Vascular Inflammation Is Negatively Autoregulated by Interaction Between CCAAT/Enhancer-Binding Protein-δ and Peroxisome Proliferator-Activated Receptor-γ
CCAAT/enhancer-binding proteins (C/EBPs) upregulate transcription of various inflammatory cytokines and acute phase proteins, such as interleukin (IL)-1β, IL-6, tumor necrosis factor-α, and cyclooxygenase-2. Recent studies have demonstrated that peroxisome proliferator-activated receptor (PPAR)-γ is present in atherosclerotic lesions, and negatively regulates expression of these genes. Interestingly, PPAR-γ gene promoter has tandem repeats of C/EBP-binding motif, and C/EBP-δ plays a pivotal role in transactivation of PPAR-γ gene. It has been well known that the interaction between C/EBPs and PPAR-γ plays a central role in maintaining adipocyte differentiation and glucometabolism; however, the relationship between PPAR-γ and C/EBPs in the vessel wall remains unclear. In the present study, we showed that a high level of C/EBP-δ expression induced by inflammation positively regulated transcription and protein expression of PPAR-γ in vascular smooth muscle cells (VSMCs). On the other hand, PPAR-γ ligands troglitazone, pioglitazone, and 15-deoxy-Δ12,14-prostaglandin J2 inhibited IL-1β-induced IL-6 expression at a transcriptional revel in VSMCs. Functional promoter analysis revealed that PPAR-γ ligands inhibited IL-1β-induced transactivation of IL-6 gene via suppression of not only nuclear factor-κB but also C/EBP-DNA binding. Moreover, PPAR-γ ligands suppressed protein expression and transcription of C/EBP-δ through dephosphorylation of signal transducer and activator of transcription 3. These findings strongly suggest that C/EBP-δ is negatively autoregulated via transactivation of PPAR-γ. This feedback mechanism probably downregulates transcription of inflammatory cytokines and acute phase proteins, and modulates inflammatory responses in the early process of atherosclerosis.
- CCAAT/enhancer-binding proteins
- peroxisome proliferator-activated receptor-γ
- vascular smooth muscle cells
- signal transducer and activator of transcription 3
Vascular inflammation plays an important role in both initiation and progression of atherosclerosis. In fact, elevated plasma levels of C-reactive protein and interleukin (IL)-6 can predict the development and mortality of cardiovascular diseases.1–3⇓⇓ This result strongly indicated that antiinflammatory agents have beneficial effects on the prevention of cardiovascular diseases. Recent studies have demonstrated that peroxisome proliferator-activated receptor (PPAR)-γ is present in macrophage foam cells, endothelial cells, and vascular smooth muscle cells (VSMCs) of human and murine atherosclerotic lesions, and suppresses expression of inflammatory cytokines and acute phase (AP) proteins.4–10⇓⇓⇓⇓⇓⇓ For example, a natural PPAR-γ ligand, 15-deoxy-Δ12,14-prostaglandin J2 (PGJ2), inhibits production of inflammatory cytokines, such as IL-1β, IL-6, and tumor necrosis factor-α (TNF-α).7 Synthetic PPAR-γ ligands, troglitazone (TRO) and BRL49653, inhibit cytokine-induced gene expression of IL-6, inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and chemokine receptor 2 (CCR2).8–10⇓⇓ Furthermore, TRO inhibits platelet-derived growth factor (PDGF)-induced migration and proliferation of VSMCs and suppresses intimal hyperplasia after balloon injury.11 However, the targets of PPAR-γ ligands and the detailed mechanisms are still unclear. Interestingly, adjacent CCAAT/enhancer-binding proteins (C/EBPs) and nuclear factor-κB (NF-κB) binding motifs are commonly identified in the promoter regions of these inflammatory cytokines and AP genes, and both C/EBP and NF-κB play a central role in the transcription of these genes.12–17⇓⇓⇓⇓⇓ Recent studies have reported that the antiinflammatory effects of PPAR-γ are mediated by a negative transcriptional control of NF-κB.8,18⇓ On the other hand, it has been well known that the interaction between C/EBPs and PPAR-γ plays a central role in maintaining adipocyte differentiation and glucometabolism19; however, the relationship between PPAR-γ and C/EBPs in the vascular wall remains to be clarified.
We have previously shown that the expression of C/EBP-β is at a low level and C/EBP-δ is negligible in quiescent VSMCs. In contrast, inflammatory cytokine, such as IL-1β, markedly induced C/EBP-β and -δ expression in VSMCs. On the other hand, the expression of C/EBP-α was negligible in either quiescent or cytokine-treated VSMCs.20 Moreover, we have demonstrated that C/EBP-δ acts as a major activator of cytokine-induced transactivation of the platelet-derived growth factor α receptor (PDGFαR) gene in VSMCs, and overexpression of C/EBP-δ specifically enhances PDGFαR gene expression and VSMC proliferation in vitro and in vivo.20–23⇓⇓⇓ These findings strongly suggest that a high level of C/EBP-δ expression is involved in the pathogenesis of vascular remodeling and atherosclerosis. Recently, we have also reported that PPAR-γ ligands inhibit cytokine-induced C/EBP-δ mRNA and protein expression in a dose-dependent manner, resulting in suppression of PDGFαR expression and proliferation activity in VSMCs.24 Unlike these inflammatory cytokines and AP proteins, there is no NF-κB binding motif in the core promoter region of the PDGFαR gene.21,22⇓
Interestingly, the PPAR-γ gene promoter has tandem repeats of C/EBP-binding motif, and C/EBP-δ plays a central role in transactivation of the PPAR-γ gene.25,26⇓ Indeed, as well as C/EBP-δ, PPAR-γ is highly expressed in the VSMCs derived from genetically hypertensive rat, but only slightly expressed in the normal artery.21,23,27⇓⇓ Given these findings, we hypothesized that C/EBP-δ is negatively autoregulated via transactivation of PPAR-γ in VSMCs, and this feedback mechanism regulates transcription of the inflammatory cytokines and AP proteins, resulting in modulation of vascular inflammation. To clarify this feedback mechanism, we here investigated the relationship between PPAR-γ and C/EBP-δ in the vascular wall.
Materials and Methods
TRO was provided from Sankyo, Co and pioglitazone (PIO) was from Takeda, Co. Recombinant rat IL-1β was purchased from R&D Systems. PGJ2 was from BIOMOL. Affinity-purified antibodies for PPAR-γ were purchased from BIOMOL, and those for C/EBPα, C/EBPβ, C/EBPδ, and p65 were from Santa Cruz Biotechnology. The double-stranded consensus oligonucleotide probes for C/EBP and NF-κB were from Santa Cruz Biotechnology.
VSMCs were isolated from the thoracic aortas of male Sprague-Dawley (SD) rats (Charles River, Kanagawa, Japan) and were maintained as previously described.22 Quiescent VSMCs were pretreated with or without a PPAR-γ ligand, TRO (0 to 10 μmol/L), PIO (10 μmol/L), or PGJ2 (0 to 5 μmol/L) for 24 hours, and then treated with or without IL-1β (10 ng/mL) as previously described.24
RNA and Protein Analysis
Total RNA, whole-cell lysates, and nuclear extracts (NEs) were prepared according to the method as previously described.20,21⇓ Northern and Western blot analysis, and immunohistochemical analysis of balloon-injured arteries were performed as previously described.24,28⇓ The production levels of IL-6 secreted from VSMCs into culture medium were measured with an ELISA kit (BioSource International) according to the manufacturer’s specifications. IL-1β-induced phosphorylation of signal transducer and activator of transcription 3 (STAT3) was determined by Western blot analysis using a PhosphoPlus STAT3 (Tyr705) Antibody Kit (Cell Signaling Technology Inc) as previously described.29
Plasmid Transfection and Luciferase Assay
C/EBP expression vector (MSV-C/EBP),21 PPAR-γ expression vector (pcDNA3-PPAR-γ),30 mock vector (Mock), or C/EBPδ promoter-luciferase fusion vector (pGL-C/EBP-δ 5) was transfected as previously described.20,31⇓ For preparation of PPAR-γ promoter luciferase fusion vector (pGL-PPAR-γ), a 615-base pair of cDNA fragment was generated by PCR using the primers 5′-ATATGAGCTCTTTATAGAATTTGGATAGCAGTA-3′ and 5′-ATATAAGCTTAACAGCATAAAACAGAGATTTGC-3′, and was subcloned into pGL3-basic vector (Promega).25 PPAR-γ, IL-6, and C/EBP-δ promoter activities were measured by luciferase assay using Dual-Luciferase Reporter Assay System (Promega) as previously described.21 Briefly, VSMCs were seeded in 60-mm dishes (5×105 cells per dish) 24 hours before transfection. Transfection was performed with cells at approximately 70% confluence with the use of Lipofectamine Plus (GIBCO BRL) according to manufacturer’s specifications. The pGL-PPAR-γ or rat IL-6 promoter-luciferase fusion vector (pGL-IL-6)32 (3 μg per dish, each) was cotransfected into VSMCs together with MSV-C/EBP (3 μg per dish) and pRL-TK vector (0.5 μg per dish) (Promega), and cells were incubated with complete medium for an additional 48 hours. Promoter activity was calculated as firefly/renilla luciferase activity ratio, and was finally expressed as a fold-activation to baseline level.
Electrophoretic Mobility Shift and Supershift Assays
Functional promoter analysis was performed by electrophoretic mobility shift assay (EMSA) and supershift assay as previously described.33 Briefly, quiescent VSMCs were pretreated with or without PPAR-γ ligands, and then NEs were obtained from the cells at 2 hours after treatment with or without IL-1β. NEs (10 μg) were incubated with the labeled C/EBP (also referred to as nuclear factor IL-6) or NF-κB probe (5.0×104 cpm, each) for 30 minutes at room temperature, and reaction mixtures were analyzed by 4% polyacrylamide gel electrophoresis. For supershift assay, NEs were incubated with each antibody for 30 minutes at room temperature before adding the labeled probe.
Analysis of variance with Bonferroni-Dunn post hoc was used to analyze differences between 2 experimental groups. All data are expressed as mean+SE, and statistical significance was defined as P<0.05.
C/EBP-δ Upregulates Transcription and Expression of PPAR-γ in VSMCs
To evaluate whether C/EBPs are required for PPAR-γ gene expression in the vascular wall, we first examined the effect of C/EBPs on transcription of PPAR-γ in VSMCs. A transient transfection study showed that overexpression of C/EBP-δ markedly increased the PPAR-γ promoter activity by a 3.0-fold. On the other hand, C/EBP-β suppressed the basal promoter activity by approximately 80% (Figure 1A). Overexpression of C/EBP-δ also enhanced PPAR-γ protein expression in VSMCs (Figure 1B). Immunohistochemical analysis of the balloon-injured carotid artery of SD rats revealed that both C/EBP-δ and PPAR-γ were drastically induced in the same area of the intimal lesion (Figure 1C).
PPAR-γ Ligands Inhibit IL-6 Expression in VSMCs
To clarify the detailed mechanisms of the antiinflammatory effect of PPAR-γ, we examined the effect of the PPAR-γ ligands TRO and PGJ2 on IL-1β-induced IL-6 expression in VSMCs. Although IL-6 mRNA and protein levels were very low in quiescent VSMCs, treatment with IL-1β drastically induced IL-6 expression, and pretreatment with PPAR-γ ligands significantly suppressed this induction in a dose-dependent manner (Figures 2A and 2B).
PPAR-γ Ligands Inhibit Transcription of IL-6 via Suppression of not Only NF-κB- but Also C/EBP-Binding
To explore the hypothesis that this inhibitory effect of PPAR-γ ligands on IL-6 production is regulated at the transcriptional level, we evaluated the effect of PPAR-γ ligands on IL-6 promoter activity in VSMCs. In the absence of PPAR-γ ligands, 1-hour treatment with IL-1β dramatically increased IL-6 promoter activity by a 6.0-fold. On the other hand, pretreatment with PPAR-γ ligands significantly reduced this transactivation of IL-6 (Figure 3). To further clarify the effect of PPAR-γ ligands on transrepression of the IL-6 gene, we performed EMSA and supershift assay for the consensus sequences for C/EBP and NF-κB (Figure 4). EMSA using a C/EBP probe revealed that DNA-protein binding activity was increased by treatment with IL-1β (lane1 versus lane 2). In contrast, pretreatment with PPAR-γ ligands significantly decreased the complex formation (lanes 3 to 5). Supershift assay using antibodies against 2 major members of C/EBP family, C/EBP-β and -δ, demonstrated that these retarded bands were supershifted by antibodies against C/EBP-β (lane 6) and -δ (lane 7). Moreover, DNA-protein binding activity of NF-κB was increased by IL-1β (lane 8 versus lane 9) and supershifted in the presence of anti-p65 antibodies (lane 13). These bindings were suppressed by pretreatment with PPAR-γ ligands (lanes 10 to 12).
PPAR-γ Ligands Negatively Regulate Transcription of C/EBP-δ via Dephosphorylation of STAT3
Transient transfection experiments demonstrated that C/EBP-δ acts as a major transcriptional activator and C/EBP-β as a suppressor of IL-6 promoter activity in VSMCs (data not shown). Therefore, we next examined the direct effect of PPAR-γ ligands on C/EBP-δ protein expression in VSMCs. In quiescent VSMCs, protein levels of C/EBP-δ were very low, whereas IL-1β markedly enhanced C/EBP-δ expression. Pretreatment with PPAR-γ ligands significantly suppressed IL-1β-induced C/EBP-δ protein expression (Figure 5).
To investigate whether PPAR-γ regulates C/EBP-δ expression at the transcriptional level, we performed promoter analysis of C/EBP-δ. Luciferase assay revealed that pretreatment with PPAR-γ ligands reduced IL-1β-mediated promoter activity of the C/EBP-δ gene. Overexpression of PPAR-γ significantly enhanced this inhibitory effect of PPAR-γ (Figure 6).
Because transactivation of C/EBP-δ is mainly regulated by phosphorylated STAT3,12,31,34⇓⇓ we evaluated the effect of PPAR-γ ligands on IL-1β-induced phosphorylation of STAT3. Stimulation with IL-1β increased protein levels of phosphorylated STAT3, and pretreatment with PPAR-γ ligands significantly suppressed this phosphorylation (Figure 7, top). In contrast, none of these treatments affected the total expression levels of the STAT3 protein (Figure 7, bottom).
C/EBP-δ expression is usually at an undetectable or minor level in normal cells or tissues, and rapidly induced by proinflammatory cytokines such as IL-1β, IL-6, and TNF-α. Therefore, C/EBP-δ is thought to be an important factor to regulate the gene transcription of AP proteins, such as serum amyloid A, COX-2, α-1 acid glycoprotein, iNOS, and granulocyte colony-stimulating factor.12 On the other hand, promoter regions of IL-1β, IL-6, and TNF-α also have C/EBP-binding motif, and C/EBP-δ upregulates transactivation of these proinflammatory cytokines.12–16⇓⇓⇓⇓ These findings suggest that C/EBP-δ plays a central role in the acceleration of inflammation (Figure 8). Several studies of mutagenesis have shown that C/EBP and NF-κB binding sites are necessary for the transactivation of the IL-6 gene, and both sites have been shown to be the target for IL-1β stimulation.12–15⇓⇓⇓ Recent studies have suggested that the antiinflammatory effects of PPAR-γ are mediated by a negative transcriptional control of NF-κB.8,18⇓ However, the effect of PPAR-γ on C/EBP has not been well defined. In the present study, we demonstrated that PPAR-γ ligands inhibited IL-1β-induced IL-6 gene expression at a transcriptional level in VSMCs (Figures 2 and 3⇑). Functional promoter analysis revealed that PPAR-γ ligands inhibit IL-1β-induced transactivation of the IL-6 gene via suppression of not only NF-κB but also C/EBP-δ-DNA binding in VSMCs (Figure 4). Interestingly, it has been reported that the cooperative interaction between C/EBPs and NF-κB regulates the synergistic induction of AP genes by proinflammatory cytokines.12–15⇓⇓⇓ Therefore, PPAR-γ ligands probably inhibit this cooperative interaction and repress transcription of AP genes and inflammatory cytokines. On the other hand, recent studies have shown that PPAR-γ ligands inhibit the gene expression of PDGFαR, CCR2, and COX-2.9,10,24⇓⇓ Unlike the IL-6 gene, there is no NF-κB binding motif in the promoter regions of these genes, and transcription of these promoters is mainly regulated by C/EBP-δ.16,17,21⇓⇓ These findings suggest that inhibition of C/EBP-δ by PPAR-γ ligands is alone sufficient to suppress transcription of these genes.
Chawla et al9 reported that PPAR-γ is required for positive effects of its ligands in modulating macrophage lipid metabolism, but that inhibitory effects on cytokine production and inflammation may be of a PPAR-γ-independent manner. On the other hand, we have previously reported that prostaglandin F2α, an agent inactivating PPAR-γ, diminishes the inhibitory effect of PPAR-γ ligands on C/EBP-δ-mediated PDGFαR expression.24 To further gain the direct evidence that the inhibitory effect of PPAR-γ ligands on C/EBP-δ is caused in a receptor-dependent manner, we examined the effect of PPAR-γ overexpression on the promoter activity of C/EBP-δ. Overexpression of PPAR-γ decreased the basal promoter activity of C/EBP-δ and enhanced the inhibitory effect of both natural and synthetic PPAR-γ ligands on the IL-1β-mediated C/EBP-δ promoter activity in VSMCs (Figure 6). These findings indicate that the effect of PPAR-γ ligands on C/EBP-δ is caused in a receptor-dependent manner.
We have previously shown that the core promoter region of the C/EBP-δ gene does not contain any obvious peroxisome proliferator response element (PPRE) motifs.31 Therefore, PPAR-γ ligands negatively regulate C/EBP-δ gene transcription by PPRE-independent fashion, presumably through interaction between PPAR-γ and other inflammatory transcription factors. Other groups and we have previously demonstrated that C/EBP-δ promoter sequences spanning −235 through −82 are sufficient for the basal promoter activity, and a core sequence involved in the basal activity is an acute-phase response element (APRE) identified at −152 through −144. DNA-binding experiments have indicated that the transactivation of C/EBP-δ gene is mainly regulated by binding of the phosphorylated STAT3 to the APRE.12,31,34⇓⇓ In the present study, we showed that PPAR-γ ligands suppress gene transactivation of C/EBP-δ by the inhibition of the STAT3 signaling pathways in VSMCs (Figure 7).
It has been reported that the PPAR-γ gene promoter itself has tandem repeats of C/EBP-binding motif, and C/EBP-δ plays a central role in transactivation of the PPAR-γ gene in adipocyte differentiation.25,26⇓ In the present study, we demonstrated that overexpression of C/EBP-δ upregulates transcription and protein expression of PPAR-γ in the vessel wall (Figure 1). These findings suggest that C/EBP-δ induced by vascular inflammation may be negatively autoregulated by transactivation of PPAR-γ. The suppression of C/EBP-δ negatively regulates transcription of inflammatory cytokines and AP genes and modulates vascular inflammation (Figure 8). These results obtained here are similar to the previous findings on the early process of adipocyte differentiation. C/EBP-β and -δ are expressed in preadipocytes and initially activate the transcription of C/EBP-α and PPAR-γ. As the expression of PPAR-γ is increased, C/EBP-β and -δ gradually decrease, and the cell maturation is finished.19
Thiazolidinediones (TZDs) were originally identified as agents that improve insulin resistance. Recently, inflammatory cytokines such as IL-1β, IL-6, and TNF-α have been called “adipocytokines” and thought to be major factors relating to insulin resistance. On the other hand, C/EBPs regulate transactivation of leptin, Na+-H+ exchanger-1. and insulin-like growth factor-1 genes.35–37⇓⇓ Furthermore, we have previously reported that C/EBP-δ is highly expressed in VSMCs derived from spontaneously hypertensive rats but not from normotensive rat strains such as SD, Wistar, and Wistar-Kyoto rats.21 These findings strongly suggest the possibility that TZDs modulate insulin resistance and hypertension via suppression of C/EBP-δ. Indeed, Okuno et al38 reported that TRO inhibits the gene expression of TNF-α and leptin, and finally improves insulin resistance.
The results obtained herein emphasize new roles of C/EBP-δ and PPAR-γ on inflammatory responses in the vessel wall. Together with the results from our previous studies, suppression of C/EBP-δ can explain the beneficial effect of PPAR-γ ligands on cardiovascular diseases including vascular inflammation and atherosclerosis.
This work was supported in part by a Grant-in-Aid for Scientific Research from Japan Society for the Promotion of Science (Nos. 12470155 and 13670718), by a Japan Heart Foundation/Pfizer Grant for Research on Hypertension and Vascular Metabolism, by grants from the Takeda Medical Research Foundation, and by the Mochida Memorial Foundation for Medical and Pharmaceutical Research. The animals used in this study were cared for in the Laboratory Animal Center at Ehime University School of Medicine. We thank Dr Steven L. McKnight for the generous gift of the MSV/CEBP plasmids, Dr Masahiko Kurabayashi for the PPAR-γ expression vector, and Dr Tosihiro Ichiki for the rat IL-6 promoter-luciferase fusion vector and IL-6 cDNA.
Original received November 6, 2001; resubmission received July 3, 2002; accepted July 17, 2002.
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