Marie-Claude Clémencet

Regulation of the peroxisomal β-oxidation-dependent pathway by peroxisome proliferator-activated receptor α and kinases

Abstract 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 × 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. PPARα, activated by hypolipidemic agents of the fibrate family or by leukotrienes; regulates lipid metabolism as well as the detoxifying enzyme-encoding genes. PPARβ/δ, which is not very well known yet, appears to be more specifically activated by fatty acids. PPARγ (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. PPARα is mainly expressed in liver and kidney, while PPARβ/δ is almost constitutively expressed. In contrast, PPARγ is largely expressed in white adipose tissue. PPAR is a transcriptional factor that requires other nuclear proteins in order to function, i.e. RXRα (9- cis -retinoic acid receptor α) 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 PPARα in the regulation of peroxisomal fatty acid β-oxidation is presented. In relation to this, we showed that PPARα activates peroxisomal β-oxidation-encoding genes such as acyl-CoA oxidase, multifunctional protein, and thiolase (Bardot et al., FEBS Lett 360: 183–186, 1995). Moreover, rat liver PPARα 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, PPARα and kinases appear to play an important role in lipid homeostasis.

Molecular cloning, gene structure and expression profile of two mouse peroxisomal 3-ketoacyl-CoA thiolase genes

BackgroundIn rats, two peroxisomal 3-ketoacyl-CoA thiolase genes (A and B) have been cloned, whereas only one thiolase gene is found in humans. The aim of this study was thus to clone the different mouse thiolase genes in order to study both their tissue expression and their associated enzymatic activity.ResultsIn this study, we cloned and characterized two mouse peroxisomal 3-ketoacyl-CoA thiolase genes (termed thiolase A and B). Both thiolase A and B genes contain 12 exons and 11 introns. Using RNA extracted from mouse liver, we cloned the two corresponding cDNAs. Thiolase A and B cDNAs possess an open reading frame of 1272 nucleotides encoding a protein of 424 amino acids. In the coding sequence, the two thiolase genes exhibited ≈97% nucleotide sequence identity and ≈96% identity at the amino acid level. The tissue-specific expression of the two peroxisomal 3-ketoacyl-CoA thiolase genes was studied in mice. Thiolase A mRNA was mainly expressed in liver and intestine, while thiolase B mRNA essentially exhibited hepatic expression and weaker levels in kidney, intestine and white adipose tissue. Thiolase A and B expressions in the other tissues such as brain or muscle were very low though these tissues were chiefly involved in peroxisomal disorders. At the enzymatic level, thiolase activity was detected in liver, kidney, intestine and white adipose tissue but no significant difference was observed between these four tissues. Moreover, thiolase A and B genes were differently induced in liver of mice treated with fenofibrate.ConclusionTwo mouse thiolase genes and cDNAs were cloned. Their corresponding transcripts are mostly expressed in the liver of mice and are differently induced by fenofibrate.

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.

Changes of peroxisomal fatty acid metabolism during cold acclimatization in hibernating jerboa (Jaculus orientalis).

Jerboa (Jaculus orientalis) is a deep hibernator originating from sub-desert highlands and represents an excellent model to help to understand the incidence of seasonal variations of food intake and of body as well as environmental temperatures on lipid metabolism. In jerboa, hibernation processes are characterized by changes in the size of mitochondria, the number of peroxisomes in liver and in the expression of enzymes linked to fatty acid metabolism. In liver and kidney, cold acclimatization shows an opposite effect on the activities of the mitochondrial acyl-CoA dehydrogenase (-50%) and the peroxisomal acyl-CoA oxidase (AOX) (+50%), while in brown and white adipose tissues, both activities are decreased down to 85%. These enzymes activities are subject to a strong induction in brown and in white adipose tissue (3.4- to 7.5-fold, respectively) during the hibernation period which is characterized by a low body temperature (around 10 degrees C) and by starvation. Expression level of AOX mRNA and protein are increased during both pre-hibernation and hibernation periods. Unexpectedly, treatment with ciprofibrate, a hypolipemic agent, deeply affects lipolysis in brown adipose tissue by increasing acyl-CoA dehydrogenase activity (3.4-fold), both AOX activity and mRNA levels (2.8- and 3.8-fold, respectively) during pre-hibernation. Therefore, during pre-hibernation acclimatization, there is a negative regulation of fatty acid degradation allowing to accumulate a lipid stock which is later degraded during the hibernation period (starvation) due to a positive regulation of enzymes providing the required energy for animal survival.

NFY interacts with the promoter region of two genes involved in the rat peroxisomal fatty acid β-oxidation: the multifunctional protein type 1 and the 3-ketoacyl-CoA B thiolase

Backgroundβ-oxidation of long and very long chain fatty acyl-CoA derivatives occurs in peroxisomes, which are ubiquitous subcellular organelles of eukaryotic cells. This pathway releases acetyl-CoA as precursor for several key molecules such as cholesterol. Numerous enzymes participating to cholesterol and fatty acids biosynthesis pathways are co-localized in peroxisomes and some of their encoding genes are known as targets of the NFY transcriptional regulator. However, until now no interaction between NFY transcription factor and genes encoding peroxisomal β-oxidation has been reported.ResultsThis work studied the interactions between NFY factor with the rat gene promoters of two enzymes of the fatty acid β-oxidation, MFP-1 (multifunctional protein type 1) and ThB (thiolase B) and their involvement in the cholesterol dependent-gene regulation. Binding of this nuclear factor to the ATTGG motif of the MFP-1 and of the ThB promoters was demonstrated by EMSA (Electrophoretic Mobility Shift Assay) and super shift assay. In contrast, in spite of the presence of putative Sp1 binding sites in these promoters, competitive EMSA did not reveal any binding. The promoter-dependent luciferase gene expression was downregulated by cholesterol in MFP-1 and ThB promoters harbouring constructs.ConclusionsThis work describes for the first time a NFY interaction with promoter sequences of the peroxisomal β-oxidation encoding genes. It suggests that cholesterol would negatively regulate the expression of genes involved in β-oxidation, which generates the initial precursor for its own biosynthesis, via at least the NFY transcription factor.

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, α 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.

Tissue-specific Expression of Two Peroxisomal 3-ketoacyl-CoA Thiolase Genes in Wild and PPARα-null Mice and Induction by Fenofibrate

Our laboratory cloned two peroxisomal 3-ketoacyl-CoA thiolase genes in mouse. These genes were named mThA (mouse peroxisomal Thiolase A) and mThB (mouse peroxisomal Thiolase B) by comparison with peroxisomal thiolase genes known in rat (Hijikata et al. 1990, Bodnar & Rachubinski, 1990). In this study, we analysed the tissue expression of the two thiolase genes on wild and on PPARa-null mice.

Gene Regulation of Peroxisomal Enzymes by Nutrients, Hormones and Nuclear Signalling Factors in Animal and Human Species

Many peroxisomal enzymes are controlled at the transcriptional level. This gene regulation is well documented in liver from rodent species and is more important upon peroxisome proliferation, although both phenomena are not always associated. Understanding of this regulation comes largely from studies on PPARs (Peroxisome Proliferator-Activated Receptors). Other transcription factors including thyroid hormone receptors, glucocorticoid receptors, LXR, also influence peroxisomal gene expression often in combination with tissue specific cofactors (co-activators or co-repressors). In human tissues and cells, inducibility of peroxisomal enzymes often has not been investigated. De Craemer (1995) reviewed peroxisome proliferation in human liver diseases caused by a large variety of drugs, metabolites, infectious agents or malignancies. Duclos et al (1997) and many others found that isolated human liver cells are almost resistant to classical peroxisome proliferators (fibrates). Overexpression of mouse PPARa in human cells does not affect expression of peroxisomal fatty acid P-oxidation enzymes while, in these circumstances, the mitochondrial counterpart is subject to regulation (Hsu et al., 2001; Lawrence et al., 2001). Indeed, PPARa activates human muscle carnitine palmitoyl transferase I-CPT I (Mascaro et al 1999). On the other hand, PPAR agonists have been shown to repress human cytochrome CYP4F2-LTB4 ω-hydroxylase promoter (Zhang et al. 2000).

Peroxisome proliferation in rodents and human: A model of cell organelle biogenesis☆

ASSAKA L.,PACOTC.,BARDOTO.elLATRUFFE N.(1991). Cell.Mo[. Biol., 37, 123-133). This peroxisomal proliferation is characterized by enhanced peroxisomes biogenesis and specific genes activation. In one hand, our current knowledges on biogenesis indicate that peroxisomal number increase derives from the fission of pre-existing peroxisomes provoked by the import in the organelle of newly svnthesized oroteins and lioids. The oolvueptides neo-svnthesized on free cytosolic poiysomes are addressed io pyroxisomes, either by C-terminal label: the SKL amino acids motif, or by N-terminal signal. Two membrane receptors for polypeptides translocation have been already identified (MANNAERTS G., Belgium and SUBRAMANlS.,USA). On the other hand. neroxisome oroliferation is associated with eenes activation under n’u’clear recep’tors control: PPAR (Peroxisome Proliferator Activated Recentor) and RXR (9-cis Retinoid Acid Receptor)