AJP - Heart Fuel your research with LabChart
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 288: H165-H174, 2005. First published September 9, 2004; doi:10.1152/ajpheart.00726.2004
0363-6135/05 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
288/1/H165    most recent
00726.2004v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (11)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Giannico, G.
Right arrow Articles by LaPointe, M. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Giannico, G.
Right arrow Articles by LaPointe, M. C.

Regulation of the membrane-localized prostaglandin E synthases mPGES-1 and mPGES-2 in cardiac myocytes and fibroblasts

Giovanna Giannico, Mariela Mendez, and Margot C. LaPointe

Hypertension and Vascular Research Division, Department of Medicine, Henry Ford Hospital, Detroit, Michigan

Submitted 20 July 2004 ; accepted in final form 3 September 2004


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The proinflammatory mediator cyclooxygenase (COX)-2 and its product PGE2 are induced in the ischemic heart, contributing to inflammatory cell infiltration, fibroblast proliferation, and cardiac hypertrophy. PGE2 synthesis coupled to COX-2 involves two membrane-localized PGE synthases, mPGES-1 and mPGES-2; however, it is not clear how these synthases are regulated in cardiac myocytes and fibroblasts. To study this, we used primary cultures of neonatal ventricular myocytes (VM) and fibroblasts (VF) treated with IL-1{beta} for 24 h. To test for involvement of MAPKs in IL-1{beta} regulation of mPGES-1 and-2, cells were pretreated with the pharmacological inhibitors of p42/44 MAPK, p38 MAPK, and c-Jun kinase (JNK). mRNA was analyzed by RT-PCR. Protein was analyzed by densitometry of Western blots. mPGES-1 was undetectable in untreated VF but induced by IL-1{beta}; inhibition of either p42/44 MAPK or JNK, but not p38 MAPK, was almost completely inhibitory. In VM, inhibition of the three MAPKs reduced IL-1{beta}-stimulated mPGES-1 protein by 70–90%. mPGES-2 was constitutively synthesized in both VM and VF and was not regulated by IL-1{beta} or MAPKs. Confocal microscopy revealed colocalization of both mPGES-1 and mPGES-2 with COX-2 in the perinuclear area of both VF and VM. Finally, PGE2 production was higher in VM than VF. Our data show that 1) mPGES-1 is induced in both VF and VM, 2) regulation of mPGES-1 by MAPK family members is different in the two cell types, 3) mPGES-2 is constitutively synthesized in both VM and VF and is not regulated, and 4) mPGES-1 and mPGES-2 are colocalized with COX-2 in both cells. Thus differences in activity of mPGES-1 and COX-2 or coupling of COX-2 with mPGES-1 may contribute to differences in PGE2 production by myocytes and fibroblasts.

prostaglandins; interleukin-1{beta}; cyclooxygenase-2; mitogen-activated protein kinase

PROSTAGLANDINS (PGs) have been implicated in normal cellular processes as well as pathophysiological conditions such as inflammation, edema, bronchoconstriction, and fever (30). PG synthesis involves the conversion of arachidonic acid to PGH2 either by constitutive cyclooxygenase (COX)-1 or by COX-2, which is induced by inflammatory and mitogenic stimuli (41). Subsequently, PGE2 is synthesized from PGH2 by three different PGE synthases (PGES): microsomal glutathione-dependent PGES (mPGES-1), which is inducible and functionally linked to COX-2 (17); glutathione-independent PGES (mPGES-2), which is constitutive and coupled to both COX isoforms (49); and cytosolic PGES (cPGES), which is preferentially coupled to COX-1 (44). Because mPGES-1 couples with COX-2 in the generation of proinflammatory PGE2, studies have recently focused on signaling pathways and transcription factors involved in its expression (4, 14, 32). Although we have previously shown in myocytes that the proinflammatory cytokine IL-1{beta} induces COX-2 and PGE2 production via p38 and p42/p44 MAPK pathways (21), there are no published studies describing the regulation of expression of mPGES in cardiac cells.

Studies have demonstrated the expression of proinflammatory cytokines (15, 40) and COX-2 in the myocardium of patients with congestive heart failure (51). COX-2 and PGE2 production are also induced in animal models of myocardial infarction, and inhibition of COX-2 improves cardiac function in these models (22, 38). PGE2 also stimulates hypertrophic growth of myocytes in vitro (27). In addition to infarction and heart failure, COX-2 and mPGES-1 are elevated in atherosclerotic plaques, contributing to instability and rupture (5).

Because the aforementioned data indicate that the induction of COX-2 and production of proinflammatory prostanoids by the heart can be deleterious, we wanted to understand the mechanisms by which PGE2 is produced by cardiac cells. For this, we examined the expression and regulation of the enzymes involved in production of PGE2 in myocytes and fibroblasts. We tested the effects of specific pharmacological MAPK inhibitors on IL-1{beta} regulation of mPGES-1 and mPGES-2. We also used confocal microscopy to ascertain the subcellular localization of mPGES-1, mPGES-2, and COX-2 in both cell types.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. Six-well and 10-cm plates were obtained from Corning (Corning, NY) and Becton Dickinson (Franklin Lakes, NJ), respectively. DMEM was obtained from GIBCO-BRL (Grand Island, NY), and FBS was obtained from Hyclone (Logan, UT). For Western blot analysis, rabbit polyclonal anti-mPGES-1 and anti-mPGES-2 were purchased from Cayman (Ann Arbor, MI), and goat polyclonal anti-COX-2 and anti-actin were purchased from Santa Cruz Biotechnologies (Santa Cruz, CA). For MAPK phosphorylation studies, anti-dual-phospho-p44/42 MAPK (Thr202/Tyr204), anti-dual-phospho-38 MAPK (Thr180/Tyr182), anti-dual-phospho-JNK/SAPK (Thr183/Tyr185), anti-p44/42 MAPK, anti-p38 MAPK, and anti-JNK/SAPK were obtained from Cell Signaling Technology (Beverly, MA). PAGE gels were purchased from Bio-Rad (Hercules, CA). IL-1{beta} was obtained from BD Biosciences (Bedford, MA), whereas the MAPK inhibitors PD-98059, SB-203580, and SP-600125 were obtained from Calbiochem (La Jolla, CA). For immunofluorescence studies, Alexa Fluor 488 donkey anti-rabbit IgG and Alexa Fluor 568 donkey anti-goat IgG were purchased from Molecular Probes (Eugene, OR), COX-2 and mPGES-1 antibodies were as described for Western blot analysis, and the mPGES-2 antibody was obtained from Santa Cruz Biotechnologies. Specimens for immunofluorescence were mounted in Fluoromount-G (Southern Biotechnologies; Birmingham, AL). All other laboratory supplies and chemicals were obtained from Sigma, Fisher, and VWR.

Cell culture. Ventricular myocyte (VM)-enriched cultures were generated from the digestion of 1- to 2-day-old Sprague-Dawley rat hearts (Charles River Laboratories; Kalamazoo, MI) as described previously (23), according to a protocol approved by the Institutional Animal Care and Use Committee of Henry Ford Hospital. VMs were plated at a density of 1 x 106 cells/well of a 6-well dish or 6 x 106 cells/10-cm dish and cultured in DMEM containing 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mmol/l glutamine, 0.1 mmol/l bromodeoxyuridine, and 10% FBS for 40 h. Twenty-four hours before the experimental treatment, cells were cultured under serum-free conditions (serum-free DMEM supplemented with 100 U/ml penicillin, 2 mmol/l glutamine, 5 mg/l insulin and transferrin, and 2.5 mg/l selenium).

Ventricular fibroblasts (VF) were generated at the preplate stage of the myocyte preparation and passaged twice before use. VFs were plated at a density of 0.5 x 105 cells/well of a 6-well dish or 1.5 x 106 cells/25-cm dish and cultured in DMEM containing 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mmol/l glutamine, and 10% FBS for 8 days until cells reached 80–90% confluence. Before the experiments were performed, cells were cultured under serum-free conditions for 48 h as described above.

Enzyme immunoassay for PGE2. Cells (in 6-well plates) were treated with or without 5 ng/ml (0.3 nmol/l) IL-1{beta} and MAPK inhibitors in 1 ml serum-free DMEM for 24 h. Aliquots of medium were diluted if necessary and assayed for PGE2 using an enzyme immunoassay kit (Cayman Chemical). PGE2 secreted into the culture medium was expressed as nanograms per well. Values from 3–5 wells were expressed as means ± SE.

Western blot analysis. To demonstrate MAPK activation by IL-1{beta}, the phosphorylated forms of p38, p42/44, and JNK/SAPK were detected in whole cell lysates by Western blot analysis (24). VMs and VFs were treated with IL-1{beta} (5 ng/ml) for 0–15 min. Lysates were prepared in buffer containing 10 mM Tris·Cl with 10 mM EDTA, 0.4% deoxycholate, 0.1% SDS, 1 mM PMSF, 1 mM Na3VO4, 1 µM okadaic acid, 20 mM {beta}-glycerophosphate, and 5 µg/ml each of pepstatin A, antipain, leupeptin, and chymostatin. Equal amounts of solubilized proteins (50 µg) in Laemmli sample buffer were resolved by electrophoresis in 7.5% acrylamide gels, transferred to Immobilon-P membranes (Millipore; Bedford, MA), and blocked at 22°C for 90 min in Tris-buffered saline (TBS)-Tween 20 buffer (TBS, pH 7.4, containing 0.1% Tween 20 and 5% skim milk). Membranes were incubated at 22°C for 90 min with the following primary antibodies: anti-dual-phospho-p44/42 MAPK (Thr202/Tyr204) diluted 1/2,000, anti-dual-phospho-38 MAPK (Thr180/Tyr182) diluted 1/1,000, and anti-dual-phospho-JNK/SAPK MAPK (Thr183/Tyr185) diluted 1/1,000. The membranes were stripped and reprobed with primary antibodies against the total MAPK isoforms (anti-p44/42 MAPK diluted 1/2,000, anti-p38 MAPK diluted 1/1,000, and anti-JNK/SAPK diluted 1/2,000 in TBS-milk buffer). Antibody detection was carried out with a chemiluminescent system (ECL, Amersham Pharmacia Biotech), visualized by exposure of Fuji RX film, and quantified by densitometry.

To clarify MAPK involvement in IL-1{beta} induction of mPGES-1 and mPGES-2, cells were pretreated with each MAPK inhibitor for 1 h, and IL-1{beta} (5 ng/ml) was then added for 24 h. Cells were lysed, and membrane-enriched preparations were generated by ultracentrifugation of the cell lysate through a 7-ml cushion of 6% sucrose at 230,000 g for 40 min. The pellet was resuspended in lysis buffer containing 60 mM PIPES, 25 mM HEPES, 0.75% Triton, 10 mM EGTA, 2 mM MgCl2, 156 µg/ml benzamidine, 1 mM iodoacetamide, 1 mM PMSF, and 5 µg/ml each of pepstatin A, antipain, leupeptin, and chymostatin. The 16-kDa mPGES-1 and the 33-kDa mPGES-2 were detected with polyclonal antibodies diluted 1:500.

Isolation of RNA and RT-PCR. We used real-time RT-PCR to assess the effect of the p42/44 MAPK inhibitor PD-98059 (25 µmol/l), the p38 MAPK inhibitor SB-203580 (10 µmol/l), and the JNK inhibitor SP-600125 (10 µmol/l) on IL-1{beta} regulation of mPGES-1 mRNA. Cells were pretreated with each inhibitor for 1 h, and IL-1{beta} was added for 24 h. Total RNA was isolated using Tri Reagent (MRC) according to the manufacturer's instructions. At the end of the procedure, samples were treated with DNAse I from Ambion's RNAqueous-4PCR kit. Reverse transcription was performed using the reagents of the Omniscript RT kit (Qiagen) with 2 µg total RNA, 1 µg random primer (Invitrogen), and 10 U/µl RNAsin (Promega) in a 20-µl vol for 1 h at 37°C. For mPGES-1 detection, real-time PCR was performed using the QuantiTect Probe PCR kit (Qiagen) and a Roche LightCycler. FRET probes and primers were designed by TIB MolBiol (Adelphia). Data analysis was performed with LightCycler software version 3.5.28. The reaction volume was 20 µl and contained 2 µl cDNA, 0.5 µM sense and antisense primers, 0.2 µM each of the LC-640 red-labeled and fluorescein-labeled probes, Taq polymerase, and dNTPs for 45 cycles. For analysis of mPGES-1 (203 bp) and {beta}-actin (342 bp) cDNA, PCR conditions were 0 s at 95°C for denaturation, 40 s at 58°C for annealing, and 40 s at 72°C for extension. The sequences of the primers/probes were as follows: mPGES-1 sense, 5'-CGCGGTGGCTGTCATCA-3'; mPGES-1 antisense, 5'-AGGGTTGGGTCCCAGGAAT-3'; mPGES-1 FL probe, 5'-GAGTGACCCAGATGTGGAGCGCT-x-3'; mPGES-1 LC probe, 5'-LC-red 640-CCTCAGAGCCCACCGCAACGAC-p-3'; {beta}-actin sense, 5'-ACCCACACTGTGCCCATCTA-3'; {beta}-actin antisense: 5'-GCCACAGGATTCCATACCCA-3'; {beta}-actin FL probe, 5'-GCCACGCTCGGTCAGGATCTTCAT-x-3'; and {beta}-actin LC probe, 5'-LC-red 705 AGGTAGTCTGTCAGGTCCCGGCCA-p-3'.

Gene-specific standard curves were generated using a linearized plasmid containing the cDNA of interest, serially diluting it to generate concentrations ranging over two to four orders of magnitude. The LightCycler software calculated the amount of cDNA in a given sample (in copies/µl) compared with the standard curve. mPGES-1 mRNA was normalized to {beta}-actin mRNA.

Confocal microscopy. Laser scanning confocal microscopy was performed using a MRC 1024 scanhead (Bio-Rad) attached to an inverted microscope (Zeiss Axiovert 100). Cells were plated on fibronectin-coated coverslips, serum starved for 24 h, and treated with IL-1{beta} as required. Cells were fixed for 15 min in 4% paraformaldehyde-PBS. Coverslips were rinsed in PBS and permeabilized/blocked with 0.1% Nonidet P-40 and 10% donkey serum in PBS for 15 min. Samples were incubated for 1 h at room temperature with primary antibodies (rabbit anti-PGES-1 and goat anti-COX-2 at 1/100 and 1/50 dilution) in the same permeabilizing/blocking solution. After the cells were gently washed three times with PBS, they were incubated with the appropriate secondary antibodies: Alexa Fluor 488 donkey anti-rabbit IgG and Alexa Fluor 568 donkey anti-goat IgG (Molecular Probes, dilution 1:2,000 and 1:1,000) in permeabilizing/blocking solution for 45 min at room temperature. The coverslips were washed three times in PBS and mounted on glass slides in Fluoromount-G. Sequential single/dual-color images were captured with a x63 oil immersion lens at 30% laser power in serial 0.3-µm optical sections in the z-axis plane of each cell. Images were analyzed with LaserSharp 2000 software (Bio-Rad). All fluorescence images were acquired using excitation lasers Kr/Ar 488 and Kr/Ar 568 and emission filters 522DF32 and 605DF36. Untreated myocytes and fibroblasts were processed with all reagents to monitor background signals for COX-2 and mPGES-1. Transmittance photographs were also taken to make sure that the immunofluorescence was associated with cellular structures.

Statistical analysis. Data are expressed as means ± SE and were analyzed by t-test or one-way ANOVA, with multiple pairwise comparisons made by the Holm-Sidak method. P < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
IL-1{beta} regulation of mPGES-1 mRNA, protein, and PGE2 production in fibroblasts. IL-1{beta} activation of MAPKs in fibroblast cell lysates was examined by Western blot analysis (Fig. 1). IL-1{beta} activated p42/44 MAPK (Fig. 1A) and JNK (Fig. 1B) within the 15-min treatment period but had a very minimal effect on p38 MAPK (Fig. 1C). In contrast, IL-1{beta} did not alter the expression of total MAPK isoforms.



View larger version (36K):
[in this window]
[in a new window]
  		Fig. 1. IL-1{beta} activation and phosphorylation of MAPKs in fibroblasts. Top: Western blots showing representative time course of p42/44 (A), c-Jun kinase (JNK)/SAPK (B) and p38 MAPK (C) phosphorylation in IL-1{beta} (0.3 nmol/l)-treated ventricular fibroblasts. Bottom: Western blots showing the effect of IL-1{beta} on total p42/44 (A), JNK/SAPK (B), and p38 MAPK (C) protein expression. The presence (+) and absence (–) of treatment with IL-1{beta} are indicated.

 
mPGES-1 mRNA was virtually undetectable in untreated cells, and IL-1{beta} induced a dramatic increase in mPGES-1 gene expression (set to 100%; Fig. 2A). Inhibition of p42/44 MAPK (PD-98059, 25 µmol/l) and JNK (SP-600125, 10 µmol/l) almost completely abolished the stimulatory effect of IL-1{beta}, whereas p38 MAPK inhibition (SB-203580, 10 µmol/l) had no effect, consistent with the minimal effect of IL-1{beta} on p38 MAPK activation. Western blot analysis of mPGES-1 protein showed identical patterns of MAPK inhibition (Fig. 2B). Secretion of PGE2 into the culture medium was also examined as a measure of mPGES activity. IL-1{beta} stimulated PGE2 secretion by 21-fold, and this was reduced by p42/44 MAPK and JNK inhibition but not by p38 MAPK inhibition (Fig. 2C), consistent with the pattern of regulation of mPGES-1 mRNA and protein.



View larger version (12K):
[in this window]
[in a new window]
  		Fig. 2. Microsomal glutathione-dependent PGE synthase (mPGES)-1 mRNA, protein, and PGE2 levels in fibroblasts. A: real-time RT-PCR analysis of mPGES-1 mRNA. mPGES-1 mRNA was normalized to {beta}-actin mRNA. The y-axis shows mPGES-1 mRNA expressed as a percentage of IL-1{beta} stimulation, and the x-axis shows the presence (+) and absence (–) of treatment (PD-98059, p42/44 MAPK inhibitor; SB-203580, p38 MAPK inhibitor; SP-600125, JNK inhibitor). Bars indicate means ± SE; n = 4. *P < 0.05 compared with cells treated with IL-1{beta} alone. B: Western blot analysis of mPGES-1 protein. Top, graph summarizing the densitometric analysis of the blots. The y-axis shows mPGES-1 protein expressed as a percentage of IL-1{beta} stimulation, and the x-axis shows treatment. Bars indicate means ± SE; n = 3. *P < 0.05 compared with cells treated with IL-1{beta} alone. Bottom, representative Western blot. C: PGE2 production. The y-axis shows PGE2 secreted into the culture medium (expressed as ng/well), and the x-axis shows treatment. Bars indicate means ± SE; n = 3–4 wells. IL-1{beta} increased PGE2 secretion from 0.06 ± 0.01 to 1.25 ± 0.8 ng/well. The average amount of protein in each well was 2.3 µg/µl.

 
mPGES-2 expression in fibroblasts. To ascertain whether mPGES-2 was present and regulated in VFs, we performed Western blot analysis of a microsomal-enriched subcellular fraction. mPGES-2 was present in untreated VFs as a 33-kDa protein. The protein level was not altered by either IL-1{beta} or the three MAPK inhibitors (Fig. 3). RT-PCR indicated that mPGES-2 mRNA was constitutively present in VFs and unaltered by IL-1{beta} (data not shown).



View larger version (27K):
[in this window]
[in a new window]
  		Fig. 3. Detection of mPGES-2 in fibroblasts. Top: densitometric analysis of the results. The y-axis shows mPGES-2 expressed as a percentage of IL-1{beta} stimulation, and the x-axis shows treatment. Bars indicate means ± SE; n = 3. *P < 0.05 compared with cells treated with IL-1{beta} alone. Bottom: representative Western blot (PD-98059, p42/44 MAPK inhibitor; SB-203580, p38 MAPK inhibitor; SP-600125, JNK inhibitor).

 
Colocalization of mPGES-1 and mPGES-2 with COX-2 in fibroblasts. We (27) have previously shown that mPGES-1 and COX-2 each have a perinuclear distribution in IL-1{beta}-stimulated myocytes. To determine whether mPGES-1 and COX-2 colocalize in fibroblasts, confocal immunofluorescent microscopy was performed using cells stained with antibodies to both proteins, and the images were merged. There were regions of overlapping expression of COX-2 and mPGES-1 mostly in the perinuclear region of IL-1{beta}-treated fibroblasts (Fig. 4, A–C). Similar results were obtained when mPGES-2 and COX-2 localization was examined (Fig. 4, D–F). Figure 4, D–F, is from IL-1{beta}-stimulated cells, because there is no COX-2 in untreated fibroblasts. IL-1{beta} did not alter the localization of mPGES-2 (data not shown).



View larger version (20K):
[in this window]
[in a new window]
  		Fig. 4. Localization of mPGES-1 and mPGES-2 with cyclooxygenase (COX)-2 in IL-1{beta}-treated fibroblasts. A: COX-2 immunofluorescence (red); B: mPGES-1 immunofluorescence (green); C: merged image (yellow). D–F: COX-2/mPGES-2 micrographs using the same colors. Negative controls are not shown.

 
IL-1{beta} regulation of mPGES-1 mRNA, protein, and PGE2 production in myocytes. Untreated VMs exhibited minimal basal phosphorylation of the three MAPKs, and 5- to 15-min treatment with IL-1{beta} resulted in increased p42/44 MAPK, p38 MAPK, and JNK phosphorylation (Fig. 5). In contrast, IL-1{beta} did not alter the expression of total MAPK isoforms in myocytes.



View larger version (47K):
[in this window]
[in a new window]
  		Fig. 5. IL-1{beta} activation and phosphorylation of MAPKs in myocytes. Top: Western blots showing representative time course of p42/44 (A), JNK (B), and p38 MAPK (C) phosphorylation in IL-1{beta} (0.3 nmol/l)-treated ventricular myocytes. Bottom: Western blots showing the effects of IL-1{beta} on total p42/44 (A), JNK/SAPK (B), and p38 MAPK (C) protein expression. +, Presence of treatment; –, absence of treatment.

 
In myocytes, mPGES-1 mRNA was undetectable in untreated cells and stimulated markedly by IL-1{beta} treatment (set to 100%; Fig. 6A). p42/44 MAPK inhibition had no statistically significant effect on IL-stimulated mPGES-1 mRNA, whereas p38 MAPK and JNK inhibition decreased it by 60–70%. Regarding mPGES-1 protein, the pattern of MAPK inhibition was similar to the mRNA results; however, MAPK inhibition had more profound effects on IL-1{beta}-stimulated mPGES-1 protein than mRNA (74%, 89%, and 92% inhibition for p42/44 MAPK, p38 MAPK, and JNK inhibitors, respectively; Fig. 6B). Myocyte secretion of PGE2 into the culture medium was also examined. IL-1{beta} increased PGE2 production over 1,000-fold, which was significantly suppressed by all three MAPK inhibitors (Fig. 6C).



View larger version (12K):
[in this window]
[in a new window]
  		Fig. 6. mPGES-1 mRNA, protein, and PGE2 levels in myocytes. A: real-time RT-PCR analysis of mPGES-1 mRNA. mPGES-1 mRNA was normalized to {beta}-actin mRNA. The y-axis shows mPGES-1 mRNA expressed as a percentage of IL-1{beta} stimulation, and the x-axis shows treatment. Bars indicate means ± SE; n = 4. *P < 0.05 compared with IL-1{beta} alone. B: Western blot analysis of mPGES-1 protein. Top, graph summarizing the densitometric analysis of the blots. The y-axis shows mPGES-1 protein expressed as a percentage of IL-1{beta} stimulation, and the x-axis shows treatment. Bars indicate means ± SE; n = 3. *P < 0.05 compared with cells treated with IL-1{beta} alone. Bottom, representative Western blot. C: PGE2 production. The y-axis shows PGE2 secreted into the culture medium (expressed as ng/well), and the x-axis shows treatment. Bars indicate mean ± SE; n = 3–5 wells. IL-1{beta} alone increased PGE2 secretion from 0.08 ± 0.01 to 139 ± 28 ng/well. The average amount of protein in each well was 7.4 µg/µl. *P < 0.05 compared with IL-1{beta} alone. There was no significant difference between the control group and either PD-98059 + IL-1{beta}, SB-203580 + IL-1{beta}, or SP-600125 + IL-1{beta} (PD-98059, p42/44 MAPK inhibitor; SB-203580, p38 MAPK inhibitor; SP-600125, JNK inhibitor).

 
mPGES-2 expression in myocytes. As noted in fibroblasts, mPGES-2 was constitutively expressed in myocytes. Its levels were not changed by IL-1{beta} treatment in the presence or absence of MAPK inhibitors (Fig. 7). Interestingly, a second band of ~40 kDa was detected and unaltered by any of the treatments. mPGES-2 mRNA was detected by RT-PCR in untreated myocytes, and its level was not changed by IL-1{beta} (data not shown).



View larger version (26K):
[in this window]
[in a new window]
  		Fig. 7. Detection of mPGES-2 in myocytes. Top: densitometric analysis of the Western blot results. The y-axis shows mPGES-2 expressed as a percentage of IL-1{beta} stimulation, and the x-axis shows treatment. Bars indicate means ± SE; n = 3. *P < 0.05 compared with IL-1{beta} alone. Bottom: representative Western blot (PD-98059, p42/44 MAPK inhibitor; SB-203580, p38 MAPK inhibitor; SP600125, JNK inhibitor).

 
Colocalization of mPGES-1 and mPGES-2 with COX-2 in myocytes. Confocal microscopy was performed in VMs to determine whether COX-2 (Fig. 8A) and mPGES-1 (Fig. 8B) are colocalized. In IL-1{beta}-stimulated myocytes, there were regions of overlapping expression of COX-2 and mPGES-1 mostly in the perinuclear area (Fig. 8C). There was some colocalization of mPGES-2 and COX-2 in the perinuclear region (Fig. 8, D–F), but the intensity appeared much less than in fibroblasts (see Fig. 4F).



View larger version (18K):
[in this window]
[in a new window]
  		Fig. 8. Localization of mPGES-1 and mPGES-2 with COX-2 in IL-1{beta}-treated myocytes. A: COX-2 immunofluorescence (red); B: mPGES-1 immunofluorescence (green); C: merged image (yellow). D–F: COX-2/mPGES-2 micrographs using the same colors. Negative controls are not shown.

 
COX-2 and mPGES-1 protein in fibroblasts and myocytes. Because mPGES-1 and COX-2 were induced by IL-1{beta} and colocalized in both myocytes and fibroblasts, we examined whether there were absolute differences in their expression by analyzing control and IL-1{beta}-treated myocyte and fibroblast samples on the same gel. COX-2 protein was expressed at high levels in all three myocyte samples, but overall expression was lower in fibroblast samples (Fig. 9A). mPGES-1 was uniformly expressed at higher levels in IL-1{beta}-treated myocytes than fibroblasts (Fig. 9C). The blot was reprobed with actin antibody to demonstrate that there were no large variations in protein loaded in each lane (Fig. 9B).



View larger version (40K):
[in this window]
[in a new window]
  		Fig. 9. Comparison of mPGES-1 and COX-2 expression in myocytes and fibroblasts. Western blots of 3 separate myocyte and fibroblast samples from IL-1{beta}-treated cells are shown. One control sample for myocytes and fibroblasts is included to demonstrate the absence of COX-2 and mPGES-1 in untreated cells. Membrane-enriched protein (25 µg) was loaded into each well for detection of mPGES-1; 50 µg total protein was used to detect COX-2. Actin (1:1,000 dilution of antibody SC 1616, Santa Cruz Biotechnology) was used to control for protein loading per well. Samples 1 and 5 are from untreated cells, and the remaining samples (samples 2–4 and 6–8) are from IL-{beta}-treated cells. Lanes 1–4 are from myocytes, and lanes 5–8 from fibroblasts. A: 72-kDa COX-2 protein; B: 43-kDa actin protein; C: 16-kDa mPGES-1 protein.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
We found that mPGES-1 was induced by the proinflammatory cytokine IL-1{beta} in both cardiac myocytes and fibroblasts and was colocalized with COX-2 mainly in a perinuclear distribution. MAPK regulation of mPGES-1 expression was different in the two cell types, with p42/44 MAPK and JNK serving as the primary regulators in fibroblasts and p38 MAPK and JNK playing a greater role than p42/44 MAPK in myocytes. A second membrane-bound form of PGES, called mPGES-2, was constitutively expressed in both myocytes and fibroblasts, colocalized with COX-2, but was not regulated. Given the induction of mPGES-1 and COX-2 in both cell types, it is likely that mPGES-1 is the major PGE2 synthase leading to the production and secretion of PGE2.

In our study, IL-1{beta} activated all three MAPKs in myocytes while preferentially activating p42/44 MAPK and JNK in fibroblasts. The time course of activation in myocytes and fibroblasts was consistent with reports of other cell types (11, 12, 52). Han et al. (14) reported that IL-1{beta} activated p38 and p42/44 MAPK in orbital fibroblasts and that inhibition of either MAPK resulted in partial repression of mPGES-1 expression, whereas both inhibitors reduced IL-1{beta} stimulation by 89%. They also demonstrated that the effect of IL-1{beta} occurred at the transcriptional level.

IL-1{beta} is a potent proinflammatory cytokine that increases expression of a wide variety of genes important for immunity and inflammation in target cells, and all three MAPKs have been implicated as mediators of its effects (20, 35, 53). Downstream effectors of the activated MAPKs include transcription factors and other kinases (8, 10, 19). Another effector of IL-1{beta} signaling is the transcription factor NF-{kappa}B (33). Transcription factors implicated in the regulation of mPGES-1 expression include NF-IL6 (46) and NF-{kappa}B (3). Interestingly, consensus binding sites for these factors have not been identified in the 5' flanking sequences of the human and mouse genes to our knowledge. On the other hand, examination of the proximal regulatory regions of the mouse and human mPGES-1 promoters revealed tandem GC box sequences (9, 32). Deletion analysis of the mouse promoter and DNA-protein binding studies indicated that the GC boxes in the proximal promoter were responsive to proinflammatory cytokines (IL-1{beta}, tumor necrosis factor-{alpha}) as well as the phorbol ester phorbol 12-myristate 13-acetate (PMA) and bound a transcription factor called Egr-1. GC boxes in the human mPGES-1 promoter also bound Egr-1 and responded to phorbol ester treatment (32). Mutation of the Egr-1 binding sites in both mouse and human promoters abolished inducible expression. Of interest, Egr-1 expression was previously found to be regulated by p38 MAPK and JNK (26, 37). Because IL-1{beta} regulation of mPGES-1 involves p38 MAPK and JNK in myocytes and p42/44 MAPK and JNK in fibroblasts, it is most likely that Egr-1 is an important mediator of IL-1{beta} induction of mPGES-1 in both cells.

Activation of more than one MAPK pathway is required for stimulation of COX-2 expression by IL-1{beta}. We have previously demonstrated that IL-1{beta} regulates COX-2 expression in myocytes via p38 and p42/44 activation (21), as shown in other reports (12, 16, 25, 36, 50). In addition to transcriptional effects, p38 MAPK stabilizes COX-2 mRNA by stimulating binding of the stabilizing factor HuR to an AU region of the proximal 3'-untranslated region (UTR) of the gene (43). In contrast to COX-2, the mPGES-1 gene does not have instability motifs (AU-rich regions) in the 3'-UTR. In fact, Han et al. (14) have shown that IL-1{beta} does not affect the half-life of mPGES-1 mRNA in orbital fibroblasts.

Although we did not perform mRNA stability studies, our results show that in myocytes (but not fibroblasts), MAPK inhibition has a greater effect on mPGES-1 protein than mRNA, suggesting a posttranscriptional effect. Posttranscriptional regulation can occur through mRNA processing, modification of mRNA stability (2, 6), and control of translation initiation (34, 48). Stabilization of mRNA affects the abundance and increases the half-life of transcripts within the cell and is a mechanism important for rapid and sustained induction of early-response genes, such as those induced by proinflammatory mediators like COX-2 and cytokines (7, 14, 47). On the other hand, it is well known that MAPKs participate in translational mechanisms. p42/44 MAPK enhances translation by promoting the recruitment of translation factors and ribosomes to mRNA, while p38 MAPK activates eukaryotic elongation factor-2 kinase (10, 39). Thus it is possible that MAPK regulation of mPGES-1 in myocytes involves both transcriptional and posttranscriptional mechanisms.

We have previously shown that cardiac myocytes secrete much more PGE2 than fibroblasts (27), which was confirmed in the present study. Even though there are differences in the MAPK regulation of mPGES-1 in myocytes and fibroblasts, it is not clear if or how this might contribute to differential PGE2 production. Because it is known that COX-2 and mPGES-1 are linked in the production of PGE2 in inflammatory conditions (31), we examined whether they were localized to the same subcellular compartment in myocytes and fibroblasts. It has been shown that COX-2 is located in the endoplasmic reticulum and nuclear membrane, as determined by confocal and immunoelectron microscopy (28, 42). Our experiments clearly show that mPGES-1 colocalized with COX-2 mostly in a perinuclear fashion in both myocytes and fibroblasts. Although these confocal studies indicate that both enzymes are in the same subcellular location, the study cannot evaluate functional coupling or whether the two enzymes are interacting physically. Future studies will evaluate whether in purified subcellular fractions, such as microsomes and nuclei, mPGES-1 can be coimmunoprecipitated with COX-2 antibodies.

Another possible explanation for the differential PGE2 production by myocytes and fibroblasts is the availability of the substrate for mPGES-1, i.e., PGH2 produced by COX-2. Boulet et al. (1) demonstrated that two different macrophage populations from mice (resident peritoneal and thioglycollate-elicited macrophages) produced 10-fold differences in PGE2 in response to lipopolysaccharide treatment. Interestingly, there were no differences in lipopolysaccharide-stimulated mPGES-1 protein between the two macrophage populations, but there was a 10-fold difference in the amount of COX-2 protein. Our studies are consistent with this, indicating that both COX-2 and mPGES-1 are more markedly induced in total protein and membrane-enriched preparations of myocytes than fibroblasts. Thus IL-1{beta} results in greater accumulation of COX-2 and mPGES-1 in myocytes than fibroblasts, accounting for the differences in PGE2 production.

A second mPGES, mPGES-2, was originally purified from the microsomal fraction of bovine hearts (49). mPGES-2 occurred in two forms: the full-length form, containing an NH2-terminal hydrophobic domain (42 kDa), and a truncated version produced by processing in the Golgi apparatus (33 kDa). Both forms were associated with membranes, although the processed form was also cytosolic, and both were enriched in the perinuclear area (29). We detected 33-kDa mPGES-2 protein in both myocytes and fibroblasts. A larger band was also detected in myocytes, probably reflecting the presence of the full-length, unprocessed protein. It is not known whether the presence of the larger form in myocytes reflects an inability to process the protein or whether the mPGES-2 form is a function of the cell culture conditions (the myocytes were examined 4 days after preparation, whereas the fibroblasts had been passaged over a 3- to 4-wk period). We found that mPGES-2 was located mostly in perinuclear regions in both myocytes and fibroblasts, confirming Murakami's findings (29). It was also colocalized with COX-2, which is consistent with their studies showing that mPGES-2 is functionally linked to both COX-1 and COX-2.

mPGES-2 in both cardiac myocytes and fibroblasts was constitutively produced and not regulated by the cytokine IL-1{beta}. This is consistent with the studies of Murakami et al. (29), who found that IL-1{beta} stimulation of a number of different murine cell lines had no effect on mPGES-2 expression. In addition, they found no changes in mPGES-2 in any of the tissues examined after lipopolysaccharide treatment of mice.

Although it is well known that PGE2 contributes to inflammation, pain, fever, tumorigenesis, and bone resorption (30), the contribution of mPGES-1 to some of these processes has only recently been examined in knockout mice. In a variety of different disease models, it has been shown that mPGES-1 is important in the development of rheumatoid arthritis, acute pain, pain hypersensitivity, and inflammation (1, 13, 18, 45). The role of PGE2 in the pathophysiology of heart disease is under investigation. We have shown that PGE2 levels and COX-2 expression are increased in the infarcted mouse heart and that COX-2 inhibition improves function while inhibiting hypertrophy and fibrosis (22). Additionally, we have shown that PGE2 promotes hypertrophy of myocytes in vitro (27). Given the coordinated upregulation of COX-2 and mPGES-1 in cardiac myocytes and fibroblasts in vitro, it is likely that mPGES-1 (and not mPGES-2) contributes to the enhanced PGE2 production in infarcted hearts. Support for this was recently reported by Murakami et al. (29), who noted mPGES-1 immunostaining in sections of infarcted human hearts. Careful investigation of different models of heart disease using mPGES-1 knockout mice is needed to more clearly delineate the conditions under which PGE2 is deleterious.

In summary, although COX-2 expression and prostanoid synthesis are known to be induced in response to ischemia, infarction, and heart failure, the functional role of mPGES-1 in the myocardium is essentially unknown. Our data show that IL-1{beta}, acting through MAPKs, regulates mPGES-1 expression in cardiac fibroblasts and myocytes in vitro. In addition, there are differences in the MAPKs involved in the two cell types, with all three MAPKS involved in myocytes and p42/44 and JNK in fibroblasts. Furthermore, in myocytes, there are different effects of MAPK inhibition at the mRNA and protein levels, suggesting posttranscriptional regulation. In contrast, mPGES-2 is constitutively produced in both cells and is not regulated. Both mPGES-1 and mPGES-2 are localized to membranes and are found mostly in the same perinuclear distribution as COX-2. In myocytes, the marked induction of COX-2, mPGES-1, and PGE2 production in response to IL-1{beta} treatment would tend to favor mPGES-1 as the major synthase involved in elevated PGE2 production. This is likely the case in fibroblasts, where PGE2 production increased 20-fold. Also, because MAPK inhibition affects both COX-2 (21) and mPGES-1 (this study), the relative contribution of each to the production of PGE2 requires further evaluation. Additional studies are needed to clarify the roles of mPGES-1 and mPGES-2 in cardiac function under physiological and pathophysiological conditions. By understanding the regulation of PGE2 production in cardiac pathophysiology, this might lead to the development of therapeutic strategies that target PGE2 rather than COX-2, thus preventing some of the systemic effects related to use of COX-2 inhibitors.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by National Heart, Lung, and Blood Institute Grant P01 HL-28982.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. C. LaPointe, Hypertension and Vascular Research Div., Dept. of Medicine, Henry Ford Hospital, 2799 W. Grand Blvd., Detroit, MI 48202-2689 (E-mail: mlapoin1{at}hfhs.org)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Boulet L, Ouellet M, Bateman KP, Ethier D, Percival MD, Riendeau D, Mancini JA, and Methot N. Deletion of microsomal prostaglandin E2 (PGE2) synthase-1 reduces inducible and basal PGE2 production and alters the gastric prostanoid profile. J Biol Chem 279: 23229–23237, 2004.[Abstract/Free Full Text]
  2. Brook M, Sully G, Clark AR, and Saklatvala J. Regulation of tumour necrosis factor alpha mRNA stability by the mitogen-activated protein kinase p38 signalling cascade. FEBS Lett 483: 57–61, 2000.[CrossRef][ISI][Medline]
  3. Catley MC, Chivers JE, Cambridge LM, Holden N, Slater DM, Staples KJ, Bergmann MW, Loser P, Barnes PJ, and Newton R. IL-1beta-dependent activation of NF-kappaB mediates PGE2 release via the expression of cyclooxygenase-2 and microsomal prostaglandin E synthase. FEBS Lett 547: 75–79, 2003.[CrossRef][ISI][Medline]
  4. Cheng S, Afif H, Martel-Pelletier J, Pelletier JP, Li X, Farrajota K, Lavigne M, and Fahmi H. Activation of peroxisome proliferator-activated receptor gamma inhibits interleukin-1beta-induced membrane-associated prostaglandin E2 synthase-1 expression in human synovial fibroblasts by interfering with Egr-1. J Biol Chem 279: 22057–22065, 2004.[Abstract/Free Full Text]
  5. Cipollone F, Fazia M, Iezzi A, Ciabattoni G, Pini B, Cuccurullo C, Ucchino S, Spigonardo F, De Luca M, Prontera C, Chiarelli F, Cuccurullo F, and Mezzetti A. Balance between PGD synthase and PGE synthase is a major determinant of atherosclerotic plaque instability in humans. Arterioscler Thromb Vasc Biol 24: 1259–1265, 2004.[Abstract/Free Full Text]
  6. Dean JL, Brook M, Clark AR, and Saklatvala J. p38 mitogen-activated protein kinase regulates cyclooxygenase-2 mRNA stability and transcription in lipopolysaccharide-treated human monocytes. J Biol Chem 274: 264–269, 1999.[Abstract/Free Full Text]
  7. Esnault S and Malter JS. Extracellular signal-regulated kinase mediates granulocyte-macrophage colony-stimulating factor messenger RNA stabilization in tumor necrosis factor-alpha plus fibronectin-activated peripheral blood eosinophils. Blood 99: 4048–4052, 2002.[Abstract/Free Full Text]
  8. Fanger GR, Gerwins P, Widmann C, Jarpe MB, and Johnson GL. MEKKs, GCKs, MLKs, PAKs, TAKs, and tpls: upstream regulators of the c-Jun amino-terminal kinases? Curr Opin Genet Dev 7: 67–74, 1997.[CrossRef][ISI][Medline]
  9. Forsberg L, Leeb L, Thoren S, Morgenstern R, and Jakobsson P. Human glutathione dependent prostaglandin E synthase: gene structure and regulation. FEBS Lett 471: 78–82, 2000.[CrossRef][ISI][Medline]
  10. Gallo KA and Johnson GL. Mixed-lineage kinase control of JNK and p38 MAPK pathways. Nat Rev Mol Cell Biol 3: 663–672, 2002.[CrossRef][ISI][Medline]
  11. Geng Y, Valbracht J, and Lotz M. Selective activation of the mitogen-activated protein kinase subgroups c-Jun NH2 terminal kinase and p38 by IL-1 and TNF in human articular chondrocytes. J Clin Invest 98: 2425–2430, 1996.[ISI][Medline]
  12. Guan Z, Buckman SY, Pentland AP, Templeton DJ, and Morrison AR. Induction of cyclooxygenase-2 by the activated MEKK1 -> SEK1/MKK4 -> p38 mitogen-activated protein kinase pathway. J Biol Chem 273: 12901–12908, 1998.[Abstract/Free Full Text]
  13. Guay J, Bateman K, Gordon R, Mancini J, and Riendeau D. Carrageenan-induced paw edema in rat elicits a predominant prostaglandin E2 (PGE2) response in the central nervous system associated with the induction of microsomal PGE2 synthase-1. J Biol Chem 279: 24866–24872, 2004.[Abstract/Free Full Text]
  14. Han R, Tsui S, and Smith TJ. Up-regulation of prostaglandin E2 synthesis by interleukin-1beta in human orbital fibroblasts involves coordinate induction of prostaglandin-endoperoxide H synthase-2 and glutathione-dependent prostaglandin E2 synthase expression. J Biol Chem 277: 16355–16364, 2002.[Abstract/Free Full Text]
  15. Herskowitz A, Choi S, Ansari AA, and Wesselingh S. Cytokine mRNA expression in postischemic/reperfused myocardium. Am J Pathol 146: 419–428, 1995.[Abstract]
  16. Hwang D, Jang BC, Yu G, and Boudreau M. Expression of mitogen-inducible cyclooxygenase induced by lipopolysaccharide: mediation through both mitogen-activated protein kinase and NF-kappaB signaling pathways in macrophages. Biochem Pharmacol 54: 87–96, 1997.[CrossRef][ISI][Medline]
  17. Jakobsson PJ, Thoren S, Morgenstern R, and Samuelsson B. Identification of human prostaglandin E synthase: a microsomal, glutathione-dependent, inducible enzyme, constituting a potential novel drug target. Proc Natl Acad Sci USA 96: 7220–7225, 1999.[Abstract/Free Full Text]
  18. Kamei D, Yamakawa K, Takegoshi Y, Mikami-Nakanishi M, Nakatani Y, Oh-Ishi S, Yasui H, Azuma Y, Hirasawa N, Ohuchi K, Kawaguchi H, Ishikawa Y, Ishii T, Uematsu S, Akira S, Murakami M, and Kudo I. Reduced pain hypersensitivity and inflammation in mice lacking microsomal prostaglandin E synthase-1. J Biol Chem 279: 33684–33695, 2004.[Abstract/Free Full Text]
  19. Kyriakis JM and Avruch J. Protein kinase cascades activated by stress and inflammatory cytokines. Bioessays 18: 567–577, 1996.[CrossRef][ISI][Medline]
  20. Kyriakis JM, Banerjee P, Nikolakaki E, Dai T, Rubie EA, Ahmad MF, Avruch J, and Woodgett JR. The stress-activated protein kinase subfamily of c-Jun kinases. Nature 369: 156–160, 1994.[CrossRef][Medline]
  21. LaPointe MC and Isenovic E. Interleukin-1beta regulation of inducible nitric oxide synthase and cyclooxygenase-2 involves the p42/44 and p38 MAPK signaling pathways in cardiac myocytes. Hypertension 33: 276–282, 1999.[Abstract/Free Full Text]
  22. LaPointe MC, Mendez M, Leung A, Tao Z, and Yang XP. Inhibition of cyclooxygenase-2 improves cardiac function after myocardial infarction in the mouse. Am J Physiol Heart Circ Physiol 286: H1416–H1424, 2004.[Abstract/Free Full Text]
  23. LaPointe MC and Sitkins JR. Phorbol ester stimulates the synthesis and secretion of brain natriuretic peptide from neonatal rat ventricular cardiocytes: a comparison with the regulation of atrial natriuretic factor. Mol Endocrinol 7: 1284–1296, 1993.[Abstract]
  24. LaPointe MC and Sitkins JR. Mechanisms of interleukin-1beta regulation of nitric oxide synthase in cardiac myocytes. Hypertension 27: 709–714, 1996.[Abstract/Free Full Text]
  25. Laporte JD, Moore PE, Panettieri RA, Moeller W, Heyder J, and Shore SA. Prostanoids mediate IL-1{beta}-induced {beta}-adrenergic hyporesponsiveness in human airway smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 275: L491–L501, 1998.[Abstract/Free Full Text]
  26. Lim CP, Jain N, and Cao X. Stress-induced immediate-early gene, egr-1, involves activation of p38/JNK1. Oncogene 16: 2915–2926, 1998.[CrossRef][ISI][Medline]
  27. Mendez M and LaPointe MC. Trophic effects of the cyclooxygenase-2 product prostaglandin E2 in cardiac myocytes. Hypertension 39: 382–388, 2002.[Abstract/Free Full Text]
  28. Morita I, Schindler M, Regier MK, Otto JC, Hori T, DeWitt DL, and Smith WL. Different intracellular locations for prostaglandin endoperoxide H synthase-1 and -2. J Biol Chem 270: 10902–10908, 1995.[Abstract/Free Full Text]
  29. Murakami M, Nakashima K, Kamei D, Masuda S, Ishikawa Y, Ishii T, Ohmiya Y, Watanabe K, and Kudo I. Cellular prostaglandin E2 production by membrane-bound prostaglandin E synthase-2 via both cyclooxygenases-1 and -2. J Biol Chem 278: 37937–37947, 2003.[Abstract/Free Full Text]
  30. Murakami M, Nakatani Y, Tanioka T, and Kudo I. Prostaglandin E synthase. Prostaglandins Other Lipid Mediat 68–69:383–399, 2002.
  31. Murakami M, Naraba H, Tanioka T, Semmyo N, Nakatani Y, Kojima F, Ikeda T, Fueki M, Ueno A, Oh-Ishi S, and Kudo I. Regulation of prostaglandin E2 biosynthesis by inducible membrane-associated prostaglandin E2 synthase that acts in concert with cyclooxygenase-2. J Biol Chem 275: 32783–32792, 2000.[Abstract/Free Full Text]
  32. Naraba H, Yokoyama C, Tago N, Murakami M, Kudo I, Fueki M, Oh-Ishi S, and Tanabe T. Transcriptional regulation of the membrane-associated prostaglandin E2 synthase gene. Essential role of the transcription factor Egr-1. J Biol Chem 277: 28601–28608, 2002.[Abstract/Free Full Text]
  33. O'Neill LA and Greene C. Signal transduction pathways activated by the IL-1 receptor family: ancient signaling machinery in mammals, insects, and plants. J Leukoc Biol 63: 650–657, 1998.[Abstract]
  34. Pyronnet S, Imataka H, Gingras AC, Fukunaga R, Hunter T, and Sonenberg N. Human eukaryotic translation initiation factor 4G (eIF4G) recruits mnk1 to phosphorylate eIF4E. EMBO J 18: 270–279, 1999.[CrossRef][ISI][Medline]
  35. Raingeaud J, Gupta S, Rogers JS, Dickens M, Han J, Ulevitch RJ, and Davis RJ. Pro-inflammatory cytokines and environmental stress cause p38 mitogen-activated protein kinase activation by dual phosphorylation on tyrosine and threonine. J Biol Chem 270: 7420–7426, 1995.[Abstract/Free Full Text]
  36. Ridley SH, Sarsfield SJ, Lee JC, Bigg HF, Cawston TE, Taylor DJ, DeWitt DL, and Saklatvala J. Actions of IL-1 are selectively controlled by p38 mitogen-activated protein kinase: regulation of prostaglandin H synthase-2, metalloproteinases, and IL-6 at different levels. J Immunol 158: 3165–3173, 1997.[Abstract]
  37. Rolli M, Kotlyarov A, Sakamoto KM, Gaestel M, and Neininger A. Stress-induced stimulation of early growth response gene-1 by p38/stress-activated protein kinase 2 is mediated by a cAMP-responsive promoter element in a MAPKAP kinase 2-independent manner. J Biol Chem 274: 19559–19564, 1999.[Abstract/Free Full Text]
  38. Saito T, Rodger IW, Hu F, Shennib H, and Giaid A. Inhibition of cyclooxygenase-2 improves cardiac function in myocardial infarction. Biochem Biophys Res Commun 273: 772–775, 2000.[CrossRef][ISI][Medline]
  39. Schramek H. MAP kinases: from intracellular signals to physiology and disease. News Physiol Sci 17: 62–67, 2002.[Abstract/Free Full Text]
  40. Shioi T, Matsumori A, Kihara Y, Inoko M, Ono K, Iwanaga Y, Yamada T, Iwasaki A, Matsushima K, and Sasayama S. Increased expression of interleukin-1 beta and monocyte chemotactic and activating factor/monocyte chemoattractant protein-1 in the hypertrophied and failing heart with pressure overload. Circ Res 81: 664–671, 1997.[Abstract/Free Full Text]
  41. Smith WL, DeWitt DL, and Garavito RM. Cyclooxygenases: structural, cellular, and molecular biology. Annu Rev Biochem 69: 145–182, 2000.[CrossRef][ISI][Medline]
  42. Spencer AG, Woods JW, Arakawa T, Singer II, and Smith WL. Subcellular localization of prostaglandin endoperoxide H synthases-1 and -2 by immunoelectron microscopy. J Biol Chem 273: 9886–9893, 1998.[Abstract/Free Full Text]
  43. Subbaramaiah K, Marmo TP, Dixon DA, and Dannenberg AJ. Regulation of cyclooxgenase-2 mRNA stability by taxanes: evidence for involvement of p38, MAPKAPK-2, and HuR. J Biol Chem 278: 37637–37647, 2003.[Abstract/Free Full Text]
  44. Tanioka T, Nakatani Y, Semmyo N, Murakami M, and Kudo I. Molecular identification of cytosolic prostaglandin E2 synthase that is functionally coupled with cyclooxygenase-1 in immediate prostaglandin E2 biosynthesis. J Biol Chem 275: 32775–32782, 2000.[Abstract/Free Full Text]
  45. Trebino CE, Stock JL, Gibbons CP, Naiman BM, Wachtmann TS, Umland JP, Pandher K, Lapointe JM, Saha S, Roach ML, Carter D, Thomas NA, Durtschi BA, McNeish JD, Hambor JE, Jakobsson PJ, Carty TJ, Perez JR, and Audoly LP. Impaired inflammatory and pain responses in mice lacking an inducible prostaglandin E synthase. Proc Natl Acad Sci USA 100: 9044–9049, 2003.[Abstract/Free Full Text]
  46. Uematsu S, Matsumoto M, Takeda K, and Akira S. Lipopolysaccharide-dependent prostaglandin E2 production is regulated by the glutathione-dependent prostaglandin E2 synthase gene induced by the Toll-like receptor 4/MyD88/NF-IL6 pathway. J Immunol 168: 5811–5816, 2002.[Abstract/Free Full Text]
  47. Wang SW, Pawlowski J, Wathen ST, Kinney SD, Lichenstein HS, and Manthey CL. Cytokine mRNA decay is accelerated by an inhibitor of p38-mitogen-activated protein kinase. Inflamm Res 48: 533–538, 1999.[CrossRef][ISI][Medline]
  48. Wang X, Flynn A, Waskiewicz AJ, Webb BL, Vries RG, Baines IA, Cooper JA, and Proud CG. The phosphorylation of eukaryotic initiation factor eIF4E in response to phorbol esters, cell stresses, and cytokines is mediated by distinct MAP kinase pathways. J Biol Chem 273: 9373–9377, 1998.[Abstract/Free Full Text]
  49. Watanabe K, Kurihara K, and Suzuki T. Purification and characterization of membrane-bound prostaglandin E synthase from bovine heart. Biochim Biophys Acta 1439: 406–414, 1999.[Medline]
  50. Wilmer WA, Tan LC, Dickerson JA, Danne M, and Rovin BH. Interleukin-1beta induction of mitogen-activated protein kinases in human mesangial cells. Role of oxidation. J Biol Chem 272: 10877–10881, 1997.[Abstract/Free Full Text]
  51. Wong SC, Fukuchi M, Melnyk P, Rodger I, and Giaid A. Induction of cyclooxygenase-2 and activation of nuclear factor-kappaB in myocardium of patients with congestive heart failure. Circulation 98: 100–103, 1998.[Abstract/Free Full Text]
  52. Wuyts WA, Vanaudenaerde BM, Dupont LJ, Demedts MG, and Verleden GM. Involvement of p38 MAPK, JNK, p42/p44 ERK and NF-kappaB in IL-1beta-induced chemokine release in human airway smooth muscle cells. Respir Med 97: 811–817, 2003.[CrossRef][ISI][Medline]
  53. Zhang S, Han J, Sells MA, Chernoff J, Knaus UG, Ulevitch RJ, and Bokoch GM. Rho family GTPases regulate p38 mitogen-activated protein kinase through the downstream mediator Pak1. J Biol Chem 270: 23934–23936, 1995.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 CirculationHome page
N. Degousee, S. Fazel, D. Angoulvant, E. Stefanski, S.-C. Pawelzik, M. Korotkova, S. Arab, P. Liu, T. F. Lindsay, S. Zhuo, et al.
Microsomal Prostaglandin E2 Synthase-1 Deletion Leads to Adverse Left Ventricular Remodeling After Myocardial Infarction
Circulation, April 1, 2008; 117(13): 1701 - 1710.
[Abstract] [Full Text] [PDF]

Home page
 Ann. Surg. Oncol.Home page
H.-W. Wang, C.-T. Hsueh, C.-F. J. Lin, T.-Y. Chou, W.-H. Hsu, L.-S. Wang, and Y.-C. Wu
Clinical Implications of Microsomal Prostaglandin E Synthase-1 Overexpression in Human Non-Small-Cell Lung Cancer
Ann. Surg. Oncol., September 1, 2006; 13(9): 1224 - 1234.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
J.-Y. Qian, A. Leung, P. Harding, and M. C. LaPointe
PGE2 stimulates human brain natriuretic peptide expression via EP4 and p42/44 MAPK
Am J Physiol Heart Circ Physiol, May 1, 2006; 290(5): H1740 - H1746.
[Abstract] [Full Text] [PDF]

Home page
 J. Histochem. Cytochem.Home page
W. E. Ackerman IV, J. M. Robinson, and D. A. Kniss
Despite Transcriptional and Functional Coordination, Cyclooxygenase-2 and Microsomal Prostaglandin E Synthase-1 Largely Reside in Distinct Lipid Microdomains in WISH Epithelial Cells
J. Histochem. Cytochem., November 1, 2005; 53(11): 1391 - 1401.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
288/1/H165    most recent
00726.2004v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed