Peroxisome Proliferator-activated Receptor-γ Activation Inhibits Interleukin-1β-mediated Platelet-derived Growth Factor-α Receptor Gene Expression via CCAAT/Enhancer-binding Protein-δ in Vascular Smooth Muscle Cells
2001; Elsevier BV; Volume: 276; Issue: 16 Linguagem: Inglês
10.1074/jbc.m011655200
ISSN1083-351X
AutoresYasunori Takata, Yutaka Kitami, Takafumi Okura, Kunio Hiwada,
Tópico(s)Adipokines, Inflammation, and Metabolic Diseases
ResumoCCAAT/enhancer-binding protein (C/EBP)-binding motifs have been identified in the promoter regions of interleukin (IL)-6, tumor necrosis factor-α, and platelet-derived growth factor-α receptor (PDGFαR). 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 PPARγ in the regulation of IL-1β-induced PDGFαR expression in VSMCs. PPARγ activators, troglitazone and 15-deoxy-Δ12,14-prostaglandin J2, inhibited IL-1β-induced PDGFαR expression and suppressed PDGF-induced proliferation activity of VSMCs. Electromobility shift and supershift assays for a C/EBP motif in the PDGFαR promoter region revealed that PPARγ activators suppressed IL-1β-induced DNA binding activity of C/EBPδ and β. PPARγ activators also suppressed IL-1β-induced C/EBPδ expression. In contrast, overexpression of C/EBPδ reversed the suppressive effect of PPARγ activators on PDGFαR expression almost completely. From these results, we conclude that the inhibitory effect of PPARγ activators on PDGFαR expression is mainly mediated by C/EBPδ suppression. Regulation of C/EBPδ by PPARγ activators probably plays critical roles in modulating inflammatory responses in the arterial wall. CCAAT/enhancer-binding protein (C/EBP)-binding motifs have been identified in the promoter regions of interleukin (IL)-6, tumor necrosis factor-α, and platelet-derived growth factor-α receptor (PDGFαR). 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 PPARγ in the regulation of IL-1β-induced PDGFαR expression in VSMCs. PPARγ activators, troglitazone and 15-deoxy-Δ12,14-prostaglandin J2, inhibited IL-1β-induced PDGFαR expression and suppressed PDGF-induced proliferation activity of VSMCs. Electromobility shift and supershift assays for a C/EBP motif in the PDGFαR promoter region revealed that PPARγ activators suppressed IL-1β-induced DNA binding activity of C/EBPδ and β. PPARγ activators also suppressed IL-1β-induced C/EBPδ expression. In contrast, overexpression of C/EBPδ reversed the suppressive effect of PPARγ activators on PDGFαR expression almost completely. From these results, we conclude that the inhibitory effect of PPARγ activators on PDGFαR expression is mainly mediated by C/EBPδ suppression. Regulation of C/EBPδ by PPARγ activators probably plays critical roles in modulating inflammatory responses in the arterial wall. Excessive or uncontrolled replication and migration of vascular smooth muscle cells (VSMCs)1are critical events involved in a number of vascular diseases including atherosclerosis, hypertension, and restenosis after balloon angioplasty (1Spaet T.H. Stemerman M.B. Veith F.J. Lejnieks I. Circ. Res. 1975; 36: 58-70Crossref PubMed Scopus (114) Google Scholar, 2Mulvany M.J. Hansen O.K. Aalkjaer C. Circ. Res. 1978; 43: 854-864Crossref PubMed Scopus (432) Google Scholar, 3Ross R. N. Engl. J. Med. 1999; 340: 115-126Crossref PubMed Scopus (19200) Google Scholar). Morphologic studies of the sequencing events in the arterial wall have revealed that macrophages are present in atherosclerotic lesions (4Jonasson L. Holm J. Skalli O. Bondjers G. Hansson G.K. Arteriosclerosis. 1986; 6: 131-138Crossref PubMed Google Scholar, 5Joris I. Zand T. Nunnari J.J. Krolikowski F.J. Majno G. Am. J. Pathol. 1983; 113: 341-358PubMed Google Scholar) and are involved in the production of several growth factors such as platelet-derived growth factors (PDGFs), basic fibroblast growth factor, tumor necrosis factor-α, and transforming growth factor-β1 (3Ross R. N. Engl. J. Med. 1999; 340: 115-126Crossref PubMed Scopus (19200) Google Scholar). Particularly, interleukin (IL)-1β is one of the major secretory products of activated macrophages and can induce proliferation and migration of fibroblasts and VSMCs (6Bitterman 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, 7Libby P. Wyler D.J. Janicka M.W. Dinarello C.A. Arteriosclerosis. 1985; 5: 186-191Crossref PubMed Google Scholar, 8Libby P. Warner S.J. Friedman G.B. J. Clin. Invest. 1988; 81: 487-498Crossref PubMed Scopus (417) Google Scholar). Previous studies have demonstrated that the mitogenic response of IL-1β for VSMCs is mediated by an indirect pathway, causing the release of PDGF-AA, which specifically binds to the PDGF α-receptor (PDGFαR) subtype on the cell surface (9Raines E.W. Dower S.K. Ross R. Science. 1989; 243: 393-396Crossref PubMed Scopus (516) Google Scholar, 10Ikeda U. Ikeda M. Oohara T. Kano S. Yaginuma T. Atherosclerosis. 1990; 84: 183-188Abstract Full Text PDF PubMed Scopus (64) Google Scholar, 11Bonin P.D. Fici G.J. Singh J.P. Exp. Cell Res. 1989; 181: 475-482Crossref PubMed Scopus (54) Google Scholar). In addition, IL-1β can also up-regulate PDGFαR expression itself in rat lung fibroblasts, thereby enhancing PDGF-mediated mitogenesis and chemotaxis of the cells (12Bonner J.C. Lindroos P.M. Rice A.B. Moomaw C.R. Morgan D.L. Am. J. Physiol. 1998; 274: L72-L80Crossref PubMed Google Scholar). Previously, we have shown that the PDGFαR promoter contains an enhancer core sequence for CCAAT/enhancer-binding protein (C/EBP), and IL-1β-mediated induction of PDGFαR expression is mainly regulated by a specific nuclear factor, C/EBPδ, in VSMCs (13Fukuoka T. Kitami Y. Okura T. Hiwada K. J. Biol. Chem. 1999; 274: 25576-25582Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). 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/EBPβ and δ play a critical role in the determination of pre-adipocyte development by activating expression of C/EBPα and PPARγ genes (14Schoonjans K. Staels B. Auwerx J. Biochim. Biophys. Acta. 1996; 1302: 93-109Crossref PubMed Scopus (907) Google Scholar, 15Lane M.D. Tang Q.Q. Jiang M.S. Biochem. Biophys. Res. Commun. 1999; 266: 677-683Crossref PubMed Scopus (238) Google Scholar). 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 (16Staels B. Koenig W. Habib A. Merval R. Lebret M. Torra I.P. Delerive P. Fadel A. Chinetti G. Fruchart J.C. Najib J. Maclouf J. Tedgui A. Nature. 1998; 393: 790-793Crossref PubMed Scopus (1053) Google Scholar, 17Ricote M. Li A.C. Willson T.M. Kelly C.J. Glass C.K. Nature. 1998; 391: 79-82Crossref PubMed Scopus (3252) Google Scholar). However, expression and function of PPARγ in VSMCs are somewhat controversial. In human VSMCs, Staelset al. (16Staels B. Koenig W. Habib A. Merval R. Lebret M. Torra I.P. Delerive P. Fadel A. Chinetti G. Fruchart J.C. Najib J. Maclouf J. Tedgui A. Nature. 1998; 393: 790-793Crossref PubMed Scopus (1053) Google Scholar) observed faint expression of PPARγ that was not involved in the negative regulation of cytokine-induced IL-6 and cyclooxygenase-2 expression; this effect was mediated by PPARγ. In contrast, Marx et al. (18Marx N. Schönbeck U. Lazar M.A. Libby P. Plutzky J. Circ. Res. 1998; 83: 1097-1103Crossref PubMed Scopus (560) Google Scholar) demonstrated that human VSMCs expressed PPARγ that inhibited matrix metalloproteinase expression and cell migration. In particular, the latter observations suggest that such a role of PPARγ may be to limit the arterial remodeling that occurs in response to hypertension, atherosclerosis, and restenosis, or even to counterbalance other vascular effects. Indeed, PPARγ activation can be shown to inhibit VSMC proliferation and migration in a variety of assays (18Marx N. Schönbeck U. Lazar M.A. Libby P. Plutzky J. Circ. Res. 1998; 83: 1097-1103Crossref PubMed Scopus (560) Google Scholar, 19Law R.E. Meehan W.P. Xi X.P. Graf K. Wuthrich D.A. Coats W. Faxon D. Hsueh W.A. J. Clin. Invest. 1996; 98: 1897-1905Crossref PubMed Scopus (461) Google Scholar, 20Goetze S. Xi X.P. Kawano H. Gotlibowski T. Fleck E. Hsueh W.A. Law R.E. J. Cardiovasc. Pharmacol. 1999; 33: 798-806Crossref PubMed Scopus (179) Google Scholar). In the present study, we demonstrated new roles of PPARγ activators, troglitazone (TRO) and a naturally occurring ligand, 15-deoxy-Δ12,14-prostaglandin J2(PGJ2), on IL-1β-induced expression of PDGFαR and clarified the underlying molecular mechanism by demonstrating a functionally important interaction between PPARγ and C/EBPδ in rat cultured VSMCs. TRO was provided from Sankyo, Co. (Tokyo, Japan). Recombinant rat IL-1β was purchased from R & D Systems (Tokyo, Japan). Prostaglandin F2α (PGF2α) and PGJ2 were from Cayman (Ann Arbor, MI). Affinity-purified antibodies for PDGFαR, PPARγ, C/EBPα, C/EBPβ, and C/EBPδ raised against peptidic epitopes corresponding with amino acid sequences of human PDGFαR (951–1,089), rat C/EBPα (253), rat C/EBPβ (258), and rat C/EBPδ (247) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). VSMCs were isolated from thoracic aortas of male Harlan Sprague-Dawley rats (Charles River Japan Inc., Kanagawa, Japan) as described previously (21Inui H. Kitami Y. Kondo T. Inagami T. J. Biol. Chem. 1993; 268: 17045-17050Abstract Full Text PDF PubMed Google Scholar) 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. Whole-cell lysates were extracted from VSMCs by the standard method (21Inui H. Kitami Y. Kondo T. Inagami T. J. Biol. Chem. 1993; 268: 17045-17050Abstract Full Text PDF PubMed Google Scholar), and nuclear extracts were prepared according to the method described by Dignamet al. (22Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9153) Google Scholar). Western blotting was essentially carried out as described previously (23Kitami Y. Inui H. Uno S. Inagami T. J. Clin. Invest. 1995; 96: 558-567Crossref PubMed Scopus (45) Google Scholar). 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 (24Okura T. Igase M. Kitami Y. Fukuoka T. Maguchi M. Kohara K. Hiwada K. Biochim. Biophys. Acta. 1998; 1403: 245-253Crossref PubMed Scopus (39) Google Scholar). 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-1β (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). The EMSA and supershift assay were performed for the C/EBP-binding motif seen in rat PDGFαR gene promoter as described previously (25Kitami Y. Fukuoka T. Hiwada K. Inagami T. Circ. Res. 1999; 84: 64-73Crossref PubMed Scopus (31) Google Scholar). VSMCs (5 × 105cells per dish) were seeded in 60-mm dishes 24 h before transfection, and then the C/EBPδ expression vector, designated MSV-C/EBPδ in our earlier studies (13Fukuoka T. Kitami Y. Okura T. Hiwada K. J. Biol. Chem. 1999; 274: 25576-25582Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 25Kitami Y. Fukuoka T. Hiwada K. Inagami T. Circ. Res. 1999; 84: 64-73Crossref PubMed Scopus (31) Google Scholar), was transfected (3 μg per dish) onto the cells with the use of LipofectAMINE Plus (Life Technologies, Inc.) according to the manufacturer's specifications. 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. We confirmed that rat cultured VSMCs used in this study expressed PPARγ mRNA and protein (data not shown). VSMCs were cultured in the presence or absence of a PPARγ activator, TRO (0–10 μmol/liter) or PGJ2 (0–5 μmol/liter), for 24 h. Then, expression levels of PDGFαR were determined by Northern (Fig. 1 A) and Western (Fig. 1 B) blotting 12 h after treatment with or without IL-1β (10 ng/ml). Previously, we have demonstrated that expression levels of PDGFαR were increased by treatment with IL-1β at doses up to 10 ng/ml in a dose-dependent manner (13Fukuoka T. Kitami Y. Okura T. Hiwada K. J. Biol. Chem. 1999; 274: 25576-25582Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Therefore, we used the dose of IL-1β at 10 ng/ml thereafter. In quiescent cells, base-line levels of PDGFαR mRNA and protein were very low. On the other hand, expression levels of PDGFαR were increased drastically by treatment with IL-1β in the absence of TRO or PGJ2, whereas induction of PDGFαR expression was significantly reduced by pretreatment with TRO or PGJ2 in a dose-dependent manner. To further gain direct evidence that the inhibitory effect of TRO or PGJ2 on IL-1β-mediated PDGFαR expression is actually caused by PPARγ-specific activation, we examined the action of PGF2α, an agent inactivating PPARγ by causing phosphorylation (Fig. 2). Incubation with PGF2α (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-1β-mediated induction of PDGFαR expression in VSMCs. 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-1β. 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-1β increased (by 1.8-fold) cell proliferation activity, and TRO or PGJ2 suppressed this effect of IL-1β almost completely. Furthermore, PDGF-AA or -BB showed an additive effect in the cell proliferation activity compared with IL-1β 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. Recently, we have demonstrated that induction of PDGFαR expression is mediated by a specific transcription factor, C/EBP, in VSMCs (13Fukuoka T. Kitami Y. Okura T. Hiwada K. J. Biol. Chem. 1999; 274: 25576-25582Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 25Kitami Y. Fukuoka T. Hiwada K. Inagami T. Circ. Res. 1999; 84: 64-73Crossref PubMed Scopus (31) Google Scholar). To clarify the effect of PPARγ 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 PDGFαR promoter region (Fig.4). Nuclear extracts were prepared from the IL-1β-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-1β (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-κB 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/EBPα, β, and δ (Fig. 4, lanes 8–10). The retarded bands (B1 and B2) seen in EMSA were supershifted by preincubation with antibodies against C/EBPβ (Fig.4, lane 9) or C/EBPδ (lane 10) but not C/EBPα (lane 8). In our earlier observations, we have shown that C/EBPδ and C/EBPβ act as the major transcriptional activator and the suppressor, respectively, of PDGFαR expression in VSMCs (25Kitami Y. Fukuoka T. Hiwada K. Inagami T. Circ. Res. 1999; 84: 64-73Crossref PubMed Scopus (31) Google Scholar). Therefore, we investigated a direct effect of PPARγ activation on C/EBPδ expression (Fig. 5). Base-line levels of C/EBPδ mRNA (Fig. 5 A) and protein (Fig. 5 B) were very low in quiescent VSMCs, whereas both levels were markedly increased by treatment with IL-1β. This IL-1β-mediated induction of C/EBPδ expression was significantly suppressed by pretreatment with TRO or PGJ2 in a dose-dependent manner. To determine whether C/EBPδ can directly modulate the inhibitory effect of PPARγ activation on PDGFαR expression, transient transfection experiments were performed using a C/EBPδ expression vector, MSV-C/EBPδ (Fig.6). Mock DNA plasmid (Fig. 6, lanes 1–3) or MSV-C/EBPδ (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-1β for 12 h. Then, whole-cell lysates were extracted from the cells, and protein levels of PDGFαR were determined by Western blotting. In the mock DNA-transfected cells (Fig. 6, lanes 1–3), protein levels of PDGFαR were markedly increased by treatment with IL-1β (lane 1 versus lane 2), whereas TRO suppressed this enhanced effect almost completely (lane 3). In the MSV-C/EBPδ-transfected VSMCs, C/EBPδ overexpression caused a significant induction of PDGFαR protein expression (Fig. 6, lane 1 versus lane 4), and an additive effect on PDGFαR expression was observed in the cells treated with IL-1β (lane 4 versus lane 5). In addition, the suppressive effect of TRO on IL-1β-mediated induction of PDGFαR expression (Fig. 6, lane 2 versus lane 3) was completely abolished by C/EBPδ overexpression (lane 5 versus lane 6). In the present study, we have produced several findings of importance, which include the following. (1Spaet T.H. Stemerman M.B. Veith F.J. Lejnieks I. Circ. Res. 1975; 36: 58-70Crossref PubMed Scopus (114) Google Scholar) PPARγ activators, TRO and PGJ2, suppress the induction of PDGFαR expression and cell proliferation activity induced by PDGFs in a ligand-dependent manner. (2Mulvany M.J. Hansen O.K. Aalkjaer C. Circ. Res. 1978; 43: 854-864Crossref PubMed Scopus (432) Google Scholar) This suppressive effect of PPARγ activators is caused by a decrease in the DNA binding activity of C/EBPδ and C/EBPβ (but not C/EBPα). (3Ross R. N. Engl. J. Med. 1999; 340: 115-126Crossref PubMed Scopus (19200) Google Scholar) PPARγ activation causes a relevant inhibition of C/EBPδ and an ensuing suppression of PDGFαR gene expression, (4Jonasson L. Holm J. Skalli O. Bondjers G. Hansson G.K. Arteriosclerosis. 1986; 6: 131-138Crossref PubMed Google Scholar) C/EBPδ overexpression can neutralize the inhibitory effect of TRO on PDGFαR expression almost completely. These results reveal a new role of PPARγ 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 (9Raines E.W. Dower S.K. Ross R. Science. 1989; 243: 393-396Crossref PubMed Scopus (516) Google Scholar, 10Ikeda U. Ikeda M. Oohara T. Kano S. Yaginuma T. Atherosclerosis. 1990; 84: 183-188Abstract Full Text PDF PubMed Scopus (64) Google Scholar, 11Bonin P.D. Fici G.J. Singh J.P. Exp. Cell Res. 1989; 181: 475-482Crossref PubMed Scopus (54) Google Scholar) have shown that the mitogenic response of IL-1β 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, PDGFαR, there is a possibility that it becomes a therapeutic target to control PDGFαR expression in the proliferative VSMCs of atherosclerotic lesions. Recently, we have reported that the rat PDGFαR promoter region contains an enhancer core sequence for C/EBP (23Kitami Y. Inui H. Uno S. Inagami T. J. Clin. Invest. 1995; 96: 558-567Crossref PubMed Scopus (45) Google Scholar), and an enhanced effect of PDGFs on VSMC proliferation activity is caused mainly by a high level of C/EBPδ expression (13Fukuoka T. Kitami Y. Okura T. Hiwada K. J. Biol. Chem. 1999; 274: 25576-25582Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 25Kitami Y. Fukuoka T. Hiwada K. Inagami T. Circ. Res. 1999; 84: 64-73Crossref PubMed Scopus (31) Google Scholar). These results strongly suggest that the C/EBP family, particularly C/EBPδ, 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 PPARγ activators, TRO, inhibits VSMC migration and proliferation induced by PDGFs (18Marx N. Schönbeck U. Lazar M.A. Libby P. Plutzky J. Circ. Res. 1998; 83: 1097-1103Crossref PubMed Scopus (560) Google Scholar, 19Law R.E. Meehan W.P. Xi X.P. Graf K. Wuthrich D.A. Coats W. Faxon D. Hsueh W.A. J. Clin. Invest. 1996; 98: 1897-1905Crossref PubMed Scopus (461) Google Scholar, 20Goetze S. Xi X.P. Kawano H. Gotlibowski T. Fleck E. Hsueh W.A. Law R.E. J. Cardiovasc. Pharmacol. 1999; 33: 798-806Crossref PubMed Scopus (179) Google Scholar) and suppresses neointimal formation of the arterial wall after balloon injury (19Law R.E. Meehan W.P. Xi X.P. Graf K. Wuthrich D.A. Coats W. Faxon D. Hsueh W.A. J. Clin. Invest. 1996; 98: 1897-1905Crossref PubMed Scopus (461) Google Scholar). In the present study, we showed that VSMC proliferation activity was increased by IL-1β alone. Moreover, both PDGF-AA and -BB enhanced the effect of IL-1β on cell proliferation activity (Fig. 3). Because PDGF-BB can bind not only PDGF-β receptor but also PDGFαR, 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-1β-mediated induction of PDGFαR 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-β receptor expression in VSMCs (data not shown), the suppressive effect of PPARγ activators on VSMC proliferation was mainly caused by down-regulation of PDGFαR expression. We have previously demonstrated that PDGFαR gene transcription is regulated mainly by C/EBPδ through a C/EBP-binding motif identified in a promoter region of the PDGFαR gene (13Fukuoka T. Kitami Y. Okura T. Hiwada K. J. Biol. Chem. 1999; 274: 25576-25582Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 25Kitami Y. Fukuoka T. Hiwada K. Inagami T. Circ. Res. 1999; 84: 64-73Crossref PubMed Scopus (31) Google Scholar). Therefore, we further determined the effect of PPARγ activators on PDGFαR and C/EBPδ expression. EMSA and supershift assay using a C/EBP probe clearly demonstrated that PPARγ activators reduced DNA binding of C/EBPδ and C/EBPβ (but not C/EBPα) (Fig. 4). Furthermore, PPARγ activators significantly suppressed IL-1β-mediated C/EBPδ expression (Fig. 5). Overexpression studies demonstrated that exogenous C/EBPδ expression modulated the inhibitory effect of TRO on PDGFαR expression (Fig. 6). These results strongly suggest that the effect of PPARγ activators on PDGFαR gene transcription is mainly mediated by suppression of C/EBPδ expression in VSMCs. Interaction between PPARγ and C/EBPs has already been investigated in several other types of cells (14Schoonjans K. Staels B. Auwerx J. Biochim. Biophys. Acta. 1996; 1302: 93-109Crossref PubMed Scopus (907) Google Scholar, 15Lane M.D. Tang Q.Q. Jiang M.S. Biochem. Biophys. Res. Commun. 1999; 266: 677-683Crossref PubMed Scopus (238) Google Scholar). However, detailed mechanisms of PPARγ activation on C/EBPδ modification in VSMCs are unknown. Our previous study has demonstrated that a core promoter region of the rat C/EBPδ gene does not contain any obvious peroxisome proliferator response element motifs (26Fukuoka T. Kitami Y. Kohara K. Hiwada K. Biochem. Biophys. Res. Commun. 1997; 231: 30-36Crossref PubMed Scopus (10) Google Scholar). This observation suggests that C/EBPδ gene transcription is not regulated directly by PPARγ through peroxisome proliferator response element motifs, but presumably through an indirect pathway, i.e. the interaction between PPARγ 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-κB signaling pathways in a peroxisome proliferator response element-independent fashion (17Ricote M. Li A.C. Willson T.M. Kelly C.J. Glass C.K. Nature. 1998; 391: 79-82Crossref PubMed Scopus (3252) Google Scholar). These findings suggest that other transcriptional factors such as nuclear factor-κB and AP-1 may also contribute to the inhibitory effect of PPARγ activation on the IL-1β-mediated PDGFαR gene transcription in VSMCs. However, further detailed studies are necessary to clarify the involvement of other factors. The expression of C/EBPδ is undetectable or minor in normal tissues; however, it is induced rapidly and drastically by treatment with inflammatory cytokines such as IL-1β, IL-6, and tumor necrosis factor-α. 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 PPARγ activator to improve insulin resistance and is known to normalize the gene expression of tumor necrosis factor-α or leptin by regulating adipocyte differentiation (27Okuno A. Tamemoto H. Tobe K. Ueki K. Mori Y. Iwamoto K. Umesono K. Akanuma Y. Fujiwara T. Horikoshi H. Yazaki Y. Kadowaki T. J. Clin. Invest. 1998; 101: 1354-1361Crossref PubMed Scopus (925) Google Scholar). As well as the PDGFαR gene, many other genes including tumor necrosis factor-α, leptin, cyclooxygenase-2, Na+/H+ exchanger-1, and IL-6 are also regulated by members of the C/EBP family (28Pope R.M. Leutz A. Ness S.A. J. Clin. Invest. 1994; 94: 1449-1455Crossref PubMed Scopus (164) Google Scholar, 29Hwang C.S. Mandrup S. MacDougald O.A. Geiman D.E. Lane M.D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 873-877Crossref PubMed Scopus (172) Google Scholar, 30Kim Y. Fischer S.M. J. Biol. Chem. 1998; 273: 27686-27694Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, 31Kolyada A.Y. Jhons C.A. Madias N.E. Am. J. Physiol. 1993; 269: C1408-C1416Crossref Google Scholar, 32Matsusaka T. Fujikawa K. Nishio Y. Mukaida N. Matsushima K. Kishimoto T. Akira S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10193-10197Crossref PubMed Scopus (877) Google Scholar). 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/EBPδ and PDGFαR 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 (23Kitami Y. Inui H. Uno S. Inagami T. J. Clin. Invest. 1995; 96: 558-567Crossref PubMed Scopus (45) Google Scholar). 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 PPARγ and C/EBPδ in regulating IL-1β-induced PDGFαR gene activation and in controlling opposing biological effects in VSMC proliferation. The functionally important interaction between C/EBPδ and PPARγ 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. 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/EBPδ plasmids. vascular smooth muscle cell platelet-derived growth factor interleukin PDGF-α receptor CCAAT/enhancer-binding protein peroxisome proliferator-activated receptor troglitazone 15-deoxy-Δ12,14-prostaglandin J2 prostaglandin F2α electromobility shift assay
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