Identification of Novel Peroxisome Proliferator-Activated Receptor α (PPARα) Target Genes in Mouse Liver Using cDNA Microarray Analysis

Abstract

Peroxisome proliferators, which function as peroxisome proliferator-activated receptor-alpha (PPARalpha) agonists, are a group of structurally diverse nongenotoxic hepatocarcinogens including the fibrate class of hypolipidemic drugs that induce peroxisome proliferation in liver parenchymal cells. Sustained activation of PPARalpha by these agents leads to the development of liver tumors in rats and mice. To understand the molecular mechanisms responsible for the pleiotropic effects of these agents, we have utilized the cDNA microarray to generate a molecular portrait of gene expression in the liver of mice treated for 2 weeks with Wy-14,643, a potent peroxisome proliferator. PPARalpha activation resulted in the stimulation of expression (fourfold or greater) of 36 genes and decreased the expression (fourfold or more decrease) of 671 genes. Enhanced expression of several genes involved in lipid and glucose metabolism and many other genes associated with peroxisome biogenesis, cell surface function, transcription, cell cycle, and apoptosis has been observed. These include: CYP2B9, CYP2B10, monoglyceride lipase, pyruvate dehydrogenase-kinase-4, cell death-inducing DNA-fragmentation factor-alpha, peroxisomal biogenesis factor 11beta, as well as several cell recognition surface proteins including annexin A2, CD24, CD39, lymphocyte antigen 6, and retinoic acid early transcript-gamma, among others. Northern blotting of total RNA extracted from the livers of PPARalpha-/- mice and from mice lacking both PPARalpha and peroxisomal fatty acyl-CoA oxidase (AOX), that were fed control and Wy-14,643-containing diets for 2 weeks, as well as time course of induction following a single dose of Wy-14,643, revealed that upregulation of genes identified by microarray procedure is dependent upon peroxisome proliferation vis-à-vis PPARalpha. However, cell death-inducing DNA-fragmentation factor-alpha mRNA, which is increased in the livers of wild-type mice treated with peroxisome proliferators, was not enhanced in AOX-/- mice with spontaneous peroxisome proliferation. These observations indicate that the activation of PPARalpha leads to increased and decreased expression of many genes not associated with peroxisomes, and that delayed onset of enhanced expression of some genes may be the result of metabolic events occurring secondary to PPARalpha activation and alterations in lipid metabolism.

Overexpression of Peroxisome Proliferator-activated Receptor-α (PPARα)-regulated Genes in Liver in the Absence of Peroxisome Proliferation in Mice Deficient in both l- and d-Forms of Enoyl-CoA Hydratase/Dehydrogenase Enzymes of Peroxisomal β-Oxidation System

“…4A). We noted marked increase in the mRNA content of Riken clone W09719 in earlier cDNA microarrays, which was identified as a PPAR␣-regulated gene in liver (19,26). Recently, this Riken clone W09719 has been found identical to SCP2/SCPx (27,28), encoding for the second thiolase enzyme (1-4).…”

“…Northern Analysis-Total RNA extracted from liver with TRIzol reagent (Invitrogen) was glyoxylated, separated on 0.8% agarose gel, transferred to nylon membrane, and then hybridized at 42°C in 50% formamide hybridization solution using 32 P-labeled cDNA probes as described (17,19). Changes in mRNA levels were estimated by densitometric scanning of autoradiograms.…”

“…4A). Northern analysis of the mRNA levels of some of the recently identified PPAR␣-regulated genes in liver, namely CD36, Ly-6D, PDK-4, SHLR, Riken clone W09719 (now identified as SCP2/SCPx), monoglyceride lipase (mLipase), and C3f (19,26) showed increases in expression in L-PBE Ϫ/Ϫ D-PBE Ϫ/Ϫ , and AOX Ϫ/Ϫ mouse livers and in the livers of wild-type mice treated with Wy-14,643, a synthetic peroxisome proliferator (Fig. 4B).…”

Functional significance of the two ACOX1 isoforms and their crosstalks with PPARα and RXRα

“…4 At the transcriptional level, PPARa, which forms a heterodimer with retinoid X receptor (RXR), 5,6 binds to peroxisome proliferator-responsive element (PPRE) present in the 5 0 -flanking region of target genes. [6][7][8] In the presence of a ligand, PPAR-RXR complexes recruit coactivators, such as steroid receptor coactivator-1 (SRC-1) and PPAR-binding protein/Mediator 1 (PBP/TRAP220/Med1) 9,10 to enhance target gene transcription. [9][10][11] The generation of PparaÀ/À mice established that PPARa is essential for hepatic peroxisome proliferation and coordinates transcriptional activation of genes coding for ACOX1, L-peroxisomal bifunctional enzyme (L-PBE), peroxisomal thiolase (PTL), cytochrome P 450 4A10 (CYP4A10) and cytochrome P 450 4A14 (CYP4A14), and other lipid metabolism-related key enzymes by structurally diverse synthetic peroxisome proliferators, as well as by several fatty acids and their polyunsaturated derivatives.…”

Time course investigation of PPARα- and Kupffer cell-dependent effects of WY-14,643 in mouse liver using microarray gene expression

“…Genomic studies investigating the effects of peroxisome proliferators in rodent liver have provided critical insight into the molecular mechanisms responsible for liver-specific effects of peroxisome proliferators in rodents and supported a non-genotoxic mechanism of their action (Cherkaoui-Malki et al, 2001;Hamadeh et al, 2002;Currie et al, 2005). However, the gap in our knowledge remains on the temporal relationship between peroxisome proliferator-modulated effects and molecular mediators of these effects, especially at the early time points where PPARα-independent events are also known to occur.…”