostaglandin E2 (PGE2), PGF2α, and interleukin-8 (IL-8) production by enzyme-linked immunosorbent assay. ERK1/2 phosphorylation in GFs was evaluated by Western blotting. In some experiments, U0126 (a MEK/ERK inhibitor) was added to the GFs culture 30 minutes before LPS and culture medium was also collected for analysis. ResultsP. gingivalis LPS (0.1–10 μg/mL) showed little cytotoxicity and morphologic changes of GFs. P. gingivalis LPS obviously stimulated the cyclooxygenase-2 (COX-2) and IL-8 messenger RNA expression of GFs after 24 hours of exposure. Moreover, P. gingivalis LPS induced PGE2, PGF2α, and IL-8 production in GFs. P. gingivalis LPS also induced ERK1/2 phosphorylation in GFs. Stimulation of PGE2 production by P. gingivalis LPS was completely attenuated by U0126, whereas U0126 only partially inhibited the LPS-induced IL-8 production in the same condition. ConclusionOur data indicate that P. gingivalis LPS stimulates gene expression of differential inflammatory mediators (COX-2 and IL-8) as well as prostanoids and IL-8 production in GFs. These events are associated with MEK/ERK signaling and crucial in the pathogenesis of inflammatory periodontal diseases.

Effects of Litchi chinensis fruit isolates on prostaglandin E(2) and nitric oxide production in J774 murine macrophage cells.

BackgroundLitchi chinensis is regarded as one of the 'heating' fruits in China, which causes serious inflammation symptoms to people.MethodsIn the current study, the effects of isolates of litchi on prostaglandin E2 (PGE2) and nitric oxide (NO) production in J774 murine macrophage cells were investigated.ResultsThe AcOEt extract (EAE) of litchi was found effective on stimulating PGE2 production, and three compounds, benzyl alcohol, hydrobenzoin and 5-hydroxymethyl-2-furfurolaldehyde (5-HMF), were isolated and identified from the EAE. Benzyl alcohol caused markedly increase in PGE2 and NO production, compared with lipopolysaccharide (LPS) as positive control, and in a dose-dependent manner. Hydrobenzoin and 5-HMF were found in litchi for the first time, and both of them stimulated PGE2 and NO production moderately in a dose-dependent manner. Besides, regulation of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) mRNA expression and NF-κB (p50) activation might be involved in mechanism of the stimulative process.ConclusionThe study showed, some short molecular compounds in litchi play inflammatory effects on human.

Signaling via Macrophage G2A Enhances Efferocytosis of Dying Neutrophils by Augmentation of Rac Activity

Phosphatidylserine (PS) and oxidized PS species have been identified as key ligands on apoptotic cells important for their recognition and removal (efferocytosis) by phagocytes, a requisite step for resolution of inflammation. We have recently demonstrated that lysophosphatidylserine (lyso-PS) generated and retained on neutrophils following short term activation of the NADPH oxidase in vitro and in vivo enhanced their clearance via signaling through the macrophage G-protein-coupled receptor G2A. Here, we investigated the signaling pathway downstream of G2A. Lyso-PS, either made endogenously in apoptosing neutrophils or supplied exogenously in liposomes along with lyso-PS(neg) apoptotic cells, signaled to macrophages in a G2A-dependent manner for their enhanced production of prostaglandin E2 (PGE2) via a calcium-dependent cytosolic phospholipase A2/cyclooxygenase-mediated mechanism. Subsequent signaling by PGE2 via EP2 receptors activated macrophage adenylyl cyclase and protein kinase A. These events, in turn, culminated in enhanced activity of Rac1, resulting in an increase in both the numbers of macrophages efferocytosing apoptotic cells and the numbers of cells ingested per macrophage. These data were surprising in light of previous reports demonstrating that signaling by PGE2 and adenylyl cyclase activation are associated with macrophage deactivation and inhibition of apoptotic cell uptake. Further investigation revealed that the impact of this pathway, either the enhancement or inhibition of efferocytosis, was exquisitely sensitive to concentration effects of these intermediaries. Together, these data support the hypothesis that lyso-PS presented on the surface of activated and dying neutrophils provides a tightly controlled, proresolution signal for high capacity clearance of neutrophils in acute inflammation.

Endothelin-1 Enhances Superoxide and Prostaglandin E2 Production of Isolated Diabetic Glomeruli

Endothelin-1 (ET-1) plays an important role in the pathogenic mechanism of diabetic nephropathy. However, the regulatory effects of ET-1 on superoxide and prostaglandin E2 (PGE2) in diabetic glomeruli are unclear. The aim of this study was to determine whether ET-1 exerts a differential effect on the production of superoxide and PGE2 in diabetic glomeruli. The regulatory effects of indomethacin, insulin, dexamethasone, and heparin were also investigated. Freshly isolated glomeruli were obtained from normal and streptozotocin-induced diabetic rats for 1 week (DM1W), 1 month (DM1M), and 3 months (DM3M), respectively. Our results showed that the basal superoxide production of isolated glomeruli was significantly higher in DM1M and DM3M than in the normal rats (p < 0.01). ET-1 stimulated superoxide production in normal, DM1W and DM1M glomeruli (p < 0.01) but not in DM3M rats. The basal production of PGE2 in isolated glomeruli did not differ between diabetic and normal rats. ET-1 also stimulated PGE2 production in diabetic rats (p < 0.05). Pretreatment with indomethacin further enhanced ET-1-stimulated superoxide production in all groups of diabetic rats (p < 0.05), while the ET-1-stimulated PGE2 production was attenuated by indomethacin. Insulin, dexamethasone and heparin had no additional effects on ET-1-mediated superoxide and PGE2 production. In conclusion, basal glomerular production of superoxide but not PGE2 was increased in the diabetic glomeruli. ET-1 further stimulated production of both superoxide and PGE2. Indomethacin could enhance ET-1-stimulated superoxide production while attenuating PGE2 production.

Development and Characterization of a Simian Virus 40 Immortalized Bovine Endometrial Stromal Cell Line

In ruminants, interferon-tau (IFNtau) is the maternal recognition signal inhibiting prostaglandin (PG) F2alpha production by endometrial epithelial cells and stimulating interferon-stimulated genes in the stroma. Stromal cells mediate the action of progesterone on epithelial cells during pregnancy. Our working hypothesis is that IFNtau acts as a molecular switch that turns on PGE(2) production in endometrial stromal cells while suppressing PGF2alpha production from epithelial cells. In this report we document immortalization and functional characterization of a bovine stromal cell line from the caruncular region of the endometrium [caruncular stromal cell (CSC)]. Primary stromal cells were immortalized by nucleofection with simian virus 40 large T antigen and integrase. The resulting cell line, CSC, expresses stromal cell-specific vimentin, estrogen, and progesterone receptors, and is amenable for transient transfection. Basal and stimulated production of PGE2 is higher than PGF2alpha and associated with cyclooxygenase (COX) 2 expression. Phorbol myristate acetate (PMA) and IFNtau up-regulate COX2 and PG production in a dose-dependent manner. When added together, low concentrations of IFNtau inhibit PMA-induced COX2 expression; whereas this inhibition is lost at high concentrations. Expression of signal transducer and activator of transcription 1 is induced by IFNtau at all concentrations studied but is not modulated by PMA. Because expression of signal transducer and activator of transcription 1 does not exhibit the biphasic response to IFNtau, we investigated the p38 MAPK pathway using the selective inhibitor SB203580. Inhibition of the p38 MAPK pathway abolishes IFNtau action on PG production. In summary, CSC appears as a good stromal cell model for investigating the molecular mechanisms related to IFNtau action and PG production in the bovine.

Effect of Compressive Force on the Production of Prostaglandin E2 and its Receptors in Osteoblastic Saos-2 Cells

In orthodontic tooth movement, prostaglandin E2 (PGE2) released from osteoblasts can alter the normal process of bone remodeling. We examined the effect of compressive force (CF) on PGE2 production, PGE receptors (Ep1–4) expression, phosphorylation of protein kinase A (p-PKA), and calcium content in Saos-2 cells. PGE2 production increased as CF strength. Applying CF of 98 or 294 Pa caused the cells to produce approximately 700 and 1400 pg/mL PGE2, respectively. CF of 98 Pa increased Ep2 gene expression, and 98 and 294 Pa CF increased Ep4. Immunohistochemical staining showed strong expression of Ep2 under 98 Pa and Ep4 under 98 and 294 Pa. The p-PKA increased as the strength of CF or PGE2 concentration. The calcium content was increased by the addition of 700 pg/mL PGE2 but was decreased by 1400 pg/mL. Thus, mechanical stress controls bone formation by stimulating PGE2 production and Ep2 and/or Ep4 expression in osteoblasts.

Agonists of peroxisome proliferators-activated receptors (PPAR) α, β/δ or γ reduce transforming growth factor (TGF)-β-induced proteoglycans' production in chondrocytes

To investigate the potency of selective agonists of peroxisome proliferators-activated receptors' (PPAR) isotypes (alpha, beta/delta or gamma) to modulate the stimulating effect of transforming growth factor-beta1 (TGF-beta1) on proteoglycans' (PGs) synthesis in chondrocytes. Rat chondrocytes embedded in alginate beads and cultured under low serum conditions were exposed to TGF-beta1 (10 ng/ml), alone or in combination with the following agonists: Wy14643 for PPARalpha, GW501516 for PPARbeta/delta, rosiglitazone (ROSI) for PPARgamma, in the presence or absence of PPAR antagonists (GW6471 for PPARalpha, GW9662 for PPARgamma). PGs' synthesis was evaluated by radiolabelled sulphate incorporation and glycosaminoglycans' (GAGs) content by Alcian blue staining of beads and colorimetric 1.9 dimethyl-methylene blue assay after beads' solubilization. Phosphorylation of Extracellular Signal-related Kinase1/2 (ERK1/2), Smad2/3 and p38-MAPK was assessed by Western Blot and production of prostaglandin E2 (PGE2) by Enzyme immuno-assay (EIA). Levels of mRNA for PPAR target genes [acyl-CoA oxidase (ACO) for PPARalpha; mitochondrial carnitin palmitoyl transferase-1 (CPT-1) for PPARbeta/delta and adiponectin for PPARgamma], aggrecan, TGF-beta1 and genes controlling GAGs' side chains' synthesis were quantified by real time polymerase chain reaction and normalized over RP29 housekeeping gene. ACO was selectively up-regulated by 100 microM of Wy14643, CPT-1 by 100 nM of GW501516 and adiponectin by 10 microM of ROSI without cell toxicity. TGF-beta1 increased PGs' synthesis by four-fold, GAGs' content and deposition by 3.5-fold and six-fold, respectively, while inducing aggrecan expression around 10-fold without modifying mRNA levels of GAGs' controlling enzymes. PPAR agonists inhibited the stimulating effect of TGF-beta1 by 24-44% on PGs' synthesis and over 75% on aggrecan, GAGs' content and deposition with the following rank order of potency: ROSI>GW501516> or =Wy14643. TGF-beta1-induced phosphorylation of Smad2/3 and ERK1/2 was reduced by ROSI over GW501516 but not by Wy14643 whereas stimulated PGE2 production was inhibited by Wy14643 over GW501516 but not by ROSI. The effect of PPAR agonists on PPAR target genes and TGF-beta1-induced aggrecan expression was reversed selectively by PPAR antagonists. In chondrocytes' beads, PPAR agonists reduced the stimulating effect of TGF-beta1 on PGs by inhibiting TGF-beta1-induced aggrecan expression in an isotype-selective manner. Thus, PPAR agonists could be deleterious in situation of cartilage repair although being protective in situation of cartilage degradation.

Temperature control: from molecular insights, regulation in king penguins and diving seals, to studies in humans

more than 100 manuscripts have been submitted in response to the special call on “Physiology and Pharmacology of Temperature Regulation,” which required spreading publication of the manuscripts over several issues of the journal. Dr. Andrej Romanovsky serves as the Special Editor for this call

Alpha-tocopheryl succinate, in contrast to alpha-tocopherol and alpha-tocopheryl acetate, inhibits prostaglandin E2 production in human lung epithelial cells

The production of prostaglandin E2 (PGE2), a key proinflammatory mediator, is regulated by the availability of its substrate, arachidonic acid (AA), and the activity of the enzyme cyclooxygenase (COX). Increased PGE2 production and COX-2 expression have been observed frequently in specimens from lung cancer patients. Agents that decrease PGE2 production may prevent the initiation and progression of lung cancer. We, therefore, tested the effects of alpha-tocopherol (alphaTOL) analogs on PGE2 production in human lung epithelial cells. Alpha-tocopheryl succinate (alphaTOS), but not alphaTOL or alpha-tocopheryl acetate (alphaTOA), inhibited the phorbol 12-myristate 13-acetate (PMA)-stimulated PGE2 production in three human lung epithelial cell lines (BEAS-2B, H460 and A549 cells). The effect of these compounds on PGE2 production was not correlated with their antioxidant activities, since alphaTOS alone did not inhibit PMA-induced generation of reactive oxygen species. alphaTOS had no effect on PMA-induced AA release or COX-2 expression, although post-incubation with alphaTOS inhibited COX activity and prostaglandin (PGE2 and PGF(2alpha)) production in PMA-stimulated cells. alphaTOS also blocked the COX activity in A549 cells with endogenous high levels of COX enzymes in the absence of PMA stimulation. In addition, the ability of alphaTOS to inhibit COX was affected by AA concentration, suggesting that alphaTOS may compete with AA for interaction with COX proteins. These results suggest that alphaTOS inhibits COX activity, thereby inhibiting PGE2 production in human lung epithelial cells, despite the lack of antioxidant activity. Administration of alphaTOS may block inflammatory responses mediated by PGE2, thereby inhibiting the initiation and progression of lung cancer.

COX-2 inhibitors suppress lung cancer cell growth by inducing p21 via COX-2 independent signals

COX-2 has been implicated in the control of human non-small cell lung carcinoma (NSCLC) cell growth. The mechanisms by which COX-2 exerts its mitogenic effects have not been entirely elucidated, but stimulation of prostaglandin E2 production and alterations in the expression of the cyclin-dependent kinase inhibitor p21(WAF-1/CIP1/MDA-6)(p2i) have been suggested. Here, we demonstrate that two COX-2 inhibitors (NS398 and Nimesulide) inhibit proliferation and induce apoptosis in NSCLC cells, and these effects were associated with induction of p21 mRNA and protein expression. However, the anti-growth effect of the COX-2 inhibitors and their ability to induce p21 were not affected by COX-2 siRNA suggesting that their actions were COX-2 independent. Instead, activation of the MEK-1/Erk pathway was necessary since COX-2 inhibitors stimulated the phosphorylation of ERKs, and their effects were blocked by PD98095, an inhibitor of this pathway. Furthermore, we show that both NS398 and Nimesulide induced p21 gene promoter activity and this was prevented by PD98095. COX-2 inhibitors increased nuclear protein binding to the Spl site in the promoter region of the p21 gene. Consistent with a role for p21, we found that p21 antisense oligonucleotides prevented the effects of COX-2 inhibitors on cell growth. In summary, our results suggest that COX-2 inhibitors suppress NSCLC cell growth by inducing the expression of the p21 gene through MEK-1/ERK signaling and DNA-protein interactions involving Spl. These observations unveil a mechanism for p21 gene regulation by COX-2 inhibitors in lung carcinoma cell growth and this pathway represents a potential target for therapy.

The shunt from the cyclooxygenase to lipoxygenase pathway in human osteoarthritic subchondral osteoblasts is linked with a variable expression of the 5-lipoxygenase-activating protein

Osteoarthritis (OA) is characterized by articular cartilage degradation and hypertrophic bone changes with osteophyte formation and abnormal bone remodeling. Two groups of OA patients were identified via the production of variable and opposite levels of prostaglandin E2 (PGE2) or leukotriene B4 (LTB4) by subchondral osteoblasts, PGE2 levels discriminating between low and high subgroups. We studied whether the expression of 5-lipoxygenase (5-LO) or 5-LO-activating protein (FLAP) is responsible for the shunt from prostaglandins to leukotrienes. FLAP mRNA levels varied in low and high OA groups compared with normal, whereas mRNA levels of 5-LO were similar in all osteoblasts. Selective inhibition of cyclooxygenase-2 (COX-2) with NS-398-stimulated FLAP expression in the high OA osteoblasts subgroup, whereas it was without effect in the low OA osteoblasts subgroup. The addition of PGE2 to the low OA osteoblasts subgroup decreased FLAP expression but failed to affect it in the high OA osteoblasts subgroup. LTB4 levels in OA osteoblasts were stimulated about twofold by 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) plus transforming growth factor-β (TGF-β), a situation corresponding to their effect on FLAP mRNA levels. Treatments with 1,25(OH)2D3 and TGF-β also modulated PGE2 production. TGF-β stimulated PGE2 production in both OA osteoblast groups, whereas 1,25(OH)2D3 alone had a limited effect but decreased the effect of TGF-β in the low OA osteoblasts subgroup. This modulation of PGE2 production was mirrored by the synthesis of COX-2. IL-18 levels were only slightly increased in a subgroup of OA osteoblasts compared with normal; however, no relationship was observed overall between IL-18 and PGE2 levels in normal and OA osteoblasts. These results suggest that the shunt from the production of PGE2 to LTB4 is through regulation of the expression of FLAP, not 5-LO, in OA osteoblasts. The expression of FLAP in OA osteoblasts is also modulated differently by 1,25(OH)2D3 and TGF-β depending on their endogenous low and high PGE2 levels.

Involvement of mitogen-activated protein kinases and protein kinase C in cadmium-induced prostaglandin E2 production in primary mouse osteoblastic cells.

We previously reported that cadmium (Cd) induced prostaglandin E2 (PGE2) biosynthesis through the activation of cytosolic phospholipase A2 (cPLA2) and induction of cyclooxygenase 2 (COX-2) in primary mouse osteoblastic cells. In the present study, we further investigated the mechanism of PGE2 production by Cd focusing on the main mitogen-activated protein kinase (MAPK) subfamilies that mediate prostaglandin synthesis, extracellular signal-regulated kinase (ERK1/2 MAPK), c-jun-amino-terminal kinase (JNK MAPK) and p38 MAPK, and protein kinase C (PKC) which is activated by Cd in several kinds of cells. Cd at 2 microM and above stimulated PGE2 production in osteoblastic cells and its production was inhibited by the kinase-specific inhibitors PD98059, SB203580, curcumin, and calphostin C. Calphostin C also inhibited the production of PGE2 by phorbol 12-myristate 13-acetate (PMA), which is a potent activator of PKC. PD98059 inhibited PGE2 production stimulated by PMA as well as Cd, indicating that activation of PKC by ERK1/2 MAPK was necessary for Cd-stimulated PGE2 production. Moreover, Cd stimulated the phosphorylation of these three MAPKs, and inhibition of the phosphorylation of ERK1/2 MAPK by calphostin C was also observed. On the other hand, Cd was found to phosphorylate cPLA2 and the phosphorylation was inhibited by PD98059, indicating that cPLA2 was activated by Cd through ERK1/2 MAPK and released arachidonic acid (AA), a substrate of COX-2, from membranous phospholipids. From these results, it was suggested that activation of each of the ERK1/2, p38, and JNK MAPK cascades in addition to that of PKC and cPLA2 played an important role in the Cd-stimulated biosynthesis of PGE2 in mouse osteoblastic cells.