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(Circulation Research. 2003;93:e38.)
© 2003 American Heart Association, Inc.

UltraRapid Communication

Regulation of the Growth Arrest and DNA Damage-Inducible Gene 45 (GADD45) by Peroxisome Proliferator-Activated Receptor in Vascular Smooth Muscle Cells

Dennis Bruemmer, Fen Yin, Joey Liu, Joel P. Berger, Toshiyuki Sakai, Florian Blaschke, Eckart Fleck, Andre J. Van Herle, Barry M. Forman, Ronald E. Law

From the Division of Endocrinology, Diabetes and Hypertension and The Gonda (Goldschmied) Diabetes Center (D.B., F.Y., J.L., F.B., A.J.V.H., R.E.L.), David Geffen School of Medicine, University of California, Los Angeles, Calif; Department of Medicine/Cardiology (D.B., F.B., E.F.), German Heart Institute, Berlin, Germany; Merck Research Laboratories (J.P.B.), Rahway, NJ; Department of Preventive Medicine (T.S.), Kyoto Prefectural University of Medicine, Kawaramachi-Hiokoji, Kamigyo-ku, Kyoto, Japan; Division of Molecular Medicine and The Gonda Diabetes and Genetic Research Center, and the Department of Diabetes, Endocrinology, and Metabolism (B.M.F.), The City of Hope National Medical Center, Beckman Research Institute, Duarte, Calif.

Correspondence to Ronald E. Law, PhD, Division of Endocrinology, Diabetes and Hypertension, David Geffen School of Medicine, University of California, Los Angeles, CA 90095. E-mail rlaw{at}mednet.ucla.edu

	Abstract

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Peroxisome proliferator-activated receptor (PPAR) is activated by thiazolidinediones (TZDs), widely used as insulin-sensitizing agents for the treatment of type 2 diabetes. TZDs have been shown to induce apoptosis in a variety of mammalian cells. In vascular smooth muscle cells (VSMCs), proliferation and apoptosis may be competing processes during the formation of restenotic and atherosclerotic lesions. The precise molecular mechanisms by which TZDs induce apoptosis in VSMCs, however, remain unclear. In the present study, we demonstrate that the TZDs rosiglitazone (RSG), troglitazone (TRO), and a novel non-TZD partial PPAR agonist (nTZDpa) induce caspase-mediated apoptosis of human coronary VSMCs. Induction of VSMC apoptosis correlated closely with an upregulation of growth arrest and DNA damage-inducible gene 45 (GADD45) mRNA expression and transcription, a well-recognized modulator of cell cycle arrest and apoptosis. Using adenoviral-mediated overexpression of a constitutively active PPAR mutant and the irreversible PPAR antagonist GW9662, we provide evidence that PPAR ligands induce caspase-mediated apoptosis and GADD45 expression through a receptor-dependent pathway. Deletion analysis of the GADD45 promoter revealed that a 153-bp region between -234 and -81 bp proximal to the transcription start site, containing an Oct-1 element, was crucial for the PPAR ligand–mediated induction of the GADD45 promoter. PPAR activation induced Oct-1 protein expression and DNA binding and stimulated activity of a reporter plasmid driven by multiple Oct-1 elements. These findings suggest that activation of PPAR can lead to apoptosis and growth arrest in VSMCs, at least in part, by inducing Oct-1–mediated transcription of GADD45. The full text of this article is available online at http://www.circresaha.org.

Key Words: peroxisome proliferator-activated receptor • apoptosis • vascular smooth muscle • octamer transcription factor

	Introduction

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The pathogenesis of atherosclerosis and postangioplasty restenosis involves an excessive proliferation of vascular smooth muscle cells (VSMCs).^1–3 Although many studies have focused on alterations in the regulation of cell growth as a fundamental feature during atherogenesis and neointimal hyperplasia after percutaneous coronary revascularization, it is becoming increasingly evident that perturbations in the regulation of cell death may be of equal importance.⁴

Peroxisome proliferator-activated receptor (PPAR) is a transcription factor belonging to the nuclear hormone receptor gene super-family.⁵ PPAR is expressed in VSMCs and prominently upregulated in response to mechanical injury of the arterial wall.^6,7 PPAR heterodimerizes with the retinoid X receptor (RXR) and binds to specific response elements termed peroxisome proliferator–response elements (PPRE).⁸ PPAR functions as a ligand-dependent transcription factor, which on ligand binding undergoes a conformational change disrupting a corepressor complex and leading to coactivator recruitment resulting in the transcriptional activation of target genes.^9,10

Thiazolidinediones (TZD) are synthetic ligands for PPAR and are commonly used as insulin-sensitizing agents in the treatment of type 2 diabetes.¹¹ Pharmacological activation of PPAR by TZD and non-TZD PPAR ligands inhibits VSMC proliferation at the G₁S phase transition of the cell cycle by suppressing mitogen-induced phosphorylation of the retinoblastoma protein, which is required for E2F release and expression of minichromosome maintenance proteins that are essential regulators of the DNA replication.^12,13 This inhibition of VSMC proliferation by PPAR ligands correlates with a potent suppression of neointima formation after balloon-injury in rat models of insulin resistance, as well as in animals with normal insulin sensitivity in vivo.^14–17 In addition, recent studies have shown that TZDs not only inhibit cell growth, but they can also induce apoptosis in diverse cell types; including endothelial cells,¹⁸ VSMCs,^19,20 monocyte-derived macrophages,²¹ and various human cancer cells.^22–25

The growth arrest and DNA damage-inducible (GADD) gene GADD45 is a member of a group of genes induced by agents that damage DNA and/or cause growth arrest.²⁶ Increased GADD45 gene expression has been detected in many mammalian cell types and has been implicated in terminal differentiation,²⁷ growth suppression,^28,29 and apoptosis.^30,31 Although the exact function of GADD45 remains unclear, evidence has emerged that GADD45 is a cell cycle–regulated nuclear protein that reaches maximal levels in the G₁ phase of the cycle.³² Through its association with Cdc2, GADD45 disrupts the interactions of Cdc2 with cyclin B1 and, thus, may induce G₂/M arrest.³³ The GADD genes, therefore, may represent a unique target for drugs that induce cell cycle arrest, apoptosis, and differentiation such as PPAR ligands.

The molecular mechanisms by which TZDs induce apoptosis in VSMCs and whether they involve a PPAR-dependent pathway remain unclear. In the present study, we demonstrate that PPAR ligands induce caspase-mediated apoptosis of human coronary VSMCs. The ability of these PPAR ligands to induce VSMC apoptosis correlated with an induction of GADD45 expression at the transcriptional level. By using adenoviral-mediated overexpression of a constitutively active form of PPAR, we provide evidence that the mechanism of action by which TZDs and non-TZDs induce apoptosis and GADD45 transcription occurs through a PPAR-dependent pathway. The PPAR ligand-responsive element in the GADD45 promoter was mapped by promoter-deletion studies to a 153-bp region between -234 and -81 bp relative to the transcription start site that harbors an Oct-1 motif. PPAR activation induced Oct-1 protein expression and increased its DNA binding and transcriptional activity. These results, therefore, identify the attenuation of Oct-1–mediated induction of GADD45 by PPAR as a novel mechanism for regulating VSMC growth and survival.

	Materials and Methods

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Cell Culture
Primary human coronary artery vascular smooth muscle cells (HCSMCs, Clonetics coronary artery smooth muscle cell systems) were commercially obtained from Cambrex Bio Science Walkersville, Inc (East Rutherford, NJ). Early passage (fourth to ninth) cells were grown to 70% to 80% confluence in SmBM-2 medium containing 5% FBS, 2 ng/mL human basic fibroblast growth factor, 0.5 ng/mL human epidermal growth factor, 50 µg/mL gentamycin, and 5 µg/mL bovine insulin. Growing cells were treated with each compound for the indicated time. Troglitazone (TRO) was kindly provided by Parke-Davis and rosiglitazone by Smith Kline Beecham. The compound nTZDpa (1-(p-chlorobenzyl)-5-chloro-3-phenylthiobenzyl-2-yl carboxylic acid) was kindly provided by Dr David E. Moller (Merck Research Laboratories, Rahway, NJ),³⁴ and the irreversible PPAR antagonist GW9662 was a kind gift from Dr Timothy M. Willson (Glaxo Smith Kline, Research Triangle Park, NC).³⁵ For all data shown, individual experiments were performed from separate lots of HCSMCs.

Annexin V Flow Cytometry
HCSMCs were treated with the PPAR ligands for 48 hours or pretreated with 20 µmol/L of the pan-caspase inhibitor Z-VAD-FMK (R&D Systems) for 3 hours before stimulation with 30 µmol/L of the PPAR ligands or vehicle (DMSO). All experiments were performing with exponentially growing cells in SmBM-2 containing growth factors and 5% FBS. Annexin V flow cytometry was performed using a commercially available annexin V fluorescein isothiocyanate (FITC) assay (Oncogene Research Products). Briefly, cells were washed with PBS and trypsinized with 0.125% trypsin until cells appeared to detach by microscopic evaluation. Cells were then released by firm tapping, resuspended in media to 1x10⁶ cells/mL, and incubated for 30 minutes with the annexin V-FITC–conjugated antibody and stained with propidium iodide. Annexin V-FITC binding was analyzed by flow cytometry collecting the fluorescence of 10 000 cells using a FACScan (Becton Dickinson) according to the manufacturer’s instructions. The total number of apoptotic cells was expressed as fold induction of annexin V-FITC–positive cells compared with vehicle (DMSO)-treated cells.

Western Immunoblotting
Cells were harvested at the indicated time after treatment with the PPAR ligand and sonicated in solubilization buffer (20 mmol/L Tris-HCl, pH 7.5; 150 mmol/L NaCl; 1 mmol/L EDTA; 1 mmol/L EGTA, 1% Triton X-100; 2.5 mmol/L sodium pyrophosphate; 1 mmol/L sodium vanadate; 10 µg/mL each aprotinin and leupeptin; 1 mmol/L phenylmethylsulfonyl fluoride). Whole cell lysates were cleared by centrifugation and protein concentrations were determined by Lowry assay. Nuclear extracts were isolated using the NuCLEAR extraction kit according to the manufacturers instructions (Sigma). Cell lysates containing equal amounts of protein were resolved by SDS-polyacrylamide gel electrophoresis. Protein was transferred to a nitrocellulose membrane (Hybond, Amersham Pharmacia Biotech). After blocking in 20 mmol/L Tris-HCl (pH 7.6) containing 150 mmol/L NaCl, 0.1% Tween-20, and 5% (wt/vol) non-fat dry milk, blots were incubated with specific antibodies for caspase-3 (Cell Signaling Technology), PPAR, or Oct-1 (Santa Cruz Biotechnology) and cohybridized with ß-actin (Santa Cruz Biotechnology) to monitor equivalent loading in different lanes. Immunoreactive bands were visualized by incubation with peroxidase-conjugated anti-mouse IgG antibody (Amersham Pharmacia Biotech). The antigen-antibody complexes were detected using ECL (Amersham Pharmacia Biotech). Quantification of the Western blots was performed by densitometry.

Isolation of RNA and Northern Blotting
Total RNA was isolated using TRIzol reagent (Life Technologies) as described by the manufacturer. Fifteen micrograms of total RNA were denatured in formamide/formaldehyde and electrophoresed through 1% formaldehyde-containing agarose gels. After electrophoresis, RNA was transferred to nylon membrane (Hybond N+, Amersham Pharmacia Biotech) by capillary blotting and fixed by UV cross-linking. Hybridization was performed using PerfectHyb Plus hybridization buffer (Sigma) as directed. cDNA for GADD45 was obtained from ATCC (No. 1150356, ATCC). The cDNAs used in the hybridization were radiolabeled with [-³²P] dCTP (ICN) using Rediprime II random prime labeling system (Amersham Pharmacia Biotech). Blots were cohybridized with cDNA encoding the constitutively expressed housekeeping gene Chinese hamster ovary gene B (CHOB) to assess equal loading of samples.

Adenoviral Infection of HCSMCs
To generate constitutively active PPAR the VP16 transactivation domain of the herpes simplex virus (HSV) was fused to the N-terminus of PPAR1 as previously described.³⁶ Recombinant type 5 adenovirus overexpressing this constitutively active PPAR mutant was generated using the Adeno X Expression System (Clontech Inc) and designated as Adx-CA-PPAR. Adenovirus overexpressing only the VP16 transactivation domain (pTet/VP16, obtained from Clontech Inc) or green fluorescent protein (GFP) were generated similarly and used as control (Adx-GFP, Adx-VP16) in all experiments. HCSMCs were infected with 1, 30, or 100 plaque forming units (PFU)/cell adenovirus in SmBM-2 containing 5% FBS for 48 hours.

RNA Stability Assay
HCSMCs grown for 48 hours in the presence of 30 µmol/L TRO or vehicle (DMSO) were treated with actinomycin D (5 µg/mL), and at different time intervals, cells were collected and RNA was isolated. Total RNA expression was examined by Northern blot analysis using radiolabeled probes for GADD45 or CHOB. Hybridization signals were quantified by densitometry, normalized to CHOB expression, and mRNA half-life (T_1/2) was calculated using the equation C=C₀e^Kdt, where C is the remaining mRNA level at time point t, C₀ is the amount of RNA at zero time of actinomycin D addition, and K_d is the mRNA decay constant.

Plasmids and Transient Transfection
The GADD45 luciferase reporter constructs and the construction of the IL-8OCx6, IL-8OmCx6, and IL-8mOCx6 have been previously described.³⁷ One microgram DNA was transfected using LipofectAMINE 2000 (Invitrogen). Twenty-four hours after transfection, cells were treated with the indicated PPAR ligand. Luciferase activity was assayed using a Dual Luciferase Reporter Assay System (Promega) according to the manufacturer’s instructions. Transfection efficiency was adjusted by normalizing firefly luciferase activities to the renilla luciferase activities generated by cotransfection with 10 ng pRL-CMV (Promega). All experiments were repeated at least three times with different cell preparations.

Electrophoretic Mobility Shift Assay
For electrophoretic mobility shift assays (EMSA), a radioactive Oct-1 consensus oligonucleotide (5'-TGTCGAATGCAAATCACT-AGAA-3', Promega), labeled with T4 polynucleotide kinase and [-³²P] ATP (ICN) was incubated with 4 µg of nuclear extract for 20 minutes in gel shift binding buffer (5 mmol/L MgCl₂, 2.5 mmol/L EDTA, 2.5 mmol/L DTT, 250 mmol/L NaCl, 50 mmol/L Tris [pH 7.5], and 0.25 µg/µL poly dIdC in 20% glycerol) and analyzed by electrophoresis using a 4% nondenaturing acrylamide gel in 0.5x TBE buffer. Gels were dried, and autoradiography was performed. For competitive and noncompetitive oligonucleotide assays 100-fold labeled double-stranded SP-1 (Promega) or unlabeled double-stranded Oct-1 were added to the nuclear extracts 20 minutes before the addition of the radiolabeled probe. For supershift experiments, 4 µg of nuclear extract were incubated with 0.5 µg of an Oct-1 antibody (Santa Cruz Biotechnology) for 20 minutes after incubation of the radiolabeled probe with nuclear proteins.

Statistics
Data were expressed as mean±SEM. Statistical significance was determined using the unpaired Student’s t test or ANOVA when comparing multiple groups. Values of P<0.05 were considered as statistically significant.

	Results

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PPAR Ligands Induce Apoptosis of Human Coronary VSMCs
To determine whether PPAR activation could induce VSMC apoptosis, human coronary VSMCs were treated with various PPAR ligands for 48 hours. Quantitative analysis of apoptosis was performed by using an annexin V-FITC–conjugated antibody allowing detection of apoptotic cells by flow cytometry. As shown in Figure 1A, treatment of HCSMCs with RSG, TRO, and nTZDpa induced apoptosis as detected by annexin V-FITC binding in a dose-dependent fashion. Quantification of annexin V-FITC–positive apoptotic cells expressed as fold increase compared with vehicle controls revealed a 2.28±0.39-fold induction for RSG, a 4.04±0.61-fold induction for TRO, and a 4.68±0.44-fold induction for nTZDpa (all at 30 µmol/L, n=4; P<0.05). The effect of the PPAR ligands on HCSMC apoptosis was more pronounced in serum-starved HCSMCs and partially suppressed by increasing the concentration of FBS to 10%, consistent with the survival-promoting activity of the various growth factors present in serum.

View larger version (35K): [in this window] [in a new window]   Figure 1. PPAR ligands induce apoptosis in vascular smooth muscle cells. A, Human coronary VSMCs were cultured in the presence of the indicated PPAR ligand (1, 10, and 30 µmol/L) for 48 hours or pretreated with the pan-caspase inhibitor Z-VAD-FMK (Z-VAD, 20 µmol/L) 3 hours before stimulation with 30 µmol/L of the indicated PPAR ligand. Apoptosis was analyzed by annexin V-FITC flow cytometry and expressed as the fold induction of the vehicle (DMSO). Data are presented as mean±SEM of 3 independently performed experiments (*P<0.05 vs vehicle; #P<0.05 vs stimulation with 30 µmol/L of the indicated PPAR ligands alone). B, Human coronary VSMCs were stimulated with 30 µmol/L of indicated PPAR ligand. Forty-eight hours after stimulation, caspase-3 protein expression was measured by Western immunoblotting using a monoclonal caspase-3 antibody. To assess loading variability, immunoblots were cohybridized with a specific antibody for ß-actin. Autoradiogram shown is representative of 3 independently performed experiments.

Because cells undergoing apoptosis execute a death program by activating cysteine proteases of the caspase family and to identify the pathway by which activation of PPAR induces apoptosis, HCSMCs were pretreated with the pan-caspase inhibitor Z-VAD-FMK that potently inhibits caspase enzymes as key executioners of apoptosis. Pretreatment of cells with 20 µmol/L Z-VAD-FMK 3 hours before stimulation with 30 µmol/L of the PPAR ligand completely prevented RSG, TRO, and nTZDpa-induced apoptosis of HCSMCs. In addition, Western blot analysis (Figure 1B) revealed increased levels of the enzymatically active 17- and 12-kDa proteolytic cleavage products of the inactive 32-kDa caspase-3 precursor in HCSMCs treated for 48 hours with either RSG, TRO, or nTZDpa. In concert, these data indicate that PPAR ligands induce apoptosis of HCSMCs through a mechanism involving activation of caspases.

GADD45 Expression Is Induced by PPAR Ligands
GADD45 is prominently induced in growth-arrested cells^28,29 and in those undergoing apoptosis.^30,31 Because PPAR activation has been shown to inhibit cell proliferation and/or to induce apoptosis in a wide variety of mammalian cells, we hypothesized that upregulation of GADD45 by PPAR ligands may be involved in their proapoptotic and antiproliferative activities. Northern blot analysis for GADD45 expression was performed on RNA extracted from HCSMCs treated with TRO for various time-points. Baseline levels of GADD45 mRNA in growing HCSMCs were low, but increased substantially after treatment with TRO (30 µmol/L), reaching maximal levels after 48 hours (Figure 2A). To determine dose-dependent effects of other PPAR ligands on GADD45 mRNA expression, HCSMCs were cultured in the presence of 1 to 30 µmol/L RSG, TRO, and nTZDpa for 48 hours. Data presented in Figure 2B demonstrate substantial dose-dependent induction of GADD45 mRNA expression after treatment with RSG, TRO, and nTZDpa (RSG 3.2±0.41-, TRO 4.9±0.75-, nTZDpa 3.4±0.53-fold increase at 30 µmol/L, n=3; P<0.05). These data suggest that ligand-dependent activation of PPAR can lead to growth arrest and induction of apoptosis in vascular smooth muscle cells, at least in part, by inducing GADD45 mRNA expression.

View larger version (32K): [in this window] [in a new window]   Figure 2. PPAR ligands induce GADD45 mRNA expression in VSMCs. A, Human coronary VSMCs were cultured in the presence of 5% FBS and growth factors and stimulated with 30 µmol/L TRO for the indicated time points. B, Human coronary VSMCs were treated with the indicated concentrations of RSG, TRO, or nTZDpa for 48 hours. RNA was analyzed for GADD45 mRNA expression by Northern blotting. Blots were cohybridized with CHOB, a constitutively expressed housekeeping gene encoding a ribosomal protein as internal control. Autoradiogram shown is representative of 3 independently performed experiments. Data are expressed as fold induction over vehicle-treated cells and presented as mean±SEM (*P<0.05 vs vehicle).

Constitutively Active PPAR Induces Caspase-Mediated Apoptosis and GADD45 mRNA in HCSMCs
The precise mechanism by which PPAR ligands exert their antiproliferative and proapoptotic effects and whether it involves PPAR mediated transactivation of target genes is not known. To determine whether activation of PPAR induces apoptosis and GADD45 expression, we used a constitutively active form of PPAR by fusing the herpes virus VP16 transactivation domain to the wild-type PPAR1 cDNA.³⁶ This constitutively active PPAR was subcloned into recombinant adenovirus to permit ubiquitous expression of this engineered nuclear receptor. As shown in Figure 3, we observed faint expression of endogenous PPAR (55 kDa) in whole-cell extracts of uninfected HCSMCs or cells infected with control Adx-VP16 or Adx-GFP. This finding is consistent with our previous observation that expression of endogenous PPAR protein in VSMCs was detectable only in nuclear fractions, not in whole-cell lysates.¹⁴ Infection of HCSMCs with Adx-CA-PPAR resulted in marked dose-dependent overexpression of constitutively active PPAR protein, which migrated slower than endogenous PPAR at 70 kDa due to the additional VP16 transactivation domain engrafted at the N-terminus. Similarly, immunoblotting for VP16 revealed fast-migrating dose-dependent expression of VP16 protein in HCSMCs infected with Adx-VP16 and slower migrating (due to the additional PPAR) VP16 in cells that were infected with Adx-CA-PPAR. Infection of HCSMCs with the control Adx-GFP or Adx-VP16 had no effect on annexin V binding, caspase-3 cleavage products, or GADD45 mRNA expression. In contrast, annexin V binding (4.57±0.34-fold induction at 100 PFU/cell, n=3; P<0.05), caspase-3 cleavage products, and GADD45 mRNA levels (3.3±0.51-fold induction at 100 PFU/cell, n=3; P<0.05) dose-dependently increased in HCSMCs overexpressing constitutively active PPAR. In concert, these data demonstrate that ligand-independent activation of PPAR specifically induces caspase-mediated apoptosis of HCSMC and GADD45 mRNA expression and provides evidence that the induction of GADD45 by PPAR ligands is mediated through a PPAR-dependent mechanism.

View larger version (51K): [in this window] [in a new window]   Figure 3. Constitutive activation of PPAR induces caspase-mediated apoptosis and GADD45 RNA expression. Human coronary VSMCs were infected for 48 hours with 1, 30, or 100 PFU/cell recombinant type 5 adenovirus overexpressing constitutively active PPAR (Adx-CA-PPAR). Uninfected cells (C) and infection with recombinant adenovirus expressing the GFP gene (Adx-GFP, 100 PFU/cell) or the VP16 transactivation domain from the herpes simplex virus (Adx-VP-16; 1, 30, or 100 PFU/cell) were used as controls. Apoptosis was analyzed by annexin V-FITC flow cytometry and expressed as fold induction over cells infected with 100 PFU/cell Adx-GFP. Western immunoblotting was performed from whole cell proteins (50 µg) for caspase-3, PPAR, and VP16 expression. To assess equivalent loading, immunoblots were cohybridized with a specific antibody for ß-actin. GADD45 RNA expression was analyzed by Northern blotting as described in Figure 2. Autoradiograms shown are representative of 3 independently performed experiments. Data are expressed as fold induction over vehicle-treated cells and presented as mean±SEM (*P<0.05 vs vehicle).

PPAR Ligands Induce GADD45 Transcription
The PPAR-mediated induction of GADD45 mRNA could result from either an effect to increase transcription and/or promote enhanced mRNA stability. GADD45 mRNA levels have been shown to be regulated by both transcriptional activation^37–39 and posttranscriptional mRNA stabilization,^40,41 depending on the cell type and specific inducer. We next investigated whether treatment with TRO affects stabilization of GADD45 mRNA. HCSMCs were treated for 48 hours with either DMSO or TRO (30 µmol/L) before actinomycin D was added at a final concentration of 5 µg/mL to suppress further transcription during the actinomycin D chase. As shown in Figure 4, treatment with TRO substantially induced GADD45 mRNA expression after 48 hours, whereas the vehicle (DMSO) had no effect on GADD45 expression. GADD45 mRNA levels in both vehicle and TRO-treated cells rapidly decreased within 2 hours. GADD45 mRNA levels were normalized to those of the house-keeping gene CHOB and the mRNA half-lives were calculated. The half-life of GADD45 mRNA in control and TRO-treated cells was calculated as 62 minutes and 72 minutes, respectively. These results demonstrate that PPAR ligands do not enhance GADD45 RNA mRNA stability in HCSMCs.

View larger version (43K): [in this window] [in a new window]   Figure 4. PPAR ligands have no effect on GADD45 mRNA stability in VSMCs. Human coronary VSMCs were grown for 48 hours in the presence of vehicle (DMSO, left) or 30 µmol/L TRO (right) and treated with actinomycin D (5 µg/mL). At different time intervals, cells were collected and RNA was isolated. Total RNA expression was examined by Northern blot analysis using radiolabeled probes for GADD45 or CHOB. Hybridization signals were quantified by densitometry and GADD45 mRNA expression was normalized to CHOB expression. Quotient from DMSO- or TRO-treated cells at time 0 was arbitrarily defined as 1.

To examine the effect of PPAR ligands on GADD45 transcription, HCSMCs were transiently transfected with a human GADD45 promoter construct containing 2543 bp of 5'-flanking DNA for the GADD45 gene inserted upstream of a luciferase reporter (pG45-Luc).³⁷ Twenty-four hours after transfection, cells where treated with either RSG, TRO, or nTZDpa at 30 µmol/L for an additional 48 hours (Figure 5, top). PPAR ligands exhibited a negligible effect on HCSMCs transfected with the control empty pGBV2 vector. RSG, TRO, and nTZDpa induced GADD45 promoter activity by 2.96±0.33-, 3.9±0.47-, and 3.21±0.38-fold (n=3, P<0.05), respectively. To further elucidate whether PPAR ligands stimulate GADD45 transcription through a receptor-dependent mechanism, HCSMCs were transfected with the GADD45 promoter construct pG45 and pretreated with the irreversible PPAR antagonist GW9662 for 3 hours. When endogenous wild-type PPAR function was pharmacologically blocked by GW9662, RSG, TRO, and nTZDpa failed to activate the GADD45 promoter (Figure 5, bottom). In combination, these results demonstrate that the induction of the GADD45 promoter activity by PPAR ligands requires functional endogenous PPAR and occurs through a receptor-dependent mechanism.

View larger version (18K): [in this window] [in a new window]   Figure 5. PPAR ligands induce transcriptional activation of the GADD45 promoter through a receptor-dependent pathway. Human coronary VSMCs were transiently transfected with 1 µg pG45-Luc or pGBV2. Transfected cells were stimulated with vehicle or the indicated PPAR ligand or were pretreated with the irreversible PPAR antagonist GW9662 (10 µmol/L) 3 hours before stimulation with the indicated PPAR ligands. Forty-eight hours after stimulation, luciferase activity was assayed. Transfection efficiency was adjusted by normalizing firefly luciferase activities to renilla luciferase activities generated by cotransfection with 10 ng pRL-CMV. All experiments were repeated at least 3 times. Data are expressed as fold induction over vehicle-treated cells and presented as mean±SEM (*P<0.05 vs vehicle; #P<0.05 vs stimulation with PPAR ligands).

Deletion Analysis of the GADD45 Promoter
To identify the PPAR-responsive elements in the GADD45 promoter, a deletion series of luciferase reporter constructs spanning the different regions of the human GADD45 promoter was used. After transfection of these reporter constructs into HCSMCs, cells were administered with the PPAR ligands RSG, TRO, or nTZDpa at 30 µmol/L. As shown in Figure 6, the full-length GADD45 promoter was strongly induced by all three PPAR ligands. Progressive 5'-deletions of the promoter that extended to -81 relative to the transcription start site exhibited almost no induction after treatment with PPAR ligands. The region between -234 and -81, therefore, contains critical elements required for activation of the GADD45 promoter by PPAR ligands. Sequence analysis indicates that these regions of the GADD45 promoter harbors an Oct-1 consensus sequence and one CCAAT motif, both of which are known to be crucial for regulating GADD45 expression.^37–39

View larger version (28K): [in this window] [in a new window]   Figure 6. Localization of the PPAR-responsive element by 5'-deletion analysis of the GADD45 promoter. Human coronary VSMCs were transiently transfected with various deletion mutants of the human GADD45 promoter. Transfected cells were stimulated with PPAR ligands as indicated for 48 hours. After stimulation, luciferase activity was assayed. Transfection efficiency was adjusted by normalizing firefly luciferase activities to renilla luciferase activities generated by cotransfection with 10 ng pRL-CMV. All experiments were repeated at least 3 times. Data are expressed as fold induction over vehicle and presented as mean±SEM.

Octamer-Binding Factor Oct-1 Is Essential for the PPAR Ligand-Mediated Induction of GADD45
To determine whether the Oct-1 and CCAAT motifs play a role in the activation of the GADD45 promoter by PPAR ligands, we used Oct-1 and CCAAT reporter plasmids, where a hexamer of a 20-bp region containing both Oct-1 and CCAAT consensus sequences was linked to a minimal IL-8 promoter construct and luciferase reporter. In Figure 7, IL-8-OCx6 was transiently transfected into HCSMCs and activated by the PPAR ligands RSG, TRO, and nTZDpa (2.16±0.15-, 2.21±0.19-, 1.86±0.14-fold, respectively n=3, P<0.05). To define the crucial element in the GADD45 promoter activated by PPAR ligands, we used a construct where either Oct-1 or CCAAT elements were mutated. Point mutations of the CCAAT motif (IL-8-OmCx6) revealed a comparable transcriptional activation by all PPAR ligands as wild-type IL-8-OCx6. In contrast, IL-8-mOCx6 harboring mutations of the Oct-1 motif were not induced by RSG, TRO, or nTZDpa. These results suggest that the Oct-1 consensus sequence is a major and essential element required for the induction of the GADD45 promoter by PPAR activation.

View larger version (24K): [in this window] [in a new window]   Figure 7. Mutations of Oct-1 motifs abrogate the activation of the GADD45 promoter after stimulation with PPAR ligands. Schematic representation of the tandem-repeat constructs of the minimal-responsive element and the mutants (left). O indicates Oct-1 consensus sequence; C, CCAAT box. Motifs containing point mutations are shown in crossed boxes. Human coronary VSMCs were transiently transfected with the indicated constructs and stimulated with PPAR ligands (10 µmol/L) as indicated for 24 hours. After stimulation, luciferase activity was assayed. Transfection efficiency was adjusted by normalizing firefly luciferase activities to renilla luciferase activities generated by cotransfection with 10 ng pRL-CMV. All experiments were repeated at least 3 times. Data are expressed as fold induction over vehicle and presented as mean±SEM (*P<0.05 vs IL8-OCx6).

PPAR Ligands Induce Oct-1 Protein Expression and Binding to the Oct-1 Consensus Sequence
To determine whether activation of PPAR directly induces Oct-1 expression in HCSMCs, cells were stimulated with RSG, TRO, and nTZDpa (30 µmol/L, 48 hours), and nuclear extracts were analyzed for Oct-1 protein expression. Oct-1 protein levels were found to be induced after stimulation with all three PPAR ligands used (Figure 8A). PPAR ligands, however, did not induce Oct-1 mRNA expression, indicating that the induction of Oct-1 expression involves a posttranscriptional mechanism (data not shown).

View larger version (83K): [in this window] [in a new window]   Figure 8. PPAR ligands Oct-1 protein expression and binding to its consensus sequence. Human coronary VSMCs were stimulated with vehicle (DMSO) or the indicated PPAR ligand (30 µmol/L) for 48 hours. A, Nuclear extracts (30 µg) were analyzed by Western immunoblotting for Oct-1 expression. B, Electrophoretic mobility shift assays were performed from 4 µg of nuclear extract using radioactive Oct-1 consensus oligonucleotide labeled with T4 polynucleotide kinase and [-32P] ATP. For competitive and noncompetitive oligonucleotide assays, 100-fold labeled double-stranded SP-1 or unlabeled double-stranded Oct-1 (cold Oct-1) was added to the nuclear extracts of nTZDpa (30 µmol/L)-treated cells before the addition of the radiolabeled probe. For supershift experiments (SS), 4 µg of nuclear extract was incubated with 0.5 µg of an Oct-1 antibody (Oct-1 ab) after incubation of the radiolabeled probe with nuclear proteins of nTZDpa (30 µmol/L)-treated cells. DNA protein complexes were analyzed by electrophoresis in a neutral polyacrylamide gel. These experiments were performed at least 3 times, and the representative results are shown in the figures.

EMSA experiments demonstrated that activation of PPAR induces Oct-1 binding to its consensus sequence. EMSA from nuclear extracts isolated from both control and PPAR ligand-stimulated HCSMCs were performed using a double-stranded Oct-1 oligonucleotide probe (Figure 8B). Specific protein-DNA complexes were undetectable in vehicle-treated cells, but increased after stimulation with 30 µmol/L RSG, TRO, and nTZDpa for 48 hours. The complex was displaced by a 100-fold molar excess of unlabeled Oct-1 oligonucleotides and supershifted in the presence of an anti-Oct-1 antibody. In contrast, excess noncompetitive double-stranded SP-1 oligonucleotides could not displace the complex. These results indicate that PPAR ligands induce Oct-1 protein expression and increase its DNA binding-activity in HCSMCs.

To further investigate whether the induction of Oct-1 protein and DNA binding by PPAR ligands is mediated through a receptor-dependent mechanism, we performed Western blot and EMSA analysis from nuclear extracts of HCSMCs infected with Adx-CA-PPAR overexpressing constitutively active PPAR. As depicted in Figures 9A and 9B, adenoviral-mediated overexpression of constitutively active PPAR resulted in a dose-dependent induction of Oct-1 protein and DNA-binding. The mechanism by which constitutively active PPAR induces Oct-1 expression occurs at a posttranscriptional level, because no induction of Oct-1 mRNA expression was observed after infection with Adx-CA-PPAR (data not shown). Infection of HCSMCs with Adx-GFP or Adx-VP-16 exhibited no effect on Oct-1 protein expression and DNA binding. Taken together, these results suggest that the induction of Oct-1 protein expression and DNA binding by PPAR ligands occurs through a receptor-dependent, posttranscriptional mechanism.

View larger version (81K): [in this window] [in a new window]   Figure 9. Constitutive activation of PPAR induces Oct-1 protein expression and binding to its consensus sequence. Human coronary VSMCs were infected with Adx-CA-PPAR (1, 30, or 100 PFU/cell) for 48 hours. Uninfected cells (C) and infection with Adx-GFP (100 PFU/cell) or Adx-VP-16 (1, 30, or 100 PFU/cell) were used as controls. A, Nuclear extracts (30 µg) were analyzed by Western immunoblotting for Oct-1 expression. B, Electrophoretic mobility shift assays were performed from 4 µg of nuclear extract as described in Figure 8. Nuclear extracts from Adx-CA-PPAR (100 PFU/cell)-infected cells were used for competitive and noncompetitive oligonucleotide assays. For supershift experiments, 4 µg of nuclear extract were incubated with 0.5 µg of an Oct-1 antibody (Oct-1 ab) after incubation of the radiolabeled probe with nuclear proteins of Adx-CA-PPAR (100 PFU/cell)-infected cells. These experiments were performed at least 3 times, and the representative results are shown in the figures.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The present study demonstrates that ligand-dependent or constitutive activation of PPAR induces caspase-mediated apoptosis of HCSMCs. The induction of apoptosis correlates with enhanced expression of GADD45 mRNA and increased transcriptional activation of GADD45. Pharmacological inhibition of PPAR by an irreversible antagonist prevents induction of the GADD45 promoter by PPAR ligands, indicating a receptor-dependent mechanism. By using 5'-deletion analysis, the PPAR-regulated elements in the GADD45 promoter have been mapped between -234 and -81, which contains one Oct-1 and one CCAAT motif. Mutation of the Oct-1 motifs in a luciferase reporter construct driven by hexamers of Oct-1 and CCAAT motifs abrogates the activation of this construct by PPAR ligands. Furthermore, activation of PPAR induces Oct-1 protein expression and binding to its consensus sequence, suggesting that PPAR-mediated transactivation of the GADD45 promoter is mediated, at least in part, through increased Oct-1 expression and activity.

Pharmacological activation of PPAR has been shown to inhibit cell growth and to induce apoptosis in VSMCs.^12,14,20 The molecular mechanisms, however, underlying the antiproliferative and proapoptotic effects of PPAR ligands on VSMCs are incompletely understood. Identification of downstream target genes directly regulated by PPAR-activation relevant to growth inhibition and apoptosis might provide insights into novel vascular actions of PPAR. We have previously reported that TZDs inhibit VSMC proliferation at the G₁S phase transition of the cell cycle by suppressing mitogen-induced phosphorylation of the retinoblastoma protein, E2F release, and expression of minichromosome maintenance proteins.^12,13 In this study, we provide further evidence for an important role of PPAR in regulating VSMC growth arrest and apoptosis. Among putative PPAR-activated target genes associated with apoptosis and growth inhibition, we observed an induction of GADD45 expression by several PPAR ligands.

GADD45 belongs to a subgroup of genes that are coordinately induced in growth-arrested cells.^28,29 Substantial evidence implicates an important role for GADD45 in regulating cell growth. Using microinjection of a GADD45 expression vector into human fibroblasts, Wang et al⁴² demonstrated that GADD45 induces G₂ cell cycle arrest. GADD45 interacts with several cell cycle–regulating proteins, and the involvement of GADD45 in cell cycle arrest at G₀/G₁ or G₂/M phase has been observed in different cell types.^42,43 Laminar shear stress on endothelial cells leads to sustained p53 activation, a concomitant upregulation of both GADD45 and the cyclin-dependent kinase inhibitor p21^cip1 and subsequent suppression of cyclin-dependent kinase activity causing hypophosphorylation of Rb that results in G₀/G₁ arrest.⁴⁴ In addition, Zhan et al^28,33 have shown that GADD45 inhibits the kinase activity of Cdc2/cyclinB1 and thus may induce a G₂/M arrest. These observations suggest that GADD45 plays a critical role in regulating cell growth and may be an important target in PPAR-mediated inhibition of VSMC cell cycle.⁴⁵

Whether GADD45 directly induces apoptosis remains controversial.²⁶ Several studies have identified GADD45 as a critical mediator of apoptosis triggered by activation of the JNK and/or p38 MAPK signaling pathways.^30,31 Takekawa and Saito³⁰ have shown that overexpression of GADD45 induces apoptosis in HeLa cells. They observed that GADD45 binds to MEKK4, an upstream protein kinase in the signaling cascade that ultimately activates both JNK and p38 MAPK to induce apoptotic cell death. Interestingly, PPAR ligands have been reported to activate both JNK and p38 MAPK in astrocytes and preadipocytes, although the effect was attributed to be independent of PPAR activation.⁴⁶ Our data support an alternative mechanism by which transcriptional upregulation of GADD45 by PPAR promotes apoptosis in VSMCs, possibly through GADD45-dependent potentiation of JNK and p38 MAPK pathways.

TZD-induced inhibition of cellular proliferation was recently observed in mouse stem cells lacking PPAR expression, indicating that antiproliferative effects of TZDs may occur through a receptor-independent pathway.⁴⁷ In contrast, Bishop-Bailey et al²⁰ recently reported that induction of VSMC apoptosis by RSG at suprapharmacological concentrations is mediated through a receptor-dependent mechanism. Although GADD45 has been demonstrated to be induced by TRO in VSMCs¹⁹ and 15-deoxy-¹²-prostaglandin J₂ in HeLa cells,⁴⁸ the mechanism by which PPAR ligands upregulate GADD45, and whether it involves a receptor dependent pathway, remains to be elucidated. To address the important issue of PPAR-dependent versus -independent mechanisms of action, we used two different experimental approaches. In our first approach, we observed that adenovirus-mediated overexpression of a constitutively active PPAR induced caspase-mediated apoptosis and GADD45 mRNA expression, similar to tested PPAR ligands. In an alternate strategy, we demonstrated that PPAR ligand–mediated induction of transcriptional activation of the GADD45 promoter was abrogated by pretreatment with an irreversible pharmacological PPAR antagonist used to block endogenous PPAR function. Our observations that the induction of GADD45 expression by PPAR ligands is mediated through a receptor-dependent pathway is in accord with studies from Satoh et al⁴⁹ showing that an induction of GADD153, another member of the GADD-gene family, by TRO was not observed in a cell line lacking PPAR expression. Data presented in this study demonstrate that the observed induction of caspase-mediated apoptosis, GADD45 mRNA expression, and GADD45 promoter activity by TZD and non-TZD PPAR ligands in HCSMCs involves a receptor-dependent pathway.

To elucidate the molecular mechanism by which PPAR controls GADD45 expression, we first examined whether the induction of GADD45 expression occurred principally at a transcriptional or posttranscriptional level. Observations from several studies have suggested that GADD45 mRNA expression, depending on the cell type and specific mechanism of induction, may be regulated either by increased GADD45 promoter activity^37–39 or enhanced mRNA stabilization.^40,41 Retinoids, which are ligands for the retinoid X receptor that heterodimerizes with PPARs to regulate target genes, have been shown to induce GADD45 mRNA through stabilization of mRNA in breast carcinoma cells.⁴⁰ GADD45 mRNA half-life, however, did not change in HCSMCs after stimulation with TRO, indicating that PPAR ligands had no significant effect on mRNA stabilization. In contrast, PPAR ligands did elicit a prominent induction of GADD45 promoter activity. PPAR can directly activate transcription of target genes by binding to PPREs and undergoing a ligand-induced conformational change which enhances interaction between the ligand binding domain (LBD) of the nuclear receptor and LXXLL motifs in transcriptional coactivators.^9,10 The lack of a consensus PPRE in the human GADD45 promoter construct used in this study, however, suggested that PPAR induces GADD45 transcription independent of direct binding to cis-regulatory elements. Using 5'-deletion constructs of the human GADD45 promoter, the PPAR-regulated element was mapped between -234 and -81 relative to the transcription start site, a region harboring both an Oct-1 and CCAAT motif. Oct-1 belongs to the POU homeodomain family of transcription factors and is ubiquitously expressed.⁵⁰

Several lines of evidence indicate that the transcription factor Oct-1 may be directly involved in the regulation of the GADD45 promoter in HCSMCs.^37–39 In colorectal carcinoma cells, JNK1 and ERK mitogen-activated protein kinases activate the GADD45 promoter through the Oct-1 element.⁵¹ In our studies, ligand-induced activation of PPAR induced transcription of a luciferase reporter driven by six tandem Oct-1 sites. Moreover, mutation of these Oct-1 sites abolished the induction of the GADD45 promoter by PPAR ligands, suggesting that Oct-1 might be involved in the PPAR-mediated transcriptional activation of the GADD45 promoter. In addition, activation of PPAR directly resulted in increased Oct-1 protein expression as well as Oct-1 binding to its consensus sequence as determined by EMSA. Regulation of Oct-1 expression by PPAR likely occurs via posttranscriptional mechanisms, because Oct-1 mRNA levels did not change in response to PPAR ligands. Our findings are similar to those of Zhao et al⁵² who found that induction of Oct-1 DNA-binding activity after DNA damage is regulated at the posttranscriptional level, whereas upregulation of GADD45 resulted from increased gene transcription.

In summary, data presented in this study demonstrate that ligand-dependent or constitutive activation of PPAR promotes caspase-mediated apoptosis in HCSMCs. The ability of PPAR activation to induce apoptosis was coupled to an induction of GADD45 expression, illustrating a novel mechanism by which PPAR can induce growth arrest and apoptosis of VSMCs. The molecular mechanism by which PPAR promotes transcriptional activation of GADD45 is dependent on Oct-1 function. These findings provide further support for the important role of PPAR in regulating VSMC cell growth and apoptosis.

	Acknowledgments

 
This study was supported by NIH Grant HL 58328 to Willa A. Hsueh. Dennis Bruemmer was supported by a research fellowship from the Gonda (Goldschmied) Diabetes Center, University of California, Los Angeles.

	Footnotes

 
J.P. Berger is an employee of Merck & Co, Inc, and potentially owns stock and/or holds stock options in the company.

Original received April 21, 2003; resubmission received July 2, 2003; accepted July 16, 2003.

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