©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Mode of Action of Peroxisome Proliferators as Hypolipidemic Drugs
SUPPRESSION OF APOLIPOPROTEIN C-III (*)

Rachel Hertz, Janette Bishara-Shieban, and Jacob Bar-Tana (§)

From the (1) Department of Human Nutrition and Metabolism, Hebrew University, Faculty of Medicine, Jerusalem 91120, Israel

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The hypolipidemic effect exerted by ,`-tetramethylhexadecanedioic acid (Medica 16) is accounted for by enhanced catabolism of plasma triglyceride-rich lipoproteins due to a decrease in plasma apolipoprotein C-III (Frenkel, B., Mayorek, N., Hertz, R., and Bar-Tana, J.(1988) J. Biol. Chem. 263, 8491-8497; Frenkel, B., Bishara-Shieban, J., and Bar-Tana, J.(1994) Biochem. J. 298, 409-414). Decrease in apolipoprotein C-III exerted by peroxisome proliferators/hypolipidemic amphipathic carboxylates (e.g. Medica 16, fibrate drugs) is shown here to result from suppression of apolipoprotein C-III gene expression. Transcriptional suppression of apolipoprotein C-III is due to transcriptional suppression of hepatic nuclear factor (HNF)-4 as well as displacement of HNF-4 from the apolipoprotein C-III promoter. HNF-4 displacement exerted by peroxisome proliferators/hypolipidemic amphipathic carboxylates is mediated by the peroxisome proliferators activated receptor (PPAR). Transcriptional suppression of HNF-4-enhanced genes (e.g. apolipoprotein C-III) along with transcriptional activation of peroxisomal and other genes by hypolipidemic drugs may account for their broad spectrum pharmacological effect.


INTRODUCTION

Aryloxyalkanoic fibrates (e.g. clofibrate (1) and bezafibrate (2) ), substituted long chain dicarboxylic acids (e.g. Medica 16 (3, 4) ), and other amphipathic carboxylates lower plasma triglycerides and cholesterol levels, and some are extensively used in humans as drugs of choice for treating hypertriglyceridemia or combined hypertriglyceridemia/hypercholesterolemia (5) . The hypolipidemic effect induced by these drugs may essentially be ascribed to enhanced catabolism of plasma triglyceride-rich lipoproteins (VLDL() and chylomicrons) with a concomitant increase in their hepatic uptake (6, 7, 8, 9) . Since apolipoprotein (apo) C-III potently inhibits plasma triglyceride-rich lipoprotein catabolism due to inhibition of their intravascular lipolysis by lipoprotein lipase as well as their liver receptor-mediated uptake (10, 11, 12, 13, 14) , and in light of the hyperlipoproteinemia induced in h-apoC-III transgenic mice (15, 16), or the hypotriglyceridemia induced in human (17) or animals (18) lacking apoC-III, a decrease in plasma apoC-III could account for the hypolipidemic effect exerted by these drugs. Medica 16 has indeed been found to dramatically decrease plasma apoC-III by 3-5-fold with a concomitant 5-10-fold increase in chylomicrons (8) and VLDL (9) plasma clearance and hepatic uptake. Hypolipidemic fibrate drugs have been similarly found to decrease plasma apoC-III.() The mode of action of hypolipidemic amphipathic carboxylates in suppressing plasma apo-C-III levels remained, however, to be investigated.

The hypolipidemic effect exerted by hypolipidemic amphipathic carboxylates is accompanied in rodents and some other species by an increase in liver size and liver peroxisomes with a concomitant induction of specific peroxisomal and other genes (e.g. peroxisomal acyl-CoA oxidase, cytochrome P-4504A1) (reviewed in Refs. 19 and 20). Induction of peroxisomal genes as well as of cytochrome P-4504A1 by hypolipidemic drugs/peroxisome proliferators (HD/PP) is due to transcriptional activation (21) mediated by binding of the peroxisome proliferator activated receptor (PPAR) homodimer or the PPAR-retinoic acid-X-receptor (RXR) heterodimer to sequence specific response elements in the 5`-flanking promoters of the concerned genes (22-26). The apparent linkage observed between the hypolipidemic and the peroxisomal effects exerted by HD/PP was claimed to indicate a causal-sequential relationship between peroxisome proliferation/induction of peroxisomal -oxidation and hypolipidemia (27, 28). This claim appeared, however, to be refuted in humans where the hypolipidemic effect exerted by hypolipidemic fibrates is unaccompanied by an increase in liver peroxisomes and peroxisomal -oxidation (29 and references therein), thus leaving open the question concerning the enigmatic linkage observed between the hypolipidemic and the peroxisomal effects exerted by HD/PP.

The mode of action of HD/PP in suppressing plasma apoC-III levels will be shown here to result from transcriptional suppression of the apoC-III gene mediated by PPAR-RXR. Transcriptional suppression of the apoC-III gene along with transcriptional activation of the respective peroxisomal genes by PPAR-RXR may rationalize the linkage between the hypolipidemic and the peroxisome proliferative effects exerted by HD/PP in rodents.


EXPERIMENTAL PROCEDURES

Animals and Cultures

Male albino rats weighing 150-200 g were fed with laboratory chow diet. 0.25% (w/w) Medica 16 or 0.2% (w/w) bezafibrate was added to their diet where indicated. HepG2, HeLa, and COS-7 cells were cultured in Dulbecco's modified Eagle's media supplemented with 10% fetal calf serum with either dimethyl sulfoxide as vehicle, Medica 16, or bezafibrate added to the culture medium as indicated.

Run-on Transcription

Rat liver nuclei were prepared according to Refs. 30 and 31 or HepG2 nuclei were prepared according to Ref. 32. Run-on transcription assays were carried out in 0.2 ml of buffer containing 25% glycerol, 50 mM Hepes (pH 7.5), 50 mM NaCl, 2.5 mM MgCl, 0.05 mM EDTA, 5 mM dithiothreitol, 1.25 mM ATP, 1.25 mM CTP, 1.25 mM GTP, 2 mM creatine phosphate, 2 µg of creatine kinase, 0.1 mM phenylmethylsulfonyl fluoride, and 250 µCi of [P]UTP, using nuclei samples amounting to 7.0 absorbance units (260 nm). Incubation was carried out at 26 °C for 30 min followed by addition of 20 units of RNase-free DNase I and then 30 µg of proteinase K. Newly synthesized [P]RNA was hybridized for 48 h at 42 °C with the respective cDNA inserts affixed to GeneScreen. Hybridization was carried out in 2 ml of 50% formamide containing 50 mM Tris-HCl (pH 8.0), 10 mM EDTA, 6 SSC, 1% SDS, 0.12% Ficoll, 0.12% polyvinylpyrrolidone, 0.12% bovine serum albumin, and 300 µg of salmon-sperm DNA. Washes were performed once in 5 SSC, 0.2% SDS, once in 2 SSC, 0.1% SDS, and once in 0.5 SSC, 0.1% SDS at 60 °C followed by a 30-min treatment with 10 µg/ml RNase A.

Transfection Assays

Transcriptional activity was measured in cells cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. Cells were transfected for 6 h with the respective CsCl-purified plasmid DNA added by calcium phosphate precipitation, washed, and further cultured for 42 h in the absence or presence of 120 µM Medica 16 added as 1000 stock in dimethyl sulfoxide. The -galactosidase expression vector pRSGAL (2 µg) added to each precipitate served as an internal control for transfection. When transfected with variable amounts of expression vectors, total amount of DNA was kept constant for each expression vector by supplementing with the parent pSG5 vector (33) . Cell extracts were prepared by freeze-thawing and assayed for -galactosidase and CAT activities. Results are expressed as -fold induction relative to CAT expression observed in cells transfected with the parent pSG5 vector. Each point represents the mean of duplicate cultures.

Gel Mobility Shift Assays

Gel mobility shift assays were carried out using rat nuclear extracts (30) , in vitro-synthesized transcription factors, or whole Cos extracts overexpressing the respectively transfected expression vectors. h-RXR and r-HNF-4 cDNAs cloned in pSG5 were linearized by XbaI and transcribed (Strategin) and translated in rabbit reticulocytes (Promega). Cos extracts enriched with PPAR, RXR, or HNF-4 were prepared from Cos-7 cells transfected for 5 h by calcium phosphate precipitation with 10 µg of either pSG5, pSG5-PPAR, pSG5-RXR, pSG5-HNF-4, or selected combinations of the above plasmids. Following transfection, cells were glycerol-shocked, incubated for 48 h, harvested, and lysed by three cycles of freezing-thawing in 100 µl/plate of lysis buffer (600 mM KCl, 10 mM Tris-HCl, pH 7.5, 1 mM dithiothreitol, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml pepstatin, and 1 µg/ml leupeptin), centrifuged at 20,000 g for 15 min, and the supernatants were then aliquoted and stored at -70 °C. For gel shift assays, programmed or unprogrammed reticulocytes lysates (2 µl) or whole Cos extracts (4 µg) as indicated were incubated for 20 min on ice in 11 mM Hepes, pH 7.9, containing 50 mM KCl, 1 mM dithiothreitol, 2.5 mM MgCl, 10% glycerol, 1 µg of poly(dI-dC) in a final volume of 20 µl. 0.1 ng of the respective P-labeled oligonucleotide was then added, and incubation was continued for an additional 20 min at room temperature. Protein-DNA complexes were resolved on a 5% nondenaturing polyacrylamide gel in 0.5 TBE.

Molecular Probes and Plasmid Constructs

A 270-nucleotide fragment from the 3`-end of m-apoC-III gene cloned into pGem-1 (pGmCIII 270) (34) , h-apoC-III cDNA (pCIII 607) and (-854/+22)h-apoC-III-CAT (35) were from T. Leff (Ann Arbor, MI). Peroxisomal acyl-CoA oxidase pMJ125 (36) was from T. Hashimoto (Nagano, Japan). HNF-4 recombinant DNA pLEN4S (37) was from F. M. Sladek (Riverside, CA). h-PABP cDNA (38) cloned into the EcoRI site of pGEM1 (Promega) was from T. Grange (Paris, France). pSG5-HNF-4 expression plasmid was constructed by inserting the BamHI fragment of pLEN4S encoding the entire 3-kilobase HNF-4 cDNA into the BamHI site of pSG5. pSG5-m-PPAR expression plasmid (22) and anti m-PPAR antiserum were from S. Green (Zeneca, Cheshire, UK). RS-h-RXR (39) was from R. Evans (La Jolla, CA). pSG5-RXR expression plasmid was constructed by inserting the EcoRI fragment of RS-h-RXR into the EcoRI site of pSG5.

HNF-4 Western Blot Analysis

Nuclear extracts of pooled (n = 3) rat liver nuclei were prepared according to Ref. 30, and 10 µg of protein were resolved by 10% SDS-PAGE and electroblotted onto nitrocellulose filters. Filters were blocked in 0.5% gelatin for 2 h at room temperature and then incubated with anti HNF-4 antisera (raised in rabbit to a synthesized peptide that corresponded to amino acids 445-455 of the rat HNF-4 protein (37) ) for 6 h at room temperature in 0.1 M sodium phosphate buffer, pH 7.4, containing 0.25% gelatin and 0.25% Tween 20. Following washes with the same buffer, filters were incubated for 1 h with goat alkaline phosphatase-conjugated anti-rabbit IgG antibodies (Promega), and the immune complexes were detected using the nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate substrate (Sigma). Equal loading of protein in each lane was verified by Coomassie blue staining.


RESULTS

The hypolipidemic effect of Medica 16 in rats is accounted for by a 3-5-fold decrease in plasma apoC-III with a concomitant 5-10-fold activation of VLDL and chylomicron uptake into the liver (8, 9) . Plasma apoC-III clearance remained unaffected by Medica 16 (9) , thus implying a decrease in the production of liver apoC-III induced by Medica 16 treatment. As shown in Fig. 1, A and B, the decrease in plasma apoC-III induced by HD/PP treatment in rats may be accounted for by a respective decrease in liver apoC-III mRNA induced either by an aryloxyalkanoic drug (bezafibrate) or substituted long chain dicarboxylic acid (Medica 16). Bezafibrate was somewhat more effective than Medica 16, resulting in 5-fold decrease in liver apoC-III mRNA following 3 days of treatment as compared with a 2-fold decrease induced by Medica 16 treatment. Similarly, a decrease in apoC-III mRNA was observed in human transformed liver cells (HepG2) incubated in the presence of either Medica 16 (Fig. 1C) or bezafibrate, thus pointing to a direct effect exerted by HD/PP on cells expressing apoC-III. Medica 16 was significantly more effective than bezafibrate in the human cells (2.5-3.0- versus 1.3-2.0-fold decrease following 3 days of treatment). Suppression of liver apoC-III mRNA was accompanied by inhibition of apoC-III transcription rate as verified by run-on transcription assays in liver nuclei derived from nontreated, Medica 16- or bezafibrate-treated rats (Fig. 2). Hence, the hypolipidemic effect exerted by HD/PP may be ascribed to suppression of transcription of the liver apoC-III gene by HD/PP.


Figure 1: ApoC-III mRNA levels in response to Medica 16 and bezafibrate. A, apoC-III mRNA in rats. Total RNA from liver of male rats treated for 6 days with either Medica 16 or bezafibrate as described under ``Experimental Procedures'' was prepared according to Ref. 54, and 25 µg were subjected to Northern blot analysis using GeneScreen membranes (DuPont NEN). Rat apoC-III mRNA was determined using the 270-base pair BamHI/EcoRI restriction fragment of pGmCIII 270 plasmid P-labeled by the random priming method (55). B, kinetics of Medica 16 () and bezafibrate () treatment in rats. Mean ± S.D. (n = 3). C, apoC-III mRNA in HepG2 cells. HepG2 cells were cultured for 72 h as described under ``Experimental Procedures'' with either vehicle (dimethyl sulfoxide) or 120 µM Medica 16. Total RNA was extracted according to Ref. 56 and subjected to Northern blot analysis. HepG2 apoC-III mRNA was probed using the 500-base pair PstI restriction fragment of h-apoC-III cDNA.




Figure 2: Rat apoC-III transcriptional activity in response to Medica 16 and bezafibrate. Male rats were treated for 5 days with either Medica 16 or bezafibrate as described under ``Experimental Procedures.'' Run-on transcription assays were carried out as described under ``Experimental Procedures.'' Newly synthesized [P]RNA was hybridized with 0.5 µg of cDNA BamHI/EcoRI 270-base pair insert of m-apoC-III and the extent of hybridization was normalized to the signal obtained with -actin cDNA. Numbers indicate relative densitometric units. Representative experiment out of 4 independent experiments.



Transcription of the apoC-III gene has previously been shown to be affected by hepatic nuclear factor (HNF)-4, a liver-abundant orphan member of the steroid/thyroid hormone superfamily receptors (41) which may bind to the C3P 5`-flanking sequence of the apoC-III gene promoter, resulting in activation of apoC-III transcription (42, 43, 44, 45) . Since liver apoC-III transcription is dependent on HNF-4 levels (44, 45) , transcriptional suppression of the apoC-III gene by HD/PP could result from suppression of HNF-4 levels and/or interference with HNF-4 binding to the apoC-III promoter.

Treatment of rats with either bezafibrate or Medica 16 resulted indeed in a decrease in liver HNF-4 mRNA with a pronounced time-dependent decrease in HNF-4 protein levels (Fig. 3). The 50% decrease in HNF-4 mRNA induced by either Medica 16 or bezafibrate could be accounted for by transcriptional suppression of the HNF-4 gene as verified in liver nuclei derived from Medica 16- or bezafibrate-treated rats or in HepG2 cells incubated in the presence of the respective HD/PP (Fig. 3A). Transcriptional suppression of HNF-4 in HepG2 cells was significantly more pronounced in the presence of Medica 16 as compared with bezafibrate and in accordance with apoC-III mRNA levels in liver cells treated with the two respective effectors. The reduced availability of HNF-4 for binding and transactivating apoC-III transcription in nuclear extracts derived from HD/PP-treated rats was further verified by analyzing the binding of respective nuclear extracts to the rat apoC-III C3P element. Bound HNF-4 in the respective extracts was identified by the extent of HNF-4 supershift in the presence of added anti HNF-4 antibodies. As shown in Fig. 3D (lanes 1, 5, and 9), nuclear extract binding to the rat apoC-III C3P element was significantly reduced in HD/PP-treated rats. Reduced binding was accounted for by the lower availability of HNF-4 in nuclear extracts derived from HD/PP-treated rats (lanes 6 and 10 as compared with 2, lanes 7 and 11 as compared with 3, and lanes 8 and 12 as compared with 4). Hence, HNF-4 suppression may account for apoC-III suppression by HD/PP.


Figure 3: HNF-4 gene expression in response to Medica 16 and bezafibrate. A, HNF-4 transcription rate. Male rats were treated for 5 days with either Medica 16 or bezafibrate as described under ``Experimental Procedures.'' HepG2 cells were cultured for 72 h with either dimethyl sulfoxide or 120 µM Medica 16. Run-on transcription assays for HNF-4 were carried out as described under ``Experimental Procedures'' using HepG2 or pooled (n = 3) rat liver nuclei. Newly synthesized [P]RNA was hybridized with the HNF-4 recombinant cDNA pLEN4S. The extent of hybridization was normalized to the signal obtained with -actin cDNA or poly(A)-binding protein (PABP). Numbers indicate relative densitometric units. Transcriptional activation of peroxisomal -oxidation genes is exemplified by hybridization with the peroxisomal acyl-CoA oxidase (AOX) pMJ125 plasmid. B, HNF-4 mRNA. HNF-4 mRNA was determined by Northern blot analysis of total RNA (20 µg) as in Fig. 1A and probed with the 3.5-kilobase EcoRI restriction fragment of the recombinant cDNA pLEN4S. C, HNF-4 protein. Male rats were treated for 1-3 days with either Medica 16 or bezafibrate as described under ``Experimental Procedures.'' HNF-4 protein in rat liver nuclear extracts was determined by Western blot analysis as described under ``Experimental Procedures.'' Western blot results were confirmed in 4 independent preparations of nuclear extracts. D, nuclear extract and HNF-4 binding to the r-apoC-III C3P element. Nuclear extracts prepared as described under ``Experimental Procedures'' from rats treated for 3 days with either Medica 16 or bezafibrate were analyzed by gel shift as described under ``Experimental Procedures'' for binding to the r-apoC-III P-labeled C3P element (filled arrowhead). HNF-4 specific binding was determined by incubating the respective extracts with anti rat HNF-4 antibodies, thus producing the HNF-4 supershifted bands (filled arrow). Lanes 1, 5, and 9, 0.05 µg of the respective nuclear extracts incubated with 1 µl of preimmune serum. Lanes 2, 6, and 10, 0.05 µg of the respective extracts incubated with 1 µl of anti-HNF-4 immune serum. Lanes 3, 7, and 11, 0.1 µg of the respective extracts incubated with 1 µl of anti-HNF-4 immune serum. Lanes 4, 8, and 12, 0.2 µg of the respective extracts incubated with 1 µl of anti-HNF-4 immune serum.



In addition to indirectly affecting apoC-III transcription by modulating HNF-4 gene expression and protein content, HD/PP exert a direct effect on apoC-III promoter as verified in cells cotransfected with a CAT reporter plasmid promoted by the 854 5`-flanking base pairs of the human apoC-III gene (42, 43) together with expression vectors for HNF-4, PPAR, and RXR. Basal expression of the (-854/+22)h-apoC-III CAT construct remained essentially unaffected by Medica 16 or bezafibrate. However, the HNF-4-enhanced transcription of this construct was repressed by PPAR and further repressed by PPAR in the presence of added Medica 16 (Fig. 4A) or bezafibrate and as a function of the relative proportions between transfected HNF-4 and PPAR. Suppression of HNF-4-activated transcription by PPAR was further affected by cotransfection with an expression vector for RXR (Fig. 4B). Thus, the 20-fold activation of apoC-III expression induced by HNF-4 could be essentially eliminated in the presence of PPAR, RXR, and Medica 16. No effect of PPAR, RXR, or both was observed in the absence of added HNF-4 (not shown), thus indicating that PPAR and RXR specifically counteracted HNF-4-enhanced transcription rather than inhibited basal transcription of the apoC-III gene. It is noteworthy that the C3P sequence of the apoC-III gene is homologous to the peroxisomal acyl-CoA oxidase promoter sequence which binds PPAR in the course of inducing transcriptional activation of peroxisomal genes by HD/PP (23, 24, 25) . Nevertheless, as noted above, basal transcription is not affected by binding of PPAR to the C3P element in the context of the -854/+22 apoC-III promoter, whereas transcriptional transactivation is induced by binding of PPAR to the C3P element in the context of peroxisomal acyl-CoA oxidase promoter (23, 24, 25) . Thus, the selective effect of PPAR binding presumably reflects the promoter context of the particular concerned gene.


Figure 4: Transcriptional activity of the apoC-III promoter in HNF-4, PPAR, and PPAR/RXR transfected cells and as a function of Medica 16. A, HepG2 cells were transfected as described under ``Experimental Procedures'' with (-854/+22)h-apoC-III-CAT construct (5 µg) and cotransfected with expression vectors for HNF-4 (as indicated) and PPAR (1 µg). Representative experiment out of 2 independent experiments. B, HeLa cells were transfected as described under ``Experimental Procedures'' with (-854/+22)h-apoC-III-CAT gene (5 µg) and cotransfected with 0.2 µg each of expression vectors for HNF-4, PPAR and RXR. Representative experiment out of three independent experiments. CAT activity determined in the presence of HNF-4 expression vector is presented as -fold induction compared to CAT activity determined in its absence. , HNF-4; , HNF-4 + RXR; , HNF-4 + PPAR; , HNF-4 + PPAR + Medica 16; , HNF-4 + PPAR + RXR; , HNF-4 + PPAR + RXR + Medica 16.



The putative binding of PPAR, PPAR-RXR, or HNF-4-PPAR to the rat (-91/-77) or human (-87/-66) apoC-III C3P element was studied by gel shift using the concerned transcription factors translated in vitro in rabbit reticulocytes or expressed in transfected Cos cells (Fig. 5). HNF-4 and the PPAR-RXR heterodimer, but not PPAR or RXR alone, were strongly bound by the rat or human apoC-III C3P element. The binding affinity of the human apoC-III C3P element to HNF-4 or PPAR-RXR was determined by mobility shift analysis using labeled h-apoC-III C3P element and increasing concentrations of nonradioactive C3P in the presence of Cos extracts derived from cells transfected with expression vectors for either HNF-4 or PPAR-RXR. The binding affinity of the h-apoC-III C3P element to HNF-4 and PPAR-RXR was found to be 0.48 ± 0.1 nM and 0.67 ± 0.06 nM, respectively. Binding of Cos extracts to the apoC-III C3P element was not affected by the presence of HD/PP, either during transfection of Cos cells with expression vectors for the respective transcription factors or during gel shift (data not shown). The respective band formed in the presence of both HNF-4 and PPAR was not shifted by an anti-PPAR antibody under conditions where binding of PPAR was completely displaced, thus indicating that no heterodimer was formed in the presence of HNF-4 and PPAR. These binding experiments thus further indicate that transcriptional suppression of the apoC-III gene by HD/PP may be mediated by displacement of HNF-4 from its apoC-III response element by PPAR-RXR binding to this promoter element.


Figure 5: Mobility shift analysis of human and rat C3P elements by PPAR, RXR, and HNF-4. The respective transcription factors were transcribed and translated in vitro in rabbit reticulocytes (A) or in transfected Cos cells (B) as described under ``Experimental Procedures.'' Mobility shift analysis was as described under ``Experimental Procedures'' using either the human (5`-agctGCAGGTGACCTTTGCCCAGCGCC-3`) or rat (5`-agctGCAGGTGACCTTTGACCAGCTc-3`) apoC-III P-labeled C3P elements, respectively. Where denoted (+Ab), anti-m-PPAR antibodies (1 µl of immune serum) were added to the incubation mixture.




DISCUSSION

Decrease in plasma apoC-III induced in rats by treatment with two structurally different hypolipidemic amphipathic carboxylates was shown here to result from transcriptional suppression of the liver apoC-III gene as verified by run-on transcription assays in liver nuclei derived from rats treated in vivo. The extent of decrease in transcription rate induced by HD/PP essentially correlates with the decrease observed in apoC-III mRNA and plasma apoC-III content induced by treatment with HD/PP. In line with the previously reported inhibition of clearance of plasma triglyceride-rich lipoproteins by apoC-III (10, 11, 12, 13, 14) , the hypolipoproteinemia induced in humans inflicted by apoC-III deficiency (17) , the hyperlipoproteinemia induced in h-apoC-III transgenic mice (16, 17) , as well as hypotriglyceridemia and protection from postprandial hypertriglyceridemia in animals lacking apoC-III (18) , transcriptional suppression of the apoC-III gene by hypolipidemic amphipathic carboxylates with a concomitant increase in plasma triglyceride-rich lipoproteins clearance may account for the hypolipidemic effect exerted by these drugs.

Transcriptional suppression of the apoC-III gene by HD/PP was found here to be related to HNF-4-enhanced transcription of the apoC-III gene and ascribed to direct and indirect complementary modes of action, namely, displacement of HNF-4 from the apoC-III promoter as well as suppressing HNF-4 levels by HD/PP. HNF-4 displacement by PPAR-RXR was implied in light of the inhibition of HNF-4-enhanced transcription by PPAR-RXR in cells transfected with a reporter plasmid promoted by the homologous apoC-III promoter. Inhibition of HNF-4-enhanced transcription by PPAR-RXR could be accounted for by binding of PPAR-RXR to the apoC-III HNF-4 element as verified by using gel shift binding assays. Since transcription of the apoC-III gene is activated by HNF-4 (44, 45, Fig. 4) while remaining unaffected by PPAR-RXR, the extent of inhibition of HNF-4 enhanced transcription by PPAR-RXR may be expected to reflect the prevailing content of the concerned transcription factors and their respective binding affinities to the apoC-III C3P element. In this respect, the PPAR-RXR heterodimer behaves similarly to other previously reported transcription factors which may compete with HNF-4 for binding to the apoC-III C3P promoter element, e.g. ARP-1, EAR-3/coup, or EAR-2 (44, 45) . It should be pointed out, however, that generalizing the direct mode of action of HD/PP as verified here in transiently transfected cells for the endogenous apoC-III promoter still remains to be complemented by studying the role played by additional regulatory sequences of the apoC-III promoter not present in the transiently transfected promoter as well as by the chromatin context of the endogenous gene. The difference between the endogenous and the transiently transfected promoter is indeed reflected in the requirement for the respective transcription factors (e.g. HNF-4, PPAR) for suppression of the transfected apoC-III gene by HD/PP. Suppression of the transiently transfected promoter by HD/PP requires cotransfection of expression vectors for the respective transcription factors (Fig. 4), whereas the endogenous promoter may be directly suppressed by HD/PP in the absence of added transcription factors ( Fig. 1and Fig. 2 ). This difference is similar to that previously reported for other members of the steroid/thyroid hormone receptors superfamily (57) where endogenous transcription factors sequestered within the chromatin may become nonaccessible to the transiently transfected promoter but still available to the endogenous promoter.

The indirect mode of action of HD/PP in suppressing apoC-III gene transcription was verified here by analyzing HNF-4 transcription rates and transcript levels as well as measuring HNF-4 protein levels in nuclear extracts of HD/PP-treated animals. Both the overall content of nuclear extract HNF-4 and that available for binding to the apoC-III C3P promoter element were found to be significantly reduced by treatment with HD/PP. Since HNF-4 expression is positively modulated by HNF-4 itself (41) , suppression of HNF-4 gene expression by HD/PP may perhaps result as well from displacement of HNF-4 from its putative C3P element in the HNF-4 promoter. Hence, the direct and indirect complementary effects of HD/PP in suppressing apoC-III gene expression may actually reflect a unified mode of action where displacement of HNF-4 from its C3P element in the apoC-III or HNF-4 gene promoters is exerted by PPAR-RXR binding.

The mode of action of HD/PP in initiating binding of the PPAR-RXR heterodimer to C3P elements of peroxisomal genes, apoC-III, and other promoters has still to be investigated, since, in contrast to other members of the steroid/thyroid hormone receptors superfamily, the putative binding of HD/PP to PPAR still remains unclear (22) . Thus, PPAR binding to its C3P element may be directly initiated by binding of the free HD/PP to a putative ligand binding site of PPAR, albeit with low binding affinity (22) , or HD/PP could indirectly affect targeting or affinity of PPAR to its C3P response elements. The extreme structural diversity of HD/PP (19, 20, 27, 40, 46) is indeed in contrast with the strict structural specificity characteristically required for ligand binding by members of the superfamily.

This proposed mode of action of amphipathic carboxylic peroxisome proliferators as hypolipidemic drugs may indeed rationalize the enigmatic linkage previously observed between the hypolipidemic and peroxisome proliferative effects exerted by HD/PP (27) . Rather than pointing to a causal-sequential linkage between peroxisome proliferation and hypolipidemia (27, 28) , the observed linkage may reflect transcriptional modulation of genes involved in the hypolipidemic and peroxisome proliferative effects by a common transduction pathway consisting of binding PPAR to C3P response elements of the respective concerned promoters. The net effect exerted by PPAR binding which may result either in transcriptional activation or inhibition of the concerned genes will depend, however, on the interplay between the various transcription factors which may bind to a particular C3P element, their prevailing nuclear concentrations, and their respective binding affinities as well as the specific promoter context of the concerned gene. Thus, binding of PPAR-RXR to a C3P element of peroxisomal acyl-CoA oxidase in the context of the h-acyl-CoA oxidase promoter, results in transcriptional activation of that gene while binding the heterodimer to a C3P element in the context of the apoC-III promoter does not modulate apoC-III transcription in the absence of added HNF-4.

Transcriptional suppression of the apoC-III gene by HD/PP mediated by modulating HNF-4 expression and its extent of binding to the apoC-III gene promoter may offer a rationale for predicting and realizing additional inhibitory effects exerted by amphipathic carboxylates acting as HD/PP. Thus, other genes reported to be transcriptionally transactivated by HNF-4 (41) should be considered as candidates for transcriptional suppression by HD/PP similar to that reported here for apoC-III. Transthyrethin is transactivated by HNF-4 and has indeed been recently reported to be inhibited by HD/PP (47) . A particular candidate of interest consists of the apoB gene recently reported to be transcriptionally activated upon HNF-4 binding to its AF-1 promoter site (48) . Transcriptional suppression of apoB by HD/PP similar to that reported here for apoC-III may result in decreased liver production of apoB with a concomitant decrease in liver VLDL production as previously reported for Medica 16 (49) . Transcriptional suppression of apoB may thus complement plasma VLDL lowering effect of HD/PP induced by apoC-III suppression. It should be pointed out, however, that additional factors and constraints other than having a C3P element, and, in particular, the specific promoter context concerned, may determine HNF-4 binding and transactivation of a particular promoter, as well as suppression of HNF-4-enhanced transcription by HD/PP.

Transcriptional suppression of HNF-4-enhanced genes together with transcriptional activation of a variety of other genes (e.g. peroxisomal -oxidation genes (19, 20) , P-4504A1 (26) , liver thyroid hormone-dependent genes (50, 51) ), all mediated by HD/PP-dependent binding of PPAR to C3P elements in the respective promoters, may form the basis for the pleiotropic effect of xenobiotic HD/PP as well as of putative natural amphipathic carboxylates (e.g. long chain fatty acids (52, 53) ) acting as broad spectrum pharmacological or physiological modulators of gene expression.


FOOTNOTES

*
This work was supported by the U.S.-Israel Binational Science Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed: Dept. of Human Nutrition and Metabolism, Hebrew University, Faculty of Medicine, P. O. Box 12272, Jerusalem 91120, Israel. Tel.: 972-2-430-785 or 972-2-758-305; Fax: 972-2-431-105.

The abbreviations used are: VLDL, very low density lipoprotein; apo, apolipoprotein; CAT, chloramphenicol acetyltransferase; HD/PP, hypolipidemic drug(s)/peroxisome proliferator(s); HNF-4, hepatic nuclear factor-4; PABP, poly(A)-binding protein; PPAR, peroxisome proliferator activated receptor; RXR, retinoic acid-X-receptor; m, mouse; h, human; r, rat.

N. Mayorek and J. Bar-Tana, unpublished observations.


ACKNOWLEDGEMENTS

We thank T. Leff, T. Hashimoto, F. M. Sladek, S. Green, R. M. Evans, and T. Grange for their kind gifts of the respective plasmids and T. Leff for critical review of the manuscript.


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