Peroxisome Proliferator-Activated Receptor δ Regulates Extracellular Matrix and Apoptosis of Vascular Smooth Muscle Cells Through the Activation of Transforming Growth Factor-β1/Smad3
Homeostasis of the extracellular matrix and apoptosis of vascular smooth muscle cells (VSMCs) are key components in the regulation of the stability of atherosclerotic plaques. Here, we demonstrate that peroxisome proliferator-activated receptor (PPAR)δ regulates extracellular matrix synthesis and degradation through transforming growth factor-β1 and its effector, Smad3. Activation of PPARδ strongly amplified the expression of types I and III collagen, fibronectin, elastin, and TIMP-3 (tissue inhibitor of metalloproteinases 3), but not of TIMP-1, matrix metalloproteinase-2 or -9. The effect of PPARδ on the expression of type III collagen was dually regulated by the direct binding of PPARδ and Smad3 to a direct repeat-1 site and a Smad-binding element, respectively, in the type III collagen gene promoter. The activation of PPARδ attenuated apoptotic cell death in VSMCs induced by oxidized low-density lipoprotein, and similar antiapoptotic effects were observed on treatment of cells with exogenous type I and/or III collagen. Administration of a PPARδ ligand GW501516 to mice also suppressed elastase-induced cell death of aortic VSMCs. These results suggest that PPARδ-induced upregulation of extracellular matrix proteins exerts an antiapoptotic effect, thereby maintaining the stability of atherosclerotic plaques. Specific ligands of PPARδ may aid in the therapeutic intervention of atherosclerosis by improving plaque stability and patient prognosis.
- extracellular matrix
- peroxisome proliferator-activated receptor δ
- transforming growth factor-β1
- vascular smooth muscle cells
Peroxisome proliferator-activated receptors (PPARs) are members of a superfamily of nuclear hormone receptors that play pivotal roles in the regulation of lipid homeostasis, energy metabolism, and inflammation, as well as cellular differentiation and proliferation.1 These receptors regulate gene expression by dimerizing with the retinoid X receptor (RXR) and binding to specific recognition sequences termed PPAR response elements (PPREs) located in the regulatory regions of target genes.2 Among 3 different isoforms identified in mammals (PPARα [NR1C1], -δ [NR1C2], and -γ [NR1C3]), the physiological function of PPARδ remains relatively unknown. PPARδ is abundantly expressed in a variety of cell lineages, including vascular smooth muscle cells (VSMCs), and has been postulated that PPARδ ligands exert antiatherosclerotic effects by increasing the availability of inflammatory suppressors.3 Although the therapeutic efficacy of PPARδ ligands in the treatment of atherosclerosis remains controversial,4,5 PPARδ has been suggested as a promising new target for the treatment of metabolic syndrome, based on its beneficial properties in modulating lipoprotein metabolism and preventing obesity.6,7
VSMCs are the structural components of atherosclerotic plaque caps, and are critical for the maintenance of plaque stability.8 Maintenance of extracellular matrix (ECM) is one of the major functions of VSMCs, and within the normal vessel wall, ECM homeostasis reflects a tightly regulated balance between matrix metalloproteinases (MMPs) and their endogenous inhibitors, tissue inhibitors of metalloproteinases (TIMPs).9 VSMCs express transforming growth factor (TGF)-β1, a cytokine with diverse functions that has been implicated in the development of atherosclerosis, restenosis, and vascular remodeling.10 TGF-β1 exerts a prominent effect on ECM deposition by enhancing the synthesis of collagens, elastin, and fibrillin-1.11 Types I and III collagen are components of atherosclerotic plaques and provide structural strength and elasticity to the plaque.12 An imbalance in the synthesis and degradation of interstitial collagen is a key event leading to the rupture of atherosclerotic plaques.13 Within atherosclerotic plaques, the balance can become shifted toward matrix degradation, particularly at the rupture-prone shoulder regions of the fibrous cap, where accumulating macrophages and phenotypically altered smooth muscle cells secrete a plethora of proteinases, including MMPs.14,15 It has been suggested that TGF-β1 regulates plaque stability through its effect on ECM deposition.16,17 In fact, numerous studies support the hypothesis that TGF-β1 stabilizes atherosclerotic lesions by attenuating inflammation in the plaque and promoting the formation of a dense fibrous cap.18,19
Recently, TGF-β1 was identified as a novel target molecule of PPARδ in VSMCs.20 Because TGF-β1 regulates ECM turnover,11 its upregulation by PPARδ in VSMCs might affect the expression of genes related to the regulation of ECM. We hypothesized that PPARδ would be a key molecule in the stabilization of atherosclerotic plaques through the modulation of ECM homeostasis. In this report, we show that the activation of PPARδ upregulates the expression of ECM-related genes directly and/or indirectly in a TGF-β1/Smad3-dependent manner. Interestingly, activation of PPARδ also attenuated oxidized low-density lipoprotein (oxLDL)- and elastase-induced apoptotic cell death in VSMCs.
Materials and Methods
Human aortic vascular smooth muscle cells (HASMCs) were cultured in Smooth Muscle Cell Medium (ScienCell Research Laboratories, Carlsbad, Calif) containing smooth muscle cell growth supplement, based on the recommendations of the manufacturer. Details on reagents and methodology are provided in the expanded Materials and Methods section in the Online Data Supplement, available at http://circres.ahajournals.org.
Activation of PPARδ Induces the mRNA Expression of Types I and III Collagen, Fibronectin, and Elastin in a TGF-β1–Dependent Manner
When HASMCs were treated with GW501516, a specific ligand for PPARδ, the mRNA levels of types I and III collagen, fibronectin, and elastin were increased in a concentration- and time-dependent manner (Figure 1A).
To verify the role of PPARδ in the upregulation of ECM-related genes, we examined the effect of GW501516 on cells that were treated with a small interfering (si)RNA against PPARδ (Figure I in the Online Data Supplement). The siRNA-mediated downregulation of PPARδ markedly suppressed the GW501516-induced expression of TGF-β1, types I and III collagen, fibronectin, and elastin (Figure 1B). The increased mRNA expression of these ECM-related genes was partially reduced in the presence of a neutralizing antibody against TGF-β. Combination of PPARδ siRNA and anti–TGF-β antibody did not affect the levels of ECM-related genes reduced by siRNA or antibody alone (Figure 1B).
Activation of PPARδ Induces the Expression of Type III Collagen in Various Cell Lines
Because the transcriptional regulation of type III collagen has been relatively poorly characterized as compared to type I collagen,21 and the type III collagen exhibited a predominant response to PPARδ ligand in HASMCs (Figure 1), we examined the regulation of type III collagen expression by PPARδ in more detail. GW501516 increased both the mRNA and protein levels of type III collagen in several different human cell lines (Figure 2A). In the presence of GW501516, there was a concentration- and time-dependent increase in type III collagen in VSMCs (Figure 2B).
TGF-β1 Mediates the Upregulation of Type III Collagen by PPARδ
We next examined the effect of actinomycin D and cycloheximide on the GW501516-induced increase in type III collagen mRNA levels. The induction of type III collagen expression by PPARδ ligand was markedly reduced in the presence of actinomycin D or cycloheximide (Figure 2C), which indicated that de novo synthesis of mRNA and protein(s) involved in the regulation of type III collagen gene expression is essential for the induction of type III collagen in GW501516-treated HASMCs.
To further characterize the upregulation of type III collagen by GW501516, VSMCs were transfected with siRNA against PPARδ. The downregulation of PPARδ by siRNA almost completely reversed the expression pattern of type III collagen protein induced by PPARδ ligand. Similarly, the addition of anti–TGF-β antibody suppressed the induction of expression of type III collagen by GW501516 (Figure 2D). These results suggested that TGF-β1 is involved in the upregulation of type III collagen by GW501516.
To determine whether the regulation of type III collagen expression through PPARδ activation and TGF-β1 occurred at the level of transcription, we carried out a transfection assay using a luciferase reporter construct driven by the type III collagen gene (COL3A1) promoter. The activation of PPARδ by GW501516 significantly increased COL3A1 promoter activity, which was consistent with the observed increase in type III collagen protein levels. However, whereas the increased promoter activity induced by GW501516 was almost completely suppressed by cotransfection with PPARδ siRNA, treatment with anti–TGF-β antibody or an inhibitor of the TGF-β type I receptor kinase (TβR-I) (ALK5 inhibitor I, Calbiochem, La Jolla, Calif) partially suppressed COL3A1 promoter activity. Combined treatment with PPARδ siRNA and anti–TGF-β antibody or TβR-I inhibitor completely abolished COL3A1 promoter activity induced by GW501516 (Figure 2E). These observations supported the hypothesis that both PPARδ and TGF-β1 are involved in the regulation of type III collagen expression in VSMCs.
Smad3, an Effector of TGF-β, and a Smad-Binding Element Mediate the PPARδ-Induced Increase in COL3A1 Promoter Activity
To elucidate the mechanism of induction of type III collagen mRNA expression by TGF-β1, we carried out a cotransfection assay using the COL3A1-luciferase reporter gene construct, and expression constructs for several different Smad isoforms, Sp-1, or c-Jun/c-Fos. As shown in Figure 3A, coexpression of Smad3 alone increased COL3A1 promoter activity, and this effect was enhanced in the presence of GW501516. The effect of Smad3 was significantly attenuated by a dominant-negative form of Smad3 (SmadΔ3) in the presence or absence of GW501516 (Figure 3B). Next, we investigated the effect of PPARδ activation on the activity of a promoter bearing a tandem array of 6 copies of the Smad-binding element (SBE) consensus motifs (p6×SBEwt/Luc).22 As shown in Figure 3C, there was a significant increase in SBE-driven luciferase activity in cells treated with GW501516 for 38 hours. Of note, treatment of cells with siRNA against PPARδ, anti–TGF-β antibody, or TβR-I inhibitor attenuated the induction of SBE-driven luciferase expression by GW501516 (Figure 3C and 3D). A reporter construct containing specific mutations in the SBE motifs (p6×SBEmut/Luc)22 was unresponsive to GW501516. These results were in agreement with the phosphorylation of Smad3 in response to PPARδ activation and suggested that the PPARδ-dependent increase in COL3A1 promoter activity is mediated by Smad3 binding to an SBE.
Both PPARδ and TGF-β1 Are Involved in the Transcriptional Regulation of Type III Collagen Expression
To identify the promoter region responsible for PPARδ-induced upregulation of type III collagen, we carried out a luciferase reporter assay using a set of truncated COL3A1 promoter constructs (Online Figure II). The response to GW501516 was markedly reduced on the deletion of sequences between nucleotides −1494 and −1030 (relative to the transcriptional start site at +1), or deletion of sequences up to nucleotides −225 of the COL3A1 promoter. Anti–TGF-β antibody had no effect on the activity of these 2 constructs. Thus, the region between nucleotides −1494 and −1030 appears to be essential for the TGF-β1–mediated increase in COL3A1 promoter activity. Further truncation of the promoter up to nucleotides −115 abolished the response to GW501516, which indicated that this region is responsible for increased promoter activity in response to GW501516.
PPARδ and Smad3 Increase COL3A1 Promoter Activity Through Sites That Contain a PPRE and SBE, Respectively
Based on the results of our analysis of the COL3A1 promoter, we speculated that the promoter elements responsible for mediating the effects of TGF-β1 and PPARδ were located between nucleotides −1494 and −1030 and −225 and −115, respectively. We searched the sequence database and identified a putative SBE at nucleotides −1116 to −1107, and a putative PPRE containing a direct repeat-1 (DR1) sequence at nucleotides −144 to −132 of the COL3A1 promoter (Figure 4A).
To determine whether the putative SBE and PPRE in the COL3A1 promoter were involved in PPARδ-mediated transcriptional activation, we introduced mutations into the SBE and DR1 site of the full-length COL3A1 promoter (1.68 kilobases). In the presence of the wild-type COL3A1 promoter, the activation of PPARδ by GW501516 resulted in a 4-fold increase in transcriptional activity. This effect was markedly or partially attenuated in the presence of PPARδ siRNA, anti–TGF-β antibody, or TβR-I inhibitor. Mutation of the putative SBE (COL3A1-SBEmt) resulted in a partial loss of promoter activity in response to GW501516. COL3A1-SBEmt activity was completely lost in the presence of siRNA against PPARδ, whereas it was unaffected by either anti–TGF-β antibody or the TβR-I inhibitor. We next introduced mutations into the putative DR1 site of the COL3A1 promoter (COL3A1-DR1mt). Surprisingly, whereas the response of COL3A1-DR1mt to GW501516 was similar to that of COL3A1-SBEmt, activity was completely lost in the presence of anti–TGF-β antibody or TβR-I inhibitor. These results indicated that the COL3A1 promoter is subject to dual regulation by PPARδ and TGF-β1, such that PPARδ binds directly to a DR1-type PPRE in the COL3A1 promoter, and also induces the expression of TGF-β1 to elicit full activation of COL3A1 expression. In support of this mechanism, double mutation of the DR1 site and the SBE completely abolished the promoter activity of COL3A1 in response to GW501516 (Figure 4B).
To gain further insight into the effector molecules involved in the TGF-β1–mediated induction of gene expression, we performed a cotransfection assay using wild-type and mutant COL3A1 promoter constructs and expression constructs for wild-type and dominant negative Smad3. The promoter activity of wild-type COL3A1 was dependent on Smad3, whereas COL3A1-SBEmt activity was unresponsive to the overexpression of Smad3 and/or SmadΔ3. The overexpression of Smad3 increased the promoter activity of COL3A1-DR1mt, and this increase was suppressed by SmadΔ3, which indicated that Smad3 plays a primary role in the PPARδ-TGF-β1–mediated augmentation of COL3A1 promoter activity (Figure 4C).
PPARδ and Smad3 Bind to the COL3A1 Promoter PPRE and SBE, Respectively
To determine whether PPARδ and Smad3 bind directly with the DR1 site and SBE of the COL3A1 promoter, we performed electrophoretic mobility shift assay using in vitro synthesized PPARδ, RXRα, and/or Smad3 proteins (Online Figure III). Neither RXRα nor PPARδ alone bound to wild-type or mutant COL3A1-DR1, whereas protein–DNA complexes were observed on the incubation of wild-type COL3A1-DR1 (DR1wt) or COL3A1-SBE (SBEwt) with PPARδ and RXRα or Smad3, respectively. The complexes were supershifted in the presence of anti-PPARδ or anti-Smad3 antibodies, and no protein–DNA complexes were observed in the presence of mutant COL3A1-DR1mt or COL3A1-SBEmt.
To confirm that Smad3 and PPARδ interact directly with the Col3a1 promoter to elicit transcriptional upregulation, we carried out a chromatin immunoprecipitation (ChIP) assay. Cells were treated with GW501516 for various lengths of time, and chromatin fragments were subjected to immunoprecipitation using anti-Smad3 or anti-PPARδ antibody. Genomic DNA from the immunoprecipitates was amplified by PCR using primers that corresponded to the putative SBE or DR1 site. As shown in Figure 4D, PCR-amplified fragments were obtained from cells treated with GW501516 but not untreated cells. The specificity of the assay was confirmed using set of primers that amplified a nonspecific region of the promoter (oligo no. 3). These results were consistent with results of the promoter assays and indicated that Smad3 and PPARδ bind to the SBE and DR1 site, respectively, that we identified in the COL3A1 promoter.
Activation of PPARδ Induces the Expression of TIMP-3, but Not TIMP-1 or MMPs, in HASMCs
To determine whether the activation of PPARδ modulated the expression of MMPs and TIMPs, which are key regulators of collagen degradation,9 cells were stimulated with GW501516 in the presence or absence of PPARδ siRNA. As shown in Figure 5A and Online Figure IV, GW501516 elevated the promoter activity and expression of TIMP-3, but not of TIMP-1, MMP-2, or MMP-9. The increased promoter activity and expression of TIMP-3 or phosphorylation of Smad3 induced by GW501516 were significantly attenuated in the presence of PPARδ siRNA. Because TGF-β1 modulates the expression of MMPs and TIMPs via Smad3,23 we also investigated the effect of blocking TGF-β signaling on the induction of TIMP-3 by GW501516. Both anti–TGF-β antibody and TβR-I inhibitor significantly reduced the GW501516-induced increase in the promoter activity and expression of TIMP-3. These findings are in line with phosphorylation status of Smad3 (Figure 5B). The overexpression of Smad3 alone elicited a dose-dependent increase in the promoter activity and expression of TIMP-3, whereas SmadΔ3 almost completely abolished both GW501516- and/or Smad3-induced expression of TIMP-3 (Figure 5C).
Expression of ECM Proteins Is Increased in the Thoracic Aorta of Mice Treated With GW501516
To determine whether the effects that we observed in cultured cells were also observed in vivo, we examined the levels of ECM proteins in the thoracic aorta of mice treated with GW501516. As shown in Figure 6, administration of GW501516 significantly increased the levels of ECM proteins in the mouse thoracic aorta. There were also significant increases in the levels of TGF-β1, types I and III collagen, fibronectin, elastin, TIMP-1, and TIMP-3, whereas the levels of MMP-2 and -9 were unaffected by GW501516. Similar results were obtained in immunohistochemical analyses (Online Figure V). These results clearly indicated that the activation of PPARδ modulates the expression of ECM proteins in vivo.
Activation of PPARδ Inhibits oxLDL-Induced Apoptosis Through the Expression of ECM Proteins
To investigate the pathophysiological role of PPARδ-mediated regulation of ECM proteins, we evaluated the effect of GW501516 on oxLDL-induced apoptosis. Although oxLDL significantly increased the number of apoptotic cells, based on Annexin V–positive staining, there was a significant decrease in apoptosis in cells treated with GW501516 for 24 hours. The PPARδ-mediated attenuation of apoptosis was reversed in the presence of PPARδ siRNA or in part by anti–TGF-β antibody, which suggested that the antiapoptotic effects of GW501516 are PPARδ- and TGF-β1–dependent (Figure 7A). Similar results were obtained when the cells were analyzed by fluorescence-activated cell sorting (Online Figure VI).
To verify the role of PPARδ-mediated expression of collagen in apoptosis, we added purified collagen directly to the culture medium. As shown in Figure 7B, the oxLDL-induced apoptosis of VSMCs was significantly reduced in the presence of type I and/or III collagen. In contrast, BSA or gelatin, a denatured collagen, failed to suppress oxLDL-induced apoptosis. To further confirm the role of PPARδ-induced collagen expression, we investigated the effect of siRNA against COL3A1 or/and COL1A1 in oxLDL-induced apoptosis. The siRNA-mediated downregulation of COL3A1 or/and COL1A1 (Online Figure VII) significantly blunted the GW501516-mediated suppression of cell death (Figure 7C). Finally, to investigate the functional consequence of the PPARδ-mediated induction of ECM proteins in vivo, we examined effects of activation of PPARδ on the elastase-induced apoptotic cell death in the thoracic aorta of mice treated with GW501516. As shown in Figure 7D, perfusion with elastase significantly increased apoptotic cell death, whereas staining of apoptotic cells was significantly attenuated in GW501516-treated aorta. These results clearly indicated that decreased apoptosis observed in cells treated with a PPARδ ligand is attributable, at least in part, to the induction of ECM proteins.
The nuclear receptor PPARδ elicits a diverse spectrum of responses through the expression of a variety of genes in vascular cells.24 Although multiple effector genes have been implicated in PPARδ action, little is known about the signaling pathways downstream of PPARδ. In the present study, we demonstrated that the activation of PPARδ by a specific ligand, GW501516, induces the expression of types I and III collagen, fibronectin, elastin, and TIMP-3, which are pivotal factors in maintaining the integrity of vascular structure.25 This induction was mediated by TGF-β1, a master cytokine involved in the regulation of ECM homeostasis.11 In mice treated with GW50156, the expression of ECM-related genes was upregulated, along with TGF-β1, in the thoracic aorta. The effect of PPARδ on the expression of type III collagen was mediated by a dual mechanism involving the direct binding of PPARδ to a DR1-type PPRE and the TGF-β1–mediated binding of Smad3 to an SBE in the COL3A1 promoter. Analysis of point mutations of the COL3A1 promoter demonstrated that the DR1 site and SBE are indispensable for the induction of type III collagen by PPARδ. Furthermore, we showed that the activation of PPARδ attenuates apoptotic cell death induced by oxLDL in a TGF-β1–dependent manner, suggesting an antiapoptotic role for PPARδ-induced collagen expression. In line with these findings, the administration of a PPARδ ligand GW501516 to mice suppressed the elastase-induced apoptotic cell death in the thoracic aorta.
PPARδ ligand specifically induced the expression of types I and III collagen, fibronectin, and elastin in a TGF-β1–dependent manner. Although all of the PPAR family members have been characterized in terms of their roles in VSMCs using specific ligands, only PPARδ induced TGF-β1.20 Given that the inhibition of TGF-β signaling in mouse models accelerates atherosclerosis and induces an unstable plaque phenotype,16 PPARδ and TGF-β1–mediated biosynthesis of ECM may contribute to the stability of atherosclerotic plaques. Indeed, VSMCs in stable lesions express greater amounts of TGF-β than unstable lesions.17 The expression levels of TGF-β and TGF-β receptors also vary in atheromatous plaques at different stages.26 Therefore, it may be possible to stabilize plaques by increasing the levels of ECM proteins, such as interstitial collagens, through PPARδ-induced expression of TGF-β1 in VSMCs.
We identified 2 cis-acting elements in the COL3A1 promoter. Previous studies have demonstrated that multiple proteins, including lysyl oxidase, are involved in the formation of protein–DNA complexes with the proximal promoter region of COL3A1.21,27 We demonstrated that PPARδ regulates the expression of COL3A1 at the transcriptional level, through the direct binding of PPARδ to a DR1 site in the COL3A1 promoter, and the TGF-β1–induced binding of Smad3 to an SBE. The PPRE identified in this study contained a canonical DR1 motif separated by an adenine. The SBE was identified as a second cis-acting element involved in the PPARδ-induced transcriptional activation of COL3A1 via TGF-β1, and we demonstrated that Smad3 forms protein–DNA complexes with the COL3A1-SBE. Thus, we have identified 2 sites, a DR1-type PPRE and an SBE in the COL3A1 promoter, that function as cis elements responsible for the PPARδ-induced activation of COL3A1 expression.
In addition to the upregulation of ECM proteins, the activation of PPARδ by GW501516 significantly increased the promoter activity and expression of TIMP-3 in a TGF-β1/Smad3-dependent manner. To identify a putative PPRE in TIMP-3 gene promoter, we searched the sequence database but could not find the homologous sequence. TIMP-3 may therefore be indirectly regulated by the PPAR signaling pathway. In any case, these findings indicate that PPARδ is involved in a dual mechanism of ECM homeostasis, which involves the acceleration of synthesis and suppression of degradation of ECM components. On the other hand, PPARδ has been shown to suppress the inflammatory reaction in vascular tissue.20 It is well recognized that the ECM captures inflammatory cells, such as macrophages, and interacts with surrounding cells, including VSMCs, to limit the bioavailability of proinflammatory mediators.28 Thus, PPARδ-induced expression of TGF-β1 may also have an antiinflammatory role, by maintaining the local ECM at sites of vascular lesions.
Of particular interest is the finding that the PPARδ-induced expression of TGF-β1 modulates apoptotic cell death through the upregulation of ECM proteins in vivo as well as in vitro. Although degraded collagen has been reported to induce apoptosis in vivo and in vitro,29,30 little is known about the antiapoptotic actions of collagens in VSMCs. Our results suggest that PPARδ may affect the fate of atherosclerotic lesions by regulating the expression of ECM proteins and suppressing VSMC apoptosis. In this context, PPARδ may serve as an antiapoptotic mediator in the maintenance vascular integrity.
In conclusion, the present findings suggest that PPARδ induces the upregulation of ECM proteins in a process that is mediated by TGF-β1 and Smad3 and that this upregulation contributes to the integrity of atherosclerotic plaques that might be prone to rapture. To our knowledge, this is the first report demonstrating that PPARδ regulates the expression of type III collagen at the transcriptional level through TGF-β1/Smad3-dependent and -independent mechanisms. Our results suggest that specific ligands for PPARδ hold promise as therapeutic interventions in atherosclerosis and may improve plaque stability and patient prognosis.
Sources of Funding
This work was supported by the Korea Science and Engineering Foundation (KOSEF), funded by the Korea government (grants R01-2008-000-10428-0 and R13-2005-012-02001-0).
Original received October 9, 2008; revision received May 13, 2009; accepted May 13, 2009.
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