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Circulation Research. 2008;102:193-200
Published online before print November 15, 2007, doi: 10.1161/CIRCRESAHA.107.158477
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(Circulation Research. 2008;102:193.)
© 2008 American Heart Association, Inc.

Molecular Medicine

Transforming Growth Factor-β1 Is a Molecular Target for the Peroxisome Proliferator-Activated Receptor {delta}

Hyo Jung Kim, Sun Ah Ham, Sung Uk Kim, Jin-Yong Hwang, Jae-Hwan Kim, Ki Churl Chang, Chihiro Yabe-Nishimura, Jin-Hoi Kim, Han Geuk Seo

From the Department of Pharmacology (H.J.K., S.A.H., K.C.C., H.G.S.) and Internal Medicine (S.U.K., J.-Y.H.), Gyeongsang Institute of Health Science, Gyeongsang National University School of Medicine, Jinju, Korea; Graduate School of Life Science & Biotechnology (Jae-Hwan Kim), Pochon CHA University, Seoul, Korea; Department of Pharmacology (C.Y.-N.), Kyoto Prefectural University of Medicine, Japan; and Department of Animal Biotechnology (Jin-Hoi Kim), Kon-Kuk University, Seoul, Korea.

Correspondence to Han Geuk Seo, Department of Pharmacology, Gyeongsang National University School of Medicine, 92 Chilam-Dong, Jinju 660-751, Korea. E-mail hgseo{at}gnu.ac.kr


*    Abstract
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 *Abstract
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The peroxisome proliferator-activated receptor (PPAR){delta} has been implicated in the pathogenesis of atherogenic disorders. However, its physiological roles and functions in vascular smooth muscle cells (VSMCs) remain relatively unclear. In the present study, we show that the gene encoding transforming growth factor (TGF)-β1 is a PPAR{delta} target in VSMCs. The PPAR{delta} activator GW501516 upregulates TGF-β1 expression in a dose- and time-dependent manner. This induction is attenuated significantly by the presence of small interfering RNA against PPAR{delta} or GW9662, an inhibitor of PPAR{delta}. Furthermore, activated PPAR{delta} induces TGF-β1 promoter activity by binding to the direct repeat-1 response element TGF-β1–direct repeat-1. Mutations in the 5' or 3' half-sites of the response element totally abrogate transcriptional activation and PPAR{delta} binding, which suggests that this site is a novel type of PPAR{delta} response element. In addition, ligand-activated PPAR{delta} attenuated the promoter activity and expression of monocyte chemoattractant protein-1 induced by interleukin-1β. These effects were significantly reduced in the presence of small interfering RNA against PPAR{delta}, anti–TGF-β1 antibody, or a TGF-β type I receptor inhibitor. Decreased monocyte chemoattractant protein-1 expression induced by PPAR{delta} was mediated by the effector of TGF-β1, Smad3. Finally, administration of GW501516 to mice upregulated TGF-β1, whereas the expression of proinflammatory genes including monocyte chemoattractant protein-1 was significantly attenuated in the thoracic aorta. Taken together, these results demonstrate the presence of a novel TGF-β1–mediated pathway in the antiinflammatory activities of PPAR{delta}.


Key Words: monocyte chemoattractant protein-1 • peroxisome proliferator-activated receptor {delta} • transforming growth factor-β1 • vascular smooth muscle cells


*    Introduction
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 *Introduction
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Transforming growth factor (TGF)-β plays a pivotal role in diverse biological processes, including the regulation of tissue remodeling, cellular differentiation and proliferation, and the modulation of inflammatory and immune responses.1,2 Three mammalian TGF-β isoforms have been identified (TGF-β1, TGF-β2, and TGF-β3), which share 60% to 80% identity at the amino acid level, and each of these is encoded by a distinct gene.3–5 TGF-β1 is the most intensively studied isoform, and it is conserved completely across human, bovine, simian, and porcine species, and the murine isoform differs by only 1 residue.3 Such extensive amino acid conservation suggests that TGF-β1 plays a significant role in maintaining cellular homeostasis. TGF-β1 is expressed in vascular smooth muscle cells (VSMCs), and it has been implicated in the development of atherosclerosis and restenosis after injury.6 TGF-β1 is a potent regulator of the inflammatory responses of the vascular system,7 and targeted disruption of the gene encoding TGF-β1 causes an excessive inflammatory response and early death.8,9 In addition, TGF-β1 inhibits expression of the monocyte chemoattractant protein (MCP)-1 via its effector, Smad3,10 and in vivo inhibition of TGF-β1 signaling exacerbates atheromatous lesions by promoting inflammation that induces unstable plaques.11,12 Thus, TGF-β1 is a key factor modulating vascular inflammation and plaque stability.13

Peroxisome proliferator-activated receptors (PPARs) are members of a nuclear hormone receptor superfamily that has been implicated in the regulation of lipid homeostasis, energy metabolism, and inflammation, as well as cellular differentiation and proliferation.14–17 These receptors regulate gene expression by forming heterodimers with the retinoid X receptor (RXR) via specific recognition sequences termed PPAR response elements (PPRE), which are located in the target genes.18 Three different isoforms have been identified: PPAR{alpha} (NR1C1); PPAR{delta} (NR1C2) (also known PPARβ, FAAR, and NUC1); and PPAR{gamma} (NR1C3). In contrast to PPAR{alpha} and PPAR{gamma}, the physiological function of PPAR{delta} remains relatively unclear. PPAR{delta} is expressed ubiquitously in a variety of cell lineages, including VSMCs.19 The promoter regions of PPAR target genes contain a direct repeat (DR1 or DR2) of the hexameric nucleotide sequence AGGTCA, separated by 1 or 2 nucleotides.16 Specifically, these PPREs are localized to regions where the binding of PPAR{delta}-RXR complexes can activate gene expression.16 A recent report indicates that PPAR{delta} ligands may possess antiatherogenic properties in vivo, whereby they attenuate the abundance of non–ligand-bound PPAR{delta} receptors in foam cells, which results in release of the transcriptional repressor BCL-6.20 Therefore, it has been postulated that PPAR{delta} may represent a therapeutic target for treatment of atherosclerosis and other inflammatory vascular disorders.21,22 A better understanding of the gene expression governed by PPAR{delta} may thus lead to illustrate its therapeutic potential.

Although multiple factors participate in the execution of PPAR{delta} action, very little is known about the effector genes that it regulates. In an attempt to elucidate this issue, we focused on identifying the transcriptional targets of PPAR{delta} in VSMCs. We report that an activator of PPAR{delta} upregulates expression of TGF-β1 mRNA and protein, and this is mediated by a functional PPRE localized in the TGF-β1 promoter. Interestingly, activation of PPAR{delta} attenuated the promoter activity and expression of interleukin (IL)-1β–induced MCP-1 in a TGF-β1–dependent manner. In mice treated with a PPAR{delta} activator, the expression of proinflammatory genes was suppressed along with upregulation of TGF-β1 in the thoracic aorta.


*    Materials and Methods
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Rat aortic VSMCs were isolated from free-floating explants of aortae, and maintained in DMEM supplemented with 10% heat-inactivated FBS. Details regarding reagents and methodology are provided in the online data supplement, available at http://circres.ahajournals.org.


*    Results
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 *Results
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A PPAR{delta} Ligand Induces TGF-β1 mRNA and Protein Expression in VSMCs
When VSMCs were treated with GW501516, a specific ligand of PPAR{delta},23 marked increases in the levels of the TGF-β1 transcript and protein were demonstrated in a concentration- and time-dependent manner (Figure 1). In the presence of 50 nmol/L GW501516, increased levels of TGF-β1 mRNA and protein were detected at 12 hours, and these reached a maximum at 38 hours.


Figure 1
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  		Figure 1. A PPAR{delta} ligand induces TGF-β1 mRNA and protein expression in VSMCs. Primary rat thoracic VSMCs were incubated for 38 hours with various concentrations of GW501516 (A and C) or treated with 50 nmol/L GW501516 for the times indicated (B and D). Northern and Western blot analysis were performed using cDNA probes (A and B) and anti–TGF-β1 antibody (C and D), respectively. An image analyzer was used to quantify band intensity, and the ratio of TGF-β1 to GAPDH or β-actin is indicated above each lane. The results are representative of 3 independent experiments.

Activation of PPAR{delta} but Not PPAR{alpha} or PPAR{gamma} Induces TGF-β1 mRNA and Protein Expression in VSMCs
To identify which PPAR isoforms were involved in induction of TGF-β1, VSMCs were treated with specific ligands for PPAR{alpha}, PPAR{gamma}, and PPAR{delta} (Figure 2). VSMCs constitutively express moderate levels of TGF-β1 mRNA and protein, and these levels increased following activation of PPAR{delta}. However, there was no significant difference in TGF-β1 expression following activation of either PPAR{alpha} or PPAR{gamma}, which suggests that only the PPAR{delta} isoform is involved in the TGF-β1 upregulation.


Figure 2
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  		Figure 2. Activation of PPAR{delta}, but not of PPAR{alpha} or -{gamma}, induces TGF-β1 mRNA and protein expression in VSMCs. Cells were incubated for 38 hours in low (50 nmol/L) or high (1.0 µmol/L) concentrations of WY-14643 (a specific agonist for PPAR{alpha}), GW501516 (a specific agonist for PPAR{delta}), troglitazone (a specific agonist for PPAR{gamma}), or 15-d-PGJ2 (15-deoxy-{delta}-12,14-prostaglandin J2) (a specific agonist for PPAR{gamma}). An image analyzer was used to quantify band intensity, and the ratio of TGF-β1 to GAPDH or β-actin is indicated above each lane. The results are representative of 3 independent experiments.

Small Interfering RNA or Inhibition of PPAR{delta} Attenuates Upregulation of TGF-β1 Expression Induced by GW501516
To examine the role of PPAR{delta} in GW501516-induced upregulation of TGF-β1, we transfected VSMCs with small interfering (si)RNA against PPAR{delta}. In the presence of PPAR{delta} siRNA, the levels of PPAR{delta} reduced significantly, whereas levels were unaffected following transfection with the nonspecific control siRNA (Figure 3A). This siRNA-mediated downregulation of PPAR{delta} counteracted the increased expression of TGF-β1 induced by a PPAR{delta} ligand (Figure 3B). Next, we investigated the effects of GW9662, which is an irreversible inhibitor of PPAR{delta}24 and observed a significant reduction in PPAR{delta}-induced upregulation of TGF-β1 (Figure 3B). We performed the same experiments using human aortic VSMCs and observed similar results (Figure 3C). These data indicate clearly that TGF-β1 expression is regulated in a PPAR{delta}-dependent manner.


Figure 3
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  		Figure 3. siRNA or PPAR{delta} inhibition attenuated upregulation of TGF-β1 induced by a PPAR{delta} ligand. A, Rat VSMCs were transfected with PPAR{delta} siRNA or control siRNA and harvested at 72 hours after transfection. Cell extracts were separated by electrophoresis and immunoblotted with anti-PPAR{delta} or anti–β-actin antibodies. B, Rat VSMCs were transfected with PPAR{delta} siRNA and grown for 72 hours, after which they were treated with GW501516 for 38 hours in the presence or absence of the GW9662. C, Human VSMCs were transfected with PPAR{delta} siRNA and grown for 72 hours, after which they were treated with GW501516 for 38 hours. An image analyzer was used to quantify band intensity, and the ratio of TGF-β1 to GAPDH or β-actin is indicated above each lane. The results are representative of 3 independent experiments.

PPAR{delta} Activation Induces TGF-β1 Expression at the Transcriptional Level
To investigate whether or not PPAR{delta} activation affects TGF-β1 promoter activity in VSMCs, transfection assays were performed using luciferase reporter constructs driven by the TGF-β1 promoter. GW501516-induced activation of PPAR{delta} significantly increased TGF-β1 promoter activity without affecting any change in PPAR{delta} protein expression. In contrast, other PPAR ligands exhibited little or no effect on the promoter activity (Figure 4A). To confirm the involvement of PPAR{delta} in the expression of TGF-β1, a TGF-β1 reporter gene was cotransfected with the PPAR{delta} expression vector (pSG5-PPAR{delta}) or an empty vector (pSG5). Transfectants were treated with GW501516 or vehicle (DMSO). An increase in TGF-β1 promoter activity was observed following PPAR{delta} cotransfection, and expression was enhanced further in the presence of GW501516 (Figure 4B). It should be noted that in the presence of GW501516, a significant increase in TGF-β1 promoter activity was observed, which indicates that the endogenous PPAR{delta} is sufficient to elicit this activity.


Figure 4
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  		Figure 4. Activation of PPAR{delta} induces TGF-β1 expression at the transcriptional level. A, Cells were treated for 38 hours with DMSO (CTR) (control), 50 nmol/L WY-14643 (WY), GW501516 (GW), troglitazone (Tro), or 15-d-PGJ2 (PJ2). Total protein was extracted and subjected to Western blot analyses using specific antibodies against each protein. TGF-β1 promoter activity was determined in cells transfected with a TGF-β1 reporter construct. The construct contained 1362 bp that were upstream of the TGF-β1 gene. Transfectants were grown for 24 hours and then treated with each PPAR ligand for 38 hours. Values are expressed as fold induction relative to the control (means±SE, n=3). *P<0.01, compared with control. Similar results were obtained in 3 independent experiments. B, Cells were transfected with a TGF-β1 reporter construct with or without a PPAR{delta} expression plasmid (pSG5-PPAR{delta}) and grown for 24 hours, followed by treatment with DMSO (open column) or 50 nmol/L GW501516 (closed column) for 38 hours. The results are expressed as the means±SE (n=3). #P<0.05, *P<0.01. C, Cells were transfected with deletion constructs of the TGF-β1 gene promoter and treated with 50 nmol/L GW501516 for 38 hours. Values (means±SE) from 4 to 6 independent transfections are expressed as fold induction relative to the control (pGL3 basic).

To determine the promoter region responsible for PPAR{delta}-induced upregulation of TGF-β1, reporter assays were performed using serially truncated constructs driven by the TGF-β1 promoter. The response to PPAR{delta} was almost completely abolished on the deletion of sequences located between –323 and –175, which indicates that the element responsible for PPAR{delta} is located between –0.32 and –0.17 kb upstream of the TGF-β1 gene (Figure 4C).

PPAR{delta} Binds to the PPRE in the TGF-β1 Gene Promoter
To investigate whether or not PPAR{delta} binds directly to the PPRE within the TGF-β1 promoter, we searched the PPRE homolog sequences using database in the region of –0.32 to –0.17 kb. Closer inspection of this region revealed the presence of a putative PPRE that is highly homologous with existing PPREs (Figure 5A). This putative PPRE site contains a DR1 sequence at positions –224 to –212 of the TGF-β1 promoter. To assess whether or not this PPRE mediates transcriptional activation by PPAR{delta}, we introduced mutations into the 1.36-kb TGF-β1 promoter construct. In the presence of the wild-type TGF-β1–DR1 site, GW501516-mediated PPAR{delta} activation resulted in a 4-fold increase in transcriptional activity. This effect was attenuated in the presence of siRNA against PPAR{delta} or GW9662, an inhibitor of PPAR{delta}. Furthermore, mutations in either the 5' or 3' motifs of TGF-β1–DR1 resulted in loss of promoter activation by the PPAR{delta} ligand (Figure 5B). Similar results were also obtained with human VSMCs, indicating that the regulation of TGF-β1 promoter activity by PPAR{delta} is conserved among species (supplemental Figure IA). These results clearly demonstrate that the TGF-β1 promoter is regulated by PPAR{delta} and that this induction is mediated via the TGF-β1–DR1 site.


Figure 5
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  		Figure 5. Activation of PPAR{delta} increases TGF-β1 promoter activity via a DR1 sequence. A, Schematic representation of the TGF-β1 gene promoter. The potential PPAR response element (DR1) was mutated by site-directed mutagenesis (underlined). B, Cells were pretreated for 72 hours with or without siRNA against PPAR{delta} and then transfected with luciferase reporter plasmids driven by the TGF-β1 promoter, as well as a SV40 β-galactosidase expression vector (pSV β-Gal). Subsequently, cells were treated for 38 hours with 50 nmol/L GW501516 and/or 5 µmol/L GW9662, and then luciferase activities were measured. Values from 4 to 6 independent transfections normalized with β-galactosidase activity are expressed as means±SE.

To determine whether or not PPAR{delta} binds directly to the TGF-β1–DR1 site, we performed electrophoretic mobility shift assays using in vitro synthesized PPAR{delta} and RXR{alpha} proteins (Figure 6A and 6B). As expected, neither RXR{alpha} nor PPAR{delta} alone could bind to either the wild-type or mutated TGF-β1–DR1 sites (Figure 6A). However, we observed binding to the TGF-β1–DR1 site when PPAR{delta} was incubated in the presence of RXR{alpha} (lane 4). The bound complex supershifted in the presence of anti-PPAR{delta} antibody (lane 5), and no protein–DNA complex was observed in the presence of the mutated TGF-β1–DR1mt5', TGF-β1–DR1mt3', or TGF-β1–DR1mt5'/3' probes. We added increasing amounts (10-, 50-, and 100-fold excess) of unlabeled oligonucleotides that covered the wild-type TGF-β1–DR1 (DR1wt), TGF-β1–DR1mt5' (DR1mt5'), TGF-β1–DR1mt3' (DR1mt3'), or TGF-β1–DR1mt5'/3' (DR1mt5'/3') sites and observed that PPAR{delta} binding to the TGF-β1–DR1wt oligo competed strongly with the TGF-β1–DR1wt site. On the other hand, the mutated TGF-β1–DR1 oligonucleotides did not affect binding between PPAR{delta} and the DR1 (Figure 6B). These data demonstrate that the PPAR{delta}-RXR{alpha} heterodimer binds to the DR1 PPRE site located at positions –224 to –212 of the TGF-β1 gene.


Figure 6
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  		Figure 6. TGF-β1 is a direct target gene for PPAR{delta}. A, A PPAR{delta}-RXR{alpha} heterodimer binds to the DR1 site of the TGF-β1 promoter. Electrophoretic mobility-shift assay was performed using end-labeled wild-type or mutated TGF-β1–DR1 probes in the presence of in vitro–translated PPAR{delta} and/or RXR{alpha}. A supershift experiment was performed using anti-PPAR{delta} antibody. B, Competition experiments were performed by adding a 10-, 50-, or 100-fold molar excess of cold TGF-β1–DR1wt (DR1wt), TGF-β1–DR1mt5' (DR1mt5'), TGF-β1–DR1mt3' (DR1mt3'), or TGF-β1–DR1mt5'/3' (DR1mt5'/3') oligonucleotides to PPAR{delta} and RXR{alpha}. The results are representative of 2 independent experiments. C, PPAR{delta} associates physically with the DR1 site of the TGF-β1 promoter. Cells were grown for the times indicated in the presence or absence of 50 nmol/L GW501516 (lane 0, untreated). Chromatin was immunoprecipitated using anti-PPAR{delta} antibody, and the genomic DNA recovered was amplified by PCR using either the oligo no. 1 (Oligo#1) or oligo no. 2 (Oligo#2) primer set. The location of these primer sets is indicated at the top, along with the PPAR{delta}-binding site (DR1). Control amplifications were performed with input chromatin obtained before immunoprecipitation. The results are representative of 3 independent experiments.

GW501516 Treatment Recruits PPAR{delta} to the TGF-β1 Promoter Region
To examine whether or not PPAR{delta} interacts directly with the TGF-β1 promoter and whether its binding correlates with TGF-β1 transcriptional upregulation, we performed a chromatin immunoprecipitation assay (Figure 6C). VSMCs were treated with or without GW501516 for 0, 4, 8, 18, or 38 hours, and chromatin fragments were immunoprecipitated using an anti-PPAR{delta} antibody. Genomic DNA contained in the immunoprecipitates was PCR amplified using the primer set oligo no. 1, corresponding to the promoter region containing the putative TGF-β1 PPRE site (see schematic representation in Figure 6C). In samples treated with GW501516, PPAR{delta} was found associated with the PCR-amplified region, whereas no reactivity was detected in untreated cells. In addition, no signal was detected from PCR amplification of the same chromatin preparations using primers for sequences located further upstream (oligo no. 2). These results are consistent with the findings obtained by electrophoretic mobility-shift assay, which indicate that PPAR{delta} binds to the PPRE identified in the TGF-β1 promoter region.

Activation of PPAR{delta} Inhibits Cytokine-Induced Expression of MCP-1 via TGF-β1
IL-1β is known to induce MCP-1 expression in VSMCs.25,26 We examined whether or not ligand-activated PPAR{delta} affects the inducible expression of MCP-1 mRNA in VSMCs. In unstimulated cells, MCP-1 mRNA is expressed at low levels, and these increase markedly following IL-1β induction (Figure 7A and 7B). Pretreatment with GW501516 attenuated this induction significantly, but siRNA against PPAR{delta} almost completely abolished this inhibitory effect (Figure 7A). Similar results were obtained with human VSMCs, indicating that the regulation of MCP-1 expression by PPAR{delta} is conserved among species (supplemental Figure IB). In macrophages, it has been reported that PPAR{delta} controls the inflammation status via its association and disassociation with the transcriptional repressor BCL-6.20 We hypothesized that in VSMCs, PPAR{delta}-induced TGF-β1 may perform an antiinflammatory role similar to BCL-6. In fact, addition of either anti–TGF-β1 antibody or TβR-1 inhibitor caused a marked attenuation in the effects of PPAR{delta} on MCP-1 mRNA expression (Figure 7B, bottom blots). Previously, IL-1β induction of MCP-1 was found to occur primarily at the level of transcription.25,26 Consistent with these findings, transient transfection assays using a MCP-1 promoter construct showed that an IL-1β–induced increase in transcriptional activity was attenuated significantly when cells were pretreated with GW501516, whereas these responses were markedly alleviated in the presence of siRNA against PPAR{delta}, an anti–TGF-β1 antibody, or a TβR-1 inhibitor (Figure 7A and 7B). In contrast, GW501516 enhanced promoter activity of PAI-1, a well-known target for TGF-β1 (Figure 7C).27


Figure 7
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  		Figure 7. Activation of PPAR{delta} inhibits cytokine-induced expression of MCP-1 via TGF-β1. A, Cells were cotransfected with siRNA against PPAR{delta} or control siRNA and MCP-1 promoter construct (–486/+6) and incubated with GW501516 for 38 hours, followed by treatment with IL-1β for 24 hours. B, Cells transfected with MCP-1 promoter construct (–486/+6) were incubated with GW501516 for 38 hours and then treated with IL-1β for 24 hours in the presence or absence of an anti–TGF-β1 antibody or a TβR-1 inhibitor. Changes in mRNA levels were analyzed by Northern blot using a 32P-labeled MCP-1 probe. An image analyzer was used to quantify band intensity, and the ratio of MCP-1 to GAPDH is indicated below each lane. C, PAI-1 promoter construct (–796/+65) was cotransfected with siRNA against PPAR{delta}. Cells were then incubated with GW501516 for 38 hours, followed by treatment with IL-1β for 24 hours. D, MCP-1 promoter construct (–486/+6) was cotransfected with the indicated Smad expression plasmids or pcDNA3.1 vector and stimulated with IL-1β for 24 hours. Values from 4 to 6 independent transfections normalized with β-galactosidase are expressed as means±SE. #P<0.01, ##P<0.05 compared with IL-1β–treated group; {dagger}P<0.01, {dagger}{dagger}P<0.05 compared with IL-1β+GW501516–treated group; *P<0.05 compared with GW501516-treated group.

Smad proteins consist of a family of intracellular effectors that mediate TGF-β1 signaling and gene expression.27 To assess whether or not the TGF-β1–mediated inhibition of MCP-1 expression is mediated by Smad, cotransfection assays with several Smad expression plasmids and a MCP-1 promoter construct were performed in the presence or absence of IL-1β. As shown in Figure 7D, Smad3 alone repressed the MCP-1 promoter activity in a manner similar to GW501516. Taken together, these findings suggest that in VSMCs, PPAR{delta} modulates MCP-1 expression at the transcriptional level via TGF-β1 and its effector Smad3.

Activation of PPAR{delta} Suppresses the TNF-{alpha}–Induced Activation of VSMCs in a TGF-β1–Dependent Manner
To further define the physiological role of PPAR{delta} activation in VSMCs, effects of GW501516 on the TNF-{alpha}–induced activation of VSMCs were investigated by cell proliferation assay. As shown in Figure 8A, TNF-{alpha} significantly induced proliferation of VSMCs, and a dose-dependent decrease in VSMC proliferation was observed in cells exposed to various concentration of GW501516 for 24 hours. Under concentrations of GW501516 used in these experiments, no effect on proliferation was observed within the adopted time frame, as determined by cell counting (supplemental Figure II). To clarify the involvement of TGF-β1 in the PPAR{delta}-mediated suppression of VSMC activation, effects of TGF-β1 antibody or a TβR-I inhibitor were examined. As shown in Figure 8B, the GW501516-mediated suppression of VSMC proliferation was blunted in the presence of TGF-β1 antibody or a TβR-I inhibitor, indicating the involvement of TGF-β1 in the PPAR{delta}-mediated suppression of VSMC activation.


Figure 8
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  		Figure 8. Activation of PPAR{delta} inhibits TNF-{alpha}–induced proliferation of VSMCs and regulates expression of proinflammatory genes in vivo. A and B, Effects of GW501516 on TNF-{alpha}–induced proliferation of VSMCs were determined by the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) (open column) and [3H]-thymidine incorporation (closed column) assay. VSMCs were incubated in DMEM containing 5% FBS (5%) in the presence or absence of each reagent for 24 hours. The results are expressed as the means±SE (n=4). *P<0.01, **P<0.05 compared with 5% FBS–treated group; #P<0.01, ##P<0.05 compared with the TNF-{alpha}–treated group; {dagger}P<0.01, {dagger}{dagger}P<0.05 compared with TNF-{alpha}+GW501516–treated group. SF indicates serum free. C and D, Expression levels of TGF-β1 and proinflammatory genes in mice treated with GW501516. Mice were treated with (closed column) or without (open column) GW501516 for 48 hours, and total RNA was extracted from the thoracic aorta. Levels of mRNAs were analyzed by Northern blot (C) and quantified (D). *P<0.01.

TGF-β1 Expression Is Increased in the Thoracic Aorta of Mice Treated With GW501516
Finally, we examined the level of TGF-β1 mRNA in the thoracic aorta of mice treated with vehicle or GW501516 to investigate whether the PPAR{delta}-mediated induction of TGF-β1 actually takes place in vivo. As shown in Figure 8C and 8D, administration of GW501516 significantly increased TGF-β1 expression, whereas levels of such proinflammatory genes as MCP-1 and macrophage inflammatory protein (MIP)-1β were significantly attenuated. Levels of TNF-{alpha} and inducible NO synthase remained unchanged in GW501516-treated aorta. These data clearly indicate that activation of PPAR{delta} modulates expression of TGF-β1 and proinflammatory genes in vivo.


*    Discussion
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 up arrowResults
 *Discussion
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Activation of the nuclear receptor PPAR{delta} has been reported to regulate the expression of a large variety of genes, and these elicit a wide spectrum of responses under different pathophysiological conditions.28–31 Although many factors participate in the execution of PPAR{delta} activity, very little is known about the actual effector genes that it regulates. Here, we demonstrated that PPAR{delta} activation by the specific ligand GW501516 induces expression of TGF-β1 mRNA and protein in VSMCs. Transfection experiments revealed that TGF-β1 expression was regulated by PPAR{delta} at the transcriptional level. Mutations of the DR1 site located in the TGF-β1 promoter region abolished PPAR{delta}-induced TGF-β1 transcription, indicating that this site is a novel type of PPRE. Furthermore, activation of PPAR{delta} resulted in the attenuation of IL-1β–induced MCP-1 expression in VSMCs and other proinflammatory gene expression in mice aorta, along with upregulation of TGF-β1.

Activation of PPAR{delta}, but not of PPAR{alpha} or -{gamma}, specifically upregulated the expression of TGF-β1 mRNA and protein in VSMCs. Induction occurs at the transcriptional level via binding of the PPAR{delta}-RXR{alpha} complex to the PPRE located in the 5'-flanking region of the TGF-β1 gene. The PPRE identified in this study contained a canonical DR1 motif separated by a cytosine. Among the PPAR isoforms that have been characterized for their roles in VSMCs using specific ligands,19,32,33 only PPAR{delta} induced TGF-β1. Therefore, it is possible that action of each nuclear receptor can be specified by expressional regulation of the particular target molecules.

To our knowledge, this is the first report demonstrating that PPAR{delta} regulates TGF-β1 expression in VSMCs. Little is known about transcriptional regulation of the TGF-β1 gene in mammalian cells. The human TGF-β1 gene possesses multiple transcriptional initiation sites and does not contain the typical TATA or CAAT boxes.3 Instead, the human TGF-β1 gene promoter contains a GC-rich region with 11 direct CCGCCC repeats.3 Several studies have demonstrated that an activator protein (AP)-1 site, located in the 5'-flanking region of the TGF-β1 gene, plays an important role in the expression modulated by such endogenous cytokines as tumor necrosis factor-{alpha}, IL-1β, and IL-13.34–36 In addition, autoinduction of the TGF-β1 gene via the AP-1 site has been reported,37 and this AP-1 site has been documented to be the most plausible element in transcriptional regulation of the TGF-β1 gene.38 In the present study, we identified DR1 as a cis element in the promoter region of the TGF-β1 gene and demonstrated that it is responsible for PPAR{delta}-induced upregulation.

Of particular interest is the possibility that PPAR{delta} induction of TGF-β1 expression may participate in the regulation of inflammation in vivo. The activation of PPAR{delta} by GW501516 caused a marked attenuation in IL-1β–induced expression of MCP-1. Furthermore, the addition of siRNA against PPAR{delta} almost completely abolished this reduction. Moreover, anti–TGF-β1 antibody and a TβR-I inhibitor antagonized PPAR{delta} activity, suggesting that the effect of PPAR{delta} on MCP-1 expression is TGF-β1 dependent. In line with these results, the administration of GW501516 to mice induced the expression of TGF-β1 in the thoracic aorta and mRNA levels of MCP-1 and MIP-1β were significantly attenuated. TGF-β1 has been reported to inhibit the activation of macrophages, cytokine-induced expression of matrix metalloproteinase (MMP)-12, inducible NO synthase, and MCP-1 in a Smad3-dependent manner.10,25,39 In contrast, little is known about the antiinflammatory activity of PPAR{delta}, except for a single report indicating that PPAR{delta} regulates MCP-1, MCP-3, and IL-1β levels in vascular lesions by physically associating or dissociating with antiinflammatory BCL-6, depending on the availability of the ligand.20 Our results suggested the possibility that PPAR{delta} may be involved in atherogenesis through the regulation of expression of inflammatory cytokines such as MCP-1 or the modulation of VSMC proliferation. In turn, this hypothesis suggests that under pathological conditions, PPAR{delta} may play a major role as an antiinflammatory mediator to maintain homeostasis within the vasculature.

Our findings indicate that TGF-β1 is a target gene for PPAR{delta}, through which it exerts its antiinflammatory activity. These results have important implications not only for the understanding of the molecular mechanisms underlying the antiinflammatory activity of PPAR{delta} but also for the transcriptional regulation of the TGF-β1 gene. It is possible that PPAR{delta} ligands may be used to effect transient activation of local TGF-β1 signaling and thus become a therapeutically relevant strategy for the treatment of atherogenesis and other inflammatory vascular disorders.


*    Acknowledgments
 
Sources of Funding

This work was supported in part by Korean Science and Engineering Foundation grant R13-2005-012-02001-0, which is funded by the Korean government; Korean Research Foundation grant KRF-2006-005-J04202); and a clinical fund of Gyeongsang National University Hospital (2006).

Disclosures

None.


*    Footnotes
 
Original received June 22, 2007; revision received October 11, 2007; accepted November 1, 2007.


*    References
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 *References
 

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