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J Biol Chem, Vol. 274, Issue 36, 25576-25582, September 3, 1999


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

Tomikazu Fukuoka, Yutaka KitamiDagger , Takafumi Okura, and Kunio Hiwada

From the Second Department of Internal Medicine, Ehime University School of Medicine, Onsen-gun, Ehime 791-0295, Japan

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-1beta (IL-1beta ) 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, PDGFalpha -receptor (PDGFalpha R), very little is known about the regulatory mechanism of PDGFalpha R gene expression in VSMC. To understand the mechanism, we studied the transcriptional control of the PDGFalpha R gene in VSMC after treatment with IL-1beta . IL-1beta (10 ng/ml) drastically increased both PDGFalpha R and CCAAT/enhancer-binding protein delta  (C/EBPdelta ) mRNA levels in a time dependent manner. A rapid induction of C/EBPdelta mRNA within 30 min was followed by slower emergence of PDGFalpha R mRNA, which reached the maximum level in 12 h, whereas C/EBPdelta mRNA was detectable at 30 min and reached the maximum level at 3 h. Electromobility shift and supershift assays revealed that IL-1beta markedly increased DNA-protein complex, which was mainly composed of C/EBPbeta and/or -delta . Both Western blotting and immunohistochemistry demonstrated that either C/EBPbeta or -delta expression was induced by IL-1beta exclusively in nuclei of VSMC. On the other hand, overexpression of C/EBPdelta specifically transactivated the promoter activity of the PDGFalpha R gene and significantly enhanced VSMC proliferation in PDGF-treated cells. We conclude that induction of PDGFalpha R expression is mainly mediated by C/EBPdelta expression in VSMC, and a high level of C/EBPdelta expression may be involved in the pathogenesis of atherosclerosis and restenosis.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 (1-3). 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 alpha , and transforming growth factor beta 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)-1beta is one of the major secretory products of activated macrophage and can induce proliferation of cultured fibroblasts and VSMC (8-11). Previous studies (12-14) have demonstrated that mitogenic activity of IL-1beta 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 alpha -receptor (PDGFalpha R) subtype on cell surface. Furthermore, recent studies (15, 16) have also demonstrated that IL-1beta can up-regulate PDGFalpha R expression in rat lung fibroblasts, thereby enhancing PDGF-mediated mitogenesis and chemotaxis of lung fibroblasts. Although the pathophysiological implications of IL-1beta -induced PDGFalpha R expression are beginning to be recognized, little is known about the molecular mechanism involved. Therefore, we have investigated the molecular mechanism of PDGFalpha R gene transcription in VSMC and obtained results indicating that IL-1beta induces PDGFalpha R gene expression via a trans-acting nuclear factor, CCAAT/enhancer-binding protein delta  (C/EBPdelta ).

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Actinomycin D and cycloheximide (CHX) were purchased from Sigma, and recombinant mouse IL-1beta was from Roche Molecular Biochemicals (Tokyo, Japan). [alpha -32P]dCTP (110 TBq/mmol) and [gamma -32P]ATP (220 TBq/mmol) were obtained from Amersham Pharmacia Biotech (Tokyo, Japan). Affinity-purified rabbit polyclonal antibodies for PDGFalpha R and C/EBPalpha , -beta , and -delta raised against peptidic epitopes corresponding to amino acid residues of human PDGFalpha R (residues 951-1,089), rat C/EBPalpha (residues 253-265), rat C/EBPbeta (residues 258-276), and rat C/EBPdelta (residues 247-268) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Expression vectors of C/EBPalpha , -beta , and -delta (designated EBPalpha , -beta , and -delta , respectively) were generous gifts of Dr. Steven L. McKnight.

Cell Culture-- 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 (17). 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 (18).

Preparation of cDNA Probes and Northern Blotting-- A 0.6-kilobase pair fragment of rat PDGFalpha R cDNA (19), 1.1-kilobase pair NcoI fragment of EBPalpha , 0.4-kilobase pair NcoI fragment of EBPbeta or 1.0-kilobase pair EcoRI-BamHI fragment of EBPdelta was used as a probe for Northern blotting. Each DNA fragment was labeled with [alpha -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 (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 -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 PDGFalpha R 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 [gamma -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 (19).

Plasmid Construction and DNA Transfection-- PDGFalpha R 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 PDGFalpha 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/EBPdelta cDNA from EBPdelta . 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 EBPalpha , -beta , -delta , or MSV (3 µg/dish each) in addition to pRL-CMV (1 µg/dish). For cell proliferation assay, EBPdelta or MSV (0.2 µg/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 -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-- Western blotting was performed by the method described previously (17, 19). Briefly, nuclear extracts (2.5 µg) prepared from VSMC were directly subjected to Western blotting for C/EBPalpha , -beta , and -delta . 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 × 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).

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 + S.E., and statistical significance is defined as p < 0.05.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Messenger RNA Induction of PDGFalpha R and C/EBPdelta by IL-1beta -- VSMC derived from Harlan Sprague-Dawley rats were incubated for 6 h in the presence of IL-1beta at various concentrations (0-40 ng/ml), and mRNA levels of both PDGFalpha R and C/EBPdelta 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-1beta at doses up to 10 ng/ml in a dose-dependent manner (data not shown). Therefore, we used the dose of IL-1beta at 10 ng/ml hereafter. To identify the kinetic relationship between C/EBP and PDGFalpha R expression during treatment with IL-1beta , mRNA levels of C/EBP members and PDGFalpha R were monitored for 48 h. As shown in Fig. 1, A and B, a high level of PDGFalpha R mRNA expression was accompanied by a similarly marked induction of C/EBPdelta mRNA in VSMC following the addition of IL-1beta . A rapid induction of C/EBPdelta mRNA within 30 min was followed by slower emergence of PDGFalpha R mRNA, which reached the maximum level (12.2-fold higher than the zero time level) in 12 h, whereas C/EBPdelta 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 PDGFalpha 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/EBPdelta induced PDGFalpha R gene transcription. In contrast, a high level of C/EBPalpha mRNA expression was observed even in a quiescent state, and IL-1beta did not significantly alter it. Although the induction of C/EBPbeta mRNA expression was also detectable at 30 min, it continued for an extended period up to 48 h.


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  		Fig. 1.   Time-course of mRNA induction of PDGFalpha R and C/EBPdelta by IL-1beta . A, VSMC were treated with IL-1beta (10 ng/ml), and total cellular RNA was extracted from the cells after 0-48 h as indicated. Total RNA (10 µg) was analyzed by Northern blotting for PDGFalpha R, C/EBPalpha , -beta , and -delta mRNA. B, densitometric analysis of experiments as described in A. The PDGFalpha R (open boxes) and C/EBPdelta (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 PDGFalpha R mRNA that was set to unity. All data are expressed as means + S.E. of four separate assays. *, p < 0.01, significant difference compared with each value of the zero time level.

Effect of IL-1beta on a Half-life Time of PDGFalpha R mRNA-- To determine whether the induction of PDGFalpha R mRNA expression after treatment with IL-1beta is due to the effect of IL-1beta on the mRNA stability, the half-life time of PDGFalpha R mRNA was determined in the presence of actinomycin D (Fig. 2). VSMC were pretreated with IL-1beta for 12 h, washed with PBS, and then exposed to a freshly prepared medium with or without IL-1beta in the presence of 5 mg/ml actinomycin D. A half-life time of the PDGFalpha R mRNA seen in cells incubated in the medium with IL-1beta was 8.6 h, and that without IL-1beta was 10.0 h, indicating that IL-1beta does not significantly affect the PDGFalpha R mRNA stability.


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  		Fig. 2.   Effect of IL-1beta on a half-life time of PDGFalpha R mRNA. After pretreatment with IL-1beta (10 ng/ml) for 12 h, VSMC were exposed to a fresh medium with (closed circles) or without IL-1beta (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 PDGFalpha 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.

Characterization of C/EBP Members That Interact with PDGFalpha 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 PDGFalpha R promoter region (Fig. 3A). Nuclear extracts were prepared from either quiescent or IL-1beta -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-1beta 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 PDGFalpha R gene, supershift assay was performed using antibodies against three major members of C/EBP family, C/EBPalpha , -beta , and -delta (Fig. 3B). In IL-1beta -treated VSMC, the band was clearly supershifted by antibodies against either C/EBPbeta or -delta but not C/EBPalpha .


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  		Fig. 3.   Electrophoretic mobility shift assay and supershift assay for C/EBP site of PDGFalpha R gene promoter. A, the end-labeled C/EBP probe (2.0 × 104 cpm) was incubated with 2 µg of nuclear extracts from either quiescent or IL-1beta -treated VSMC (IL-1beta , -/+). For the competition experiment, a 100-fold molar excess of unlabeled competitor (Comp.) for C/EBP or nuclear factor-1 (NF-1) was incubated with nuclear extracts for 30 min before addition of the labeled C/EBP probe. B, nuclear extracts (2 µg) from either quiescent or IL-1beta -treated VSMC were incubated with 1 µl of preimmune serum (PI) or specific antibodies (Ab.) against, C/EBPalpha (alpha ), C/EBPbeta (beta ), C/EBPdelta (delta ), or 2 µl of antibody mix (1:1) against both C/EBPbeta and -delta (beta  + delta ) for 30 min before addition of the labeled C/EBP probe. The position of the supershifted band is marked with an asterisk.

Effects of C/EBP Overexpression on PDGFalpha R Gene Promoter and Cell Proliferation Activities-- To further clarify the direct evidence for C/EBP family in regulating the transcription of the rat PDGFalpha R gene, we evaluated the ability of the C/EBP family to transactivate the basal promoter of PDGFalpha R in a luciferase fusion construct (Fig. 4A). The wild-type PDGFalpha R promoter/firefly luciferase construct, -1,381/+68 WT, was cotransfected with a mock vector, MSV, or an expression vector for each C/EBP member, EBPalpha , -beta , or -delta . Forced expression of C/EBPdelta 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 PDGFalpha R gene promoter activity. Furthermore, cell proliferation activity following treatment with PDGF-AA or -BB was determined in the transfected VSMC with MSV or EBPdelta (Fig. 4B). Proliferation activity following treatment with PDGF-AA or -BB was significantly enhanced in the transfected cells with EBPdelta compared with those with MSV, the extent of enhancement being on the order of 1.6- or 1.5-fold, respectively.


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  		Fig. 4.   Effect of C/EBP overexpression on PDGFalpha R gene promoter or cell proliferation activities. A, 1 day before transfection, VSMC were seeded onto 60-mm dishes (5 × 105 cells/dish). PGL3-Basic or -1,381/+68 WT (3 µg/dish each) was cotransfected with an overexpression vector, EBPalpha , -beta , or -delta (closed column), or a mock vector, MSV (open column) (3 µg/dish each), together with pRL-CMV (1 µg/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 -1,381/+68 WT cotransfected with MSV that was set to unity. All data are expressed as means + S.E. of four separate assays. *, p < 0.01, significant difference compared with the value of -1,381/+68 WT cotransfected with MSV. B, 1 day before transfection, VSMC were seeded onto 96-well plates (1 × 104 cells/well). MSV (open column) or EBPdelta (closed column) (0.2 µg/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 (A450 - A690) 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 + S.E. of five separate assays. *, p < 0.01, significant difference between VSMC transfected with MSV and C/EBPdelta . dagger , p < 0.01, significant difference between VSMC following treatment with PDGF-AA and -BB.

Effect of CHX against C/EBPdelta Induction by IL-1beta -- To see if C/EBPdelta gene expression is activated by IL-1beta without any other de novo protein synthesis, the ability of IL-1beta to induce C/EBPdelta gene expression was determined in the presence of CHX (10 µg/ml) (Fig. 5). Although induction of PDGFalpha R mRNA expression by IL-1beta was markedly reduced in the presence of CHX, that of C/EBPdelta mRNA expression was even greater than in the absence of CHX. On the other hand, CHX alone did not cause the superinduction of C/EBPdelta mRNA expression.


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  		Fig. 5.   Effects of IL-1beta on the mRNA expression of PDGFalpha R and C/EBPdelta in the absence or presence of CHX. A, total RNA (10 µg) was prepared from quiescent or IL-1beta -treated VSMC (IL-1beta , -/+) in the absence or presence of CHX (10 µg/ml) (CHX, -/+) and analyzed by Northern blotting for PDGFalpha R and C/EBPdelta mRNA. A dose of 10 ng/ml IL-1beta was used for 6 h to stimulate VSMC. B, densitometric analysis of experiments as described in A. The PDGFalpha R (open boxes) and C/EBPdelta (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 PDGFalpha R mRNA that was set to unity. All data are expressed as means + S.E. of four separate assays. *, p < 0.01, significant difference compared with each value of base-line level.

Immunohistochemistry of Cultured VSMC-- In Fig. 6, protein levels of PDGFalpha R and C/EBPalpha , -beta , and -delta expression were evaluated by immunohistochemistry in quiescent or IL-1beta -treated VSMC. Although protein levels of PDGFalpha R were very low or almost negligible in quiescent VSMC, IL-1beta drastically induced immunoreactive PDGFalpha R expression exclusively in the cytoplasm of cells. A high level of C/EBPalpha protein expression was observed in both quiescent and IL-1beta -treated VSMC and was localized mainly in the cytoplasm. Positive staining of immunoreactive C/EBPdelta protein was not detected in quiescent VSMC, whereas that of immunoreactive C/EBPbeta protein was identified specifically in the peripheral portion of the nuclei. Furthermore, either C/EBPbeta or -delta protein expression was markedly induced by the treatment with IL-1beta and was localized exclusively and homogeneously in the nuclei.


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  		Fig. 6.   Immunohistochemistry of PDGFalpha R, C/EBPalpha , -beta , and -delta proteins in quiescent or IL-1beta -treated VSMC. Quiescent or IL-1beta -treated VSMC were subjected to immunohistochemical evaluation for PDGFalpha R (-/+), C/EBPalpha (-/+), C/EBPbeta (-/+), or C/EBPdelta (-/+) using Vectastain Elite ABC kit. IL-1beta -treated VSMC were stimulated by IL-1beta (10 ng/ml) for 12 h. All slides were counterstained with hematoxylin, and black or brown color indicates positive staining for each immunoreactive protein. Original magnification, × 200.

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 or IL-1beta -treated VSMC were directly subjected to Western blotting using specific antibodies against C/EBPalpha , -beta , and -delta (Fig. 7). Although nuclear extracts from quiescent VSMC contained only a recognizable level of C/EBPbeta protein but not C/EBPalpha or -delta protein, either C/EBPbeta (36 kDa) or C/EBPdelta (33 kDa) protein was markedly induced in the nuclear extracts from IL-1beta -treated VSMC for 12 h. The expression level of C/EBPalpha (42 kDa) protein was almost negligible in the nuclear extracts from either quiescent or IL-1beta -treated VSMC.


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  		Fig. 7.   Western blotting for C/EBPalpha , -beta , and -delta proteins in nuclear extracts from quiescent or IL-1beta -treated VSMC. A, nuclear extracts (2.5 µg) were obtained from quiescent or IL-1beta -treated VSMC (IL-1beta , -/+) and directly subjected to Western blotting for C/EBPalpha , -beta , or -delta . IL-1beta -treated cells were stimulated by IL-1beta (10 ng/ml) for 8 h. Specific antibodies against each C/EBP member recognized 42-kDa protein for C/EBPalpha , 36 kDa for C/EBPbeta , or 33 kDa for C/EBPdelta , respectively. B, densitometric analysis of experiments as described for A. Band intensities of C/EBPalpha (open boxes), C/EBPbeta (hatched boxes), and C/EBPdelta (closed boxes) protein expression were presented as arbitrary OD units. All data are expressed as means + S.E. of four separate assays. *, p < 0.01, significant difference compared with each value of IL-1beta (-).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

C/EBPdelta 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 (25-27). Previous studies revealed that C/EBPdelta 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-1beta , IL-6, and tumor necrosis factor alpha  (28-30). Therefore, C/EBPdelta 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, PDGFalpha R, are also known to be up-regulated by the treatment with IL-1beta 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 beta 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 beta -receptor (PDGFbeta R) gene is mainly regulated by the CCAAT box located at position -67 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 PDGFalpha R and PDGFbeta R genes.

In the present study, we have clearly demonstrated that an increase in the level of PDGFalpha R mRNA in VSMC upon treatment with IL-1beta 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/EBPdelta mRNA expression was drastically induced in the IL-1beta -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/EBPdelta induction by IL-1beta turned on within 30 min and peaking at 3 h that was followed by a slower (3-h) emergence (peaking at 12 h) of PDGFalpha R mRNA, indicating that C/EBPdelta expression is directly related to the transactivation of the PDGFalpha R gene in VSMC. Furthermore, we have determined the ability of IL-1beta to induce C/EBPdelta gene expression in the presence of CHX to see if C/EBPdelta gene expression is activated by IL-1beta without any other de novo protein synthesis (Fig. 5). Although either CHX alone or with IL-1beta did not induce PDGFalpha R mRNA, CHX with IL-1beta allowed a marked C/EBPdelta mRNA induction. This indicates that C/EBPdelta activates the endogenous PDGFalpha R gene expression in VSMC without de novo synthesis of other proteins.

Recently, we have reported that a C/EBP binding site seen in PDGFalpha R gene promoter region acts as a major regulatory element responsible for its restricting expression in a strain-dependent manner (33). Kolyada et al. (34) have reported that the C/EBP family is involved in the transcriptional regulation of the Na+/H+ 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 PDGFalpha R and PDGFbeta R, also has a C/EBP binding site in the promoter region, and either PU.1 (Spi-1) or C/EBPalpha 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/EBPdelta , is a major determinant of PDGFalpha R gene transcription in VSMC. Supershift assay and Western blotting indicated that IL-1beta 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 PDGFalpha R promoter region, are C/EBPbeta and -delta (Figs. 3B and 7). Although C/EBPbeta was induced by IL-1beta , it was also detected even in quiescent VSMC (Figs. 6 and 7). On the other hand, C/EBPdelta was identified exclusively in the nuclei after treatment with IL-1beta and was actually capable of interacting with the C/EBP binding site of the PDGFalpha R gene. Furthermore, overexpression studies have demonstrated that C/EBPdelta but not C/EBPalpha or -beta specifically transactivated PDGFalpha 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 EBPdelta compared with those with MSV (Fig. 4B). Since PDGF-BB can bind to not only PDGFbeta R but also PDGFalpha R, enhanced effect on the cell proliferation following treatment with PDGF-BB is mediated by the action through the PDGFalpha R (but not PDGFbeta R) up-regulated by C/EBPdelta 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-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/EBPdelta gene to understand the regulatory mechanism of C/EBPdelta gene transcription by IL-1beta in VSMC (36). A similar study with respect to the molecular mechanism of rat C/EBPdelta gene transcription in human hepatoma cell lines, HepG2, has demonstrated that the C/EBPdelta 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/EBPdelta gene transcription, suggesting that a similar mechanism may exist on the transactivation of the C/EBPdelta gene by IL-1beta 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 PDGFalpha R in VSMC. The results obtained herein show a direct evidence for new significant roles of the C/EBP family, especially C/EBPdelta , on vascular growth and development and also provide important information to understand the mechanism underlying pathogenesis of vascular remodeling and ensuing atherosclerosis or restenosis.

    ACKNOWLEDGEMENTS

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 EBPalpha , -beta , and -delta 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.

    FOOTNOTES

* This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Culture and Sports, Japan 08457210, 08670797, and 09670723; Japan Heart Foundation Grant for Research on Hypertension and Vascular Metabolism; and the Takeda Medical Research Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom all correspondence should be addressed: Second Dept. of Internal Medicine, Ehime University School of Medicine, Ehime 791-0295, Japan. Tel.: 81-89-960-5303; Fax: 81-89-960-5306; E-mail: kitamiyk@m.ehime-u.ac.jp.

    ABBREVIATIONS

The abbreviations used are: VSMC, vascular smooth muscle cell(s); PDGF, platelet-derived growth factor; IL, interleukin; PDGFalpha R, platelet-derived growth factor alpha -receptor; PDGFbeta R, platelet-derived growth factor beta -receptor; C/EBP, CCAAT/enhancer-binding protein; CHX, cycloheximide.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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