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J. Biol. Chem., Vol. 275, Issue 42, 33142-33150, October 20, 2000

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Suppression of Rat Thromboxane Synthase Gene Transcription by Peroxisome Proliferator-activated Receptor  in Macrophages via an Interaction with NRF2^*

Yukio Ikeda, Akira Sugawara, Yoshihiro Taniyama, Akira Uruno, Kazuhiko Igarashi^§, Shuji Arima, Sadayoshi Ito, and Kazuhisa Takeuchi^¶

From the  Division of Nephrology, Endocrinology, and Vascular Medicine, Department of Medicine, Tohoku University Graduate School of Medicine, Sendai 980-8574, Japan and the ^§ Department of Biochemistry, Hiroshima University School of Medicine, Hiroshima 734-8551, Japan

Received for publication, March 20, 2000, and in revised form, June 22, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

We have studied the transcription regulation of the rat thromboxane synthase (TXS) gene by peroxisome proliferator-activated receptor  (PPAR) in macrophages. The transcription activity of a cloned 5'-flanking region (1.6 kilobases) of the rat TXS gene (5'FL-TXS) was examined by luciferase reporter gene assay. TXS mRNA expression and the transcription activity of 5'FL-TXS were inhibited by PPAR ligands, 15-deoxy-^12,14-prostaglandin J₂ (PGJ₂), and the thiazolidinedione troglitazone (TRO) in a dose-dependent manner. Overexpression of PPAR also significantly suppressed transcription, and further addition of PGJ₂ or TRO augmented the suppression. Deletion analysis showed that the element responsible for the PPAR effect is located in a region containing the nuclear factor E2 (NF-E2)/AP-1 site (98/88), which was indicated to be the major promoter of the TXS gene. By electrophoretic mobility shift assay using the NF-E2/AP-1 site and nuclear extracts from macrophages, we observed a specific protein-DNA complex formation, which was inhibited by a specific antibody against the transcription factor NRF2 (NF-E2-related factor 2). Moreover, the complex was decreased with PGJ₂, TRO, or in vitro translated PPAR. The transcription suppression by PPAR was confirmed using this truncated NRF2-binding element (98/88) by the reporter gene assay. Finally, a direct interaction between PPAR and NRF2 was confirmed by glutathione S-transferase pull-down assay. In conclusion, the NRF2-binding site (98/88) is the major promoter of 5'FL-TXS which can be suppressed by activated PPAR via a protein-protein interaction with NRF2 in macrophages.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Thromboxane A₂ is a labile metabolite of arachidonic acid and a potent inducer of platelet aggregation and vasoconstriction (1) and cell proliferation (2). Thromboxane A₂ has been known to play a pathophysiological role in atherosclerosis (3) and glomerulonephritis (4). Thromboxane synthase (TXS)¹ catalyzes the conversion of prostaglandin H₂ to thromboxane A₂ and is expressed in lung, liver, kidney, and blood cells including macrophages (5). The structures of human (6), mouse (7), and rat (8) TXS have been reported and are indicated to belong to a cytochrome P450 subfamily. We also have observed that TXS mRNA is induced in an inflammatory model of hydronephrosis characterized by infiltration of macrophages in the renal interstitium (8). Although the transcriptional regulatory regions of the human (9) and mouse TXS (10) genes have been cloned, analysis of their gene transcription has not yet been extensively performed, and any role of the peroxisome proliferator-activated receptor (PPAR) in TXS gene transcription has never been examined.

PPAR is one of the nuclear receptors that serves as a transcription factor (11). Three different types of PPAR isoforms are present: PPAR, PPAR, and PPAR. PPARs heterodimerize with the retinoid X receptor (RXR) (12) and regulate transcription through binding to the PPAR-response element, which is present in the transcriptional regulatory region in several genes. PPARs appear to exhibit distinct patterns of tissue distribution and have different ligands, suggesting that they have their own functions in different tissues. PPAR was initially identified in differentiated adipocytes, and its role has been determined in relation to the pathogenesis of insulin resistance because the thiazolidinedione class of antidiabetic drugs (insulin sensitizers), such as troglitazone (TRO), was identified with a ligand of PPAR (13) as well as 15-deoxy-^12,14-prostaglandin J₂ (PGJ₂) (14). Recently, however, PPAR has been shown to be expressed not only in adipocytes, but also in vascular tissues, such as vascular smooth muscle cells (VSMCs) and endothelial cells, and in macrophages in atheromatous plaques (15). Moreover, PPAR activators inhibit matrix metalloproteinase-9 expression in VSMCs (16) and thrombin-induced endothelin-1 production in endothelial cells (17). In monocytes/macrophages, PPAR activators suppress production of inflammatory cytokines (18) and stimulate the expression of scavenger receptors and the uptake of oxidized low density lipoprotein, leading to differentiation of monocytes/macrophages to foam cells (19). Intimal hyperplasia of a balloon-injured rat aorta was also inhibited by TRO (20). These observations suggest that PPAR plays a role in vascular metabolism.

In this study, we assessed the role of PPAR in TXS gene regulation in macrophages since both TXS and PPAR are expressed in macrophages and are possibly involved in atherogenesis. We first cloned a 5'-flanking region of the rat TXS gene (5'FL-TXS), and identified its major promoter in macrophages. We then observed suppression of TXS gene transcription by PPAR and revealed the mechanism. In conclusion, PPAR can inhibit TXS gene transcription by a possible direct protein-protein interaction between NRF2 and PPAR via the NRF2-binding site (98/88) in 5'FL-TXS.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Cloning of 5'FL-TXS-- 5'FL-TXS was cloned by the polymerase chain reaction (PCR) using Genome Walker kits (CLONTECH). Briefly, the first PCR was conducted with a combination of adaptor primer 1 (provided in the kit) and TXS cDNA primer 1 (5'-TCT TGA GAA CGC TGA TGT GGA GTA C-3') using a rat genomic library provided in the kit as a template. Nested PCR was then carried out with a combination of adaptor primer 2 (provided in the kit) and TXS cDNA primer 2 (5'-ATT TCA GGA GGG CCA AGA GAA CCA C-3') using the first PCR product as a template. The resultant PCR product was subcloned into pBluescript SK(+) between the SalI and XhoI sites. Sequencing of the insert was performed in both directions by the previously reported PCR cycle sequence method (21) with an automatic sequence analyzer (ABI PRISM 310 Genetic Analyzer).

Rapid Amplification of 5'-cDNA Ends-- This was carried out using Marathon-Ready cDNAs (CLONTECH). Briefly, PCR was conducted with adaptor primer 1 (provided in the kit) and TXS cDNA primer 2 using a rat kidney cDNA library as a template. Nested PCR was then performed with adaptor primer 2 and TXS cDNA primer 3 (5'-ACG GTA CCA ACT TCG AAC TTG A-3') using the first PCR product as a template. The resultant PCR products were cloned into pCR2.1-TOPO with the TOPO TA cloning kit (Invitrogen). Ten clones were sequenced, and their 5'-ends were identified.

Synthesis of Chimeric Luciferase Expression Vectors and Other Constructs-- To examine the transcription function of 5'FL-TXS, we constructed chimeric expression vectors containing fragments of 5'FL-TXS fused upstream of firefly luciferase cDNA. Briefly, the fragment of 5'FL-TXS was inserted between MluI and XhoI of the luciferase reporter vector Pica Gene Vector 2 (Nippon Gene). Deletion mutants of 5'FL-TXS were synthesized based on an exonuclease III deletion protocol (22). The 5'-end of each deletion fragment was determined by sequencing. The internal mutation was introduced in the full-length fragment (1598 bp) using the Transformer^TM site-directed mutagenesis kit (CLONTECH). To analyze the transcription function of a putative NF-E2/AP-1 site, an oligonucleotide (104/86, 5'-TAA AGT TGC TGA TTC ATT G-3') containing the NF-E2/AP-1 site (98/88) in 5'FL-TXS was inserted into the HindIII/XhoI fragment in the pT109 vector upstream of the thymidine kinase promoter to give NF-E2/AP-1-tkLuc.

Mouse PPAR1 cDNA in pCMX was kindly provided by Dr. K. Umezono (Kyoto University, Kyoto, Japan) (23). Mouse RXR cDNA (kindly provided by Dr. R. M. Evans, Salk Institute, San Diego, CA) (24) was subcloned into pcDNA1/Amp (Invitrogen). Human NRF2 cDNA (1807 bp) (25) was subcloned into the pGEX-4T-2 vector (Amersham Pharmacia Biotech) and is designated as pGEX-NRF2. The full-length human NRF2 cDNA was inserted into pcDNA1/Amp to synthesize the human NRF2 expression vector pcDNA1-hNRF2.

Cell Culture, Transfection, and Luciferase Assay-- Cultured rat VSMCs (22, 26) were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and penicillin/streptomycin. Cells (10⁶ cells/well) were cultured and transfected by the lipofection method (3.0 µg of DNA/well) using N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium salts liposomal transfection reagent (Roche Molecular Biochemicals). Cultured rat macrophages (NR8383) were maintained in Ham's F-12K medium containing 2 mM L-glutamine, 1.5 g/liter sodium bicarbonate, 15% fetal bovine serum, and penicillin/streptomycin. Cells (5 × 10⁵ cells/well) were cultured and transfected by the lipofection method (3.0 µg/well). Cultured rat lung endothelial cells (RLECs) (27) were maintained in RPMI 1640 medium containing 10% fetal calf serum and penicillin/streptomycin. Cells (10⁶ cells/well) were cultured and transfected by a modification of the DEAE-dextran method. Briefly, 3.0 µg of DNA were mixed with 27 µl of 10 mg/ml DEAE-dextran and 0.85 µl of 100 ng of chloroquine, and the serum-free medium was added to a 1-ml final volume/well. Cells were incubated with the mixture for 90 min and treated with 10% dimethyl sulfoxide/phosphate-buffer saline). Afterward, the medium was removed and replaced. As an internal control to normalize transfection efficiency, 1.0 µg of cytomegalovirus/-galactosidase expression vector was cotransfected. After transfection, the cells were cultured with a hyposerum medium containing 1% fetal bovine serum or fetal calf serum for 18 h and then treated with 0.5-5.0 µM PGJ₂ (Cayman Chemical Co., Inc.) or 1.0-20 µM TRO (kindly provided by Sankyo Co., Ltd.) for 12 h or with 1.0 µM dexamethasone (Wako), 700 IU/ml recombinant rat interferon- (Genzyme Corp.), 40 ng/ml recombinant rat interleukin-6 (BIOSOURCE International), and 100 ng/ml tumor necrosis factor- (Genzyme Corp.) for 24 h. In the overexpression study, 1.0 µg/well plasmid of PPAR1 cDNA, human NRF2 cDNA, or RXR cDNA was cotransfected. Luciferase and -galactosidase expression was analyzed by previously reported methods (22, 26).

Determination of TXS mRNA Levels-- To analyze TXS mRNA expression levels, Northern blot analysis and reverse transcription (RT)-PCR were performed. Briefly, 7 × 10⁶ cells were seeded in 100-mm dishes and maintained. Twelve hours after stimulation with PGJ₂ or TRO, the cells were collected, and the total mRNAs were isolated by Trizol reagent (Life Technologies, Inc.). In the Northern blot analysis, the isolated RNAs (10 µg each) were electrophoresed on a 1.5% formaldehyde-agarose gel and transferred to a nylon membrane (Hybond-N, Amersham Pharmacia Biotech) for hybridization with the radiolabeled rat TXS cDNA probe (706 bp). Integrity of RNA was verified by hybridization with the -actin probe. Semiquantitative RT-PCR was performed with the One Step RNA PCR kit (TaKaRa) with a forward primer (5'-TTC ACA GGC TTG GCT GAT GAG AGG TGT CAT-3') and a reverse primer (5'-GGC TTC TCA AGT TCG AAG TCA GTG GTA CCG-3') to amplify TXS mRNA. Constitutively expressed rat glyceraldehyde-3-phosphate dehydrogenase mRNA was also amplified with a forward primer (5'-TCC CTC AAG ATT GTC AGC AA-3') and a reverse primer (5'-AGA TCC ACA ACG GAT ACA TT-3'). PCR was performed under the following conditions: 94 °C for 30 s, 57 °C for 30 s, and 72 °C for 1 min for 30 cycles. Under these PCR conditions, a linear correlation between the densitometric intensity units of the PCR product and amounts of template was confirmed. TXS mRNA levels were determined by a ratio between the densitometric intensity units of the PCR products from TXS and glyceraldehyde-3-phosphate dehydrogenase mRNAs on an ethidium bromide-stained 2% agarose gel. Densitometric units of the PCR products were determined using a computerized analysis system (Luminous Imager 2.0, Aisin Cosmos R&D, Ltd.). The endogenous expression of both PPAR and RXR mRNAs in macrophages was also confirmed by RT-PCR with a pair of primers (forward primer, 5'-ATG AGG AAG GTG TCG ATG GG-3'; and reverse primer, 5'-TTT GCC AAG CTG CTG CTC-3') for RXR and a pair of primers (forward primer, 5'-ACT CTG GAT TCA GCT GGT CG-3'; and reverse primer, 5'-GTT CAT GCT TGT GAA GGA TGC-3') for PPAR under the following conditions: 94 °C for 30 s, 50 °C for 30 s, and 72 °C for 30 s for 40 cycles to detect RXR mRNA and 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 1 min for 30 cycles to detect PPAR mRNA. The endogenous expression of NRF2 in macrophages was confirmed by detecting NRF2 mRNA by RT-PCR with a pair of primers (forward primer, 5'-ATG GAT TTG ATT GAC ATA CTT-3'; and reverse primer, 5'-CTA GTT TTT CTT AAC ATC T-3') under the following conditions: 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min for 20 cycles.

Electrophoretic Mobility Shift Assay (EMSA)-- Macrophages were maintained in 100-mm dishes with 1% hypomedium and treated with 1.0 µM PGJ₂ or 10 µM TRO for 12 h. Nuclear extracts were prepared from untreated or treated cells according to the previously described method (24), and stored at 70 °C until used for the experiments. PPAR and RXR were synthesized in vitro using the TNT Quick Coupled Transcription/Translation system (Promega). The de novo translation product was confirmed on an SDS-polyacrylamide gel. EMSA was performed based on a previously reported method (24). Briefly, nuclear extracts from macrophages were incubated in a 20-µl reaction mixture containing 22.5 mM HEPES (pH 7.9), 2.6 mM MgCl₂, 13.3% glycerol, 50 mM KCl, 0.125 mM EDTA, 0.5 mM dithiothreitol, 0.04 µg/µl sheared salmon sperm DNA, and 20,000 cpm end-labeled probe for 30 min at room temperature. After incubation, samples were subjected to electrophoresis through a 4.5% polyacrylamide gel in 45 mM Tris borate and 1 mM EDTA for 1.5 h at 4 °C and analyzed by autoradiography. Double-stranded oligonucleotides (104/86, 5'-TAA AGT TGC TGA TTC ATT G-3') containing the NF-E2/AP-1 site (98/88) in 5'FL-TXS were labeled with ³²P by a fill-in reaction with the Klenow fragment. Unlabeled oligonucleotides containing the NF-E2/AP-1 site as well as oligonucleotides harboring the mutated sequence of the NF-E2/AP-1 site (5'-TAA AGT TGC Ttg TTC ATT G-3') were used as competitors. To characterize the protein binding to the NF-E2/AP-1 site, antisera against transcription factors were incubated in the binding reactions at 10-fold dilution for 30 min on ice before the addition of the radiolabeled oligonucleotides. The antibodies used in this study were raised against NRF1 (c-19, Santa Cruz Biotechnology, Inc.), NRF2 (ECH) (28), BACH1/2 (29), NF-E2 (p45) (30), and MAF (31). Antibodies against c-Fos (sc-52) and c-Jun (sc-45) were obtained from Santa Cruz Biotechnology, Inc. In the pre-absorption experiment in EMSA, anti-NRF2 serum was incubated with GST or GST-NRF2 fusion proteins loaded onto glutathione-Sepharose beads for 2 h at 4 °C. After incubation, the mixtures were briefly centrifuged, and supernatants were incubated with the nuclear extracts and probe.

In Vitro Binding Assay-- GST fusion proteins were synthesized by the GST Gene Fusion system (Amersham Pharmacia Biotech). The GST fusion proteins were loaded onto glutathione-Sepharose beads, which were washed and resuspended in binding buffer (20 mM HEPES (pH 7.7), 75 mM KCl, 0.1 mM EDTA, 2.5 mM MgCl₂, 0.05% Nonidet P-40, 2 mM dithiothreitol, and 10% glycerol). The beads were incubated with 5 µl of in vitro translated ³⁵S-labeled PPAR or RXR protein for 1 h at 4 °C, followed by washing seven times with binding buffer in the presence or absence of TRO (10 µM). They were then resuspended in 30 µl of 2× SDS sample buffer, and the supernatant was analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography.

Statistical Analysis-- Data were analyzed using Stat View 4.0 (Abacus Concepts, Inc.). Analysis of variance was adopted to compare means among groups.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Structure of 5'FL-TXS-- We cloned a fragment of the 5'-flanking region of the rat TXS gene (1.6 kilobase) and revealed its sequence structure (Fig. 1). This sequence has 75% homology to murine 5'FL-TXS (10). The transcription start site was indicated to be 136 bases upstream from the protein coding region (indicated with an asterisk). There are several putative transcription factor-binding sites: the TATA box, GATA-1 box, AP-1-binding site, AP-2-binding site, glucocorticoid-responsive element, -interferon-activated site, shear stress-responsive element, and NF-E2/AP-1-binding site. There are two putative binding sites for PU.1, which is known as a macrophage- and B cell-specific transcription factor. Since Southern blot analysis with genomic DNA gave a single hybridization band upon BamHI and EcoRI digestion, respectively (data not shown), and the gene had already been mapped in a single chromosome region (32), the fragment we have cloned was suggested to be the unique 5'-flanking region for rat TXS.

View larger version (57K): [in this window] [in a new window]   Fig. 1.   Nucleotide sequence of 5'FL-TXS. The 5'-transcription start site is indicated by the asterisk (position +1). Putative transcription factor-binding sites are underlined. SRE, sterol regulatory element; GRE, glucocorticoid-responsive element; GAS, -interferon-activated site; SSRE, shear stress-responsive element; PU.1, PU.1-binding site; NF-E2/AP-1, overlapping NF-E2 and AP-1-binding site; AP-1, AP-1-binding site; AP-2, AP-2-binding site; NF-KB, nuclear factor B-binding site; GATA-1, GATA-1-binding site.

Tissue-dependent TXS mRNA Expression and Transcription Activity of 5'FL-TXS-- We first compared TXS mRNA expression levels by the semiquantitative RT-PCR method among macrophages, RLECs, and VSMCs (Fig. 2A). RT-PCR products for TXS mRNA (466 bp) were most abundant in macrophages, followed by RLECs. In VSMCs, however, TXS mRNA could not be detected under the PCR conditions we adopted. Nonetheless, it was detected when the number of PCR cycles was increased.

View larger version (25K): [in this window] [in a new window]   Fig. 2.   TXS mRNA expression and transcription activity of 5'FL-TXS in different tissues. A, total RNA was isolated from the different cell types and subjected to semiquantitative RT-PCR using TXS- and glyceraldehyde-3-phosphate dehydrogenase-specific primers. The resultant PCR products were resolved on an ethidium bromide-stained 2% agarose gel. Isolated mRNA was treated (RT(+)) or not (RT()) with reverse transcriptase. RT-PCR products for TXS mRNA (466 bp) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA (308 bp) are indicated by arrows. B, the 1598Luc construct was transfected into macrophages (M), RLECs, and VSMCs. Bars represent means ± S.E. (n = 6) of luciferase expression after normalization of transfection efficiency by -galactosidase expression expressed as relative luciferase activity. , p < 0.001 compared with luciferase expression in VSMCs.

For transcription study, 1598Luc was transfected into three kinds of cells, and luciferase expression was compared (Fig. 2B). The luciferase expression in macrophages was highest among the three kinds of cells (5.6 times higher than that in VSMCs), followed by RLECs (2.0 times higher than that in VSMCs), in proportion to mRNA expression levels among these cells. To further characterize the tissue-specific transcription activity of this 5'-flanking region, deletion analysis of the 5'-flanking region was performed in these cells (Fig. 3). The pattern of transcription activity was similar in VSMCs and RLECs, and the 304Luc construct had the greatest transcription activity. 96Luc still possessed transcription activity, but the transcription activity of 34Luc was absent in both RLECs and VSMCs.

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Transcription activities of various lengths of 5'FL-TXS in the different tissues. Chimeric constructs containing deleted fragments of 5'FL-TXS were transfected into the different cell types: macrophages (M; A), RLECs (B), and VSMCs (C). Bars represent means ± S.E. (n = 6) of luciferase expression expressed as relative luciferase activity (RLA). RSV, Rous sarcoma virus.

On the other hand, the transcription activity of 5'FL-TXS in macrophages was different. The luciferase expression decreased with 1083Luc, but this was then reversed, suggesting that there would be a silencer element between positions 1083 and 773. Distinct from VSMCs and RLECs, significant luciferase expression was detected with 168Luc, whereas 96Luc and 34Luc transcription activities were not detected. It is therefore suggested that the major promoter region in 5'FL-TXS in macrophages is different from that in RLECs and VSMCs and is located between positions 168 and 96 or their surrounding regions.

As 5'FL-TXS has several putative cis-acting elements, the effect of some potential stimulators on transcription activity was examined in macrophages using 1598Luc (Fig. 4). PGJ₂ (1.0 µM; PPAR activator) significantly suppressed transcription activity. Interferon- (700 IU/ml) and dexamethasone (1.0 µM) also inhibited transcription, whereas tumor necrosis factor- (100 ng/ml) and interleukin-6 (40 ng/ml) stimulated it.

View larger version (12K): [in this window] [in a new window]   Fig. 4.   Effects of cytokines, glucocorticoid, and PGJ2 on the transcription activity of 5'FL-TXS in macrophages. 1598Luc was transfected into macrophages. Transfected cells were stimulated with several agents 18 h after transfection. Bars represent means ± S.E. (n = 6) of luciferase expression expressed as relative luciferase activity (RLA). Recombinant human tumor necrosis factor- (TNF-; 100 ng/ml), dexamethasone (DEX; 1.0 µM), interferon- (IFN-; 700 IU/ml), recombinant human interleukin-6 (IL-6; 40 ng/ml), and PGJ2 (1.0 µM) were used. and , p < 0.001 and p < 0.05 compared with the vehicle treatment, respectively. RLA, relative luciferase activity.

Effects of PGJ₂ and TRO on TXS mRNA Expression-- Northern blot analysis showed that TXS mRNA expression levels were significantly reduced by PGJ₂ and TRO in a dose-dependent manner (Fig. 5A). The endogenous expression of both PPAR and RXR was also verified in macrophages by RT-PCR products for PPAR mRNA (250 bp) and RXR mRNA (113 bp), respectively (Fig. 5B). PGJ₂ and TRO may affect gene expression, possibly via activation of PPAR associated with RXR.

View larger version (35K): [in this window] [in a new window]   Fig. 5.   PPAR activators PGJ2 and TRO inhibit TXS mRNA expression. A, Northern blot analysis was performed using total RNA from macrophages treated with PGJ2 (1.0 and 5.0 µM) and TRO (10 and 20 µM). Representative blots for TXS (upper panels) and -actin (lower panels) are shown. Bars represent densitometric units of TXS mRNA from six repeated experiments. , p < 0.01 compared with the vehicle treatment. B, endogenous PPAR and RXR mRNA expression was confirmed in macrophages. RT-PCR was performed using specific primers for PPAR and RXR. RT(), RT-PCR without reverse transcriptase treatment. RT-PCR products for PPAR mRNA (250 bp) and RXR mRNA (113 bp) are indicated by arrows.

Transcription Inhibition of PPAR in 5'FL-TXS-- First, to examine the effect of PPAR activators, macrophages transfected with 1598Luc were stimulated with PGJ₂ or TRO. PGJ₂ (Fig. 6A) and TRO (Fig. 6B) significantly suppressed luciferase expression in a dose-dependent manner. Second, to examine the role of PPAR in transcription suppression, 1598Luc was cotransfected with PPAR cDNA into macrophages. As shown in Fig. 6C, cotransfection with PPAR significantly decreased the transcription activity of 5'FL-TXS, which was enhanced by PGJ₂ (1.0 µM) or TRO (10 µM). These results suggest that PGJ₂ and TRO can inhibit TXS gene expression mediated by PPAR at the transcriptional level in macrophages.

View larger version (19K): [in this window] [in a new window]   Fig. 6.   PGJ2 and TRO treatment and PPAR overexpression inhibit the transcription activity of 5'FL-TXS. PGJ2 (A) and TRO (B) inhibited the transcription activity of 5'FL-TXS in a dose-dependent manner. 1598Luc was transfected into macrophages. Transfected cells were stimulated with PGJ2 (0.5, 1.0, and 5.0 µM) and TRO (1.0, 10, and 20 µM). Bars represent means ± S.E. (n = 6) of luciferase expression after normalization of transfection efficiency by -galactosidase expression expressed as relative luciferase activity (RLA). , p < 0.01 compared with the vehicle treatment. C, overexpression of PPAR potentiated the inhibitory effect by PGJ2 and TRO. 1598Luc and the PPAR expression vector were cotransfected into macrophages. Transfected cells were stimulated with PGJ2 (1.0 µM) and TRO (10 µM). Bars represent means ± S.E. (n = 6) of luciferase expression expressed as relative luciferase activity. , p < 0.01 compared with the vehicle treatment; , p < 0.01 compared with luciferase expression by PPAR cDNA cotransfection.

Element Responsible for the Inhibitory Effect of PPAR on 5'FL-TXS-- We next tried to localize the potential element responsible for the PPAR action. Macrophages transfected with deletion mutants were stimulated with PGJ₂ (1.0 µM) or TRO (10 µM). Both agents significantly suppressed luciferase expression with 506Luc, 423Luc, 304Luc, and 168Luc, but no response was observed with 96Luc (Fig. 7). These results suggest that the element responsible for the transcription inhibitory effect of PPAR activators is located between positions 168 and 96 or their surrounding regions, where the major promoter was shown to be present in macrophages (Fig. 3A). By sequence homology search, we found a putative NF-E2/AP-1 site at positions 98 to 88, whose sequence was incidentally disrupted in 96Luc (Fig. 8A), and no other potential cis-acting element including the PPAR-response element was identified between positions168 and 96. We thus focused on the NF-E2/AP-1 site in the following experiments.

View larger version (19K): [in this window] [in a new window]   Fig. 7.   PGJ2 and TRO response elements in 5'FL-TXS. Various deletion constructs of 5'FL-TXS (506Luc, 423Luc, 304Luc, 168Luc, and 96Luc) were transfected into macrophages. Transfected cells were stimulated with PGJ2 (1.0 µM) and TRO (10 µM). Bars represent means ± S.E. (n = 6) of luciferase expression expressed as relative luciferase activity (RLA). , p < 0.01 compared with the vehicle. NS, not significant.

View larger version (17K): [in this window] [in a new window]   Fig. 8.   The 98/88 element as the major promoter of 5'FL-TXS. A, shown is sequence 168 to 68 in 5'FL-TXS. The putative NF-E2/AP-1 site (98/88) is underlined. B, the mutation in sequence 98 to 88 abrogated the promoter activity of 5'FL-TXS. Bars represent means ± S.E. (n = 4) of luciferase expression expressed as relative luciferase activity (RLA). , p < 0.001 compared with the wild-type construct.

Characterization of the NF-E2/AP-1 Site in 5'FL-TXS-- We conducted a mutation analysis to determine the role of the NF-E2/AP-1 site in transcription of the TXS gene. As a result, the transcription activity of 1598mLuc was markedly suppressed (Fig. 8B). Together with the previous observation that 96Luc (lacking the putative NF-E2/AP-1 site) (Fig. 8A) did not show significant transcription activity (Fig. 3A), it was thus confirmed that the NF-E2/AP-1 site is a major promoter in macrophages.

We then examined a protein-DNA interaction in EMSA using radiolabeled oligonucleotides of the NF-E2/AP-1 site and nuclear proteins from macrophages (Fig. 9A). We observed a protein-DNA complex (arrow), which was decreased with oligonucleotides containing the intact NF-E2/AP-1 site, but not with an excess of oligonucleotides harboring the mutated NF-E2/AP-1 site. Next, to identify the specific protein bound to this element, we treated the nuclear extracts with antisera against some candidate transcription factors (Fig. 9B). The addition of the anti-NRF2 serum, which had previously been shown to react with the NRF2·DNA complex (33), pronouncedly inhibited protein-DNA complex formation (lane 4). The inhibitory effect of anti-NRF2 serum was abrogated by preincubation of the serum with GST-NRF2 fusion protein (lane 10), but not with GST alone (lane 9), indicating the specific activity of anti-NRF2 serum. We also detected retarded bands in lane 4 (anti-NRF2), lane 5 (anti-p45), and lane 6 (anti-BACH) at the upper position of the NRF2·DNA complex. However, these bands comigrated with a band induced by preimmune serum treatment (lane 7). These results suggest that NRF2 is involved in the protein-DNA complex, but involvement of other NF-E2-related factors could not be excluded, even though the retarded bands appear to be nonspecific. To support the interaction between NRF2 and NF-E2/AP-1 in macrophages, endogenous expression of NRF2 was confirmed by detecting expression of NRF2 mRNA (1770 bp) by RT-PCR (Fig. 9C).

View larger version (53K): [in this window] [in a new window]   Fig. 9.   Possible binding of NRF2 to the fragment containing the 98/88 site (NF-E2/AP-1). A, EMSA was performed using the radiolabeled oligonucleotides containing the NF-E2/AP-1 site and nuclear extracts from macrophages. Lane 1, incubation without nuclear extracts; lanes 2-8, incubation with nuclear extracts. A competition experiment was performed using unlabeled oligonucleotides containing the intact NF-E2/AP-1 site (98/88) at a 10-, 50-, or 100-fold molar excess (lanes 3-5, respectively) or the mutated NF-E2/AP-1 site at a 10-, 50-, or 100-fold molar excess (lanes 6-8, respectively). B, EMSA was performed using various antibodies. Lanes 1 and 11, incubation without nuclear extracts; lanes 2, 8, and 12, nuclear extracts alone; lanes 7 and 13, preimmune serum; lane 3, anti-NRF1 serum; lane 4, anti-NRF2 serum; lane 5, anti-NF-E2 serum (anti-p45); lane 6, anti-BACH serum; lane 9, pre-absorption of anti-NRF2 serum with GST protein (GST); lane 10, pre-absorption of anti-NRF2 serum with GST-NRF2 protein (GST-NRF2); lane 14, anti-small MAF serum (anti-Maf); lane 15, anti-c-Fos serum; lane 16, anti-c-Jun serum. Arrows indicate specific bands. C, endogenous NRF2 mRNA expression was confirmed in macrophages. The RT-PCR product of NRF2 mRNA was detected at 1770 bp (arrow). RT(+), RT-PCR with treatment of reverse transcriptase; RT(), RT-PCR without treatment of reverse transcriptase.

Effect of PPAR on the Protein-DNA Interaction at the NF-E2/AP-1 Site-- We further examined a possible interaction of PPAR with the protein-DNA complex in EMSA. PGJ₂ (1.0 µM) and TRO (10 µM) inhibited complex formation (Fig. 10A). Moreover, the addition of in vitro translated PPAR reduced the intensity of the protein-DNA complex in a dose-dependent manner, but RXR had no effect (Fig. 10B).

View larger version (54K): [in this window] [in a new window]   Fig. 10.   PGJ2, TRO, and in vitro synthesized PPAR inhibit formation of the NRF2·DNA complex. A, EMSA was performed using the radiolabeled oligonucleotides containing the NF-E2/AP-1 site and nuclear extracts from macrophages stimulated with PGJ2 (1.0 µM) and TRO (10 µM). The arrow indicates specific bands. B, EMSA was performed in the presence of in vitro translated RXR at 1.0 or 2.0 µl (lanes 3 and 4, respectively), PPAR at 1.0 or 2.0 µl (lanes 5 and 6, respectively), or combination of both PPAR and RXR at 0.5 or 1.0 µl each (lanes 7 and 8, respectively). Lysate volumes were adjusted to be equal using lysate programmed with empty vector plasmid. Lane 1, incubation without nuclear extracts; lanes 2-9, incubation with nuclear extracts; lane 9, 2.0 µl of lysate programmed with empty vector plasmid (RL).

Next, to further verify the transcription function of the NF-E2/AP-1 site, the truncated element containing the NF-E2/AP-1 site in 5'FL-TXS was fused upstream of the thymidine kinase promoter and luciferase cDNA (NF-E2/AP-1-tkLuc), and this construct was cotransfected with PPAR, RXR, or human NRF2 cDNA into macrophages. As shown in Fig. 11, the luciferase expression of NF-E2/AP-1-tkLuc significantly increased compared with that of the control vector (pT109) alone (bar 1 versus bar 12), and it was enhanced ~2-fold by cotransfection with human NRF2 cDNA (bar 1 versus bar 2). On the other hand, the luciferase expression of a control vector was not affected by cotransfection (bar 12 versus bar 13). The enhanced luciferase expression of NF-E2/AP-1-tkLuc with human NRF2 cDNA was attenuated by either PGJ₂ (1.0 µM) (bar 2 versus bar 6) or TRO (10 µM) (bar 2 versus bar 7). The luciferase expression of NF-E2/AP-1-tkLuc was also suppressed by cotransfection with PPAR cDNA (bar 2 versus bar 4) or PPAR and RXR cDNAs (bar 2 versus bar 5). Moreover, the suppression was further enhanced with the addition of PGJ₂ (bars 8 and 10) or TRO (bars 9 and 11). However, cotransfection with RXR cDNA did not affect the luciferase expression (bar 2 versus bar 3).

View larger version (28K): [in this window] [in a new window]   Fig. 11.   PPAR inhibits transcription activation by NRF2 at the NF-E2/AP-1 site. NF-E2/AP-1-tkLuc containing oligonucleotides of the NF-E2/AP-1 site was cotransfected with or without cDNA for human NRF2, RXR, or PPAR into macrophages. Transfected cells were also stimulated with PGJ2 (1.0 µM) and TRO (10 µM). Bars represent means ± S.E. (n = 6) of luciferase expression expressed as relative luciferase activity (RLA). *, p < 0.01 compared with bar 1; , p < 0.01 compared with bar 2; , p < 0.01 compared with their controls (bars 4-7); , not significant compared with bar 2; , not significant compared with bar 12.

Physical Interaction between PPAR and NRF2-- Finally, we performed a GST pull-down assay to confirm the direct interaction between NRF2 and PPAR. As shown in Fig. 12A, full-length NRF2 (amino acids 1-589) interacted with PPAR, and the interaction was enhanced in the presence of TRO (10 µM). On the other hand, RXR did not interact with NRF2 (Fig. 12B). These results suggest that PPAR physically interacts with NRF2 and probably interferes with the transcription function of NRF2.

View larger version (24K): [in this window] [in a new window]   Fig. 12.   PPAR, but not RXR, interacts with NRF2. GST pull-down assays using the GST-NRF2 fusion protein and either in vitro translated 35S-labeled PPAR or RXR protein were performed. Arrowhead indicate PPAR and RXR protein. Where indicated, TRO was present at 10 µM. The Input lane represents the 100% volume of in vitro translated PPAR or RXR used in the pull-down assays.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

TXS gene transcription analysis with deletion mutants (Fig. 3A) and a construct harboring the mutated NF-E2/AP-1 site (1598mLuc) (Fig. 8B) suggests that the major promoter in macrophages whose sequence is homologous to the NF-E2/AP-1 site is present in the 98/88 region. Consistent with our observation, Lee et al. (9) have shown that the NF-E2/AP-1 site in the human TXS gene plays a crucial role in TXS gene transcription in the human promyelocytic leukemia cell line HL-60. They have also shown that transcription via the NF-E2/AP-1 site is activated by overexpression of NF-E2 (p45) and have concluded that the NF-E2 site is important for enhancing the TXS gene promoter activity in HL-60 cells, although they have not identified the transcription factor binding to the NF-E2/AP-1 site. In the present study, we observed inhibition of protein-DNA complex formation with anti-NRF2 serum in EMSA using nuclear extracts from macrophages, and the specificity of the inhibitory effect of anti-NRF2 serum was confirmed by a pre-absorption experiment. The results indicated that the NF-E2/AP-1 site (98/88) in 5'FL-TXS is possibly bound by NRF2 as an endogenous transcription factor in macrophages. This is the first evidence that NRF2 is involved in the regulation of the TXS gene promoter in macrophages. However, we do not exclude a possibility that other factors, including BACH and p45, are also involved in protein-DNA complex formation. In megakaryocytes, the human TXS promoter was also shown to be bound by NF-E2 (p45) (34). Further analysis is needed to identify other factors than NRF2 that interact with the NF-E2/AP-1 site in macrophages.

NRF2 is a transcription factor with a leucine zipper structure and one of the Cap'n'Collar family transcription factors, including p45, NRF1, NRF3, BACH1, and BACH2 (36). Recently, it has been reported that NRF2 is involved in the induction of heme oxygenase-1 gene expression by oxidative stress (37, 38). Indeed, in NRF2-deficient macrophages, an important role of NRF2 has been demonstrated in the oxidative stress-induced response of heme oxygenase-1 gene expression (39). Moreover, NRF2 is also involved in gene transcription of phase II detoxifying enzyme genes, including the NAD(P)H:quinone oxidoreductase-1 gene (40) and the -glutamylcysteine synthetase subunit gene (41), via an interaction with the antioxidant response element or the electrophile response element, which possesses the sequence related to an NF-E2/AP-1-binding site. Thus, NRF2 is now known to play an important role in the gene transcription of oxidative stress-induced genes and phase II detoxifying enzymes. The TXS gene (belonging to a cytochrome P450 subfamily) is another new target gene transactivated by NRF2.

The NF-E2/AP-1 site was shown to be the major promoter of the TXS gene in macrophages. On the other hand, in RLECs and VSMCs, the major promoter appears to be located in the region where a putative TATA box is present and downstream of the NF-E2/AP-1 site. Moreover, the transcription activity of 5'FL-TXS is suggested to be most potent in macrophages, followed by RLECs and VSMCs, in proportion to the mRNA expression levels in these cells. Consistent with our present results, it has also been shown that the TXS protein is abundantly expressed in macrophages (5). 5'FL-TXS is thus suggested to exert different transcription activities dependent on the tissues.

PPAR has been shown to transactivate some genes, most typically the lipoprotein lipase gene (42), dependent on the PPAR-response element. Accumulated observations have shown, however, that activation of PPAR can suppress transcription of some genes, including the angiotensin AT₁ receptor (43) and endothelin-1 (17) genes. In the present study, we focused on the molecular mechanism of transcription suppression by PPAR in the TXS gene. Treatment with PPAR activators PGJ₂ and TRO inhibited transcription of 5'FL-TXS as well as mRNA expression levels. Moreover, overexpression of PPAR also inhibited transcription of 5'FL-TXS, and the addition of PGJ₂ or TRO augmented the transcription inhibition. We thus confirmed the transcription suppression of 5'FL-TXS by PPAR activation. Next, we tried to identify the element responsible for the transcription inhibition. The negative regulation by PPAR was not observed in 5'-flanking region constructs lacking the major promoter (98/88) in macrophages (Fig. 7). It was therefore hypothesized that PPAR would be involved in the transcription activity of the NF-E2/AP-1 site. Using a reporter construct of the truncated NF-E2/AP-1 site, we examined the transcription suppression by PPAR. As shown in Fig. 11, the transcription activity of the NF-E2/AP-1 site was stimulated by cotransfection with NRF2 cDNA, and the stimulation was inhibited by PGJ₂ as well as by TRO. Overexpression of PPAR also inhibited the transcription by NRF2, and further addition of PGJ₂ or TRO augmented the inhibition. These inhibitory responses in gene transcription were identical when full-length 5'FL-TXS was used. In EMSA with radiolabeled oligonucleotides containing the NF-E2/AP-1 site and nuclear extracts from macrophages, the formation of the protein-DNA complex was markedly inhibited by PGJ₂ or TRO. Moreover, we observed pronounced inhibition of complex formation by in vitro synthesized PPAR, but not by RXR (Fig. 10). The results clearly indicate that activation of PPAR inhibits the protein-DNA complex formation caused by the NF-E2/AP-1 site and nuclear proteins including NRF2. Finally, in the GST pull-down assay, PPAR was shown to physically interact with NRF2. This is the first report of the direct interaction of PPAR with NRF2 that possibly leads to gene transcription suppression. Taken together, the results suggest that PPAR can interact with NRF2 and may suppress transcription of the TXS gene, probably interfering with the binding of NRF2 to the NF-E2/AP-1 site.

Thromboxane inhibitors have been reported to inhibit the progression of experimental diabetic nephropathy in rats (44) and to ameliorate microalbuminuria in diabetic patients (45). In inflammatory kidneys, infiltrating macrophages express abundant TXS protein (46), and enhanced thromboxane synthesis in monocytes/macrophages leads to glomerular injury (47). It is thus suggested that thromboxane synthesis from infiltrating macrophages plays an important role in inflammatory diseases. Thiazolidinediones are now widely used for the treatment of type 2 diabetes mellitus. It has also been reported that the agents would possess other clinical benefits such as inhibiting neointimal formation following balloon injury (20), decreasing blood pressure (48), and ameliorating microalbuminuria (49), although the causative mechanisms are still in need of examination. The present observations may provide an insight into the role of PPAR in vascular and renal diseases in terms of regulation of gene expression.

In summary, we have revealed the structure of the functional transcriptional regulatory region of the rat TXS gene. In macrophages, 5'FL-TXS is primarily dependent on the NRF2-binding element (98/88) in basal transcription. Activation of PPAR may possibly suppress TXS gene expression at a transcriptional level. The NRF2-binding element is responsible for the transcription suppression by PPAR. In EMSA, the formation of the protein-DNA complex caused by the NRF2-binding element and nuclear extracts from macrophages was clearly inhibited by PPAR. The GST pull-down assay indicated the direct interaction between PPAR and NRF2. RXR was not implicated in the mechanism. In conclusion, activation of PPAR can suppress transcription of the TXS gene via a possible interaction with NRF2 in macrophages.

	ACKNOWLEDGEMENTS

We thank Dr. Nobuyuki Takahashi for critical reading of this manuscript and Dr. Akira Takeshita for suggestions regarding the GST pull-down assay.

	FOOTNOTES

^* This work was supported in part by Grants-in-aid 09470236 and 12671020 from the Ministry of Education, Science, and Culture; the Research Foundation for Community Medicine; the Takeda Research Foundation; the Research Foundation for Metabolic Disorder; Takeda Yakuhin, ko-gyo, Co., Ltd., Japan; and Sankyo Co., Ltd., Japan.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.

The nucleotide sequence reported in this paper has been submitted to the DDBJ/GenBank^TM/EBI Data Bank with accession number AB015876.

^¶ To whom correspondence should be addressed: Molecular Biology Unit, Div. of Nephrology, Endocrinology, and Vascular Medicine, Dept. of Medicine, Tohoku University Graduate School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai 980-8574, Japan. Tel.: 81-22-717-7163; Fax: 81-22-717-7168; E-mail: kazut2i@mail.cc.tohoku.ac.jp.

Published, JBC Papers in Press, August 4, 2000, DOI 10.1074/jbc.M002319200

	ABBREVIATIONS

The abbreviations used are: TXS, thromboxane synthase; PPAR, peroxisome proliferator-activated receptor; RXR, retinoid X receptor; PGJ₂, 15-deoxy-^12,14-prostaglandin J₂; TRO, troglitazone; VSMCs, vascular smooth muscle cells; 5'FL-TXS, 5'-flanking region of the rat TXS gene; PCR, polymerase chain reaction; bp, base pairs; NF-E2, nuclear factor E2; RLECs, rat lung endothelial cells; RT, reverse transcription; EMSA, electrophoretic mobility shift assay; GST, glutathione S-transferase.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES