Transcriptional Regulation of the Platelet-derived Growth Factor α Receptor Gene via CCAAT/Enhancer-binding Protein-δ in Vascular Smooth Muscle Cells*

Inflammatory cytokines stimulate the proliferation of vascular smooth muscle cells (VSMC) and play a pivotal role in the pathogenesis of vascular diseases including atherosclerosis and restenosis. Mitogenic response of interleukin-1β (IL-1β) on VSMC is thought to be mediated by induction of endogenous platelet-derived growth factor (PDGF), especially PDGF-AA. Although the action of PDGF-AA is mediated by its specific receptor, PDGFα-receptor (PDGFαR), very little is known about the regulatory mechanism of PDGFαR gene expression in VSMC. To understand the mechanism, we studied the transcriptional control of the PDGFαR gene in VSMC after treatment with IL-1β. IL-1β (10 ng/ml) drastically increased both PDGFαR and CCAAT/enhancer-binding protein δ (C/EBPδ) mRNA levels in a time dependent manner. A rapid induction of C/EBPδ mRNA within 30 min was followed by slower emergence of PDGFαR mRNA, which reached the maximum level in 12 h, whereas C/EBPδ mRNA was detectable at 30 min and reached the maximum level at 3 h. Electromobility shift and supershift assays revealed that IL-1β markedly increased DNA-protein complex, which was mainly composed of C/EBPβ and/or -δ. Both Western blotting and immunohistochemistry demonstrated that either C/EBPβ or -δ expression was induced by IL-1β exclusively in nuclei of VSMC. On the other hand, overexpression of C/EBPδ specifically transactivated the promoter activity of the PDGFαR gene and significantly enhanced VSMC proliferation in PDGF-treated cells. We conclude that induction of PDGFαR expression is mainly mediated by C/EBPδ expression in VSMC, and a high level of C/EBPδ expression may be involved in the pathogenesis of atherosclerosis and restenosis.

Inflammatory cytokines stimulate the proliferation of vascular smooth muscle cells (VSMC) and play a pivotal role in the pathogenesis of vascular diseases including atherosclerosis and restenosis. Mitogenic response of interleukin-1␤ (IL-1␤) on VSMC is thought to be mediated by induction of endogenous platelet-derived growth factor (PDGF), especially PDGF-AA. Although the action of PDGF-AA is mediated by its specific receptor, PDGF␣-receptor (PDGF␣R), very little is known about the regulatory mechanism of PDGF␣R gene expression in VSMC. To understand the mechanism, we studied the transcriptional control of the PDGF␣R gene in VSMC after treatment with IL-1␤. IL-1␤ (10 ng/ml) drastically increased both PDGF␣R and CCAAT/enhancer-binding protein ␦ (C/EBP␦) mRNA levels in a time dependent manner. A rapid induction of C/EBP␦ mRNA within 30 min was followed by slower emergence of PDGF␣R mRNA, which reached the maximum level in 12 h, whereas C/EBP␦ mRNA was detectable at 30 min and reached the maximum level at 3 h. Electromobility shift and supershift assays revealed that IL-1␤ markedly increased DNA-protein complex, which was mainly composed of C/EBP␤ and/or -␦. Both Western blotting and immunohistochemistry demonstrated that either C/EBP␤ or -␦ expression was induced by IL-1␤ exclusively in nuclei of VSMC. On the other hand, overexpression of C/EBP␦ specifically transactivated the promoter activity of the PDGF␣R gene and significantly enhanced VSMC proliferation in PDGF-treated cells. We conclude that induction of PDGF␣R expression is mainly mediated by C/EBP␦ expression in VSMC, and a high level of C/EBP␦ expression may be involved in the pathogenesis of atherosclerosis and restenosis.
Excessive or uncontrolled replication and migration of vascular smooth muscle cells (VSMC) 1 are critical events involved in a number of vascular diseases including atherosclerosis, hypertension, and restenosis that often occurs after balloon angioplasty (1)(2)(3). Morphologic studies of the sequencing events in the arterial wall of animals with artificially induced hypercholesterolemia showed that macrophages are present in all processes of the formation of atherosclerotic lesions (4 -7). The normal function of the macrophage is to act not only as an antigen-presenting cell to T lymphocytes but also as a source of several growth factors such as platelet-derived growth factor (PDGF), basic fibroblast growth factor, tumor necrosis factor ␣, and transforming growth factor ␤ 1 , which are generally not expressed in the normal artery, whereas they are up-regulated in the lesions of atherosclerosis (3). Thus, the macrophage is thought to be a principal inflammatory mediator of cells in the atheromatous plaque microenvironment.
Interleukin (IL)-1␤ is one of the major secretory products of activated macrophage and can induce proliferation of cultured fibroblasts and VSMC (8 -11). Previous studies (12)(13)(14) have demonstrated that mitogenic activity of IL-1␤ for fibroblasts and VSMC is mediated indirectly via an autocrine loop by causing the release of PDGF-AA, which then specifically binds to the PDGF ␣-receptor (PDGF␣R) subtype on cell surface. Furthermore, recent studies (15,16) have also demonstrated that IL-1␤ can up-regulate PDGF␣R expression in rat lung fibroblasts, thereby enhancing PDGF-mediated mitogenesis and chemotaxis of lung fibroblasts. Although the pathophysiological implications of IL-1␤-induced PDGF␣R expression are beginning to be recognized, little is known about the molecular mechanism involved. Therefore, we have investigated the molecular mechanism of PDGF␣R gene transcription in VSMC and obtained results indicating that IL-1␤ induces PDGF␣R gene expression via a trans-acting nuclear factor, CCAAT/enhancer-binding protein ␦ (C/EBP␦). were performed on cells at 3-10 passages from primary culture. In preparation for experiments, confluent cells, which exhibited a typical hill and valley pattern of smooth muscle cells in culture, were made quiescent by placing them in a defined serum-free medium containing insulin (10 g/ml), transferrin (10 g/ml), and sodium selenite (10 ng/ml) for 48 h. This medium has been shown to maintain VSMC in a quiescent and noncatabolic state for an extended period of time (18).
Preparation of cDNA Probes and Northern Blotting-A 0.6-kilobase pair fragment of rat PDGF␣R cDNA (19), 1.1-kilobase pair NcoI fragment of EBP␣, 0.4-kilobase pair NcoI fragment of EBP␤ or 1.0-kilobase pair EcoRI-BamHI fragment of EBP␦ was used as a probe for Northern blotting. Each DNA fragment was labeled with [␣-32 P]dCTP using the random primer method. Total cellular RNA extraction from VSMC and Northern blot analysis were carried out by the methods described previously (19,20).
Electromobility Shift and Supershift Assays-Nuclear extracts were prepared from VSMC according to the method described by Dignam et al. (21). After protein concentrations were determined using Bio-Rad Protein Assay Reagent, nuclear extracts were divided into small aliquots, quickly frozen in liquid nitrogen, and stored at Ϫ80°C. For electrophoretic mobility shift assay and supershift assay, a doublestranded oligodeoxynucleotide probe for the consensus sequence of C/EBP was generated by annealing two complementary oligodeoxynucleotides corresponding to the nucleotide sequence spanning Ϫ165 to Ϫ138 in the 5Ј-flanking region of the rat PDGF␣R gene, 5Ј-CCCCAGATTGCATAAGAGCAAAAAGCCA-3Ј. Another doublestranded oligodeoxynucleotide probe for the consensus sequence of nuclear factor-1, 5Ј-CCTTTGGCATGCTGCCAATAT G-3Ј, was purchased from Promega (Madison, WI) and used as an unrelated competitor. The C/EBP probe was end-labeled with [␥-32 P]ATP using T 4 -polynucleotide kinase. Nuclear extracts (2 g) were incubated with 2.0 ϫ 10 4 cpm of the labeled C/EBP probe for 30 min at room temperature in a 10-l binding buffer containing 12 mM Hepes-KOH, pH 7.9, 60 mM KCl, 4 mM MgCl 2 , 1 mM EDTA, 1 mM dithiothreitol, 10% glycerol, and 50 g/ml of poly(dI-dC)(dI-dC) (Amersham Pharmacia Biotech). For competition experiments or supershift assay, a 100-fold molar excess of unlabeled probe or 1-2 l of antibodies against each subtype of the C/EBP family was added to nuclear extracts, respectively, and was incubated for 30 min at room temperature before addition of the labeled C/EBP probe. Then all reaction mixtures were analyzed by 5% polyacrylamide gel electrophoresis under nondenaturing conditions, and the gel was dried and processed as described previously (19).
Plasmid Construction and DNA Transfection-PDGF␣R promoter/ firefly luciferase fusion vector, which was designated as Ϫ1,381/ϩ68 WT, was prepared by insertion of the basal promoter region spanning positions Ϫ1,381 through ϩ68 of the PDGF␣R gene (19) onto pGL3-Basic vector (Promega, Madison, WI). Mock vector, which was designated as MSV, was prepared by deletion of the coding region of C/EBP␦ cDNA from EBP␦. pRL-CMV (Promega), which can drive Renilla luciferase activity, was used as an internal control to normalize transfection efficiency. One day before transfection, VSMC were seeded onto 60-mm dishes (5 ϫ 10 5 cells/dish) or 96-well plates (1 ϫ 10 4 cells/well) for luciferase or cell proliferation assay, respectively. DNA transfection was performed with cells at approximately 70% confluency according to the manufacturer's specifications of Lipofectamine Plus (Life Technologies, Inc., Tokyo, Japan). For luciferase assay, pGL3-Basic or Ϫ1,381/ ϩ68 WT (3 g/dish each) was used for cotransfection with EBP␣, -␤, -␦, or MSV (3 g/dish each) in addition to pRL-CMV (1 g/dish). For cell proliferation assay, EBP␦ or MSV (0.2 g/well each) was used for transfection of VSMC.
Luciferase and Cell Proliferation Assays-Promoter activity was determined by the Dual-Luciferase Reporter Assay System (Promega) as described previously (22,23). After normalization for transfection efficiency in reference to sequentially determined Renilla luciferase activity, each promoter activity was presented as a relative luciferase activity in reference to the activity of Ϫ1,381/ϩ68 WT cotransfected with MSV that was set to unity. Cell proliferation reagent WST-1 (Roche Molecular Biochemicals) was used for VSMC proliferation assay according to the manufacturer's specifications. One day after transfection, PDGF-AA or -BB (50 ng/ml each) was directly added to the culture medium, and cells were incubated for an additional 24-h. Then WST-1 reagent (0.1 volume of culture medium) was added to each well, and cells were incubated for 30 min at 37°C. Finally, the absorbance (A 450 Ϫ A 690 ) of each well was measured by an enzyme-linked immunosorbent assay reader, and cell proliferation activity was presented as a relative activity in reference to the activity of MSV-transfected VSMC after treatment with PDGF-AA that was set to unity.
Western Blotting-Western blotting was performed by the method described previously (17,19). Briefly, nuclear extracts (2.5 g) prepared from VSMC were directly subjected to Western blotting for C/EBP␣, -␤, and -␦. After boiling with sample buffer, SDS-polyacrylamide gel electrophoresis was performed using a 12.5% gel according to Laemmli (24), and proteins in the gel were transferred to a polyvinylidene difluoride membrane (Trans-Blot Transfer Medium; Bio-Rad) by electroblotting for 1 h at 100 V. The membrane was treated with diluted primary antibodies against each C/EBP member, and immunoreactive proteins were detected by autoradiography using a chemiluminescence detection system (the ECL Western blotting analysis system; Amersham Pharmacia Biotech). Immunohistochemistry-VSMC were cultured for 24 h in wells of chamber slides (Lab-Tek II Chamber Slide; Nalege Nunc International, Naperville, IL) at a density of 1 ϫ 10 4 cells/well and then fixed with 100% cold acetone for 30 min. All slides were treated with PBS containing 0.3% H 2 O 2 for 10 min at 37°C, and the following steps were performed according to the manufacturer's specifications for the Vectastain Elite ABC Kit (Vector Laboratories, Inc., Burlingame, CA). After blocking for 30 min at 37°C with diluted normal goat serum, slides were covered with diluted primary antibodies for 1 h at room temperature. After exposure to a solution containing diluted biotinylated secondary antibodies, the slides were treated with a Vectastain Elite ABC Reagent. Positive staining cells were visualized with a solution of 3,3Ј-diaminobenzidine tetrahydrochloride substrate kit (Vector Laboratories, Inc.). The slides were counterstained with hematoxylin, dehydrated with ethanol gradient and with 100% xylene, and then mounted in mounting medium (Mount-Quick; Daido Sangyo Co., Ltd., Tokyo, Japan).
Statistical Analysis-Analysis of variance with Bonferroni-Dunn post hoc analysis was used to analyze differences between two experimental groups. All data are expressed as mean ϩ S.E., and statistical significance is defined as p Ͻ 0.05.

Messenger RNA Induction of PDGF␣R and C/EBP␦ by IL-
1␤-VSMC derived from Harlan Sprague-Dawley rats were incubated for 6 h in the presence of IL-1␤ at various concentrations (0 -40 ng/ml), and mRNA levels of both PDGF␣R and C/EBP␦ were determined by Northern blotting. Although baseline levels of both mRNA expression were very low or almost negligible in quiescent VSMC, they were markedly increased by the treatment with IL-1␤ at doses up to 10 ng/ml in a dose-dependent manner (data not shown). Therefore, we used the dose of IL-1␤ at 10 ng/ml hereafter. To identify the kinetic relationship between C/EBP and PDGF␣R expression during treatment with IL-1␤, mRNA levels of C/EBP members and PDGF␣R were monitored for 48 h. As shown in Fig. 1, A and B, a high level of PDGF␣R mRNA expression was accompanied by a similarly marked induction of C/EBP␦ mRNA in VSMC following the addition of IL-1␤. A rapid induction of C/EBP␦ mRNA within 30 min was followed by slower emergence of PDGF␣R mRNA, which reached the maximum level (12.2-fold higher than the zero time level) in 12 h, whereas C/EBP␦ mRNA reached the maximum level (10.5-fold higher than the zero time level) at 3 h, continued at least for 12 h, and then decreased gradually to a basal level within 48 h. The significant induction of PDGF␣R mRNA expression began to increase at 3 h and continued at least for 24 h. This time course indicates a causal relationship in which C/EBP␦ induced PDGF␣R gene transcription. In contrast, a high level of C/EBP␣ mRNA expression was observed even in a quiescent state, and IL-1␤ did not significantly alter it. Although the induction of C/EBP␤ mRNA expression was also detectable at 30 min, it continued for an extended period up to 48 h.
Effect of IL-1␤ on a Half-life Time of PDGF␣R mRNA-To determine whether the induction of PDGF␣R mRNA expression after treatment with IL-1␤ is due to the effect of IL-1␤ on the mRNA stability, the half-life time of PDGF␣R mRNA was determined in the presence of actinomycin D (Fig. 2). VSMC were pretreated with IL-1␤ for 12 h, washed with PBS, and then exposed to a freshly prepared medium with or without IL-1␤ in the presence of 5 mg/ml actinomycin D. A half-life time of the PDGF␣R mRNA seen in cells incubated in the medium with IL-1␤ was 8.6 h, and that without IL-1␤ was 10.0 h, indicating that IL-1␤ does not significantly affect the PDGF␣R mRNA stability.
Characterization of C/EBP Members That Interact with PDGF␣R Gene Promoter-Electrophoretic mobility shift assay was performed by using a labeled C/EBP probe containing the consensus sequence of C/EBP recognition site of rat PDGF␣R promoter region (Fig. 3A). Nuclear extracts were prepared from either quiescent or IL-1␤-treated VSMC. Although the C/EBP probe was not shifted by nuclear extracts from quiescent VSMC, it was clearly shifted by nuclear extracts from VSMC treated with IL-1␤ for 12 h, generating a band of the DNAprotein complex. The DNA-protein complex was markedly competed out by a 100-fold molar excess of unlabeled C/EBP probe but not by a 100-fold molar excess of unlabeled nuclear factor-1 probe. To determine the specific subtype of C/EBP that is bound by the probe and actually involved in the transcriptional activation of the PDGF␣R gene, supershift assay was performed using antibodies against three major members of C/EBP family, C/EBP␣, -␤, and -␦ (Fig. 3B). In IL-1␤-treated VSMC, the band was clearly supershifted by antibodies against either C/EBP␤ or -␦ but not C/EBP␣. The PDGF␣R (open boxes) and C/EBP␦ (closed boxes) mRNA expression was normalized in reference to 28 S RNA expression and finally presented as relative units in reference to the zero time level of PDGF␣R mRNA that was set to unity. All data are expressed as means ϩ S.E. of four separate assays. *, p Ͻ 0.01, significant difference compared with each value of the zero time level.

FIG. 2. Effect of IL-1␤ on a half-life time of PDGF␣R mRNA.
After pretreatment with IL-1␤ (10 ng/ml) for 12 h, VSMC were exposed to a fresh medium with (closed circles) or without IL-1␤ (open circles) in the presence of actinomycin D (5 mg/ml), and then total cellar RNA was extracted from the cells after 0 -24 h as indicated. After normalization by the expression level of 28 S rRNA, the PDGF␣R mRNA level thus corrected was finally presented as percentage changes in reference to the zero time level that was set to 100%. All data are expressed as means of two separate assays.
to transactivate the basal promoter of PDGF␣R in a luciferase fusion construct (Fig. 4A). The wild-type PDGF␣R promoter/ firefly luciferase construct, Ϫ1,381/ϩ68 WT, was cotransfected with a mock vector, MSV, or an expression vector for each C/EBP member, EBP␣, -␤, or -␦. Forced expression of C/EBP␦ specifically transactivated the promoter activity of Ϫ1,381/ϩ68 WT, the extent of stimulation being on the order of 9.8-fold compared with that of Ϫ1,381/ϩ68 WT cotransfected with MSV. On the other hand, forced expression of other C/EBP members did not significantly affect PDGF␣R gene promoter activity. Furthermore, cell proliferation activity following treatment with PDGF-AA or -BB was determined in the transfected VSMC with MSV or EBP␦ (Fig. 4B). Proliferation activity following treatment with PDGF-AA or -BB was significantly enhanced in the transfected cells with EBP␦ compared with those with MSV, the extent of enhancement being on the order of 1.6-or 1.5-fold, respectively.
Effect of CHX against C/EBP␦ Induction by IL-1␤-To see if C/EBP␦ gene expression is activated by IL-1␤ without any other de novo protein synthesis, the ability of IL-1␤ to induce C/EBP␦ gene expression was determined in the presence of CHX (10 g/ml) (Fig. 5). Although induction of PDGF␣R mRNA expression by IL-1␤ was markedly reduced in the presence of CHX, that of C/EBP␦ mRNA expression was even greater than in the absence of CHX. On the other hand, CHX alone did not cause the superinduction of C/EBP␦ mRNA expression.
Immunohistochemistry of Cultured VSMC-In Fig. 6, protein levels of PDGF␣R and C/EBP␣, -␤, and -␦ expression were evaluated by immunohistochemistry in quiescent or IL-1␤treated VSMC. Although protein levels of PDGF␣R were very low or almost negligible in quiescent VSMC, IL-1␤ drastically induced immunoreactive PDGF␣R expression exclusively in the cytoplasm of cells. A high level of C/EBP␣ protein expression was observed in both quiescent and IL-1␤-treated VSMC and was localized mainly in the cytoplasm. Positive staining of immunoreactive C/EBP␦ protein was not detected in quiescent VSMC, whereas that of immunoreactive C/EBP␤ protein was identified specifically in the peripheral portion of the nuclei.
Furthermore, either C/EBP␤ or -␦ protein expression was markedly induced by the treatment with IL-1␤ and was localized exclusively and homogeneously in the nuclei.
Western Blotting for Nuclear Extracts from VSMC-To determine a quantitative evaluation of functionally active C/EBP members as nuclear proteins, nuclear extracts from quiescent FIG. 4. Effect of C/EBP overexpression on PDGF␣R gene promoter or cell proliferation activities. A, 1 day before transfection, VSMC were seeded onto 60-mm dishes (5 ϫ 10 5 cells/dish). PGL3-Basic or Ϫ1,381/ϩ68 WT (3 g/dish each) was cotransfected with an overexpression vector, EBP␣, -␤, or -␦ (closed column), or a mock vector, MSV (open column) (3 g/dish each), together with pRL-CMV (1 g/dish). After normalization for transfection efficiency in reference to sequentially determined Renilla luciferase activity, each promoter activity was finally presented as relative luciferase activity in reference to that of Ϫ1,381/ϩ68 WT cotransfected with MSV that was set to unity. All data are expressed as means ϩ S.E. of four separate assays. *, p Ͻ 0.01, significant difference compared with the value of Ϫ1,381/ϩ68 WT cotransfected with MSV. B, 1 day before transfection, VSMC were seeded onto 96-well plates (1 ϫ 10 4 cells/well). MSV (open column) or EBP␦ (closed column) (0.2 g/well each) was transfected to VSMC, and cells were incubated for 24 h. Then PDGFG-AA or -BB (50 ng/ml each) was directly added to the culture medium, and cells were incubated for an additional 24 h. After WST-1 reagent (0.1 volume of culture medium) was added to each well and cells were incubated for 30 min at 37°C, the absorbance (A 450 Ϫ A 690 ) of each well was measured by an enzyme-linked immunosorbent assay reader. Cell proliferation activity was finally presented as a relative activity in the reference to the activity of MSV-transfected VSMC after treatment with PDGF-AA that was set to unity. All data are expressed as means ϩ S.E. of five separate assays. *, p Ͻ 0.01, significant difference between VSMC transfected with MSV and C/EBP␦. †, p Ͻ 0.01, significant difference between VSMC following treatment with PDGF-AA and -BB.

FIG. 5. Effects of IL-1␤ on the mRNA expression of PDGF␣R and C/EBP␦ in the absence or presence of CHX.
A, total RNA (10 g) was prepared from quiescent or IL-1␤-treated VSMC (IL-1␤, Ϫ/ϩ) in the absence or presence of CHX (10 g/ml) (CHX, Ϫ/ϩ) and analyzed by Northern blotting for PDGF␣R and C/EBP␦ mRNA. A dose of 10 ng/ml IL-1␤ was used for 6 h to stimulate VSMC. B, densitometric analysis of experiments as described in A. The PDGF␣R (open boxes) and C/EBP␦ (closed boxes) mRNA expression was normalized in reference to 28 S RNA expression and finally presented as relative units in reference to the base-line level of PDGF␣R mRNA that was set to unity. All data are expressed as means ϩ S.E. of four separate assays. *, p Ͻ 0.01, significant difference compared with each value of base-line level.
or IL-1␤-treated VSMC were directly subjected to Western blotting using specific antibodies against C/EBP␣, -␤, and -␦ (Fig. 7). Although nuclear extracts from quiescent VSMC contained only a recognizable level of C/EBP␤ protein but not C/EBP␣ or -␦ protein, either C/EBP␤ (36 kDa) or C/EBP␦ (33 kDa) protein was markedly induced in the nuclear extracts from IL-1␤-treated VSMC for 12 h. The expression level of C/EBP␣ (42 kDa) protein was almost negligible in the nuclear extracts from either quiescent or IL-1␤-treated VSMC. DISCUSSION C/EBP␦ has been originally identified in the liver as one of the closely related members of C/EBP family that belongs to the basic leucine zipper transcriptional factors (25)(26)(27). Previous studies revealed that C/EBP␦ expression was usually at an undetectable or minor level in normal cells or tissues and was rapidly induced by lipopolysaccharide and inflammatory cytokines such as IL-1␤, IL-6, and tumor necrosis factor ␣ (28 -30).
Therefore, C/EBP␦ is thought to be an important factor to regulate the gene transcription of acute phase reactive proteins (28,31). Both PDGF-A and its specific receptor, PDGF␣R, are also known to be up-regulated by the treatment with IL-1␤ in several cells including VSMC and pulmonary fibroblasts, causing the cell migration and/or proliferation in the pathologic conditions (12,15,16). However, little has been known about detailed molecular mechanisms of its gene up-regulation. Recently, Khachigian et al. (32) have demonstrated that major vascular growth-related genes such as PDGF-A chain, PDGF-B chain, transforming growth factor ␤ 1 , and tissue factor are transactivated by the interaction of two specific regulatory nuclear factors, Sp-1 and Egr-1, suggesting that a common mechanism may exist in the transcriptional regulation of these genes. Interestingly, we have recently demonstrated that the PDGF ␤-receptor (PDGF␤R) gene is mainly regulated by the CCAAT box located at position Ϫ67 of its promoter region in VSMC (22,23), strongly suggesting that a common mechanism via the CCAAT box also exists in the transcriptional regulation of vascular growth-related receptor genes such as PDGF␣R and PDGF␤R genes.
In the present study, we have clearly demonstrated that an increase in the level of PDGF␣R mRNA in VSMC upon treatment with IL-1␤ is mainly due to the transcriptional activation of the gene but not due to stabilization of mRNA (Fig. 2). As anticipated, Northern blotting revealed that C/EBP␦ mRNA expression was drastically induced in the IL-1␤-treated VSMC in good accord with results obtained from the supershift assay (Fig. 3B), immunohistochemistry (Fig. 6), and Western blotting (Fig. 7). As shown in Fig. 1, this mechanism is supported by a rapid C/EBP␦ induction by IL-1␤ turned on within 30 min and peaking at 3 h that was followed by a slower (3-h) emergence (peaking at 12 h) of PDGF␣R mRNA, indicating that C/EBP␦ expression is directly related to the transactivation of the PDGF␣R gene in VSMC. Furthermore, we have determined the ability of IL-1␤ to induce C/EBP␦ gene expression in the presence of CHX to see if C/EBP␦ gene expression is activated by IL-1␤ without any other de novo protein synthesis (Fig. 5). Although either CHX alone or with IL-1␤ did not induce PDGF␣R mRNA, CHX with IL-1␤ allowed a marked C/EBP␦ mRNA induction. This indicates that C/EBP␦ activates the endogenous PDGF␣R gene expression in VSMC without de novo synthesis of other proteins.
Recently, we have reported that a C/EBP binding site seen in PDGF␣R gene promoter region acts as a major regulatory element responsible for its restricting expression in a straindependent manner (33). Kolyada et al. (34) have reported that the C/EBP family is involved in the transcriptional regulation of the Na ϩ /H ϩ exchanger gene in hepatocytes. Hohaus et al. (35) have reported that the c-fms gene, which belongs to the class III receptor tyrosine kinase family together with PDGF␣R and PDGF␤R, also has a C/EBP binding site in the promoter region, and either PU.1 (Spi-1) or C/EBP␣ mainly regulates the cell type-specific gene expression in hematopoietic cells. Taken together, these data strongly support the hypothesis that the C/EBP family, especially C/EBP␦, is a major determinant of PDGF␣R gene transcription in VSMC. Supershift assay and Western blotting indicated that IL-1␤ markedly induced specific DNA-binding proteins, which are identified as C/EBP family, and inducible C/EBP isoforms, interacting with the C/EBP binding site of PDGF␣R promoter region, are C/EBP␤ and -␦ (Figs. 3B and 7). Although C/EBP␤ was induced by IL-1␤, it was also detected even in quiescent VSMC (Figs. 6 and 7). On the other hand, C/EBP␦ was identified exclusively in the nuclei after treatment with IL-1␤ and was actually capable of interacting with the C/EBP binding site of the PDGF␣R gene. Furthermore, overexpression studies have demonstrated that C/EBP␦ but not C/EBP␣ or -␤ specifically transactivated PDGF␣R promoter activity in VSMC (Fig. 4A), and that cell proliferation activity following treatment with PDGF-AA or -BB was significantly enhanced in the transfected VSMC with EBP␦ compared with those with MSV (Fig. 4B). Since PDGF-BB can bind to not only PDGF␤R but also PDGF␣R, enhanced effect on the cell proliferation following treatment with PDGF-BB is mediated by the action through the PDGF␣R (but not PDGF␤R) up-regulated by C/EBP␦ overexpression. Moreover, -fold enhancement of cell proliferation was significantly higher in the VSMC after treatment with PDGF-BB compared with those with PDGF-AA. This result was in agreement with our previous study (17) in which we evaluated the mitogenic activity after treatment with PDGF-AA or -BB by measuring radioactive incorporation of [methyl-3 H]thymidine and found that it was significantly higher in the cells after treatment with PDGF-BB compared with those with PDGF-AA.
Previously, we have isolated and characterized the promoter region of the rat C/EBP␦ gene to understand the regulatory mechanism of C/EBP␦ gene transcription by IL-1␤ in VSMC (36). A similar study with respect to the molecular mechanism of rat C/EBP␦ gene transcription in human hepatoma cell lines, HepG2, has demonstrated that the C/EBP␦ gene is activated by IL-6 through the regulatory domain, which is recognized by acute phase response factor/signal transducers and activators of transcription 3 (37). Especially, phosphorylation of acute phase response factor/signal transducers and activators of transcription 3 by IL-6 increased its DNA binding activity and caused an induction of C/EBP␦ gene transcription, suggesting that a similar mechanism may exist on the transactivation of the C/EBP␦ gene by IL-1␤ and giving support to the hypothesis that de novo synthesis of other proteins is not necessary for its action.
In conclusion, the present study is aimed at delineating the molecular mechanism in the tissue-specific gene expression of PDGF␣R in VSMC. The results obtained herein show a direct evidence for new significant roles of the C/EBP family, especially C/EBP␦, on vascular growth and development and also provide important information to understand the mechanism underlying pathogenesis of vascular remodeling and ensuing atherosclerosis or restenosis.