Peroxisome Proliferator-activated Receptor-gamma Activation Inhibits Interleukin-1beta -mediated Platelet-derived Growth Factor-alpha Receptor Gene Expression via CCAAT/Enhancer-binding Protein-delta in Vascular Smooth Muscle Cells*

Yasunori Takata, Yutaka KitamiDagger, Takafumi Okura, and Kunio Hiwada

From The Second Department of Internal Medicine, Ehime University School of Medicine, Ehime 791-0295, Japan

Received for publication, December 26, 2000, and in revised form, January 16, 2001



    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES

CCAAT/enhancer-binding protein (C/EBP)-binding motifs have been identified in the promoter regions of interleukin (IL)-6, tumor necrosis factor-alpha , and platelet-derived growth factor-alpha receptor (PDGFalpha R). Recently, peroxisome proliferator-activated receptors (PPARs) have been suggested to be important immunomodulatory mediators. Although many studies have demonstrated that the interaction between C/EBPs and PPARs plays a central role in lipid metabolism, expression and function of these factors are unknown in vascular smooth muscle cells (VSMCs). In the present study, we clarified a functional relationship between C/EBPs and PPARgamma in the regulation of IL-1beta -induced PDGFalpha R expression in VSMCs. PPARgamma activators, troglitazone and 15-deoxy-Delta 12,14-prostaglandin J2, inhibited IL-1beta -induced PDGFalpha R expression and suppressed PDGF-induced proliferation activity of VSMCs. Electromobility shift and supershift assays for a C/EBP motif in the PDGFalpha R promoter region revealed that PPARgamma activators suppressed IL-1beta -induced DNA binding activity of C/EBPdelta and beta . PPARgamma activators also suppressed IL-1beta -induced C/EBPdelta expression. In contrast, overexpression of C/EBPdelta reversed the suppressive effect of PPARgamma activators on PDGFalpha R expression almost completely. From these results, we conclude that the inhibitory effect of PPARgamma activators on PDGFalpha R expression is mainly mediated by C/EBPdelta suppression. Regulation of C/EBPdelta by PPARgamma activators probably plays critical roles in modulating inflammatory responses in the arterial wall.



    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Excessive or uncontrolled replication and migration of vascular smooth muscle cells (VSMCs)1 are critical events involved in a number of vascular diseases including atherosclerosis, hypertension, and restenosis after balloon angioplasty (1-3). Morphologic studies of the sequencing events in the arterial wall have revealed that macrophages are present in atherosclerotic lesions (4, 5) and are involved in the production of several growth factors such as platelet-derived growth factors (PDGFs), basic fibroblast growth factor, tumor necrosis factor-alpha , and transforming growth factor-beta 1 (3). Particularly, interleukin (IL)-1beta is one of the major secretory products of activated macrophages and can induce proliferation and migration of fibroblasts and VSMCs (6-8). Previous studies have demonstrated that the mitogenic response of IL-1beta for VSMCs is mediated by an indirect pathway, causing the release of PDGF-AA, which specifically binds to the PDGF alpha -receptor (PDGFalpha R) subtype on the cell surface (9-11). In addition, IL-1beta can also up-regulate PDGFalpha R expression itself in rat lung fibroblasts, thereby enhancing PDGF-mediated mitogenesis and chemotaxis of the cells (12). Previously, we have shown that the PDGFalpha R promoter contains an enhancer core sequence for CCAAT/enhancer-binding protein (C/EBP), and IL-1beta -mediated induction of PDGFalpha R expression is mainly regulated by a specific nuclear factor, C/EBPdelta , in VSMCs (13).

Peroxisome proliferator-activated receptors (PPARs) belong to the nuclear receptor superfamily of ligand-dependent transcription factors, and cross-regulation between the C/EBP family and the PPAR family is very important in maintaining adipocyte differentiation. Especially, C/EBPbeta and delta  play a critical role in the determination of pre-adipocyte development by activating expression of C/EBPalpha and PPARgamma genes (14, 15). Recent studies have shown that in addition to its proposed roles in the regulation of adipocyte differentiation and glucometabolism, PPARs probably regulate inflammatory responses by interaction with other transcription factors in several types of cells (16, 17). However, expression and function of PPARgamma in VSMCs are somewhat controversial. In human VSMCs, Staels et al. (16) observed faint expression of PPARgamma that was not involved in the negative regulation of cytokine-induced IL-6 and cyclooxygenase-2 expression; this effect was mediated by PPARgamma . In contrast, Marx et al. (18) demonstrated that human VSMCs expressed PPARgamma that inhibited matrix metalloproteinase expression and cell migration. In particular, the latter observations suggest that such a role of PPARgamma may be to limit the arterial remodeling that occurs in response to hypertension, atherosclerosis, and restenosis, or even to counterbalance other vascular effects. Indeed, PPARgamma activation can be shown to inhibit VSMC proliferation and migration in a variety of assays (18-20).

In the present study, we demonstrated new roles of PPARgamma activators, troglitazone (TRO) and a naturally occurring ligand, 15-deoxy-Delta 12,14-prostaglandin J2 (PGJ2), on IL-1beta -induced expression of PDGFalpha R and clarified the underlying molecular mechanism by demonstrating a functionally important interaction between PPARgamma and C/EBPdelta in rat cultured VSMCs.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- TRO was provided from Sankyo, Co. (Tokyo, Japan). Recombinant rat IL-1beta was purchased from R & D Systems (Tokyo, Japan). Prostaglandin F2alpha (PGF2alpha ) and PGJ2 were from Cayman (Ann Arbor, MI). Affinity-purified antibodies for PDGFalpha R, PPARgamma , C/EBPalpha , C/EBPbeta , and C/EBPdelta raised against peptidic epitopes corresponding with amino acid sequences of human PDGFalpha R (951-1,089), rat C/EBPalpha (253), rat C/EBPbeta (258), and rat C/EBPdelta (247) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Cell Culture-- VSMCs were isolated from thoracic aortas of male Harlan Sprague-Dawley rats (Charles River Japan Inc., Kanagawa, Japan) as described previously (21) and were 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. In preparation for all experiments, subconfluent cells at 3-8 passages from primary culture were made quiescent by placing them in Dulbecco's modified Eagle's medium supplemented with 0.1% fetal calf serum for 2 days.

Protein Extraction and Western Blotting-- Whole-cell lysates were extracted from VSMCs by the standard method (21), and nuclear extracts were prepared according to the method described by Dignam et al. (22). Western blotting was essentially carried out as described previously (23).

RNA Extraction and Measurement of mRNA-- Total RNA was extracted from VSMCs with the use of ISOGEN (Nippon Gene, Tokyo, Japan). Measurement of mRNA was done by Northern blotting. Northern hybridization, autoradiography, and densitometric analysis were performed as described previously (24).

Measurement of BrdUrd Incorporation-- VSMCs were seeded on 96-well plates (1 × 104 cells per well) and were cultured in the presence or absence of TRO (10 µmol/liter) or PGJ2 (5 µmol/liter) for 24 h. Then, cells were treated with IL-1beta (10 ng/ml) for 4 h and were stimulated with PDGF (20 ng/ml) for 12 h. BrdUrd incorporation was finally determined by the Cell Proliferation enzyme-linked immunosorbent assay system (Amersham Pharmacia Biotech).

Electromobility Shift Assay (EMSA), Supershift Assay, and Plasmid Transfection-- The EMSA and supershift assay were performed for the C/EBP-binding motif seen in rat PDGFalpha R gene promoter as described previously (25). VSMCs (5 × 105 cells per dish) were seeded in 60-mm dishes 24 h before transfection, and then the C/EBPdelta expression vector, designated MSV-C/EBPdelta in our earlier studies (13, 25), was transfected (3 µg per dish) onto the cells with the use of LipofectAMINE Plus (Life Technologies, Inc.) according to the manufacturer's specifications.

Statistical Analysis-- Analysis of variance with Boferroni-Dunn post hoc was used to analyze differences between two experimental groups. All data are expressed as means + S.E., and statistical significance was defined as p < 0.05.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

TRO or PGJ2 Inhibits IL-1beta -mediated PDGFalpha R Expression-- We confirmed that rat cultured VSMCs used in this study expressed PPARgamma mRNA and protein (data not shown). VSMCs were cultured in the presence or absence of a PPARgamma activator, TRO (0-10 µmol/liter) or PGJ2 (0-5 µmol/liter), for 24 h. Then, expression levels of PDGFalpha R were determined by Northern (Fig. 1A) and Western (Fig. 1B) blotting 12 h after treatment with or without IL-1beta (10 ng/ml). Previously, we have demonstrated that expression levels of PDGFalpha R were increased by treatment with IL-1beta at doses up to 10 ng/ml in a dose-dependent manner (13). Therefore, we used the dose of IL-1beta at 10 ng/ml thereafter. In quiescent cells, base-line levels of PDGFalpha R mRNA and protein were very low. On the other hand, expression levels of PDGFalpha R were increased drastically by treatment with IL-1beta in the absence of TRO or PGJ2, whereas induction of PDGFalpha R expression was significantly reduced by pretreatment with TRO or PGJ2 in a dose-dependent manner.


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Fig. 1.   Inhibitory effect of TRO and PGJ2 on IL-1beta -mediated PDGFalpha R expression. Quiescent VSMCs were cultured in the presence or absence of a PPARgamma activator, TRO or PGJ2, at the indicated concentrations for 24 h. After stimulation with (+) or without IL-1beta (-) for 12 h, mRNA (A) and protein (B) levels of PDGFalpha R were determined by Northern or Western blotting, respectively. 28S, ethidium bromide staining of 28 S ribosomal RNA. Similar results were obtained from three independent experiments.

PGF2alpha Diminishes the Inhibitory Effect of TRO or PGJ2 on IL-1beta -mediated PDGFalpha R Expression-- To further gain direct evidence that the inhibitory effect of TRO or PGJ2 on IL-1beta -mediated PDGFalpha R expression is actually caused by PPARgamma -specific activation, we examined the action of PGF2alpha , an agent inactivating PPARgamma by causing phosphorylation (Fig. 2). Incubation with PGF2alpha (200 nmol/liter) diminished the inhibitory effect of TRO (10 µmol/liter) (Fig. 2, lane 3 versus lane 4) or PGJ2 (5 µmol/liter) (lane 5 versus lane 6) on IL-1beta -mediated induction of PDGFalpha R expression in VSMCs.


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Fig. 2.   Effect of PPARgamma -specific inactivation by PGF2alpha on IL-1-beta -mediated PDGFalpha R expression. VSMCs were pretreated with (lanes 4 and 6) or without (lanes 1-3 and 5) PGF2alpha (200 nmol/liter) for 12 h. Then, cells were treated with (+) or without IL-1beta (-) for 12 h in the presence (+) or absence (-) of TRO (10 µmol/liter) or PGJ2 (5 µmol/liter), and whole-cell lysates obtained from each treated VSMC were analyzed by Western blotting for PDGFalpha R. Similar results were obtained from three independent experiments.

TRO or PGJ2 Suppresses PDGF-mediated VSMC Proliferation-- In Fig. 3, VSMCs were cultured in the presence and absence of TRO (10 µmol/liter) or PGJ2 (5 µmol/liter) and were stimulated with or without IL-1beta . Then, cell proliferation activity was determined as BrdUrd incorporation 12 h after treatment with PDGF (20 ng/ml). In quiescent cells, pretreatment with TRO or PGJ2 did not affect base-line levels of cell proliferation activity. On the other hand, IL-1beta increased (by 1.8-fold) cell proliferation activity, and TRO or PGJ2 suppressed this effect of IL-1beta almost completely. Furthermore, PDGF-AA or -BB showed an additive effect in the cell proliferation activity compared with IL-1beta alone, the extent of activation being on the order of 1.4- or 2.2-fold, respectively. This additive effect of PDGFs was significantly suppressed by pretreatment with TRO or PGJ2.


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Fig. 3.   Suppressive effect of TRO or PGJ2 on PDGF-mediated VSMC proliferation. VSMCs were cultured in the absence (open bars) or presence (closed bars) of TRO (10 µmol/liter) or PGJ2 (5 µmol/liter) (hatched bars) for 24 h and then stimulated with (+) or without (-) IL-1beta for 4 h. Then, VSMC proliferation activity was measured as BrdUrd incorporation 12 h after treatment with (+) or without (-) 20 ng/ml of PDGF-AA or -BB and was finally presented as a fold activation in reference to the control activity seen in quiescent VSMCs without PPARgamma activators. All data are expressed as means + S.E. of six separate assays. *, p < 0.05, significant difference versus control activity; and dagger , p < 0.05, significant difference among the three treatment groups represented by open, closed, and hatched bars.

TRO or PGJ2 Inhibits C/EBP Binding Activity-- Recently, we have demonstrated that induction of PDGFalpha R expression is mediated by a specific transcription factor, C/EBP, in VSMCs (13, 25). To clarify the effect of PPARgamma activation on DNA binding of C/EBP members, EMSA and supershift assays were performed using a labeled C/EBP probe containing a consensus sequence seen in the rat PDGFalpha R promoter region (Fig. 4). Nuclear extracts were prepared from the IL-1beta -stimulated VSMCs after pretreatment with or without TRO (10 µmol/liter) or PGJ2 (5 µmol/liter). Intensities of two specifically retarded bands (B1 and B2) were increased by treatment with IL-1beta (Fig. 4, lane 1 versus lane 2), whereas both band intensities were reduced by pretreatment with TRO (lane 3) or PGJ2 (lane 4) almost completely. Competition experiments indicated that the retarded bands were competed out exclusively by adding a 100-fold molar excess of unlabeled C/EBP probe (Fig. 4, lane 6) but not of an unrelated probe for a nuclear factor-kappa B consensus sequence (lane 7). To determine the nature of C/EBP members that actually contribute to the DNA binding, a supershift assay was performed using specific antibodies against three major members of the C/EBP family: C/EBPalpha , beta , and delta  (Fig. 4, lanes 8-10). The retarded bands (B1 and B2) seen in EMSA were supershifted by preincubation with antibodies against C/EBPbeta (Fig. 4, lane 9) or C/EBPdelta (lane 10) but not C/EBPalpha (lane 8).


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Fig. 4.   Effect of TRO or PGJ2 on DNA binding activity of C/EBP. Nuclear extracts prepared from either quiescent (lane 1) or IL-1beta -treated VSMC for 2 h (lanes 2-10) after pretreatment with TRO (10 µmol/liter) (lane 3) or PGJ2 (5 µmol/liter) (lane 4) or without a PPARgamma activator (lanes 1, 2, and 5-10). Competition experiments were carried out by adding a 100-fold molar excess of unlabeled C/EBP probe (lane 6) or unlabeled nuclear factor-kappa B probe (lane 7). Supershift assay was performed using antibodies against three major members of the C/EBP family: C/EBPalpha (lane 8), C/EBPbeta (lane 9), or C/EBPdelta (lane 10). B1 and B2, specifically retarded bands; S, supershifted band.

TRO or PGJ2 Suppresses IL-1beta -mediated C/EBPdelta Expression-- In our earlier observations, we have shown that C/EBPdelta and C/EBPbeta act as the major transcriptional activator and the suppressor, respectively, of PDGFalpha R expression in VSMCs (25). Therefore, we investigated a direct effect of PPARgamma activation on C/EBPdelta expression (Fig. 5). Base-line levels of C/EBPdelta mRNA (Fig. 5A) and protein (Fig. 5B) were very low in quiescent VSMCs, whereas both levels were markedly increased by treatment with IL-1beta . This IL-1beta -mediated induction of C/EBPdelta expression was significantly suppressed by pretreatment with TRO or PGJ2 in a dose-dependent manner.


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Fig. 5.   Suppressive effect of TRO or PGJ2 on IL-1beta -mediated C/EBPdelta expression. Quiescent VSMCs were cultured in the presence or absence of a PPARgamma activator, TRO or PGJ2, at the indicated concentrations for 24 h. After stimulation with (+) or without (-) IL-1beta for 12 h, mRNA (A) and protein (B) levels of C/EBPdelta were determined by Northern or Western blotting, respectively. 28S, ethidium bromide staining of 28 S ribosomal RNA. Similar results were obtained from three independent experiments.

C/EBPdelta Overexpression Neutralizes the Inhibitory Effect of TRO on PDGFalpha R Expression-- To determine whether C/EBPdelta can directly modulate the inhibitory effect of PPARgamma activation on PDGFalpha R expression, transient transfection experiments were performed using a C/EBPdelta expression vector, MSV-C/EBPdelta (Fig. 6). Mock DNA plasmid (Fig. 6, lanes 1-3) or MSV-C/EBPdelta (lanes 4-6) was transfected onto VSMCs. Transfected cells were treated with or without TRO (10 µmol/liter) for 24 h and were stimulated with or without IL-1beta for 12 h. Then, whole-cell lysates were extracted from the cells, and protein levels of PDGFalpha R were determined by Western blotting. In the mock DNA-transfected cells (Fig. 6, lanes 1-3), protein levels of PDGFalpha R were markedly increased by treatment with IL-1beta (lane 1 versus lane 2), whereas TRO suppressed this enhanced effect almost completely (lane 3). In the MSV-C/EBPdelta -transfected VSMCs, C/EBPdelta overexpression caused a significant induction of PDGFalpha R protein expression (Fig. 6, lane 1 versus lane 4), and an additive effect on PDGFalpha R expression was observed in the cells treated with IL-1beta (lane 4 versus lane 5). In addition, the suppressive effect of TRO on IL-1beta -mediated induction of PDGFalpha R expression (Fig. 6, lane 2 versus lane 3) was completely abolished by C/EBPdelta overexpression (lane 5 versus lane 6).


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Fig. 6.   Effect of C/EBPdelta overexpression on TRO-mediated suppression of PDGFalpha R expression. VSMCs were seeded in 60-mm dishes (5 × 105 cells per dish) 24 h before transfection. At ~70-80% confluence, 3 µg of mock DNA plasmid (lanes 1-3) or MSV-C/EBPdelta vector (lanes 4-6) was transfected onto cells with the use of LipofectAMINE Plus. One day after transfection, cells were treated with TRO (10 µmol/liter) (lanes 3 and 6) for 24 h and then stimulated with IL-1beta for 12 h (lanes 2, 3, 5, and 6). Whole-cell lysates were prepared from each treated VSMC and were analyzed by Western blotting for PDGFalpha R.


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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In the present study, we have produced several findings of importance, which include the following. (1) PPARgamma activators, TRO and PGJ2, suppress the induction of PDGFalpha R expression and cell proliferation activity induced by PDGFs in a ligand-dependent manner. (2) This suppressive effect of PPARgamma activators is caused by a decrease in the DNA binding activity of C/EBPdelta and C/EBPbeta (but not C/EBPalpha ). (3) PPARgamma activation causes a relevant inhibition of C/EBPdelta and an ensuing suppression of PDGFalpha R gene expression, (4) C/EBPdelta overexpression can neutralize the inhibitory effect of TRO on PDGFalpha R expression almost completely. These results reveal a new role of PPARgamma activators on VSMC growth and proliferation and also provide us with important information to understand the underlying mechanism in the pathogenesis and progression of atherosclerosis and restenosis.

Previous studies (9-11) have shown that the mitogenic response of IL-1beta for fibroblasts and VSMCs is mediated by an indirect pathway, causing the release of endogenous PDGFs, especially PDGF-AA, via an autocrine or paracrine loop. Because the action of PDGF-AA is mediated by its specific receptor, PDGFalpha R, there is a possibility that it becomes a therapeutic target to control PDGFalpha R expression in the proliferative VSMCs of atherosclerotic lesions. Recently, we have reported that the rat PDGFalpha R promoter region contains an enhancer core sequence for C/EBP (23), and an enhanced effect of PDGFs on VSMC proliferation activity is caused mainly by a high level of C/EBPdelta expression (13, 25). These results strongly suggest that the C/EBP family, particularly C/EBPdelta , becomes a novel candidate gene that regulates vascular growth and development and also plays an important role in the pathogenesis of vascular remodeling and atherosclerosis.

Some recent studies have demonstrated that one of the PPARgamma activators, TRO, inhibits VSMC migration and proliferation induced by PDGFs (18-20) and suppresses neointimal formation of the arterial wall after balloon injury (19). In the present study, we showed that VSMC proliferation activity was increased by IL-1beta alone. Moreover, both PDGF-AA and -BB enhanced the effect of IL-1beta on cell proliferation activity (Fig. 3). Because PDGF-BB can bind not only PDGF-beta receptor but also PDGFalpha R, the enhanced effect of PDGF-BB is mediated by the action through both subtypes of PDGF receptors. Furthermore, pretreatment with TRO or PGJ2 significantly reduced IL-1beta -mediated induction of PDGFalpha R expression in a dose-dependent manner (Fig. 1) and suppressed cell proliferation activity after treatment with PDGFs (Fig. 3). Because TRO or PGJ2 did not alter PDGF-beta receptor expression in VSMCs (data not shown), the suppressive effect of PPARgamma activators on VSMC proliferation was mainly caused by down-regulation of PDGFalpha R expression.

We have previously demonstrated that PDGFalpha R gene transcription is regulated mainly by C/EBPdelta through a C/EBP-binding motif identified in a promoter region of the PDGFalpha R gene (13, 25). Therefore, we further determined the effect of PPARgamma activators on PDGFalpha R and C/EBPdelta expression. EMSA and supershift assay using a C/EBP probe clearly demonstrated that PPARgamma activators reduced DNA binding of C/EBPdelta and C/EBPbeta (but not C/EBPalpha ) (Fig. 4). Furthermore, PPARgamma activators significantly suppressed IL-1beta -mediated C/EBPdelta expression (Fig. 5). Overexpression studies demonstrated that exogenous C/EBPdelta expression modulated the inhibitory effect of TRO on PDGFalpha R expression (Fig. 6). These results strongly suggest that the effect of PPARgamma activators on PDGFalpha R gene transcription is mainly mediated by suppression of C/EBPdelta expression in VSMCs.

Interaction between PPARgamma and C/EBPs has already been investigated in several other types of cells (14, 15). However, detailed mechanisms of PPARgamma activation on C/EBPdelta modification in VSMCs are unknown. Our previous study has demonstrated that a core promoter region of the rat C/EBPdelta gene does not contain any obvious peroxisome proliferator response element motifs (26). This observation suggests that C/EBPdelta gene transcription is not regulated directly by PPARgamma through peroxisome proliferator response element motifs, but presumably through an indirect pathway, i.e. the interaction between PPARgamma and other inflammatory transcription factors. Indeed, several recent studies have indicated that PPARs repress gene transcription by interfering with signal transducers and activators of transcription, AP-1, and nuclear factor-kappa B signaling pathways in a peroxisome proliferator response element-independent fashion (17). These findings suggest that other transcriptional factors such as nuclear factor-kappa B and AP-1 may also contribute to the inhibitory effect of PPARgamma activation on the IL-1beta -mediated PDGFalpha R gene transcription in VSMCs. However, further detailed studies are necessary to clarify the involvement of other factors.

The expression of C/EBPdelta is undetectable or minor in normal tissues; however, it is induced rapidly and drastically by treatment with inflammatory cytokines such as IL-1beta , IL-6, and tumor necrosis factor-alpha . Recently, these cytokines have been called "adipocytokines," and have been thought to be major factors relating to atherosclerosis and insulin resistance. TRO was originally identified as a PPARgamma activator to improve insulin resistance and is known to normalize the gene expression of tumor necrosis factor-alpha or leptin by regulating adipocyte differentiation (27). As well as the PDGFalpha R gene, many other genes including tumor necrosis factor-alpha , leptin, cyclooxygenase-2, Na+/H+ exchanger-1, and IL-6 are also regulated by members of the C/EBP family (28-32). Therefore, C/EBPs are supposed to regulate various target genes and play a pivotal role in the pathological conditions of many types of cells including adipocytes and VSMCs. We have previously demonstrated that gene expression of both C/EBPdelta and PDGFalpha R is markedly elevated in cultured VSMCs prepared from spontaneously hypertensive rats but not from normotensive rat strains such as Harlan Sprague-Dawley, Wistar, and Wistar-Kyoto rats (23). These results strongly suggest the possibility that C/EBPs play important roles in the pathogenesis of atherosclerosis, insulin resistance, and hypertension.

The results obtained herein show evidence for new roles of the two transcriptional factors PPARgamma and C/EBPdelta in regulating IL-1beta -induced PDGFalpha R gene activation and in controlling opposing biological effects in VSMC proliferation. The functionally important interaction between C/EBPdelta and PPARgamma is probably involved in the regulation of inflammatory responses in the early process of vascular remodeling and resultant atherosclerosis and in the success of maintaining homeostasis in the arterial wall.

    ACKNOWLEDGEMENT

We are deeply indebted to Dr. Steven L. McKnight (Department of Biochemistry, The University of Texas South Medical Center, Dallas, TX) for the generous gift of MSV-C/EBPdelta plasmids.

    FOOTNOTES

* This work was supported in part by Grants-in-aid for Scientific Research 11838012 and 12470155 from the Ministry of Education, Science, Sports, and Culture of Japan and by grants from the 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 correspondence should be addressed: The Second Department 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.

Published, JBC Papers in Press, January 22, 2001, DOI 10.1074/jbc.M011655200

    ABBREVIATIONS

The abbreviations used are: VSMC, vascular smooth muscle cell; PDGF, platelet-derived growth factor; IL, interleukin; PDGFalpha R, PDGF-alpha receptor; C/EBP, CCAAT/enhancer-binding protein; PPAR, peroxisome proliferator-activated receptor; TRO, troglitazone; PGJ2, 15-deoxy-Delta 12,14-prostaglandin J2; PGF2alpha , prostaglandin F2alpha ; EMSA, electromobility shift assay.

    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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