© 1997 American Heart Association, Inc.
Articles |
Studies on the Mechanism of Fibrate-Inhibited Expression of Plasminogen Activator Inhibitor-1 in Cultured Hepatocytes From Cynomolgus Monkey
the Gaubius Laboratory, Leiden, The Netherlands.
Correspondence to Dr T. Kooistra, Gaubius Laboratory, TNO-PG, PO Box 2215, 2301 CE Leiden, The Netherlands.
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Abstract |
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Fibrates are widely used drugs in hyperlipidemic disorders. In addition to lowering serum triglyceride levels, fibrates have also been shown to reduce elevated plasma plasminogen activator inhibitor-1 (PAI-1) levels in vivo. We demonstrate that fibrates suppress PAI-1 synthesis in cultured cynomolgus monkey hepatocytes in a concentration-dependent way (0.1 to 1.0 mmol/L) and independent of their lipid-lowering effect. Different fibrates showed different potency in suppressing PAI-1 production: gemfibrozil and clofibric acid, at a concentration of 1 mmol/L, reduced PAI-1 synthesis over 24 hours to 52±20% and 60±5%, while clofibrate and bezafibrate lowered PAI-1 synthesis to only 86±17% and 84±15% of control values, respectively. These changes in PAI-1 production by fibrates correlated with changes in PAI-1 mRNA levels and were also visible at the level of gene transcription. Fibrates did not lower basal PAI-1 synthesis but attenuated an acceleration of PAI-1 production during culture. The suppressing effect of fibrates on PAI-1 synthesis could not be mimicked with activators or inhibitors of protein kinase C (PKC). Furthermore, fibrates did not inhibit the increase in PAI-1 synthesis induced by epidermal growth factor or transforming growth factor-ß. These results make mechanisms involving PKC modulation or growth factor receptor inactivation as a mode of action of fibrates unlikely. The suppressing effect of fibrates on PAI-1 synthesis could involve the nuclear receptor peroxisome proliferator–activated receptor (PPAR) and its heterodimeric partner, the retinoid X receptor (RXR). The alpha forms of PPAR and RXR were both found to be expressed in cynomolgus monkey hepatocytes. The ligand for RXR
, 9-cis retinoic acid, suppressed PAI-1 synthesis to the same extent as gemfibrozil, while a combination of gemfibrozil and 9-cis retinoic acid had no more effect on PAI-1 synthesis than any of these compounds alone at optimal concentrations. In conclusion, fibrates downregulate an induced PAI-1 production in cynomolgus monkey hepatocytes independent of a decrease in triglyceride levels. A possible involvement of PPAR/RXR in this downregulation is discussed.
Key Words: fibrates • plasminogen activator inhibitor-1 • hepatocytes • peroxisome proliferator–activated receptor • 9-cis retinoic acid
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Introduction |
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Fibrates are a class of hypolipidemic drugs widely used in the treatment of diet-resistant hyperlipidemia. In humans, fibrates effectively lower elevated serum triglycerides and increase high-density lipoprotein cholesterol. Fibrates also moderately lower low-density lipoprotein cholesterol levels in patients with hypercholesterolemia.1 In addition to these lipoprotein profile–altering effects, some fibrates also exert a favorable influence on plasma levels of hemostatic risk factors, such as PAI-1. These combined actions of fibrates may be beneficial in reducing the risk of coronary heart disease.1
The mechanism by which fibrates reduce plasma PAI-1 levels is unknown. Several reports have documented a correlation between plasma triglyceride levels and PAI-1 levels.2 Also, lowering triglyceride levels by diet or drugs has been shown to be associated with a decrease in PAI-1 levels,2 3 4 5 thus suggesting a relationship between triglyceride and PAI-1 levels.2 6 On the other hand, several lines of evidence suggest a mechanism of action of fibrates independent of their triglyceride-lowering effect. For example, the fibrate gemfibrozil was shown to significantly lower PAI-1 antigen levels, while in the same study no change in PAI-1 antigen levels by fenofibrate was observed, although both fibrates were equipotent in lowering triglyceride levels.7 Furthermore, it was shown that gemfibrozil reduces PAI-1 secretion in vitro in the human hepatoma cell line Hep G2, suggesting a direct effect of this drug on PAI-1 expression.8
In the present report we demonstrate that fibrates directly, ie, independent of lowering triglyceride levels, suppress PAI-1 expression in primary cultures of hepatocytes from cynomolgus monkey. We have further used this in vitro model of cultured monkey hepatocytes to compare the efficacy of various fibrates to lower PAI-1 production and to study the mechanism(s) by which fibrates exert their action. Studies were designed to evaluate the role of a number of signal-transduction pathways reported to be affected by fibrates and/or to be involved in PAI-1 gene expression. First, we tested the role of PKC, the activity of which has been shown to be modulated by fibrates9 10 and to be important for PAI-1 expression in Hep G2.11 Second, fibrates have been reported to affect the phosphorylation of growth factor receptors and thereby their signal transduction activity.12 We have evaluated whether fibrates interfere with the response of hepatocytes to EGF and TGF-ß, growth factors that have been found to be inducers of PAI-1 expression in Hep G2.13 14 Third, we examined a possible role of the nuclear hormone receptor PPAR, which is activated by fibrates.15 PPAR has been reported to downregulate gene expression through squelching of c-jun,16 a transcription factor that we found to be critical in PAI-1 gene expression in the human hepatoma cell line Hep G2.16A
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Methods |
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Materials
Clofibric acid, clofibrate, dexamethasone, and MTT were obtained from Sigma Chemical Co. Gemfibrozil was a gift of Dr B. Bierman, Warner-Lambert (Hoofddorp, The Netherlands). Bezafibrate was obtained from Boehringer Mannheim BV. Stock solutions of fibrates (1 mol/L) and PMA (100 µmol/L) were prepared in DMSO and ethanol, respectively, and kept at -20°C. Before use, fibrate stocks were diluted in incubation medium and kept for 2 hours at 37°C to ensure complete dissolution of the fibrates. Human EGF was obtained from Campro, TGF-ß from Harbor Bioproducts, tyrphostin from Brunschwig, and insulin (Actrapid Penfill 1.5) from Novo Nordisk Farma BV. 9-cis retinoic acid was a gift from Drs M. Klaus and C. Apfel, Hoffmann-LaRoche Ltd (Basel, Switzerland). The specific PKC inhibitor Ro 31-8220 was a gift of Dr G. Lawton, Hoffmann-LaRoche (Welwyn Garden City, UK). Deoxycytidine 5[
-32P]triphosphate (3 Ci/mol), [35S]methionine (>1000 Ci/mmol), and the Megaprime kit were obtained from Amersham Nederland BV. The Tintelize enzyme immunoassay kit for determination of PAI-1 antigen was from Biopool. Bradford protein reagent was from Biorad. Other materials used in the methods described below have been specified in detail in relating references or were purchased from standard commercial sources.
Isolation and Culture of Cynomolgus Monkey Primary Hepatocytes
Simian hepatocytes were isolated from livers of both male and female cynomolgus monkeys (Macaca fascicularis, 1.5 to 3 years old), which were obtained from the National Institute of Public Health and Environmental Protection (RIVM), Bilthoven, The Netherlands. The animals were bred at the RIVM and served as donors for kidneys used in the production of poliomyelitis vaccine at that institute. The isolation procedure was essentially as described for human hepatocytes,17 18 with a few modifications as described by Kaptein et al.19 Total cell yields varied from 0.5 to 1.5x109 viable cells. Viability, based on the ability of hepatocytes to exclude trypan blue dye (0.11%, wt/vol), was at least 65%. The cells were seeded in culture dishes at a density of 2x105 viable cells per square centimeter and were maintained in Williams' medium E supplemented with 10% (vol/vol) heat-inactivated fetal bovine serum, 135 nmol/L insulin, 50 nmol/L dexamethasone, 2 mmol/L L-glutamine (Flow Laboratories), 100 IU/mL penicillin, 100 µg/mL streptomycin, 100 µg/mL kanamycin, at 37°C in a 5% CO2/95% air atmosphere. After 16 hours, the nonadherent cells were washed from the plates and the remaining cells refreshed with the same medium as described above. After 8 hours, the medium was changed to incubation medium in which the amount of insulin was lowered to 10 nmol/L. Experiments were started 24 hours after hepatocyte isolation.
Conditioned media were obtained by incubating cells at 37°C for various times with incubation medium containing the appropriate concentration of the test compound or stock solvent (DMSO or ethanol; final concentration 0.1% [vol/vol]) as control. For prolonged incubations, the media were refreshed every 24 hours. Conditioned media were centrifuged for 4 minutes at 5000g in a Beckman Microfuge centrifuge to remove cells and cellular debris, and the samples were kept at -20°C until use. The cells were washed twice with ice-cold phosphate-buffered saline and were used for isolation of RNA or nuclei.
Northern Blot Analysis
Total RNA was isolated from at least 2x106 simian hepatocytes according to Chomczynski and Sacchi.20 RNA was fractionated by electrophoresis in a 1% (wt/vol) agarose gel under denaturing conditions using 1 mol/L formaldehyde,21 and blotted to Hybond-N filter according to the manufacturer's instructions. The filters were hybridized overnight at 63°C (with the PAI-1, GAPDH, PPAR, and RXR probe) or at 42°C (with the ACO probe) in hybridization mix, consisting of 7% (wt/vol) SDS, 0.5 mol/L Na2HPO4/NaH2PO4 buffer (pH 7.2), and 1 mmol/L EDTA, containing 3 ng of [-32P]CTP-labeled probe per milliliter. After hybridization with GAPDH or PAI-1 probe, the filters were washed twice with 2x SSC (1x SSC being 0.15 mol/L NaCl, 0.015 mol/L Na3 citrate), 1% (wt/vol) SDS, and twice with 1x SSC, 1% (wt/vol) SDS for 20 minutes at 63°C. In the case of hybridizations with PPAR or RXR probe, the filters were washed four times for 20 minutes each with 2x SSC, 1% (wt/vol) SDS at 63°C, while blots hybridized with ACO probe were washed three times for 15 minutes each with 2x SSC, 1% (wt/vol) SDS and one time for 15 minutes with 0.5x SSC, 1% (wt/vol) SDS at 42°C. The filters were then exposed to Kodak XAR-5 X-ray film with an intensifying screen at -80°C. The relative intensities of the bands present were determined on a Fujix Bas 1000 phosphoimager.
cDNA Probes
cDNA probes used were a 2.5-kb EcoRI fragment of the human PAI-1 cDNA22 ; a 1.2-kb Pst I fragment of the rat GAPDH cDNA, provided by Dr R. Offringa23 ; a 1.3-kb Nru I/BamHI fragment of the human PPAR cDNA, provided by Dr F.J. Gonzalez24 ; a 1.4-kb EcoRI/Bgl II fragment of the human RXR cDNA, provided by Dr J. Grippo (Hoffmann-LaRoche, Nutley, NJ); a 2.0-kb Sac I fragment of the rat ACO cDNA, provided by Dr T. Osumi25 ; and a 1.2-kb Pst I fragment of the hamster actin cDNA, provided by Dr W. Quax.26
Assays
PAI-1 antigen levels in conditioned media of cynomolgus monkey hepatocyte cultures were determined with an adapted Tintelize PAI-1 assay from Biopool. This assay normally does not recognize cynomolgus monkey PAI-1 antigen, but when the coating antibody is replaced by a goat anti-human PAI-1 polyclonal antiserum (5 µg/mL; Biopool), both monkey and human PAI-1 antigen can be determined. Monkey PAI-1 antigen values were calibrated by using the human PAI-1 calibration sample present in the kit. Overall protein synthesis was determined by measuring the incorporation of [35S]methionine into the 10% (wt/vol) trichloroacetic acid–precipitable fraction of radiolabeled conditioned medium and cell extract.27 Cell viability was assessed by the MTT assay,28 which is based on the cellular reduction of MTT by mitochondrial dehydrogenase of viable cells to a blue formazan product that can be measured spectrophotometrically at 545 nm. Protein was measured using the Bradford protein assay.29 Nuclear run-on assays were performed according to Groudine et al,29 with some minor modifications as described by Twisk et al.30
Statistical Analysis
Statistical significance of differences was calculated using Student's t test for paired data, with the level of significance selected to be P<.05 and P<.01. Values are expressed as mean±SD.
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Results |
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Dose Dependency and Time Course of the Effect of Different Fibrates on PAI-1 Synthesis
Cynomolgus monkey hepatocytes were incubated with three concentrations (0.1, 0.3, and 1 mmol/L) of four different fibrates (gemfibrozil, clofibric acid, clofibrate, and bezafibrate) for two consecutive periods of 24 hours. As shown in Fig 1
, all four fibrates dose dependently lowered PAI-1 levels, reaching 52±20% (gemfibrozil), 60±5% (clofibric acid), 86±17% (clofibrate), and 84±15% (bezafibrate) of control values after 24 hours at a 1 mmol/L concentration. During the second 24-hour incubation period, the fibrates did not lower PAI-1 levels markedly further than in the first 24-hour incubation period (Fig 1). Similar results were obtained when experiments were performed with medium containing 10% (vol/vol) lipoprotein-depleted serum or 1% (vol/vol) human serum albumin instead of 10% (vol/vol) fetal calf serum, indicating that fibrates can suppress PAI-1 synthesis independent of changes in triglyceride levels (data not shown). The decreases in PAI-1 production by fibrates were not due to diminished cell viability (as tested with the MTT test) or changes in overall protein synthesis (as assessed by simultaneous measurement of the incorporation of [35S]methionine into trichloroacetic acid–precipitable products). Fibrate concentrations higher than 1 mmol/L, however, were found to lower overall protein synthesis (data not shown). These results indicate that different fibrates possess different potency to reduce PAI-1 expression.
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Fig 2 shows a representative time course of the suppressive effect of gemfibrozil (1 mmol/L) on PAI-1 antigen accumulation. Both in the absence and presence of gemfibrozil, PAI-1 antigen levels increase linearly in time during the first 16 hours. However, between 16 and 24 hours of incubation, PAI-1 shows an accelerated increase under control conditions, whereas with gemfibrozil, PAI-1 continues to accumulate at a constant rate. This results in approximately a twofold higher PAI-1 antigen level in conditioned medium of control cells than in the conditioned medium of gemfibrozil-treated cells after 24 hours. The accelerated increase in PAI-1 synthesis varied between 32% and 75% in five independent experiments and was comparable in magnitude to the observed inhibition of PAI-1 synthesis by gemfibrozil. Similar results were obtained with clofibric acid, and analysis of the attenuating effect of fibrates on PAI-1 synthesis in Hep G2 cells showed that these effects could also be attributed to a diminishing effect of fibrates on the induction of PAI-1 synthesis during culture of Hep G2 cells (data not shown). Apparently, fibrates do not inhibit basal PAI-1 synthesis but prevent the accelerated production of PAI-1, as occurred during control incubation conditions.
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) or absence (
) of 1 mmol/L gemfibrozil, and the conditioned media were analyzed for PAI-1 antigen as described in the "Methods" section. The experiment shown is representative of five independent experiments in duplicate. Values shown are mean±range.
Effect of Gemfibrozil on PAI-1 mRNA Levels and Transcription
To determine whether the accelerated increase in PAI-1 antigen levels and inhibition thereof by fibrates were reflected at the mRNA level, we performed Northern blot analysis. As shown in Fig 3, there is no marked difference in PAI-1 mRNA levels between control and 1 mmol/L gemfibrozil-treated hepatocytes at 8 hours. At 16 hours, however, PAI-1 mRNA levels are strongly elevated in control but not in gemfibrozil-treated hepatocytes, and this difference is maintained at 24 hours. These mRNA data explain the different PAI-1 protein production rates in control and gemfibrozil-treated hepatocytes between 16 and 24 hours (Fig 2
). The transient, about twofold induction of PAI-1 mRNA by gemfibrozil at 4 hours was consistently found in three independent experiments and could be suppressed by an inhibitor of PKC (see below). No such induction was observed with the other three fibrates (data not shown).
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To determine whether the differences in PAI-1 mRNA levels in control and gemfibrozil-treated hepatocytes were the result of different PAI-1 transcription rates, we performed a nuclear run-on assay. Fig 4 shows that hepatocytes incubated with gemfibrozil have a twofold lower PAI-1 transcription rate than control cells.
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Role of Protein Kinase C in PAI-1 Expression
The transient increase in PAI-1 mRNA levels observed with gemfibrozil at 4 hours (Fig 3) is probably due to activation of PKC, since a similar rapid increase in PAI-1 mRNA levels was also seen with the specific PKC activator PMA, and both effects could be suppressed by the PKC inhibitor Ro 31-8220 (Fig 5). To assess whether modulation of PKC activity also plays a role in the suppression of the accelerated increase in PAI-1 synthesis between 16 and 24 hours (see Fig 2), we examined the effect of PMA and Ro 31-8220 on PAI-1 synthesis over a 24-hour period (Table). Opposed to gemfibrozil, PMA increased PAI-1 levels, thus reflecting the strong induction of PAI-1 mRNA levels at 4 hours (Fig 3). The PKC inhibitor Ro 31-8220 suppressed the effect of PMA but had no effect on PAI-1 production under control conditions. Together with the finding that clofibric acid, which did not transiently activate PKC after 4 hours, was almost as effective as gemfibrozil in suppressing PAI-1 synthesis after a 24-hour incubation period, these results indicate that fibrates suppress PAI-1 synthesis through a mechanism independent of PKC.
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Effect of Fibrates on the Induction of PAI-1 by EGF and TGF-ß
As shown in Fig 6, EGF (5 ng/mL) and TGF-ß (5 ng/mL) induce PAI-1 mRNA levels about threefold in primary hepatocytes after a 4- and 6-hour incubation period, respectively. The EGF-mediated induction of PAI-1 mRNA levels was suppressed >50% by the tyrosine protein kinase inhibitor tyrphostin (30 µg/mL; data not shown), a compound known to interfere with EGF receptor–mediated signaling.31 The induction of PAI-1 mRNA could not be inhibited, however, with 1 mmol/L gemfibrozil added to the cells 1 hour before the addition of EGF or TGF-ß (Fig 6). Similar negative results were obtained with 1 mmol/L clofibric acid, clofibrate, and bezafibrate (data not shown). Because the effect of fibrates on PAI-1 synthesis became apparent after 16 hours, we repeated the experiment after a 16-hour preincubation with the fibrates. Again, no quenching effect on PAI-1 mRNA induction by EGF or TGF-ß was observed (data not shown). These data indicate that fibrates do not interfere with growth factor receptor–mediated signaling.
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Studies on a Role of PPAR in the Inhibition of PAI-1 Synthesis by Gemfibrozil
We next considered a possible role for PPAR in the inhibition of PAI-1 synthesis by gemfibrozil. As shown in Fig 7, cynomolgus monkey hepatocytes express the mRNAs of PPAR and RXR, another steroid hormone receptor with which PPAR interacts to form heterodimers. Since the RXR ligand, 9-cis retinoic acid, has been demonstrated to enhance PPAR action,32 we tested the effect of gemfibrozil, 9-cis retinoic acid, and combinations thereof on PAI-1 synthesis. As shown in Fig 8, 10 µmol/L 9-cis retinoic acid was almost as effective as 1 mmol/L gemfibrozil in inhibiting PAI-1 synthesis: in three independent experiments, 9-cis retinoic acid and gemfibrozil decreased PAI-1 synthesis to 65±4% and 59±6% of control values (mean±SD), respectively. The inhibitions of PAI-1 synthesis by 9-cis retinoic acid and gemfibrozil in these experiments were of the same magnitude as the corresponding accelerated increases in PAI-1 synthesis under control conditions (data not shown). These data suggest that 9-cis retinoic acid, like gemfibrozil, prevents the accelerated increase in PAI-1 production but does not affect the uninduced PAI-1 synthesis rate. Combinations of suboptimal concentrations of the two ligands cooperatively decreased PAI-1 synthesis (data not shown), but the maximal effect never exceeded the inhibiting effect seen with the optimal concentration of gemfibrozil, as illustrated in Fig 8, for 1 µmol/L 9-cis retinoic acid and 1 mmol/L gemfibrozil. Taken together, these data suggest that 9-cis retinoic acid and gemfibrozil interfere with the same PAI-1 stimulatory pathway. They do not necessarily imply, however, that 9-cis retinoic acid and gemfibrozil act via the same regulatory pathway or mechanism.
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Discussion |
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In this study, we demonstrated a suppressive effect of fibrates on PAI-1 synthesis and PAI-1 mRNA levels in cultured cynomolgus monkey hepatocytes independent of changes in concentrations of triglycerides in the culture medium. We found that fibrates inhibit PAI-1 synthesis in a dose-dependent way and that different fibrates differ in their capacity to suppress PAI-1 production. Fibrates appeared to attenuate the accelerated increase in PAI-1 synthesis occurring under basal culture conditions. The changes in PAI-1 protein synthesis correlated closely with changes in PAI-1 mRNA and could also be demonstrated at the transcriptional level. Therefore, fibrates act selectively on a pathway that stimulates the activation of PAI-1 gene transcription. The regulatory mechanism by which this suppressive effect is brought about remains unknown but could involve PPAR
/RXR. Our finding that the suppression of PAI-1 synthesis by fibrates is a direct effect, ie, independent of lowering triglyceride levels, is in agreement with in vivo studies which show that simply reducing triglycerides is not sufficient to lower PAI-1 in patient populations with elevated triglyceride and PAI-1 levels.1 33 34 Furthermore, our in vitro observation that fibrates inhibit an induction of PAI-1 synthesis rather than basal PAI-1 expression is comparable to findings reported for the human hepatoma cell line Hep G28 and is in agreement with the in vivo situation in which fibrates lower only elevated PAI-1 levels in patients.1 Similarly, our in vitro finding that different fibrates which are equipotent in lowering triglyceride levels can differ in their efficacy to lower PAI-1 synthesis parallels in vivo results. For example, we found in our cultured cynomolgus monkey hepatocytes that gemfibrozil and clofibric acid were potent PAI-1 suppressors, while clofibrate and bezafibrate were not. Similarly, gemfibrozil but not bezafibrate was found to decrease enhanced PAI-1 levels in type IV hypertriglyceridemic patients.7 35 36
Our observation that PAI-1 synthesis in the simian hepatocytes is induced during basal culture conditions resembles similar findings in Hep G2 cells.37 PAI-1 synthesis in Hep G2 was found to be induced by an autocrine factor, secreted during cell culture. The nature of this factor has not been identified as yet but had no similarity to any steroid, retinoid, growth factor, or cytokine, factors known to induce PAI-1.38 Similarly, the factor(s) responsible for the accelerated production of PAI-1 in cynomolgus monkey hepatocytes remained elusive.
The obscurity of the PAI-1 inducing factor hampered a rational approach to understanding the mechanism by which fibrates suppress PAI-1 expression. We found that gemfibrozil rapidly and transiently increased PAI-1 mRNA expression in a manner comparable to the PKC activator PMA, albeit much weaker, and this effect could be inhibited with the PKC inhibitor Ro 31-8220. Activation of PKC by fibrates has also been reported in rat hepatocytes.9 10 However, specific activation or inhibition of PKC did not prevent the accelerated increase in PAI-1 during culture. Also, clofibric acid, which did not activate PKC, was as effective as gemfibrozil in suppressing PAI-1 production. These results make it unlikely that fibrates exert their PAI-1 suppressing action by interference with a PKC-dependent pathway. We could also exclude an effect of fibrates on the signal transduction pathways activated by growth factors like EGF and TGF-ß. We found that EGF and TGF-ß did induce PAI-1 mRNA levels in simian hepatocytes, but this action could not be prevented by fibrates. Apparently, not every PAI-1 induction is inhibited by fibrates. This is also true in vivo. For example, gemfibrozil did not lower elevated PAI-1 levels in patients with a history of venous thrombosis33 or in men with combined hyperlipoproteinemia.34
The finding that a number of fibrates are potent activators of the nuclear receptor PPAR suggested the possibility that this receptor mediates the beneficial action of fibrates on PAI-1. Indeed, PPAR and its heterodimeric partner RXR are both expressed in cultured cynomolgus monkey hepatocytes, and the ligand for RXR, 9-cis retinoic acid, also suppressed PAI-1 production. Two mechanisms have been described as to how PPAR/RXR can interfere with gene transcription and that might be relevant for PAI-1 expression. PPAR/RXR can antagonize transcriptional activation by competing with another transcription factor for binding to the same cis-acting element, as described by Keller et al.39 Second, inhibition can be due to mechanisms involving DNA-independent negative interferences such as squelching. Sakai et al16 recently showed that PPAR downregulates transcription of the glutathione transferase P gene in rat hepatocytes through squelching of the transcription factor c-jun. Interference with c-jun activity has also been reported for the RXR receptor.40 In this context it is of interest that c-jun is an important factor in PAI-1 gene transcription in the human hepatoma cell line Hep G2.16A (Arts and Kooistra, unpublished data). If PPAR activation is important for inhibition of PAI-1 expression by fibrates, then gemfibrozil and clofibric acid should be much stronger activators of PPAR activity in our system than bezafibrate or clofibrate. As a measure of PPAR activity, we examined the expression of the ACO gene. ACO is a peroxisomal target of PPARs, and its level has been found to be elevated manyfold in the livers of fibrate-treated rodents.42 43 In contrast to rodents, however, ACO mRNA levels in cynomolgus monkey hepatocytes were only slightly increased on treatment with fibrates (maximal increases of 150% were found after 24 hours' incubation with 1 mmol/L gemfibrozil). This finding is in agreement with previous reports showing that fibrates poorly induce peroxisome proliferation and peroxisomal ß-oxidation enzymes like ACO in both human and cynomolgus monkey hepatocytes.44 45 Ultimately, transfection experiments with dominant negative PPAR/RXR mutants, antisense technology, or the use of PPAR/RXR knockout mice are needed to answer the question of whether PPAR and RXR are indeed involved in mediating the fibrate-induced decrease in PAI-1 production.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported by a grant from the Netherlands Heart Foundation (90.267) and by a grant from the Netherlands Organisation for Scientific Research (NWO; 900-523-181). Gemfibrozil was a gift of Dr B. Bierman, Warner-Lambert (Hoofddorp, The Netherlands. The 9-cis retinoic acid was a gift from Drs M. Klaus and C. Apfel, Hoffmann-LaRoche Ltd (Basel, Switzerland). The specific PKC inhibitor Ro 31-8220 was a gift of Dr G. Lawton, Hoffmann-LaRoche (Welwyn Garden City, UK). A 1.2-kb Pst I fragment of the rat GAPDH cDNA was provided by Dr R. Offringa; a 1.3-kb Nru I/BamHI fragment of the human PPAR
cDNA by Dr F.J. Gonzalez; a 1.4-kb EcoRI/Bgl II fragment of the human RXR cDNA by Dr J. Grippo (Hoffmann-LaRoche, Nutley, NJ); a 2.0-kb Sac I fragment of the rat ACO cDNA by Dr T. Osumi; and a 1.2-kb Pst I fragment of the hamster actin cDNA by Dr W. Quax. We gratefully acknowledge Elly de Wit, Diana Neele, and Allard Kaptein for technical help with the simian hepatocyte isolations.
Received April 3, 1996;
revision received May 23, 1996;
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References |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Schonfeld G. The effects of fibrates on lipoprotein and hemostatic coronary risk factors. Atherosclerosis. 1994;111:161-174.[Medline] [Order article via Infotrieve]
2.
Mussoni L, Mannucci L, Sirtori M, Camera M, Maderna P, Sironi L, Tremoli E. Hypertriglyceridemia and regulation of fibrinolytic activity. Arterioscler Thromb. 1992;12:19-27.
3. Andersen P, Smith P, Seljeflot I, Brataker S, Arnesen H. Effects of gemfibrozil on lipids and haemostasis after myocardial infarction. Thromb Haemost. 1990;63:174-177.[Medline] [Order article via Infotrieve]
4. Avellone G, Di Garbo V, Cordova R, Ranelli G, De Simone R, Bompiani G. Effect of gemfibrozil treatment on fibrinolysis system in patients with hypertriglyceridemia. Curr Ther Res. 1992;52:338-347.
5.
Tenkanen L, Manttari M, Manninen V. Some coronary risk factors related to the insulin resistance syndrome and treatment with gemfibrozil. Circulation. 1995;92:1779-1785.
6.
Brown SL, Sobel BE, Fujii S. Attenuation of the synthesis of plasminogen activator inhibitor type 1 by niacin: a potential link between lipid lowering and fibrinolysis. Circulation. 1995;92:767-772.
7. Keber I, Lavre J, Suc S, Keber D. The decrease of plasminogen activator inhibitor after normalization of triglycerides during treatment with fibrates. Fibrinolysis. 1994;8:57-59.
8.
Fujii S, Sobel BE. Direct effects of gemfibrozil on the fibrinolytic system: diminution of synthesis of plasminogen activator inhibitor type I. Circulation. 1992;85:1888-1893.
9. Watanabe T, Okawa S, Itoga H, Imanaka T, Suga T. Involvement of calmodulin- and protein kinase C–related mechanism in an induction process of peroxisomal fatty acid oxidation–related enzymes by hypolipidemic peroxisome proliferators. Biochim Biophys Acta. 1992;1135:84-90.[Medline] [Order article via Infotrieve]
10. Bieri F. Peroxisome proliferators and cellular signalling pathways: a review. Biol Cell. 1993;77:43-46.[Medline] [Order article via Infotrieve]
11.
Bosma PJ, Kooistra T. Different induction of two plasminogen activator inhibitor 1 mRNA species by phorbol ester in human hepatoma cells. J Biol Chem. 1991;266:17845-17849.
12. Orellana A, Holuigue L, Hidalgo PC, Faundez V, Gonzalez A, Bronfman M. Ciprofibrate, a carcinogenic peroxisome proliferator, increases the phosphorylation of epidermal-growth-factor receptor in isolated rat hepatocytes. Eur J Biochem. 1993;215:903-906.[Medline] [Order article via Infotrieve]
13.
Lucore CL, Fujii S, Wun T-C, Sobel BE, Billadello JJ. Regulation of the expression of type 1 plasminogen activator inhibitor in Hep G2 cells by epidermal growth factor. J Biol Chem. 1988;263:15845-15848.
14.
Westerhausen DR, Hopkins WE, Billadello JJ. Multiple transforming growth factor-ß–inducible elements regulate expression of the plasminogen activator inhibitor type 1 gene in Hep G2 cells. J Biol Chem. 1991;266:1092-1100.
15. Auwerx J. Regulation of gene expression by fatty acids and fibric acid derivatives: an integrative role for peroxisome proliferator activated receptors. Horm Res. 1992;38:269-277.[Medline] [Order article via Infotrieve]
16.
Sakai M, Matsushimahibiya Y, Nishizawa M, Nishi S. Suppression of rat glutathione transferase P expression by peroxisome proliferators: interaction between jun and peroxisome proliferator–activated receptor alpha. Cancer Res. 1995;55:5370-5376.
16A. Arts J, Grimbergen J, Bosma PJ, Rahmsdorf HJ, Kooistra T. Role of c-Jun and proximal phorbol 12-myristate 13-acetate-(PMA)-responsive elements in the regulation of basal and PMA-stimulated plasminogen-activator inhibitor-1 gene expression in HepG2. Eur J Biochem. 1996;241:393-402.[Medline] [Order article via Infotrieve]
17.
Princen HMG, Huijsmans CMG, Kuipers F, Vonk RJ, Kempen HJM. Ketoconazole blocks bile acid synthesis in hepatocyte monolayer cultures and in vivo in rat by inhibiting cholesterol 7-hydroxylase. J Clin Invest. 1986;78:1064-1071.
18. Kooistra T, Bosma PJ, Tons HAM, van den Berg AP, Meyer P, Princen HMG. Plasminogen activator inhibitor 1: biosynthesis and mRNA level are increased by insulin in cultured human hepatocytes. Thromb Haemost. 1989;62:723-728.[Medline] [Order article via Infotrieve]
19.
Kaptein A, De Wit ECM, Princen HMG. Retinoids stimulate ApoA-I synthesis by induction of gene transcription in primary hepatocyte cultures from cynomolgus monkey (Macaca fascicularis). Arterioscler Thromb. 1993;13:1505-1514.
20. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987;162:156-159.[Medline] [Order article via Infotrieve]
21. Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory; 1989.
22. Van den Berg EA, Sprengers ED, Jaye M, Burgess W, Maciag T, van Hinsbergh VWM. Regulation of plasminogen activator inhibitor-1 mRNA in human endothelial cells. Thromb Haemost. 1988;60:63-67.[Medline] [Order article via Infotrieve]
23.
Fort P, Marty L, Piechaczyk M, El Sabrouty S, Dani C, Jeanteur P, Blanchard JM. Various rat adult tissues express only one major mRNA species from the glyceraldehyde-3-phosphate-dehydrogenase multigenic family. Nucleic Acids Res. 1985;13:1431-1442.
24. Sher T, Yi H-F, McBride OW, Gonzalez FJ. cDNA cloning, chromosomal mapping, and functional characterization of the human peroxisome proliferator activated receptor. Biochemistry. 1993;32:5598-5604.[Medline] [Order article via Infotrieve]
25.
Miyazawa S, Osumi T, Hashimoto T, Ohno K, Miura S, Fujiki Y. Peroxisome targeting signal of rat liver acyl-coenzyme A oxidase residues at the carboxy terminus. Mol Cell Biol. 1989;9:83-91.
26. Dodemont HJ, Soriano P, Quax WJ, Ramaekers F, Lenstra JA, Groenen MAM, Bernardi G, Bloemendal H. The genes coding for cytoskeletal proteins actin and vimentin in warm-blooded vertebrates. EMBO J. 1982;1:167-171.[Medline] [Order article via Infotrieve]
27. Kooistra T, van den Berg J, Tons A, Platenburg G, Rijken DC, van den Berg E. Butyrate stimulates tissue-type plasminogen-activator synthesis in cultured human endothelial cells. Biochem J. 1987;247:605-612.[Medline] [Order article via Infotrieve]
28.
De Vries EGE, Meijer C, Timmer-Bosscha H, Berendsen HH, De Leij L, Scheper RJ, Mulder NH. Resistance mechanisms in three human small cell lung cancer cell lines established from one patient during clinical follow-up. Cancer Res. 1989;49:4175-4178.
29. Bradford MM. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248-254.[Medline] [Order article via Infotrieve]
30.
Groudine M, Peretz M, Weintraub H. Transcriptional regulation of hemoglobin switching on chicken embryos. Mol Cell Biol. 1981;1:281-288.
31.
Twisk J, Lehmann EM, Princen HMG. Differential feedback regulation of cholesterol 7-hydroxylase mRNA and transcriptional activity by rat bile acids in primary monolayer cultures of rat hepatocytes. Biochem J. 1993;290:685-691.
32. Margolis B, Rhee SG, Felder S, Mervic M, Lyall R, Levitzki A, Ullrich A, Zilberstein A, Schlessinger J. EGF induces tyrosine phosphorylation of phospholipase C-II: a potential mechanism for EGF receptor signalling. Cell. 1989;57:1101-1107.[Medline] [Order article via Infotrieve]
33. Kliewer SA, Umesono K, Noonan DJ, Heyman RA, Evans RM. Convergence of 9-cis retinoic acid and peroxisome proliferator signalling pathways through heterodimer formation of their receptors. Nature. 1992;358:771-774.[Medline] [Order article via Infotrieve]
34. Haire WD. Gemfibrozil predictably lowers triglycerides but does not significantly change plasminogen activator inhibitor activity in hypertriglyceridemic patients with a history of thrombosis. Thromb Res. 1991;64:493-501.[Medline] [Order article via Infotrieve]
35.
Broijersen A, Eriksson M, Wiman B, Angelin B, Hjemdahl P. Gemfibrozil treatment of combined hyperlipoproteinemia: no improvement of fibrinolysis despite marked reduction of plasma triglyceride levels. Arterioscler Thromb Vasc Biol. 1996;16:511-516.
36. Almer L-O, Kjellstrom T. The fibrinolytic system and coagulation during bezafibrate treatment of hypertriglyceridemia. Atherosclerosis. 1986;61:81-85.[Medline] [Order article via Infotrieve]
37. Pazzucconi F, Mannucci L, Mussoni L, Gianfranceschi G, Maderna P, Werba P, Franceschini G, Sirtori CR, Tremoli E. Bezafibrate lowers plasma lipids, fibrinogen and platelet aggregability in hypertriglyceridaemia. Eur J Clin Pharmacol. 1992;43:219-223.[Medline] [Order article via Infotrieve]
38. Bergonzelli GE, Kruithof EKO. Constitutive plasminogen activator inhibitor 1 (PAI-1) biosynthesis in human Hep G2 hepatoma cells is maintained by an autocrine factor. Thromb Haemost. 1991;66:222-225.[Medline] [Order article via Infotrieve]
39. Bergonzelli GE, Kruithof EKO. Partial characterisation of a plasminogen activator inhibitor 1 stimulating factor produced by human hepatoma cells. Fibrinolysis. 1993;7:335-340.
40.
Keller H, Givel F, Perroud M, Wahli W. Signalling cross-talk between peroxisome proliferator–activated receptor/retinoid X receptor and estrogen receptor through estrogen response elements. Mol Endocrinol. 1995;9:794-804.
41.
Salbert G, Fanjul A, Piedrafita J, Lu XP, Kim S-J, Tran P, Pfahl M. Retinoic acid receptors and retinoid X receptor- downregulate the transforming growth factor-ß1 promoter by antagonizing AP-1 activity. Mol Endocrinol. 1993;7:1347-1356.
42. Dreyer C, Krey G, Keller H, Givel F, Helftenbein G, Wahli W. Control of the peroxisomal ß-oxidation pathway by a novel family of nuclear hormone receptors. Cell. 1992;68:879-887.[Medline] [Order article via Infotrieve]
43. Tugwood JD, Isseman I, Anderson RG, Bundell KR, McPheat WL, Green S. The mouse peroxisome proliferator activated receptor recognizes a response element in the 5' flanking sequence of the rat acyl-CoA oxidase gene. EMBO J. 1992;11:433-439.[Medline] [Order article via Infotrieve]
44. Blaauboer BJ, van Holsteijn CWM, Bleumink R, Mennes WC, van Pelt FNAM, Yap SH, van Pelt JF, van Iersel AAJ, Timmerman A, Schmid BP. The effect of beclobric acid and clofibric acid on peroxisomal ß-oxidation and peroxisome proliferation in primary cultures of rat, monkey and human hepatocytes. Biochem Pharmacol. 1990;40:521-528.[Medline] [Order article via Infotrieve]
45. Dirven HAAM, van den Broek PHH, Peeters MCE, Peters JGP, Mennes WC, Blaauboer BJ, Noordhoek J, Jongeneelen J. Effects of the peroxisome proliferator mono(2-ethylhexyl)phthalate in primary hepatocyte cultures derived from rat, guinea pig, rabbit and monkey. Biochem Pharmacol. 1993;45:2425-2434.[Medline] [Order article via Infotrieve]
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