HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 277, Issue 30, 26808-26814, July 26, 2002

This Article Abstract Full Text (PDF) All Versions of this Article: 277/30/26808    most recent M203748200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Fu, M. Articles by Chen, Y. E. Search for Related Content PubMed PubMed Citation Articles by Fu, M. Articles by Chen, Y. E. Social Bookmarking                      What's this?

Early Growth Response Factor-1 Is a Critical Transcriptional Mediator of Peroxisome Proliferator-activated Receptor-1 Gene Expression in Human Aortic Smooth Muscle Cells^*

Mingui Fu^§, Jifeng Zhang^§, Yiming Lin, Xiaojun Zhu, Markus U. Ehrengruber^¶, and Yuqing E. Chen^**

From the  Cardiovascular Research Institute, Morehouse School of Medicine, Atlanta, Georgia 30310, the ^¶ Brain Research Institute, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland, and the  Cardiovascular Research Institute, Peking University Health Science Center, 38 Xue Yuan Road, Beijing, 100083 People's Republic of China

Received for publication, April 18, 2002, and in revised form, May 7, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

To explore the molecular mechanisms of PPAR1 gene expression in vascular smooth muscle cells (VSMC), we hypothesized that early growth-response factor-1 (Egr-1) might be a transcriptional mediator of the growth factor- and cytokine-induced PPAR1 gene expression since a putative Egr-1 binding element was found in the human PPAR1 promoter. In this study, we document that overexpression of Egr-1 activates the human PPAR1 promoter in both VSMC and HepG2 cells. Using Northern blot analysis, we observed that growth factors and cytokines such as PDGF, bFGF, Ang II, TNF, and IL-1 induce Egr-1 expression prior to PPAR1 up-regulation in human VSMC. In addition, overexpression of a constitutively active form of Egr-1 by adenoviral gene transfer in VSMC dramatically induced PPAR1 gene expression by 6-8-fold, and overexpression of NAB2, a potent negative feedback regulator of Egr-1, abrogated the growth factor- and cytokine-induced PPAR1 expression in VSMC. Furthermore, we demonstrate with gel mobility shift and transient transfection assays that the putative Egr-1 element in the human PPAR1 promoter specifically binds Egr-1 protein and becomes trans-activated by Egr-1. Taken together, our data demonstrate for the first time that Egr-1 is necessary and sufficient to activate human PPAR1 gene expression in VSMC.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Peroxisome proliferator-activated receptors (PPARs)¹ are a family of ligand-activated nuclear transcriptional factors (1, 2) that include three members, PPAR, , and /. PPAR is found predominantly in adipose tissue where it plays a crucial role in adipocyte differentiation, fat storage, and glucose homeostasis (3). PPAR has two subtypes, PPAR1 and PPAR2, which are generated from the same gene by using different promoters and alternative splicing. The two PPAR transcripts differ in their 5'-ends where PPAR2 encodes 30 additional amino acids to the start codon of PPAR1 (4, 5). Due to the use of different promoters, PPAR1 and PPAR2 have distinct tissue distributions. PPAR2 is selectively expressed in adipose tissue, whereas PPAR1 is broadly distributed in many tissues including adipose tissue, heart, spleen, colon, and vasculature (6, 7).

It is well documented that PPAR2 gene expression is directly regulated by CCAAT/enhancer-binding proteins (C/EBPs) (8). Two C/EBP binding sites were identified in the promoter of the PPAR2 gene, whereas no such sites exist in the PPAR1 promoter (9, 10). In recent years the role of PPAR1 in vasculature has been intensively investigated; however, the transcriptional regulation of the PPAR1 gene remains poorly understood. Although our previous study demonstrated that only PPAR1 exists in human aortic smooth muscle cells (HASMC) and that platelet-derived growth factor (PDGF) up-regulates PPAR1 gene expression by PI3 kinase/Akt and MAPK-mediated signal pathways (11), the precise mechanism of PPAR1 transcriptional regulation is still unknown. We now analyzed the human PPAR1 promoter and found a putative binding site for early growth-response gene-1 (Egr-1) between nt 184 and 173 of the human PPAR1 promoter, suggesting that Egr-1 may be a key mediator to regulate PPAR1 gene expression in VSMC.

Egr-1 is a DNA-binding protein containing three zinc finger motifs that regulates gene transcription by interacting with a consensus G+C-rich sequence, 5'-GCG(T/G)GGGCG-3', also termed the GSG motif (12). Structure analysis of Egr-1 identified an inhibitory domain (34 amino acids, called R1) at the 5' zinc finger binding domain. Two corepressors, NGFI-A-binding proteins 1 and 2 (NAB1 and NAB2) can markedly decrease Egr-1 transcriptional activity by binding to this domain (13). NAB2 is a target gene of Egr-1, thus providing a negative feedback to limit Egr-1 activity (14), and both NAB1 and NAB2 provide useful tools to study Egr-1 function in situ. Egr-1 is a critical mediator of cell proliferation, differentiation, and apoptosis and is rapidly and transiently induced by many growth factors and cytokines (15). In the vasculature Egr-1 is capable of activating the transcription of several genes implicated in the pathogenesis of vascular diseases, including PDGF-A, PDGF-B, FGF-2, TNF-, interleukin-2 (IL-2), tissue factor, and urokinase-type plasminogen activator (u-PA) (16). In turn, many of these gene products also stimulate the expression of Egr-1. These positive feedback loops serve to amplify and sustain gene transcription through Egr-1-mediated mechanisms.

In this study we demonstrate that Egr-1 is the transcriptional mediator of growth factor- and cytokine-induced PPAR1 gene expression in VSMC.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- Human recombinant PDGF-BB, bFGF, AngII, TNF, and IL-1 were purchased from Sigma. PMA (phorbol 12-myristate 13-acetate) and cycloheximide were also purchased from Sigma. LipofectAMINE2000 was from Invitrogen. [-³²P]ATP and [-³²P]dCTP were obtained from PerkinElmer Life Sciences. Gel mobility shift assay and luciferase assay kits were from Promega. Rabbit anti-PPAR polyclonal antibody (sc-7196), rabbit anti-Egr-1 polyclonal antibody (sc-189), rabbit anti-NAB2 polyclonal antibody (sc-12153), goat anti-actin polyclonal antibody, and rabbit anti-c-Fos polyclonal antibody (sc-52) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Egr-1 (sc-189x) and c-Fos (sc-52x) antibodies for supershift mobility assay were also from Santa Cruz Biotechnology.

Plasmids and Adenoviral Recombinants-- The reporter construct pGL3-1p3000 containing ~3 kb of regulatory sequence of the human PPAR1 gene was a generous gift of Dr. J. Auwerx (IGBMC, Centre Universitaire de Strasbourg, France) (4). The Egr-1 expression vector pcDNA3-Egr-1 was constructed in our laboratory. Briefly, we designed two human Egr-1 primers, 5'-tgaagcttCTCCAGCCTGCTCGTCCAGGATG-3', which contains the HindIII linker (lowercase letters) and human Egr-1 sequence nt 251-273 of Genbank^TM NM_001964 (capital letters), and 5'-cttctagaATGGCCATCTCCTCCTCCTG-TCCT-3', which contains the XbaI linker (lowercase letters) and human Egr-1 sequence nt 1955-1978 of Genbank^TM NM_001964 (capital letters). Using these two primers, we amplified an ~1.7-kb fragment from human vascular smooth muscle cells by reverse transcriptase (RT)-PCR. This fragment was digested by HindIII and XbaI and cloned into pcDNA3.1(+). After sequencing this fragment, the resultant plasmid was designated as pcDNA3.1-Egr-1. To generate reporter constructs, synthetic three-tandem repeats of the putative Egr-1 binding sequence, 5'-ctactgtgcgcgggcggcggc-3' of nt 189 to 169 from the human PPAR1 promoter (GenBank^TM number NT_005927.8) or its mutated sequence, 5'-ctactgtgcAcAAAcAgcggc-3', were inserted into the promoter region of the TK-luciferase vector (Promega). The resultant plasmids were designed as pEgr-1WT×3-TK-Luc and pEgr-1Mut×3-TK-Luc respectively. The adenoviral recombinants containing constitutively active (NAB-insensitive) Egr-1 (AdGFP Egr-1*), wild type Egr-1 (Ad Egr-1), AdGFP NAB2, and the control adenoviruses AdGFP and AdLacZ were described previously (14).

Cell Culture-- HASMC was purchased from BioWhittaker (San Diego, CA) and cultured in smooth muscle cell growth medium-2 containing 5% fetal bovine serum, 2 ng/ml human basic fibroblast growth factor, 0.5 ng/ml human epidermal growth factor, 50 µg/ml gentamicin, 50 ng/ml amphotericin-B, and 5 µg/ml bovine insulin. For all experiments, early passages (5-7) of HASMC were grown to 80-90% confluence and rendered quiescent by serum starvation in Optium-MEM (Invitrogen) for 48 h. The HepG2 cell line and the rat aortic smooth muscle cell line (A7r5) were purchased from the American Type Culture Collection and cultured in Dulbecco's modified Eagle's medium supplemented with 10%(v/v) fetal bovine serum in a 5% CO₂ humidified atmosphere at 37 °C. Cycloheximide was added to the cells 30 min prior to the stimulation with growth factors or cytokines. Adenoviral infection was performed as previously described (14).

Northern Blot and Western Blot Analyses-- Both Northern blot and Western blot analyses were performed as previously described (11).

Transient Transfection and Luciferase Assays-- HepG2 cells and A7r5 cells, grown to 80% confluence in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, were transiently transfected using LipofectAMINE2000 (Invitrogen) with reporter and expression plasmids as described in the figure legends. The green fluorescence protein (GFP) expression plasmid (CLONTECH CA) was co-transfected as control for transfection efficiency. The total amount of transfected DNA was kept constant by using a corresponding empty vector. 24 h after transfection cells were cultured for 24 h in serum-free medium and incubated for 6-12 h in the same medium containing appropriate reagents for experiments. A reporter luciferase assay kit (Promega) was used to measure the luciferase activity from cells according to the manufacturer's instructions with a luminometer (Victor II, PerkinElmer Life Sciences). Luciferase activity was normalized by GFP activity.

Gel Mobility Shift Assay-- Nuclear extracts were isolated from untreated HASMC or HASMC infected with Ad Egr-1 or AdLacZ using a nuclear protein isolation kit (Pierce). Nucleotide sequences of the sense strand of the double-stranded oligonucleotides were as follows: Oligo-Egr1, 5'-GGATCCAGCGGGGGCGAGCGGGGGCGA-3' containing two consensus Egr-1 binding sites (underlined) from Santa Cruz Biotechnology; Oligo-Egr-1WT, 5'-GGGCCTACTGTGCGCGGGCGGCGGCCGAGCCC-3' containing the putative Egr-1 binding sequence (underlined) in nt 193 to 162 of the human PPAR1 promoter; Oligo-Egr-1Mut, 5'-GGGCCTACTGTGCtCaaaCaGCGGCCGAGCCC-3' in which the Egr-1 site within the human PPAR1 promoter was mutated (lowercase letters). Binding reactions for gel shift assays were performed in 20 µl of 10 mM HEPES, pH 8, 0.1 mM EDTA, 50 mM NaCl, 50 mM KCl, 5 mM MgCl₂, 4 mM spermidine, 2 mM dithiothreitol, 1 µg of bovine serum albumin, 1 µg of poly deoxyinosinic-deoxycytidylic (dI-dC), and ³²P-labeled oligonucleotide probe (50,000 cpm) with 8 µg of nuclear extract for 30 min at room temperature. The samples were then loaded onto a 5% nondenaturating polyacrylamide gel. The gels were subjected to electrophoresis, drying, and autoradiography. For supershift assays, 2 µg of the appropriate antibody was preincubated with the crude nuclear extract for 10 min at room temperature before the labeled probe was added. For the competition assay, unlabeled competitor DNA fragments were preincubated with the parallel samples 10 min before labeled probe was added.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Sequence Analysis of the Human PPAR1 Promoter-- To explore the molecular mechanisms of the transcriptional regulation of the human PPAR1 gene, the 5-kb sequence of the human PPAR1 promoter was analyzed. A 308-bp fragment of the published human PPAR1 promoter (GenBank^TM number AF012873) was used to search the human genome data base and was matched to a ~19,000-kb DNA fragment from chromosome 3 (Genbank^TM number NT_005927) to which PPAR1 was mapped. Using TRANSFAC4.0 (17) we analyzed the 5-kb PPAR1 promoter, which is relative to the transcription initiation site reported by Fajas et al. (4). No canonical TATA box was found in the PPAR1 promoter region close to the transcription initiation site. Interestingly, a putative Egr-1 binding site, which overlapped with an Sp1 binding site, was found between nt 184 and 173 relative to the transcription start site in the PPAR1 promoter. Other transcription factor binding sites such as NF-AT, AP1, MyoD, and NF-B were also identified in the human PPAR1 promoter. A portion (~1.3 kb) sequence of the human PPAR1 promoter is shown in Fig. 1. To investigate which transcription factors may be involved in the regulation of the PPAR1 gene transcription, we transiently cotransfected pGL3-1p3000, a luciferase reporter construct driven by ~3 kb of the PPAR1 promoter with several expression plasmids such as pcDNA3.1-Egr-1, pcDNA3.1-FosB, pcDNA3.1-JunD, pcDNA3.1-p65, and pcDNA3.1-Smad3 into HepG2 cells or A7r5 cells. As shown in Fig. 2, overexpression of pcDNA3.1-Egr-1 increased PPAR1 promoter activity by ~2.5- and ~2-fold in HepG2 and A7r5 cells, respectively. However, all other transcriptional factors tested failed to activate the human PPAR1 promoter (data not shown).

View larger version (50K): [in this window] [in a new window]   Fig. 1.   Nucleotide sequences of the 5'-flanking promoter region of the human PPAR1 gene. The putative cis-acting regulatory elements are underlined. The transcription start site is designated as +1.

View larger version (14K): [in this window] [in a new window]   Fig. 2.   Overexpression of Egr-1 activates the human PPAR1 promoter. HepG2 cells (A) and A7r5 cells (B) were co-transfected with pGL3-P3000 and pcDNA3.1-Egr-1 or pcDNA3.1 expression plasmids. GFP reporter plasmid was used as a control for transfection efficiency. The luciferase activities normalized by GFP fluorescence were expressed relative to pcDNA3.1 (mean ± S.E., n = 6).

Egr-1 Is a Key Mediator of PPAR1 Gene Expression Induced by Growth Factors and Cytokines-- Our previous study demonstrated that PDGF stimulation up-regulates PPAR1 gene expression in HASMC (11), but the precise mechanism of PPAR1 transcriptional regulation remained unknown. The identification of a functional Egr-1 binding site in the human PPAR1 promoter (above) led us to hypothesize that Egr-1 may be a key regulator of PPAR1 gene expression in VSMC. Using Northern blot analyses, we found that PPAR expression was significantly induced in HASMC by stimulation with PDGF-BB (20 ng/ml), bFGF (25 ng/ml), Ang II (3 × 10⁷ mol/liter), TNF (10 ng/ml), IL-1 (10 ng/ml) or PMA (100 ng/ml) (Fig 3). Interestingly, Egr-1 expression was significantly induced at 15 min and reached the maximum level at 1 h stimulation with the above factors (Fig 3). This temporal pattern of Egr-1 and PPAR gene expression suggests that Egr-1 is a key mediator of growth factor- and cytokine-induced PPAR gene expression in HASMC.

View larger version (44K): [in this window] [in a new window]   Fig. 3.   Time course of PDGF-, bFGF-, Ang II-, TNF-, IL-1-, and PMA-induced expression of PPAR1 and Egr-1 in HASMC. Cells were incubated with PDGF (20 ng/ml), bFGF (25 ng/ml), Ang II (3 × 107 mol/liter), TNF (10 ng/ml), IL-1 (10 ng/ml), and PMA (100 ng/ml) for different periods as indicated. PPAR and Egr-1 mRNA levels were analyzed by Northern blotting. Equal loading was confirmed by assaying for GAPDH.

To define whether Egr-1 mediates the transcriptional regulation of PPAR1 gene expression in VSMC, we used an adenoviral vector containing a constitutively active Egr-1 in which the corepressor (NAB)-binding domain is mutated (I293F). It has been documented that this mutant (named Egr-1*) of Egr-1 is NAB-insensitive and has much higher transcriptional activity than wild type Egr-1 (13, 14). In addition, the adenovirus AdGFP Egr-1* expresses the GFP reporter gene to monitor successful infection. Using Western blot analysis, we confirmed that Egr-1 was overexpressed in HASMC, which were infected with AdGFP Egr-1* (Fig. 4A). Interestingly, as shown in Fig. 4B, PPAR mRNA levels were dramatically up-regulated by ~8-fold in HASMC infected with AdGFP Egr-1* (15 plaque-forming units (pfu)/cell), whereas the control adenovirus AdGFP did not affect PPAR gene expression. In addition, we used different titers of AdGFP Egr-1* (1, 5, and 10 pfu/cell) to infect the cells. As shown in Fig. 4C, Egr-1* overexpression increased PPAR1 gene expression in a concentration-dependent manner. Taken together our data demonstrate that Egr-1 activates PPAR1 gene expression in HASMC.

View larger version (52K): [in this window] [in a new window]   Fig. 4.   Overexpression of Egr-1 up-regulates PPAR1 gene expression in HASMC. A, cells were infected with different titers of AdGFP Egr-1* (0-10 pfu/cell) for 24 h. Western blot analysis was performed to evaluate the Egr-1 protein levels. Equal loading was confirmed by assessing for -actin. B, cells were infected with or without AdGFP Egr-1* (15 pfu/cell) or AdGFP (15 pfu/cell) for 24 h and then cultured in Optium-MEM for another 24 h to render them quiescent. Representative Northern blots show the mRNA levels of PPAR1 and GAPDH. C, cells were infected with different titers of AdGFP Egr-1* (0 - 10 pfu/cell) for 24 h and then cultured in Optium-MEM for another 24 h to render them quiescent. Representative Northern blots show the mRNA levels of PPAR1 and GAPDH. The relative PPAR1 mRNA levels normalized by GAPDH are shown in the middle of panels B and C. Three independent experiments show similar results.

NAB2 is a corepressor of Egr-1; it binds to the R1 region of Egr-1 protein and inhibits the transcriptional function of Egr-1 (13). To further determine whether Egr-1 is the mediator of the growth factor- and cytokine-induced PPAR1 gene expression in HASMC, cells infected with the NAB2 adenovirus (AdGFP NAB2) or control virus (AdGFP) at 5 pfu/cell were stimulated with PDGF-BB, bFGF, AngII, TNF, IL-1, or PMA for 2 h respectively. Then, total RNA was isolated from these cells for Northern blot analyses. As shown in Fig. 5, overexpression of NAB2 abrogated PPAR1 induction by all of above growth factors and cytokines. Taken together, these results provide the first evidence that Egr-1 is a critical mediator of PPAR1 gene expression induced by growth factors and cytokines.

View larger version (44K): [in this window] [in a new window]   Fig. 5.   Overexpression of NAB2 abrogates the PPAR1 gene expression induced by PDGF-, bFGF-, Ang II-, TNF-, IL-1-, and PMA in HASMC. A, cells were infected with NAB2 adenovirus (AdGFP NAB2, 5 pfu/cell) or control adenovirus (AdGFP, 5 pfu/cell) for 24 h and then cultured in Optium-MEM for another 24 h to render them quiescent. The cells were then incubated with PDGF (20 ng/ml), bFGF (25 ng/ml), Ang II (3 × 107 mol/liter), TNF (10 ng/ml), IL-1 (10 ng/ml), and PMA (100 ng/ml) for 2 h. Representative Northern blots show the mRNA levels of PPAR1 and GAPDH. The relative PPAR1 mRNA levels normalized by GAPDH are shown in the middle of the panels in A. Three independent experiments show similar results. B, cells were infected with different titers of AdGFP NAB2 (0-10 pfu/cell) for 24 h. Western blots were performed to evaluate the protein levels of NAB2 and -actin.

Inducible PPAR1 Gene Expression Requires de Novo Protein Synthesis-- To determine whether the inducible PPAR1 gene expression is dependent on de novo protein synthesis in HASMC, the cells were pretreated with cycloheximide (10 µg/ml), a potent protein synthesis inhibitor, or vehicle for 30 min and then stimulated with PDGF-BB (10 ng/ml), PMA (100 ng/ml), or vehicle for 2 h. As shown in Fig. 6, both PDGF- and PMA-induced PPAR1 mRNA expression were dramatically decreased by cycloheximide. Interestingly, cycloheximide alone marginally increased PPAR1 gene expression in HASMC. Taken together, our data demonstrate that inducible PPAR1 gene expression requires de novo protein synthesis, which provides further support for Egr-1 being a critical mediator of PPAR1 up-regulation through growth factors and cytokines.

View larger version (61K): [in this window] [in a new window]   Fig. 6.   Effect of cycloheximide on the PDGF- and PMA-induced PPAR1 expression in HASMC. Cells were pretreated with or without cycloheximide (10 µg/ml) for 30 min and then stimulated with PDGF (20 ng/ml) or PMA (100 ng/ml) for 2 h. Representative Northern blot shows the mRNA levels of PPAR1 and GAPDH. The relative PPAR1 mRNA levels normalized by GAPDH are shown in the middle of the two panels. Three independent experiments show similar results.

Functional Relevance of the Egr-1 Binding Site in the Human PPAR1 Promoter-- To examine whether the putative Egr-1 binding site in the human PPAR1 promoter is a functional response element, we inserted three copies of the wild type and a mutated Egr-1 element into a vector encoding luciferase under the control of a minimal thymidine kinase (TK) promoter (Fig. 7A). Cotransfection of the wild type reporter (pEgr-1WTx3-Luc) with pcDNA3.1-Egr-1 into HepG2 cells (Fig. 7B) or A7r5 cells (Fig. 7C) significantly enhanced the luciferase activity, whereas the mutant reporter failed to respond to overexpressed Egr-1. These results indicate that the putative Egr-1 binding site in the human PPAR1 promoter is a functional response element for Egr-1. In addition, PDGF significantly increased luciferase activity driven by the wild type Egr-1 elements in A7r5 cells, but it failed to activate the luciferase reporter downstream of the mutated Egr-1 elements (Fig. 7D). These results suggest that the Egr-1 binding site can mediate PDGF-induced transcriptional events.

View larger version (26K): [in this window] [in a new window]   Fig. 7.   Functional study of the putative Egr-1 element in the human PPAR1 promoter. A, schematic diagram of the pEgr-1WT×3-TK-Luc (left) and pEgr-1Mut×3-TK-Luc (right) reporter constructs. B and C, pEgr-1WT×3-TK-Luc or pEgr-1Mut×3-TK-Luc reporter constructs were cotransfected with or without an Egr-1 expression plasmid in HepG2 cells (B) or A7r5 cells (C). A GFP reporter plasmid was used as a control for transfection efficiency. The luciferase activities normalized to the GFP activity are expressed relative to pEgr-1WT×3-TK-Luc plus pcDNA3.1 (mean ± S.E., n = 6). D, Egr-1WT×3-TK-Luc or Egr-1Mut×3-TK-Luc reporter constructs were cotransfected with GFP expression plasmid in A7r5 cells. After 48 h, the cells were stimulated with vehicle or PDGF at 20 ng/ml for 6 h. Luciferase activities normalized by GFP activity are expressed relative to pEgr-1WT×3-TK-Luc with no PDGF stimulation (mean ± S.E., n = 6).

Interaction of Egr-1 with the Putative Egr-1 Element in the Proximal PPAR1 Promoter-- Having demonstrated that the putative Egr-1 element is responsible for transcriptional induction of PPAR1 by PDGF, we next examined whether this element specifically binds to Egr-1. The following three double-stranded oligonucleotides were radiolabeled and subjected to a gel mobility shift assay: 1) an Egr-1 probe containing two consensus Egr-1 binding sites (purchased from Santa Cruz Biotechnology), 2) a fragment of the human PPAR1 promoter containing the wild type Egr-1 binding site (Egr1-WT), and 3) a fragment of the human PPAR1 promoter with a mutated Egr-1 binding site (Egr-1Mut). As shown in Fig. 8A, a DNA-protein complex was retarded when the Santa Cruz Egr-1 probe or the Egr-1WT probe were incubated with nuclear extracts from HASMC infected with adenovirus expressing the wild type Egr-1 (AdEgr-1). This complex, however, did not form when the Egr-1Mut probe containing no functional Egr-1 site was used. In addition, only a faint band was observed when the Santa Cruz Egr-1 probe was incubated with nuclear extracts from untreated HASMC or HASMC that had been infected with control adenovirus expressing E. coli -galactosidase (AdLacZ; Fig. 8A, lanes 3, 7, and 11). To confirm that the aforementioned complex was specifically formed by the interaction between Egr-1 protein and the Egr-1 binding element, we performed a competitive gel mobility shift assay. As shown in Fig. 8B, complex formation was abrogated by a 10-, 50-, and 200-fold excess of unlabeled Egr-1WT probe. Furthermore, an anti-Egr-1 antibody also abolished this complex formation, whereas an anti-c-Fos antibody did not affect it (Fig. 8C). Taken together, these results demonstrate that Egr-1 protein specifically binds to the putative Egr-1 site in the human PPAR1 promoter.

View larger version (61K): [in this window] [in a new window]   Fig. 8.   Egr-1 specifically binds to the putative Egr-1 element in the human PPAR1 promoter. A, nuclear extracts were isolated from untreated HASMC (lanes 2, 6, 10), HASMC infected with the control adenovirus (AdLacZ at 5 pfu/cell, lanes 3, 7, 11), and HASMC infected with an adenovirus encoding wild type Egr-1 (Ad Egr-1 at 5 pfu/cell, lanes 4, 8, 12). The Egr-1 probe from Santa Cruz Biotechnology containing two consensus Egr-1 binding sites (lanes 1, 2, 3, 4), Egr-1 wild type element (Egr-1WT) from human PPAR1 gene promoter containing one Egr-1 binding site (lanes 5, 6, 7, 8), and mutated Egr-1WT (Egr-1Mut) containing no Egr-1 binding site (lanes 9, 10, 11, 12) were radiolabeled with [32P]ATP. B, competitive gel mobility shift assay. 10-, 50-, and 200-fold molar excess of unlabeled Egr-1WT was preincubated with nuclear extracts from HASMC infected with Ad Egr-1 for 10 min before adding the 32P-labeled Egr-1WT. C, gel mobility supershift assay. 2 µg of the indicated antibodies (anti-Egr-1 or -c-Fos) were preincubated with nuclear extracts from HASMC infected with AdEgr-1 for 10 min prior to the addition of the 32P-Egr-1 probe.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

It has been well documented that all major cells of the vasculature including endothelial cells, VSMC, and monocytes/macrophages express PPAR and that PPAR levels are substantially elevated in the neointima of injured vessels and in atherosclerotic lesions (18, 19). However, the regulation and downstream effects of PPAR expression in pathologic vasculature are poorly defined. We previously demonstrated that PPAR1 rather than PPAR2 is expressed in vascular HASMC and that PDGF can up-regulate PPAR1 gene expression via PI3 kinase/Akt and MAPK signal pathways in VSMC (11). However, the precise transcriptional mechanisms of PPAR1 regulation remained unknown. In the present study we analyzed the human PPAR1 promoter and found a putative Egr-1 binding site at nt 184 to 173. In addition, we demonstrated that Egr-1 is the major transcriptional mediator of growth factor- and cytokine-induced PPAR1 gene expression in VSMC.

It has been noted that the Egr-1 protein in the vasculature is rapidly and transiently induced by various growth factors and other extracellular signals (20, 21, 22). In this report, we confirmed that growth factors and cytokines including PDGF, bFGF, Ang II, TNF, IL-1, and PMA up-regulated Egr-1 mRNA expression as early as 15 min upon stimulation, reaching maximal levels after 1 h. In addition, our data show that growth factor- and cytokine-induced Egr-1 mRNA expression in VSMC occurs prior to PPAR1 mRNA expression, suggesting that Egr-1 may be the transcriptional mediator of PPAR1 expression. By overexpressing a constitutively active Egr-1 mutant in VSMC using adenoviral gene transfer, PPAR1 mRNA expression was dramatically induced by 6-8-fold, whereas overexpression of NAB2, a potent repressor of Egr-1, abrogated growth factor- and cytokine-induced PPAR1 expression in HASMC. In addition, we demonstrate that the putative Egr-1 element in the human PPAR1 promoter specifically binds to Egr-1 protein and is functional. Taken together, our data provide the first evidence that Egr-1 is both necessary and sufficient to activate human PPAR1 expression in VSMC.

It has been known that gene transcription activation/repression not only involves transcriptional factors but also co-activators/co-repressors. Several cofactors of Egr-1 have been described. These include proteins such as p300 that has a required intrinsic containing histone acetyltransferases (HATs) activity for co-activation. The activities of these HATs loosen the interaction between histones and DNA, allowing the transcription factors to access promoters and response elements of DNA and initiate transcription. Although studies on chromatin remodeling complexes are necessary to further understand the molecular mechanisms of PPAR gene expression mediated by Egr-1, this is beyond the scope of the present study.

PPAR has two subtypes, PPAR1 and PPAR2, which are generated from the same gene by two different promoters and alternate splicing. PPAR2 has an N-terminal extension of 30 amino acids and is expressed selectively in adipose tissue, whereas PPAR1 is present in many tissues including adipose tissue, heart, spleen, colon, and vasculature (4, 5). The experiments in this study were performed in human VSMC. It remains to be tested whether PPAR1 expression is also Egr-1 dependent in the other tissues or cell types. As Egr-1 is coexpressed in tissues that express PPAR1 (12), it is possible that Egr-1 also mediates PPAR1 expression in those tissues and cell types. In support of this hypothesis, we document that Egr-1 also activates the PPAR1 gene in HepG2 cells (see Fig. 2A). With regard to species selectivity, it remains to be noted that the mouse PPAR1 promoter also contains an Egr-1 binding site (GenBank^TM number S79403).

To date, Egr-1 is capable of activating the transcription of several genes implicated in the pathogenesis of vascular diseases including PDGF-A (23), PDGF-B (24), FGF-2 (25), TNF- (26), IL-2 (27), tissue factor (28), u-PA (29), etc. In turn, many of these gene products also stimulate the expression of Egr-1. These positive feedback loops serve to amplify and sustain gene transcription through Egr-1-mediated mechanisms. Recently, there is increasing evidence that PPAR activation in vascular cells inhibits production of cytokines, including TNF- (30) and IL-2 (31), and down-regulates expression of several growth factor receptors including Ang II type I receptor (32, 33), PDGF receptor (34), epidermal growth factor (EGF) receptor, and vascular endothelial growth factor receptor (35, 36). As Egr-1 mediates PPAR1 expression in VSMC (this study), we hypothesize that PPAR1 up-regulation through Egr-1 exerts a negative feedback to inhibit expression or/and signaling pathways of growth factors or cytokines (Fig. 9). We are currently examining this hypothesis in vivo by using Egr-1-deficient mice. Interestingly, a recent paper documented that chronic down-regulation of CREB rather than its rapid activation actually drives VSMC growth, although, CREB has been shown to undergo rapid and transient activation by various growth factors in VSMC (37). Therefore, increased vascular CREB content could be another mechanism leading to inhibit VSMC proliferation and migration in addition to PPAR. It will be interesting to study the relationship between PPAR and CREB in vasculature.

View larger version (32K): [in this window] [in a new window]   Fig. 9.   Model for mechanisms by which PPAR1 exerts a negative feedback on the growth factor- and cytokine-induced Egr-1 expression. Many growth factors and cytokines can induce Egr-1 expression in the vasculature. In return, Egr-1 is capable of activating the transcription of several of these genes including PDGF-A, PDGF-B, FGF-2, and TNF-, which all have been implicated in the pathogenesis of vascular diseases. The positive feedback loops serve to amplify and sustain gene transcription through Egr-1-mediated mechanisms. From the present study, we propose that PPAR1 up-regulation through Egr-1 may exert a negative feedback to inhibit the expression and/or signaling pathways of growth factors or cytokines.

Although we defined Egr-1 as a key mediator of inducible PPAR1 expression, we cannot exclude that other transcription factors may also be involved in the regulation of PPAR1 gene expression. Egr-1 binding sites are always overlapping with Sp1 binding sites. In vitro studies using recombinant proteins suggest that Egr-1 and Sp1 can displace each other from many promoters, and their binding depends on an equilibrium between their concentration within the nucleus and affinity for the binding site (16). We also note that the Egr-1 binding site in the human PPAR1 promoter overlaps with an Sp1 binding site. Transient transfection studies show that Sp1 has a moderate effect on the human PPAR1 promoter; however, Sp1 mRNA is almost undetectable by Northern blot analysis in human VSMC (data not shown). It will still be interesting to examine the relationship between Egr-1 and Sp1 in regulating the human PPAR1 promoter in other types of cells.

In conclusion, our results provide the first evidence that Egr-1 is both necessary and sufficient for human PPAR1 expression and is a critical mediator of growth factor- and cytokine-induced PPAR1 expression in vasculature. Up-regulation of PPAR1 may function as a negative feedback to inhibit expression or/and signaling pathways of growth factors or cytokines in VSMC. This study is thus important for the understanding of the biological roles of PPAR1 in vasculature.

	FOOTNOTES

^* This work was supported in part by a starting grant from Morehouse Cardiovascular Research Institute (Enhancement of Cardiovascular and Related Research Areas, NHLBI/National Institutes of Health 5 UH1 HL03676-02), an institutional grant (NIGMS/National Institutes of Health S06GM08248), National Institutes of Health Grant R01HL068878 (to Y. E. C.), American Heart Association grant (to Y. E. C.), China State Major Basic Research Development Program (G2000056905), and Swiss National Science Foundation Grant 31-57125.99 (to M. U. E.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ These authors contributed equally to this work.

^** To whom correspondence should be addressed: Cardiovascular Research Inst., Morehouse School of Medicine, 720 Westview Dr. SW, Atlanta, GA 30310. Tel.: 404-752-1821; Fax: 404-752-1042; E-mail: echen@msm.edu.

Published, JBC Papers in Press, May 13, 2002, DOI 10.1074/jbc.M203748200

	ABBREVIATIONS

The abbreviations used are: PPAR, peroxisome proliferator-activated receptor; VSMC, vascular smooth muscle cells; Egr-1, early growth-response factor-1; HASMC, human aortic smooth muscle cells; PDGF, platelet-derived growth factor; IL-2, interleukin-2; TNF, tumor necrosis factor ; FGF, fibroblast growth factor; u-PA, urokinase-type plasminogen activator; GFP, green fluorescence protein; nt, nucleotides; PMA, phorbol 12-myristate 13-acetate; pfu, plaque-forming units; TK, thymidine kinase; CREB, cAMP-response element-binding protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Ang II, angiotensin II; MAPK, mitogen-activated protein kinase; NAB2, NGFI-A-binding protein 2.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				Y.-L. Chen, P.-C. Lin, S.-P. Chen, C.-C. Lin, N.-M. Tsai, Y.-L. Cheng, W.-L. Chang, S.-Z. Lin, and H.-J. Harn Activation of Nonsteroidal Anti-Inflammatory Drug-Activated Gene-1 via Extracellular Signal-Regulated Kinase 1/2 Mitogen-Activated Protein Kinase Revealed a Isochaihulactone-Triggered Apoptotic Pathway in Human Lung Cancer A549 Cells J. Pharmacol. Exp. Ther., November 1, 2007; 323(2): 746 - 756. [Abstract] [Full Text] [PDF]

				T.-C. Ho, S.-L. Chen, Y.-C. Yang, C.-L. Liao, H.-C. Cheng, and Y.-P. Tsao PEDF induces p53-mediated apoptosis through PPAR gamma signaling in human umbilical vein endothelial cells Cardiovasc Res, November 1, 2007; 76(2): 213 - 223. [Abstract] [Full Text] [PDF]

				X. Zhu, Y. Lin, M. Bacanamwo, L. Chang, R. Chai, I. Massud, J. Zhang, M. T. Garcia-Barrio, W. E. Thompson, and Y. E. Chen Interleukin-1 {beta}-induced Id2 gene expression is mediated by Egr-1 in vascular smooth muscle cells Cardiovasc Res, October 1, 2007; 76(1): 141 - 148. [Abstract] [Full Text] [PDF]

				S.-F. Yan, E. Harja, M. Andrassy, T. Fujita, and A. M. Schmidt Protein Kinase C {beta}/Early Growth Response-1 Pathway: A Key Player in Ischemia, Atherosclerosis, and Restenosis J. Am. Coll. Cardiol., October 27, 2006; 48(9_Suppl_A): A47 - A55. [Abstract] [Full Text] [PDF]

				I. E. Konstantinov, S. Arab, J. Li, J. G. Coles, C. Boscarino, A. Mori, E. Cukerman, F. Dawood, M. M.H. Cheung, M. Shimizu, et al. The remote ischemic preconditioning stimulus modifies gene expression in mouse myocardium J. Thorac. Cardiovasc. Surg., November 1, 2005; 130(5): 1326 - 1332. [Abstract] [Full Text] [PDF]

				F. J. Davis, J. B Pillai, M. Gupta, and M. P. Gupta Concurrent opposite effects of trichostatin A, an inhibitor of histone deacetylases, on expression of {alpha}-MHC and cardiac tubulins: implication for gain in cardiac muscle contractility Am J Physiol Heart Circ Physiol, March 1, 2005; 288(3): H1477 - H1490. [Abstract] [Full Text] [PDF]

				P. Collin-Osdoby Regulation of Vascular Calcification by Osteoclast Regulatory Factors RANKL and Osteoprotegerin Circ. Res., November 26, 2004; 95(11): 1046 - 1057. [Abstract] [Full Text] [PDF]

				S. Cheng, H. Afif, J. Martel-Pelletier, J.-P. Pelletier, X. Li, K. Farrajota, M. Lavigne, and H. Fahmi Activation of Peroxisome Proliferator-activated Receptor {gamma} Inhibits Interleukin-1{beta}-induced Membrane-associated Prostaglandin E2 Synthase-1 Expression in Human Synovial Fibroblasts by Interfering with Egr-1 J. Biol. Chem., May 21, 2004; 279(21): 22057 - 22065. [Abstract] [Full Text] [PDF]

				C. K. Sung, H. She, S. Xiong, and H. Tsukamoto Tumor necrosis factor-{alpha} inhibits peroxisome proliferator-activated receptor {gamma} activity at a posttranslational level in hepatic stellate cells Am J Physiol Gastrointest Liver Physiol, May 1, 2004; 286(5): G722 - G729. [Abstract] [Full Text] [PDF]

				E. Harja, L. G. Bucciarelli, Y. Lu, D. M. Stern, Y. S. Zou, A. M. Schmidt, and S.-F. Yan Early Growth Response-1 Promotes Atherogenesis: Mice Deficient in Early Growth Response-1 and Apolipoprotein E Display Decreased Atherosclerosis and Vascular Inflammation Circ. Res., February 20, 2004; 94(3): 333 - 339. [Abstract] [Full Text] [PDF]

				A. Haider, I. Lee, J. Grabarek, Z. Darzynkiewicz, and N. R. Ferreri Dual Functionality of Cyclooxygenase-2 as a Regulator of Tumor Necrosis Factor-Mediated G1 Shortening and Nitric Oxide-Mediated Inhibition of Vascular Smooth Muscle Cell Proliferation Circulation, August 26, 2003; 108(8): 1015 - 1021. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 277/30/26808    most recent M203748200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Fu, M. Articles by Chen, Y. E. Search for Related Content PubMed PubMed Citation Articles by Fu, M. Articles by Chen, Y. E. Social Bookmarking                      What's this?