JBC PeproTech; Our Business is Cytokines!

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M102839200 on June 20, 2001

J. Biol. Chem., Vol. 276, Issue 36, 33471-33477, September 7, 2001
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
276/36/33471    most recent
M102839200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Gervois, P.
Right arrow Articles by Staels, B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Gervois, P.
Right arrow Articles by Staels, B.

Negative Regulation of Human Fibrinogen Gene Expression by Peroxisome Proliferator-activated Receptor alpha  Agonists via Inhibition of CCAAT Box/Enhancer-binding Protein beta *

Philippe GervoisDagger §**, Ngoc Vu-DacDagger , Robert Kleemann§, Maaike Kockx§, Guillaume DuboisDagger , Bernard Laine, Vladimir Kosykh||, Jean-Charles FruchartDagger , Teake Kooistra§, and Bart StaelsDagger **

From the Dagger  Département d'Athérosclerose, U.545 INSERM, Institut Pasteur de Lille and Faculté de Pharmacie, Université de Lille II, 59019 Lille, France, the § Gaubius Laboratory, TNO-Prevention and Health, P. O. Box 2215, 2301 CE Leiden, The Netherlands,  U.459 INSERM, Laboratoire de Biologie Cellulaire, Faculté de Médecine H. Warembourg, 59045 Lille Cédex, France, and the || Cardiology Research Complex, 721552 Moscow, Russia

Received for publication, March 30, 2001, and in revised form, June 11, 2001

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha  (PPARalpha ). 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-beta gene in human primary hepatocytes and hepatoma HepG2 cells. Fibrates diminish basal and IL-6-induced fibrinogen-beta promoter activity, and this effect is enhanced in the presence of co-transfected PPARalpha . Site-directed mutagenesis experiments demonstrate that PPARalpha activators decrease human fibrinogen-beta 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-beta gene transcription and alleviates the repressive effect of PPARalpha . Co-immunoprecipitation experiments demonstrate that PPARalpha and GRIP1/TIF2 physically interact in vivo in human liver. These data demonstrate that PPARalpha agonists repress human fibrinogen gene expression by interference with the C/EBPbeta pathway through titration of the coactivator GRIP1/TIF2. We observed that the anti-inflammatory action of PPARalpha 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 PPARalpha and reveal the direct implication of PPARalpha in the modulation of the inflammatory gene response in the liver.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 (1-3). Fibrinogen is synthesized in hepatocytes and secreted into the blood as a dimeric molecule, with each half composed of three nonidentical polypeptides (Aalpha , Bbeta , and gamma ) linked by disulfide bonds. The three polypeptides are encoded by three distinct genes clustered on the long arm of chromosome 4 (4). The three genes are arranged in the order of gamma , alpha , beta , with the gene for the beta -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 Aalpha and Bbeta genes (5, 6) and interleukin 6 (IL-6)1 responsive elements 5' to all three genes (7-12). 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-beta -chain synthesis is considered to be the rate-limiting chain for assembly and secretion of mature fibrinogen (13, 14). IL-6 induction of human fibrinogen-beta transcription involves two juxtaposed specific elements (7, 8, 15). 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 (16). 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 (17, 18). Fibrates exert their effects on lipid and lipoprotein metabolism via activation of the nuclear receptor peroxisome proliferator-activated receptor alpha  (PPARalpha ) (19). We have previously demonstrated the involvement of PPARalpha as a mediator of the negative regulation of fibrinogen gene expression by fibrates (20), but the exact molecular mechanism remained unresolved.

PPARalpha 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 (21), 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 (22, 23). Furthermore, it has been reported that fibrates decrease plasma concentrations of inflammatory cytokines, such as tumor necrosis factor alpha  and IL-6, in patients with angiographically established atherosclerosis (24, 25). Interestingly, PPARalpha has been demonstrated to act as a negative regulator of the vascular inflammatory gene response by antagonizing the activity of the transcription factors NF-kappa B and AP-1 (26). In line with these findings, PPARalpha knockout mice exhibit a prolonged inflammatory response compared with wild type mice (27).

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 PPARalpha is also crucial for the negative regulation of the human fibrinogen-beta gene expression by PPARalpha agonists and that this occurs under both basal and inflammatory conditions. Evidence is provided that the suppressive effect of PPARalpha requires the integrity of the C/EBP response element and is independent of the IL-6 response element. PPARalpha 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-beta transcription and that the sequestration of GRIP1/TIF2 by PPARalpha constitutes a molecular mechanism by which negative regulation of fibrinogen-beta by PPARalpha agonists takes place. Finally, we observed that the PPARalpha inhibitory effect may be extended to acute phase response genes other than the fibrinogen-beta gene.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Reagents-- 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.

Cell Culture-- 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 (28).

Fibrinogen Measurement-- Fibrinogen concentrations in conditioned medium were measured by an enzyme-linked immunosorbent assay procedure as previously described (20).

RNA Extraction-- Total RNA extraction and Northern blot analysis were performed as described (29) using a 1930-base pair EcoRI/PstI fragment of the human fibrinogen-beta and human acidic ribosomal phosphoprotein 36B4 (30) cDNA probes. Probes for human fibrinogen-alpha , fibrinogen-gamma , and serum amyloid A were generated by reverse transcription-polymerase chain reaction.

Plasmids-- pSG5-hPPARalpha and pSG5-hPPARalpha Delta LBD were described previously (31). phFib-beta was generated by amplification and cloning of a 400-base pair genomic fragment corresponding to nucleotides -400 to +13 of the human fibrinogen-beta promoter pGL3 reporter vector. Specific mutations in either the IL-6 or C/EBPbeta response elements previously described (8) were generated by site-directed mutagenesis using the quick mutagenesis kit (Stratagene) and phFib-beta as template, giving rise to phFib-beta M1 and phFib-beta M3 plasmids, respectively.

Transfections-- 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).

Cell Extracts-- 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 mM benzamidine, 5 µg/ml aprotinin, and 0.5 mM phenylmethylsulfonyl 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.

Western Blotting-- 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.

In Vitro Protein-Protein Interaction Assay (GST Pull-down)-- 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 mM dithiothreitol, 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.

In Vivo Protein-Protein Interaction Assay (Co-immunoprecipitation)-- For binding of endogenous hPPARalpha 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-hPPARalpha 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.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Fibrates Down-regulate Fibrinogen-beta Expression and Reduce Fibrinogen Secretion in Human Hepatocytes-- The regulation of fibrinogen biosynthesis is mainly transcriptional and is stimulated by IL-6. Fibrinogen-beta is considered the rate-limiting chain for fibrinogen biosynthesis. Therefore, we studied the effect of fibrates on the regulation of human fibrinogen-beta 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-beta mRNA. When cells were treated with both IL-6 and fenofibric acid, fibrinogen expression was strongly lowered.


View larger version (42K):
[in this window]
[in a new window]
  		Fig. 1.   Effect of fenofibric acid on fibrinogen-beta expression in human primary hepatocytes under basal and IL-6-stimulated conditions. Human hepatocytes were isolated and treated for 3 h with 100 µM fenofibric acid or vehicle (DMSO, dimethyl sulfoxide) and subsequently stimulated with IL-6 for 21 h (25 ng/ml) as indicated. Total RNA (10 µg) was subjected to Northern blot analysis using human fibrinogen-beta (Fib-beta ) (top panel) or 36B4 (bottom panel) cDNA probes.

In order to check whether this inhibitory effect is not restricted to fenofibric acid, fibrinogen-beta 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-beta expression in both the presence and the absence of IL-6 (Fig. 2a). 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. 2b). Treatment of HepG2 cells with increasing concentrations of Wy 14,643 resulted in a dose-dependent inhibition of basal and IL-6-induced fibrinogen-beta mRNA levels (Fig. 3). These experiments demonstrate that PPARalpha agonists suppress human fibrinogen-beta mRNA levels and fibrinogen secretion under both basal and IL-6-stimulated conditions.


View larger version (24K):
[in this window]
[in a new window]
  		Fig. 2.   Effect of fibrates on fibrinogen-beta expression under basal and IL-6-stimulated conditions in HepG2 cells. Cells were treated for 3 h with fenofibric acid (250 µM), ciprofibrate (250 µM), Wy 14,643 (250 µM), bezafibrate (250 µM), or vehicle (DMSO, Me2SO) and then stimulated for 21 h with IL-6 (25 ng/ml) or vehicle as indicated. a, total RNA (10 µg) was subjected to Northern blot analysis using human fibrinogen-beta (top panel) or 36B4 (bottom panel) cDNA probes. b, fibrinogen concentrations were measured in culture medium by enzyme-linked immunosorbent assay and expressed as mean ± S.D.


View larger version (46K):
[in this window]
[in a new window]
  		Fig. 3.   Dose-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-beta (top panel) or 36B4 (bottom panel) cDNA probes. DMSO, Me2SO.

Fibrinogen-beta Repression by Fibrates Occurs at the Transcriptional Level through Activation of PPARalpha -- To elucidate whether fibrates can suppress the expression of the fibrinogen-beta -chain gene at the transcriptional level, a 400-base pair promoter fragment of the human fibrinogen-beta gene, which contains the essential regulatory elements for basal and inducible promoter activity (8), was cloned in front of a luciferase reporter gene giving rise to phFib-beta . 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. 4a, 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-beta transcription induced by IL-6 (Fig. 4a). Co-transfection of PPARalpha reduced fibrinogen-beta promoter activity in both control and IL-6-treated cells, an effect that was further enhanced by the presence of Wy 14,643 (Fig. 4b). These results indicate that the repressive effect of fibrates on human fibrinogen-beta expression occurs at the transcriptional level through activation of PPARalpha .


View larger version (14K):
[in this window]
[in a new window]
  		Fig. 4.   Effect of fibrates on human fibrinogen-beta promoter activity. HepG2 cells were transfected with the human fibrinogen-beta promoter reporter plasmid (1 µg) and stimulated (black columns) or not (white columns) with IL-6 (25 ng/ml) for 24 h. a, cells treated with 10 µM of various PPARalpha agonists or vehicle as indicated. b, cells were co-transfected with phFibbeta and 200 ng of pSG5-hPPARalpha or pSG5 vectors and treated with Wy 14,643 (10 µM) or vehicle for 24 h. Luciferase activities are expressed as mean ± S.D. DMSO, Me2SO.

PPARalpha Functions as a Repressor of C/EBPbeta -mediated Fibrinogen-beta Gene Transactivation-- IL-6 induction of fibrinogen-beta promoter is mediated by two distinct cis-acting elements (7, 8). 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 PPARalpha -mediated repression of fibrinogen-beta gene transcription, we performed transient transfection experiments using the 400-base pair fibrinogen promoter reporter constructs carrying mutations in either the C/EBPbeta or IL-6 response elements (Fig. 5). As described above, phFib-beta activity was repressed by activated PPARalpha in both the presence and absence of IL-6. Mutation of the IL-6 RE core site in the fibrinogen-beta promoter construct (phFib-beta M1) led to a decreased basal transcriptional activity and to a loss of IL-6 responsiveness. Furthermore, its activity was unaffected by activated PPARalpha . Mutation in the C/EBP binding site of fibrinogen-beta promoter (phFib-beta M3) resulted in a weaker basal transcriptional activity and in a diminished IL-6 inducibility. Interestingly, activated PPARalpha did not influence transcriptional activity of phFib-beta 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-beta promoter activity, whereas the C/EBP binding site alone does not respond to IL-6 but rather controls the overall transcriptional activity. Furthermore, PPARalpha does not interfere directly with the IL-6 pathway but diminishes the overall activity of fibrinogen-beta promoter by antagonizing C/EBPbeta -mediated activation of fibrinogen-beta gene transcription.


View larger version (16K):
[in this window]
[in a new window]
  		Fig. 5.   Mapping of the PPARalpha response element in the human fibrinogen-beta promoter. HepG2 cells were transfected with 1 µg of either human fibrinogen-beta promoter reporter construct phFib-beta , phFib-beta -M1, or phFib-beta -M3 reporter constructs as indicated, and subsequently stimulated with (black columns) or without (white columns) IL-6 (25 ng/ml). +, cells co-transfected with pSG5-hPPARalpha and activated with Wy 14,643 (10 µM); -, cells were co-transfected with pSG5 vector and vehicle-treated. Luciferase activities are expressed as mean ± S.D.

To determine whether PPARalpha could directly interfere with the C/EBPbeta -mediated activation of fibrinogen-beta transcription, we analyzed the effect of PPARalpha on C/EBPbeta -induced fibrinogen-beta promoter activity. As described above, overexpression of PPARalpha decreases basal and IL-6-induced activity of fibrinogen-beta promoter, an effect that was enhanced in the presence of Wy 14,643 (Fig. 6). Transfection of C/EBPbeta resulted in a 3-fold induction of basal fibrinogen-beta 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/EBPbeta and increasing amounts of PPARalpha led to a dose-dependent inhibition of fibrinogen-beta transactivation, an effect that was amplified by the presence of Wy 14,643 (Fig. 6). Interestingly, PPARalpha also repressed in a dose-dependent manner fibrinogen-beta transcriptional activity induced by C/EBPbeta in the presence of IL-6 stimulation. Again, this action of PPARalpha was much more pronounced in the presence of Wy 14,643. Taken together, these results demonstrate that PPARalpha counteracts C/EBPbeta -induced activation of the fibrinogen-beta promoter.


View larger version (23K):
[in this window]
[in a new window]
  		Fig. 6.   Inhibition of C/EBPbeta -induced fibrinogen-beta promoter activity by PPARalpha . HepG2 cells were transfected with the human fibrinogen-beta promoter reporter plasmid (1 µg) in the presence of PPARalpha and/or C/EBPbeta expression vectors. Increasing amounts of pSG5-hPPARalpha (0.5×, 1×, and 2×) were added to a constant amount (100 ng) of pC/EBPbeta . Cells were stimulated (black columns) or not (white columns) with IL-6 (25 ng/ml) and treated with 10 µM Wy 14,643 (+) or Me2SO (-). Luciferase activities are expressed as mean ± S.D.

GRIP1/TIF2 Alleviates the Repressive Effect of PPARalpha on Fibrinogen-beta Transcription-- Next, we addressed the question whether PPARalpha inhibits C/EBP induction of fibrinogen-beta 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 PPARgamma and C/EBPalpha as reported by Hollenberg et al. (36), it is unlikely that PPARalpha and C/EBPbeta directly interact. Therefore, we hypothesized that interference with coactivator might be a mechanism of gene repression by PPARalpha . 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 alpha 1-acid glycoprotein gene, another acute phase protein (32). In addition, GRIP1/TIF2 has been described as a transcriptional mediator for the ligand-dependent activation function of nuclear receptors (33). We first analyzed whether fibrinogen-beta transcriptional activity could be affected by GRIP1/TIF2. Transfection of GRIP1/TIF2 in HepG2 cells increased fibrinogen-beta reporter activity and enhanced stimulation of fibrinogen transcription in the presence of IL-6 (Fig. 7A). GRIP1/TIF2 had no effect on fibrinogen-beta promoter mutated in its C/EBP binding site (phFib-beta M3), showing that C/EBP binding site integrity is required. To investigate whether GRIP1/TIF2 plays a role in PPARalpha -mediated repression of fibrinogen transcription, GRIP1/TIF2 and PPARalpha expression were co-transfected together with the fibrinogen-beta reporter vector. Wy 14,643 treatment failed to repress transcription when GRIP1/TIF2 was overexpressed (Fig. 7B). In addition, co-transfection of increasing amounts of GRIP1/TIF2 expression vector with a constant amount of PPARalpha expression vector led to the abolishment of PPARalpha inhibitory effect on fibrinogen-beta transactivation; this also occurred in the presence of PPARalpha ligand. Transfected cell extract subjected to electrophoresis demonstrated that the abolishment of PPARalpha action by GRIP1/TIF2 was not linked to an indirect effect on PPARalpha expression vector (Fig. 7C). These data highlight that GRIP1/TIF2 potentiates C/EBP-mediated fibrinogen-beta transcription and strongly suggest that PPARalpha exerts its repressive effect through titration of this co-factor.


View larger version (37K):
[in this window]
[in a new window]
  		Fig. 7.   GRIP1/TIF2 inhibits the repression of human fibrinogen promoter activity by PPARalpha . A, HepG2 cells were transfected with 1 µg of either human fibrinogen-beta promoter phFib-beta or phFib-beta -M3 reporter constructs as indicated and subsequently stimulated with (black columns) or without (white columns) IL-6 (25 ng/ml). +, cells co-transfected with pSG5-GRIP1/TIF2; -, cells were co-transfected with pSG5 vector. B, HepG2 cells were transfected with the human fibrinogen-beta promoter reporter plasmid (1 µg) in the presence of PPARalpha and/or GRIP1/TIF2 expression vectors. Increasing amounts of pSG5-GRIP1/TIF2 (0.5×, 1×, and 2×) were added to a constant amount (100 ng) of pSG5-hPPARalpha . Cells were treated with 10 µM Wy 14,643 or vehicle (Me2SO). Luciferase activities are expressed as mean ± S.D. C, analysis of PPARalpha protein levels by Western blotting of protein extract from HepG2 cells transfected with a constant amount of PPARalpha and increasing amounts of GRIP1/TIF2 expression vectors.

PPARalpha Physically Interacts with GRIP1/TIF2-- To evaluate whether sequestration of GRIP1/TIF2 by PPARalpha also occurs under physiological conditions, association between endogenous proteins expressed at physiological levels in regular human hepatocytes was assessed by co-immunoprecipitation. Endogenous PPARalpha -GRIP1/TIF2 complexes were precipitated from human liver protein extracts (Fig. 8a). Specific co-immunoprecipitation of GRIP1/TIF2 was detected by anti-GRIP1/TIF2 Western blot when anti-PPARalpha but not control antibody was used for precipitation (Fig. 8a).


View larger version (12K):
[in this window]
[in a new window]
  		Fig. 8.   GRIP1/TIF2 and PPARalpha interact in vitro and in vivo. a, co-immunoprecipitation was performed on protein extract prepared from fresh human liver. Anti-PPARalpha antibody was used to precipitate endogenous PPARalpha and GRIP-1. b, GST pull-down assay using GST-GRIP1/TIF2(536-1121) and in vitro synthesized 35S-labeled PPARalpha in the presence or absence of Wy 14,643 as indicated. The input represents 20% of the amount of PPARalpha protein.

We also performed in vitro experiments to investigate whether protein-protein interaction between PPARalpha and GRIP1/TIF2 is ligand-dependent. GST pull-down experiments revealed that interaction between GRIP1/TIF2 and PPARalpha is enhanced in the presence of PPARalpha ligand (Fig. 8b), which is in agreement with the transfection results. Because PPARalpha ligands can potentiate the interaction between PPARalpha and GRIP1/TIF2, we analyzed whether PPARalpha ligand binding domain (LBD) is required for the transcriptional repression of fibrinogen-beta promoter activity by PPARalpha . We therefore compared the activity of wild type PPARalpha with that of the recently identified PPARalpha truncated isoform (31) lacking the entire LBD (PPARalpha -Delta LBD) on fibrinogen-beta transcription. Transcriptional activity of fibrinogen-beta was not affected by co-transfection of PPARalpha -Delta LBD, in contrast to PPARalpha wild type (data not shown). Altogether, these experiments demonstrate that PPARalpha negatively regulates fibrinogen-beta through sequestration of GRIP1/TIF2 and that functional interference between PPARalpha and GRIP1/TIF2 requires the presence of the LBD of PPARalpha .

PPARalpha Down-regulates Positive Acute Phase Response Genes Other Than Fibrinogen-beta -- To determine a broader regulatory pattern of the PPARalpha regulatory process, we examined the effect of Wy 14,643 on the expression of other acute phase genes, such as serum amyloid A, fibrinogen-alpha , and fibrinogen-gamma , in HepG2 cells. The expression of serum amyloid A and fibrinogen-alpha were decreased following treatment with Wy 14,643 in a dose-dependent manner under both basal and IL-6-stimulated conditions (Fig. 9). By contrast, both basal and IL-6-stimulated expression of fibrinogen-gamma gene (which contains an IL-6 response element but lacks a C/EBP response element) were unaffected by treatment with Wy 14,643. These results demonstrate that the PPARalpha repressive action is not restricted to the isolated case of fibrinogen-beta but also applies to other acute phase genes containing a C/EBP response element.


View larger version (61K):
[in this window]
[in a new window]
  		Fig. 9.   Dose-dependent effect of Wy 14,643 on gene expression of acute phase proteins 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 serum amyloid A (SAA), fibrinogen-alpha (Fbgalpha ), fibrinogen-gamma (Fbggamma ), or 36B4 cDNA probes.

Because glucocorticoids potentiate the action of IL-6 on the regulation of acute phase genes, we sought to determine whether PPARalpha could modulate this effect. HepG2 cells were stimulated by IL-6 and dexamethasone in the presence of Wy 14,643. As shown in Fig. 10, the low basal expression of the genes tested was only slightly affected by dexamethasone treatment. By contrast, the IL-6-stimulated expression of serum amyloid A, fibrinogen-alpha , fibrinogen-beta , and fibrinogen-gamma was enhanced by the presence of dexamethasone. Interestingly, the potentiation of IL-6 action by dexamethasone was counteracted by treatment with Wy 14,643 (Fig. 10, right panel). Altogether, these results indicate that PPARalpha also controls the expression of acute phase genes stimulated by IL-6 in combination with dexamethasone.


View larger version (46K):
[in this window]
[in a new window]
  		Fig. 10.   Effect of Wy 14,643 on gene expression of acute phase proteins induced by dexamethasone and IL-6. Cells were treated for 3 h with Wy 14,643 (100 µM) and incubated for 21 h with or without IL-6 (25 ng/ml) in the presence or absence of dexamethasone (10 µM) as indicated. Total RNA (10 µg) was subjected to Northern blot analysis using human serum amyloid A (SAA), fibrinogen-alpha (Fbgalpha ), fibrinogen-beta (Fbgbeta ), fibrinogen-gamma (Fbggamma ), or 36B4 cDNA probes.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

High circulating plasma fibrinogen levels are associated with an increased risk for myocardial infarction and stroke. Therefore, factors that down-regulate fibrinogen expression may be of importance in the prevention of cardiovascular diseases. In previous studies, we showed that PPARalpha regulates basal levels of plasma fibrinogen and established that PPARalpha mediates the fibrate-induced suppression of fibrinogen expression in rodents (20). We now report that PPARalpha also negatively regulates fibrinogen expression in humans under both basal and IL-6-stimulated conditions. We furthermore elucidate the molecular mechanism of this action. We demonstrated that PPARalpha interferes with the C/EBPbeta but not the IL-6 pathway. Remarkably, we identified GRIP1/TIF2 as a positive factor of fibrinogen-beta gene regulation and found that titration of GRIP1/TIF2 by PPARalpha may explain the negative regulation of fibrinogen-beta expression by fibrates. Finally, we observed that the inhibitory effect of PPARalpha can be extended to other acute phase response genes containing a C/EBP response element.

Because fibrates suppress fibrinogen-beta expression at the transcriptional level through activation of PPARalpha under both basal and IL-6-induced conditions, we sought to identify the cis-regulatory elements involved in PPARalpha action. Functional analysis of the promoter of the human fibrinogen-beta -chain in HepG2 cells revealed the existence of two response elements that are crucial for full induction by IL-6 (7). The first element, present in several promoters of acute phase response genes (34, 35), is the so-called IL-6 RE. Transfection of a fibrinogen-beta promoter construct carrying mutations in the IL-6 RE led to a loss of IL-6 inducibility. The second important response element consists of a C/EBP binding site. As previously reported, we found that C/EBPbeta induces fibrinogen-beta transcriptional activity (7). Site-directed mutagenesis of the C/EBPbeta binding site did not affect IL-6 inducibility but rendered the fibrinogen-beta promoter unresponsive to activated PPARalpha . These observations indicate that PPARalpha does not directly interfere with the IL-6 transduction pathway but rather interferes with the C/EBPbeta activation function of the fibrinogen-beta promoter. Notably, whereas combination of C/EBPbeta expression and IL-6 stimulation conferred full induction of fibrinogen-beta transcription, co-transfection of PPARalpha reduced the overall promoter activity in a dose-dependent manner but was unable to abolish IL-6 inducibility. Functional antagonism between PPAR and C/EBP factors has also been described for the regulation of the leptin promoter (36). In this study, it was observed that PPARgamma down-regulates leptin expression by inhibiting C/EBPalpha -mediated transactivation, although direct interaction between PPAR and C/EBP was not shown.

Our studies on the regulatory mechanisms of fibrinogen expression have focused on the beta -chain gene because of findings from whole animal and cell culture studies indicating that the beta -chain synthesis is rate-limiting compared with alpha - and gamma -chains (14, 37). However, the possibility cannot be excluded that under certain experimental conditions or in different species, regulation of alpha  and gamma  fibrinogen genes are equally important (9). In that respect, it may be significant that also the alpha -fibrinogen gene appears to be regulated by a C/EBP-dependent pathway (7, 8, 10). By contrast, the fact that no C/EBP-like binding site gene has been identified in gamma -fibrinogen might explain why its expression is hardly suppressed by fibrates (20). Interestingly, we observed that fibrinogen-alpha expression is down-regulated by PPARalpha ligand, whereas fibrinogen-gamma expression was unaffected by PPARalpha activation in both the presence and the absence of IL-6, thus corroborating our findings about the requirement of C/EBP binding site in the repressive action of PPARalpha .

Because it has recently been reported that TIF1beta co-operates with C/EBPbeta to induce the expression of alpha 1-acid glycoprotein gene (32), another acute phase response gene, we asked ourselves whether titration of such a coactivator could also be the mechanism by which PPARalpha exerts its repressive effect on fibrinogen-beta transcription. Results from transient transfection experiments revealed that GRIP1/TIF2 enhances fibrinogen-beta transcription and thereby counteracts the inhibitory effect of PPARalpha . Moreover, co-immunoprecipitated PPAR/GRIP complexes were shown to exist in vivo in human liver, and GST-pull down experiments showed that the functional interference PPAR/GRIP1/TIF2 was due to a direct ligand-stimulated physical interaction. We therefore identified a novel positive regulator of fibrinogen-beta transcription and demonstrated that PPARalpha mediates the repressive effect of fibrates on fibrinogen expression through sequestration of the coactivator GRIP1/TIF2.

Our findings that PPARalpha can diminish the functional activity of C/EBP through binding to the coactivator GRIP1/TIF2 and thereby down-regulates fibrinogen expression may be also of relevance for other acute phase response genes (APRGs) because numerous APRGs are regulated by C/EBP transcription factors (38, 39). Indeed, for some of these genes, e.g. alpha 1-glycoprotein and fibrinogen-alpha , it has been shown that PPARalpha activators decrease their mRNA levels. Here we report that PPARalpha inhibitory effect is not restricted to fibrinogen-beta gene but may be extended to serum amyloid A and fibrinogen-alpha under both basal and inflammatory conditions.

Our observation that the stimulatory effect of IL-6 on the expression of acute phase genes is potentiated in the presence of dexamethasone is in agreement with previous reports (38, 40, 41). This potentiating effect of dexamethasone has been ascribed to interaction between C/EBP, glucocorticoid receptor, and TIF in the context of alpha 1-acid glycoprotein gene (32). Our finding that PPARalpha activation also prevents the dexamethasone-potentiated IL-6 effect on several acute phase genes is in accordance with a mechanism in which PPARalpha binds the coactivator GRIP1/TIF2, whether it is complexed with glucocorticoid receptor or not.

Involvement of PPARalpha in various other anti-inflammatory mechanisms suggests that PPARalpha may also have a role in the regulation of APRGs that are not under control of C/EBP-dependent signaling pathways. For example, AP-1 and NF-kappa B sites also participate in the activation of certain APRGs (42, 43). Because PPARalpha can also interfere negatively with AP-1 and NF-kappa B transcription complex (25, 26, 44), it is possible that PPARalpha is also implicated in the negative regulation of APRGs regulated by these transcription factors. This hypothesis is corroborated by the findings that exposure of rodents to peroxisome proliferators leads to the down-regulation of diverse liver-specific genes, including acute phase response genes (45). Involvement of PPARalpha in several cytokine signaling pathways through different mechanisms suggests a general role for PPARalpha in the regulation of acute phase response in the liver. PPARalpha has also been reported to be a negative regulator of vascular gene response by inhibition of inflammatory mediators involved in atherogenesis (25, 26). Indeed, PPARalpha activators lower plasma concentrations of IL-6, tumor necrosis factor alpha , and interferon gamma  in patients with atherosclerosis (24, 25) and suppress cytokine-stimulated IL-6 production in human aortic smooth muscle cells (25), inducible nitric-oxide synthase activity in murine macrophages (46), and vascular cell adhesion molecule-1 expression in endothelial cells (47). Altogether, these data indicate that PPARalpha plays an important role in the control of inflammation.

The positive association between plasma fibrinogen and cardiovascular diseases is dependent on a relatively small variation in fibrinogen concentrations. Small changes in PPARalpha activity may be sufficient to bring about such a small variation in fibrinogen concentrations. First, PPARalpha activity is modulated by the level of expression of PPARalpha , which is reported to vary among individuals (31). Second, PPARalpha activity depends on the quality and the quantity of PPARalpha activators. Existing drugs, such as fibrates, and also certain naturally occurring dietary fatty acids are activators of PPARalpha and may thereby influence fibrinogen expression. These data highlight the necessity to further understand the regulation of fibrinogen synthesis to allow a rational approach to lower fibrinogen plasma levels.

The results of the present study demonstrate that fibrates down-regulate human fibrinogen-beta via negative interference with C/EBPbeta as a result of titration of GRIP1/TIF2 by PPARalpha and that the repressive action of PPARalpha may also be applicable to other acute phase response genes. This novel mode of action of PPARalpha agonists further adds to the anti-inflammatory potential of PPARalpha and is complementary to its beneficial effect on lipid and lipoprotein metabolism. These observations underscore the importance of PPARalpha as an attractive target for therapeutic strategies for preventing cardiovascular diseases.

    ACKNOWLEDGEMENTS

We thank Sebastien Playe and Odile Vidal for excellent technical assistance and Jean Dallongeville for helpful discussions. We are grateful to M. Stallcup for providing the GRIP1/TIF2 plasmid constructs.

    FOOTNOTES

* This work was supported by grants from the Fondation pour la Recherche Médicale and by European Community Marie Curie Fellowship QLK1-CT-1999-51206 (to P. G.), Netherlands Organization for Scientific Research Grant NOW:902-23-181 (to M. K.), and Netherlands Heart Fondation Grant NHS 99.110 (to R. K.).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.

** To whom correspondence may be addressed. Tel.: 33(0)3-20-87-73-88; Fax: 33-(0)3-20-87-73-60; E-mail: Bart.Staels@pasteur-lille.fr or philippe.gervois{at}pasteur-lille.fr.

Published, JBC Papers in Press, June 20, 2001, DOI 10.1074/jbc.M102839200

    ABBREVIATIONS

The abbreviations used are: IL, interleukin; APRG, acute phase response gene; C/EBP, CCAAT box/enhancer-binding protein; GRIP, glucocorticoid receptor-interacting protein; GST, glutathione S-transferase; LBD, ligand binding domain; PBS, phosphate-buffered saline; PPAR, peroxisome proliferator-activated receptor; RE, response element; TIF, transcriptional intermediary factor.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Heinrich, J., Balleisen, L., Schulte, H., Assmann, G., and van de Loo, J. (1994) Arterioscler. Thromb. 14, 54-59
2. Meade, T. W., Mellows, S., Brozovic, M., Miller, G. J., Chakrabarti, R. R., North, W. R., Haines, A. P., Imeson, J. D., and Thompson, S. G. (1986) Lancet 2, 533-537
3. Wilhelmsen, L., Svardsudd, K., Korsan-Bengtsen, K., Larsson, B., Welin, L., and Tibblin, G. (1984) N. Engl. J. Med. 311, 501-505
4. Kant, J. A., Fornace, A. J., Saxe, D., Simon, M. I., McBride, O. W., and Crabtree, G. R. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 2344-2348
5. Courtois, G., Baumhueter, S., and Crabtree, G. R. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 7937-7941
6. Morgan, J. G., Courtois, G., Fourel, G., Chodosh, L. A., Campbell, L., Evans, E., and Crabtree, G. R. (1988) Mol. Cell. Biol. 8, 2628-2637
7. Anderson, G. M., Shaw, A. R., and Shafer, A. (1993) J. Biol. Chem. 268, 22650-22655
8. Dalmon, J., Laurent, M., and Courtois, G. (1993) Mol. Cell. Biol. 13, 1183-1193
9. Hu, C. H., Harris, J. E., Davie, E. W., and Chung, D. W. (1995) J. Biol. Chem. 270, 28342-28349
10. Liu, Z., and Fuller, G. M. (1995) J. Biol. Chem. 270, 7580-7586
11. Mizuguchi, J., Hu, C. H., Cao, Z., Loeb, K. R., Chung, D. W., and Davie, E. W. (1995) J. Biol. Chem. 270, 28350-28356
12. Zhang, Z., Fuentes, N. L., and Fuller, G. M. (1995) J. Biol. Chem. 270, 24287-24291
13. Roy, S. N., Mukhopadhyay, G., and Redman, C. M. (1990) J. Biol. Chem. 265, 6389-6393
14. Yu, S., Sher, B., Kudryk, B., and Redman, C. M. (1983) J. Biol. Chem. 258, 13407-13410
15. Huber, P., Laurent, M., and Dalmon, J. (1990) J. Biol. Chem. 265, 5695-5701
16. Sirtori, C. R., and Colli, S. (1993) Cardiovasc. Drugs Ther. 7, 823
17. Hokanson, J. E., and Austin, M. A. (1996) J. Cardiovasc. Risk 3, 213-219
18. Anonymous. (1994) Circulation 89, 1329-1345
19. Gervois, P., Pineda-Torra, I., Fruchart, J.-C., and Staels, B. (2000) Clin. Chem. Lab. Med. 38, 3-11
20. Kockx, M., Gervois, P., Poulain, P., Derudas, B., Peters, J. M., Gonzalez, F. J., Princen, H. M. G., Kooistra, T., and Staels, B. (1999) Blood 93, 2991-2998
21. Staels, B., Dallongeville, J., Auwerx, J., Schoonjans, K., Leitersdorf, E., and Fruchart, J.-C. (1998) Circulation 98, 2088-2093
22. Frick, M. H., Syvanne, M., Nieminen, M. S., Kauma, H., Majahalme, S., Virtanen, V., Kesaniemi, Y. A., Pasternack, A., and Taskinen, M. R. (1997) Circulation 96, 2137-2143
23. Ruotolo, G., Ericsson, C. G., Tettamanti, C., Karpe, F., Grip, L., Svane, B., Nilsson, J., de Faire, U., and Hamsten, A. (1998) J. Am. Coll. Cardiol. 32, 1648-1656
24. Madej, A., Okopien, B., Kowalski, J., Zielinski, M., Wysocki, J., Szygula, B., Kalina, Z., and Herman, Z. S. (1998) Int. J. Clin. Pharmacol. Ther. 36, 345-349
25. Staels, 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., and Tedgui, A. (1998) Nature 393, 790-793
26. Delerive, P., De Bosscher, K., Besnard, S., Vanden Berghe, W., Peters, J. M., Gonzalez, F. J., Fruchart, J.-C., Tedgui, A., Haegeman, G., and Staels, B. (1999) J. Biol. Chem. 274, 32048-32054
27. Devchand, P. R., Keller, H., Peters, J. M., Vazquez, M., Gonzalez, F. J., and Wahli, W. (1996) Nature 384, 39-43
28. Staels, B., Vu-Dac, N., Kosykh, V. A., Saladin, R., Fruchart, J.-C., Dallongeville, J., and Auwerx, J. (1995) J. Clin. Invest. 95, 705-712
29. Staels, B., and Auwerx, J. (1992) Development 115, 1035-1043
30. Masiakowski, P., Breathnach, R., Bloch, J., Gannon, F., Krust, A., and Chambon, P. (1982) Nucleic Acids Res. 10, 7895-7903
31. Gervois, P., Pineda Torra, I., Chinetti, G., Grotzinger, T., Dubois, G., Fruchart, J.-C., Fruchart-Najib, J., Leitersdorf, E., and Staels, B. (1999) Mol. Endocrinol. 13, 1535-1549
32. Chang, C. J., Chen, Y. L., and Lee, S. C. (1998) Mol. Cell. Biol. 18, 5880-5887
33. Voegel, J. J., Heine, M. J., Zechel, C., Chambon, P., and Gronemeyer, H. (1996) EMBO J. 15, 3667-3675
34. Fowlkes, D. M., Mullis, N. T., Comeau, C. M., and Crabtree, G. R. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 2313-2316
35. Hattori, M., Abraham, L. J., Northemann, W., and Fey, G. H. (1987) Proc. Natl. Acad. Sci. U. S. A. 87, 2364-2368
36. Hollenberg, A. N., Susulic, V. S., Madura, J. P., Zhang, B., Moller, D. E., Tontonoz, P., Sarraf, P., Spiegelman, B. M., and Lowel, B. B. (1997) J. Biol. Chem. 272, 5283-5290
37. Yu, S., Kong, V., Kudryk, B., and Redman, C. (1987) Thromb. Res. 46, 281-293
38. Baumann, H., Jahreis, G. P., Morella, K. K., Won, K. A., Pruitt, S. C., Jones, V. E., and Prowse, K. R. (1991) J. Biol. Chem. 266, 20390-20399
39. Baumann, H., Morella, K. K., Campos, S. P., Cao, Z., and Jahreis, G. P. (1992) J. Biol. Chem. 267, 19744-19751
40. Baumann, H., Firestone, G. L., Burgess, T. L., Gross, K. W., Yamamoto, K. R., and Held, W. A. (1983) J. Biol. Chem. 258, 563-570
41. Baumann, H., Prowse, K. R., Marinkovic, S., Won, K. A., and Jahreis, G. P. (1989) Ann. N. Y. Acad. Sci. 557, 280-295
42. Nonaka, M., and Huang, Z. M. (1990) Mol. Cell. Biol. 10, 6283-6289
43. Ron, D., Brasier, A. R., McGehee, R. J., and Habener, J. F. (1992) J. Clin. Invest. 89, 223-233
44. Ricote, M., Li, A. C., Willson, T. M., Kelly, C. J., and Glass, C. K. (1998) Nature 391, 79-82
45. Corton, J. C., Fan, L. Q., Brown, S., Anderson, S. P., Bocos, C., Cattley, R. C., Mode, A., and Gustafsson, J. A. (1998) Mol. Pharmacol. 54, 463-473
46. Colville-Nash, P. R., Qureshi, S. S., Willis, D., and Willoughby, D. A. (1998) J. Immunol. 161, 978-984
47. Marx, N., Sukhova, G. K., Collins, T., Libby, P., and Plutzky, J. (1999) Circulation 99, 3125-3131

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.


This article has been cited by other articles:


Home page
 Arterioscler. Thromb. Vasc. Bio.Home page
C. J.F. de Wolf, R. M.J. Cupers, R. M. Bertina, and H. L. Vos
Interleukin-6 Induction of Protein S Is Regulated Through Signal Transducer and Activator of Transcription 3
Arterioscler. Thromb. Vasc. Biol., September 1, 2006; 26(9): 2168 - 2174.
[Abstract] [Full Text] [PDF]

Home page
 Ann Rheum DisHome page
J Westra, J Bijzet, B Doornbos-van der Meer, M H van Rijswijk, and P C Limburg
Differential influence of p38 mitogen activated protein kinase (MAPK) inhibition on acute phase protein synthesis in human hepatoma cell lines
Ann Rheum Dis, July 1, 2006; 65(7): 929 - 935.
[Abstract] [Full Text] [PDF]