Targeted Overexpression of CCAAT/Enhancer-Binding Protein-δ Evokes Enhanced Gene Transcription of Platelet-Derived Growth Factor α-Receptor in Vascular Smooth Muscle Cells
Abstract— Platelet-derived growth factor (PDGF) is thought to play a significant role in various models of vascular remodeling, particularly in the early process of vascular diseases. Its action is mediated by its specific receptor, the PDGF receptor. The PDGF α-receptor (PDGFαR) plays an important role in the growth and proliferation of vascular smooth muscle cells (VSMCs), and its gene expression is thought to be regulated by several potential transcriptional nuclear factors. However, the detailed mechanisms of tissue-specific transactivation of the PDGFαR gene in VSMCs remain to be clarified. We have previously demonstrated that the rat PDGFαR gene contains an enhancer core sequence for CCAAT/enhancer-binding proteins (C/EBPs) in its promoter region, and we have also suggested that C/EBP-δ is the principal factor involved in the induction of tissue-specific transcriptional activity of the PDGFαR gene in VSMCs. To explore the definitive roles of C/EBP-δ protein on PDGFαR gene transcription in VSMCs, we developed C/EBP-δ transgenic rats by using a chimeric fusion gene of the mouse smooth muscle α-actin promoter and an entire coding region of rat C/EBP-δ cDNA. This report describes the first successful targeted overexpression of C/EBP-δ capable of inducing PDGFαR gene transcription and modifying cell proliferative activity to PDGFs. Targeted overexpression of C/EBP-δ evokes high levels of PDGFαR gene expression, susceptibility to VSMC growth, and proliferation of VSMCs to PDGFs. The results obtained reveal evidence of a new role and new functional significance of C/EBP-δ on VSMC growth via the PDGFαR during the process of vascular remodeling and atherosclerosis.
- platelet-derived growth factor α-receptor
- vascular smooth muscle cells
- CCAAT/enhancer-binding protein-&dgr
- transgenic rats
- gene expression
Vascular smooth muscle cells (VSMCs) acquire the ability to proliferate and to remodel the extracellular matrix when they are subjected to particular pathophysiological challenges.1 The signals controlling this process are complex and may involve the production of growth factors that act locally to orchestrate the response. Platelet-derived growth factor (PDGF) is one of the major mitogens, and it is responsible for VSMC growth and proliferation through its specific receptor, the PDGF receptor (PDGFR). In recent years, PDGF and its receptor have been proposed to play significant roles in various models of vascular remodeling in vitro and particularly in the early process of vascular diseases, including coronary artery disease and atherosclerosis.2,3 In fact, VSMCs are capable of producing and releasing PDGF-A chain in a growth-dependent manner, which may contribute to the autocrine and paracrine growth-stimulating mechanism of blood vessels through the PDGF α-receptor (PDGFαR).4
Several potential transcriptional factors are thought to be involved in the regulation of PDGFαR gene expression. However, the trans-acting factors regulating the expression of PDGFαR in VSMCs remain to be identified. We have previously reported that the basal transcriptional activity of the PDGFαR gene is predominantly driven by a transcriptional factor, CCAAT/enhancer-binding protein (C/EBP)-δ, in VSMCs.5 We have also demonstrated that PDGFαR gene transcription differs between hypertensive and normotensive animals6 and that regulation of this transcription occurs in a cytokine-dependent manner,7 suggesting that PDGFαR gene transcription may be controlled by a genetically and pathophysiologically enhanced C/EBP-δ expression in VSMCs. To prove this hypothesis, overexpression studies of C/EBP-δ using transient or permanent transfection experiments are important. However, because the transfection efficiency onto primary cultured VSMCs is very low, it is difficult to establish a cell line that consistently overexpresses exogenous DNA.
In the present study, we generated transgenic rats overexpressing C/EBP-δ through a mouse smooth muscle (SM) α-actin promoter and succeeded in creating the first rat model of SM-specific C/EBP-δ overexpression capable of transactivating PDGFαR gene expression. Using cultured VSMCs derived from the transgenic rats, we also demonstrated that C/EBP-δ overexpression made PDGFs susceptible to cell proliferation activity.
Materials and Methods
Recombinant human PDGF-AA and -BB were purchased from Upstate Biotechnology, Inc, and affinity-purified goat IgG capable of neutralizing PDGF-AA activity was purchased from R & D Systems, Inc. Affinity-purified rabbit polyclonal antibodies for rat PDGFαR, rat PDGF β-receptor (PDGFβR), and rat C/EBP-δ were obtained from Santa Cruz Biotechnology, Inc. [α-32P]dCTP, [γ-32P]ATP, and [125I]PDGF-BB were purchased from Amersham-Pharmacia Biotechnology, and [125I]PDGF-AA was purchased from Biomedical Technologies.
Generation of Transgenic Rats
The SM α-actin–C/EBP-δ chimeric gene was constructed by fusing a 3.6-kb fragment of the mouse SM α-actin promoter (designated SMP8)8,9 to an entire coding region of rat C/EBP-δ cDNA, followed by the simian virus 40 early polyadenylation signal fragment (Figure 1). The male pronuclei of fertilized eggs from Sprague-Dawley rats were microinjected with a 4.8-kb linearized DNA (designated SMP8–C/EBP-δ) at the transgenic animal facility of Nippon SLC Corp. Microinjected eggs were implanted into the oviduct of pseudopregnant female rats and carried to term. Positive founders for SMP8–C/EBP-δ were identified by polymerase chain reaction and bred to wild-type Sprague-Dawley rats for propagation of the line. The inserted transgene was identified by Southern blotting of genomic DNA from the tail. Heterozygotes and nontransgenic progeny from F1 were selected by polymerase chain reaction of genomic DNA.
VSMCs were isolated from thoracic aortas of transgenic rats and their nontransgenic littermates as previously described10 and were maintained in DMEM supplemented with 10% FBS. In preparation for all experiments, subconfluent cells at 2 to 4 passages from primary culture were made quiescent by placing them in DMEM supplemented with ITS medium (Sigma) for 2 days.11
RNA Isolation and Northern Blotting
Total RNA was isolated from tissues and cultured VSMCs by a single-step extraction method of ISOGEN (Nippon Gene). Northern hybridization, autoradiography, and densitometric analysis were performed as previously described.6,7
Western blotting was performed by the method previously described.5,7 Briefly, nuclear extracts (2.5 μg) or whole-cell lysates (10 μg) prepared from VSMCs were subjected to Western blotting of C/EBP-δ or PDGFαR, respectively. SDS-PAGE and transfer to a polyvinylidine difluoride (PVDF) membrane (Bio-Rad Laboratories) were performed by using standard methods. The membrane was treated with diluted primary antibodies against C/EBP-δ or PDGFαR, and the immunoreactive proteins were detected by autoradiography with the use of an ECL chemiluminescence detection system (Amersham-Pharmacia Biotechnology).
PDGFR Binding Assay
VSMCs (2×105 per well) in 24-well plates were cultured for 12 hours and then rendered quiescent for 2 days. For the saturation binding assay, cells were incubated with different concentrations (1 to 100 ng/mL) of [125I]PDGF-AA or [125I]PDGF-BB for 3 to 4 hours at 4°C in the absence or presence of unlabeled PDGF-AA or -BB (500 ng/mL each), respectively. Cells were then rinsed 4 times with ice-cold DMEM containing 10 mmol/L HEPES-NaOH buffer (pH 7.4) and 0.5% BSA and solubilized in a 1-mL buffer of 20 mmol/L HEPES-NaOH (pH 7.4) containing 1% Triton X-100 and 10% glycerol, and radioactivity associated with the cells was counted. Specific binding was defined as the difference between total and nonspecific binding, and saturation binding data were subjected to Scatchard analysis to obtain dissociation constants (Kd values) and the maximum number of binding sites (Bmax).
Measurement of BrdU Incorporation
VSMCs (1×104 per well) in 96-well plates were cultured for 12 hours. After cells were made quiescent for 2 days, they were treated with 20 ng/mL of PDGF-AA or -BB for 12 hours. To test the effect of neutralizing antibodies for PDGF-AA on baseline levels of VSMC proliferation activity, quiescent cells were cultured in the presence of affinity purified anti–PDGF-AA IgG (50 μg/mL) for 12 hours. Bromodeoxyuridine (BrdU) incorporation was determined by using a Cell Proliferation ELISA System (Amersham-Pharmacia Biotechnology) according to the manufacturer’s specifications.
EMSA and Supershift Assay
Nuclear extracts were prepared from VSMCs according to the method described by Dignam et al.12 Construction of a double-stranded oligonucleotide C/EBP probe, electrophoretic mobility shift assay (EMSA), and supershift assay were performed as previously described.5–7
ANOVA with the Bonferroni-Dunn post hoc test was used to analyze differences between the two experimental groups, and all data were expressed as mean±SD. Values of P<0.05 were considered to indicate statistical significance.
Generation of C/EBP-δ Transgenic Rats and Transgenic mRNA Expression in Tissues
Six SMP8–C/EBP-δ founder animals were obtained from a total of 14 rats screened. Mating of hemizygous rats with control rats produced ≈50% transgenic offspring with equal sex distribution. Tail DNAs isolated from two selected transgenic rat lines (Tg1 and Tg2) were subjected to Southern blotting analysis to identify the insert transgene and to compare its copy number (Figure 2A). The transgene copy number of Tg1 was significantly higher than that of Tg2, whereas C/EBP-δ mRNA levels in aortic media obtained from Tg2 were almost the same as those in media obtained from Tg1 (Figure 2B). Accordingly, we used heterozygotes and nontransgenic progeny from Tg2 thereafter.
C/EBP-δ mRNA levels were evaluated by Northern blotting in several tissues, including aortic media, heart ventricle, liver, and kidney tissues dissected from a 12-week-old transgenic male rat and a nontransgenic male littermate (Figure 3). In the transgenic rat, C/EBP-δ mRNA was detected at a high level in the aortic media but was not detected in the other tissues. On the other hand, C/EBP-δ mRNA was not detectable by Northern blotting in any of the tissues dissected from the nontransgenic littermate.
C/EBP-δ and PDGFR Expression in VSMCs
To elucidate significant roles of C/EBP-δ on PDGFαR gene expression in VSMCs, VSMCs were prepared from a 12-week-old male transgenic rat and a nontransgenic littermate. Expression levels of C/EBP-δ, PDGFαR, and PDGFβR mRNAs were measured by Northern blotting (Figure 4A). Both C/EBP-δ and PDGFαR mRNAs were detected at high levels in VSMCs from the transgenic rat. In contrast, these mRNAs were detected at very low levels in VSMCs from the nontransgenic littermate. There were no significant differences in PDGFβR mRNA levels between the transgenic and nontransgenic VSMCs. Protein levels of C/EBP-δ and PDGFαR were evaluated by Western blotting in the nuclear extracts and whole-cell lysates, respectively (Figure 4B). Both proteins were highly expressed in transgenic VSMCs compared with nontransgenic cells, which was consistent with the results obtained from Northern blotting.
Figure 5 shows the Scatchard analysis of data obtained from the saturation binding assay for PDGF-BB and -AA. The Kd values for PDGF-BB were estimated to be ≈19 ng/mL both in nontransgenic and transgenic VSMCs. The binding sites for PDGF-BB in nontransgenic and transgenic VSMCs were estimated to be 250 000 and 300 000 sites per cell, respectively. On the other hand, the Kd values for PDGF-AA were estimated to be ≈22 ng/mL in both nontransgenic and transgenic cells. The binding sites for PDGF-AA in nontransgenic and transgenic VSMCs were estimated to be 4200 and 68 000 site per cell, respectively.
DNA-Binding Activity of Exogenously Expressed C/EBP-δ
To test the DNA-binding ability of C/EBP-δ protein overexpressed in transgenic VSMCs, an EMSA and a supershift assay were performed by using a C/EBP probe (Figure 6). Although the labeled C/EBP probe was shifted by nuclear extracts from transgenic and nontransgenic VSMCs generating two specific bands (B1 and B2), these band intensities of transgenic cells were markedly stronger than those of nontransgenic cells (Figure 6, lane 1 versus lane 2). Supershift assay for the nuclear extracts from transgenic VSMCs demonstrated that band B1 was almost completely supershifted and band B2 was partially supershifted by antibodies against rat C/EBP-δ (Figure 6, lane 3).
Cell Proliferation to PDGFs
Finally, cell proliferation to PDGFs was evaluated by BrdU incorporation to identify a physiological function of C/EBP-δ in VSMCs. In Figure 7, BrdU incorporation was measured in nontransgenic and transgenic cells 12 hours after treatment with PDGF-AA or -BB (20 ng/mL each). Baseline levels of BrdU incorporation in transgenic cells were significantly higher (1.9-fold) than those in nontransgenic cells. To explain the difference in baseline levels of BrdU incorporation between nontransgenic and transgenic VSMCs, we examined the effect of neutralizing antibodies for PDGF-AA on cell proliferation activity. Neutralizing antibodies had no effect on proliferative activity in nontransgenic cells, whereas the antibodies significantly suppressed baseline levels of proliferative activity in transgenic cells to a level almost equal to that in nontransgenic cells. Furthermore, PDGF-AA significantly enhanced (by 1.8-fold) proliferative activity only in transgenic cells but not in nontransgenic cells, and PDGF-BB significantly increased proliferative activity both in nontransgenic and transgenic VSMCs compared with the corresponding baseline levels (a 2.8-fold and 3.3-fold increase, respectively).
C/EBP-δ was originally identified in the liver as a basic leucine zipper-type transcriptional factor belonging to the C/EBP family, whose members are all closely related.13–15 C/EBP-δ expression is very low or undetectable in normal tissues and can be induced by proinflammatory cytokines, such as interleukin (IL)-1β and IL-6.7,16 Two C/EBP members, C/EBP-β (also called NF-IL6) and C/EBP-δ (also called NF-IL6β), are rapidly induced during inflammation17 and mediate transcriptional induction of most class I acute-phase protein genes, such as haptoglobin,18 α1-acid glycoprotein,19 complement component 3,20 C-reactive protein,21 and serum amyloid A.22 Previously, we have demonstrated that C/EBP-δ acts as a major transcriptional activator and that C/EBP-β probably acts as a transcriptional suppressor for PDGFαR gene expression in VSMCs.5–7 We have also reported that a high level of C/EBP-δ expression is a major determinant for markedly elevated differential gene expression of PDGFαRs in VSMCs of genetically hypertensive rats6 and for IL-1β–mediated PDGFαR gene induction in VSMCs of Sprague-Dawley rats, a normotensive rat strain.7 However, these earlier experiments have not provided conclusive evidence of a direct link between C/EBP-δ expression and transactivation of the PDGFαR gene in VSMCs.
Studies based on mice carrying null mutations are a useful tool for investigating the diversity of transcriptional regulation of various genes. In the case of the PDGFαR gene, naturally occurring deficient mice carrying the deletional mutation of the patch (Ph) locus have already been reported.23 Patch homozygotes Ph(−/−) have been shown to lack the PDGFαR genomic sequences, to display gross anatomic abnormalities, and to die midway through gestation. On the other hand, mice carrying null mutations of either C/EBP-β or -δ have been generated by homologous recombinations, and these two types of deficient mice have failed to reveal any dramatic defects or anatomic abnormalities.24 However, it is impossible to reach a definitive conclusion regarding the overall role of C/EBP-β and -δ in the regulation of the PDGFαR gene promoter on the basis of these data alone, inasmuch as C/EBP-β or -δ might well be able to compensate for the absence of the other. In fact, double-knockout mice with both C/EBP-β and C/EBP-δ deficiencies24 have been shown to have major defects and abnormalities in various tissues and to die midway through gestation in the same manner as PDGFαR-deficient mice. In this context, the overexpression study becomes another powerful tool to elucidate the physiological function of C/EBP members on tissue-specific transcriptional control of the PDGFαR gene in VSMCs. In the present study, SM cell–specific transgenic rats were generated to clarify the potentially important function of C/EBP-δ on PDGFαR gene transcription and VSMC proliferation activity to PDGF-AA through the PDGFαR. The results clearly showed that C/EBP-δ was the principal factor involved in inducing the tissue-specific transcriptional activity of the PDGFαR gene in VSMCs.
To examine the tissue-specific function of C/EBP-δ in VSMCs, we used the SM α-actin promoter to target C/EBP-δ expression. Because SM α-actin gene expression is primarily restricted to SM cell–rich tissues of adult rodents and rabbits, its promoter seemed well suited to the task. In fact, the SMP-8 promoter used in the present study directed robust levels of transgene expression specifically to aortic media (Figure 3). Wang and colleagues8,9 have previously generated two lines of transgenic mice overexpressing insulin growth factor-I and insulin-like growth factor–binding protein-4 under control of the same promoter, SMP8, and have demonstrated that the selective overexpression of the transgenes is observed in SM cell–rich tissues such as those of the aorta. These results indicate that the SMP8 promoter can drive tissue-specific transcriptional activity in SM-rich tissues of adult rodents.
High levels of exogenous C/EBP-δ expression and the ensuing endogenous PDGFαR induction were confirmed by Northern blotting and Western blotting analyses of transgenic VSMCs (Figure 4). Because PDGF-AA can bind only to PDGFαR, analysis of both receptor binding and cell proliferation to PDGF-AA (Figures 5 and 7⇑) demonstrated that only transgenic VSMCs expressed high levels of functionally active PDGFαR on the cell surface. Obviously, the DNA-binding analyses (Figure 6) indicated that exogenously induced C/EBP-δ in the nuclear extracts from transgenic VSMCs can act as a functionally active regulatory factor for PDGFαR gene transcription. In the proliferation assay experiments, we found that there was a significant increase in basal proliferative activity of transgenic VSMCs without the addition of exogenous PDGFs (Figure 7). Because our previous report6 demonstrated that VSMCs, even if they are quiescent cells, consistently produce and release PDGF-AA, they should be stimulated by endogenous PDGF-AA via an autocrine or a paracrine loop when an adequate amount of PDGFαR is expressed on their surfaces. Generally, wild-type VSMCs are known to express only a few levels of PDGFαR at a quiescent state, whereas the transgenic VSMCs reported in the present study express high levels of PDGFαR. Therefore, there was an ≈2.0-fold increase in basal proliferative activity of transgenic VSMCs compared with nontransgenic cells. We demonstrated that this increase can be specifically blocked by the addition of neutralizing antibodies for PDGF-AA, strongly supporting our above hypothesis. Northern and Western blotting demonstrated that there were no significant differences in PDGFβR expression between nontransgenic and transgenic VSMCs. However, there were significant differences between nontransgenic and transgenic cells in binding sites for PDGF-BB (250 000 versus 300 000 sites per cell, respectively) and in the fold stimulation of cell proliferation activity to PDGF-BB (2.8- versus 3.3-fold, respectively). Moreover, the fold stimulation of cell proliferation activity to PDGF-BB was significantly higher than that to PDGF-AA in transgenic VSMCs (3.3-fold versus 1.9-fold, respectively). Because PDGF-BB binds not only PDGFβR but also PDGFαR, these discrepancies can be explained by the binding specificity for PDGFs to PDGFRs.
In conclusion, the present study, in conjunction with our previous work, indicates that PDGFαR gene expression is controlled by a genetically or a pathophysiologically enhanced C/EBP-δ expression in the vessel wall. Accordingly, the suppression of C/EBP-δ expression in VSMCs may become a potential therapeutic candidate for the prevention of the initiation or progression of vascular remodeling.
This study was supported in part by grants-in-aid for scientific research from the Ministry of Education, Science, Culture, and Sports of Japan (Nos. 11838012 and 12470155), by a Japan Heart Foundation grant for research on hypertension and vascular metabolism, and by grants from the Takeda Medical Research Foundation and the Mochida Memorial Foundation for medical and pharmaceutical research. The animals used in this study were cared for in the Laboratory Animal Center at Ehime University School of Medicine, and the authors extend their special thanks to Ken-ichi Okugawa at the Animal Center for his technical assistance.
Original received January 25, 2001; resubmission received June 29, 2001; accepted July 18, 2001.
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