Janardan K. Reddy

Isolation and Characterization of PBP, a Protein That Interacts with Peroxisome Proliferator-activated Receptor*

In an attempt to identify cofactors that could possibly influence the transcriptional activity of peroxisome proliferator-activated receptors (PPARs), we used a yeast two-hybrid system with Gal4-PPARγ as bait to screen a mouse liver cDNA library and have identified steroid receptor coactivator-1 (SRC-1) as a PPAR transcriptional coactivator. We now report the isolation of a cDNA encoding a 165-kDa PPARγ-binding protein, designated PBP which also serves as a coactivator. PBP also binds to PPARα, RARα, RXR, and TRβ1, and this binding is increased in the presence of specific ligands. Deletion of the last 12 amino acids from the carboxyl terminus of PPARγ results in the abolition of interaction between PBP and PPARγ. PBP modestly increased the transcriptional activity of PPARγ, and a truncated form of PBP (amino acids 487–735) acted as a dominant-negative repressor, suggesting that PBP is a genuine coactivator for PPAR. In addition, PBP contains two LXXLL signature motifs considered necessary and sufficient for the binding of several coactivators to nuclear receptors. In situhybridization and Northern analysis showed that PBP is expressed in many tissues of adult mice, including the germinal epithelium of testis, where it appeared most abundant, and during ontogeny, suggesting a possible role for this cofactor in cellular proliferation and differentiation.

PPARalpha: energy combustion, hypolipidemia, inflammation and cancer.

The peroxisome proliferator-activated receptor α (PPARα, or NR1C1) is a nuclear hormone receptor activated by a structurally diverse array of synthetic chemicals known as peroxisome proliferators. Endogenous activation of PPARα in liver has also been observed in certain gene knockout mouse models of lipid metabolism, implying the existence of enzymes that either generate (synthesize) or degrade endogenous PPARα agonists. For example, substrates involved in fatty acid oxidation can function as PPARα ligands. PPARα serves as a xenobiotic and lipid sensor to regulate energy combustion, hepatic steatosis, lipoprotein synthesis, inflammation and liver cancer. Mainly, PPARα modulates the activities of all three fatty acid oxidation systems, namely mitochondrial and peroxisomal β-oxidation and microsomal co-oxidation, and thus plays a key role in energy expenditure. Sustained activation of PPARα by either exogenous or endogenous agonists leads to the development of hepatocellular carcinoma resulting from sustained oxidative and possibly endoplasmic reticulum stress and liver cell proliferation. PPARα requires transcription coactivator PPAR-binding protein (PBP)/mediator subunit 1(MED1) for its transcriptional activity.

Hydrogen peroxide generation in peroxisome proliferator-induced oncogenesis.

Peroxisome proliferators are a structurally diverse group of non-genotoxic chemicals that induce predictable pleiotropic responses including the development of liver tumors in rats and mice. These chemicals interact variably with peroxisome proliferator-activated receptors (PPARs), which are members of the nuclear receptor superfamily. Evidence derived from mice with PPARalpha gene disruption indicates that of the three PPAR isoforms (alpha, beta/delta and gamma), the isoform PPARalpha is essential for the pleiotropic responses induced by peroxisome proliferators. Peroxisome proliferator-induced activation of PPARalpha leads to profound transcriptional activation of genes encoding for the classical peroxisomal beta-oxidation system and cytochrome P450 CYP 4A isoforms, CYP4A1 and CYP4A3, among others. Livers with peroxisome proliferation manifest substantial increases in the expression of H(2)O(2)-generating peroxisomal fatty acyl-CoA oxidase, the first enzyme of the classical peroxisomal fatty acid beta-oxidation system, and of microsomal cytochrome P450 4A1 and 4A3 genes. Disproportionate increases in H(2)O(2)-generating enzymes and H(2)O(2)-degrading enzyme catalase and reductions in glutathione peroxidase activity by peroxisome proliferators, lead to increased oxidative stress in liver cells. Sustained oxidative stress resulting from chronic increases in H(2)O(2)-generating enzymes manifests as massive accumulation of lipofuscin in hepatocytes, and increased levels of 8-hydroxydeoxyguanosine adducts in liver DNA; this supports the hypothesis that oxidative stress plays a critical role in the development of liver tumors induced by these non-genotoxic chemical carcinogens. Evidence also indicates that cells stably overexpressing H(2)O(2)-generating fatty acyl-CoA oxidase or urate oxidase, when exposed to appropriate substrate(s), reveal features of neoplastic conversion including growth in soft agar and formation of tumors in nude mice. Mice with disrupted fatty acyl-CoA oxidase gene (AOX(-/-) mice), which encodes the first enzyme of the PPARalpha regulated peroxisomal beta-oxidation system, exhibit profound spontaneous peroxisome proliferation, including development of liver tumors, indicative of sustained activation of PPARalpha by the unmetabolized substrates of acyl-CoA oxidase. With the exception of fatty acyl-CoA oxidase, all PPARalpha responsive genes including CYP4A1 and CYP4A3 are up-regulated in the livers of these AOX(-/-) mice. Thus, the substrates of acyl-CoA oxidase serve as endogenous ligands for this receptor leading to a receptor-enzyme cross-talk, because acyl-CoA oxidase gene is transcriptionally regulated by PPARalpha. Peroxisome proliferators induce only a transient increase in liver cell proliferation and this may serve as an additional contributory factor, rather than play a primary role in liver tumor development. Thus, sustained activation of PPARalpha by either synthetic or natural ligands leads to reproducible pleiotropic responses culminating in the development of liver tumors. This phenomenon of peroxisome proliferation provides fascinating challenges in exploring the molecular mechanisms of cell specific transcription, and in identifying the PPARalpha responsive target genes, as well as events involved in their regulation. Genetically altered animals and cell lines should enable investigations on the role of H(2)O(2)-producing enzymes in neoplastic conversion.

Hepatic peroxisome (microbody) proliferation in rats fed plasticizers and related compounds

Abstract Male rats were fed the plasticizers di-(2-ethylhexyl) phthalate (DEHP), di-(2-ethylhexyl) adipate (DEHA), di-(2-ethylhexyl) sebacate (DEHS), adipic acid, and diethyl phthalate at a dietary concentration of 2% for 3 weeks. Hepatic peroxisome proliferation in association with an increase in liver size, increase of hepatic activities of the peroxisome-associated enzymes catalase and carnitine acetyltransferase, and hypolipidemia were observed in animals treated with DEHP, DEHA, and DEHS but not in animals fed adipic acid and diethyl phthalate. To relate structure to biological activity, additional groups of rats were fed 2-ethylhexyl alcohol (a metabolite of DEHP), hexyl alcohol. 2-ethylhexanoic acid, hexanoic acid, 2-ethylhexyl aldehyde, hexylaldehyde, and 2-ethylhexyl amine at a 2% dose level. The changes induced by 2-ethylhexyl alcohol and 2-ethylhexanoic acid were comparable to those induced by DEHP, DEHA, and DEHS, suggesting that 2-ethylhexyl alcohol is the active part of the molecule responsible for peroxisome proliferation. 2-Ethylhexyl aldehyde induced a moderate increase in peroxisome population. No effect on hepatic peroxisomes or their associated enzymes was induced by the straight-chained analogs hexyl alcohol, hexanoic acid, and hexyl aldehyde. The hepatic effects of plasticizers capable of inducing peroxisome proliferation are similar to those resulting from treatment with clofibrate and other hypolipidemic drugs.

Peroxisomal and mitochondrial fatty acid beta-oxidation in mice nullizygous for both peroxisome proliferator-activated receptor alpha and peroxisomal fatty acyl-CoA oxidase. Genotype correlation with fatty liver phenotype.

Fatty acid β-oxidation occurs in both mitochondria and peroxisomes. Long chain fatty acids are also metabolized by the cytochrome P450 CYP4A ω-oxidation enzymes to toxic dicarboxylic acids (DCAs) that serve as substrates for peroxisomal β-oxidation. Synthetic peroxisome proliferators interact with peroxisome proliferator activated receptor α (PPARα) to transcriptionally activate genes that participate in peroxisomal, microsomal, and mitochondrial fatty acid oxidation. Mice lacking PPARα (PPARα−/−) fail to respond to the inductive effects of peroxisome proliferators, whereas those lacking fatty acyl-CoA oxidase (AOX−/−), the first enzyme of the peroxisomal β-oxidation system, exhibit extensive microvesicular steatohepatitis, leading to hepatocellular regeneration and massive peroxisome proliferation, implying sustained activation of PPARα by natural ligands. We now report that mice nullizygous for both PPARα and AOX (PPARα−/− AOX−/−) failed to exhibit spontaneous peroxisome proliferation and induction of PPARα-regulated genes by biological ligands unmetabolized in the absence of AOX. In AOX−/− mice, the hyperactivity of PPARα enhances the severity of steatosis by inducing CYP4A family proteins that generate DCAs and since they are not metabolized in the absence of peroxisomal β-oxidation, they damage mitochondria leading to steatosis. Blunting of microvesicular steatosis, which is restricted to few liver cells in periportal regions in PPARα−/−AOX−/− mice, suggests a role for PPARα-induced genes, especially members of CYP4A family, in determining the severity of steatosis in livers with defective peroxisomal β-oxidation. In age-matched PPARα−/− mice, a decrease in constitutive mitochondrial β-oxidation with intact constitutive peroxisomal β-oxidation system contributes to large droplet fatty change that is restricted to centrilobular hepatocytes. These data define a critical role for both PPARα and AOX in hepatic lipid metabolism and in the pathogenesis of specific fatty liver phenotype.

Hepatocellular and hepatic peroxisomal alterations in mice with a disrupted peroxisomal fatty acyl-coenzyme A oxidase gene.

Peroxisomal genetic disorders, such as Zellweger syndrome, are characterized by defects in one or more enzymes involved in the peroxisomal β-oxidation of very long chain fatty acids and are associated with defective peroxisomal biogenesis. The biologic role of peroxisomal β-oxidation system, which consists of three enzymes: fatty acyl-CoA oxidase (ACOX), enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase (HD), and thiolase, has been examined in mice by disrupting ACOX gene, which encodes the first and rate-limiting enzyme of this system. Homozygous (ACOX −/−) mice lacked the expression of ACOX protein and accumulate very long chain fatty acids in blood. However, these homozygous mice are viable, but growth-retarded and infertile. During the first 3-4 months of age, the livers of ACOX −/− mice reveal severe microvesicular fatty metamorphosis of hepatocytes. In such steatotic cells, peroxisome assembly is markedly defective; as a result, they contain few or no peroxisomes. Few hepatocytes in 1-3-month-old ACOX −/− mice contain numerous peroxisomes, and these peroxisome-rich hepatocytes show no fatty change. At this stage, the basal mRNA levels of HD, thiolase, and other peroxisome proliferator-induced target genes were elevated in ACOX −/− mouse liver, but these mice, when treated with a peroxisome proliferator, showed no increases in the number of hepatic peroxisomes and in the mRNAs levels of these target genes. Between 4 and 5 months of age, severe steatosis resulted in scattered cell death, steatohepatitis, formation of lipogranulomas, and focal hepatocellular regeneration. In 6-7-month-old animals, the newly emerging hepatocytes, which progressively replaced steatotic cells, revealed spontaneous peroxisome proliferation. These livers showed marked increases in the mRNA levels of the remaining two genes of the β-oxidation system, suggesting that ACOX gene disruption leads to increased endogenous ligand-mediated transcription levels. These observations demonstrate links among peroxisomal β-oxidation, development of severe microvesicular fatty liver, peroxisome assembly, cell death, and cell proliferation in liver.

Peroxisome proliferator-activated receptors, coactivators, and downstream targets

Peroxisomes in liver parenchymal cells proliferate in response to structurally diverse nonmutagenic compounds designated as peroxisome proliferators (PP). Sustained induction of peroxisome proliferation and peroxisomal fatty acid β-oxidation system in rats and mice leads to the development of liver tumors. Two mechanistic issues are important for consideration: elucidation of the upstream events responsible for the tissue and species specific induction of the characteristic pleiotropic responses by PPs; and delineation of the downstream events associated with peroxisome proliferation, and their role in the development of liver tumors in species that are sensitive to the induction of peroxisome proliferation. The induction of peroxisome proliferation is mediated by PP-activated receptor α (PPARα), a member of a group of transcription factors that regulate the expression of genes associated with lipid metabolism and adipocyte differentiation. Three isotypes of this family of nuclear receptors, namely PPARα, PPARγ, and PPARδ (also called β), have been identified as products of separate genes. Although PPARα is responsible for the PP-induced pleiotropic responses, PPARγ seems to be involved in adipogenesis and differentiation, but the events associated with PPARγ do not directly involve peroxisomes and peroxisome proliferation. PPARs heterodimerize with 9-cis retinoic acid receptor (RXR), and bind to PP response element(s) (PPREs) on the target gene promoter to initiate inducible transcriptional activity. Tissue and species responses to PPs depend on pharmacokinetics, relative abundance of PPAR isotypes, nature of PPRE in the upstream regions of target genes, the extent of competition or cross-talk among nuclear transcription factors for PPAR heterodimerization partner retinoid X receptor and the modulating role of coactivators and corepressors on ligand-dependent transcription of PPARs. Using PPAR as bait in the yeast two-hybrid system, the authors recently cloned mouse steroid receptor coactivator-1 (SRC-1) and PPAR-binding protein (PBP), and identified them as PPAR coactivators. Both SRC-1 and PBP contain LXXLL signature motifs, considered necessary and sufficient for the binding of coactivators to nuclear receptors. A multifaceted approach, which includes the identification of additional coactivators that may be responsible for cell specific transcriptional activation of PPAR-mediated target genes, and generation of genetically modified animals (transgenic and gene disrupted), will be necessary to gain more insight into the upstream and downstream targets responsible for the induction of early and delayed PP-induced pleiotropic responses. In this context, it is important to note that mice deficient in fatty acyl-CoA oxidase, the first and rate-limiting enzyme of the peroxisomal β-oxidation system, revealed that this enzyme is indispensable for the physiological regulation of PPARα, and the absence of this enzyme leads to sustained transcriptional activation of genes regulated by this receptor.

Oxidative DNA damage caused by persistent peroxisome proliferation: its role in hepatocarcinogenesis

Peroxisome proliferators are considered as a novel class of hepatocarcinogenic agents because of their non-mutagenic nature and their ability to cause a significant increase in the levels of hydrogen peroxide generating peroxisomal fatty acid beta-oxidation enzyme system in the liver. Sustained increase in the number of peroxisomes in liver has been shown to induce oxidative stress in the liver. Increased levels of H2O2 generation, hydroxyl free-radical formation, lipid peroxidation and accumulation of lipofuscin are found in the livers of rats following long-term treatment with peroxisome proliferators. Recent evidence indicates the presence of 8-hydroxydeoxyguanosine in the liver DNA of rats chronically treated with a peroxisome proliferator suggesting that this may be the basis for carcinogenesis by this class of non-mutagenic carcinogens.

Evaluation of Selected Hypolipidemic Agents for the Induction of Peroxisomal Enzymes and Peroxisome Proliferation in the Rat Liver

There is a considerable interest in developing potent and safe hypolipidemic drugs for the prevention and management of coronary heart disease in man. In rodents, many of these hypolipidemic compounds induce hepatomegaly, proliferation of peroxisomes and a polypeptide with an approximate mol. wt. of 80000 in liver cells. In the present study, we have examined 10 hypolipidemic compounds for the induction of peroxisome proliferation associated 80000 mol. wt. polypeptide (polypeptide PPA-80), peroxisomal enzymes and peroxisome proliferation in rat liver, in view of the emerging evidence that hepatic peroxisome proliferators as a class are carcinogenic in rats and mice. All ten compounds, fenofibrate (isopropyl-[4-( p-chlorobenzoyl)2-phenoxy-2-methyl] propionate; LS 2265 (taurine derivative of fenofibrate); bezafibrate (2-{4-(2-[4-chlorobenzamido)ethyl] phenoxy}-methyl propionic acid; gemfibrozil (5-2[2,5-dimethylphenoxy]2-2-dimethylpentanoic acid); methyl clofenapate (methyl-2-[4-(p-chlorophenyl)phenoxy]-2-methyl propionate); DG 5685 (5-[4-phenoxybenzyl]trans-2-(3-pyridyl)1,3-dioxane), DH6463 (5-[4-phenoxybenzyl] trans-2-(3-pyrimidinyl)-1,3-dioxane); tiadenol(bis[hydroxyethylthio]-7, 10-decane); ciprofibrate (2,-[4-{2,2-dichlorocyclopropyl}-phenoxy]2-methyl propionic acid) and RMI-14,514 ([5-tetradecycloxyl-2-furancarboxylic acid), produced a marked but variable increase in the activities of peroxisomal enzymes catalase, carnitine acetyltransferase, heat-labile enoyl-CoA hydratase and the fatty acid β-oxidation system and in the amount of polypeptide PPA-80 as demonstrated by SDS-polyacrylamide gel electrophoresis. The peptide map patterns of polypeptide PPA-80 in liver induced by these compounds were strikingly similar. The ultrastructural studies demonstrate that fenofibrate, ciprofibrate, LS 2265, DG 5685 and DH 6463 can induce proliferation of peroxisomes in liver cells of rats, and further confirm the previous reports of hepatic peroxisome proliferative activity of methyl clofenapate, tiadenol, bezafibrate, gemfibrozil and RMI-14514, as shown morphologically. Whether these structurally unrelated chemicals or their metabolite(s) directly activate the peroxisome specific genes to induce this multi-enzyme system or they exert their action on peroxisomes indirectly by causing fatty acid overload in hepatocytes remains to be elucidated. These chemicals offer a simple and reproducible means of stimulating peroxisomal enzymes in liver and should serve as useful tools, for evaluating the implications of hepatic peroxisome proliferation and in elucidating the mechanism of peroxisome proliferator-induced carcinogenesis.

Identification of novel peroxisome proliferator-activated receptor alpha (PPARalpha) target genes in mouse liver using cDNA microarray analysis.

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.