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Originally published In Press as doi:10.1074/jbc.M406063200 on July 9, 2004

J. Biol. Chem., Vol. 279, Issue 39, 40289-40295, September 24, 2004
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Fibroblast Growth Factor-2 Induction of Platelet-derived Growth Factor-C Chain Transcription in Vascular Smooth Muscle Cells Is ERK-dependent but Not JNK-dependent and Mediated by Egr-1*

Valerie C. Midgley and Levon M. Khachigian{ddagger}

From the Centre for Vascular Research, The University of New South Wales and the Department of Haematology, The Prince of Wales Hospital, Sydney, New South Wales 2052, Australia

Received for publication, June 1, 2004 , and in revised form, July 9, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Platelet-derived growth factors (PDGFs) play an integral role in normal tissue growth and maintenance as well as many human pathological states including atherosclerosis, fibrosis, and tumorigenesis. The PDGF family of ligands is comprised of A, B, C, and D chains. Here, we provide the first functional characterization of the PDGF-C promoter. We examined 797 bp of the human PDGF-C promoter and identified several putative recognition elements for Sp1, Ets Egr-1, and Smad. The proximal region of the PDGF-C promoter bears a remarkable resemblance to a comparable region of the PDGF-A promoter (1). Binding and transient transfection analysis in primary vascular smooth muscle cells revealed that PDGF-C, like PDGF-A, is under the transcriptional control of the zinc finger nuclear protein Egr-1 (early growth response-1). Electrophoretic mobility shift analysis using both smooth muscle cell nuclear extracts and recombinant protein revealed that Egr-1 and Sp1 bind this region of the PDGF-C promoter (Oligo C, –35 to –1). Egr-1 competes with Sp1 for overlapping binding sites even when the former is at a stoichiometric disadvantage. Reverse transcriptase PCR and supershift analysis demonstrate that fibroblast growth factor-2 (FGF-2) stimulates both Egr-1 and PDGF-C mRNA expression in a time-dependent and transient manner and that FGF-2-inducible Egr-1 binds the proximal PDGF-C promoter. FGF-2-inducible PDGF-C expression was completely abrogated using catalytic DNA (DNAzymes) targeting Egr-1 but not by its scrambled counterpart. Moreover, using pharmacological inhibitors we demonstrate the critical role of ERK but not JNK in FGF-2-inducible PDGF-C expression. These findings thus demonstrate that PDGF-C transcription, activated by FGF-2, is mediated by Egr-1 and its upstream kinase ERK.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Platelet-derived growth factors (PDGFs)1 are important regulators of cell proliferation and survival in many types of mesenchymal cells, including smooth muscle cells (SMCs), connective tissue cells, and fibroblasts. Studies over the last two decades have implicated PDGF-A and -B, the classical PDGFs, in pathophysiologic processes such as atherosclerosis, restenosis, fibrosis, and tumorigenesis (2). Since their discovery 4 years ago, PDGF-C and PDGF-D (3–5) have been implicated in angiogenesis (6), embryogenesis (7–10), and platelet activation (11).

Neointima formation in atherosclerosis or post-angioplasty restenosis is mediated by a complex series of signaling and transcriptional events involving smooth muscle cells (SMCs), the hallmark cell type in these vascular occlusive disorders. Studies have shown that PDGF-C, like PDGF-A and -B, is a potent mitogen for SMCs (12, 13) and is expressed by SMCs in the intact arterial wall (13). Moreover, PDGF-C has recently been identified as a platelet {alpha}-granule secretory protein (11) similar to the classical PDGFs, suggesting common functions between PDGF ligand family members.

PDGF-A and PDGF-B also share common mechanisms of gene regulation. The promoters of each gene are controlled, at least in part, by the nuclear activity of the zinc finger proteins Egr-1 and Sp1 (1, 14, 15). Egr-1 and Sp1 have affinity for overlapping G + C-rich binding sites in the proximal region of the PDGF-A and PDGF-B promoters. Interplay between these factors mediates inducible PDGF-A and PDGF-B transcription in cells exposed to chemical stresses such as phorbol 12-myristate 13-acetate and environmental stresses such as altered fluid shear stress and mechanical injury (1, 14, 15). Egr-1/Sp1 interplay is also a feature common to the inducible expression of other vascular genes such as tissue factor (16). Whether Egr-1 and/or Sp1 play a role in PDGF-C and PDGF-D promoter regulation is not known, because neither promoter has yet been characterized.

Here we provide the first functional characterization of the PDGF-C promoter. We show that PDGF-C transcription is governed by Egr-1, which binds the proximal region of the PDGF-C promoter, competes with Sp1 for overlapping binding sites, and mediates FGF-2-inducible PDGF-C expression in an ERK but not a JNK-dependent manner.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
 REFERENCES
 
Cell Culture—Primary rat aortic SMCs were cultured in Waymouth's MB752/1 medium (Invitrogen), pH 7.4, supplemented with 10 units/ml penicillin, 10 µg/ml streptomycin, and 10% fetal calf serum at 37 °C and 5% CO2. At confluence, the cells were rinsed twice with phosphate-buffered saline solution and passaged by incubating with 0.05% trypsin and 0.02% EDTA in Hanks' balanced salt solution (BioWhittaker) for 7–10 min at 37 °C. The cells were resuspended in growth medium. All experiments were performed using cells between passages 5 and 8. Cells were growth-arrested for 24 h prior to the addition of fibroblast growth factor-2 (FGF-2; Promega) at a final concentration of 25 ng/ml.

Plasmid Construction—The 1288-bp region upstream from the PDGF-C ATG start site was amplified from human genomic DNA (Roche Applied Science) by PCR using the primers 5'-ATGCTAGCCCTGAACACAAGCCACAAGA-3' (forward) and 5'-CGCTCGAGTTGTTGCTGGAAAACTGGAA-3' (reverse). The fragment was cleaved by NheI and XhoI and ligated into the firefly luciferase-based reporter vector pGL3-basic (Promega) to create construct pPDGF-C-797. The integrity of the reporter construct was confirmed by nucleotide sequencing.

Transient Transfections—Primary SMCs at 70% confluence (100-mm Petri dishes) were incubated in serum-free medium for 6 h prior to transfection. Transfections were carried out using FuGENE6 (Roche Applied Science), according to the manufacturer's guidelines, with 5 µg of reporter construct pPDGF-C-797 or pGL3-basic and 3 µg of pCB6 or pCB6-Egr-1. Cells were harvested 24h later, and the corresponding luciferase activity was quantified using the Dual Luciferase Reporter Assay (Promega). A double transfection was performed for transfections involving DNAzymes (ED5 and ED5SCR) (27) targeting Egr-1. Briefly the cells were growth arrested for 6 h prior to the initial transfection using FuGENE6 and DNAzyme (0.4µM, final concentration). Eighteen hours later, fresh serum-free media was added together with FGF-2 at a final concentration of 25 ng/ml. Minutes later, a second but identical transfection mix was added to the cells and incubated for a further 6 h prior to harvesting.

Preparation of Nuclear Extracts—SMCs treated with or without FGF-2 were washed twice and scraped in 10 ml of cold PBS, pH 7.4. The cells were centrifuged at 1200 rpm for 10 min at 4 °C, and the pellet was resuspended in 100 µl of cold Buffer A (10 mM HEPES, pH 8, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol, 20 mM sucrose, and 0.5% Nonidet P-40) and incubated on ice for 5 min. The suspension was centrifuged at 1400 rpm for 40 s, and the pellet of nuclei was lysed with 20 µl of cold Buffer C (20 mM HEPES, pH 8, 1.5 mM MgCl2, 420 mM NaCl, 0.2 mM EDTA, and 1 mM dithiothreitol) by mixing gently for 20 min at 4 °C. After re-centrifugation at 1400 rpm for 1 min, 20 µl of supernatant was combined with 20 µl of cold Buffer D (20 mM HEPES, pH 8, 100 mM KCl, 0.2 mM EDTA, 20% glycerol, and 1 mM dithiothreitol) and stored at –80 °C until use. All buffers contained protease inhibitors.

Electrophoretic Mobility Shift Assay (EMSA)—Binding reactions were carried out in volume of 20 µl containing 10 mM Tris-HCl, 50 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 5% glycerol, 1 µg of poly(dI-dC), 1 µg of salmon DNA (Sigma), 32P-labeled oligonucleotide probe (100,000 cpm), and 6–10 µg of nuclear extract. The reaction was incubated for 30 min at 22 °C. In supershift studies, 1 µl of the indicated affinity-purified rabbit anti-peptide antibodies (Santa Cruz Biotechnology) were incubated with the extracts for 15 min prior to the addition of the probe. In interplay experiments, binding reactions were carried out for 15 min on ice with the addition of bovine serum albumin (2 µg) and human recombinant Sp1 (12.5–100 ng) (Promega) or human recombinant Egr-1 (20–80 ng) (Alexis Biochemicals). Samples were resolved by electrophoresis on a 6% non-denaturing polyacrylamide gel and vacuum dried at 80 °C for 1 h before visualization by autoradiography.

Total RNA Preparation and mRNA Expression Analysis—Total RNA was prepared using TRIzol® (Invitrogen) according the manufacturer's instructions. cDNA was generated using 5 µg of total RNA, oligo(dT), and Superscript II reverse transcriptase (Promega) according the manufacturer's instructions. Thermal cycling conditions were as follows: PDGF-C, 98 °C for 30 s, 62.5 °C for 30 s, and 72 °C for 2 min for 40 cycles; {beta}-actin, 98 °C for 30 s, 61 °C for 30 s, and 72 °C for 1 min for 18 cycles; and Egr-1, 98 °C for 30 s, 52 °C for 30 s, and 72 °C for 1 min for 30 cycles. The relative primers are as follows: PDGF-C, 5'-TCCAGCAACAAGGAACAGAAC-3' (forward) and 5'-CTGAAGGGGGTAGCTCTGAA-3' (reverse); {beta}-actin, 5'-TGACGGGGTCACCCACACTGTGCCCATCTA-3' (forward) and 5'-CTAGAAGCATTTGCGGTGGACGATGGAGGG-3' (reverse); Egr-1, 5'-GCATGTAACGCGGCCA-3' (forward) and 5'-CCGAACGGGTCAGAGAT-3' (reverse). The mitogen-activated kinase kinase inhibitors PD98059 and SP600125 were purchased from Calbiochem.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
REFERENCES
 
Putative Binding Sites in the Human PDGF-C Promoter— Numerous studies have investigated the biological roles of PDGF-C since its discovery 4 years ago (5–12, 17–23). However, to date there are no reports of the functional characterization of the PDGF-C promoter in any cell type. We isolated a 1.395-kb fragment of human genomic PDGF-C DNA from a commercial source (Roche Applied Science) using primers engineered to contain restriction sites for NheI or XhoI. The 5' primer targeted position –797 (as the extreme 5' nucleotide upstream from the predicted transcriptional start site defined by the University of California Santa Cruz genome data base), whereas the 3' primer targeted position 598 bp spanning the entire 5' untranslated region. The 797-bp promoter region lacks a consensus TATA box but contains putative binding sites for Sp1, Egr-1, Ets-1, and Smad (Fig. 1A). The nucleotide sequence immediately adjacent to the 5' untranslated region is extremely G + C-rich. The 1288-bp PDGF-C genomic sequence is ~80% homologous between human and mouse. To begin to investigate the transcriptional regulation of PDGF-C, we subcloned this fragment into the corresponding restriction sites in the firefly luciferase-based reporter construct pGL3-basic.



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  		FIG. 1.
The PDGF-C proximal promoter region. A, putative nucleotide recognition elements for transcription factors in the human PDGF-C promoter. The 797-bp region of the PDGF-C promoter lacks a consensus TATA but has putative sites for Smad, Ets, Sp1, and Egr-1. The 35 nucleotides immediately adjacent (5' side) to the transcriptional start site are indicated by italics, boldface, and underlining. B, a striking similarity between the proximal promoter sequences of PDGF-C and PDGF-A. The 35-bp (-35/-1) G + C-rich region of the proximal PDGF-C promoter (Oligo C, –35 to –1) shares ~65% homology to a comparable region of the PDGF-A promoter (Oligo A, –76 to –47) which binds Egr-1 and Sp1 (1).

 
Identification of a G + C-rich region in the PDGF-C Proximal Promoter that Physically Interacts with Egr-1 and Sp1—Upon examination of the PDGF-C promoter (Fig. 1A), we discovered that a 35-bp G + C-rich region (spanning bp –35 to –1) possesses striking similarity to a comparably sized region (–6 to –47) in the proximal human PDGF-A promoter (Fig. 1B). This region of the PDGF-A promoter contains overlapping binding elements for Egr-1 and Sp1 (1), which led us to hypothesize that the proximal region of the PDGF-C promoter might also support the interaction of Egr-1 and Sp1 (Fig. 1B). Accordingly, we performed EMSA using a 32P-labeled, double-stranded oligonucleotide spanning bp –35 to –1 of the PDGF-C promoter (32P-Oligo C –35 to –1) and nuclear extracts of SMCs that had been growth-arrested in serum-free media for 24 h. Six nucleoprotein complexes formed, i.e. three major (complexes N1, N5, and N6) and three minor (complexes N2, N3, and N4) (Fig. 2A). Cold competition analysis using a 50-fold molar excess of unlabeled Oligo A (–76 to –47) bearing the proximal PDGF-A promoter sequence inhibited the formation of all six complexes (Fig. 2A). In contrast, the same fold molar excess of unlabeled Oligo Ets (bearing a consensus Ets binding site) failed to alter the profile of these complexes (Fig. 2A). Unlabeled Oligo C (–35 to –1) inhibited formation of the same complexes as Oligo A (compare Fig. 2, A and B). The fact that the formation of complex N5 was not completely inhibited at this molar excess of either unlabeled oligonucleotide as compared with other complexes (Fig. 2, A and B) indicates that the protein components of these complexes bind the DNA with different affinities.



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  		FIG. 2.
Egr-1 and Sp1 specifically bind the PDGF-C promoter. A, interaction of Sp1 and Egr-1 with the G + C-rich element in the PDGF-C proximal promoter as demonstrated by competition studies with Oligo A (PDGF-A). EMSA was performed using 32P-Oligo C (–35 to –1) and nuclear extracts (NE) from serum-starved SMCs. Cold competition with Oligo C established the specificity of the six nucleoprotein complexes formed (N1–N6). Oligo Ets (30) had no effect. B, Egr-1 and Sp1 bind the PDGF-C promoter. Supershift analysis demonstrating Sp1 and Egr-1 but not Sp3 binding to this region of the PDGF-C proximal promoter. {alpha}, antibody.

 
The capacity of Oligo A (–76 to –47) to specifically compete with Oligo C (–35 to –1) suggests the possibility that similar proteins bind to each oligonucleotide. Supershift analysis using antibodies raised against Egr-1 and Sp1 revealed that the 32P-Oligo C (–35 to –1) complex N1 contains Sp1 (Fig. 2B) and that complex N3 contains Egr-1 (Fig. 2B). In contrast, the nucleoprotein complex profile was not affected by Sp3 antibodies (Fig. 2B).

To further confirm the physical interaction of Egr-1 and Sp1 with the PDGF-C promoter, we performed EMSA using human recombinant Sp1 and/or Egr-1 and 32P-labeled Oligo C (–35 to –1). Both recombinant proteins bound to the oligonucleotide (Fig. 3A). The formation of multiple complexes (Fig. 3A; S1–S2 for Sp1 and E1–E4 for Egr-1) suggests either the existence of multiple sites for each protein in this region of the promoter, self-association, or binding by partially degraded species. Because Egr-1 and Sp1 bind to overlapping binding sites in the PDGF-A promoter, we performed protein-protein competition analysis using EMSA and the two recombinant proteins with 32P-Oligo C (–35 to –1). When increasing amounts of Egr-1 were added to a reaction containing fixed amounts of Sp1, we observed a dose-dependent reduction in Sp1 binding as Egr-1 binding became more apparent (Fig. 3B). No "super" species was observed when Sp1 and Egr-1 were co-incubated with the oligonucleotide (Fig. 3B), indicating that these proteins do not simultaneously occupy the promoter fragment. This data (Fig. 3B), supported by cross-competition studies with Oligo A (–76 to –47) and Oligo C (–35 to –1) (Fig. 2B) indicates the existence of interplay in the proximal PDGF-C promoter. Moreover, because Egr-1 is induced by multiple pro-atherogenic stimuli (14, 15, 24), PDGF-C transcription may be positively regulated by Egr-1 in SMCs exposed to pathophysiologically relevant stimuli.



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  		FIG. 3.
Competition between Egr-1 and Sp1 in the proximal PDGF-C promoter. A, human recombinant Sp1 and Egr-1 bind to the PDGF-C proximal promoter. EMSA using 32P-Oligo C (–35 to –1) demonstrates the specific binding of hrSp1 (100 ng; complexes S1–S2) or rhEgr-1 (80 ng; complexes E1–E4). B, competition between Egr-1 and Sp1 for binding to 32P-Oligo C (–35 to –1) demonstrated by EMSA. Sp1 (100 ng) was competed with increasing amounts of Egr-1 (10–80 ng) in binding reactions as described under "Experimental Procedures."

 
Egr-1 Activation of the PDGF-C Promoter—To determine the functional consequence of the interaction of endogenous and recombinant Egr-1 with the PDGF-C promoter, we performed transient co-transfection analysis using pPDGF-C-797 and pCB6-Egr-1, which generates exogenous Egr-1 (Fig. 4). pCB6-Egr-1 activated the PDGF-C promoter >5-fold as compared with the backbone, pCB6, alone (Fig. 4). In contrast, no difference was observed when pCB6-Egr-1 or pCB6 was co-transfected with pGL3-basic, the empty luciferase vector (Fig. 4).



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  		FIG. 4.
Egr-1 activates the PDGF-C promoter. Transient transfection analysis was performed in SMCs transfected with pGL3-basic (5 µg) or pPDGF-C-797 (5 µg) and with pCB6 (3 µg) or pCB6-Egr-1 (3 µg). Error bars represent the mean ± S.E. The data are representative of two or more independent determinations.

 
FGF-2 Induces Egr-1 and PDGF-C mRNA Expression in SMCs—FGF-2 is a key mediator of cell growth and differentiation under physiological and pathological conditions (25). Previously, we have shown that Egr-1 is under the transcriptional control of FGF-2 (26). We therefore postulated that FGF-2 mediates Egr-1-dependent PDGF-C activation. Serum-starved SMCs were treated with FGF-2 (25 ng/ml) for various times prior to the assessment of steady-state mRNA levels by semiquantitative reverse transcriptase PCR (Fig. 5). PDGF-C mRNA expression increased in response to FGF-2 in a time-dependent manner, maximally at 4 h (Fig. 5, top section). FGF-2 induced Egr-1 mRNA expression maximally at 2 h and prior to returning to basal levels by 24 h (Fig. 5, middle section). {beta}-Actin levels demonstrate unbiased loading between the samples (Fig. 5, bottom section). Interestingly, low levels of basal PDGF-C expression in the absence of FGF-2 coincide with weak Egr-1 expression (Fig. 5) and Egr-1 DNA binding (Fig. 2, A and B, and Fig. 6). These findings, which illustrate the tight temporal relationship between Egr-1 and FGF-2 expression, demonstrate for the first time that FGF-2 is an agonist of PDGF-C expression.



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  		FIG. 5.
FGF-2 induces PDGF-C and Egr-1 expression. FGF-2 transiently induces PDGF-C mRNA expression within 2 h. Reverse transcriptase PCR was performed for PDGF-C, Egr-1, and {beta}-actin as described under "Experimental Procedures." RNA was isolated from serum-starved SMCs treated with or without 25 ng/ml FGF-2 for 2, 4, 8, or 24 h. {beta}-Actin expression demonstrates unbiased loading.

 



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  		FIG. 6.
Induction of Egr-1 DNA-binding activity by FGF-2. EMSA was performed using 32P-Oligo C (–35 to –1) and nuclear extracts (NE) from serum-starved SMCs treated with or without FGF-2 (25 ng/ml) for 2 or 4 h. Complexes N1–N6 are indicated by arrows. Supershift analysis demonstrates the specificity of the FGF-2-induced nucleoprotein complex (IND) as Egr-1. Nuclear extracts were corrected for protein concentration prior to assay. {alpha}, antibody.

 
FGF-2 Induces Egr-1 Binding to the PDGF-C Proximal Promoter in SMCs—EMSA using nuclear extracts of SMCs treated with FGF-2 (25 ng/ml) and 32P-Oligo C (–35 to –1) revealed the formation of complexes N1–N6 (Fig. 6). Of these, only one complex (N3) was transiently induced (Fig. 6, IND) upon exposure to FGF-2 (Fig. 6). Complex N3 was clearly apparent within 2 h; then intensity levels fell within the subsequent 2 h (Fig. 6). Supershift analysis revealed that the nucleoprotein complex was eliminated with antibodies to Egr-1 (Fig. 6) but not to Sp1 (Fig. 6). The weak intensity of complex N3 in Fig. 2, A and B, reflects the use of extracts from growth-quiescent (unstimulated) SMCs. These data indicate that FGF-2 stimulates the synthesis and nuclear accumulation of Egr-1, which then translocates to the nucleus and binds the proximal PDGF-C promoter coincident with increased PDGF-C expression.

Egr-1 DNAzymes Block FGF-2-inducible PDGF-C mRNA Expression—To directly establish whether FGF-2-inducible PDGF-C expression is critically dependent upon the activation of Egr-1, we assessed PDGF-C levels in SMCs that had previously been transfected with the Egr-1 DNAzyme ED5. DNAzymes are sequence-specific "gene knock-down" agents, which we used previously to inhibit neointima formation in rat carotid arteries after balloon injury (27) and in pig coronary arteries following stenting (28). Serum-starved SMCs were transfected either with ED5 (27), which targets and cleaves the translational start site of rat Egr-1 mRNA (27), or ED5SCR, the scrambled counterpart to ED5. After treatment with FGF-2 (25 ng/ml) for 6 h, we observed no activation of PDGF-C expression in cells transfected with ED5 (Fig. 7). In contrast, PDGF-C expression was still induced in SMCs transfected with ED5SCR and exposed to FGF-2 (Fig. 7). PDGF-C expression under these latter conditions did not differ from that of cells stimulated with FGF-2 alone (Fig. 7).



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  		FIG. 7.
DNAzyme targeting Egr-1 mRNA prevents FGF-2-inducible PDGF-C expression. Reverse transcriptase PCR was performed after the isolation of RNA from serum-starved SMCs that had been transfected twice with or without the DNAzyme ED5 (0.4 µM) or its scrambled counterpart ED5SCR (0.4 µM), followed by treatment with FGF-2 (25 ng/ml) for 6 h as described under "Experimental Procedures." {beta}-Actin expression demonstrates unbiased loading.

 
FGF-2 Induces PDGF-C via an ERK but Not a JNK-dependent Signaling Pathway—FGF-2-induced signal transduction is known to involve the activation of multiple mitogen-activated protein kinases such as ERK and JNK. These pathways are important for many fundamental cellular processes including proliferation, differentiation, and survival. To determine the signaling pathway mediating FGF-2-inducible PDGF-C expression, we incubated serum-starved SMC with either PD98059 (10 µM) or SP600125 (25 nM) for 1 h prior to treatment with FGF-2 (25 ng/ml). FGF-2-inducible PDGF-C mRNA expression was blocked by the MEK/ERK inhibitor PD98059 (Fig. 8) but not by the JNK inhibitor SP600125 (Fig. 8). These findings, consistent with our previous demonstration that FGF-2 stimulates Egr-1 expression through the specific activation of ERK (26), therefore indicates the involvement of the ERK-Egr-1 axis in FGF-2-inducible PDGF-C expression.



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  		FIG. 8.
FGF-2 inducible PDGF-C expression is blocked by ERK inhibitors but by not inhibitors of JNK. Reverse transcriptase PCR was performed after the isolation of RNA from serum-starved SMCs that had been exposed to PD98059 (10 µM) or SP600125 (25 nM) for 1 h prior to stimulation with FGF-2 (25 ng/ml) for 6 h, as described under "Experimental Procedures." {beta}-Actin expression demonstrates unbiased loading.

 
The formation of the neointima in occlusive vascular disorders such as atherosclerosis and post-angioplasty restenosis is mediated by a complex series of signaling and transcriptional events that alter the SMC phenotype. Growth factors such as FGF-2 are constitutively expressed by SMC and endothelial cells in the normal and diseased artery wall and serve as reservoirs of mitogens that are released upon vascular injury. The release of FGF-2 may trigger the expression of other mitogens, thereby starting a cascade of autocrine/paracrine growth and consequential intimal thickening. The PDGF-C chain is expressed by SMCs in the intact artery wall and is a potent mitogen for this cell type (12, 13). In this study, we have demonstrated that FGF-2 stimulates PDGF-C expression in SMCs via the ERK-dependent zinc finger transcription factor Egr-1, which is itself rapidly and transiently induced by arterial injury (7, 15, 26, 29). As such, this study, through the use of agonists, overexpression, and novel knockdown approaches, is the first to provide key insights into the transcriptional mechanisms regulating PDGF-C activation in any cell type. It also points out the remarkable similarity between the proximal G + C-rich regions of the PDGF-A (bp –76 to –47) and PDGF-C (–35 to –1) promoters and how Egr-1/Sp1 interplay is a common theme to both these promoters, as it is to those of PDGF-B, u-PA, PAI-1, and tissue factor (15). Because of its ability to activate multiple pathophysiologically important genes involved in cell growth, migration, cell adhesion, and coagulation, strategies targeting Egr-1 may be useful in the treatment and/or prevention of vascular occlusive disorders.


   FOOTNOTES
 
* This work was supported by a program grant from the National Health and Medical Research Council and grants from the Australian Research Council, the National Heart Foundation, and an Research Infrastructure Grant from the New South Wales Department of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} A National Health and Medical Research Council Principal Research Fellow and to whom correspondence should be addressed. Tel.: 61-2-9385-2537; Fax: 61-2-9385-1389; E-mail: L.Khachigian{at}unsw.edu.au.

1 The abbreviations used are: PDGF, platelet-derived growth factor; Egr-1, early growth response-1; EMSA, electrophoretic mobility shift assay; ERK, extracellular signal-regulated kinase; FGF, fibroblast growth factor; JNK, c-Jun N-terminal kinase; MEK, mitogen-activated kinase/ERK kinase; SMC, smooth muscle cell. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 

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