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

1999; Elsevier BV; Volume: 274; Issue: 36 Linguagem: Inglês

10.1074/jbc.274.36.25576

ISSN

1083-351X

Autores

Tomikazu Fukuoka, Yutaka Kitami, Takafumi Okura, Kunio Hiwada,

Tópico(s)

Inflammatory mediators and NSAID effects

Resumo

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-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. vascular smooth muscle cell(s) platelet-derived growth factor interleukin platelet-derived growth factor α-receptor platelet-derived growth factor β-receptor CCAAT/enhancer-binding protein cycloheximide Excessive or uncontrolled replication and migration of vascular smooth muscle cells (VSMC)1are critical events involved in a number of vascular diseases including atherosclerosis, hypertension, and restenosis that often occurs after balloon angioplasty (1Mulvany M.J. Hansen P.K. Aalkjaer C. Circ. Res. 1978; 43: 854-864Crossref PubMed Scopus (434) Google Scholar, 2Spaet T.H. Stemerman M.B. Veith F.J. Lejnieks I. Circ. Res. 1975; 36: 58-70Crossref PubMed Scopus (115) Google Scholar, 3Ross R. Nature. 1993; 362: 801-809Crossref PubMed Scopus (10004) Google Scholar). 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 (4Jonasson L. Holm J. Skalli O. Bondjers G. Hansson G.K. Arteriosclerosis. 1986; 6: 131-138Crossref PubMed Google Scholar, 5Fowler S. Berberian P.A. Shio H. Goldfischer S. Wolinsky H. Circ. Res. 1980; 46: 520-530Crossref PubMed Google Scholar, 6Gown A.M. Tsukada T. Ross R. Am. J. Pathol. 1986; 125: 191-207PubMed Google Scholar, 7Joris I. Zand T. Nunnari J.J. Krolikowski F.J. Majno G. Am. J. Pathol. 1983; 113: 341-358PubMed Google Scholar). 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 α, and transforming growth factor β1, which are generally not expressed in the normal artery, whereas they are up-regulated in the lesions of atherosclerosis (3Ross R. Nature. 1993; 362: 801-809Crossref PubMed Scopus (10004) Google Scholar). Thus, the macrophage is thought to be a principal inflammatory mediator of cells in the atheromatous plaque microenvironment. Interleukin (IL)-1β is one of the major secretory products of activated macrophage and can induce proliferation of cultured fibroblasts and VSMC (8Schmidt J.A. Mizel S.B. Cohen D. Green I. J. Immunol. 1982; 128: 2177-2182PubMed Google Scholar, 9Bitterman P.B. Wewers M.D. Rennard S.I. Adelberg S. Crystal R.G. J. Clin. Invest. 1986; 77: 700-708Crossref PubMed Scopus (175) Google Scholar, 10Libby P. Wyler D.J. Janicka M.W. Dinarello C.A. Arteriosclerosis. 1985; 5: 186-191Crossref PubMed Google Scholar, 11Libby P. Warner S.J.C. Friedman G.B. J. Clin. Invest. 1988; 81: 487-498Crossref PubMed Scopus (417) Google Scholar). Previous studies (12Raines E.W. Dower S.K. Ross R. Science. 1989; 243: 393-396Crossref PubMed Scopus (518) Google Scholar, 13Ikeda U. Ikeda M. Oohara T. Kano S. Yaginuma T. Atherosclerosis. 1990; 84: 183-188Abstract Full Text PDF PubMed Scopus (65) Google Scholar, 14Bonin P.D. Fici G.J. Singh J.P. Exp. Cell. Res. 1989; 181: 475-482Crossref PubMed Scopus (54) Google Scholar) have demonstrated that mitogenic activity of IL-1β 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 α-receptor (PDGFαR) subtype on cell surface. Furthermore, recent studies (15Bonner J.C. Lindroos P.M. Rice A.B. Moomaw C.R. Morgan D.L. Am. J. Physiol. 1998; 274: L72-L80Crossref PubMed Google Scholar, 16Lindroos P.M. Coin P.G. Badgett A. Morgan D.L. Bonner J.C. Am. J. Respir. Cell. Mol. Biol. 1998; 16: 283-292Crossref Scopus (59) Google Scholar) have also demonstrated that IL-1β can up-regulate PDGFαR expression in rat lung fibroblasts, thereby enhancing PDGF-mediated mitogenesis and chemotaxis of lung fibroblasts. Although the pathophysiological implications of IL-1β-induced PDGFαR expression are beginning to be recognized, little is known about the molecular mechanism involved. Therefore, we have investigated the molecular mechanism of PDGFαR gene transcription in VSMC and obtained results indicating that IL-1β induces PDGFαR gene expression via a trans-acting nuclear factor, CCAAT/enhancer-binding protein δ (C/EBPδ). Actinomycin D and cycloheximide (CHX) were purchased from Sigma, and recombinant mouse IL-1β was from Roche Molecular Biochemicals (Tokyo, Japan). [α-32P]dCTP (110 TBq/mmol) and [γ-32P]ATP (220 TBq/mmol) were obtained from Amersham Pharmacia Biotech (Tokyo, Japan). Affinity-purified rabbit polyclonal antibodies for PDGFαR and C/EBPα, -β, and -δ raised against peptidic epitopes corresponding to amino acid residues of human PDGFαR (residues 951–1,089), rat C/EBPα (residues 253–265), rat C/EBPβ (residues 258–276), and rat C/EBPδ (residues 247–268) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Expression vectors of C/EBPα, -β, and -δ (designated EBPα, -β, and -δ, respectively) were generous gifts of Dr. Steven L. McKnight. VSMC were isolated from the thoracic aorta of male Harlan Sprague-Dawley rats (Charles River Japan Inc., Kanagawa, Japan), weighing 280–320 g, by the method described previously (17Inui H. Kitami Y. Kondo T. Inagami T. J. Biol. Chem. 1993; 268: 17045-17050Abstract Full Text PDF PubMed Google Scholar). Cells were seeded onto 100-mm dishes at a density of 1 × 106 per dish and maintained in Dulbecco's modified Eagle's medium with 10% heat-inactivated fetal calf serum at 37 °C in a humidified atmosphere of 95% air, 5% CO2. VSMC were passaged every 4–7 days, and experiments 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 μg/ml), transferrin (10 μg/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 (18Libby P. O'Brien K.V. J. Cell. Physiol. 1983; 115: 217-223Crossref PubMed Scopus (184) Google Scholar). A 0.6-kilobase pair fragment of rat PDGFαR cDNA (19Kitami Y. Inui H. Uno S. Inagami T. J. Clin. Invest. 1995; 96: 558-567Crossref PubMed Scopus (45) Google Scholar), 1.1-kilobase pair Nco I fragment of EBPα, 0.4-kilobase pair Nco I fragment of EBPβ or 1.0-kilobase pair Eco RI–Bam HI fragment of EBPδ was used as a probe for Northern blotting. Each DNA fragment was labeled with [α-32P]dCTP using the random primer method. Total cellular RNA extraction from VSMC and Northern blot analysis were carried out by the methods described previously (19Kitami Y. Inui H. Uno S. Inagami T. J. Clin. Invest. 1995; 96: 558-567Crossref PubMed Scopus (45) Google Scholar, 20Kitami Y. Hiwada K. Kokubu T. J. Hypertens. 1989; 7: 727-731Crossref PubMed Scopus (20) Google Scholar). Nuclear extracts were prepared from VSMC according to the method described by Dignam et al. (21Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9164) Google Scholar). 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 −80 °C. For electrophoretic mobility shift assay and supershift assay, a double-stranded oligodeoxynucleotide probe for the consensus sequence of C/EBP was generated by annealing two complementary oligodeoxynucleotides corresponding to the nucleotide sequence spanning −165 to −138 in the 5′-flanking region of the rat PDGFαR gene, 5′-CCCCAGATTGCATAAGAGCAAAAAGCCA-3′. Another double-stranded oligodeoxynucleotide probe for the consensus sequence of nuclear factor-1, 5′-CCTTTGGCATGCTGCCAATAT G-3′, was purchased from Promega (Madison, WI) and used as an unrelated competitor. The C/EBP probe was end-labeled with [γ-32P]ATP using T4-polynucleotide kinase. Nuclear extracts (2 μg) were incubated with 2.0 × 104 cpm of the labeled C/EBP probe for 30 min at room temperature in a 10-μl binding buffer containing 12 mm Hepes-KOH, pH 7.9, 60 mm KCl, 4 mm MgCl2, 1 mm EDTA, 1 mm dithiothreitol, 10% glycerol, and 50 μg/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 μl 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 (19Kitami Y. Inui H. Uno S. Inagami T. J. Clin. Invest. 1995; 96: 558-567Crossref PubMed Scopus (45) Google Scholar). PDGFαR promoter/firefly luciferase fusion vector, which was designated as −1,381/+68 WT, was prepared by insertion of the basal promoter region spanning positions −1,381 through +68 of the PDGFαR gene (19Kitami Y. Inui H. Uno S. Inagami T. J. Clin. Invest. 1995; 96: 558-567Crossref PubMed Scopus (45) Google Scholar) onto pGL3-Basic vector (Promega, Madison, WI). Mock vector, which was designated as MSV, was prepared by deletion of the coding region of C/EBPδ cDNA from EBPδ. 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 × 105 cells/dish) or 96-well plates (1 × 104 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 −1,381/+68 WT (3 μg/dish each) was used for cotransfection with EBPα, -β, -δ, or MSV (3 μg/dish each) in addition to pRL-CMV (1 μg/dish). For cell proliferation assay, EBPδ or MSV (0.2 μg/well each) was used for transfection of VSMC. Promoter activity was determined by the Dual-Luciferase Reporter Assay System (Promega) as described previously (22Kitami Y. Fukuoka T. Okura T. Takata Y. Maguchi M. Igase M. Kohara K. Hiwada K. J. Hypertens. 1998; 16: 437-445Crossref PubMed Scopus (6) Google Scholar, 23Takata Y. Kitami Y. Fukuoka T. Okura T. Hiwada K. Hypertension. 1999; 33: 298-302Crossref PubMed Google Scholar). 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 −1,381/+68 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 (A450 −A690) 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 was performed by the method described previously (17Inui H. Kitami Y. Kondo T. Inagami T. J. Biol. Chem. 1993; 268: 17045-17050Abstract Full Text PDF PubMed Google Scholar, 19Kitami Y. Inui H. Uno S. Inagami T. J. Clin. Invest. 1995; 96: 558-567Crossref PubMed Scopus (45) Google Scholar). Briefly, nuclear extracts (2.5 μg) prepared from VSMC were directly subjected to Western blotting for C/EBPα, -β, and -δ. After boiling with sample buffer, SDS-polyacrylamide gel electrophoresis was performed using a 12.5% gel according to Laemmli (24Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar), 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). 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 × 104 cells/well and then fixed with 100% cold acetone for 30 min. All slides were treated with PBS containing 0.3% H2O2 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 3,3′-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). Analysis of variance with Bonferroni-Dunn post hoc analysis was used to analyze differences between two experimental groups. All data are expressed as mean + S.E., and statistical significance is defined as p < 0.05. VSMC derived from Harlan Sprague-Dawley rats were incubated for 6 h in the presence of IL-1β at various concentrations (0–40 ng/ml), and mRNA levels of both PDGFαR and C/EBPδ were determined by Northern blotting. Although base-line levels of both mRNA expression were very low or almost negligible in quiescent VSMC, they were markedly increased by the treatment with IL-1β at doses up to 10 ng/ml in a dose-dependent manner (data not shown). Therefore, we used the dose of IL-1β at 10 ng/ml hereafter. To identify the kinetic relationship between C/EBP and PDGFαR expression during treatment with IL-1β, mRNA levels of C/EBP members and PDGFαR were monitored for 48 h. As shown in Fig. 1, A and B, a high level of PDGFαR mRNA expression was accompanied by a similarly marked induction of C/EBPδ mRNA in VSMC following the addition of IL-1β. A rapid induction of C/EBPδ mRNA within 30 min was followed by slower emergence of PDGFαR mRNA, which reached the maximum level (12.2-fold higher than the zero time level) in 12 h, whereas C/EBPδ 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 PDGFαR 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/EBPδ induced PDGFαR gene transcription. In contrast, a high level of C/EBPα mRNA expression was observed even in a quiescent state, and IL-1β did not significantly alter it. Although the induction of C/EBPβ mRNA expression was also detectable at 30 min, it continued for an extended period up to 48 h. To determine whether the induction of PDGFαR mRNA expression after treatment with IL-1β is due to the effect of IL-1β on the mRNA stability, the half-life time of PDGFαR mRNA was determined in the presence of actinomycin D (Fig. 2). VSMC were pretreated with IL-1β for 12 h, washed with PBS, and then exposed to a freshly prepared medium with or without IL-1β in the presence of 5 mg/ml actinomycin D. A half-life time of the PDGFαR mRNA seen in cells incubated in the medium with IL-1β was 8.6 h, and that without IL-1β was 10.0 h, indicating that IL-1β does not significantly affect the PDGFαR mRNA stability. Electrophoretic mobility shift assay was performed by using a labeled C/EBP probe containing the consensus sequence of C/EBP recognition site of rat PDGFαR promoter region (Fig.3 A). Nuclear extracts were prepared from either quiescent or IL-1β-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-1β for 12 h, generating a band of the DNA-protein 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 PDGFαR gene, supershift assay was performed using antibodies against three major members of C/EBP family, C/EBPα, -β, and -δ (Fig. 3 B). In IL-1β-treated VSMC, the band was clearly supershifted by antibodies against either C/EBPβ or -δ but not C/EBPα. To further clarify the direct evidence for C/EBP family in regulating the transcription of the rat PDGFαR gene, we evaluated the ability of the C/EBP family to transactivate the basal promoter of PDGFαR in a luciferase fusion construct (Fig.4 A). The wild-type PDGFαR promoter/firefly luciferase construct, −1,381/+68 WT, was cotransfected with a mock vector, MSV, or an expression vector for each C/EBP member, EBPα, -β, or -δ. Forced expression of C/EBPδ specifically transactivated the promoter activity of −1,381/+68 WT, the extent of stimulation being on the order of 9.8-fold compared with that of −1,381/+68 WT cotransfected with MSV. On the other hand, forced expression of other C/EBP members did not significantly affect PDGFαR gene promoter activity. Furthermore, cell proliferation activity following treatment with PDGF-AA or -BB was determined in the transfected VSMC with MSV or EBPδ (Fig. 4 B). Proliferation activity following treatment with PDGF-AA or -BB was significantly enhanced in the transfected cells with EBPδ compared with those with MSV, the extent of enhancement being on the order of 1.6- or 1.5-fold, respectively. To see if C/EBPδ gene expression is activated by IL-1β without any other de novo protein synthesis, the ability of IL-1β to induce C/EBPδ gene expression was determined in the presence of CHX (10 μg/ml) (Fig. 5). Although induction of PDGFαR mRNA expression by IL-1β was markedly reduced in the presence of CHX, that of C/EBPδ mRNA expression was even greater than in the absence of CHX. On the other hand, CHX alone did not cause the superinduction of C/EBPδ mRNA expression. In Fig.6, protein levels of PDGFαR and C/EBPα, -β, and -δ expression were evaluated by immunohistochemistry in quiescent or IL-1β-treated VSMC. Although protein levels of PDGFαR were very low or almost negligible in quiescent VSMC, IL-1β drastically induced immunoreactive PDGFαR expression exclusively in the cytoplasm of cells. A high level of C/EBPα protein expression was observed in both quiescent and IL-1β-treated VSMC and was localized mainly in the cytoplasm. Positive staining of immunoreactive C/EBPδ protein was not detected in quiescent VSMC, whereas that of immunoreactive C/EBPβ protein was identified specifically in the peripheral portion of the nuclei. Furthermore, either C/EBPβ or -δ protein expression was markedly induced by the treatment with IL-1β and was localized exclusively and homogeneously in the nuclei. To determine a quantitative evaluation of functionally active C/EBP members as nuclear proteins, nuclear extracts from quiescent or IL-1β-treated VSMC were directly subjected to Western blotting using specific antibodies against C/EBPα, -β, and -δ (Fig.7). Although nuclear extracts from quiescent VSMC contained only a recognizable level of C/EBPβ protein but not C/EBPα or -δ protein, either C/EBPβ (36 kDa) or C/EBPδ (33 kDa) protein was markedly induced in the nuclear extracts from IL-1β-treated VSMC for 12 h. The expression level of C/EBPα (42 kDa) protein was almost negligible in the nuclear extracts from either quiescent or IL-1β-treated VSMC. C/EBPδ 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 (25Williams S.C. Cantwell C.A. Johnson P.F. Genes Dev. 1991; 5: 1553-1567Crossref PubMed Scopus (439) Google Scholar, 26Cao Z. Umek R.M. McKnight S.L. Genes Dev. 1991; 5: 1538-1552Crossref PubMed Scopus (1350) Google Scholar, 27Kageyama R. Sasai Y. Nakanishi S. J. Biol. Chem. 1991; 266: 15525-15531Abstract Full Text PDF PubMed Google Scholar). Previous studies revealed that C/EBPδ 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-1β, IL-6, and tumor necrosis factor α (28Juan T.S-C. Wilson D.R. Wilde M.D. Darlington G.J. Proc. Natl. Acad Sci. U. S. A. 1993; 90: 2584-2588Crossref PubMed Scopus (126) Google Scholar, 29Kinoshita S. Akira S. Kishimoto T. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 1473-1476Crossref PubMed Scopus (262) Google Scholar, 30Alam T. An M.R. Papaconstantinou J. J. Biol. Chem. 1992; 267: 5021-5024Abstract Full Text PDF PubMed Google Scholar). Therefore, C/EBPδ is thought to be an important factor to regulate the gene transcription of acute phase reactive proteins (28Juan T.S-C. Wilson D.R. Wilde M.D. Darlington G.J. Proc. Natl. Acad Sci. U. S. A. 1993; 90: 2584-2588Crossref PubMed Scopus (126) Google Scholar, 31Baumann H. Morella K.K. Campos S.P. Cao Z. Jahreis G.P. J. Biol. Chem. 1992; 267: 19744-19751Abstract Full Text PDF PubMed Google Scholar). Both PDGF-A and its specific receptor, PDGFαR, are also known to be up-regulated by the treatment with IL-1β in several cells including VSMC and pulmonary fibroblasts, causing the cell migration and/or proliferation in the pathologic conditions (12Raines E.W. Dower S.K. Ross R. Science. 1989; 243: 393-396Crossref PubMed Scopus (518) Google Scholar, 15Bonner J.C. Lindroos P.M. Rice A.B. Moomaw C.R. Morgan D.L. Am. J. Physiol. 1998; 274: L72-L80Crossref PubMed Google Scholar, 16Lindroos P.M. Coin P.G. Badgett A. Morgan D.L. Bonner J.C. Am. J. Respir. Cell. Mol. Biol. 1998; 16: 283-292Crossref Scopus (59) Google Scholar). However, little has been known about detailed molecular mechanisms of its gene up-regulation. Recently, Khachigian et al. (32Khachigian L.M. Lindner V. Williams A.J. Collins T. Science. 1996; 271: 1427-1431Crossref PubMed Scopus (478) Google Scholar) have demonstrated that major vascular growth-related genes such as PDGF-A chain, PDGF-B chain, transforming growth factor β1, 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 β-receptor (PDGFβR) gene is mainly regulated by the CCAAT box located at position −67 of its promoter region in VSMC (22Kitami Y. Fukuoka T. Okura T. Takata Y. Maguchi M. Igase M. Kohara K. Hiwada K. J. Hypertens. 1998; 16: 437-445Crossref PubMed Scopus (6) Google Scholar, 23Takata Y. Kitami Y. Fukuoka T. Okura T. Hiwada K. Hypertension. 1999; 33: 298-302Crossref PubMed Google Scholar), strongly suggesting that a common mechanism via the CCAAT box also exists in the transcriptional regulation of vascular growth-related receptor genes such as PDGFαR and PDGFβR genes. In the present study, we have clearly demonstrated that an increase in the level of PDGFαR mRNA in VSMC upon treatment with IL-1β 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/EBPδ mRNA expression was drastically induced in the IL-1β-treated VSMC in good accord with results obtained from the supershift assay (Fig. 3 B), immunohistochemistry (Fig. 6), and Western blotting (Fig. 7). As shown in Fig. 1, this mechanism is supported by a rapid C/EBPδ induction by IL-1β turned on within 30 min and peaking at 3 h that was followed by a slower (3-h) emergence (peaking at 12 h) of PDGFαR mRNA, indicating that C/EBPδ expression is directly related to the transactivation of the PDGFαR gene in VSMC. Furthermore, we have determined the ability of IL-1β to induce C/EBPδ gene expression in the presence of CHX to see if C/EBPδ gene expression is activated by IL-1β without any other de novo protein synthesis (Fig. 5). Although either CHX alone or with IL-1β did not induce PDGFαR mRNA, CHX with IL-1β allowed a marked C/EBPδ mRNA induction. This indicates that C/EBPδ activates the endogenous PDGFαR gene expression in VSMC without de novo synthesis of other proteins. Recently, we have reported that a C/EBP binding site seen in PDGFαR gene promoter region acts as a major regulatory element responsible for its restricting expression in a strain-dependent manner (33Kitami Y. Fukuoka T. Hiwada K. Inagami T. Circ. Res. 1999; 84: 64-73Crossref PubMed Scopus (31) Google Scholar). Kolyada et al. (34Kolyada A.Y. Johns C.A. Madias N.E. Am. J. Physiol. 1995; 269: C1408-C1416Crossref PubMed Google Scholar) have reported that the C/EBP family is involved in the transcriptional regulation of the Na+/H+ exchanger gene in hepatocytes. Hohaus et al. (35Hohaus S. Petrovick M.S. Voso M.T. Sun Z. Zhang D.E. Tenen D.G. Mol. Cell. Biol. 1995; 15: 5830-5845Crossref PubMed Google Scholar) have reported that the c-fms gene, which belongs to the class III receptor tyrosine kinase family together with PDGFαR and PDGFβR, also has a C/EBP binding site in the promoter region, and either PU.1 (Spi-1) or C/EBPα 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/EBPδ, is a major determinant of PDGFαR gene transcription in VSMC. Supershift assay and Western blotting indicated that IL-1β 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 PDGFαR promoter region, are C/EBPβ and -δ (Figs. 3 B and 7). Although C/EBPβ was induced by IL-1β, it was also detected even in quiescent VSMC (Figs. 6 and 7). On the other hand, C/EBPδ was identified exclusively in the nuclei after treatment with IL-1β and was actually capable of interacting with the C/EBP binding site of the PDGFαR gene. Furthermore, overexpression studies have demonstrated that C/EBPδ but not C/EBPα or -β specifically transactivated PDGFαR promoter activity in VSMC (Fig. 4 A), and that cell proliferation activity following treatment with PDGF-AA or -BB was significantly enhanced in the transfected VSMC with EBPδ compared with those with MSV (Fig.4 B). Since PDGF-BB can bind to not only PDGFβR but also PDGFαR, enhanced effect on the cell proliferation following treatment with PDGF-BB is mediated by the action through the PDGFαR (but not PDGFβR) up-regulated by C/EBPδ 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 (17Inui H. Kitami Y. Kondo T. Inagami T. J. Biol. Chem. 1993; 268: 17045-17050Abstract Full Text PDF PubMed Google Scholar) in which we evaluated the mitogenic activity after treatment with PDGF-AA or -BB by measuring radioactive incorporation of [methyl-3H]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/EBPδ gene to understand the regulatory mechanism of C/EBPδ gene transcription by IL-1β in VSMC (36Fukuoka T. Kitami Y. Kohara K. Hiwada K. Biochem. Biophys. Res. Commun. 1997; 231: 30-36Crossref PubMed Scopus (10) Google Scholar). A similar study with respect to the molecular mechanism of rat C/EBPδ gene transcription in human hepatoma cell lines, HepG2, has demonstrated that the C/EBPδ 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 (37Yamada T. Tobita K. Osada S. Nishihara T. Imagawa M. J. Biochem. (Tokyo). 1997; 121: 731-738Crossref PubMed Scopus (43) Google Scholar). 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/EBPδ gene transcription, suggesting that a similar mechanism may exist on the transactivation of the C/EBPδ gene by IL-1β 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 PDGFαR in VSMC. The results obtained herein show a direct evidence for new significant roles of the C/EBP family, especially C/EBPδ, on vascular growth and development and also provide important information to understand the mechanism underlying pathogenesis of vascular remodeling and ensuing atherosclerosis or restenosis. We are deeply indebted to Dr. Steven L. McKnight (Department of Biochemistry, University of Texas South Medical Center, Dallas, TX) for the generous gift of EBPα, -β, and -δ plasmids. We are also grateful to Dr. Tadashi Inagami (Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN) for critical reading of the manuscript.

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