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UltraRapid Communication |
in Vascular Smooth Muscle Cells
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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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
ligandmediated 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-1mediated 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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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 proliferatorresponse 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 G1
S 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.1417 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.2225
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 cycleregulated 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-1mediated induction of GADD45 by PPAR
as a novel mechanism for regulating VSMC growth and survival.
| Materials and Methods |
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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-FITCconjugated 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 manufacturers instructions. The total number of apoptotic cells was expressed as fold induction of annexin V-FITCpositive 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 PPAR
1 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 (T1/2) was calculated using the equation C=C0eKdt, 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 Kd 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 manufacturers 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 Students t test or ANOVA when comparing multiple groups. Values of P<0.05 were considered as statistically significant.
| Results |
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Ligands Induce Apoptosis of Human Coronary VSMCs
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-FITCconjugated 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-FITCpositive 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.
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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 cells28,29 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.
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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 PPAR
1 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.
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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 activation3739 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.
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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.
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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.3739
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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.
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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).
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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.
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| Discussion |
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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 G1
S 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 cycleregulating 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 p21cip1 and subsequent suppression of cyclin-dependent kinase activity causing hypophosphorylation of Rb that results in G0/G1 arrest.44 In addition, Zhan et al28,33 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
ligandmediated 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 activity3739 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.3739 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 |
|---|
| Footnotes |
|---|
Original received April 21, 2003; resubmission received July 2, 2003; accepted July 16, 2003.
| References |
|---|
|
|
|---|
2. Schwartz SM. Perspectives series: cell adhesion in vascular biology: smooth muscle migration in atherosclerosis and restenosis. J Clin Invest. 1997; 99: 28142816.[Medline] [Order article via Infotrieve]
3. Dzau VJ, Braun-Dullaeus RC, Sedding DG. Vascular proliferation and atherosclerosis: new perspectives and therapeutic strategies. Nat Med. 2002; 8: 12491256.[CrossRef][Medline] [Order article via Infotrieve]
4. Geng YJ, Libby P. Progression of atheroma: a struggle between death and procreation. Arterioscler Thromb Vasc Biol. 2002; 22: 13701380.
5. Schoonjans K, Martin G, Staels B, Auwerx J. Peroxisome proliferator-activated receptors, orphans with ligands and functions. Curr Opin Lipidol. 1997; 8: 159166.[Medline] [Order article via Infotrieve]
6. Law RE, Goetze S, Xi XP, Jackson S, Kawano Y, Demer L, Fishbein MC, Meehan WP, Hsueh WA. Expression and function of PPAR
in rat and human vascular smooth muscle cells. Circulation. 2000; 101: 13111318.
7. Marx N, Schonbeck U, Lazar MA, Libby P, Plutzky J. Peroxisome proliferator-activated receptor gamma activators inhibit gene expression and migration in human vascular smooth muscle cells. Circ Res. 1998; 83: 10971103.
8. Tugwood JD, Issemann I, Anderson RG, Bundell KR, McPheat WL, Green S. The mouse peroxisome proliferator activated receptor recognizes a response element in the 5' flanking sequence of the rat acyl CoA oxidase gene. EMBO J. 1992; 11: 433439.[Medline] [Order article via Infotrieve]
9. Nolte RT, Wisely GB, Westin S, Cobb JE, Lambert MH, Kurokawa R, Rosenfeld MG, Willson TM, Glass CK, Milburn MV. Ligand binding and co-activator assembly of the peroxisome proliferator-activated receptor-gamma. Nature. 1998; 395: 137143.[CrossRef][Medline] [Order article via Infotrieve]
10. Uppenberg J, Svensson C, Jaki M, Bertilsson G, Jendeberg L, Berkenstam A. Crystal structure of the ligand binding domain of the human nuclear receptor PPAR
. J Biol Chem. 1998; 273: 3110831112.
11. Lehmann JM, Moore LB, Smith-Oliver TA, Wilkison WO, Willson TM, Kliewer SA. An antidiabetic thiazolidinedione is a high affinity ligand for peroxisome proliferator-activated receptor gamma (PPAR
). J Biol Chem. 1995; 270: 1295312956.
12. Wakino S, Kintscher U, Kim S, Yin F, Hsueh WA, Law RE. Peroxisome proliferator-activated receptor gamma ligands inhibit retinoblastoma phosphorylation and G1
S transition in vascular smooth muscle cells. J Biol Chem. 2000; 275: 2243522441.
13. Bruemmer D, Yin F, Liu J, Berger JP, Kiyono T, Chen J, Fleck E, Van Herle AJ, Forman BM, Law RE. Peroxisome proliferator-activated receptor
inhibits expression of minichromosome maintenance proteins in vascular smooth muscle cells. Mol Endocrinol. 2003; 17: 10051018.
14. Law RE, Meehan WP, Xi XP, Graf K, Wuthrich DA, Coats W, Faxon D, Hsueh WA. Troglitazone inhibits vascular smooth muscle cell growth and intimal hyperplasia. J Clin Invest. 1996; 98: 18971905.[Medline] [Order article via Infotrieve]
15. Igarashi M, Takeda Y, Ishibashi N, Takahashi K, Mori S, Tominaga M, Saito Y. Pioglitazone reduces smooth muscle cell density of rat carotid arterial intima induced by balloon catheterization. Horm Metab Res. 1997; 29: 444449.[Medline] [Order article via Infotrieve]
16. Shinohara E, Kihara S, Ouchi N, Funahashi T, Nakamura T, Yamashita S, Kameda-Takemura K, Matsuzawa Y. Troglitazone suppresses intimal formation following balloon injury in insulin-resistant Zucker fatty rats. Atherosclerosis. 1998; 136: 275279.[CrossRef][Medline] [Order article via Infotrieve]
17. Aizawa Y, Kawabe J, Hasebe N, Takehara N, Kikuchi K. Pioglitazone enhances cytokine-induced apoptosis in vascular smooth muscle cells and reduces intimal hyperplasia. Circulation. 2001; 104: 455460.
18. Bishop-Bailey D, Hla T. Endothelial cell apoptosis induced by the peroxisome proliferator-activated receptor (PPAR) ligand 15-deoxy-
12,14-prostaglandin J2. J Biol Chem. 1999; 274: 1704217048.
19. Okura T, Nakamura M, Takata Y, Watanabe S, Kitami Y, Hiwada K. Troglitazone induces apoptosis via the p53 and Gadd45 pathway in vascular smooth muscle cells. Eur J Pharmacol. 2000; 407: 227235.[CrossRef][Medline] [Order article via Infotrieve]
20. Bishop-Bailey D, Hla T, Warner TD. Intimal smooth muscle cells as a target for peroxisome proliferator-activated receptor-
ligand therapy. Circ Res. 2002; 91: 210217.
21. Chinetti G, Griglio S, Antonucci M, Torra IP, Delerive P, Majd Z, Fruchart JC, Chapman J, Najib J, Staels B. Activation of proliferator-activated receptors
and
induces apoptosis of human monocyte-derived macrophages. J Biol Chem. 1998; 273: 2557325580.
22. Elstner E, Muller C, Koshizuka K, Williamson EA, Park D, Asou H, Shintaku P, Said JW, Heber D, Koeffler HP. Ligands for peroxisome proliferator-activated receptor
and retinoic acid receptor inhibit growth and induce apoptosis of human breast cancer cells in vitro and in BNX mice. Proc Natl Acad Sci U S A. 1998; 95: 88068811.
23. Takahashi N, Okumura T, Motomura W, Fujimoto Y, Kawabata I, Kohgo Y. Activation of PPAR
inhibits cell growth and induces apoptosis in human gastric cancer cells. FEBS Lett. 1999; 455: 135139.[CrossRef][Medline]
[Order article via Infotrieve]
24. Chang TH, Szabo E. Induction of differentiation and apoptosis by ligands of peroxisome proliferator-activated receptor
in non-small cell lung cancer. Cancer Res. 2000; 60: 11291138.
25. Eibl G, Wente MN, Reber HA, Hines OJ. Peroxisome proliferator-activated receptor gamma induces pancreatic cancer cell apoptosis. Biochem Biophys Res Commun. 2001; 287: 522529.[CrossRef][Medline] [Order article via Infotrieve]
26. Sheikh MS, Hollander MC, Fornance AJ Jr. Role of Gadd45 in apoptosis. Biochem Pharmacol. 2000; 59: 4345.[CrossRef][Medline] [Order article via Infotrieve]
27. Liebermann DA, Hoffman B. Myeloid differentiation (MyD) primary response genes in hematopoiesis. Oncogene. 2002; 21: 33913402.[CrossRef][Medline] [Order article via Infotrieve]
28. Zhan Q, Lord KA, Alamo I Jr, Hollander MC, Carrier F, Ron D, Kohn KW, Hoffman B, Liebermann DA, Fornace AJ Jr. The gadd and MyD genes define a novel set of mammalian genes encoding acidic proteins that synergistically suppress cell growth. Mol Cell Biol. 1994; 14: 23612371.
29. Jin S, Antinore MJ, Lung FD, Dong X, Zhao H, Fan F, Colchagie AB, Blanck P, Roller PP, Fornace AJ, Jr., Zhan Q. The GADD45 inhibition of Cdc2 kinase correlates with GADD45-mediated growth suppression. J Biol Chem. 2000; 275: 1660216608.
30. Takekawa M, Saito H. A family of stress-inducible GADD45-like proteins mediate activation of the stress-responsive MTK1/MEKK4 MAPKKK. Cell. 1998; 95: 521530.[CrossRef][Medline] [Order article via Infotrieve]
31. Harkin DP, Bean JM, Miklos D, Song YH, Truong VB, Englert C, Christians FC, Ellisen LW, Maheswaran S, Oliner JD, Haber DA. Induction of GADD45 and JNK/SAPK-dependent apoptosis following inducible expression of BRCA1. Cell. 1999; 97: 575586.[CrossRef][Medline] [Order article via Infotrieve]
32. Hall PA, Kearsey JM, Coates PJ, Norman DG, Warbrick E, Cox LS. Characterisation of the interaction between PCNA and Gadd45. Oncogene. 1995; 10: 24272433.[Medline] [Order article via Infotrieve]
33. Zhan Q, Antinore MJ, Wang XW, Carrier F, Smith ML, Harris CC, Fornace AJ Jr. Association with Cdc2 and inhibition of Cdc2/Cyclin B1 kinase activity by the p53-regulated protein Gadd45. Oncogene. 1999; 18: 28922900.[CrossRef][Medline] [Order article via Infotrieve]
34. Berger JP, Petro AE, Macnaul KL, Kelly LJ, Zhang BB, Richards K, Elbrecht A, Johnson BA, Zhou G, Doebber TW, Biswas C, Parikh M, Sharma N, Tanen MR, Thompson GM, Ventre J, Adams AD, Mosley R, Surwit RS, Moller DE. Distinct properties and advantages of a novel peroxisome proliferator-activated protein
selective modulator. Mol Endocrinol. 2003; 17: 662676.
35. Leesnitzer LM, Parks DJ, Bledsoe RK, Cobb JE, Collins JL, Consler TG, Davis RG, Hull-Ryde EA, Lenhard JM, Patel L, Plunket KD, Shenk JL, Stimmel JB, Therapontos C, Willson TM, Blanchard SG. Functional consequences of cysteine modification in the ligand binding sites of peroxisome proliferator activated receptors by GW9662. Biochemistry. 2002; 41: 66406650.[CrossRef][Medline] [Order article via Infotrieve]
36. Wang N, Verna L, Chen NG, Chen J, Li H, Forman BM, Stemerman MB. Constitutive activation of peroxisome proliferator-activated receptor-
suppresses pro-inflammatory adhesion molecules in human vascular endothelial cells. J Biol Chem. 2002; 277: 3417634181.
37. Takahashi S, Saito S, Ohtani N, Sakai T. Involvement of the Oct-1 regulatory element of the gadd45 promoter in the p53-independent response to ultraviolet irradiation. Cancer Res. 2001; 61: 11871195.
38. Fan W, Jin S, Tong T, Zhao H, Fan F, Antinore MJ, Rajasekaran B, Wu M, Zhan Q. BRCA1 regulates GADD45 through its interactions with the OCT-1 and CAAT motifs. J Biol Chem. 2002; 277: 80618067.
39. Jin S, Fan F, Fan W, Zhao H, Tong T, Blanck P, Alomo I, Rajasekaran B, Zhan Q. Transcription factors Oct-1 and NF-YA regulate the p53-independent induction of the GADD45 following DNA damage. Oncogene. 2001; 20: 26832690.[CrossRef][Medline] [Order article via Infotrieve]
40. Rishi AK, Sun RJ, Gao Y, Hsu CK, Gerald TM, Saeed Sheikh M, Dawson MI, Reichert U, Shroot B, Fornace AJ Jr, Brewer G, Fontana JA. Post-transcriptional regulation of the DNA damage-inducible gadd45 gene in human breast carcinoma cells exposed to a novel retinoid CD437. Nucleic Acids Res. 1999; 27: 31113119.
41. Sakaue M, Adachi H, Jetten AM. Post-transcriptional regulation of MyD118 and GADD45 in human lung carcinoma cells during 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid-induced apoptosis. Mol Pharmacol. 1999; 55: 668676.
42. Wang XW, Zhan Q, Coursen JD, Khan MA, Kontny HU, Yu L, Hollander MC, OConnor PM, Fornace AJ Jr, Harris CC. GADD45 induction of a G2/M cell cycle checkpoint. Proc Natl Acad Sci U S A. 1999; 96: 37063711.
43. Smith ML, Chen IT, Zhan Q, Bae I, Chen CY, Gilmer TM, Kastan MB, OConnor PM, Fornace AJ Jr. Interaction of the p53-regulated protein Gadd45 with proliferating cell nuclear antigen. Science. 1994; 266: 13761380.
44. Lin K, Hsu PP, Chen BP, Yuan S, Usami S, Shyy JY, Li YS, Chien S. Molecular mechanism of endothelial growth arrest by laminar shear stress. Proc Natl Acad Sci U S A. 2000; 97: 93859389.
45. Wakino S, Kintscher U, Liu Z, Kim S, Yin F, Ohba M, Kuroki T, Schonthal AH, Hsueh WA, Law RE. Peroxisome proliferator-activated receptor
ligands inhibit mitogenic induction of p21(Cip1) by modulating the protein kinase C pathway in vascular smooth muscle cells. J Biol Chem. 2001; 276: 4765047657.
46. Lennon AM, Ramauge M, Dessouroux A, Pierre M. MAP kinase cascades are activated in astrocytes and preadipocytes by 15-deoxy-
(1214)-prostaglandin J(2) and the thiazolidinedione ciglitazone through peroxisome proliferator activator receptor
independent mechanisms involving reactive oxygenated species. J Biol Chem. 2002; 277: 2968129685.
47. Palakurthi SS, Aktas H, Grubissich LM, Mortensen RM, Halperin JA. Anticancer effects of thiazolidinediones are independent of peroxisome proliferator-activated receptor gamma and mediated by inhibition of translation initiation. Cancer Res. 2001; 61: 62136218.
48. Ohtani-Fujita N, Minami S, Mimaki S, Dao S, Sakai T. p53-Independent activation of the gadd45 promoter by
12-prostaglandin J2. Biochem Biophys Res Commun. 1998; 251: 648652.[CrossRef][Medline]
[Order article via Infotrieve]
49. Satoh T, Toyoda M, Hoshino H, Monden T, Yamada M, Shimizu H, Miyamoto K, Mori M. Activation of peroxisome proliferator-activated receptor-
stimulates the growth arrest and DNA-damage inducible 153 gene in non-small cell lung carcinoma cells. Oncogene. 2002; 21: 21712180.[CrossRef][Medline]
[Order article via Infotrieve]
50. Sturm RA, Das G, Herr W. The ubiquitous octamer-binding protein Oct-1 contains a POU domain with a homeo box subdomain. Genes Dev. 1988; 2: 15821599.
51. Tong T, Fan W, Zhao H, Jin S, Fan F, Blanck P, Alomo I, Rajasekaran B, Liu Y, Holbrook NJ, Zhan Q. Involvement of the MAP kinase pathways in induction of GADD45 following UV radiation. Exp Cell Res. 2001; 269: 6472.[CrossRef][Medline] [Order article via Infotrieve]
52. Zhao H, Jin S, Fan F, Fan W, Tong T, Zhan Q. Activation of the transcription factor Oct-1 in response to DNA damage. Cancer Res. 2000; 60: 62766280.
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