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• W2086342197 abstract "Fibrinogen is a coagulation factor and an acute phase reactant up-regulated by inflammatory cytokines, such as interleukin 6 (IL-6). Elevated plasma fibrinogen levels are associated with coronary heart diseases. Fibrates are clinically used hypolipidemic drugs that act via the nuclear receptor peroxisome proliferator-activated receptor α (PPARα). In addition, most fibrates also reduce plasma fibrinogen levels, but the molecular mechanism is unknown. In this study, we demonstrate that fibrates decrease basal and IL-6-stimulated expression of the human fibrinogen-β gene in human primary hepatocytes and hepatoma HepG2 cells. Fibrates diminish basal and IL-6-induced fibrinogen-β promoter activity, and this effect is enhanced in the presence of co-transfected PPARα. Site-directed mutagenesis experiments demonstrate that PPARα activators decrease human fibrinogen-β promoter activity via the CCAAT box/enhancer-binding protein (C/EBP) response element. Co-transfection of the transcriptional intermediary factor glucocorticoid receptor-interacting protein 1/transcriptional intermediary factor 2 (GRIP1/TIF2) enhances fibrinogen-β gene transcription and alleviates the repressive effect of PPARα. Co-immunoprecipitation experiments demonstrate that PPARα and GRIP1/TIF2 physically interact in vivo in human liver. These data demonstrate that PPARα agonists repress human fibrinogen gene expression by interference with the C/EBPβ pathway through titration of the coactivator GRIP1/TIF2. We observed that the anti-inflammatory action of PPARα is not restricted to fibrinogen but also applies to other acute phase genes containing a C/EBP response element; it also occurs under conditions in which the stimulating action of IL-6 is potentiated by dexamethasone. These findings identify a novel molecular mechanism of negative gene regulation by PPARα and reveal the direct implication of PPARα in the modulation of the inflammatory gene response in the liver. Fibrinogen is a coagulation factor and an acute phase reactant up-regulated by inflammatory cytokines, such as interleukin 6 (IL-6). Elevated plasma fibrinogen levels are associated with coronary heart diseases. Fibrates are clinically used hypolipidemic drugs that act via the nuclear receptor peroxisome proliferator-activated receptor α (PPARα). In addition, most fibrates also reduce plasma fibrinogen levels, but the molecular mechanism is unknown. In this study, we demonstrate that fibrates decrease basal and IL-6-stimulated expression of the human fibrinogen-β gene in human primary hepatocytes and hepatoma HepG2 cells. Fibrates diminish basal and IL-6-induced fibrinogen-β promoter activity, and this effect is enhanced in the presence of co-transfected PPARα. Site-directed mutagenesis experiments demonstrate that PPARα activators decrease human fibrinogen-β promoter activity via the CCAAT box/enhancer-binding protein (C/EBP) response element. Co-transfection of the transcriptional intermediary factor glucocorticoid receptor-interacting protein 1/transcriptional intermediary factor 2 (GRIP1/TIF2) enhances fibrinogen-β gene transcription and alleviates the repressive effect of PPARα. Co-immunoprecipitation experiments demonstrate that PPARα and GRIP1/TIF2 physically interact in vivo in human liver. These data demonstrate that PPARα agonists repress human fibrinogen gene expression by interference with the C/EBPβ pathway through titration of the coactivator GRIP1/TIF2. We observed that the anti-inflammatory action of PPARα is not restricted to fibrinogen but also applies to other acute phase genes containing a C/EBP response element; it also occurs under conditions in which the stimulating action of IL-6 is potentiated by dexamethasone. These findings identify a novel molecular mechanism of negative gene regulation by PPARα and reveal the direct implication of PPARα in the modulation of the inflammatory gene response in the liver. interleukin acute phase response gene CCAAT box/enhancer-binding protein glucocorticoid receptor-interacting protein glutathione S-transferase ligand binding domain phosphate-buffered saline peroxisome proliferator-activated receptor response element transcriptional intermediary factor Elevated plasma fibrinogen levels have been consistently associated with occlusive vascular disorders, and several investigations have prospectively related fibrinogen to myocardial infarction and stroke outcomes (1Heinrich J. Balleisen L. Schulte H. Assmann G. van de Loo J. Arterioscler. Thromb. 1994; 14: 54-59Crossref PubMed Google Scholar, 2Meade T.W. Mellows S. Brozovic M. Miller G.J. Chakrabarti R.R. North W.R. Haines A.P. Imeson J.D. Thompson S.G. Lancet. 1986; 2: 533-537Abstract PubMed Scopus (1910) Google Scholar, 3Wilhelmsen L. Svardsudd K. Korsan-Bengtsen K. Larsson B. Welin L. Tibblin G. N. Engl. J. Med. 1984; 311: 501-505Crossref PubMed Scopus (1557) Google Scholar). Fibrinogen is synthesized in hepatocytes and secreted into the blood as a dimeric molecule, with each half composed of three nonidentical polypeptides (Aα, Bβ, and γ) linked by disulfide bonds. The three polypeptides are encoded by three distinct genes clustered on the long arm of chromosome 4 (4Kant J.A. Fornace A.J. Saxe D. Simon M.I. McBride O.W. Crabtree G.R. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 2344-2348Crossref PubMed Scopus (244) Google Scholar). The three genes are arranged in the order of γ, α, β, with the gene for the β-chain transcribed in the opposite direction. The three genes contain promoter elements with TATA and CAAT boxes and a number of regulatory elements that confer liver-specific and cytokine-induced expression, including hepatic nuclear factor 1 in the promoter region of Aα and Bβ genes (5Courtois G. Baumhueter S. Crabtree G.R. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 7937-7941Crossref PubMed Scopus (204) Google Scholar, 6Morgan J.G. Courtois G. Fourel G. Chodosh L.A. Campbell L. Evans E. Crabtree G.R. Mol. Cell. Biol. 1988; 8: 2628-2637Crossref PubMed Scopus (38) Google Scholar) and interleukin 6 (IL-6)1 responsive elements 5′ to all three genes (7Anderson G.M. Shaw A.R. Shafer A. J. Biol. Chem. 1993; 268: 22650-22655Abstract Full Text PDF PubMed Google Scholar, 8Dalmon J. Laurent M. Courtois G. Mol. Cell. Biol. 1993; 13: 1183-1193Crossref PubMed Google Scholar, 9Hu C.H. Harris J.E. Davie E.W. Chung D.W. J. Biol. Chem. 1995; 270: 28342-28349Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 10Liu Z. Fuller G.M. J. Biol. Chem. 1995; 270: 7580-7586Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 11Mizuguchi J. Hu C.H. Cao Z. Loeb K.R. Chung D.W. Davie E.W. J. Biol. Chem. 1995; 270: 28350-28356Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 12Zhang Z. Fuentes N.L. Fuller G.M. J. Biol. Chem. 1995; 270: 24287-24291Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Induction of fibrinogen biosynthesis in response to trauma and inflammation is mainly mediated by IL-6 and occurs at the transcriptional level. In humans, fibrinogen-β-chain synthesis is considered to be the rate-limiting chain for assembly and secretion of mature fibrinogen (13Roy S.N. Mukhopadhyay G. Redman C.M. J. Biol. Chem. 1990; 265: 6389-6393Abstract Full Text PDF PubMed Google Scholar, 14Yu S. Sher B. Kudryk B. Redman C.M. J. Biol. Chem. 1983; 258: 13407-13410Abstract Full Text PDF PubMed Google Scholar). IL-6 induction of human fibrinogen-β transcription involves two juxtaposed specific elements (7Anderson G.M. Shaw A.R. Shafer A. J. Biol. Chem. 1993; 268: 22650-22655Abstract Full Text PDF PubMed Google Scholar, 8Dalmon J. Laurent M. Courtois G. Mol. Cell. Biol. 1993; 13: 1183-1193Crossref PubMed Google Scholar, 15Huber P. Laurent M. Dalmon J. J. Biol. Chem. 1990; 265: 5695-5701Abstract Full Text PDF PubMed Google Scholar). The first element is an IL-6 response element, and the second is a binding site for the CCAAT box/enhancer-binding protein (C/EBP) family of transcription factors. These two distinct elements are both required for maximal induction by IL-6. Among drugs affecting plasma fibrinogen levels, fibric acid derivatives are reported as negative regulators of fibrinogen (16Sirtori C.R. Colli S. Cardiovasc. Drugs Ther. 1993; 7: 823Crossref Scopus (21) Google Scholar). The rationale behind the use of fibrates in reducing cardiovascular events is based on their ability to attenuate hypertriglyceridemia and hypercholesterolemia, both of which are established risk factors for cardiovascular diseases (17Hokanson J.E. Austin M.A. J. Cardiovasc. Risk. 1996; 3: 213-219Crossref PubMed Google Scholar, 18AnonymousCirculation. 1994; 89: 1329-1345Google Scholar). Fibrates exert their effects on lipid and lipoprotein metabolism via activation of the nuclear receptor peroxisome proliferator-activated receptor α (PPARα) (19Gervois P. Pineda-Torra I. Fruchart J.-C. Staels B. Clin. Chem. Lab. Med. 2000; 38: 3-11Crossref PubMed Scopus (215) Google Scholar). We have previously demonstrated the involvement of PPARα as a mediator of the negative regulation of fibrinogen gene expression by fibrates (20Kockx M. Gervois P. Poulain P. Derudas B. Peters J.M. Gonzalez F.J. Princen H.M.G. Kooistra T. Staels B. Blood. 1999; 93: 2991-2998Crossref PubMed Google Scholar), but the exact molecular mechanism remained unresolved. PPARα belongs to the PPAR subfamily of nuclear receptors that activate gene expression in response to ligands following dimerization with the retinoid X receptor. PPAR/retinoid X receptor heterodimers bind to specific sequences localized in the promoter region of target genes termed peroxisome proliferator response elements. Several lines of evidence suggest that in addition to their hypolipidemic effect (21Staels B. Dallongeville J. Auwerx J. Schoonjans K. Leitersdorf E. Fruchart J.-C. Circulation. 1998; 98: 2088-2093Crossref PubMed Scopus (1426) Google Scholar), fibrates may exert direct anti-atherogenic activity through an anti-inflammatory activity at the level of the vascular wall. For instance, several clinical studies, such as BECAIT and LOCAT, revealed that fibrate treatment causes a slower progression of coronary atherosclerosis that is independent of any significant lowering of atherogenic lipoprotein concentrations (22Frick M.H. Syvanne M. Nieminen M.S. Kauma H. Majahalme S. Virtanen V. Kesaniemi Y.A. Pasternack A. Taskinen M.R. Circulation. 1997; 96: 2137-2143Crossref PubMed Scopus (355) Google Scholar,23Ruotolo G. Ericsson C.G. Tettamanti C. Karpe F. Grip L. Svane B. Nilsson J. de Faire U. Hamsten A. J. Am. Coll. Cardiol. 1998; 32: 1648-1656Crossref PubMed Scopus (157) Google Scholar). Furthermore, it has been reported that fibrates decrease plasma concentrations of inflammatory cytokines, such as tumor necrosis factor α and IL-6, in patients with angiographically established atherosclerosis (24Madej A. Okopien B. Kowalski J. Zielinski M. Wysocki J. Szygula B. Kalina Z. Herman Z.S. Int. J. Clin. Pharmacol. Ther. 1998; 36: 345-349PubMed Google Scholar, 25Staels B. Koenig W. Habib A. Merval R. Lebret M. Pineda Torra I. Delerive P. Fadel A. Chinetti G. Fruchart J.C. Najib J. Maclouf J. Tedgui A. Nature. 1998; 393: 790-793Crossref PubMed Scopus (1060) Google Scholar). Interestingly, PPARα has been demonstrated to act as a negative regulator of the vascular inflammatory gene response by antagonizing the activity of the transcription factors NF-κB and AP-1 (26Delerive P. De Bosscher K. Besnard S. Vanden Berghe W. Peters J.M. Gonzalez F.J. Fruchart J.-C. Tedgui A. Haegeman G. Staels B. J. Biol. Chem. 1999; 274: 32048-32054Abstract Full Text Full Text PDF PubMed Scopus (987) Google Scholar). In line with these findings, PPARα knockout mice exhibit a prolonged inflammatory response compared with wild type mice (27Devchand P.R. Keller H. Peters J.M. Vazquez M. Gonzalez F.J. Wahli W. Nature. 1996; 384: 39-43Crossref PubMed Scopus (1214) Google Scholar). In the present work, we delineated the molecular mechanism of fibrinogen gene regulation in more detail, and we extended our previous observations in rodents to the human situation. We demonstrate that the nuclear receptor PPARα is also crucial for the negative regulation of the human fibrinogen-β gene expression by PPARα agonists and that this occurs under both basal and inflammatory conditions. Evidence is provided that the suppressive effect of PPARα requires the integrity of the C/EBP response element and is independent of the IL-6 response element. PPARα does not interact directly with C/EBP. Instead, we found that transcriptional intermediary factor glucocorticoid receptor-interacting protein 1/transcriptional intermediary factor 2 (GRIP1/TIF2) is a novel positive regulator of fibrinogen-β transcription and that the sequestration of GRIP1/TIF2 by PPARα constitutes a molecular mechanism by which negative regulation of fibrinogen-β by PPARα agonists takes place. Finally, we observed that the PPARα inhibitory effect may be extended to acute phase response genes other than the fibrinogen-β gene. Fenofibric acid was a kind gift of Dr. A. Edgar (Laboratoires Fournier, Daix, France); ciprofibrate and bezafibrate were from Sanofi-Synthelabo (Aramon, France) and Roche Molecular Biochemicals, respectively. Wy 14,643 was from Chemsyn (Lenexa, KS). Human recombinant IL-6 was purchased from Tebu (Le Perray-en-Yvelines, France). Dexamethasone was from Sigma. Human hepatocytes, isolated by collagenase perfusion, and HepG2 cells, obtained from the European Collection of Animal Cell Culture (Porton Down, Salisbury, United Kingdom) were cultured exactly as described previously (28Staels B. Vu-Dac N. Kosykh V.A. Saladin R. Fruchart J.-C. Dallongeville J. Auwerx J. J. Clin. Invest. 1995; 95: 705-712Crossref PubMed Google Scholar). Fibrinogen concentrations in conditioned medium were measured by an enzyme-linked immunosorbent assay procedure as previously described (20Kockx M. Gervois P. Poulain P. Derudas B. Peters J.M. Gonzalez F.J. Princen H.M.G. Kooistra T. Staels B. Blood. 1999; 93: 2991-2998Crossref PubMed Google Scholar). Total RNA extraction and Northern blot analysis were performed as described (29Staels B. Auwerx J. Development. 1992; 115: 1035-1043PubMed Google Scholar) using a 1930-base pairEcoRI/PstI fragment of the human fibrinogen-β and human acidic ribosomal phosphoprotein 36B4 (30Masiakowski P. Breathnach R. Bloch J. Gannon F. Krust A. Chambon P. Nucleic Acids Res. 1982; 10: 7895-7903Crossref PubMed Scopus (702) Google Scholar) cDNA probes. Probes for human fibrinogen-α, fibrinogen-γ, and serum amyloid A were generated by reverse transcription-polymerase chain reaction. pSG5-hPPARα and pSG5-hPPARαΔLBD were described previously (31Gervois P. Pineda Torra I. Chinetti G. Grotzinger T. Dubois G. Fruchart J.-C. Fruchart-Najib J. Leitersdorf E. Staels B. Mol. Endocrinol. 1999; 13: 1535-1549PubMed Google Scholar). phFib-β was generated by amplification and cloning of a 400-base pair genomic fragment corresponding to nucleotides –400 to +13 of the human fibrinogen-β promoter pGL3 reporter vector. Specific mutations in either the IL-6 or C/EBPβ response elements previously described (8Dalmon J. Laurent M. Courtois G. Mol. Cell. Biol. 1993; 13: 1183-1193Crossref PubMed Google Scholar) were generated by site-directed mutagenesis using the quick mutagenesis kit (Stratagene) and phFib-β as template, giving rise to phFib-β M1 and phFib-β M3 plasmids, respectively. HepG2 cells were transiently transfected using the calcium phosphate precipitation method with reporter and expression plasmids, as stated in the figure legends. The total amount of DNA was kept constant by complementation with corresponding empty vector mock DNA. After a 4-h incubation period, cells were washed with phosphate-buffered saline (PBS) and refed with Dulbecco's modified Eagle's medium supplemented with 0.2% fetal calf serum and Wy 14,643 or vehicle and IL-6 as indicated in the figure legends. Cells were harvested after 24-h incubation and collected for the determination of the luciferase activity performed using a luciferase assay system (Promega Corp., Madison, WI). HepG2 cells were washed twice with ice-cold PBS, scraped off in 1 ml of ice-cold PBS, and collected by centrifugation for 5 min at 500 × g at 4 °C. The pellet was resuspended in 100 μl of ice-cold lysis buffer (1% Nonidet P-40, 0.5% sodium desoxycholate, 0.1% SDS in PBS), protease inhibitors were freshly added (5 μg/ml leupeptin, 5 μg/ml pepstatin, 5 mg/ml EDTA-Na2, 1 mmbenzamidine, 5 μg/ml aprotinin, and 0.5 mmphenylmethylsulfonyl fluoride), and the suspension was vigorously vortexed. The cell extract was centrifuged (5 min at 10,000 ×g and 4 °C), and the supernatant was transferred to new tubes, aliquoted, and stored at −80 °C. Electrophoresis of samples was performed on 10% SDS-polyacrylamide gels (Minigel system, Bio-Rad) under reducing conditions (10 mm dithiothreitol). Proteins were blotted onto nitrocellulose membrane. Nonspecific binding sites were blocked with 10% skim milk powder diluted in TNT buffer (20 mm Tris, 55 mm NaCl, 0.1% Tween), overnight at 4 °C. The membrane was probed with primary antibody diluted in 5% skim milk-TNT for 4 h at room temperature. Membrane was washed and incubated with peroxidase-conjugated anti-rabbit antibody, followed by a subsequent six washes of 10 min. The bands were visualized using SuperSignal® West Dura substrate. 0.5 μg of GST-GRIP1/TIF2(536–1121) bound to glutathione-Sepharose 4B beads was incubated with 5 μl of in vitro synthesized [35S]methionine-labeled protein in the presence or absence of 100 μm Wy-14643 (dissolved in Me2SO) in a total volume of 200 μl of incubation buffer (20 mm Hepes, pH 7.8, 100 mm KCl, 10 mm MgCl2, 10% glycerol, 0.1% Nonidet P-40, 0.1% Triton X-100, 0.1% bovine serum albumin, 1 mmdithiothreitol, 1 μg/ml of each aprotinin, leupeptin, and pepstatin) and gently rotated at 4 °C. After centrifugation, the beads were washed four times for 15 min with incubation buffer without bovine serum albumin, resuspended in 30 μl of 1× Laemmli buffer, boiled for 5 min, and centrifuged. The supernatant was loaded on a SDS-polyacrylamide gel electrophoresis gel. After drying the gel, input and bound proteins were analyzed with a phosphorimager apparatus equipped with ImageQuant software. For binding of endogenous hPPARα to GRIP1/TIF2 a freshly isolated piece of human liver of about 1 g was homogenized in ice-cold PBS containing proteinase inhibitor mixture (Roche Molecular Biochemicals) and stored at −80 °C. Thawed homogenates were centrifuged at 10,000 × g for 10 min to recover soluble proteins. Samples were diluted 10-fold in PBS/protease inhibitors and rotated at 4 °C for 6 h in the presence of 4 μg/ml primary rabbit anti-hPPARα antibody (Santa Cruz Biotechnology) or rabbit preimmune serum, respectively. Complexes were immunoprecipitated by antibody/protein A-Sepharose (Amersham Pharmacia Biotech) at 4 °C for 2 h and washed once in PBS/protease inhibitors and three times in protease inhibitor-containing 50 mm Tris-HCl, pH 8.0, 170 mm NaCl, 0.5% Nonidet P-40, 50 mm NaF. Co-immunoprecipitated proteins were analyzed by immunoblotting. The regulation of fibrinogen biosynthesis is mainly transcriptional and is stimulated by IL-6. Fibrinogen-β is considered the rate-limiting chain for fibrinogen biosynthesis. Therefore, we studied the effect of fibrates on the regulation of human fibrinogen-β expression in primary hepatocytes under basal and IL-6-induced conditions. In the absence of IL-6, basal fibrinogen mRNA levels were decreased by treatment with fenofibric acid, whereas control 36B4 mRNA was unaffected (Fig.1). The addition of IL-6 led to enhanced expression of fibrinogen-β mRNA. When cells were treated with both IL-6 and fenofibric acid, fibrinogen expression was strongly lowered. In order to check whether this inhibitory effect is not restricted to fenofibric acid, fibrinogen-β expression was analyzed in HepG2 cells treated with various other fibrates for 24 h. Treatment with each fibrate resulted in a down-regulation of fibrinogen-β expression in both the presence and the absence of IL-6 (Fig.2 a). The lowering effect also occurred at the protein level because secretion of fibrinogen was diminished by fibrate treatment; it also occurred in the presence of IL-6 (Fig. 2 b). Treatment of HepG2 cells with increasing concentrations of Wy 14,643 resulted in a dose-dependent inhibition of basal and IL-6-induced fibrinogen-β mRNA levels (Fig. 3). These experiments demonstrate that PPARα agonists suppress human fibrinogen-β mRNA levels and fibrinogen secretion under both basal and IL-6-stimulated conditions.Figure 3Dose-dependent effect of Wy 14,643 on fibrinogen expression in HepG2 cells. Cells were treated for 3 h with increasing concentrations of Wy 14,643 (3, 10, 30, and 100 μm) and stimulated for 21 h or not with IL-6 (25 ng/ml) as indicated. Total RNA (10 μg) was subjected to Northern blot analysis using human fibrinogen-β (top panel) or 36B4 (bottom panel) cDNA probes. DMSO,Me2SO.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To elucidate whether fibrates can suppress the expression of the fibrinogen-β-chain gene at the transcriptional level, a 400-base pair promoter fragment of the human fibrinogen-β gene, which contains the essential regulatory elements for basal and inducible promoter activity (8Dalmon J. Laurent M. Courtois G. Mol. Cell. Biol. 1993; 13: 1183-1193Crossref PubMed Google Scholar), was cloned in front of a luciferase reporter gene giving rise to phFib-β. This reporter construct was transiently transfected into HepG2 cells that were subsequently treated with different fibrates in the presence or absence of IL-6. As shown in Fig.4 a, basal promoter activity decreased when cells were treated with fibrates and was enhanced 6-fold when cells were incubated with IL-6. Furthermore, prior treatment of the cells with fibrates resulted in a consistent inhibition of fibrinogen-β transcription induced by IL-6 (Fig. 4 a). Co-transfection of PPARα reduced fibrinogen-β promoter activity in both control and IL-6-treated cells, an effect that was further enhanced by the presence of Wy 14,643 (Fig. 4 b). These results indicate that the repressive effect of fibrates on human fibrinogen-β expression occurs at the transcriptional level through activation of PPARα. IL-6 induction of fibrinogen-β promoter is mediated by two distinct cis-acting elements (7Anderson G.M. Shaw A.R. Shafer A. J. Biol. Chem. 1993; 268: 22650-22655Abstract Full Text PDF PubMed Google Scholar, 8Dalmon J. Laurent M. Courtois G. Mol. Cell. Biol. 1993; 13: 1183-1193Crossref PubMed Google Scholar). One of these elements is an IL-6 response element (RE)-like site, and the other is a consensus binding site for members of the C/EBP family of transcription factors. To delineate whether one of these elements is involved in PPARα-mediated repression of fibrinogen-β gene transcription, we performed transient transfection experiments using the 400-base pair fibrinogen promoter reporter constructs carrying mutations in either the C/EBPβ or IL-6 response elements (Fig. 5). As described above, phFib-β activity was repressed by activated PPARα in both the presence and absence of IL-6. Mutation of the IL-6 RE core site in the fibrinogen-β promoter construct (phFib-β M1) led to a decreased basal transcriptional activity and to a loss of IL-6 responsiveness. Furthermore, its activity was unaffected by activated PPARα. Mutation in the C/EBP binding site of fibrinogen-β promoter (phFib-β M3) resulted in a weaker basal transcriptional activity and in a diminished IL-6 inducibility. Interestingly, activated PPARα did not influence transcriptional activity of phFib-β M3 in either the absence or presence of IL-6. These results point to a crucial role of the IL6-RE in both basal and IL-6-induced fibrinogen-β promoter activity, whereas the C/EBP binding site alone does not respond to IL-6 but rather controls the overall transcriptional activity. Furthermore, PPARα does not interfere directly with the IL-6 pathway but diminishes the overall activity of fibrinogen-β promoter by antagonizing C/EBPβ-mediated activation of fibrinogen-β gene transcription. To determine whether PPARα could directly interfere with the C/EBPβ-mediated activation of fibrinogen-β transcription, we analyzed the effect of PPARα on C/EBPβ-induced fibrinogen-β promoter activity. As described above, overexpression of PPARα decreases basal and IL-6-induced activity of fibrinogen-β promoter, an effect that was enhanced in the presence of Wy 14,643 (Fig.6). Transfection of C/EBPβ resulted in a 3-fold induction of basal fibrinogen-β promoter activity, and luciferase activity was further increased upon addition of IL-6. In the absence of IL-6, co-transfection of a constant amount of C/EBPβ and increasing amounts of PPARα led to a dose-dependent inhibition of fibrinogen-β transactivation, an effect that was amplified by the presence of Wy 14,643 (Fig. 6). Interestingly, PPARα also repressed in a dose-dependent manner fibrinogen-β transcriptional activity induced by C/EBPβ in the presence of IL-6 stimulation. Again, this action of PPARα was much more pronounced in the presence of Wy 14,643. Taken together, these results demonstrate that PPARα counteracts C/EBPβ-induced activation of the fibrinogen-β promoter. Next, we addressed the question whether PPARα inhibits C/EBP induction of fibrinogen-β promoter activity through direct protein-protein interaction or by competition for a common co-factor. Because GST fusion protein pull-down assays and yeast two hybrid analysis failed to detect a direct interaction between PPARγ and C/EBPα as reported by Hollenberg et al.(36Hollenberg A.N. Susulic V.S. Madura J.P. Zhang B. Moller D.E. Tontonoz P. Sarraf P. Spiegelman B.M. Lowel B.B. J. Biol. Chem. 1997; 272: 5283-5290Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar), it is unlikely that PPARα and C/EBPβ directly interact. Therefore, we hypothesized that interference with coactivator might be a mechanism of gene repression by PPARα. Interestingly, a member of the TIF family has been previously identified as a co-factor interacting with both C/EBP and glucocorticoid receptor to regulate expression of the α1-acid glycoprotein gene, another acute phase protein (32Chang C.J. Chen Y.L. Lee S.C. Mol. Cell. Biol. 1998; 18: 5880-5887Crossref PubMed Google Scholar). In addition, GRIP1/TIF2 has been described as a transcriptional mediator for the ligand-dependent activation function of nuclear receptors (33Voegel J.J. Heine M.J. Zechel C. Chambon P. Gronemeyer H. EMBO J. 1996; 15: 3667-3675Crossref PubMed Scopus (953) Google Scholar). We first analyzed whether fibrinogen-β transcriptional activity could be affected by GRIP1/TIF2. Transfection of GRIP1/TIF2 in HepG2 cells increased fibrinogen-β reporter activity and enhanced stimulation of fibrinogen transcription in the presence of IL-6 (Fig.7 A). GRIP1/TIF2 had no effect on fibrinogen-β promoter mutated in its C/EBP binding site (phFib-β M3), showing that C/EBP binding site integrity is required. To investigate whether GRIP1/TIF2 plays a role in PPARα-mediated repression of fibrinogen transcription, GRIP1/TIF2 and PPARα expression were co-transfected together with the fibrinogen-β reporter vector. Wy 14,643 treatment failed to repress transcription when GRIP1/TIF2 was overexpressed (Fig. 7 B). In addition, co-transfection of increasing amounts of GRIP1/TIF2 expression vector with a constant amount of PPARα expression vector led to the abolishment of PPARα inhibitory effect on fibrinogen-β transactivation; this also occurred in the presence of PPARα ligand. Transfected cell extract subjected to electrophoresis demonstrated that the abolishment of PPARα action by GRIP1/TIF2 was not linked to an indirect effect on PPARα expression vector (Fig.7 C). These data highlight that GRIP1/TIF2 potentiates C/EBP-mediated fibrinogen-β transcription and strongly suggest that PPARα exerts its repressive effect through titration of this co-factor. To evaluate whether sequestration of GRIP1/TIF2 by PPARα also occurs under physiological conditions, association between endogenous proteins expressed at physiological levels in regular human hepatocytes was assessed by co-immunoprecipitation. Endogenous PPARα-GRIP1/TIF2 complexes were precipitated from human liver protein extracts (Fig.8 a). Specific co-immunoprecipitation of GRIP1/TIF2 was detected by anti-GRIP1/TIF2 Western blot when anti-PPARα but not control antibody was used for precipitation (Fig. 8 a). We also performed in vitro experiments to investigate whether protein-protein interaction between PPARα and GRIP1/TIF2 is ligand-dependent. GST pull-down experiments revealed that interaction between GRIP1/TIF2 and PPARα is enhanced in the presence of PPARα ligand (Fig. 8 b), which is in agreement with the transfection results. Because PPARα ligands can potentiate the interaction between PPARα and GRIP1/TIF2, we analyzed whether PPARα ligand binding domain (LBD) is required for the transcriptional repression of fibrinogen-β promoter activity by PPARα. We therefore compared the activity of wild type PPARα with that of the recently identified PPARα truncated isoform (31Gervois P. Pineda Torra I. Chinetti G. Grotzinger T. Dubois G. Fruchart J.-C. Fruchart-Najib J. Leitersdorf E. Staels B. Mol. Endocrinol. 1999; 13: 1535-1549PubMed Google Scholar) lacking the entire LBD (PPARα-ΔLBD) on fibrinogen-β transcription. Transcriptional activity of fibrinogen-β was not affected by co-transfection of PPARα-ΔLBD, in contrast to PPARα wild type (data not shown). Altogether, these experiments demonstrate that PPARα negatively regulates fibrinogen-β through sequ" @default.
• W2086342197 created "2016-06-24" @default.
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• W2086342197 date "2001-09-01" @default.
• W2086342197 modified "2023-09-29" @default.
• W2086342197 title "Negative Regulation of Human Fibrinogen Gene Expression by Peroxisome Proliferator-activated Receptor α Agonists via Inhibition of CCAAT Box/Enhancer-binding Protein β" @default.
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