The Peroxisomal 3-keto-acyl-CoA thiolase B Gene Expression Is under the Dual Control of PPARα and HNF4α in the Liver.

PPARα and HNF4α are nuclear receptors that control gene transcription by direct binding to specific nucleotide sequences. Using transgenic mice deficient for either PPARα or HNF4α, we show that the expression of the peroxisomal 3-keto-acyl-CoA thiolase B (Thb) is under the dependence of these two transcription factors. Transactivation and gel shift experiments identified a novel PPAR response element within intron 3 of the Thb gene, by which PPARα but not HNF4α transactivates. Intriguingly, we found that HNF4α enhanced PPARα/RXRα transactivation from TB PPRE3 in a DNA-binding independent manner. Coimmunoprecipitation assays supported the hypothesis that HNF4α was physically interacting with RXRα. RT-PCR performed with RNA from liver-specific HNF4α-null mice confirmed the involvement of HNF4α in the PPARα-regulated induction of Thb by Wy14,643. Overall, we conclude that HNF4α enhances the PPARα-mediated activation of Thb gene expression in part through interaction with the obligate PPARα partner, RXRα.

Peroxisome-proliferator-activated receptors as physiological sensors of fatty acid metabolism: molecular regulation in peroxisomes.

The enzymes required for the beta-oxidation of fatty acyl-CoA are present in peroxisomes and mitochondria. Administration of hypolipidaemic compounds such as clofibrate to rodents leads to an increase in the volume and density of peroxisomes in liver cells. These proliferators also induce simultaneously the expression of genes encoding acyl-CoA oxidase, enoyl-CoA hydratase-hydroxyacyl-CoA dehydrogenase (multifunctional enzyme) and thiolase (3-ketoacyl-CoA thiolase). All these enzymes are responsible for long-chain and very-long-chain fatty acid beta-oxidation in peroxisomes. Similar results were observed when rat hepatocytes, or liver-derived cell lines, were cultured with a peroxisome proliferator. The increased expression of these genes is due to the stimulation of their transcription rate. These results show that the peroxisome proliferators act on the hepatic cells and regulate the transcription through various cellular components and pathways, including peroxisome-proliferator-activated receptor alpha (PPARalpha). After activation by specific ligands, either fibrates or fatty acid derivatives, PPARalpha binds to a DNA response element: peroxisome-proliferator-responsive element (PPRE), which is a direct repeat of the following consensus sequence: TGACCTXTGACCT, found in the promoter region of the target genes. PPARalpha is expressed mainly in liver, intestine and kidney. PPARalpha is a transcriptional factor, which requires other nuclear proteins for function including retinoic acid X receptor (RXRalpha) and other regulatory proteins. From our results and others we suggest the role of PPARalpha in the regulation of the peroxisomal fatty acid beta-oxidation. In this regard, we showed that although PPARalpha binds to thiolase B gene promoter at -681 to -669, a better response is observed with hepatic nuclear factor 4 ("HNf-4"). Moreover, rat liver PPARalpha regulatory activity is dependent on its phosphorylated state. In contrast, a protein-kinase-C-mediated signal transduction pathway seems to be modified by peroxisome proliferators, leading to an increase in the phosphorylation level of specific proteins, some of which have been shown to be involved in the phosphoinositide metabolism.

Regulation of the peroxisomal beta-oxidation-dependent pathway by peroxisome proliferator-activated receptor alpha and kinases.

The first PPAR (peroxisome proliferator-activated receptor) was cloned in 1990 by Issemann and Green (Nature 347:645-650). This nuclear receptor was so named since it is activated by peroxisome proliferators including several drugs of the fibrate family, plasticizers, and herbicides. This receptor belongs to the steroid receptor superfamily. After activation by a specific ligand, it binds to a DNA response element, PPRE (peroxisome proliferator response element), which is a DR-1 direct repeat of the consensus sequence TGACCT x TGACCT. This mechanism leads to the transcriptional activation of target genes (Motojima et al., J Biol Chem 273:16710-16714, 1998). After the first discovery, several isoforms were characterized in most of the vertebrates investigated. PPAR alpha, activated by hypolipidemic agents of the fibrate family or by leukotrienes; regulates lipid metabolism as well as the detoxifying enzyme-encoding genes. PPAR beta/delta, which is not very well known yet, appears to be more specifically activated by fatty acids. PPAR gamma (subisoforms 1, 2, 3) is activated by the prostaglandin PGJ2 or by antidiabetic thiazolidinediones (Vamecq and Latruffe, Lancet 354:411-418, 1999). This latter isoform is involved in adipogenesis. The level of PPAR expression is largely dependent on the tissue type. PPAR alpha is mainly expressed in liver and kidney, while PPAR beta/delta is almost constitutively expressed. In contrast, PPAR gamma is largely expressed in white adipose tissue. PPAR is a transcriptional factor that requires other nuclear proteins in order to function, i.e. RXRalpha (9-cis-retinoic acid receptor alpha) in all cases in addition to other regulatory proteins. Peroxisomes are specific organelles for very long-chain and polyunsaturated fatty acid catabolism. From our results and those of others, the inventory of the role of PPAR alpha in the regulation of peroxisomal fatty acid beta-oxidation is presented. In relation to this, we showed that PPAR alpha activates peroxisomal beta-oxidation-encoding genes such as acyl-CoA oxidase, multifunctional protein, and thiolase (Bardot et al., FEBS Lett 360:183-186, 1995). Moreover, rat liver PPAR alpha regulatory activity is dependent on its phosphorylated state (Passilly et al., Biochem Pharmacol 58:1001-1008, 1999). On the other hand, some signal transduction pathways such as protein kinase C are modified by peroxisome proliferators that increase the phosphorylation level of some specific proteins (Passilly et al. Eur J Biochem 230:316-321, 1995). From all these findings, PPAR alpha and kinases appear to play an important role in lipid homeostasis.

Relationship between signal transduction and PPAR alpha-regulated genes of lipid metabolism in rat hepatic-derived Fao cells.

The goal of this study was to characterize phosphorylated proteins and to evaluate the changes in their phosphorylation level under the influence of a peroxisome proliferator (PP) with hypolipidemic activity of the fibrate family. The incubation of rat hepatic derived Fao cells with ciprofibrate leads to an overphosphorylation of proteins, especially one of 85 kDa, indicating that kinase (or phosphatase) activities are modified. Moreover, immunoprecipitation of 32P-labeled cell lysates shows that the nuclear receptor, PP-activated receptor, alpha isoform, can exist in a phosphorylated form, and its phosphorylation is increased by ciprofibrate. This study shows that PP acts at different steps of cell signaling. These steps can modulate gene expression of enzymes involved in fatty acid metabolism and lipid homeostasis, as well as in detoxication processes.

Carcinogenic aspect of xenobiotic molecules belonging to the peroxisome proliferator family.

It is known that a short-term exposure of rat, mice or incubation of hepatic cells with fibrate molecules leads to increase in peroxisome number and cell hyperplasia. Further, long-term incubation of cells (at least a year) show transformed characteristics with foci and nodules. To explain the hepatocarcinogenic effect of peroxisome proliferators in rodents we studied the effect of peroxisome proliferators on rat liver oncogenes expression. Earlier, we reported an increase in liver and kidney mRNA level of c-myc and N-myc. Since several metabolic genes are activated by PPAR (peroxisome proliferators activated receptor) through a PPRE (peroxisome proliferator response element), we suggest the involvment of PPAR in oncogene activation, because of the presence of PPRE in the N-myc 5'-upstream region. We showed by flow cytometric analysis that ciprofibrate increased the size of rat Fao derived cell line and the activity of palmitoyl CoA oxidase, a peroxisome proliferation enzyme marker for studying peroxisome proliferation was increased. The above effects which can contribute to hepatocarcinogenesis seem to be restricted to rat and mice, which show strong response to peroxisome proliferators. Indeed, no changes are observed in weak responsive species such as humans (using hepatic derived cell lines) and guinea pig. These data provide arguments for the non-carcinogenic effect of this xenobiotic class in human especially when sensitive, or normal individuals are exposed either to hypolipidaemic agents of the fibrate family.

Differential regulation by a peroxisome proliferator of the different multifunctional proteins in guinea pig: cDNA cloning of the guinea pig D-specific multifunctional protein 2.

After our previous report on the cloning of two cDNA species in guinea pig, both encoding the same hepatic 79 kDa multifunctional protein 1 (MFP-1) [Caira, Cherkaoui-Malki, Hoefler and Latruffe (1996) FEBS Lett. 378, 57-60], here we report the cloning of a cDNA encoding a second multifunctional peroxisomal protein (MFP-2) in guinea-pig liver. This 2356 nt cDNA encodes a protein of 735 residues (79.7 kDa) whose sequence shows 83% identity with rat MFP-2 [Dieuaide-Noubhani, Novikov, Baumgart, Vanhooren, Fransen, Goethals, Vandekerckhove, Van Veldhoven and Mannaerts (1996) Eur. J. Biochem. 240, 660-666]. In parallel, we studied the effect of ciprofibrate, a hypolipaemic agent also known as peroxisome proliferator in rodent, on the expression of MFP-1 and MFP-2 (2.6 kb) in rats and guinea pigs. By Northern blotting analysis we demonstrated that three MFP-1-related mRNA species are expressed in the guinea-pig liver. The expression of two of them (3.5 and 2.6 kb) is slightly increased by ciprofibrate, whereas the 3.0 kb MFP-1 mRNA is, unlike the rat one, strongly down-regulated in guinea pigs treated with ciprofibrate. In a similar way, the hepatic expression of the guinea-pig 2.6 kb MFP-2 mRNA is also down-regulated in guinea pigs treated with ciprofibrate. These results demonstrate (1) that in contrast with the unique 3.0 kb MFP-1 rat mRNA, at least three hepatic MFP-1-related mRNA species are co-expressed in guinea pig; and (2) that, opposed to the accepted idea of non-responsiveness of the guinea pig to ciprofibrate, this drug affects MFP-1 and MFP-2 gene expression in this species. Also, the mRNA species for acyl-CoA oxidase and thiolase, two other enzymes of the peroxisomal beta-oxidation pathway that are induced severalfold in responsive species are down-regulated in guinea pig. This paper is the first, to our knowledge, reporting the down-regulation of the expression of genes encoding enzymes involved in the peroxisomal beta-oxidation of fatty acids (MFP-1) and bile acid synthesis (MFP-2) in mammals.

Delayed effects of ciprofibrate on rat liver peroxisomal properties and proto-oncogene expression.

Peroxisome proliferators (PPs) are non-genotoxic carcinogens in rodents. Their reversible effects on rat liver have been studied with ciprofibrate and fenofibrate. We found that with the hypolipemic drug fenofibrate a pause of 28 days is sufficient for a return to normal status, whereas with the highly potent PP ciprofibrate, the stimulation of ACO mRNA levels remains after its withdrawal. We investigated the effects of the renewal of the treatment with PPs on other peroxisomal parameters and proto-oncogene expression using Wistar rats. Interestingly, c-myc expression was enhanced even upon drug withdrawal, and was more stimulated by the second exposure to ciprofibrate, while c-fos expression was unaltered. However, only slight differences in c-Ha-ras expression were observed. Therefore, the effects of PPs in the Wistar rats are not totally reversible within 28 days following withdrawal, depending on the drug used. These delayed effects of ciprofibrate could be a key to our understanding the hepatocarcinogenic effect of PPs in rodents.

Monoclonal antibody TeM 106 reacts with a tonoplast intrinsic protein of 106 kDa from Brassica oleracea L.

A monoclonal antibody, designated TeM 106, that recognizes an intrinsic protein from the vacuole membrane (tonoplast) of cauliflower (Brassica oleracea L. var. botrytis) is described. Mice were immunized with a tonoplast fraction that had been purified from differentiating meristematic cells from the cauliflower head. Hybridomas were generated and screened by means of Enzyme Linked Immuno Sorbent Assays for differential reactivity to tonoplast over non-related proteins (bovine serum albumin). One out of 14 reactive murine clones was selected on the basis of its stability, secretory efficiency, and high affinity of the secreted antibodies. TeM 106 is an IgM which was shown by indirect immunofluorescence microscopy of frozen thin sections to bind specifically to the tonoplast of highly vacuolated cells as well as to the tonoplast of small vacuoles in meristematic cells. The molecular specificities of TeM 106 were preliminarily determined using electrophoretic transfer procedures (immunoblotting). TeM 106 reacted with a single protein band of 106,000 M(r) from the tonoplast of cauliflower. Using two-dimensional gel electrophoresis, it was shown that the epitope is borne by a single polypeptide. The antigen is a glycopeptide containing mannose and/or glucose residues in the oligosaccharide side chain but the epitope, resistant to the metaperiodate oxidation, is contained in the polypeptide backbone. Salt elution experiments indicated that the antigen, unlike several proteins from the tonoplast, is not eluted from the membrane by KCl treatments and is, therefore, tentatively considered as a tonoplast intrinsic protein, designated TIP 106.

The analysis of modified peroxisome proliferator responsive elements of the peroxisomal bifunctional enzyme in transfected HepG2 cells reveals two regulatory motifs.

Peroxisome proliferators (PPs) are non-genotoxic carcinogens in rodents. They can induce the expression of numerous genes via the heterodimerization of two members of the steroid hormone receptor superfamily, called the peroxisome proliferator-activated receptor (PPAR) and the 9-cis retinoic acid receptor (RXR). Many of the PP responsive genes possess a peroxisome proliferator response element (PPRE) formed by two TGACCT-related motifs. The bifunctional enzyme (HD) PPRE contains 3 such motifs, creating DR1 and DR2 sequences. PPAR and RXR regulate transcription via the DR1 element while DR2 modulates the expression of the gene via auxiliary factors in HepG2 cells.

Antibodies to the tonoplast from the storage parenchyma cells of beetroot recognize a major intrinsic protein related to TIPs.

A vacuole membrane (= tonoplast) subfraction was purified by sedimentation in a discontinuous sucrose gradient (0.6 M/0.3 M) from a crude vacuole fraction isolated from red beetroot storage cells. The vacuole membrane-enriched fraction was injected into the popliteal lymph nodes of rabbits to raise polyclonal antibodies. The resultant serum was tested by indirect immunofluorescence microscopy on cryosections of shoot meristem. Antibodies specifically bound to the tonoplast but not the cytoplasm or the nucleus. By immunogold microscopy of ultrathin frozen sections, the anti-tonoplast antibodies were shown to label the luminal (exoplasmic) surface of the vacuole membrane and vesicular elements of the Golgi complex. Almost all of the antibodies were directed against a polypeptide with an M(r) of 27,000 as determined by Western blotting. A 30,000 M(r) polypeptide was occasionally detected in tonoplast-enriched fractions. It was weakly labeled by the crude immune serum, but not by the serum purified by affinity on the M(r) 27,000 band. The 27 kDa polypeptide which accounted for 10 to 15% of the total membrane proteins was partially characterized. The M(r) 27,000, but not the M(r) 30,000 polypeptide, had Triton X-114 binding properties and was extracted by chloroform:methanol, indicating its high hydrophobicity. On the basis of their partitioning in detergent/aqueous phases it is suggested that the 27 kDa and the 30 kDa polypeptides belong to the tonoplast and to the vacuolar sap, respectively. Neither polypeptide binds to Con A or is sensitive to Endo F digestion, suggesting that they are either unglycosylated polypeptides or glycopeptides with modified oligosaccharides. When separated by SDS-PAGE under non-reducing conditions, the 27 kDa antigen displays a small increase in apparent relative mobility. Its NH2-terminal amino acid sequence shares homology with plant members of the MIP channel family. As suggested by its relative molecular mass, its high hydrophobicity and its NH2-terminal amino acid sequence, the M(r) 27,000 polypeptide from the tonoplast of beetroot may be a new member of the TIP family. It appears to represent a useful specific marker for the vacuole membrane at all developmental stages of the storage parenchyma cells.

Effects of two peroxisome proliferators (ciprofibrate and fenofibrate) on peroxisomal membrane proteins and dihydroxyacetone-phosphate acyl-transferase activity in rat liver.

The effects of ciprofibrate and fenofibrate, which are more potent peroxisome proliferators than clofibrate, on the activities of dihydroxyacetone-phosphate acyl-transferase (DHAP-AT) and glycerol-3-phosphate acyl-transferase (G3P-AT) were studied at the two pH optima 5.5 and 7.4 in subcellular fractions of rat liver, and in solubilized peroxisomal membranes (PMP) as well. Protein was also analyzed by gel electrophoresis. 1) Under the conditions of the specific activity of peroxisomal acyl-CoA oxidase (CN(-)-ACO) being increased (8 to 9-fold), there was no specific induction of the DHAP-AT activity when measured at pH 5.5 in purified peroxisomes and PMP. However, the total activities of DHAP-AT in these two fractions were increased by 6 to 11 times, as a result of hepatomegaly and peroxisome proliferation. In contrast, they were only slightly enhanced (x 1.1 to 2.2-fold) when determined at pH 7.4. The magnitude of the effects of a fibrate treatment was, therefore, dependent on the pH of the incubation medium. 2) Experiments of reversibility of enzyme induction reinforced the finding that the peroxisomal DHAP-AT activity is not specifically induced by ciprofibrate and fenofibrate. 3) Our results suggest the existence of a peroxisomal G3P-AT, non-inducible by fibrates, in the rat liver. 4) Induction of peroxisomal membrane-associated polypeptides with apparent molecular masses of 26- and 36-kDa was evidenced in stained electrophoretic gels of protein.