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J. Biol. Chem., Vol. 282, Issue 25, 18162-18172, June 22, 2007
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Activation*
1
From the
Institute of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, Taipei 100, Taiwan, the Department of Internal Medicine, National Taiwan University Hospital, Taipei 100, Taiwan, the ¶Institute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan, the ||National Core Facilities for Proteomics Research, Taipei 115, Taiwan, and the **Institute for Translational Medicine and Therapeutics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104
Received for publication, March 16, 2007 , and in revised form, April 19, 2007.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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13-reductase (PGR-2), which catalyzes the reaction converting 15-keto-PGE2 to 13,14-dihydro-15-keto-PGE2. The expression of PGR-2 is up-regulated in the late phase of 3T3-L1 adipocyte differentiation and predominantly distributed in adipose tissue. Overexpression of PGR-2 in cells decreases peroxisome proliferator-activated receptor 
(PPAR)-dependent transcription and prohibits 3T3-L1 adipocyte differentiation without affecting expression of PPAR. Interestingly, we found that 15-keto-PGE2 can act as a ligand of PPAR to increase co-activator recruitment, thus activating PPAR-mediated transcription and enhancing adipogenesis of 3T3-L1 cells. Overexpression of 15-hydroxyprostaglandin dehydrogenase, which catalyzes the oxidation reaction of PGE2 to form 15-keto-PGE2, significantly increased PPAR-mediated transcription in a PGE2-dependent manner. Reciprocally, overexpression of wild-type PGR-2, but not the catalytically defective mutant, abolished the effect of 15-keto-PGE2 on PPAR activation. These results demonstrate a novel link between catabolism of PGE2 and regulation of ligand-induced PPAR activation. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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(PPAR)2 plays important roles in adipogenesis, lipid and glucose homeostasis, and macrophage function (1–4). PPAR is a transcription factor in the nuclear receptor family, binding to the promoter of target genes by forming heterodimer with retinoid X receptor (5). Upon ligand binding, PPAR releases bound corepressors and recruits co-activator for transcriptional activation (6, 7). High-affinity synthetic agonists of PPAR, thiazolidinediones, have been widely used as antidiabetic drugs because of their effects in the regulation of lipid metabolism and their anti-inflammatory effects in adipose tissue (8, 9). Several naturally occurring ligands, many associated with the promotion or resolution of inflammation (10–13), including 15-deoxy-12,14prostaglandin J2 (15d-PGJ2) (14, 15), components of oxidized low density lipoprotein such as 9-hydroxyoctadecadienoic acid (HODE), 13-HODE, and 15-hydroxyeicosatetraenoic acid (10, 16), lysophosphatidic acid (17), and nitrolinoleic acid (11), have the capacity to activate PPAR. The production of prostaglandin E2 (PGE2) is elevated in many syndromes of inflammation (13, 18). However, little is known about whether the catabolism of PGE2 is associated with modulation of PPAR activity (19).
PGE2, a short-lived mediator, is inactivated via an oxidation reaction catalyzed by NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (PGDH), which generates 15-keto-PGE2, which is, in turn, further catabolized by a reaction catalyzed by NADPH/NADH-dependent 15-oxoprostaglandin-13-reductase (PGR) (20). It has been shown that adipose tissue possesses high activity of both PGDH and PGR, indicating that PGE2 catabolism is highly active in adipocytes (21). 3T3-L1 preadipocyte cell line has been used as a model for characterizing the events responsible for adipocyte differentiation (22), and PGE2 is the most abundant prostaglandin produced in 3T3-L1 fibroblasts through the release of arachidonic acid from endogenous phospholipids stores or upon addition of exogenous arachidonic acid (23). Although the levels of PGE2 decreased upon stimulation of 3T3-L1 adipocyte differentiation, PGE2 is a major prostaglandin produced in adipocytes (23, 24).
In this report, we used a differential display to identify a novel gene encoding prostaglandin reductase, designated as PGR-2. It is highly expressed in the late phase of 3T3-L1 adipocyte differentiation and is also abundant in adipose tissues. PGR-2 is capable of catabolizing 15-keto-PGE2, and its overexpression represses the transcriptional activity of PPAR. Following these observations, we further established that 15-keto-PGE2, an intermediate metabolite within the PGE2 catabolic pathway, can function as a PPAR ligand, stimulating mouse fibroblasts differentiation into adipocytes. Correspondingly, overexpression of PGDH also increased PGE2-dependent activation of PPAR. Our findings provide new insights into the potential importance of PGE2 catabolism in the regulation of PPAR activity.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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(E-8, Santa Cruz Biotechnology), anti-mouse actin (Chemicon), anti-FLAG (M5; Sigma), and anti-aP2 (Alpha Diagnostic International). Rabbit polyclonal antibodies against the recombinant GST-PGR-2 fusion protein were prepared and purified using GST protein-bound glutathione-Sepharose 4B (Amersham Biosciences). Recombinant human PPAR-LBD (His-tagged) was purchased from Invitrogen. Expression vectors for GAL4-DBD fusion of PPAR-LBDs (GAL4-PPARs) UASG x 4-TK-LUC reporter genes were generously provided by R. M. Evans (Howard Hughes Medical Institute, The Salk Institute for Biological Studies, La Jolla, CA). PPRE x 3-TK-LUC and TK-LUC reporter genes were kindly provided by C. K. Glass (University of California San Diego, La Jolla, CA). pGEX-5X3-SRC1568–781 plasmid was kindly provided by B. Desvergne (University of Lausanne, Lausanne, Switzerland). Cell Culture—3T3-L1 fibroblasts were maintained in Dulbecco's modified Eagle's medium (DMEM; high glucose) plus 10% calf serum. Two days after confluence, differentiation was induced by the addition of DMEM containing 10% fetal bovine serum (FBS), 172 nM insulin, 1 µM dexamethasone, and 0.5 mM methylisobutylxanthine for 2 days. The medium was then replaced with DMEM containing 10% FBS and 0.4 µM insulin for an additional 2 days of incubation followed by switching to DMEM plus 10% FBS for full differentiation in 2–3 days. The degree of differentiation was monitored by using Oil-Red O staining and triglyceride (TG) assay (GPO-Trinder; Sigma). 293T cells were cultured in DMEM in the presence of 10% FBS.
Reverse Transcription-Polymerase Chain Reaction—Total RNA was isolated from 3T3-L1 cells at various times throughout differentiation and was reverse transcribed with Supertranscript II (Invitrogen). Semiquantitative PCR amplification was performed for PGR-2 (sense primer, 5'-TAA GTC AGA TGA ATG AGA ACA G-3'; antisense primer, 5'-AAC CAC TGA CTC AGC TGT AG-3') and 36B4 (sense primer, 5'-CAT GAT GCG CAA GGC TAT CAG-3'; antisense primer, 5'-GAA GGT GTA CTC AGT CTC CA-3').
Cloning of Mouse PGR-2 cDNA—Mouse PGR-2 was cloned from cDNA of 3T3-L1 adipocyte using PCR amplification (sense primer, 5'-CGG TAT AGC TTG GGA CGC TA-3'; antisense primer, 5'-TGC ATG TTA AGA ATC TTT GTG G-3') and ligated into a pGEM-T easy vector (Promega) to generate the pTE-PGR-2 construct. The coding region of PGR-2 was then subcloned to the pCMV-Tag2B expression vector (Stratagene). A PCR reaction was carried out for the construction of pFLAG-PGR-2 using pTE-PGR-2 as a template and two primers (forward primer, 5'-AAC TGA AGC TTC AAG TGA TGA TCA TA-3', where the start codon is underlined; and reverse primer, 5'-AGC TCT CCC ATA TGG TCG ACC T-3') to generate a HindIII-SalI DNA fragment of PGR-2. This DNA fragment was cloned into the HindIII-SalI sites of pCMV-Tag2B, yielding pFLAG-PGR-2. The HindIII-XhoI fragment of the pFLAG-PGR-2 was inserted into the SmaI-XhoI sites of pGEX-4T-3 vector (Amersham Biosciences) to yield the pGEX-PGR-2 construct, which was used for the generation of the GST-PGR-2 fusion protein. The HindIII-SalI fragment of the pFLAG-PGR-2 was inserted into the EcoRV-SalI sites of pEGFP-C1 vector (BD Biosciences-Clontech) to create the pEGFP-PGR-2 construct. Site-directed mutagenesis of pGEX-PGR-2, pFLAG-PGR-2, and pEGFP-PGR-2 was performed to generate the PGR-2/Y259F catalytically defective mutant using the QuikChange kit (Stratagene) with the following mutagenic primers (mutated sites are underlined): forward primer, 5'-GGT CAG ATT TCT CAG TTC AGT AAC GAT GTG CCC-3'; reverse primer, 5'-GGG CAC ATC GTT ACT GAA CTG AGA AAT CTG ACC-3'.
Cloning of Mouse PGDH cDNA—NAD+-dependent PGDH was cloned from the cDNA of mouse kidney using PCR amplification (sense primer, 5'-AGT CGG ATC CAT GCA CGT GAA CG-3', where the start codon is underlined; antisense primer, 5'-CAG TCT CGA GTT ATG GAG CTT TTA C-3') to generate a BamHI-XhoI DNA fragment of PGDH. This DNA fragment was cloned into the BamHI-XhoI sites of pCMV-Tag2B to yield the pFLAG-PGDH construct.
Expression and Purification of Recombinant PGR-2—An Escherichia coli strain XL1-blue was transformed with the plasmid pGEX-PGR-2 to generate the recombinant protein of PGR-2 for the enzyme assay, and the recombinant protein was induced with 0.1 mM isopropyl-1-thio-
-D-galactoside at 25 °C overnight. GST-PGR-2 recombinant protein was purified with a glutathione-Sepharose column according to the manufacturer's instructions (Amersham Biosciences).
Enzymatic Assay for PGR-2—PGR-2 activity assay was carried out in a mixture containing 0.1 M Tris-HCl (pH 7.4), 0.5 mM NADPH, and the substrate 15-keto-PGE1, 15-keto-PGE2, 15-keto-PGF1
, or 15-keto-PGF2 in a total volume of 100 µl. The reaction was started by adding 5 µg of purified GST-PGR-2 protein and incubated at 37 °C for 30 min. NADPH remaining after the reaction was oxidized by adding 200 µl of color reagent (790 µM indonitrotetrazolium chloride, 60 µM phenazene methosulfate, and 1% Tween 20) at 37 °C in the dark for 10 min followed by adding 700 µl of phthalate buffer (pH 3.0) to stop the reaction. The colorimetric reaction is based on the fact that indonitrotetrazolium can react with NADPH in the presence of phenazene methosulfate to produce formazans (25). The absorbance of formazans was measured at 490 nm with a spectrophotometer. One unit of the enzyme was defined as the amount of enzyme catalyzing the production of 1 µmol NADP+/min. The apparent Km and Vmax values were based on Michaelis-Menten kinetics and calculated by Eadie-Hofstee regression using GraphPad Prism software (version 4.0). All values presented are the means of three or more measurements.
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Prostaglandin Analysis—The in vitro PGR-2 reaction products were analyzed by nano-ESI-MS and MS/MS. The analytes were directed to the homemade nanosprayer applied with –3.5 kV on the QSTAR-XL hybrid quadrupole time-of-flight mass spectrometer (Applied Biosystems/MDS Sciex). All of the data were acquired and processed using AnalystQS 1.1 with Bioanalyst 1.1 extension. The instrument was calibrated using the fragment ions that resulted from the collision-induced dissociation of Glu-fibrinopeptide B. Full scan mass spectra (MS) were recorded in the negative ion mode in the range of m/z 100–500. Product ion mass spectra (MS/MS) were obtained for m/z 351 (13,14-dihydro-15-keto-PGE2) and m/z 349 (15-keto-PGE2) for further identification of the analytes.
Samples were dried under nitrogen, dissolved in 200 µlof 20% acetonitrile, and filtered before analysis for characterization of intracellular PGE2 metabolites. The instrument used was a Thermo-Finnigan Quantum Ultra AM tandem quadrupole interfaced to a Shimadzu Prominence HPLC system. A 150 mm x 2mm x 3 µm Luna C18 (2) 100A HPLC column (Phenomenex) was maintained at 40 °C. The mobile phase was generated from HPLC-grade water (A) and 5% methanol/95% acetonitrile (B), each containing 0.005% acetic acid adjusted to pH 5.7 with ammonium hydroxide. The flow rate was 200 µl/min using a gradient starting at 20% B and ramping to 60% B in 20 min. Transitions monitored were: [2H4]-PGE2, m/z 355 
275 at a collision energy (CE) of 18 eV; PGE2, m/z 351 271, CE 18 eV; [2H4]-15-keto-PGE2, m/z 353 165, CE 20 eV; 15-keto-PGE2, m/z 349 161, CE 20 eV; [2H4]-13,14-dihydro-15-keto-PGE2, m/z 355 223, CE 18 eV; 13,14-dihydro-15-keto-PGE2, m/z 351 219, CE 18 eV. The collision gas was argon, 1.5 millitorr. Source collision-induced dissociation was 12 eV. All compounds were conclusively identified by comparison with synthetic standards (Cayman Chemical).
Transfection and Reporter Assays—One day before transfection, 3T3-L1 fibroblasts were plated at 70–80% confluence, and 293T cells were plated at 30% confluence. After overnight growth at 37 °C, cells were incubated with transfection mixture containing plasmid DNA and Lipofectamine at a 1:6 ratio (Invitrogen). After 24 h of transfection, cells were harvested for determination of luciferase and -galactosidase activity. Each experiment was repeated three or more times.
Transient Transfection in 3T3-L1 Preadipocytes—Enforced expression of GFP-PGR-2 was performed in post-confluent 3T3-L1 preadipocytes. Cells were transfected with pEGFP-C1, pEGFP-PGR-2, and pEGFP-PGR-2/Y259F construct using Lipofectamine 2000 (Invitrogen) for 5 h according to the manufacturer's instructions. Cells were then washed gently and treated with the standard induction medium for adipocyte differentiation.
Ligand Binding Assays—Ligand binding assays were performed with GST-mPPAR2 fusion protein, which was expressed in E. coli and purified by glutathione-Sepharose affinity chromatography. The protein-bound Sepharose beads were used directly in a binding reaction with 100 nM [3H]BRL49653 in a buffer containing 10 mM Tris-HCl (pH 7.4), 50 mM KCl, and 10 mM dithiothreitol. Competitors or solvent (dimethyl sulfoxide) were added as indicated. Following incubation for 2 h at 4 °C, the PPAR-Sepharose beads were washed with 10 volumes of ice-cold binding buffer and pulled by centrifugation. The amount of [3H]BRL49653 bound to the beads was measured with a scintillation counter. Each experiment was repeated three or more times.
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-LBD was preincubated with ligands for 30 min on ice and then mixed with immobilized GST or GST-SRC1 in the pulldown buffer (1x phosphate-buffered saline, 10% glycerol, 0.5% Nonidet P-40). The reactions were incubated overnight at 4 °C after which beads were washed three times in pulldown buffer and boiled in 2x sample buffer. Proteins bound to beads were separated by 11% SDS-PAGE. Blots were then developed with antibodies against PPAR or GST. Statistical Analyses—Results are expressed as the means ± S.D. Statistical analyses were performed with Student's t test for comparison of each effect versus control. A p value <0.05 was considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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13-reductase (LTB4DH/PGR or PGR-1) (26–28) (Fig. 1). Based on this similarity, this new gene was designated PGR-2.
Expression of PGR-2 was increased during the differentiation of 3T3-L1 cells at both the mRNA (Fig. 2A) and protein levels, with a time lag of 
1 day following the expression of PPAR (Fig. 2B). Among the mouse tissues examined, adipose tissue showed the highest expression of PGR-2 mRNA (Fig. 2C). This expression pattern was distinct from that of PGR-1, which is most highly expressed in liver and kidney (26).
Functional Characterization of PGR-2 as a 15-Oxoprostaglandin-13-reductase—To analyze the biochemical function of PGR-2, we cloned and sequenced PGR-2 cDNA from 3T3-L1 adipocytes (see "Experimental Procedures"). We expressed and purified the recombinant mouse PGR-2 as a GST fusion protein from the E. coli system to perform an enzymatic assay (Fig. 3A). Based on the presence of a conserved domain of putative NADP(H)-dependent oxidoreductase in its amino acid sequence, we measured its prostaglandin reductase activity using different prostaglandins as the substrate. Toward this end, we developed a colorimetric assay by measuring formazan formation from the reduction of the tetrazolium salt in detecting the prostaglandin reductase activity (See "Experimental Procedures").
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, and 15-keto-PGF1 were promising substrates, whereas 13,14-dihydro-15-ketoprostaglandins were not. Neither dehydrogenase activity nor LTB4DH activity was associated with PGR-2 (Table 1). Kinetic studies revealed that PGR-2, requiring NADPH but not NADH as a cofactor, had the highest efficiency in converting 15-keto-PGE2 into 13,14-dihydro-15-keto-PGE2 (Kcat/Km = 229.8 mM–1 min–1 compared with 222.9 mM–1 min–1 for 15-keto-PGF1, 215.1 mM–1 min–1 for 15-keto-PGE, and 136.0 mM–1 min–11 for 15-keto-PGF2 (as summarized in Table 2)). The reaction product obtained from the incubation of 15-keto-PGE2 and NADPH with purified PGR-2 was further analyzed by ESI-MS/MS to verify 13,14-dihydro-15-keto-PGE2 formation in the reaction (Fig. 3C). The molecular anion ([M – H]– = m/z 351) from the reaction product gave the same fragments in the MS/MS spectrum as standard 13,14-dihydro-15-keto-PGE2, which yielded ions that were 2 proton mass units higher than corresponding ions in the 15-keto-PGE2 MS/MS spectrum (Fig. 3, B and C).
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The Role of PGR-2 Expression in Adipogenesis—Because the expressed level of PGR-2 was not increased until 3T3-L1 cells had attained late stage differentiation to adipocytes, we then determined whether constitutive expression of PGR-2 would influence adipocyte differentiation. 3T3-L1 cells were transiently transfected with a wild-type expression vector and a catalytically defective GFP-PGR-2, and differentiation was then induced. Based on structural prediction, the conserved Tyr-259 residue in PGR-2 (Fig. 1) might function as the Tyr-245 of PGR-1 that participates in the hydrogen bond network around the 2'-hydroxyl group of the nicotine amide ribose, which interacts with two water molecules in stabilizing an enolate intermediate for the catalysis of 15-keto-PGE2 reduction (28). The Y259F mutant of PGR-2 was then generated and showed a significant decrease in catalytic efficiency in the reduction reaction of 15-keto-PGE2 (Fig. 5A). We found that ectopic expression of the wild type but not Y259F of GFP-PGR-2 dramatically inhibited adipocyte differentiation, as evaluated by the accumulation of lipid droplets with Oil-Red O staining (Fig. 5B), and decreased intracellular TG synthesis by 53% as compared with control cells (Fig. 5C). Furthermore, ectopic expression of wild-type GFP-PGR-2, but not the Y259F mutant, significantly reduced the induction of the PPAR target gene, aP2, whereas the expression level of PPAR remained unaltered (Fig. 5D). These results clearly demonstrate that constitutive expression of functional PGR-2 impairs PPAR-mediated adipocyte differentiation without affecting the protein expression level of PPAR.
15-Keto-PGE2 Is a PPAR Ligand—The effect of overexpression of PGR-2 on the suppression of adipocyte differentiation (Fig. 5, B–D) and the induction pattern of PGR-2 expression (Fig. 2, A and B) indicates that catabolism of 15-keto-PGE2 by PGR-2 needs to be suppressed during the early phase of adipocyte differentiation. Because PGE2 is a major prostaglandin produced in preadipocytes (23, 24) and there is a temporal difference between the expression of PGR-2 and PPAR during adipogenesis of 3T3-L1 cells (Fig. 2B), we raised the question of whether 15-keto-PGE2, rather than PGE2, has a functional role in modulating PPAR activity during adipocyte differentiation. We transiently transfected the PPRE-based reporter gene (PPRE-TK-Luc) into 3T3-L1 that had been induced for differentiation for 2 days (PPAR expression was induced and the level of PGR-2 was still low) to determine the effect of 15-keto-PGE2 on PPRE-dependent transcription in the early differentiation stage of 3T3-L1 cells. The addition of 15-keto-PGE2 to the culture medium of transfected cells was capable of activating transcription of the PPRE-based reporter gene as efficiently as BRL49653, a synthetic ligand of PPAR (Fig. 6A). In parallel experiments, the control reporter gene (TK-Luc) did not respond to 15-keto-PGE2 or BRL49653. The results suggest that 15-keto-PGE2 can activate PPRE-dependent transcription.
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in vitro using ligand competition assays. 15-Keto-PGE2, similar to unlabeled BRL49653, was able to compete for the binding of [3H]BRL49653 to PPAR (Fig. 6B), whereas neither PGE2, 13,14-dihydro-15-keto-PGE2, nor 15-keto-PGF2 (another substrate of PGR-2) had competitive capacity. Fig. 6C further demonstrates the competitive inhibition of [3H]BRL49653 binding to PPAR by 15-keto-PGE2 in a dose-dependent manner (the estimated Kd is around 30 µM). We did not test for 15-keto-PGE1 and 15-keto-PGF1, because the physiological concentrations of PGE1 and PGF1 are much lower than that of PGE2 (29).
Because ligand-induced transactivation of PPAR is achieved by the recruitment of coactivators such as SRC1 and TIF2 (30), we performed an in vitro coactivator-dependent receptor ligand assay by incubating the purified recombinant GST-SRC1 with PPAR-LBD proteins. Similar to BRL49653 as a positive control, inclusion of 15-keto-PGE2 in the assay enabled PPAR-LBD to be pulled down by GST-SRC1, indicating that 15-keto-PGE2 is capable of acting as a ligand in inducing the interaction between SRC1 and PPAR-LBD (Fig. 6D). The same effect of 15-keto-PGE2 could be seen when the coactivator TIF2 was used for the interaction assay (data not shown). No interaction was detected when GST protein was used for the binding assay. Taken together, these results indicate that 15-keto-PGE2 is a PPAR ligand that activates its transcriptional function by promoting co-activator recruitment.
Catabolic Conversion of PGE2 by Expression of Prostaglandin Dehyrogenase Activates PPAR—To substantiate the potential role of 15-keto-PGE2 generation in PPAR activation, we next tested whether overexpression of PGDH could convert PGE2 to an activator for PPAR-mediated transactivation. 293T cells were transfected both with the GAL4 luciferase reporter plasmid (UASG) and a vector expressing the ligand-binding domain (LBD) of the mouse PPAR fused to yeast GAL4 DNA-binding domain with or without the PGDH expression vector (Fig. 7A). Overexpression of PGDH had no effect on the transactivation activities of GAL4-PPAR. Treatment of cells with 10 µM PGE2 significantly increased GAL4-PPAR-mediated transcriptional activity in cells expressing PGDH, but not in the cells transfected with the control vector (Fig. 7B). Consistent with the catabolic function of PGDH for PGE2, treatment with 15-keto-PGE2 also activated GAL4-PPAR in the cells regardless of PGDH expression (Fig. 7B). Furthermore, activation of PPAR activity in cells expressing PGDH was increased with increasing concentration of PGE2 (Fig. 7C). These results support our notion that metabolic conversion of PGE2 to 15-keto-PGE2 is able to activate PPAR.
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-mediated Transcription and Enhances Adipogenesis in 3T3-L1 Cells—The GAL4 luciferase reporter plasmid UASG was transfected to 3T3-L1 fibroblasts with GAL4-PPAR, -PPAR, or -PPAR
to determine the effect of 15-keto-PGE2 on the transcriptional function of different subtypes of PPAR in vivo. Treatment of transfected cells with 15-keto-PGE2 increased the transactivation activities of GAL4-PPAR and, to a lesser degree, GAL4-PPAR but not of GAL4-PPAR (Fig. 8A). Moreover, GAL4-PPAR was more effectively activated by increasing concentrations of 15-keto-PGE2 than GAL4-PPAR (Fig. 8B).
It has been reported that treatment of 3T3-L1 preadipocytes with dexamethasone and insulin (DI) is unable to induce adipocyte differentiation unless methylisobutylxanthine (MIX) is added to DI to stimulate the generation of endogenous PPAR ligands via the cAMP signaling pathways (31, 32). This system allows us to evaluate the effect of supplementation of PPAR ligands on the promotion of adipogenesis in the absence of methylisobutylxanthine. As shown in Fig. 8C, differentiation did not occur when preadipocytes were incubated in the presence of DI or MIX alone. As expected, the addition of MIX into DI medium gave rise to adipocyte differentiation of 3T3-L1 cells, as manifested by the accumulation of lipid droplets with Oil-Red O staining (Fig. 8C) and the quantitative analysis of the intracellular TG content (Fig. 8D). Interestingly, the addition of 15-keto-PGE2 or BRL49653 into DI medium also resulted in adipocyte differentiation to a degree comparable with the effect of MIX in DI medium (Fig. 8, C and D). We further examined the ability of 15-keto-PGE2 to induce expression of PPAR target genes during adipogenesis. Similar to MIX treatment, both 15-keto-PGE2 and BRL49653 treatment strongly induced the expression of aP2, an adipocyte-specific marker, after 6 days of adipocyte differentiation (Fig. 8E). Previous studies have shown that PPAR ligands are involved in a positive feedback loop to maintain a relatively high expression of PPAR (31). Consistently, expression of PPAR was increased in cells cultured in a DI medium supplemented with 15-keto-PGE2, BRL49653, or MIX. Moreover, the addition of DI with 15-keto-PGE2 or BRL49653 already induced a prominent aP2 expression in 2 days of treatment, in contrast to a rather weak aP2 induction following the addition of MIX to DI medium (Fig. 8F). The differences in aP2 induction in response to 15-keto-PGE2, BRL49653, and MIX treatment during the early phase of induction of adipogenesis suggest that direct ligand-induced activation of PPAR by 15-keto-PGE2 and BRL49653 gave an earlier induction of aP2 than MIX treatment, which requires more time for endogenous ligand generation. Therefore, these data support the proposition that 15-keto-PGE2 can act as a PPAR ligand for adipocyte differentiation.
Overexpression of PGR-2 Suppressed 15-Keto-PGE2-mediated Transactivation of PPAR—The enzymatic function of PGR-2 in the reduction of 15-keto-PGE2 led us to determine whether overexpression of PGR-2 could inhibit 15-keto-PGE2-induced activation of PPAR. We expressed FLAG-PGR-2 and GAL4-PPAR in 293T cells at the protein levels comparable with the endogenous levels of PGR-2 and PPAR in differentiated 3T3-L1 adipocytes (Fig. 9A). The addition of 15-keto-PGE2 to the culture medium without coexpression of FLAG-PGR-2 caused an 8-fold increase in GAL4-PPAR-mediated transcriptional activation of UASG luciferase reporter activity in 293T cells (Fig. 9B). Ectopic expression of FLAG-PGR-2 markedly suppressed 15-keto-PGE2-mediated activation of GAL4-PPAR in the reporter activity in a dose-dependent manner (Fig. 9B). Coexpression of Y259F mutant with GAL4-PPAR no longer decreased the 15-keto-PGE2-activated transcriptional activity of PPAR (Fig. 9B), indicating that the catalytic activity of PGR-2 is required for negative modulation of 15-keto-PGE2-dependent transcriptional activation of PPAR (Fig. 9C).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-dependent transcription and adipogenesis. We have shown that catabolic conversion of PGE2 to 15-keto-PGE2 by overexpression of PGDH enables PPAR activation. In addition, we identified PGR-2 as an enzyme that catalyzes the reaction in converting 15-keto-PGE2 to 13,14-dihydro-15-keto-PGE2. The amount of PGR-2 expression is abundant in adipose tissue and is up-regulated during 3T3-L1 adipocyte differentiation. Ectopic overexpression of PGR-2 in 3T3-L1 cells dramatically inhibits adipocyte differentiation. These data illuminate the role of PGE2 catabolism in PPAR-dependent transcription.
PGE2 is the major prostaglandin produced by preadipocytes (23), and its catabolism in adipose tissue is highly active (21). Using in vitro ligand binding assays, we have proved that 15-keto-PGE2, a metabolite of PGE2, is able to compete for binding of a synthetic ligand, BRL49653, to PPAR. Furthermore, the interaction between SRC1 and PPAR is induced upon binding of 15-keto-PGE2 to PPAR, indicating its ligand function in coactivator recruitment. In addition, in vivo transactivation assays have demonstrated that 15-keto-PGE2 preferentially activates PPAR-LBD-mediated transcription as compared with PPAR and PPAR. More importantly, the addition of 15-keto-PGE2 to 3T3-L1 cells can replace the agonist of PPAR, thereby inducing differentiation of adipocytes efficiently. Altogether, these results show that 15-keto-PGE2 can function as a PPAR ligand.
PPAR was suggested as a nuclear prostanoid receptor when 15d-PGJ2, the dehydration product of PGD2, was first identified as a possible endogenous PPAR ligand (14, 15). Indeed, 15d-PGJ2 can drive PPAR-derived adipocyte differentiation but only at concentrations considerably in excess of those formed endogenously (24, 32). Other compounds formed naturally also possess the capacity to activate PPAR; among them are 9-HODE, 13-HODE, and 15-hydroxyeicosatetraenoic acid (10, 16), lysophosphatidic acid (17), and nitrolinoleic acid (11). However, it remains to be determined whether the concentrations necessary (IC50 values in the in vitro ligand binding assay ranging from 1 to 50 µM) are ever attained in vivo.
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Here, we have provided diversified lines of evidence consistent with the capacity of 15-keto-PGE2 to activate PPAR-dependent adipogenesis. An outstanding question is whether this occurs at concentrations of the metabolite actually formed in vivo. At present, it remains a technical challenge to quantify 15-keto-PGE2 in adipocytes. However, it has been reported that lipid metabolites containing the ,-unsaturated ketone, including 15-keto-PGE2, bind to PPAR covalently, activating PPAR. Accordingly, it is possible that this covalent binding mechanism enables 15-keto-PGE2 to activate PPAR in vivo at lower concentrations than would otherwise be the case (34). Further studies will be needed to determine whether PPAR is actually covalently modified by endogenous concentrations of 15-keto-PGE2 and whether this modified form accumulates during 3T3-L1 adipocyte differentiation.
This is one of the few examples of a biological functionality attributable to a prostaglandin metabolite rather than to the parent compound. For example, the role of PGDH in closure of the ductus arteriosus (35) is attributable to its capacity to inactivate the parent moiety. The present observations raise the possibility that the increased PGDH but unaltered PPAR expression in white adipose tissue of ob/ob mice (36, 37) might reflect a role for 15-keto-PGE2 in adipogenesis in this model, whereas the recognition that PPAR can exert its anti-inflammatory effects by ligand-dependent transrepression of inflammatory response genes (38, 39) raises the possibility that macrophage-derived 15-keto-PGE2 might also modulate adipocyte differentiation and function. Because PGDH is responsible for terminating pro-inflammatory PGE2 signaling, it will be interesting to know whether the anti-inflammatory capacity of PPAR in adipose tissue is regulated by catabolic conversion of PGE2 into 15-keto-PGE2.
In summary, this study demonstrates for the first time a new link between the catabolism of PGE2 and the regulation of the PPAR function. Future studies will determine the importance of this system in the regulation of adipogenesis in vivo.
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FOOTNOTES |
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1 To whom correspondence should be addressed: Inst. of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, No. 1, Sec. 1, Jen-Ai Rd., Taipei 100, Taiwan. Tel.: 886-2-23123456, ext. 8229; Fax: 886-2-23958904; E-mail: ZFCHANG{at}ha.mc.ntu.edu.tw.
2 The abbreviations used are: PPAR, peroxisome proliferator-activated receptor ; PPRE, PPAR response element; 15d-PGJ2, 15-deoxy-12,14prostaglandin J2; HODE, hydroxyoctadecadienoic acid; PGR, 15-oxoprostaglandin-13-reductase; PG, prostaglandin; PGDH, 15-hydroxyprostaglandin dehydrogenase; TG, triglyceride; LBD, ligand-binding domain; GST, glutathione S-transferase; FBS, fetal bovine serum; DMEM, Dulbecco's modified Eagle's medium; HPLC, high pressure liquid chromatography; MS, mass spectrometry; MS/MS, tandem MS; ESI, electrospray ionization; DI, dexamethasone and insulin medium; MIX, methylisobutylxanthine; LTB4DH, leukotriene B4-12-hydroxydehydrogenase.
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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