GC Factor 2 Represses Platelet-Derived Growth Factor A-Chain Gene Transcription and Is Itself Induced by Arterial Injury

Cell Culture

Bovine aortic endothelial cells were purchased from Cell Applications, Inc, and grown in DMEM (pH 7.4) containing 10% FBS, 50 μg/mL streptomycin, and 50 IU/mL penicillin at 37°C in a humidified atmosphere of 5% CO₂. Rat aortic smooth muscle cells were obtained from Cell Applications, Inc, and grown in Waymouth’s medium (pH 7.4) containing 10% FBS and antibiotics. Cells were routinely passaged with trypsin/EDTA and not used in experiments beyond passage 6.

Transient Transfection Analysis and Chloramphenicol Acetyltransferase (CAT) Assay

Endothelial cells in 100-mm-diameter dishes at 70% confluency were transfected with the indicated amounts of cesium chloride–purified plasmid DNA using a modification of a calcium phosphate precipitation method.¹⁹ After transfection, the cells were incubated overnight in an atmosphere of 3% CO₂, washed twice with PBS (pH 7.4) and incubated for an additional 24 hours at 5% CO₂. Cell lysates were assessed for CAT activity as previously described.² Conditioned medium was sampled at the time the cells were harvested and assayed for human growth hormone (Bioclone) to correct for transfection efficiency. Total protein concentration in the lysates was assessed using a modified Bradford assay (Bio-Rad) and used to normalize transfection data.

Electrophoretic Mobility Shift Assay (EMSA)

Binding reactions for gel shift assays were performed in 20 μL of 10 mmol/L Tris-HCl, 50 mmol/L MgCl₂, 1 mmol/L EDTA, 1 mmol/L DTT, 5% glycerol, 1 mmol/L PMSF, 0.75 μg salmon sperm DNA, 5% sucrose, 0.75 μg poly dI-dC, 10 μg of BSA, ³²P-labeled oligonucleotide probe (150 000 cpm), and 1.5, 3, or 6 μg of in vitro transcribed/translated GCF2 protein.¹⁷ The reaction was incubated for 35 minutes at 22°C. In competition gel shifts, 6 μg of nuclear extract was prepared as described² and combined with 6 μg of GCF2 protein. As a control, an equal amount of BSA protein was substituted for GCF2. Bound complexes were separated from free probe by loading the samples onto a 5% nondenaturing polyacrylamide gel (PAGE) and electrophoresing at 200 V for 2 to 3 hours. The gels were dried and subjected to autoradiography overnight at −80°C.

Northern Blot Analysis

Endothelial cells or smooth muscle cells were grown to 70% confluency in 100-mm-diameter Petri dishes and transfected with 10 μg pcDNA3 or GCF2-pcDNA3 using FuGENE6 reagent (Boehringer Mannheim) at a FuGENE6:DNA ratio of 3:1 (vol:μg), per the manufacturer’s instructions. After 24 hours, total RNA was extracted with TRIzol reagent (Life Technologies), and 20 μg of each sample was resolved by electrophoresis on 1% formaldehyde/agarose/MOPS gels. RNA was transferred overnight onto Hybond nylon membranes before hybridization with a ³²P-labeled 260-bp fragment of PDGF-A cDNA in ExpressHyb (Clontech). Vacuum-dried gels were autoradiographed for 1 week at −80°C, then stripped and reprobed with a ³²P-labeled 1.1-kb fragment of GCF2 cDNA, and later with a ³²P-labeled fragment of β-actin cDNA followed by reexposure.

Injury and Western Blot Analysis

Endothelial cells or smooth muscle cells were rendered growth-quiescent by incubation overnight in medium containing 1% or 0.1% FBS, respectively. The cells were injured by repeated scraping with a sterile stainless steel comb and solubilized in RIPA buffer. Extracts were resolved by 10% SDS-PAGE and transferred to nylon membranes by electrophoretic blotting. Nonspecific binding sites on the membrane were blocked with nonfat skim milk before probing with affinity-purified rabbit polyclonal antibodies to PDGF-A (Genzyme) or GCF2. This was followed by chemiluminescent detection using HRP-linked swine anti-rabbit secondary antiserum (NEN-DuPont).

Assays of Cell Proliferation

Cells were seeded into 96-well titer plates (Nunc-InterMed) at 2500 cells per well and allowed to grow for 48 hours in complete medium. The cells were transfected with 1 μg of the indicated plasmid in complete growth medium using FuGENE6 (Boehringer Mannheim) in accordance with manufacturer’s instructions. At the time of transfection, and again after 24 hours, the cells were rinsed with PBS (pH 7.4) and trypsinized, and the suspension was quantitated using an automated particle counter (Coulter Electronics).

Rat Carotid Artery Injury and Immunohistochemical Analysis

Sprague-Dawley rats (400 g) were obtained from the Biology Research Unit, Baker Medical Research Institute, Melbourne, Australia. Left common carotid arteries of anesthetized rats (ketamine 80 mg/kg; xylene 10 mg/kg) were subjected to balloon catheter injury using a 2F Fogarty catheter as previously described.²⁰ Briefly, the saline-inflated balloon catheter was passed along the length of the common carotid four times. Various times after injury (0, 4 hours, 6 hours, 24 hours, and 10 days), rats were anesthetized with sodium pentobarbitone (100 mg/kg). The vessels were perfused with saline and gently dissected free of surrounding tissue, frozen in OCT compound (Tissue-Tek), and sectioned (5 μm). Sections were fixed in 4% formalin for 2 minutes, then absolute ethanol for 10 minutes. For immunohistochemical analysis,²¹ sections were incubated with affinity-purified rabbit polyclonal IgG raised against PDGF-AA (1:100) (Genzyme) or in vitro transcribed/translated GCF2 (1:200).¹⁷ Secondary detection was performed using the avidin-biotin complex (ABC) kit.²² Deendothelialization was confirmed by vital staining with Evans blue (60 mg/kg IV). All surgical procedures were approved by the Baker Medical Research Institute and Alfred Hospital Animal Experimentation Committee.

GCF2 Represses PDGF-A Promoter-Dependent Expression

To determine whether GCF2 can influence expression driven by the PDGF-A promoter, we transfected endothelial cells with a GCF2 expression vector driven by the cytomegaloviral (CMV) promoter (construct GCF2-pcDNA3), together with a CAT reporter construct, ΔSac, bearing 643 bp of PDGF-A promoter sequence. CAT activity was normalized for transfection efficiency after assessment of human growth hormone levels secreted into the culture medium. Overexpression of GCF2 in this cotransfection setting strongly inhibited reporter expression driven by the PDGF-A promoter (Figure 1⇓). GCF2 repression of PDGF-A promoter-driven CAT activity was also observed in rat vascular smooth muscle cells (data not shown). In contrast, overexpression of a transcriptional activator of PDGF-A, Egr-1, with the same backbone (pcDNA3) stimulated CAT activity 2-fold (Figure 1⇓).

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Figure 1.

GCF2 represses the activity of the PDGF-A promoter. Endothelial cells were transfected with 15 μg of PDGF-A promoter CAT construct ΔSac and 5 μg of pcDNA3, GCF2-pcDNA3, or Egr-1-pcDNA3 as indicated. The cells were cotransfected with 2 μg of pTKGH to correct for transfection efficiency. CAT reporter activity was normalized to levels of growth hormone (GH) secreted into the conditioned medium. Error bars represent SEM from at least 2 independent determinations.

Repression by GCF2 Is Mediated by Nucleotide Elements Located in the Proximal Region of the PDGF-A Promoter

GCF2 binds preferentially to nucleotide sequences rich in guanines and cytosines.¹⁷ The proximal region of the PDGF-A promoter is intensely G+C-rich.¹ We hypothesized that GCF2 repression of the PDGF-A promoter may involve elements located in the proximal region. Transient cotransfection experiments revealed that expression driven by construct f28, which contains 71 bp of PDGF-A promoter sequence,² was inhibited by GCF2 coexpression (Figure 2⇓). However, when GCF2-pcDNA3 was cotransfected with construct f36, which bears 55 bp of promoter sequence,² repression by GCF2 was no longer observed (Figure 2⇓). These findings suggest that GCF2 repression of the PDGF-A promoter is mediated by nucleotide elements located between the 5′ promoter end points of f28 and f36. The GCF2 binding site in the epidermal growth factor receptor promoter has a core binding sequence of 5′-AGC CCC CGG CG-3′ (or 5′-CG CCG GGG GCT-3′).¹⁷ The nucleotide sequence between the f28 and f36 5′ end points bears the 5′-CGG GGG C-3′ motif (Figure 2⇓). Separate experiments revealed that a CAT reporter construct driven by 153 bp of the PDGF-B promoter,²³ which does not bear the GCF2 core binding sequence, was unaffected by cotransfection with GCF2-pcDNA3 (data not shown).

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Figure 2.

GCF2 repression of the PDGF-A promoter is mediated by a G+C-rich sequence in the proximal region of the promoter. Cells were transfected with 15 μg of PDGF-A promoter CAT construct f28 or f36 and either 5 μg of pcDNA3 or GCF2-pcDNA3. The cells were cotransfected with 2 μg of pTKGH to correct for transfection efficiency. CAT reporter activity was normalized to levels of growth hormone in the supernatant. Error bars represent SEM from at least 2 independent determinations. A schematic representation of f28 and f36 is also provided.²

GCF2 Interacts With Proximal PDGF-A Promoter

To determine whether GCF2 binds directly to the PDGF-A promoter, we incubated in vitro transcribed/translated GCF2 with a ³²P-labeled probe, Oligo A, whose sequence spans the 5′ end points of f28 and f36, and resolved the adducts by electrophoresis under nondenaturing conditions. GCF2 bound to the oligonucleotide in a dose-dependent manner as two distinct nucleoprotein complexes (Figure 3A⇓). The upper and more intense complex represents dimeric GCF2, whereas the lower complex is the monomeric form.¹⁷ Neither complex was observed when an oligonucleotide bearing an unrelated sequence, ³²P-LKmdc, was substituted for ³²P-Oligo A (Figure 3A⇓). Furthermore, neither complex formed when ³²P-Oligo Am was substituted for ³²P-Oligo A (Figure 3A⇓). Oligo Am was generated by altering the 5′-CGG GGG C-3′ repeated motif in Oligo A by insertion of additional guanines (5′-CGG GGG gC-3′) thus disrupting the promoter sequence. When wild-type ³²P-Oligo A was incubated with GCF2Δ510, an in vitro transcribed/translated form of GCF2 that carries a deletion in its DNA binding domain,¹⁷ nucleoprotein complex formation was no longer observed (Figure 3B⇓). Additional experiments revealed that wild-type GCF2 failed to interact with a ³²P-labeled G+C-rich RNA oligomer composed of 23 bases (data not shown). Taken together, these findings demonstrate that GCF2 interacts with the proximal PDGF-A promoter in a dose-dependent and specific manner.

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Figure 3.

GCF2 interacts with the proximal PDGF-A promoter. A, Specific and dose-dependent interaction of GCF2. EMSA was performed with in vitro transcribed/translated GCF2 protein and the ³²P-labeled oligonucleotides indicated in the figure as described in Materials and Methods. B, DNA binding domain of GCF2 is critical for interaction with proximal PDGF-A promoter. In vitro transcribed/translated GCF2Δ510 lacks the complete 309-bp DNA binding domain of GCF2 (1382-1690).¹⁷ Arrows indicate monomeric and dimeric forms of bound GCF2.¹⁷ The nucleotide sequence of Oligo A is 5′-GGG GGG GGC GGG GGC GGG GGC GGG GGA GG-3′ and LKmdc is 5′-GCT GTC TCC ACC CAT TGT TCG CAC TCT-3′; Oligo Am is 5′-GGG GGG GGC GgG GGG CgG GGG GCg GGG GGA GG-3′ (sense strand shown; bases indicated in lowercase denote insertions).

Competition Between Several Zinc Finger Binding Proteins and GCF2 for Binding Sites in the Proximal PDGF-A Promoter

The preceding findings demonstrate the capacity of GCF2 to bind to the PDGF-A promoter and inhibit promoter-dependent reporter gene expression. We next addressed the question of the mechanism underlying GCF2 repression. Previous studies by our group have shown that a number of zinc finger transcription factors interact with the GCF2 binding site and mediate basal and inducible expression of the PDGF-A gene. These nuclear factors include Sp1 and Egr-1.² We explored the possibility that GCF2 competes with these factors for binding to the PDGF-A promoter. Endothelial extracts were incubated with ³²P-Oligo A in the absence or presence of GCF2 or a corresponding amount of BSA, before resolution of ³²P-labeled nucleoprotein complexes by electrophoresis. This produced the characteristic profile of ³²P-labeled nucleoproteins composed of Sp1, Egr-1, Sp3, and other nuclear proteins^{2 3 24} (Figure 4⇓). Inclusion of GCF2 in the binding mixture markedly attenuated the intensity of the Sp1, Egr-1, and Sp3 complexes, among other complexes (Figure 4⇓). In contrast, these complexes were unaffected by substitution of GCF2 with an identical amount of BSA (Figure 4⇓). Thus, GCF2 competes with these positive regulatory transcription factors for overlapping binding sites in the PDGF-A promoter.

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Figure 4.

GCF2 competes with several zinc finger transcription factors for binding to the proximal PDGF-A promoter. Nuclear extracts of endothelial cells in log phase were incubated with ³²P-Oligo A, in the absence or presence of 1 μg in vitro transcribed/translated GCF2 or BSA before resolution by electrophoresis and autoradiography.

Mechanical Injury Stimulates PDGF-A and GCF2 Expression In Vitro

To determine the potential pathophysiological relevance of the above findings to vascular disorders, we examined the relationship between GCF2 and PDGF-A expression in an in vitro model of cell injury. Endothelial cell monolayers were scraped repeatedly with a sterile stainless steel comb,^{3 9} and lysates were assessed for levels of PDGF-A. PDGF-A was weakly expressed in unmanipulated endothelial cells (Figure 5A⇓). However, within 4 hours of injury, PDGF-A levels increased 3-fold (Figure 5A⇓, open bars), consistent with a previous report of inducible PDGF-A expression in aortic endothelium within 4 hours of mechanical injury.²⁵ By 8 hours, PDGF-A levels increased almost 10-fold and returned to preinjury levels by 12 hours (Figure 5A⇓).

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Figure 5.

Mechanical injury induces endothelial expression of GCF2 and PDGF-A. Vascular endothelial cells were grown to confluence and rendered quiescent by incubation in 1% FBS for 24 hours. Monolayers were injured by repeated scraping with a sterile stainless steel comb. Cell lysates were resolved by SDS-PAGE, before transfer to nylon membranes and Western immunoblot analysis with polyclonal antibodies raised against PDGF-AA (A) or GCF2 (B). After chemiluminescent detection, bands were quantitated by scanning densitometry. Open bars denote PDGF-A; closed bars, GCF2. Gels were stained with Coomassie blue to demonstrate equal protein loading.

We next determined whether GCF2 was induced by injury in this model. GCF2 is basally expressed in resting cells, and levels increased 4 hours and 8 hours after injury (Figure 5B⇑). By 12 hours, however, GCF2 levels increased 3-fold (Figure 5B⇑, closed bars), at which time PDGF-A levels had returned to background (Figure 5A⇑). The inverse temporal correlation between GCF2 and PDGF-A after peak expression of the growth factor in this model suggests a regulatory role for GCF2 in the expression of PDGF-A after injury.

Mechanical Injury of Arteries In Vivo Stimulates GCF2 Expression

To provide evidence for a similar relationship between these genes in the artery wall, levels of PDGF-A and GCF2 were assessed in the arterial media by immunohistochemical analysis various times after balloon catheter injury of the rat carotid²⁰ . PDGF-A antigen was weakly, if at all, expressed in the unmanipulated artery wall (Figure 6A⇓). Within 4 hours of injury, PDGF-A was clearly expressed in the media (Figure 6B⇓, arrow) as previously reported.^{26 27} Expression was only transient, because PDGF-A immunoreactivity was not detected at 6 hours (Figure 6C⇓) or after 10 days (Figure 6D⇓), when a significant neointima had formed, a hallmark of this model.²⁰

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Figure 6.

Induction of PDGF-A expression by mechanical injury to the rat carotid artery wall. Immunohistochemistry was performed on 5-μm cross sections of balloon-injured rat carotid arteries using affinity-purified rabbit polyclonal antibodies raised against PDGF-A. A, Uninjured artery. B, Four hours after injury. C, Six hours after injury. D, Ten days after injury. E, Four hours after injury, without PDGF-A IgG (primary) antibody but with secondary (goat anti-rabbit IgG) antibody and components of ABC kit. Brown staining (indicated by arrow) indicates specific signal. Sections were counterstained with hematoxylin to identify cell nuclei. Magnification ×200.

In contrast to PDGF-A, GCF2 was basally expressed in normal artery wall (Figure 7A⇓). Injury dramatically increased GCF2 expression in the media within 4 hours (Figure 7B⇓). Unlike PDGF-A, GCF2 levels remained elevated over the next 18 hours (Figures 7B⇓ through 7D⇓). Indeed, intense GCF2 expression even 24 hours after injury (Figure 7D⇓) contrasts dramatically with the rapid decline in PDGF-A levels between 4 and 6 hours (Figures 6B⇑ through 6C⇑). Ten days after injury, GCF2 was still expressed at levels comparable to those in the uninjured vessel wall (compare Figure 7E⇓ to 7A⇓). Staining for both GCF2 and PDGF-A was specific, because neither the secondary antibody nor components of the immunohistochemical detection system produced positive staining in the absence of primary antibody. These findings provide a spatial and inverse temporal correlation between GCF2 and PDGF-A after peak PDGF-A expression after injury to the vessel wall.

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Figure 7.

Sustained activation of GCF2 by injury. Immunohistochemical analysis was performed on 5-μm cross sections of balloon-injured rat carotid arteries using affinity-purified rabbit polyclonal antibodies raised against in vitro transcribed/translated GCF2. A, Uninjured artery. B, Four hours after injury. C, Six hours after injury. D, Twenty-four hours after injury. E, Ten days after injury. F, Uninjured artery without GCF2 (primary) antibody but with secondary (goat anti rabbit IgG) antibody and components of ABC kit. Brown staining indicates specific signal. The blue stain is a consequence of Evans blue infusion before sacrifice. Sections were counterstained with hematoxylin to identify cell nuclei. Magnification ×200.

We performed Western blot analysis for GCF2 protein with lysates of cultured smooth muscle cells prepared various times after mechanical injury to verify findings from immunostaining of the injured artery wall (Figure 7⇑). GCF2 protein was inducibly expressed within 1 hour of injury; levels continued to increase at 4 hours and 6 hours (Figure 8⇓). These data are consistent with the temporal pattern of inducible GCF2 protein expression in medial smooth muscle cells of the injured vessel wall (Figure 7⇑).

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Figure 8.

GCF2 protein is inducibly expressed by cultured smooth muscle cells after mechanical injury. Quiescent confluent smooth muscle cells were injured by repeated scraping, and lysates were resolved by SDS-PAGE. Western immunoblot analysis was performed with GCF2 antibodies. Bands were visualized by chemiluminescent detection. Gels were stained with Coomassie blue to demonstrate equal protein loading.

Overexpression of GCF2 Attenuates Endogenous PDGF-A Expression

The temporal relationship between GCF2 and PDGF-A in these models suggests that GCF2 may downregulate the expression of endogenous PDGF-A. Because our earlier studies used PDGF-A promoter reporter constructs in an overexpression setting, we performed Northern blot analysis using total RNA of two different vascular cell types 24 hours after transfection with GCF2-pcDNA3 or the backbone alone. Because this approach requires high transfection efficiency, we used a lipid-based formulation that enabled transfection of virtually the entire cell population (data not shown). PDGF-A mRNA, as expected, was expressed at higher levels in smooth muscle cells than endothelial cells²⁸ (Figure 9A⇓). In both cell types, however, endogenous PDGF-A expression was completely inhibited by GCF2-pcDNA3 transfection (Figure 9A⇓). To confirm the expression of GCF2 on transfection of this plasmid, the blot was stripped and reprobed with a ³²P-labeled GCF2 cDNA fragment. Strong hybridization was observed in both cell types with an mRNA species of ≈2.3 kb¹⁷ (Figure 9A⇓), consistent with the size of GCF2 cDNA insert present in the vector. In contrast, β-actin mRNA levels were unaffected by GCF2 overexpression (Figure 9A⇓). These findings demonstrate the capacity of GCF2 to suppress native PDGF-A expression in two vascular cell types. To demonstrate expression of GCF2 protein, as well as mRNA, after transfection with GCF2-pcDNA3, we prepared nuclear extracts of cells transfected with this construct as well as the backbone alone. EMSA, using these extracts together with ³²P-labeled Oligo A, indicates that in vivo expressed GCF2 protein can interact with its binding site in the proximal PDGF-A promoter (Figure 9B⇓).

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Figure 9.

Overexpression of GCF2 represses PDGF-A mRNA expression. A, Total RNA (20 μg) from endothelial cells (ECs) or smooth muscle cells (SMCs) transfected with 10 μg pcDNA3 or GCF2-pcDNA3 using FuGENE6 was resolved by electrophoresis, before transfer to a nylon membrane and hybridization with ³²P-labeled fragment of PDGF-A cDNA. After autoradiography, the blot was stripped and reprobed with a ³²P-labeled fragment of GCF2 cDNA. After a second exposure, the blot was stripped and reprobed with a ³²P-labeled fragment of β-actin cDNA. B, Nuclear extracts of endothelial cells transfected with 10 μg pcDNA3 or GCF2-pcDNA3 using FuGENE6 were incubated with ³²P-Oligo A, and the products were resolved by electrophoresis and subsequent autoradiography. The filled arrow indicates GCF2 binding activity; empty arrow, complex formation by extract proteins; and NE, nuclear extract. Free label was run off the gel for improved resolution.

Overexpression of GCF2 Inhibits Smooth Muscle Cell Replication

Finally, we determined whether exogenous GCF2 had the ability to influence cell proliferation. Endothelial cells or smooth muscle cells transfected with either GCF2-pcDNA3 or pcDNA3 were resuspended by trypsinization and quantitated. Endothelial cell replication after 24 hours was unaffected by GCF2 transfection (Figure 10A⇓). However, proliferation was inhibited in smooth muscle cells transfected with GCF2 (Figure 10B⇓). Cell growth in this group 24 hours after transfection was 37% less than the population of cells transfected with pcDNA3 alone (Figure 10B⇓). In contrast, proliferation of smooth muscle cells transfected with construct GCF2Δ428-pcDNA3, which lacks the GCF2 DNA binding domain, was not inhibited (Figure 10B⇓). Interestingly, addition of PDGF-AA (30 ng/mL) rescued the cells from growth inhibition by GCF2 (Figure 10B⇓). Morphological inspection of both cell types as well as trypan blue exclusion experiments revealed that differences in cell number were not due to cytotoxicity (data not shown). Therefore, although GCF2 suppressed endogenous PDGF-A expression in endothelial cells and smooth muscle cells, proliferation of only the latter cell type was attenuated as a consequence.

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Figure 10.

GCF2 overexpression inhibits smooth muscle cell proliferation. ECs or SMCs were seeded in 96-well plates and left undisturbed for 48 hours. Cells were transfected with 1 μg pcDNA3, GCF2-pcDNA3, or GCF2Δ428-pcDNA3 using FuGENE6 in complete growth medium. Cell numbers per well were determined immediately before (day 0) or 24 hours after (day 1) the addition of serum (±PDGF-AA, 30 ng/mL), by quantitation of trypsinized suspensions by Coulter counter. Cell numbers were not assessed on subsequent days because of artifact introduced by contact inhibition. SFM (empty bars) indicates cells that were incubated with serum-free medium and exposed to the same amount of FuGENE6 as those transfected with DNA. Mean+SEM of 3 experiments performed in triplicate is shown. **P<0.0001 relative to the pcDNA3 value by Student paired t test.