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Molecular Endocrinology, doi:10.1210/me.2002-0410
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Molecular Endocrinology 17 (6): 1005-1018
Copyright © 2003 by The Endocrine Society

Peroxisome Proliferator-Activated Receptor {gamma} Inhibits Expression of Minichromosome Maintenance Proteins in Vascular Smooth Muscle Cells

Dennis Bruemmer, Fen Yin, Joey Liu, Joel P. Berger, Tohru Kiyono, Jasmine Chen, Eckart Fleck, Andre J. Van Herle, Barry M. Forman and Ronald E. Law

Division of Endocrinology, Diabetes and Hypertension and The Gonda (Goldschmied) Diabetes Center (D.B., F.Y., J.L., A.J.V.H., R.E.L.), David Geffen School of Medicine, University of California, Los Angeles, California 90095; Department of Medicine/Cardiology (D.B., E.F.), German Heart Institute, Berlin, D-13353, Germany; Merck Research Laboratories (J.P.B.), Rahway, New Jersey 07065; Virology Division (T.K.), National Cancer Center Research Institute, Tokyo,104-0045, Japan; and Division of Molecular Medicine and The Gonda Diabetes and Genetic Research Center, and the Department of Diabetes, Endocrinology, and Metabolism (J.C., B.M.F.), The City of Hope National Medical Center, Beckman Research Institute, Duarte, California 91010

Address all correspondence and requests for reprints to: Ronald E. Law, Ph.D, University of California, Los Angeles School of Medicine, Division of Endocrinology, Diabetes and Hypertension, Warren Hall, Suite 24-130, 900 Veteran Avenue, Los Angeles, California 90095. E-mail: rlaw{at}mednet.ucla.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Using a cDNA array consisting only of cell cycle genes, we found that a novel nonthiazolidinedione partial peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) agonist (nTZDpa) inhibited expression of minichromosome maintenance (MCM) proteins 6 and 7 in vascular smooth muscle cells. MCM proteins are required for the initiation and elongation stages of DNA replication and are regulated by the transcription factor E2F. Mitogen-induced MCM6 and MCM7 mRNA expression was potently inhibited by nTZDpa and to a lesser degree by the full PPAR{gamma} agonist, rosiglitazone. Inhibition of MCM6 and MCM7 expression by nTZDpa and rosiglitazone paralleled their effect to inhibit phosphorylation of the retinoblastoma protein and cell proliferation. Transient transfection experiments revealed that the nTZDpa inhibited mitogen-induced MCM6 and MCM7 promoter activity, implicating a transcriptional mechanism. Adenoviral-mediated E2F overexpression reversed the suppressive effect of nTZDpa on MCM6 and MCM7 expression. Furthermore, activity of a luciferase reporter plasmid driven by multiple E2F elements was inhibited by nTZDpa, indicating that their down-regulation by nTZDpa involves an E2F-dependent mechanism. Overexpression of dominant-negative PPAR{gamma} or addition of a PPAR{gamma} antagonist, GW 9662, blocked nTZDpa inhibition of MCM7 transcription. Adenovirus-mediated overexpression of constitutively active PPAR{gamma} inhibited MCM7 expression in a similar manner as the nTZDpa. These findings provide strong evidence that activation of PPAR{gamma} attenuates MCM7 transcription and support the important role of this nuclear receptor in regulating vascular smooth muscle cell proliferation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
PEROXISOME PROLIFERATOR-activated receptor {gamma} (PPAR{gamma}) ligands have been shown to inhibit growth of vascular and cancer cells by interfering with the expression and function of multiple cell cycle regulators (1, 2, 3, 4, 5, 6). In rat aortic vascular smooth muscle cells (VSMC), we have previously reported that the thiazolidinedione (TZD) PPAR{gamma} ligands, troglitazone (TRO) and rosiglitazone (RSG), inhibit exit from G1 into S phase by attenuating retinoblastoma protein phosphorylation (4). Decreased phosphorylation of retinoblastoma protein by TRO and RSG likely resulted from their ability to elevate levels of the cyclin-dependent kinase inhibitor p27kip1 and reduce the activity of cyclin D- and cyclin E-dependent kinases (4). TZDs, however, have also been reported to inhibit VSMC proliferation in the absence of an effect on p27kip1 (7). The mechanism of action for the antiproliferative effects of PPAR{gamma} ligands, therefore, may involve the targeting of additional cell cycle regulators.

Despite having a two-log lower affinity for PPAR{gamma}, TRO suppressed VSMC proliferation to the same extent as RSG (4). Although TRO is much weaker than RSG in inducing PPAR{gamma}-mediated transactivation of target genes, it can effectively antagonize the activity of agonists that display stronger affinity for the nuclear receptor (8). Thus, TRO behaves as a partial agonist for PPAR{gamma}, as evidenced by its reduced activity or inactivity toward a subset of promoters and target genes regulated by other PPAR{gamma} ligands. In contrast, RSG functions more as a full agonist. Although the insulin-sensitizing property of PPAR{gamma} agonists correlates closely with their binding affinity to the receptor (9, 10), it is unknown whether this relationship also exists for their antiproliferative effects. In fact, it remains controversial whether their effects on the cell cycle results from PPAR{gamma} activation (6).

To date, no studies have examined the effect of PPAR{gamma} ligands on the expression of genes active in the S phase of the cell cycle and required for DNA replication. A biased DNA array containing 96 genes known to control the cell cycle was used to screen for nuclear targets that potentially mediate the antiproliferative activity of a novel non-TZD partial PPAR{gamma} agonist (nTZDpa) on VSMC. Using that approach, we found that nTZDpa inhibited expression of genes encoding the minichromosome maintenance (MCM) proteins 6 and 7.

MCM proteins play a central role in the regulation of the initiation of DNA replication and ensure that DNA replicates only once during each cell cycle (11, 12). In eukaryotes, MCM2–MCM7 are recruited onto replication origins during the G1 phase of the cell cycle and assembled into a heteromeric hexamer (13, 14). Formation of this prereplication complex, a process often referred to as "replication licensing" (15), establishes the competence of this origin for initiation of DNA replication in the subsequent S phase (16, 17). The promoter regions of MCM6 and MCM7 contain several elements for E2F, indicating that this transcription factor may be primarily responsible for the coordinated increase in MCM mRNA during the G1->S phase transition (18, 19). Entry into S phase requires increased phosphorylation of the retinoblastoma protein (Rb), which releases sequestered E2F to permit transcription of genes encoding the replicative machinery for DNA synthesis, including MCM proteins (20).

We report here that PPAR{gamma} agonists, both partial and full, inhibit the mitogen-induced expression of MCM6 and MCM7, two important regulators of DNA replication, at the transcriptional level by blocking E2F release from Rb and suppressing their E2F-dependent transactivation. Regulation of MCM expression during the cell cycle by nTZDpa is mediated through PPAR{gamma}, based on gain-of-function and loss-of-function studies using mutant forms of PPAR{gamma}, a pharmacological antagonist of PPAR{gamma}, and fibroblasts that lack PPAR{gamma} expression.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Characterization of a Non-TZD PPAR{gamma} Ligand as a Partial Agonist
Transcriptional activation of PPAR{gamma} by the TZD full PPAR{gamma} agonist RSG and a novel acyl indole non-TZD partial PPAR{gamma} agonist nTZDpa (chemical structures are depicted in Fig. 1Go) in rat aortic VSMC (RASMC) was assessed by ligand-induced activation of a luciferase reporter gene driven by three copies of the peroxisome proliferator-response element (PPRE) from the acyl-coenzyme A (CoA) oxidase gene linked to the minimal thymidine kinase promoter. RSG induced a strong ligand-dependent transcriptional response in a dose-dependent manner with an EC50 of 28 nM. In contrast, the nTZDpa served as a partial PPAR{gamma} agonist (EC50 = 31 nM), activating the receptor only to approximately 24% of the maximum efficacy attained with the full PPAR{gamma} agonist RSG (data not shown). These data identify nTZDpa as partial PPAR{gamma} agonist, which, as previously reported, does not activate PPAR{delta} or PPAR{alpha} at concentrations as high as 10 µM (21).



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  		Figure 1. Chemical Structure of RSG and nTZDpa

 
RSG and nTZDpa Differentially Inhibit RASMC Growth, Bromodeoxyuridine (BrdU) Incorporation, and Rb Phosphorylation
Our previous studies demonstrated that TRO, although it exhibits an approximately 100-fold lesser binding affinity to PPAR{gamma} than the full agonist RSG, had similar activity to inhibit cell cycle progression (4). This finding suggests that different PPAR{gamma} ligands may have an antiproliferative activity that does not directly correlate with their binding affinity to or transcriptional activation of PPAR{gamma}. To determine the dose-dependent effects of full and partial PPAR{gamma} agonists on mitogen-induced RASMC growth, quiescent cells were treated with RSG or the partial PPAR{gamma} agonist nTZDpa and stimulated with platelet-derived growth factor (PDGF) plus insulin. At the concentrations used, neither of the PPAR{gamma} ligands exerted cytotoxic effects, as evidenced by the lack of cell detachment and no uptake of the vital dye trypan blue. RASMC cell proliferation and DNA synthesis were assessed by performing cell counts (Fig. 2AGo) and analyzing BrdU incorporation (Fig. 2BGo). RSG (10 µM) significantly inhibited mitogen-induced RASMC growth (54.8 ± 5.7% inhibition, n = 6, P < 0.05) and DNA synthesis (44.3 ± 3.8% inhibition, n = 6, P < 0.05) in a dose-dependent fashion. A more substantial growth-inhibitory effect was observed for nTZDpa, which inhibited mitogen-induced RASMC growth (100% inhibition, n = 6, P < 0.05) and BrdU incorporation (93.8 ± 7.1% inhibition, n = 6, P < 0.05).



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  		Figure 2. RSG and nTZDpa Differentially Inhibit Mitogen-Induced RASMC Growth, DNA Synthesis, and Rb Phosphorylation

Quiescent RASMC were preincubated with vehicle (DMSO), RSG, or nTZDpa at the indicated concentrations 30 min before stimulation with PDGF (20 ng/ml) plus insulin (1 µM). After 48 h, cell proliferation was assayed by performing cell counts using a hematocytometer (A) or incorporation of the thymidine analog BrdU (B) using a commercially available immunoassay. C, Twenty-four hours after stimulation, whole-cell protein (40 µg) was subjected to immunoblotting using a specific antibody for Ser807/811 pRb. To assess loading variability, immunoblots were cohybridized with a specific antibody for ß-actin. The autoradiogram shown is representative of three independently performed experiments. Quantification was performed by densitometry and normalization to ß-actin. Results are expressed as mean percent ± SEM of the maximal mitogenic induction of cell proliferation, DNA synthesis or Rb phosphorylation at Ser807/811 from at least three separate experiments (*, P < 0.05 vs. PDGF plus insulin and vehicle).

 
Cell cycle progression and growth require increased phosphorylation of Rb to release the S-phase transcription factor, E2F, which regulates expression of genes encoding the enzymatic machinery for DNA synthesis (20). We, therefore, next examined the effect of the full PPAR{gamma} agonist RSG and the partial PPAR{gamma} agonist nTZDpa on mitogen-induced Rb phosphorylation in RASMC. As shown in Fig. 2CGo, Western blotting results revealed that treatment with RSG (10 µM) partially inhibited PDGF plus insulin-induced Rb phosphorylation (49.1 ± 3.6% inhibition, n = 3, P < 0.05). The partial PPAR{gamma} agonist nTZDpa (10 µM), however, was more efficacious in inhibiting mitogen-induced Rb phosphorylation (74.8 ± 5.2% inhibition, n = 3, P < 0.05). Thus, these findings indicate that a partial PPAR{gamma} agonist nTZDpa is a more effective inhibitor of RASMC proliferation, DNA synthesis, and Rb phosphorylation in RASMC than the full PPAR{gamma} agonist RSG.

nTZDpa Inhibits MCM6 and MCM7 mRNA Expression in Growing RASMC
To identify cell cycle genes regulated by the partial PPAR{gamma} agonist nTZDpa, we employed a biased DNA gene array containing genes known to control cell proliferation. Growing RASMC were treated with nTZDpa (5 µM), which resulted in marked down-regulation of MCM6 and MCM7 mRNA (Fig. 3Go), as visualized on a DNA array of cell cycle regulatory genes. MCM2–MCM5 were also displayed on the array; their mRNA levels, however, were substantially lower than those for MCM6 and MCM7 and did not reach the threshold for detection.



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  		Figure 3. Inhibition of MCM6 and MCM7 mRNA by nTZDpa in Growing RASMC

Growing RASMC were treated with vehicle (DMSO) or nTZDpa (5 µM) and harvested after 24 h. Total RNA was isolated, reverse transcribed into biotin-16-dUTP-labeled single-strand cDNA, and hybridized with cDNA array membranes containing 96 DNA-oligonucleotides from genes regulating the cell cycle. Chemiluminescence was visualized by autoradiography. MCM6 (left) and MCM7 (right) are indicated in the boxed area. The autoradiogram depicted is representative of three separate experiments. Due to a lower level of expression, the autoradiogram for MCM7 represents a longer exposure of the array membrane.

 
Mitogen-Induced MCM6 and MCM7 mRNA Expression in RASMC Is Attenuated by PPAR{gamma} Ligands
The S phase of the cell cycle and DNA replication requires the function of MCM gene products (22). To investigate the effects of PPAR{gamma} ligands on PDGF plus insulin-induced MCM6 and MCM7 mRNA expression, RASMC were treated with PDGF (20 ng/ml) and insulin (1 µM) to induce growth-regulated expression of MCM6 and MCM7 in the presence or absence of the PPAR{gamma} ligands RSG and nTZDpa (0.1–10 µM). Northern analysis revealed that both PPAR{gamma} ligands, RSG and nTZDpa, dose-dependently inhibited PDGF plus insulin-induced MCM6 and MCM7 mRNA expression (Fig. 4Go). However, compared with the full PPAR{gamma} agonist RSG (44.3 ± 4.1% and 64.1 ± 5.4% inhibition of MCM6 and MCM7 mRNA expression, respectively, n = 3, P < 0.05), nTZDpa exhibited a more substantial inhibitory effect on mitogen-induced MCM6 and MCM7 expression (100% and 97.6 ± 6.1% inhibition of MCM6 and MCM7 mRNA expression, respectively, n = 3, P < 0.05). Reduced MCM6 and MCM7 mRNA levels by nTZDpa could reflect either decreased transcription and/or mRNA stability. Because nTZDpa exhibited the most substantial effect in inhibiting mitogen-induced MCM6 and MCM7 expression, all subsequent experiments focused on that PPAR{gamma} ligand.



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  		Figure 4. RSG and nTZDpa Differentially Inhibit PDGF Plus Insulin-Induced MCM6 and MCM7 mRNA Expression

Quiescent RASMC were preincubated with vehicle (DMSO), RSG, or nTZDpa at the indicated concentrations 30 min before addition of PDGF (20 ng/ml) plus insulin (1 µm). Twelve hours after stimulation, cells were harvested and total RNA was analyzed for MCM6 (white bars) and MCM7 (black bars) mRNA expression by Northern blotting. Blots were cohybridized with CHOB, a constitutively expressed housekeeping gene encoding a ribosomal protein as internal control. The autoradiogram shown is representative of three independently performed experiments. Quantification was performed by densitometry and normalization to CHOB. Results are expressed as mean ± SEM from three separate experiments (*, P < 0.05 vs. PDGF plus insulin and vehicle).

 
nTZDpa Inhibits PDGF Plus Insulin-Induced MCM6 and MCM7 Promoter Activity
To examine the effect of the nTZDpa on MCM6 and MCM7 transcription, we transiently transfected RASMC with human MCM6 and MCM7 promoter fragments driving expression of a luciferase reporter gene (Fig. 5Go). The employed MCM6 promoter fragment contains a 789-bp DNA segment (-754 to +35) and the MCM7 promoter fragment a 505-bp DNA segment (-558 to -54). PDGF plus insulin induced MCM6 and MCM7 promoter activity (3.6 ± 0.2-, 4.4 ± 0.6-fold induction vs. quiescent cells, n = 3, P < 0.005), which was attenuated by nTZDpa in a dose-dependent fashion [82.1 ± 7.9%, 84.7 ± 7.3% inhibition vs. PDGF plus insulin after 24 h (10 µM), n = 3, P < 0.005]. These data suggest that nTZDpa inhibits mitogen-induced MCM6 and MCM7 expression at the transcriptional level.



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  		Figure 5. nTZDpa Inhibits Mitogen-Induced MCM6 and MCM7 Transcription

RASMC were transiently transfected with 1 µg pHsMCM6-Luc(-754) (white bars) and pHsMCM7-Luc(-558) (black bars) promoter fragments driving expression of the luciferase reporter gene. Transfected cells were serum-starved for 24 h. Thirty minutes before mitogenic stimulation with PDGF (20 ng/ml) plus insulin (1 µM), cells were pretreated with the indicated concentrations of the nTZDpa. Twenty-four 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 three times with different cell preparations. Data are expressed as mean ± SEM (*, P < 0.005 vs. PDGF plus insulin and vehicle).

 
nTZDpa Inhibits PDGF Plus Insulin-Induced E2F-Dependent Transactivation
The MCM6 promoter contains five E2F sites, whereas three E2F sites have been identified in the MCM7 promoter (18, 19). These E2F sites are critical for the transcriptional activation of the MCM6 and MCM7 promoters during the cell cycle. To determine the mechanism by which the nTZDpa down-regulates the transcriptional activity of the MCM6 and MCM7 promoters, we investigated the effect of nTZDpa on the activity of a luciferase reporter plasmid driven by multiple E2F binding sites (Fig. 6Go). The mitogen-induced promoter activity of this reporter plasmid was also inhibited by nTZDpa [86.9 ± 6.8% inhibition vs. PDGF plus insulin after 24 h (10 µM), n = 3, P < 0.005]. These data suggest that the inhibition of mitogen-induced MCM6 and MCM7 expression by nTZDpa occurs, at least in part, by suppressing their E2F-dependent transcriptional activation.



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  		Figure 6. nTZDpa Inhibits Mitogen-Induced E2F Transcriptional Activation

RASMC were transiently transfected with pE2F-TA-Luc and serum-starved for 24 h. Thirty minutes before mitogenic stimulation with PDGF (20 ng/ml) plus insulin (1 µM), cells were pretreated with the indicated concentrations of the nTZDpa. Twenty-four 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 three times with different cell preparations. Data are expressed as mean ± SEM (*, P < 0.005 vs. PDGF plus insulin and vehicle).

 
Adenoviral Overexpression of E2F-1 Reverses the Inhibitory Effect of nTZDpa on Mitogenic Induction of MCM6 and MCM7
To further investigate the role of E2F in the attenuation of PDGF plus insulin-induced MCM6 and MCM7 induction by nTZDpa, we used an adenoviral expression vector to overexpress E2F independent of Rb phosphorylation during the cell cycle. E2F-1 recombinant adenovirus (Adx-E2F) was employed to overexpress E2F-1 in serum-starved quiescent RASMC (Fig. 7BGo). In Adx-green fluorescent protein (GFP)-infected cells, stimulation with PDGF plus insulin resulted in a marked increase of MCM7 mRNA (Fig. 7AGo, 4.7 ± 0.9-fold induction after stimulation with PDGF plus insulin, P < 0.05, n = 3) and protein levels (Fig. 7BGo, 3.1 ± 0.6-fold induction after stimulation with PDGF plus insulin, P < 0.05, n = 3). Treatment with nTZDpa (10 µM) completely blocked mitogenic induction of MCM7 mRNA and potently suppressed MCM7 protein expression by 61.1 ± 5.6% (P < 0.05, n = 3). Upon infection of quiescent cells with Adx-E2F, the level of MCM7 (mRNA and protein) was increased as E2F activated S-phase gene expression. This finding demonstrates that MCM7 expression in RASMC is regulated principally by E2F. Compared with Adx-GFP-infected cells, adenoviral-mediated E2F-1 overexpression reversed the inhibitory effect of nTZDpa on mitogen-induced MCM7 mRNA (Fig. 7AGo) and protein expression (Fig. 7BGo). Similarly, the nTZDpa had no effect on MCM7 expression in quiescent cells infected with Adx-E2F (Fig. 7CGo). Comparable results were obtained for MCM6 (data not shown). The inhibitory effect of nTZDpa on PDGF plus insulin-induced MCM6 and MCM7 expression, therefore, is primarily mediated through an inhibition of E2F release from Rb.



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  		Figure 7. The Inhibitory Effect of nTZDpa on MCM7 Expression Is Reversed by Adenoviral-Mediated Overexpression of E2F

Quiescent RASMC were infected with 100 PFU/cell of Adx overexpressing human E2F-1 driven by the CMV immediate-early promoter (Adx-E2F) for 24 h. Infection of RASMC with 100 PFU/cell of recombinant type 5 Adx-GFP served as control. A, Cells were pretreated with the nTZDpa (10 µm) for 30 min before stimulation with PDGF (20 ng/ml) plus insulin (1 µm). Twelve hours after stimulation, cells were harvested and total RNA was analyzed by Northern blotting for MCM7 and CHOB mRNA, a constitutively expressed housekeeping gene encoding a ribosomal protein as internal control mRNA expression. Quantification was performed by densitometry of three independently performed experiments and normalization to CHOB (*, P < 0.05 vs. Adx-GFP 100 PFU/cell and PDGF plus insulin). B, Twenty-four hours after stimulation, whole-cell proteins (40 µg) were analyzed by immunoblotting using a specific MCM7 antibody. Overexpression of human E2F-1 was monitored by immunoblotting with a specific human E2F-1 antibody. Quantification was performed by densitometry of three independently performed experiments and normalization to ß-actin (*, P < 0.05 vs. 100 PFU/cell of Adx-GFP and PDGF plus insulin). C, Serum-starved RASMC were infected for 24 h with 20 or 100 PFU/cell of Adx-E2F or Adx-GFP and treated with the nTZDpa (10 µM) as indicated for 12 h. Northern blots were analyzed for MCM7 mRNA expression and cohybridized with CHOB. Quantification was performed by densitometry of three independently performed experiments and normalization to CHOB (*, P < 0.05 vs. uninfected control). Data are expressed as mean ± SEM, and all autoradiograms shown are representative of three independently performed experiments.

 
Inhibition of MCM7 Transcription by nTZDpa Is PPAR{gamma} Dependent
The precise mechanism by which PPAR{gamma} ligands inhibit cell growth and whether it involves PPAR{gamma}- mediated transactivation of target genes is not known. Several different approaches were employed to determine whether the inhibition of MCM7 transcription by nTZDpa is mediated through a PPAR{gamma}-dependent mechanism. As a first approach, to block endogenous wild-type PPAR{gamma} function, RASMC were cotransfected with the MCM7 promoter and a D/N L468A/E471A PPAR{gamma} mutant (D/N-PPAR{gamma}) expression plasmid. Ligand-induced transcriptional activation of PPAR{gamma} is severely impaired in cells overexpressing D/N-PPAR{gamma} (23). In an alternate, but complementary, strategy, endogenous wild-type PPAR{gamma} was pharmacologically blocked by an irreversible PPAR{gamma} antagonist, GW9662 (10 µM; Ref. 24). Both D/N-PPAR{gamma} and the PPAR{gamma} antagonist prevented nTZDpa (10 µM) from inhibiting MCM7 promoter activity, consistent with a PPAR{gamma}-dependent mechanism of action (Fig. 8AGo).



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  		Figure 8. Inhibition of Mitogen-Induced MCM7 Transcription Is PPAR{gamma} Dependent

A, RASMC were transiently transfected with pHsMCM7-Luc(-558) or cotransfected with pHsMCM7-Luc(-558) and a D/N-PPAR{gamma} expression vector (D/N-PPAR{gamma}). Transfected cells were serum-starved for 24 h, pretreated with the indicated concentration of nTZDpa or GW9662, an irreversible PPAR{gamma} antagonist, and stimulated with PDGF (20 ng/ml) plus insulin (1 µM). B, NIH3T3 fibroblasts were transfected with pHsMCM7-Luc(-558) or cotransfected with pHsMCM7-Luc(-558) and a wild-type PPAR{gamma}1 expression vector (WT-PPAR{gamma}1). Transfected cells were serum-starved for 24 h, pretreated with the indicated concentration of nTZDpa, and stimulated with PDGF (20 ng/ml) plus insulin (1 µM). Twenty-four 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 three times with different cell preparations. Data are expressed as mean ± SEM [*, P < 0.05 vs. RASMC transfected with pHsMCM7-Luc(-558) alone and stimulated with PDGF plus insulin].

 
PPAR{gamma} expression is not detectable in NIH3T3 fibroblasts, and this cell type was used to provide further evidence for a PPAR{gamma}-dependent pathway (25). As shown in Fig. 8BGo, the nTZDpa (10 µM) had no effect on mitogen-induced MCM7 transcription in NIH3T3 cells. However, when NIH3T3 fibroblasts were cotransfected with a PPAR{gamma}1 expression vector, nTZDpa inhibited mitogen-induced MCM7 transcription by 84.1 ± 5.1% (P < 0.05, n = 3). These data provide further evidence supporting that the effects of nTZDpa on MCM7 transcription are mediated through PPAR{gamma}.

Constitutively Active PPAR{gamma} Suppresses MCM7 Expression
To directly demonstrate that PPAR{gamma} regulated MCM7 mRNA expression and transcription, we employed a constitutively active form of PPAR{gamma} created by fusing a herpes simplex virus (HSV) VP16 transactivation domain to the wild-type PPAR{gamma}1 cDNA (26). This constitutively active PPAR{gamma} was subcloned into a recombinant adenovirus to permit ubiquitous expression of this nuclear receptor. Infection of RASMC with the control Adx-GFP had no effect on the mitogenic induction of MCM7 mRNA and protein expression (Fig. 9Go, A and B). As shown in Fig. 9BGo, we observed faint expression of endogenous PPAR{gamma} in whole-cell extracts of RASMC infected with Adx-GFP, which migrated at approximately 55 kDa. This is consistent with our previous findings that detection of endogenous PPAR{gamma} protein was detectable only in nuclear fractions (1). Infection of RASMC with Adx-constitutively active (CA)-PPAR{gamma} resulted in marked overexpression of constitutively active PPAR{gamma} protein, which migrated more slowly than endogenous PPAR{gamma}, at approximately 70 kDa (Fig. 9BGo), due to the additional VP16 transactivation domain engrafted at the N terminus. In cells overexpressing the constitutively active PPAR{gamma}, MCM7 mRNA (Fig. 9AGo) and protein (Fig. 9BGo) induction by mitogens was markedly inhibited. Similar results were observed for MCM6 (data not shown). Taken together, these data show that constitutive activation of PPAR{gamma} specifically attenuates mitogen-induced MCM7 mRNA and protein expression in RASMC.



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  		Figure 9. Constitutive Activation of PPAR{gamma} Inhibits Mitogen-Induced MCM7 Expression

A, Quiescent RASMC were infected for 24 h with 100 PFU/cell of recombinant type 5 Adx overexpressing CA PPAR{gamma} (Adx-CA-PPAR{gamma}). Recombinant Adx expressing the GFP gene was used as a control vector (Adx-GFP). After further starvation for 24 h, cells were stimulation with PDGF (20 ng/ml) plus insulin (1 µM). Twelve hours after stimulation, cells were harvested and total RNA was analyzed for MCM7 and CHOB mRNA expression by Northern blotting. The autoradiograms shown are representative of three independently performed experiments. B, Twenty-four hours after stimulation, cells were harvested and whole-cell proteins (40 µg) were assayed by Western immunoblotting for expression of PPAR{gamma} and MCM7. To assess equivalent loading, immunoblots were cohybridized with a specific antibody for ß-actin The autoradiograms shown are representative of three independently performed experiments. C, RASMC were transiently transfected with pHsMCM7-Luc(-558) alone or cotransfected with pHsMCM7-Luc(-558) and a CA PPAR{gamma} expression vector (CA-PPAR{gamma}) or pHsMCM7-Luc(-558) and a VP16 expression vector. Transfected cells were serum-starved for 24 h and stimulated with PDGF (20 ng/ml) plus insulin (1 µM) or FBS (10%). 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 three times with different cell preparations. Data are expressed as mean ± SEM [*, P < 0.05 vs. pHsMCM7-Luc(-558) transfected alone].

 
We next examined whether constitutive activation of PPAR{gamma} inhibits MCM7 transcription in a similar manner as nTZDpa. In Fig. 9CGo, we investigated the effect of the CA-PPAR{gamma} on mitogen-induced MCM7 promoter activity. Mitogenic stimulation [PDGF plus insulin or 10% fetal bovine serum (FBS)] of RASMC transfected with pHsMCM7-Luc(-558) resulted in significant transcriptional activation of MCM7 promoter activity. Cotransfection of pHsMCM7-Luc(-558) with a CA-PPAR{gamma} plasmid expression vector completely inhibited this mitogenic induction. HSV infection blocks G1 events in the cell cycle (27, 28), and the HSV-VP16 transactivation domain was employed to generate the constitutively active PPAR{gamma}. The viral gene products responsible for the G1 arrest induced by HSV have not been completely defined. As a control for a potential spurious effect of the engrafted VP16 transactivation domain to inhibit MCM promoter activity, RASMC were cotransfected with the pHsMCM7-Luc(-558) and a VP16 expression vector. The VP16 expression vector had no effect on MCM7 promoter activity. In fact, mitogenic induction of MCM7 promoter activity was further induced by the VP16 expression vector. Comparable results were obtained for a MCM6 promoter reporter construct (data not shown). Thus, these data demonstrate that the inhibition of MCM6 and MCM7 transcriptional activity was mediated by constitutively active PPAR{gamma}. Moreover, inhibition of MCM7 transcription by constitutively active PPAR{gamma} demonstrates that activation of PPAR{gamma} by means independent of pharmacological ligands can inhibit the function of cell cycle regulators. In combination with other findings, this underscores an important biological role for PPAR{gamma} in controlling VSMC cell cycle progression.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Using DNA array analysis focusing exclusively on cell cycle genes, we identified MCM6 and MCM7 as novel VSMC targets for nTZDpa. RSG, a full PPAR{gamma} agonist, also inhibited MCM6 and MCM7 expression, albeit to a lesser extent than nTZDpa. The nTZDpa effectively inhibited the mitogenic induction of MCM6 and MCM7 expression by suppressing their E2F-dependent transcriptional activation. Four distinct gain-of-function or loss-of-function strategies were employed to demonstrate that nTZDpa inhibited MCM transcription through PPAR{gamma}.

DNA replication is an integrated step of the cell cycle, and MCM proteins are central components of the initiation of chromosomal DNA replication (11, 12). Their recruitment onto replication origins during the G1 phase of the cell cycle is required for the formation of a prereplicative complex (22). However, the expression and regulation of MCM genes in vascular cells has not been investigated. PPAR{gamma}, as a ligand-activated transcription factor, regulates transcription of target genes through binding to specific PPREs located in the promoter of target genes (29). We have observed that nTZDpa attenuated mitogen-induced expression of MCM6 and MCM7 in RASMC at a transcriptional level. Examination of the 5' flanking regions of the MCM6 and MCM7 promoters did not reveal the presence of PPRE consensus motif. Suppression of MCM6 and MCM7 transcription by nTZDpa, therefore, may involve an indirect effect of PPAR{gamma} to interfere with the activity of other transcription factors that interact with the MCM promoter.

Nuclear receptors can inhibit transcription through several mechanisms that do not involve their direct binding to the DNA of the repressed gene (30). Such DNA-binding-independent mechanisms for inhibiting transcription are collectively termed "transrepression" (30, 31). PPAR{gamma} activates transcription of target genes by binding to PPREs and undergoing a ligand-induced conformational change, which enhances interaction between the ligand binding domain of the nuclear receptor and LXXLL motifs in transcriptional coactivators. (32, 33). These transcriptional coactivators may be rate limiting for cellular transcription, and their sequestration at ligand binding domains may lead to PPAR{gamma}-dependent, indirect repression of genes devoid of PPREs (34). Nuclear receptors can also transrepress genes through other mechanisms, including direct protein-protein interaction, i.e. PPAR{alpha} with nuclear factror-{kappa}B (35) or by induction of genes encoding proteins that inhibit transcription factor activity, i.e. glucocorticoid receptor induction of inhibitor of nuclear factor-{kappa}B (36).

Phosphorylation of Rb results in a conformational change that releases the entrapped S-phase transcription factor, E2F, enabling it to transactivate target genes encoding the enzymatic machinery for DNA synthesis (20). The MCM6 and MCM7 promoters contain functional E2F binding sites, suggesting that E2F is responsible for the coordinate expression of MCMs during progression through the cell cycle (18, 19). Consistent with a central role for E2F in regulating MCM expression, we have found that nTZDpa inhibited E2F-dependent transactivation. Moreover, adenovirus-mediated overexpression of E2F independent of Rb phosphorylation abrogated the inhibitory effect of TZDpa on MCM expression, consistent with nTZDpa inhibiting MCM6 and MCM7 transcription by preventing E2F release from Rb. Blockade of E2F release from Rb thus defines a novel mechanism for transrepression of gene expression by PPAR{gamma}. In addition to an inhibition of E2F release from Rb, PPAR{gamma} ligands may have also direct effects on E2F function. In a simian virus 40-transformed cell line, Altiok et al. (37) showed that the TZD pioglitazone induced cell cycle arrest by stimulating the phosphorylation of E2F and its heterodimeric partner DP-1, which inhibited both their DNA-binding and transcriptional activities. Our finding that RSG and nTZDpa inhibited two E2F-regulated S-phase genes identifies E2F as a major target for the antiproliferative effects of various PPAR{gamma} ligands in vascular cells.

The precise details of how PPAR{gamma} ligands block G1->S progression and inhibit DNA synthesis are not fully elucidated. Using HeLa cells, which lack PPAR{gamma}, Wang et al. (38) observed an inhibition of S-phase entry by the natural PPAR{gamma} ligand 15-deoxy-{Delta}-12,14prostaglandin-J2 only after transfection with a PPAR{gamma} expression vector. However, recent findings in PPAR{gamma}-null stem cells have suggested that PPAR{gamma} ligands may have receptor-independent antiproliferative activities (6). The efficacy of PPAR{gamma} ligands to inhibit Rb phosphorylation and cell proliferation exhibits a right-shifted dose response compared with their activity to promote the differentiation of 3T3L1 preadipocytes into adipocytes (3, 39). Right shifting of the dose response for the antiproliferative activity of PPAR{gamma} ligands provides pharmacological evidence supporting a receptor-independent mechanism of action. In contrast, the fact that small molecules of distinct chemical classes, TZDs and nTZDs, share common properties as both PPAR{gamma} ligands and antiproliferative agents is most easily explained by interactions with a common cellular target, namely PPAR{gamma}. Moreover, Bishop-Bailey et al. (40) have recently shown that a PPAR{gamma}-selective antagonist prevented the induction of apoptosis in quiescent VSMC by suprapharmacological concentrations (30–100 µm) of the full agonist RSG. Those investigators interpreted their findings as indicating that right shifting of certain biological effects of PPAR{gamma} ligands may still be nuclear receptor mediated.

In this study, we observed that inhibition of MCM6 and MCM7 expression by nTZDpa occurred at concentrations approximately 2 logs higher than its EC50 for PPAR{gamma} activation. Despite this right shifting of the dose response for nTZDpa’s effects on VSMC, our findings strongly favor a PPAR{gamma}-dependent mechanism of action. Both a small molecule antagonist, GW9662, and overexpression of D/N-PPAR{gamma} were effective in blocking nTZDpa-mediated repression of MCM7 transcription. In NIH3T3 fibroblasts, which do not express detectable levels of PPAR{gamma} (25), nTZDpa was similarly ineffective. Thus, in these experimental systems corresponding to a loss of PPAR{gamma} function, nTZDpa failed to repress MCM7 transcription. When PPAR{gamma} function was regained in NIH3T3 fibroblasts by overexpressing wild-type PPAR{gamma}, nTZDpa repression of transcription from the E2F-driven MCM7 promoter was restored. RSG has a similar EC50 as nTZDpa for PPAR{gamma} but is a much more efficacious ligand based on the maximal transcriptional activation achieved in cell-based transfection studies (21). With respect to Rb phosphorylation and MCM expression, however, nTZDpa was superior to RSG in its inhibitory activity. Negative regulation of a subset of genes that control the cell cycle, like MCMs, may be more important than binding affinity or transcriptional activation in explaining or predicting the antiproliferative activity of different PPAR{gamma} ligands.

Additional evidence that MCMs are bona fide targets for PPAR{gamma} is provided by experiments using an adenovirus to overexpress a constitutively active form of PPAR{gamma} in VSMC. Engraftment of the VP16 transactivation domain from HSV renders the transcriptional activity of this engineered species of PPAR{gamma} independent of exogenous synthetic ligands and endogenous natural ligands. We observed that adenoviral overexpression of constitutively active PPAR{gamma} potently inhibited the mitogen-induced expression of MCM7 mRNA and protein similar to effects observed with the nTZDpa. Inhibition of MCM expression by the constitutively active PPAR{gamma} is unlikely to be attributed to a nonspecific effect of the VP16 transactivation domain because MCM7 transcription was suppressed by the constitutively active PPAR{gamma} but not by overexpression of the VP16 transactivation domain alone. In concert, these observations support the conclusion that the inhibitory effect of constitutively active PPAR{gamma} and nTZDpa on MCM expression is mediated through a PPAR{gamma}-dependent mechanism. Although PPAR{alpha} and PPAR{delta} are expressed at higher levels than PPAR{gamma} in VSMC (41, 42), our results do not support their involvement in nTZDpa effects on MCM6 and MCM7, particularly because nTZDpa does not activate PPAR{alpha} or PPAR{delta} at concentrations as high as 10 µM (21).

PPAR{gamma} ligands inhibit intimal hyperplasia in rat models of restenosis after balloon injury in both insulin-resistant and insulin-sensitive animals (1, 43, 44, 45, 46). Neointima formation after arterial injury results to a great extent from proliferation of intimal VSMC (47, 48). Concomitant with the phenotypic shift from quiescent VSMC resident in the uninjured vessel wall to proliferating VSMC present in the neointima, there is a substantial up-regulation of PPAR{gamma} expression (3, 40). High-level expression of functional PPAR{gamma} in intimal VSMC, therefore, provides an attractive therapeutic target to exploit the antiproliferative properties of TZD and non-TZD ligands (40). Early clinical trials with TRO have demonstrated an almost 50% reduction in neointimal tissue formation after coronary stent implantation to treat diabetes-associated macrovascular disease (49, 50). Identifying novel PPAR{gamma} ligands based on their direct vascular activity, as opposed to their portfolio of metabolic effects on glucose and lipids, may yield compounds with even greater efficacy against proliferative vascular diseases. Data presented in this study underscore the potential of that approach by demonstrating that a partial agonist for PPAR{gamma} has greater vascular activity than a full agonist. Ultimately, partial PPAR{gamma} agonists may emerge as a new class of cardiovascular drugs that are devoid of some or all of the adverse effects seen with current TZDs, such as edema.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Cell Culture and Treatment with Growth Factor and Reagents
RASMC were prepared from the thoracic aorta of 2- to 3-month-old Sprague-Dawley rats by using the explant technique. Cells of three to eight passages were used for the experiments. Murine NIH3T3 fibroblasts were purchased from ATCC (Manassas, VA). Cells were cultured in DMEM containing 10% FBS (Irvine Scientific, Santa Ana, CA), 100 U/ml penicillin, 100 mg/ml streptomycin, and 2 mM L-glutamine. For the cDNA SuperArray, growing cells (10% FBS in DMEM) were treated with each compound for 24 h. In all other experiments, cells were grown to 60–70% confluency and made quiescent by serum starvation (0.4% FBS) for at least 24 h. Each compound examined was added 30 min before the addition of rat recombinant PDGF (Sigma, St. Louis, MO) and insulin (Eli Lilly \|[amp ]\| Co., Indianapolis, IN) at the final concentration of 20 ng/ml and 1 µM, respectively. For all data shown, each individual experiment was performed using an independent preparation of RASMC. The compound nTZDpa [1-(p-chlorobenzyl)-5-chloro-3-phenylthiobenzyl-2-yl carboxylic acid] was a generous gift from Merck Research Laboratories (Rahway, NJ; Ref. 21). RSG was kindly provided by Smith Kline Beecham (King of Prussia, PA). The irreversible PPAR{gamma} antagonist GW9662 was a kind gift from Dr. Timothy M. Willson (GlaxoSmithKline, Research Triangle Park, NC; Ref. 24).

Cell Growth Assay and BrdU Proliferation Assay
RASMC were plated at 1.0 x 106 cells on 60-mm plates and maintained under starvation in DMEM containing 0.4% FBS. After 48 h, cells were pretreated with the PPAR{gamma} ligands for 30 min and stimulated with growth factors (20 ng/ml PDGF-BB plus 1 µmol/liter insulin) for 48 h. Cells were harvested, and cell proliferation was measured by counting the cells in a hematocytometer. For analysis of DNA synthesis, incorporation of the thymidine analog BrdU was measured using a commercially available immunoassay from Oncogene Research Products (Cambridge, MA) according to the manufacturer’s instructions. Data were based on six different experiments from three different preparations of RASMC.

Western Immunoblotting
Cells were harvested at the indicated time after the addition of growth factors and sonicated in solubilization buffer (20 mM Tris-HCl, pH 7.5; 150 mM NaCl; 1 mM EDTA; 1 mM EGTA, 1% Triton X-100; 2.5 mM sodium pyrophosphate; 1 mM sodium vanadate; 10 µg/ml each aprotinin and leupeptin; 1 mM phenylmethylsulfonyl fluoride). Cell lysates were cleared by centrifugation, and protein concentrations were determined by Lowry assay. Cell lysates containing equal amounts of protein were resolved by SDS-PAGE. Protein was transferred to a nitrocellulose membrane (Hybond, Amersham Pharmacia Biotech, Piscataway, NJ). After blocking in 20 mM Tris-HCl (pH 7.6) containing 150 mM NaCl, 0.1% Tween-20, and 5% (wt/vol) nonfat dry milk, blots were incubated with specific antibodies for MCM6 (sc-9843), MCM7 (sc-9966), PPAR{gamma} (sc-7196; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), phospho-Rb Ser 807/811 (no. 9308S; Cell Signaling Technology, Beverly, MA) or E2F-1 (05-379; Upstate Biotechnology, Lake Placid, NY). Immunoblots were cohybridized with ß-actin (sc-1616; Santa Cruz Biotechnology, Inc.) to monitor equivalent loading in different lanes. Immunoreactive bands were visualized by incubation with peroxidase-conjugated antimouse IgG antibody (Amersham Pharmacia Biotech) or antigoat IgG antibody (Santa Cruz Biotechnology, Inc.). The antigen-antibody complexes were detected using enhanced chemiluminescence (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, Inc., Rockville, MD) as described by the manufacturer. Fifteen micrograms of total RNA were denatured in formamide and formaldehyde and electrophoresed through 1% formaldehyde-containing agarose gels. After electrophoresis, the RNA was transferred to nylon membrane (Hybond N+, Amersham Pharmacia Biotech) by capillary blotting and then fixed by UV cross-linking. Hybridization was performed using PerfectHyb Plus hybridization buffer (Sigma) as directed. cDNA for MCM6 was kindly provided by Hiroshi Nojima (Department of Molecular Genetics, Osaka University, Osaka, Japan; Ref. 18). cDNA for MCM7 was used as previously described (19). Probes for MCM6 and MCM7 used in the hybridization were radiolabeled with [{alpha}-32P]deoxycytidine triphosphate (ICN Biomedicals, Irvine, CA) using the Rediprime II random prime labeling system (Amersham Pharmacia Biotech). Blots were cohybridized with Chinese hamster ovary gene B (CHOB), a constitutively expressed housekeeping gene encoding a ribosomal protein, to assess equal loading of samples.

cDNA Array Assay
To analyze cell cycle gene regulation, we employed a commercially available biased cDNA array corresponding to 96 cell-cycle-regulatory human genes and 12 housekeeping genes (Cell Cycle GEArray Q series version 1, SuperArray, Bethesda, MD) according to the manufacturer’s instructions. Briefly, growing RASMC were treated with the nTZDpa (5 µM) for 24 h. Ten micrograms of total RNA were reverse transcribed into biotin-16-deoxy-UTP-labeled single-strand cDNA by Moloney murine eeukemia virus reverse transcriptase. After prehybridization, membranes were hybridized with biotin-labeled cDNA and incubated with alkaline-phosphatase-conjugated streptavidin. Chemiluminescence was visualized by autoradiography. Five housekeeping genes were included to confirm the integrity of RNA and correct loading of different samples.

Adenoviral Infection of RASMC
To generate constitutively active PPAR{gamma}, the VP16 transactivation domain of the HSV was fused to the N terminus of PPAR{gamma}1 as previously described (26). Recombinant type 5 adenovirus overexpressing this constitutively active PPAR{gamma} mutant was generated using the Adeno X Expression System (CLONTECH Laboratories, Inc., Palo Alto, CA) and designated as Adx-CA-PPAR{gamma}. Recombinant type 5 adenovirus expressing GFP gene was generated similarly and used as a control vector (Adx-GFP) in all experiments. Adenovirus encoding human E2F-1, driven by the cytomegalovirus (CMV) immediate-early promoter (Adx-E2F), was provided by Dr. Robb MacLellan (University of California, Los Angeles; Ref. 51). RASMC were infected with 20 or 100 plaque-forming units (PFU)/cell in DMEM containing 0.4% FBS for 24 h. After further starvation for 24 h, cells were pretreated with the nTZDpa for 30 min and the mitogen was added for 24 h.

Plasmids and Transient Transfection
The acyl-CoA oxidase PPRE-Tk-luciferase reporter construct was kindly provided by Dr. Peter Tontonoz (University of California, Los Angeles; Ref. 52). The full-length PPAR{gamma}1 expression vector was obtained from Dr. Alex Elbrecht (Merck Research Laboratories, Rahway, NJ; Ref. 53). To generate CA-PPAR{gamma}, the VP16 transactivation domain of the HSV was fused to the N terminus of PPAR{gamma}1 as previously described (26). D/N-PPAR{gamma} was constructed by mutating the Leu468 and Glu471 of the full-length PPAR{gamma}1 into Ala. Mutations at these sites create a D/N form of PPAR{gamma} (23). The human MCM6 promoter pHSMCM6-Luc(-754) driven by a luciferase reporter plasmid was kindly provided by Dr. Hiroshi Nojima. The human MCM7 promoter pHsMCM7-Luc(-558) luciferase reporter plasmid was used as previously as described (19). The pE2F-TA-Luc luciferase reporter containing four E2F enhancer elements was from CLONTECH Laboratories, Inc. (pE2F-Luc, Mercury Pathway Profiling Luciferase System 4).

To determine ligand-induced PPAR{gamma} transcriptional activity, 200 ng DNA of the acyl-CoA oxidase PPRE-Tk-luciferase reporter construct were cotransfected with 400 ng DNA of full-length PPAR{gamma}1 using LipofectAMINE 2000 (Invitrogen, Rockville, MD) because transcriptional activation of endogenous PPAR{gamma} in RASMC was modest. Twenty-four hours after transfection, cells were starved in DMEM containing 0.4% FBS for 24 h and then stimulated for 24 h with the PPAR{gamma} agonists. For analysis of MCM6, MCM7, and E2F transcriptional activation, 500 ng/ml of the pHSMCM6-Luc(-754), pHsMCM7-Luc(-558), or pE2F-TA-Luc reporter plasmid was transfected. To determine a PPAR{gamma}-dependent mechanism, pHsMCM7-Luc(-558) was cotransfected with either 500 ng/ml of the CA-PPAR{gamma} or D/N-PPAR{gamma} expression vector in RASMC or with the wild-type PPAR{gamma}1 expression vector in NIH3T3 fibroblasts. Twenty-four hours after the transfection, cells were starved in DMEM containing 0.4% FBS for 24 h. Cells were then pretreated with the indicated concentrations of the nTZDpa 30 min before the addition PDGF-BB (20 ng/ml) and insulin (1 µm) and stimulated for 24 h. Luciferase activity was assayed using a Dual Luciferase Reporter Assay System (Promega Corp., Madison, WI) 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 Corp.). All experiments were repeated at least three times with different cell preparations.

Statistics
Statistical significance was determined using the Student’s t test, and P < 0.05 was considered to be statistically significant. Data were expressed as mean ± SEM.


   ACKNOWLEDGMENTS
 
We are indebted to Dr. Hiroshi Nojima for providing the human MCM6 cDNA and the human MCM6 promoter reporter plasmid. The authors thank Dr. Timothy M. Willson for kindly providing the PPAR{gamma} antagonist GW9662 and Dr. David E. Moller for providing the nTZDpa compound.


   FOOTNOTES
 
This study was supported by a NIH Grant HL-58328 (to W.A.H.). D.B. was supported by a grant from MSD Sharp & Dohme (Acute Coronary Syndrome 2000) and by a research fellowship from the Gonda (Goldschmied) Diabetes Center, University of California, Los Angeles.

Abbreviations: Adx, Adenovirus; BrdU, bromodeoxyuridine; CA, constitutively active; CHOB, Chinese hamster ovary gene B; CMV, cytomegalovirus; CoA, coenzyme A; D/N, dominant negative; FBS, fetal bovine serum; GFP, green fluorescent protein; HSV, herpes simplex virus; MCM, minichromosome maintenance; nTZDpa, non-TZD partial PPAR{gamma} agonist; PDGF, platelet-derived growth factor; PFU, plaque-forming units; PPAR{gamma}, peroxisome proliferator-activated receptor {gamma}; PPRE, peroxisome proliferator-response element; RASMC, rat aortic VSMC; Rb, retinoblastoma protein; RSG, rosiglitazone; TRO, troglitazone; TZD, thiazolidinedione; VSMC, vascular smooth muscle cells.

Received for publication December 9, 2002. Accepted for publication March 26, 2003.


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NURSA Molecule Pages Link:

Nuclear Receptors:   PPARγ
Ligands:   GW 9662  |  Rosiglitazone



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