Activation of Peroxisome Proliferator-Activated Receptor Suppresses Telomerase Activity in Vascular Smooth Muscle Cells
http://www.100md.com
Daisuke Ogawa, Takashi Nomiyama, Takafum
参见附件。
the Division of Endocrinology and Molecular Medicine (D.O., T. Nomiyama, T. Nakamachi, E.B.H., J.F.S., D.B.), University of Kentucky College of Medicine, Lexington
Department of Metabolic Disorders (J.P.B.), Merck Research Laboratories, Rahway, NJ
Takeda Pharmaceuticals North America (R.E.L.), Lincolnshire, Ill.
Abstract
Activation of the peroxisome proliferator-activated receptor (PPAR) , the molecular target for insulin sensitizing thiazolidinediones used in patients with type 2 diabetes, inhibits vascular smooth muscle cell (VSMC) proliferation and prevents atherosclerosis and neointima formation. Emerging evidence indicates that telomerase controls key cellular functions including replicative lifespan, differentiation, and cell proliferation. In the present study, we demonstrate that ligand-induced and constitutive PPAR activation inhibits telomerase activity in VSMCs. Telomerase reverse transcriptase (TERT) confers the catalytic activity of telomerase, and PPAR ligands inhibit TERT expression through a receptor-dependent suppression of the TERT promoter. 5'-deletion analysis, site-directed mutagenesis, and transactivation studies using overexpression of Ets-1 revealed that suppression of TERT transcription by PPAR is mediated through negative cross-talk with Ets-1–dependent transactivation of the TERT promoter. Chromatin immunoprecipitation assays further demonstrated that PPAR ligands inhibit Ets-1 binding to the TERT promoter, which is mediated at least in part through an inhibition of Ets-1 expression by PPAR ligands. In VSMCs overexpressing TERT, the efficacy of PPAR ligands to inhibit cell proliferation is lost, indicating that TERT constitutes an important molecular target for the antiproliferative effects of PPAR ligands. Finally, we demonstrate that telomerase activation during the proliferative response after vascular injury is effectively inhibited by PPAR ligands. These findings provide a previously unrecognized mechanism for the antiproliferative effects of PPAR ligands and support the concept that PPAR ligands may constitute a novel therapeutic approach for the treatment of proliferative cardiovascular diseases.
Key Words: telomerase peroxisome proliferator-activated receptor vascular smooth muscle cell proliferation
Introduction
Peroxisome proliferator-activated receptor (PPAR) is a ligand-activated transcription factor belonging to the nuclear hormone receptor superfamily.1 Synthetic ligands include thiazolidinedione (TZD) PPAR ligands, like rosiglitazone (RSG) or pioglitazone (PIO), which are clinically used as insulin sensitizers in patients with type 2 diabetes. TZD PPAR agonists not only improve insulin resistance in patients with type 2 diabetes but also exert a broad spectrum of pleiotropic vascular effects in vitro and in animal models.2 PPAR is expressed in macrophages, endothelial cells, and vascular smooth muscle cells (VSMCs) and regulates gene expression of key proteins involved in glucose/lipid metabolism, vascular inflammation, and proliferation.3 TZD PPAR ligands prevent the development of atherosclerosis and intimal hyperplasia after balloon injury in animal models.4–6 Early clinical studies further demonstrate that PPAR ligands prevent the progression of intima/media thickening used as sensitive measurement of atherosclerosis7,8 and the development of restenosis after coronary stent implantation.9,10 These beneficial cardiovascular effects are further supported by the recently published randomized controlled PROactive trial, which demonstrated a significant 16% reduction of the main secondary end points all-cause mortality, myocardial infarction, and stroke in type 2 diabetic patients treated with PIO.11
Proliferation of VSMCs is a critical process for the development of atherosclerosis and the primary mechanism resulting in the failure of procedures used to treat occlusive atherosclerotic diseases, such as postangioplasty restenosis, transplant vasculopathy, and vein bypass graft failure.12 PPAR ligands have been demonstrated to inhibit VSMC proliferation, an effect that may importantly contribute to their beneficial cardiovascular effects.13 The molecular mechanisms by which PPAR ligands inhibit VSMC proliferation involves an inhibition of cell cycle progression and phosphorylation of the retinoblastoma protein (Rb).14 Rb serves as a G1 gatekeeper, and Rb phosphorylation results in the release of the E2F transcription factor controlling S phase gene expression. The inhibition of Rb phosphorylation by PPAR is mediated by preventing degradation of the cyclin-dependent kinase inhibitor (CDKI) p27Kip1 and blocking cyclin A and D1 expression, essential for Rb phosphorylation and cell cycle progression.14,15
Telomerase controls key cellular functions including replicative lifespan, differentiation, and cell proliferation.16 Telomerase activity is required for VSMC proliferation, and disruption of telomerase activity reduces atherosclerosis and neointima formation, indicating that telomerase may serve as a novel pharmacological target for the treatment of vascular diseases.17–19 Telomeres consist of TTAGGG repeats at the end of chromosomes, and telomere length decreases with increasing cell division and senescence.20 Mice lacking telomerase function exhibit reduced tumorigenesis and decreased cell proliferation in highly proliferative organs, suggesting that telomerase activity is involved in the regulation of cell proliferation.21 Human telomerase consists of three subunits: the telomerase RNA (TR), which provides the template for telomere synthesis, the telomerase-associated protein 1 (TEP1) and the telomerase reverse transcriptase (TERT), which contains the catalytic activity of telomerase.22 TERT is the limiting factor for telomerase activity, and overexpression of TERT has been demonstrated to induce cell proliferation, whereas TERT antisense oligonucleotides inhibit VSMC proliferation.16,17,23 Thus, telomerase, and specifically TERT, may play an important role for the regulation of VSMC growth and neointima formation.
In the present study, we investigated the regulation of telomerase activity by ligand-induced and constitutive activation of PPAR. We report that activation of PPAR suppresses telomerase activity in VSMCs in vitro and in vivo. These studies identify telomerase as an important molecular target for the antiproliferative effects of PPAR and outline a previously unrecognized mechanism by which PPAR regulates VSMC proliferation.
Materials and Methods
Cell Culture
Rat aortic VSMCs were isolated and cultured as described previously.24 Cells were grown to 60% to 70% confluence and serum deprived in 0.1% FBS for at least 48 hours. Quiescent VSMCs were pretreated with the PPAR ligands RSG (1 to 10 μmol/L), PIO (1 to 10 μmol/L), or a non-TZD partial PPAR agonist25 (nTZDpa; 0.1 to 5 μmol/L) for 24 hours. After pretreatment, cells were stimulated in the presence of vehicle or the ligand with rat recombinant platelet-derived growth factor (PDGF)–BB (Sigma) and insulin (Eli Lilly & Co.) at the final concentration of 20 ng/mL and 1 μmol/L, respectively. The combination of PDGF and insulin as mitogen was used based on previous publications demonstrating that PDGF and insulin exert additive effects on VSMC proliferation.26 RSG was kindly provided by GlaxoSmithKline (King of Prussia, Pa). PIO was provided by Takeda Chemical Industry. nTZDpa25 was a generous gift from Merck (Rahway, NJ). For all data shown, individual experiments were repeated at least three times with different lots or preparation of cells.
Telomeric Repeat Amplification Protocol Assay
Telomerase activity was analyzed using a commercially available polymerase chain reaction (PCR)–based assay according to manufacturer instructions (TeloTAGGG Telomerase PCR ELISA Plus; Roche Applied Sciences). After stimulation, cells were harvested in provided lysis reagent, and whole-cell proteins (3 μg) were used for elongation/amplification. After amplification, PCR products were resolved on a 12% nondenaturing polyacrylamide gel and vacuum-transferred to nylon membranes. The characteristic 6-bp telomerase-specific ladder was detected using a Biotin-Luminescent Detection Kit (Roche Applied Sciences). In addition, telomerase activity was quantified by ELISA according to manufacturer instructions.
Adenoviral Constructs and Infections
The adenovirus overexpressing constitutively active PPAR was generated by fusing VP16 transactivation domain of the herpes simplex virus to the N terminus of PPAR1 as described previously.27 Overexpression of this mutant results in a potent ligand-independent activation of PPAR.28 Recombinant type 5 adenovirus-expressing green fluorescent protein (GFP) or the VP-16 transactivation domain were used as a control vector in all experiments.27 VSMCs were infected with 1, 25, or 100 plaque-forming units (PFU)/cell in DMEM containing 0.1% FBS for 24 hours. After further starvation for 24 hours, cells were stimulated with PDGF/insulin for 48 hours and analyzed for telomerase activity.
Northern Blotting
Isolation of total RNA and Northern blotting was performed as described previously.24 cDNA for TERT was kindly provided by Dr Tohru Kiyono (National Cancer Center Research Institute, Tokyo, Japan).29,30 Blots were cohybridized with cDNA encoding the constitutively expressed housekeeping gene 36B4 to assess equal loading of samples. Quantification was performed by densitometry and normalization to 36B4 mRNA expression.
Western Blot Analysis
Western blotting was performed as described recently.24 Primary antibodies were obtained from the following supplies: PPAR sc-7196 (Santa Cruz Biotechnology), VP16 3844-1 (BD Biosciences), TERT 582000 (EMD Biosciences), telomerase-associated protein 1 (TEP1) sc-13052 (Santa Cruz Biotechnology), and -actin ab6276 (Abcam Inc). Quantification of the Western blots was performed by densitometry and normalization to -actin.
Plasmids, Transient Transfections, and Luciferase Assay
The TERT promoter constructs were kindly provided by Dr Satoru Kyo (Kanazawa University School of Medicine, Japan).31 Wild-type and dominant-negative Ets-1 expression vectors were generously provided by Dr Naoyuki Taniguchi (Osaka University Medical School, Japan).32 The two side-by-side Ets-1 consensus motifs at -23 from the transcription initiation site were mutated from –23CTTCCTTTCCG–12 to –23CTTAATTTAAG–12 using the QuickChange II Site–directed mutagenesis kit (Stratagene) and the following primer pairs: forward 5'-CCAGCCCCTCCCCTTAATTTAAGCGGCCCCGCCCTCTC-3', reverse 5'-GAGAGGGCGGGGCCGCTTAAATTAAGGGGAGGGGCTGG -3'.
VSMCs were transfected for 8 hours with 1 μg of reporter DNA using LipofectAMINE (Invitrogen). After transfection, cells were cultured in 0.1% FBS for 24 hours followed by pretreatment with vehicle (dimethyl sulfoxide [DMSO]) or the indicated PPAR ligand for 24 hours. Subsequently, cells were stimulated with PDGF (20 ng/mL) and insulin (1 μmol/L), and luciferase activity was assayed after 24 hours using a Dual Luciferase Reporter Assay (Promega). Transfection efficiency was normalized to renilla luciferase activities generated by cotransfection with 10 ng/well pRL-CMV (Promega, Madison, WI). All experiments were repeated at least three times with different cell preparations.
Chromatin Immunoprecipitation Assays
Chromatin immunoprecipitation (ChIP) assays were performed using a ChIP assay kit (Upstate) according to manufacturer instructions. Briefly, quiescent VSMCs were pretreated with the indicated ligand for 24 hours and stimulated with PDGF/insulin. Cells were harvested after 3 hours, and soluble chromatin was prepared. Chromatin was immunoprecipitated with antibodies (2 μg) directed against Ets-1 (sc-111; Santa Cruz Biotechnology). Final DNA extractions were PCR amplified using primer pairs that cover the two Ets-1 consensus sequences at -23 in the TERT promoter as follows: forward 5'-CTTCCAGCTCCGCCTCCT-3'; reverse 5'-CAGCGCTGCCTGAAACTC -3'.
Confocal Microscopy
Quiescent VSMCs grown on plastic chamber-slides coated with poly-L-lysine were incubated with the PPAR ligand or vehicle (DMSO) for 24 hours before stimulation with PDGF/insulin for another 24 hours. Cells were fixed for 10 minutes with 10% formalin and incubated for 12 hours with the TERT antibody (582000; EMD Biosciences). An fluorescein isothiocyanate–conjugated secondary antibody and 4',6-diamidino-2-phenylindole staining was used for visualization. The data represent the mean of three independent experiments.
Cell Proliferation Assays
Small interfering RNA (siRNA) experiments were performed using the SMARTpool technology (Dharmacon RNA Technologies), which provides a mix of four different proprietary siRNAs specific for rat TERT. VSMCs were plated at a density of 1.0x105 cells on six-well plates and transfected with 250 nmol/L of TERT siRNA or scrambled siRNA using LipofectAMINE (Invitrogen). Experiments using TERT overexpression were performed by transfecting VSMCs with an expression vector overexpressing TERT (pCLXSN-TERT) or an empty control vector (pCLXSN; Imgenex Corp.). The pCLXSN-TERT expression vector under the control of a cytomegalovirus immediate-early long terminal repeat (CMV-IE/LTR) fusion promoter was kindly provided by Dr Tohru Kiyono (National Cancer Center Research Institute, Tokyo, Japan).29,30 After transfection, cells were maintained under serum deprivation for 24 hours, pretreated with vehicle or PIO (10 μmol/L) for an additional 24 hours, followed by mitogenic stimulation with PDGF (20 ng/mL) and insulin 1 μmol/L) for 72 hours. Cells were harvested and cell proliferation was measured by counting the cells using a hematocytometer. Experiments were performed in triplicate using three different preparations of rat aortic VSMCs.
Endovascular Femoral Artery Wire Injury
C57BL/6 male mice, 4 to 6 weeks of age, were purchased from Jackson Laboratory (Bar Harbor, Me). Mice were maintained in a temperature-controlled barrier facility with a 12-hour light/dark cycle and were given free access to food and water. Eight- to 10-week-old mice were treated with vehicle (n=8) or PIO (10 mg/kg per day; n=8) by daily intraperitoneal injection until euthanasia. After a treatment period of three days, endovascular wire injury was performed on the right femoral artery using a 0.25-mm SilverSpeed-10 hydrophilic guide wire (kindly provided by Sara Mueller, Micro Therapeutics Inc., Irvine, Calif), as described by Roque et al.33 Sham surgery without injury was performed on the contralateral left side. Mice were euthanized two days after injury, and the femoral arteries were isolated. Telomerase activities were analyzed by Southern blotting and ELISA as described above using 3 μg protein isolated from tissues homogenized in provided lysis buffer. All procedures on mice were approved by the institutional animal care and use committee at the University of Kentucky.
Statistical Analysis
ANOVA and paired or unpaired t test were performed for statistical analysis as appropriate. P values <0.05 were considered to be statistically significant. Results are expressed as mean±SEM.
Results
PPAR Ligands Inhibit Mitogen-Induced Telomerase Activity
To investigate the regulation of telomerase activity by PPAR ligands, quiescent VSMCs were treated with various PPAR agonists before stimulation with PDGF and insulin. As depicted in Figure 1A and 1B, mitogenic stimulation of serum-deprived VSMCs resulted in a substantial induction of VSMC telomerase activity, which was significantly inhibited by all used PPAR ligands. Whereas RSG inhibited telomerase activity at a dose of 10 μmol/L, PIO suppressed telomerase activity at a dose of 5 μmol/L and completely inhibited mitogen-induced telomerase activity at 10 μmol/L. The partial PPAR agonist nTZDpa completely inhibited telomerase activity at a dose of 5 μmol/L. The inhibition of telomerase activity was dose dependent over a concentration range known to inhibit VSMC proliferation14 and occurred in the absence of an effect of the different PPAR ligands on PDGF receptor expression (data not shown), suggesting a direct effect on telomerase activity.
Overexpression of Constitutively Active PPAR Inhibits Mitogen-Induced Telomerase Activity
To further determine whether the observed inhibition of telomerase activity by PPAR ligands is mediated through a receptor-dependent pathway, we used an adenoviral construct to overexpress a constitutively active PPAR mutant. As shown in Figure 2A, infection of VSMCs with adenovirus overexpressing constitutively active PPAR (Adx-CA-PPAR) resulted in marked dose-dependent overexpression of constitutively active PPAR protein. Consistent with our previous observation that expression of endogenous PPAR in VSMCs is almost exclusively detectable in the nuclear fraction, we observed faint expression of endogenous PPAR (55 kDa) in whole-cell extracts.34 Immunoblotting for VP16 revealed fast-migrating VP16 protein in VSMCs infected with Adx-VP16 and, because of the additional PPAR, slower-migrating VP16 in cells infected with Adx-CA-PPAR. Whereas telomerase activity was slightly increased in VSMCs infected with Adx-VP16, overexpression of constitutively active PPAR dose-dependently decreased mitogen-induced telomerase activity (Figure 2A and 2B). In concert, these data demonstrate that ligand-independent activation of PPAR suppresses telomerase activity in VSMCs, supporting the notion that the inhibition of telomerase activity by PPAR ligands is likely mediated through a PPAR-dependent mechanism.
PPAR Ligands Inhibit Mitogen-Induced TERT Expression
TERT constitutes the catalytic subunit of telomerase, and overexpression of TERT stimulates cell proliferation.16,23 To determine the mechanism by which PPAR ligands inhibit telomerase activity, we next analyzed the regulation of TERT expression by PPAR ligands. Stimulation of quiescent VSMCs with PDGF/insulin significantly induced TERT mRNA and protein expression (5.94±0.74 and 6.2±0.94-fold induction, respectively; P<0.01), which was dose-dependently inhibited by PIO and nTZDpa (Figure 3A and 3B). Expression of the TEP1 and the telomeric RNA component was not regulated in response to stimulation with PDGF/insulin or by treatment with the PPAR agonists (supplemental Figure IA and IB, available online at http://circres.ahajournals.org).
To further investigate whether the inhibition of mitogen-induced TERT expression by PPAR ligands is a result of decreased TERT promoter activity, VSMCs were transiently transfected with a 3.3-kb TERT promoter construct. As depicted in Figure 4A, PPAR agonists dose-dependently suppressed PDGF/insulin-induced TERT promoter activity. Similarly, cotransfection of the TERT promoter with an expression vector overexpressing a constitutively active PPAR mutant completely suppressed PDGF/insulin-induced TERT transcriptional activity in the absence of a PPAR ligand. In contrast, cotransfection of the TERT promoter with the empty vector or the VP16 transactivation domain vector exerted no effect to suppress mitogen-induced TERT promoter activity (Figure 4B). In concert, these findings indicate that PPAR ligands inhibit telomerase activity by suppressing mitogen-induced TERT transcription and further support the concept that the inhibition of TERT expression by PPAR ligands is mediated through a receptor-dependent mechanism.
PPAR Ligands Suppress TERT Transcription by Negative Cross-Talk With Ets-1–Dependent Transactivation of the TERT Promoter
The mechanism by which PPAR activation suppresses TERT transcriptional activation is unlikely to be direct because sequence analysis of the 5'-flanking region of the TERT promoter did not reveal the presence of PPAR response element (PPRE). To identify potential regulatory elements in the TERT promoter that support mitogen-induced TERT transcription and mediate the suppression of TERT promoter activity by PPAR, we used a series of 5'-deletion constructs of the TERT promoter (Figure 5A). PDGF/insulin-induced transcriptional activity of a -32 TERT promoter construct was completely suppressed by the PPAR ligand. In contrast, the +18 TERT promoter construct was not inducible by mitogens and exhibited a transcriptional activity only slightly above the background signal. The -32–bp proximal promoter that was determined as the minimal responsive element for PDGF/insulin contains two side-by-side consensus motifs for Ets-1 transcription factors at -23 from the transcription start site. Previous studies demonstrated that these sites are important for TERT transcription and that PPAR ligands interfere with Ets-1–mediated regulation of gene expression.31,35 Based on this evidence, we performed site-directed mutagenesis of both Ets-1 consensus sites, which resulted in a significant loss of basal and mitogen-induced TERT transcriptional activity (Figure 5B). Furthermore, PPAR ligands exerted no efficacy to suppress TERT promoter activity. To corroborate that PPAR activation suppresses TERT transcription through negative interference with Ets-1–dependent transactivation of the TERT promoter, we used expression vectors to overexpress wild-type and dominant-negative Ets-1. Cotransfection with an Ets-1 expression vector significantly induced basal TERT promoter activity (2.63±0.31-fold induction versus empty vector; P<0.05) and resulted in a complete loss of the effect of the PPAR agonist to suppress mitogen-induced TERT promoter activity (Figure 5C). Furthermore, cotransfection with a dominant-negative Ets-1 expression vector significantly repressed both basal and mitogen-induced TERT promoter activity. These data indicate that mitogens induce TERT promoter activation via a mechanism involving the Ets-1 site and that PPAR agonists repress mitogen-induced TERT expression by inhibiting Ets-1–dependent transactivation of the TERT promoter.
PPAR Activation Suppresses Ets-1 Binding to the -23 Consensus Motif in the TERT Promoter
To confirm that Ets-1 binds to the endogenous TERT promoter and that PPAR agonists interfere with this binding in vivo, we next performed ChIP assays. PCR amplification using primer pairs that cover the Ets-1 sites at -23 in the TERT promoter demonstrated that this TERT promoter sequence immunoprecipitated with Ets-1 after stimulation with PDGF and insulin. The time course experiments revealed maximal induction of Ets-1 binding to this site after a stimulation period of 3 hours (Figure 6A). Induction of Ets-1 binding to the proximal TERT promoter was substantially inhibited by both PIO (10 μmol/L) and nTZDpa (5 μmol/L; Figure 6B). To further determine potential mechanisms involved in the suppression of Ets-1 binding to the TERT promoter, we next analyzed whether PPAR ligands inhibit Ets-1 expression. Both PPAR ligands dose-dependently suppressed PDGF/insulin-induced Ets-1 mRNA expression in VSMCs (Figure 7). Therefore, inhibition of Ets-1 binding to the TERT promoter by PPAR agonists is mediated at least in part through an inhibition of Ets-1 expression.
PPAR Ligands Inhibit TERT Nuclear Translocation
Telomerase activity has further been reported to be regulated by post-translational modifications involving TERT nuclear translocation.36 Because the physiological role of telomerase in the synthesis of telomeric repeats occurs in the nucleus and nuclear translocation of TERT may play a role in the regulation of telomerase function, we performed experiments to determine the subcellular localization of TERT in VSMCs. As shown in Figure 8A, in quiescent VSMCs, most of the TERT protein was localized in the cytoplasm, whereas stimulation with PDGF/insulin resulted in a profound nuclear accumulation of TERT (Figure 8B). Interestingly, pretreatment with the PPAR agonist significantly attenuated mitogen-induced translocation of TERT into the nucleus. Together, these data demonstrate that PPAR ligands inhibit mitogen-induced TERT nuclear translocation, which may, in addition to the transcriptional suppression of TERT, play a role in the inhibition of telomerase activity by PPAR ligands in VSMCs.
Overexpression of TERT Reverses the Inhibitory Effect of PPAR Ligands on VSMC Proliferation
We next investigated whether TERT expression is required for mitogen-induced VSMC proliferation and whether inhibition of TERT contributes to the antiproliferative effects of PPAR agonists. TERT knockdown in VSMCs using siRNA (supplemental Figure IIA) resulted in a complete loss of mitogen-induced VSMC proliferation (supplemental Figure IIB). Consistent with recent observations by Cao et al,23 these data demonstrate that TERT expression is required for VSMC proliferation. To further determine whether the inhibition of TERT by PPAR ligands contributes to their antiproliferative efficacy, we next analyzed the effect of PIO on VSMC proliferation in cells overexpressing TERT. As depicted in Figure 9, PIO significantly inhibited mitogen-induced VSMC proliferation in cells transfected with control vector. Interestingly, overexpression of TERT resulted in a 3.1-fold induction of basal VSMC proliferation. However, in cells overexpressing TERT, treatment with the PPAR ligand resulted in a significant loss of the antiproliferative efficacy of the PPAR ligand. These data indicate that the inhibition of VSMC proliferation by PPAR ligands is at least in part mediated through an inhibition of TERT expression.
PPAR Ligands Inhibit Telomerase Activation During the Proliferative Response After Vascular Injury
To finally determine the regulation of telomerase activity during the proliferative response after vascular injury and to define whether the observed suppression of telomerase activity by PPAR ligands is applicable in vivo, we performed endovascular femoral artery denudation injuries in mice. Endothelial denudation in this model is associated with significant VSMC proliferation and subsequent neointima formation similar to that found after angioplasty in humans.33 Concomitant with the phenotypic shift from quiescent VSMCs resident in the uninjured vessel wall to proliferating smooth muscle cells present in the neointima, we observed a profound induction of telomerase activity in response to injury (Figure 10A and 10B). Time course studies demonstrated maximal telomerase activation 48 hours after injury (data not shown). Telomerase activation during the proliferative response was substantially suppressed in mice treated with PIO (84.8±7.9% inhibition versus vehicle-treated mice; P<0.001). Together, these results demonstrate that PPAR ligands suppress telomerase activation in response to vascular injury in vivo and indicate that telomerase may serve as a novel target for the inhibition of neointimal VSMC proliferation by PPAR ligands.
Discussion
PPAR activation by TZD has been demonstrated previously to inhibit VSMC proliferation and to prevent in-stent restenosis after angioplasty and atherosclerosis in early clinical trials.7–10 Understanding of the molecular mechanisms responsible for their beneficial efficacy in cardiovascular disease provides an important basis for the future development of TZD in the treatment of vascular diseases. VSMC proliferation is dependent on telomerase activity, and inhibition of telomerase has been demonstrated recently to inhibit neointima formation and atherosclerosis.17–19,23 In the present study, we investigated the regulation of telomerase by PPAR ligands, and we demonstrate that ligand-induced and constitutive activation of PPAR suppresses telomerase activity, providing a previously unrecognized mechanism by which PPAR ligands inhibit VSMC proliferation.
The absence of telomerase activity in most somatic cells has been associated with telomere shortening, reduced proliferation, and senescence.20–22 Specific inhibition of telomerase activity in VSMCs by targeting TERT expression has been demonstrated recently to inhibit VSMC proliferation, suggesting that telomerase is crucial for VSMC proliferation.23 Our results demonstrate that mitogen-induced telomerase activity in VSMCs is inhibited by PPAR ligands. Moreover, inhibition of VSMC proliferation by PPAR ligands was prevented in cells overexpressing TERT, characterizing telomerase as an important antiproliferative target for PPAR ligands. This concept is further supported by the close correlation between the efficacies of the used PPAR ligands to inhibit telomerase activity and their respective effects on VSMC proliferation. The finding that nTZDpa was the most potent inhibitor of telomerase activity among the used PPAR agonists is consistent with our characterization of this compound as a potent inhibitor of VSMC proliferation.15,24 The precise molecular mechanisms for the observed different efficacies to inhibit telomerase activity remain subject for future investigation. However, a potentially involved mechanism includes the concept of selective PPAR modulation in which binding of different PPAR ligands to the receptor results in a selective receptor conformation and subsequent differential corepressor release and coactivator recruitment.37,38 As a result of these differential interactions, each PPAR ligand receptor complex may lead to a differential pattern of gene expression.
Inhibition of telomerase activity by PPAR ligands occurred within 48 hours and, therefore, precedes inhibition of VSMC proliferation by PPAR ligands observed after 72 hours.39 These results indicate that inhibition of VSMC proliferation by targeting telomerase activity appears be independent of progressive telomere shortening, which occurs after multiple population doublings. This concept is further supported by several recent studies confirming that telomerase activity affects cell proliferation independent of its activity to extend telomeres.40,41 Interestingly, telomerase activity is regulated in a cell cycle–dependent manner and specific inhibition of telomerase induces cell cycle arrest, which is associated with an induction of the CDKI p27Kip1.42 The mechanism by which telomerase promotes cell cycle progression likely involves the G1S phase checkpoint because inhibition of telomerase activity attenuates Rb phosphorylation and suppresses E2F transactivity.43 Consistent with this concept, we recently identified inhibition of cell cycle progression through the CDKI–Rb-E2F pathway as a key mechanism responsible for the antiproliferative effects of PPAR ligands.24 However, it should be noted that specific inhibition of the cell cycle and phosphorylation of Rb has also been reported to inhibit telomerase activity,44 and further studies are necessary to determine the precise mechanisms by which telomerase regulates cell proliferation.
TERT has been recognized as the rate-limiting component of telomerase activity, and inhibition of TERT inhibits proliferation of VSMCs.23 To investigate potential regulatory mechanisms by which PPAR suppresses telomerase activity, we analyzed the regulation of TERT by PPAR ligands. PPAR activation suppressed TERT expression in VSMCs at the transcriptional level. The analysis of the TERT promoter did not reveal the presence of any putative PPRE response elements, indicating that the suppression of TERT transcription by PPAR ligands likely involves an indirect mechanism through the regulation of other transcription factors supporting TERT transcription. The TERT promoter contains two Ets-1 motifs at -23 and -18, and it has recently been suggested that Ets-1 confers the transcriptional activation of the TERT promoter in response to mitogenic stimulation.31 Moreover, Ets-1 mediates a variety of growth signals in neointimal VSMC proliferation45 and atherosclerosis,46 and PPAR ligands have been reported to inhibit Ets-1 expression.35 Our results from 5'-deletion analysis, site-directed mutagenesis of the Ets-1 binding sites and transactivation studies using wild-type and dominant-negative Ets-1 expression vectors indicated that PPAR ligands interfere with Ets-1–mediated activation of the TERT promoter. Moreover, ChIP assays revealed that PPAR ligands inhibit Ets-1 binding to the TERT promoter, which was mediated at least in part through an inhibition of Ets-1 expression. Based on these data, inhibition of telomerase activity by PPAR ligands is likely mediated through an inhibition of Ets-1–dependent transactivation of the TERT promoter.
In addition to a transcriptional regulation of TERT, recent studies have indicated that telomerase is regulated through post-translational modification of TERT involving Akt-dependent phosphorylation and subsequent nuclear translocation.36,47 We analyzed whether PPAR ligands interfere with the post-translational activation of TERT and observed that PPAR ligands inhibit mitogen-induced TERT nuclear translocation. Minamino et al recently demonstrated that proliferating VSMCs express TERT primarily in phosphorylated form, which is located predominantly in the nucleus, and that inhibition of TERT phosphorylation inhibits VSMC proliferation.17 Based on this evidence, inhibition of TERT nuclear translocation may provide an additional mechanism by which PPAR ligands regulate VSMC proliferation. In line with this concept are further studies demonstrating that PPAR ligands inhibit Akt activity in various cell types including VSMCs,48–50 providing a potential mechanism by which PPAR ligands inhibit TERT nuclear translocation. However, additional studies are warranted to determine whether inhibition of Akt-dependent TERT phosphorylation precedes the inhibition of TERT nuclear translocation by PPAR ligands.
In response to mechanical, biochemical, or immunological injury, a large number of growth factors are secreted resulting in VSMC proliferation and subsequent intimal hyperplasia, contributing to the pathogenesis of vascular disease. Consistent with the emerging role of telomerase in regulating VSMC proliferation,17,23 we observed a profound induction of telomerase activity two days after vascular injury. This induction of vascular telomerase activity was almost completely suppressed by treatment with the PPAR ligand PIO. Based on the recent observation that specific targeting of telomerase activity inhibits restenosis after angioplasty in a rat model18 and atherosclerosis in a murine model,19 suppression of vascular telomerase activity by PPAR ligands is likely to causally contribute to the beneficial vascular effects of PPAR ligands observed in animal models of atherosclerosis and restenosis.4–6
In summary, the present studies demonstrate that PPAR activation inhibits telomerase activity in VSMCs. Transcriptional suppression of Ets-1–dependent activation of the TERT promoter and inhibition of TERT nuclear translocation appear to be the key mechanisms by which PPAR ligands inhibit telomerase activity. Based on the previous observations that telomerase inhibition by targeting TERT expression results in an inhibition of VSMC proliferation,23 our studies provide evidence for a previously unrecognized mechanism by which PPAR activation inhibits VSMC proliferation and neointima formation in response to vascular injury. Therefore, suppression of telomerase activity by PPAR ligands may contribute at least in part to the beneficial cardiovascular effects of TZD PPAR ligands.
Acknowledgments
This study was supported in part by the American Diabetes Association (research award 1-06-RA-17 to D.B.) and the American Heart Association (scientist development grant 0435239N to D.B.). T. Nomiyama was supported by a fellowship grant from the Manpei Suzuki Diabetes Foundation, Japan.
Footnotes
J.P.B. is an employee of Merck Research Laboratories, and R.E.L. is an employee of Takeda Pharmaceuticals North America.
Original received September 5, 2005; resubmission received February 10, 2006; revised resubmission received March 7, 2006; accepted March 9, 2006.
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the Division of Endocrinology and Molecular Medicine (D.O., T. Nomiyama, T. Nakamachi, E.B.H., J.F.S., D.B.), University of Kentucky College of Medicine, Lexington
Department of Metabolic Disorders (J.P.B.), Merck Research Laboratories, Rahway, NJ
Takeda Pharmaceuticals North America (R.E.L.), Lincolnshire, Ill.
Abstract
Activation of the peroxisome proliferator-activated receptor (PPAR) , the molecular target for insulin sensitizing thiazolidinediones used in patients with type 2 diabetes, inhibits vascular smooth muscle cell (VSMC) proliferation and prevents atherosclerosis and neointima formation. Emerging evidence indicates that telomerase controls key cellular functions including replicative lifespan, differentiation, and cell proliferation. In the present study, we demonstrate that ligand-induced and constitutive PPAR activation inhibits telomerase activity in VSMCs. Telomerase reverse transcriptase (TERT) confers the catalytic activity of telomerase, and PPAR ligands inhibit TERT expression through a receptor-dependent suppression of the TERT promoter. 5'-deletion analysis, site-directed mutagenesis, and transactivation studies using overexpression of Ets-1 revealed that suppression of TERT transcription by PPAR is mediated through negative cross-talk with Ets-1–dependent transactivation of the TERT promoter. Chromatin immunoprecipitation assays further demonstrated that PPAR ligands inhibit Ets-1 binding to the TERT promoter, which is mediated at least in part through an inhibition of Ets-1 expression by PPAR ligands. In VSMCs overexpressing TERT, the efficacy of PPAR ligands to inhibit cell proliferation is lost, indicating that TERT constitutes an important molecular target for the antiproliferative effects of PPAR ligands. Finally, we demonstrate that telomerase activation during the proliferative response after vascular injury is effectively inhibited by PPAR ligands. These findings provide a previously unrecognized mechanism for the antiproliferative effects of PPAR ligands and support the concept that PPAR ligands may constitute a novel therapeutic approach for the treatment of proliferative cardiovascular diseases.
Key Words: telomerase peroxisome proliferator-activated receptor vascular smooth muscle cell proliferation
Introduction
Peroxisome proliferator-activated receptor (PPAR) is a ligand-activated transcription factor belonging to the nuclear hormone receptor superfamily.1 Synthetic ligands include thiazolidinedione (TZD) PPAR ligands, like rosiglitazone (RSG) or pioglitazone (PIO), which are clinically used as insulin sensitizers in patients with type 2 diabetes. TZD PPAR agonists not only improve insulin resistance in patients with type 2 diabetes but also exert a broad spectrum of pleiotropic vascular effects in vitro and in animal models.2 PPAR is expressed in macrophages, endothelial cells, and vascular smooth muscle cells (VSMCs) and regulates gene expression of key proteins involved in glucose/lipid metabolism, vascular inflammation, and proliferation.3 TZD PPAR ligands prevent the development of atherosclerosis and intimal hyperplasia after balloon injury in animal models.4–6 Early clinical studies further demonstrate that PPAR ligands prevent the progression of intima/media thickening used as sensitive measurement of atherosclerosis7,8 and the development of restenosis after coronary stent implantation.9,10 These beneficial cardiovascular effects are further supported by the recently published randomized controlled PROactive trial, which demonstrated a significant 16% reduction of the main secondary end points all-cause mortality, myocardial infarction, and stroke in type 2 diabetic patients treated with PIO.11
Proliferation of VSMCs is a critical process for the development of atherosclerosis and the primary mechanism resulting in the failure of procedures used to treat occlusive atherosclerotic diseases, such as postangioplasty restenosis, transplant vasculopathy, and vein bypass graft failure.12 PPAR ligands have been demonstrated to inhibit VSMC proliferation, an effect that may importantly contribute to their beneficial cardiovascular effects.13 The molecular mechanisms by which PPAR ligands inhibit VSMC proliferation involves an inhibition of cell cycle progression and phosphorylation of the retinoblastoma protein (Rb).14 Rb serves as a G1 gatekeeper, and Rb phosphorylation results in the release of the E2F transcription factor controlling S phase gene expression. The inhibition of Rb phosphorylation by PPAR is mediated by preventing degradation of the cyclin-dependent kinase inhibitor (CDKI) p27Kip1 and blocking cyclin A and D1 expression, essential for Rb phosphorylation and cell cycle progression.14,15
Telomerase controls key cellular functions including replicative lifespan, differentiation, and cell proliferation.16 Telomerase activity is required for VSMC proliferation, and disruption of telomerase activity reduces atherosclerosis and neointima formation, indicating that telomerase may serve as a novel pharmacological target for the treatment of vascular diseases.17–19 Telomeres consist of TTAGGG repeats at the end of chromosomes, and telomere length decreases with increasing cell division and senescence.20 Mice lacking telomerase function exhibit reduced tumorigenesis and decreased cell proliferation in highly proliferative organs, suggesting that telomerase activity is involved in the regulation of cell proliferation.21 Human telomerase consists of three subunits: the telomerase RNA (TR), which provides the template for telomere synthesis, the telomerase-associated protein 1 (TEP1) and the telomerase reverse transcriptase (TERT), which contains the catalytic activity of telomerase.22 TERT is the limiting factor for telomerase activity, and overexpression of TERT has been demonstrated to induce cell proliferation, whereas TERT antisense oligonucleotides inhibit VSMC proliferation.16,17,23 Thus, telomerase, and specifically TERT, may play an important role for the regulation of VSMC growth and neointima formation.
In the present study, we investigated the regulation of telomerase activity by ligand-induced and constitutive activation of PPAR. We report that activation of PPAR suppresses telomerase activity in VSMCs in vitro and in vivo. These studies identify telomerase as an important molecular target for the antiproliferative effects of PPAR and outline a previously unrecognized mechanism by which PPAR regulates VSMC proliferation.
Materials and Methods
Cell Culture
Rat aortic VSMCs were isolated and cultured as described previously.24 Cells were grown to 60% to 70% confluence and serum deprived in 0.1% FBS for at least 48 hours. Quiescent VSMCs were pretreated with the PPAR ligands RSG (1 to 10 μmol/L), PIO (1 to 10 μmol/L), or a non-TZD partial PPAR agonist25 (nTZDpa; 0.1 to 5 μmol/L) for 24 hours. After pretreatment, cells were stimulated in the presence of vehicle or the ligand with rat recombinant platelet-derived growth factor (PDGF)–BB (Sigma) and insulin (Eli Lilly & Co.) at the final concentration of 20 ng/mL and 1 μmol/L, respectively. The combination of PDGF and insulin as mitogen was used based on previous publications demonstrating that PDGF and insulin exert additive effects on VSMC proliferation.26 RSG was kindly provided by GlaxoSmithKline (King of Prussia, Pa). PIO was provided by Takeda Chemical Industry. nTZDpa25 was a generous gift from Merck (Rahway, NJ). For all data shown, individual experiments were repeated at least three times with different lots or preparation of cells.
Telomeric Repeat Amplification Protocol Assay
Telomerase activity was analyzed using a commercially available polymerase chain reaction (PCR)–based assay according to manufacturer instructions (TeloTAGGG Telomerase PCR ELISA Plus; Roche Applied Sciences). After stimulation, cells were harvested in provided lysis reagent, and whole-cell proteins (3 μg) were used for elongation/amplification. After amplification, PCR products were resolved on a 12% nondenaturing polyacrylamide gel and vacuum-transferred to nylon membranes. The characteristic 6-bp telomerase-specific ladder was detected using a Biotin-Luminescent Detection Kit (Roche Applied Sciences). In addition, telomerase activity was quantified by ELISA according to manufacturer instructions.
Adenoviral Constructs and Infections
The adenovirus overexpressing constitutively active PPAR was generated by fusing VP16 transactivation domain of the herpes simplex virus to the N terminus of PPAR1 as described previously.27 Overexpression of this mutant results in a potent ligand-independent activation of PPAR.28 Recombinant type 5 adenovirus-expressing green fluorescent protein (GFP) or the VP-16 transactivation domain were used as a control vector in all experiments.27 VSMCs were infected with 1, 25, or 100 plaque-forming units (PFU)/cell in DMEM containing 0.1% FBS for 24 hours. After further starvation for 24 hours, cells were stimulated with PDGF/insulin for 48 hours and analyzed for telomerase activity.
Northern Blotting
Isolation of total RNA and Northern blotting was performed as described previously.24 cDNA for TERT was kindly provided by Dr Tohru Kiyono (National Cancer Center Research Institute, Tokyo, Japan).29,30 Blots were cohybridized with cDNA encoding the constitutively expressed housekeeping gene 36B4 to assess equal loading of samples. Quantification was performed by densitometry and normalization to 36B4 mRNA expression.
Western Blot Analysis
Western blotting was performed as described recently.24 Primary antibodies were obtained from the following supplies: PPAR sc-7196 (Santa Cruz Biotechnology), VP16 3844-1 (BD Biosciences), TERT 582000 (EMD Biosciences), telomerase-associated protein 1 (TEP1) sc-13052 (Santa Cruz Biotechnology), and -actin ab6276 (Abcam Inc). Quantification of the Western blots was performed by densitometry and normalization to -actin.
Plasmids, Transient Transfections, and Luciferase Assay
The TERT promoter constructs were kindly provided by Dr Satoru Kyo (Kanazawa University School of Medicine, Japan).31 Wild-type and dominant-negative Ets-1 expression vectors were generously provided by Dr Naoyuki Taniguchi (Osaka University Medical School, Japan).32 The two side-by-side Ets-1 consensus motifs at -23 from the transcription initiation site were mutated from –23CTTCCTTTCCG–12 to –23CTTAATTTAAG–12 using the QuickChange II Site–directed mutagenesis kit (Stratagene) and the following primer pairs: forward 5'-CCAGCCCCTCCCCTTAATTTAAGCGGCCCCGCCCTCTC-3', reverse 5'-GAGAGGGCGGGGCCGCTTAAATTAAGGGGAGGGGCTGG -3'.
VSMCs were transfected for 8 hours with 1 μg of reporter DNA using LipofectAMINE (Invitrogen). After transfection, cells were cultured in 0.1% FBS for 24 hours followed by pretreatment with vehicle (dimethyl sulfoxide [DMSO]) or the indicated PPAR ligand for 24 hours. Subsequently, cells were stimulated with PDGF (20 ng/mL) and insulin (1 μmol/L), and luciferase activity was assayed after 24 hours using a Dual Luciferase Reporter Assay (Promega). Transfection efficiency was normalized to renilla luciferase activities generated by cotransfection with 10 ng/well pRL-CMV (Promega, Madison, WI). All experiments were repeated at least three times with different cell preparations.
Chromatin Immunoprecipitation Assays
Chromatin immunoprecipitation (ChIP) assays were performed using a ChIP assay kit (Upstate) according to manufacturer instructions. Briefly, quiescent VSMCs were pretreated with the indicated ligand for 24 hours and stimulated with PDGF/insulin. Cells were harvested after 3 hours, and soluble chromatin was prepared. Chromatin was immunoprecipitated with antibodies (2 μg) directed against Ets-1 (sc-111; Santa Cruz Biotechnology). Final DNA extractions were PCR amplified using primer pairs that cover the two Ets-1 consensus sequences at -23 in the TERT promoter as follows: forward 5'-CTTCCAGCTCCGCCTCCT-3'; reverse 5'-CAGCGCTGCCTGAAACTC -3'.
Confocal Microscopy
Quiescent VSMCs grown on plastic chamber-slides coated with poly-L-lysine were incubated with the PPAR ligand or vehicle (DMSO) for 24 hours before stimulation with PDGF/insulin for another 24 hours. Cells were fixed for 10 minutes with 10% formalin and incubated for 12 hours with the TERT antibody (582000; EMD Biosciences). An fluorescein isothiocyanate–conjugated secondary antibody and 4',6-diamidino-2-phenylindole staining was used for visualization. The data represent the mean of three independent experiments.
Cell Proliferation Assays
Small interfering RNA (siRNA) experiments were performed using the SMARTpool technology (Dharmacon RNA Technologies), which provides a mix of four different proprietary siRNAs specific for rat TERT. VSMCs were plated at a density of 1.0x105 cells on six-well plates and transfected with 250 nmol/L of TERT siRNA or scrambled siRNA using LipofectAMINE (Invitrogen). Experiments using TERT overexpression were performed by transfecting VSMCs with an expression vector overexpressing TERT (pCLXSN-TERT) or an empty control vector (pCLXSN; Imgenex Corp.). The pCLXSN-TERT expression vector under the control of a cytomegalovirus immediate-early long terminal repeat (CMV-IE/LTR) fusion promoter was kindly provided by Dr Tohru Kiyono (National Cancer Center Research Institute, Tokyo, Japan).29,30 After transfection, cells were maintained under serum deprivation for 24 hours, pretreated with vehicle or PIO (10 μmol/L) for an additional 24 hours, followed by mitogenic stimulation with PDGF (20 ng/mL) and insulin 1 μmol/L) for 72 hours. Cells were harvested and cell proliferation was measured by counting the cells using a hematocytometer. Experiments were performed in triplicate using three different preparations of rat aortic VSMCs.
Endovascular Femoral Artery Wire Injury
C57BL/6 male mice, 4 to 6 weeks of age, were purchased from Jackson Laboratory (Bar Harbor, Me). Mice were maintained in a temperature-controlled barrier facility with a 12-hour light/dark cycle and were given free access to food and water. Eight- to 10-week-old mice were treated with vehicle (n=8) or PIO (10 mg/kg per day; n=8) by daily intraperitoneal injection until euthanasia. After a treatment period of three days, endovascular wire injury was performed on the right femoral artery using a 0.25-mm SilverSpeed-10 hydrophilic guide wire (kindly provided by Sara Mueller, Micro Therapeutics Inc., Irvine, Calif), as described by Roque et al.33 Sham surgery without injury was performed on the contralateral left side. Mice were euthanized two days after injury, and the femoral arteries were isolated. Telomerase activities were analyzed by Southern blotting and ELISA as described above using 3 μg protein isolated from tissues homogenized in provided lysis buffer. All procedures on mice were approved by the institutional animal care and use committee at the University of Kentucky.
Statistical Analysis
ANOVA and paired or unpaired t test were performed for statistical analysis as appropriate. P values <0.05 were considered to be statistically significant. Results are expressed as mean±SEM.
Results
PPAR Ligands Inhibit Mitogen-Induced Telomerase Activity
To investigate the regulation of telomerase activity by PPAR ligands, quiescent VSMCs were treated with various PPAR agonists before stimulation with PDGF and insulin. As depicted in Figure 1A and 1B, mitogenic stimulation of serum-deprived VSMCs resulted in a substantial induction of VSMC telomerase activity, which was significantly inhibited by all used PPAR ligands. Whereas RSG inhibited telomerase activity at a dose of 10 μmol/L, PIO suppressed telomerase activity at a dose of 5 μmol/L and completely inhibited mitogen-induced telomerase activity at 10 μmol/L. The partial PPAR agonist nTZDpa completely inhibited telomerase activity at a dose of 5 μmol/L. The inhibition of telomerase activity was dose dependent over a concentration range known to inhibit VSMC proliferation14 and occurred in the absence of an effect of the different PPAR ligands on PDGF receptor expression (data not shown), suggesting a direct effect on telomerase activity.
Overexpression of Constitutively Active PPAR Inhibits Mitogen-Induced Telomerase Activity
To further determine whether the observed inhibition of telomerase activity by PPAR ligands is mediated through a receptor-dependent pathway, we used an adenoviral construct to overexpress a constitutively active PPAR mutant. As shown in Figure 2A, infection of VSMCs with adenovirus overexpressing constitutively active PPAR (Adx-CA-PPAR) resulted in marked dose-dependent overexpression of constitutively active PPAR protein. Consistent with our previous observation that expression of endogenous PPAR in VSMCs is almost exclusively detectable in the nuclear fraction, we observed faint expression of endogenous PPAR (55 kDa) in whole-cell extracts.34 Immunoblotting for VP16 revealed fast-migrating VP16 protein in VSMCs infected with Adx-VP16 and, because of the additional PPAR, slower-migrating VP16 in cells infected with Adx-CA-PPAR. Whereas telomerase activity was slightly increased in VSMCs infected with Adx-VP16, overexpression of constitutively active PPAR dose-dependently decreased mitogen-induced telomerase activity (Figure 2A and 2B). In concert, these data demonstrate that ligand-independent activation of PPAR suppresses telomerase activity in VSMCs, supporting the notion that the inhibition of telomerase activity by PPAR ligands is likely mediated through a PPAR-dependent mechanism.
PPAR Ligands Inhibit Mitogen-Induced TERT Expression
TERT constitutes the catalytic subunit of telomerase, and overexpression of TERT stimulates cell proliferation.16,23 To determine the mechanism by which PPAR ligands inhibit telomerase activity, we next analyzed the regulation of TERT expression by PPAR ligands. Stimulation of quiescent VSMCs with PDGF/insulin significantly induced TERT mRNA and protein expression (5.94±0.74 and 6.2±0.94-fold induction, respectively; P<0.01), which was dose-dependently inhibited by PIO and nTZDpa (Figure 3A and 3B). Expression of the TEP1 and the telomeric RNA component was not regulated in response to stimulation with PDGF/insulin or by treatment with the PPAR agonists (supplemental Figure IA and IB, available online at http://circres.ahajournals.org).
To further investigate whether the inhibition of mitogen-induced TERT expression by PPAR ligands is a result of decreased TERT promoter activity, VSMCs were transiently transfected with a 3.3-kb TERT promoter construct. As depicted in Figure 4A, PPAR agonists dose-dependently suppressed PDGF/insulin-induced TERT promoter activity. Similarly, cotransfection of the TERT promoter with an expression vector overexpressing a constitutively active PPAR mutant completely suppressed PDGF/insulin-induced TERT transcriptional activity in the absence of a PPAR ligand. In contrast, cotransfection of the TERT promoter with the empty vector or the VP16 transactivation domain vector exerted no effect to suppress mitogen-induced TERT promoter activity (Figure 4B). In concert, these findings indicate that PPAR ligands inhibit telomerase activity by suppressing mitogen-induced TERT transcription and further support the concept that the inhibition of TERT expression by PPAR ligands is mediated through a receptor-dependent mechanism.
PPAR Ligands Suppress TERT Transcription by Negative Cross-Talk With Ets-1–Dependent Transactivation of the TERT Promoter
The mechanism by which PPAR activation suppresses TERT transcriptional activation is unlikely to be direct because sequence analysis of the 5'-flanking region of the TERT promoter did not reveal the presence of PPAR response element (PPRE). To identify potential regulatory elements in the TERT promoter that support mitogen-induced TERT transcription and mediate the suppression of TERT promoter activity by PPAR, we used a series of 5'-deletion constructs of the TERT promoter (Figure 5A). PDGF/insulin-induced transcriptional activity of a -32 TERT promoter construct was completely suppressed by the PPAR ligand. In contrast, the +18 TERT promoter construct was not inducible by mitogens and exhibited a transcriptional activity only slightly above the background signal. The -32–bp proximal promoter that was determined as the minimal responsive element for PDGF/insulin contains two side-by-side consensus motifs for Ets-1 transcription factors at -23 from the transcription start site. Previous studies demonstrated that these sites are important for TERT transcription and that PPAR ligands interfere with Ets-1–mediated regulation of gene expression.31,35 Based on this evidence, we performed site-directed mutagenesis of both Ets-1 consensus sites, which resulted in a significant loss of basal and mitogen-induced TERT transcriptional activity (Figure 5B). Furthermore, PPAR ligands exerted no efficacy to suppress TERT promoter activity. To corroborate that PPAR activation suppresses TERT transcription through negative interference with Ets-1–dependent transactivation of the TERT promoter, we used expression vectors to overexpress wild-type and dominant-negative Ets-1. Cotransfection with an Ets-1 expression vector significantly induced basal TERT promoter activity (2.63±0.31-fold induction versus empty vector; P<0.05) and resulted in a complete loss of the effect of the PPAR agonist to suppress mitogen-induced TERT promoter activity (Figure 5C). Furthermore, cotransfection with a dominant-negative Ets-1 expression vector significantly repressed both basal and mitogen-induced TERT promoter activity. These data indicate that mitogens induce TERT promoter activation via a mechanism involving the Ets-1 site and that PPAR agonists repress mitogen-induced TERT expression by inhibiting Ets-1–dependent transactivation of the TERT promoter.
PPAR Activation Suppresses Ets-1 Binding to the -23 Consensus Motif in the TERT Promoter
To confirm that Ets-1 binds to the endogenous TERT promoter and that PPAR agonists interfere with this binding in vivo, we next performed ChIP assays. PCR amplification using primer pairs that cover the Ets-1 sites at -23 in the TERT promoter demonstrated that this TERT promoter sequence immunoprecipitated with Ets-1 after stimulation with PDGF and insulin. The time course experiments revealed maximal induction of Ets-1 binding to this site after a stimulation period of 3 hours (Figure 6A). Induction of Ets-1 binding to the proximal TERT promoter was substantially inhibited by both PIO (10 μmol/L) and nTZDpa (5 μmol/L; Figure 6B). To further determine potential mechanisms involved in the suppression of Ets-1 binding to the TERT promoter, we next analyzed whether PPAR ligands inhibit Ets-1 expression. Both PPAR ligands dose-dependently suppressed PDGF/insulin-induced Ets-1 mRNA expression in VSMCs (Figure 7). Therefore, inhibition of Ets-1 binding to the TERT promoter by PPAR agonists is mediated at least in part through an inhibition of Ets-1 expression.
PPAR Ligands Inhibit TERT Nuclear Translocation
Telomerase activity has further been reported to be regulated by post-translational modifications involving TERT nuclear translocation.36 Because the physiological role of telomerase in the synthesis of telomeric repeats occurs in the nucleus and nuclear translocation of TERT may play a role in the regulation of telomerase function, we performed experiments to determine the subcellular localization of TERT in VSMCs. As shown in Figure 8A, in quiescent VSMCs, most of the TERT protein was localized in the cytoplasm, whereas stimulation with PDGF/insulin resulted in a profound nuclear accumulation of TERT (Figure 8B). Interestingly, pretreatment with the PPAR agonist significantly attenuated mitogen-induced translocation of TERT into the nucleus. Together, these data demonstrate that PPAR ligands inhibit mitogen-induced TERT nuclear translocation, which may, in addition to the transcriptional suppression of TERT, play a role in the inhibition of telomerase activity by PPAR ligands in VSMCs.
Overexpression of TERT Reverses the Inhibitory Effect of PPAR Ligands on VSMC Proliferation
We next investigated whether TERT expression is required for mitogen-induced VSMC proliferation and whether inhibition of TERT contributes to the antiproliferative effects of PPAR agonists. TERT knockdown in VSMCs using siRNA (supplemental Figure IIA) resulted in a complete loss of mitogen-induced VSMC proliferation (supplemental Figure IIB). Consistent with recent observations by Cao et al,23 these data demonstrate that TERT expression is required for VSMC proliferation. To further determine whether the inhibition of TERT by PPAR ligands contributes to their antiproliferative efficacy, we next analyzed the effect of PIO on VSMC proliferation in cells overexpressing TERT. As depicted in Figure 9, PIO significantly inhibited mitogen-induced VSMC proliferation in cells transfected with control vector. Interestingly, overexpression of TERT resulted in a 3.1-fold induction of basal VSMC proliferation. However, in cells overexpressing TERT, treatment with the PPAR ligand resulted in a significant loss of the antiproliferative efficacy of the PPAR ligand. These data indicate that the inhibition of VSMC proliferation by PPAR ligands is at least in part mediated through an inhibition of TERT expression.
PPAR Ligands Inhibit Telomerase Activation During the Proliferative Response After Vascular Injury
To finally determine the regulation of telomerase activity during the proliferative response after vascular injury and to define whether the observed suppression of telomerase activity by PPAR ligands is applicable in vivo, we performed endovascular femoral artery denudation injuries in mice. Endothelial denudation in this model is associated with significant VSMC proliferation and subsequent neointima formation similar to that found after angioplasty in humans.33 Concomitant with the phenotypic shift from quiescent VSMCs resident in the uninjured vessel wall to proliferating smooth muscle cells present in the neointima, we observed a profound induction of telomerase activity in response to injury (Figure 10A and 10B). Time course studies demonstrated maximal telomerase activation 48 hours after injury (data not shown). Telomerase activation during the proliferative response was substantially suppressed in mice treated with PIO (84.8±7.9% inhibition versus vehicle-treated mice; P<0.001). Together, these results demonstrate that PPAR ligands suppress telomerase activation in response to vascular injury in vivo and indicate that telomerase may serve as a novel target for the inhibition of neointimal VSMC proliferation by PPAR ligands.
Discussion
PPAR activation by TZD has been demonstrated previously to inhibit VSMC proliferation and to prevent in-stent restenosis after angioplasty and atherosclerosis in early clinical trials.7–10 Understanding of the molecular mechanisms responsible for their beneficial efficacy in cardiovascular disease provides an important basis for the future development of TZD in the treatment of vascular diseases. VSMC proliferation is dependent on telomerase activity, and inhibition of telomerase has been demonstrated recently to inhibit neointima formation and atherosclerosis.17–19,23 In the present study, we investigated the regulation of telomerase by PPAR ligands, and we demonstrate that ligand-induced and constitutive activation of PPAR suppresses telomerase activity, providing a previously unrecognized mechanism by which PPAR ligands inhibit VSMC proliferation.
The absence of telomerase activity in most somatic cells has been associated with telomere shortening, reduced proliferation, and senescence.20–22 Specific inhibition of telomerase activity in VSMCs by targeting TERT expression has been demonstrated recently to inhibit VSMC proliferation, suggesting that telomerase is crucial for VSMC proliferation.23 Our results demonstrate that mitogen-induced telomerase activity in VSMCs is inhibited by PPAR ligands. Moreover, inhibition of VSMC proliferation by PPAR ligands was prevented in cells overexpressing TERT, characterizing telomerase as an important antiproliferative target for PPAR ligands. This concept is further supported by the close correlation between the efficacies of the used PPAR ligands to inhibit telomerase activity and their respective effects on VSMC proliferation. The finding that nTZDpa was the most potent inhibitor of telomerase activity among the used PPAR agonists is consistent with our characterization of this compound as a potent inhibitor of VSMC proliferation.15,24 The precise molecular mechanisms for the observed different efficacies to inhibit telomerase activity remain subject for future investigation. However, a potentially involved mechanism includes the concept of selective PPAR modulation in which binding of different PPAR ligands to the receptor results in a selective receptor conformation and subsequent differential corepressor release and coactivator recruitment.37,38 As a result of these differential interactions, each PPAR ligand receptor complex may lead to a differential pattern of gene expression.
Inhibition of telomerase activity by PPAR ligands occurred within 48 hours and, therefore, precedes inhibition of VSMC proliferation by PPAR ligands observed after 72 hours.39 These results indicate that inhibition of VSMC proliferation by targeting telomerase activity appears be independent of progressive telomere shortening, which occurs after multiple population doublings. This concept is further supported by several recent studies confirming that telomerase activity affects cell proliferation independent of its activity to extend telomeres.40,41 Interestingly, telomerase activity is regulated in a cell cycle–dependent manner and specific inhibition of telomerase induces cell cycle arrest, which is associated with an induction of the CDKI p27Kip1.42 The mechanism by which telomerase promotes cell cycle progression likely involves the G1S phase checkpoint because inhibition of telomerase activity attenuates Rb phosphorylation and suppresses E2F transactivity.43 Consistent with this concept, we recently identified inhibition of cell cycle progression through the CDKI–Rb-E2F pathway as a key mechanism responsible for the antiproliferative effects of PPAR ligands.24 However, it should be noted that specific inhibition of the cell cycle and phosphorylation of Rb has also been reported to inhibit telomerase activity,44 and further studies are necessary to determine the precise mechanisms by which telomerase regulates cell proliferation.
TERT has been recognized as the rate-limiting component of telomerase activity, and inhibition of TERT inhibits proliferation of VSMCs.23 To investigate potential regulatory mechanisms by which PPAR suppresses telomerase activity, we analyzed the regulation of TERT by PPAR ligands. PPAR activation suppressed TERT expression in VSMCs at the transcriptional level. The analysis of the TERT promoter did not reveal the presence of any putative PPRE response elements, indicating that the suppression of TERT transcription by PPAR ligands likely involves an indirect mechanism through the regulation of other transcription factors supporting TERT transcription. The TERT promoter contains two Ets-1 motifs at -23 and -18, and it has recently been suggested that Ets-1 confers the transcriptional activation of the TERT promoter in response to mitogenic stimulation.31 Moreover, Ets-1 mediates a variety of growth signals in neointimal VSMC proliferation45 and atherosclerosis,46 and PPAR ligands have been reported to inhibit Ets-1 expression.35 Our results from 5'-deletion analysis, site-directed mutagenesis of the Ets-1 binding sites and transactivation studies using wild-type and dominant-negative Ets-1 expression vectors indicated that PPAR ligands interfere with Ets-1–mediated activation of the TERT promoter. Moreover, ChIP assays revealed that PPAR ligands inhibit Ets-1 binding to the TERT promoter, which was mediated at least in part through an inhibition of Ets-1 expression. Based on these data, inhibition of telomerase activity by PPAR ligands is likely mediated through an inhibition of Ets-1–dependent transactivation of the TERT promoter.
In addition to a transcriptional regulation of TERT, recent studies have indicated that telomerase is regulated through post-translational modification of TERT involving Akt-dependent phosphorylation and subsequent nuclear translocation.36,47 We analyzed whether PPAR ligands interfere with the post-translational activation of TERT and observed that PPAR ligands inhibit mitogen-induced TERT nuclear translocation. Minamino et al recently demonstrated that proliferating VSMCs express TERT primarily in phosphorylated form, which is located predominantly in the nucleus, and that inhibition of TERT phosphorylation inhibits VSMC proliferation.17 Based on this evidence, inhibition of TERT nuclear translocation may provide an additional mechanism by which PPAR ligands regulate VSMC proliferation. In line with this concept are further studies demonstrating that PPAR ligands inhibit Akt activity in various cell types including VSMCs,48–50 providing a potential mechanism by which PPAR ligands inhibit TERT nuclear translocation. However, additional studies are warranted to determine whether inhibition of Akt-dependent TERT phosphorylation precedes the inhibition of TERT nuclear translocation by PPAR ligands.
In response to mechanical, biochemical, or immunological injury, a large number of growth factors are secreted resulting in VSMC proliferation and subsequent intimal hyperplasia, contributing to the pathogenesis of vascular disease. Consistent with the emerging role of telomerase in regulating VSMC proliferation,17,23 we observed a profound induction of telomerase activity two days after vascular injury. This induction of vascular telomerase activity was almost completely suppressed by treatment with the PPAR ligand PIO. Based on the recent observation that specific targeting of telomerase activity inhibits restenosis after angioplasty in a rat model18 and atherosclerosis in a murine model,19 suppression of vascular telomerase activity by PPAR ligands is likely to causally contribute to the beneficial vascular effects of PPAR ligands observed in animal models of atherosclerosis and restenosis.4–6
In summary, the present studies demonstrate that PPAR activation inhibits telomerase activity in VSMCs. Transcriptional suppression of Ets-1–dependent activation of the TERT promoter and inhibition of TERT nuclear translocation appear to be the key mechanisms by which PPAR ligands inhibit telomerase activity. Based on the previous observations that telomerase inhibition by targeting TERT expression results in an inhibition of VSMC proliferation,23 our studies provide evidence for a previously unrecognized mechanism by which PPAR activation inhibits VSMC proliferation and neointima formation in response to vascular injury. Therefore, suppression of telomerase activity by PPAR ligands may contribute at least in part to the beneficial cardiovascular effects of TZD PPAR ligands.
Acknowledgments
This study was supported in part by the American Diabetes Association (research award 1-06-RA-17 to D.B.) and the American Heart Association (scientist development grant 0435239N to D.B.). T. Nomiyama was supported by a fellowship grant from the Manpei Suzuki Diabetes Foundation, Japan.
Footnotes
J.P.B. is an employee of Merck Research Laboratories, and R.E.L. is an employee of Takeda Pharmaceuticals North America.
Original received September 5, 2005; resubmission received February 10, 2006; revised resubmission received March 7, 2006; accepted March 9, 2006.
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