Putative Peroxisome Proliferator Response Element Research Articles

WY-14643 Regulates CYP1B1 Expression through Peroxisome Proliferator-Activated Receptor α-Mediated Signaling in Human Breast Cancer Cells.

Human cytochrome P450 1B1 (CYP1B1)-mediated biotransformation of endobiotics and xenobiotics plays an important role in the progression of human breast cancer. In this study, we investigated the effects of WY-14643, a peroxisome proliferator-activated receptor α (PPARα) agonist, on CYP1B1 expression and the related mechanism in MCF7 breast cancer cells. We performed quantitative reverse transcription-polymerase chain reaction, transient transfection, and chromatin immunoprecipitation to evaluate the effects of PPARα on peroxisome proliferator response element (PPRE)-mediated transcription. WY-14643 increased the protein and mRNA levels of CYP1B1, as well as promoter activity, in MCF-7 cells. Moreover, WY-14643 plus GW6471, a PPARα antagonist, significantly inhibited the WY-14643-mediated increase in CYP1B1 expression. PPARα knockdown by a small interfering RNA markedly suppressed the induction of CYP1B1 expression by WY-14643, suggesting that WY-14643 induces CYP1B1 expression via a PPARα-dependent mechanism. Bioinformatics analysis identified putative PPREs (−833/−813) within the promoter region of the CYP1B1 gene. Inactivation of these putative PPREs by deletion mutagenesis suppressed the WY-14643-mediated induction of CYP1B1 promoter activation. Furthermore, WY-14643 induced PPARα to assume a form capable of binding specifically to the PPRE-binding site in the CYP1B1 promoter. Our findings suggest that WY-14643 induces the expression of CYP1B1 through activation of PPARα.

PPARγ-mediated G-protein coupled receptor 120 signaling pathway promotes transcriptional activation of miR-143 in adipocytes

MicroRNAs (miRNAs), the small noncoding RNAs, regulate various biological processes such as adipogenesis. MicroRNA-143 (miR-143) promotes adipocyte differentiation, and is correlated with obesity in mice fed a high-fat diet. However, the transcriptional regulation of miR-143 is largely unknown. In this study, we identified that miR-143 is a target of peroxisome proliferator-activated receptor γ (PPARγ), a key transcription factor in adipogenesis. Four putative peroxisome proliferator response elements (PPREs) were identified in the miR-143 promoter region. Using chromatin immune-precipitation, we observed that PPARγ was bound with two PPRE regions of the miR-143 promoter in 3T3-L1 adipocytes. A luciferase reporter assay showed that the PPRE1 region (-1330/-1309) of the miR-143 promoter played an important role in PPARγ transcriptional activation. In addition, we determined that G-protein coupled receptor 120 (GPR 120), which functions as an omega 3 fatty acid receptor, affected miR-143 expression in adipocytes. GPR120 silencing in adipocytes inhibited the expression of PPARγ and miR-143, whereas GPR120 overexpression led to increased expressions of PPARγ and miR-143. Silencing of PPARγ inhibited the induction of miR-143 by GPR-120. These results suggested that a PPARγ-mediated GPR120 signaling pathway promotes transcriptional activation of miR-143 in adipocytes.

Transcriptional activation of PIK3R1 by PPARγ in adipocytes.

Phosphatidylinositol 3-kinase (PI3K) plays an important role in the metabolic actions of insulin and is required for adipogenesis. Regulatory subunit 1 of PI3K (PIK3R1) is a critical component of the PI3K signaling pathway. Peroxisome proliferator-activated receptor gamma (PPARγ) is a key regulator of adipogenesis. Although the PPARγ agonist rosiglitazone induces the expression of PIK3R1, the transcriptional regulation of PIK3R1 in adipocytes remains unknown. In this study, we investigated whether PIK3R1 is a direct target of PPARγ. The level of PIK3R1 expression in 3T3-L1 cells was increased after the induction of adipocyte differentiation and was also induced by overexpression of PPARγ. Furthermore, the upregulation of PPARγ-mediated PIK3R1 expression enhanced the insulin-stimulated AKT activation in 3T3-L1 cells. Two putative peroxisome proliferator response elements (PPREs) in the PIK3R1 promoter were identified as PPARγ binding sites. By chromatin immunoprecipitation, we observed that PPARγ interacts with the two PPRE regions of the PIK3R1 promoter in mature adipocytes. In addition, luciferase reporter assays showed that the -1183/-1161 and -573/-551 regions of the PIK3R1 promoter contain essential elements for PPARγ binding. Taken together, these results suggest that PPARγ is essential for the transcriptional activity of PIK3R1 during adipogenesis.

지방분화시 PPARγ에 의한 microRNA-17의 발현 조절

MicroRNAs comprise a family of small noncoding RNAs that modulate physiological processes, including adipogenesis. MicroRNA-17 (miR-17) promotes adipocyte differentiation and enhances lipid accumulation. The transcriptional regulation of miR-17 during adipogenesis remains unknown. In this study, we investigated whether miR-17 is a target of peroxisome proliferator-activated receptor γ (PPARγ), which is a key regulator of adipogenesis. The levels of miR-17 and the expression of PPARγ increased after the induction of adipocyte differentiation. Three putative peroxisome proliferator response elements (PPREs) were identified in the miR-17 promoter region. Using chromatin immunoprecipitation and luciferase reporter assays, we observed the interaction of PPARγ with the miR-17 promoter. Mutagenesis experiments showed that the -677/-655 region of the miR-17 promoter could function as a PPRE site. These results suggest that PPARγ is essential for transcriptional activation of the miR-17 gene, thereby contributing to understanding the molecular mechanism of adipogenesis in adipocytes.

Transcriptional activation of melanocortin 2 receptor accessory protein by PPARγ in adipocytes

Adrenocorticotropic hormone (ACTH) in rodents decreases lipid accumulation and body weight. Melanocortin receptor 2 (MC2R) and MC2R accessory protein (MRAP) are specific receptors for ACTH in adipocytes. Peroxisome proliferator-activated receptor γ (PPARγ) plays a role in the transcriptional regulation of metabolic pathways such as adipogenesis and β-oxidation of fatty acids. In this study we investigated the transcriptional regulation of MRAP expression during differentiation of 3T3-L1 cells. Stimulation with ACTH affected lipolysis in murine mature adipocytes via MRAP. Putative peroxisome proliferator response element (PPRE) was identified in the MRAP promoter region. In chromatin immunoprecipitation and reporter assays, we observed binding of PPARγ to the MRAP promoter. The mutagenesis experiments showed that the −1209/−1198 region of the MRAP promoter could function as a PPRE site. These results suggest that PPARγ is required for transcriptional activation of the MRAP gene during adipogenesis, which contributes to understanding of the molecular mechanism of lipolysis in adipocytes.

FAM3A is a target gene of peroxisome proliferator-activated receptor gamma

BackgroundTo date, the biological function of FAM3A, the first member of FAM3 gene family, remains unknown. We aimed to investigate whether the expression of FAM3A in liver cells is regulated by peroxisome proliferator-activated receptors (PPARs). Methods and resultsThe transcriptional activity of human and mouse FAM3A gene promoters was determined by luciferase reporter assay system. PPARγ agonist rosiglitazone induced FAM3A expression in primary cultured mouse hepatocytes and human HepG2 cells. PPARγ antagonism blocked rosiglitazone-induced FAM3A expression, whereas PPARγ overexpression stimulated FAM3A expression in HepG2 cells. In contrast, PPARα agonist fenofibrate or PPARβ agonist GW0742 failed to affect FAM3A expression in HepG2 cells. The transcriptional activities of human and mouse FAM3A promoters were markedly stimulated by PPARγ activation, but not by PPARα and PPARβ activation. Chromatin immunoprecipitation (ChIP) assay revealed a direct binding of PPARγ to the putative peroxisome proliferator response element (PPRE) located at −1258/−1246 in the human FAM3A promoter. Site-directed mutagenesis of this PPRE-like motif abolished PPARγ's stimulatory effect on the transcriptional activity of human FAM3A promoter. In vivo, oral rosiglitazone treatment upregulated FAM3A expression in the livers of C57BL/6 mice and db/db mice. Moreover, upregulation of FAM3A by PPARγ activation was correlated with increased level of phosphorylated Akt (pAkt) in liver cells. ConclusionsFAM3A as a novel target gene of PPARγ. Upregulation of FAM3A by PPARγ activation is correlated with increased pAkt level in liver cells. General significanceUpregulation of FAM3A might contribute to PPARγ's metabolic effects in the liver.

The mouse gene encoding the carnitine biosynthetic enzyme 4-N-trimethylaminobutyraldehyde dehydrogenase is regulated by peroxisome proliferator-activated receptor α

Genes involved in carnitine uptake and synthesis, such as organic cation transporter-2 (OCTN2) and γ-butyrobetaine dioxygenase (BBD), have been shown to be regulated by peroxisome proliferator-activated receptor (PPAR)α directly. Whether other genes encoding enzymes involved in the carnitine synthesis pathway, such as 4-N-trimethylaminobutyraldehyde dehydrogenase (TMABA-DH) and trimethyllysine dioxygenase (TMLD), are also direct PPARα target genes is less clear. In silico-analysis of the mouse TMLD promoter and first intron and the TMABA-DH promoter revealed several putative peroxisome proliferator response elements (PPRE) with high similarity to the consensus PPRE. Luciferase reporter gene assays using either a 2kb TMLD promoter or a 4kb TMLD first intron reporter constructs revealed no functional PPRE. In contrast, reporter gene assays using wild-type and mutated 5´-truncation TMABA-DH promoter reporter constructs showed that one PPRE located at position −132 in the proximal promoter is probably functional. Using gel shift assays we observed in vitro-binding of PPARα to this PPRE. Moreover, using chromatin immunoprecipitation assays we found that PPARα also binds in vivo to a nucleotide sequence spanning the PPRE at −132, which confirms that this PPRE is functional. In conclusion, the present study shows that the mouse TMABA-DH gene is a direct PPARα target gene. Together with the recent identification of the mouse BBD and the mouse OCTN2 genes as PPARα target genes this finding confirm that PPARα plays a key role in the regulation of carnitine homeostasis by controlling genes involved in carnitine synthesis and carnitine uptake.

Mouse γ-butyrobetaine dioxygenase is regulated by peroxisome proliferator-activated receptor α through a PPRE located in the proximal promoter

Convincing evidence from studies with peroxisome proliferator-activated receptor (PPAR)α-deficient mice suggested that the carnitine biosynthetic enzyme γ-butyrobetaine dioxygenase (BBD) is regulated by PPARα. However, the identification of BBD as a direct PPARα target gene as well as its exact regulation remained to be demonstrated. In silico-analysis of the mouse BBD promoter revealed seven putative peroxisome proliferator response elements (PPRE) with high similarity to the consensus PPRE. Luciferase reporter gene assays using mutated and non-mutated serial 5′-truncation BBD promoter reporter constructs revealed that one PPRE located at −75 to −87 relative to the transcription start site in the proximal BBD promoter is probably functional. Using gel shift assays we observed in vitro-binding of PPARα/RXRα heterodimer to this PPRE confirming that it is functional. In conclusion, the present study clearly shows that mouse BBD is a direct PPARα target gene and that transcriptional up-regulation of mouse BBD by PPARα is likely mediated by binding of the PPARα/RXR heterodimer to one PPRE located in its proximal promoter region. The results confirm emerging evidence from recent studies that PPARα plays a key role in the regulation of carnitine homeostasis by controlling genes involved in both, carnitine synthesis and carnitine uptake.

Peroxisome Proliferator-activated Receptor α Is Responsible for the Up-regulation of Hepatic Glucose-6-phosphatase Gene Expression in Fasting and db/db Mice

Glucose-6-phosphatase (G6Pase) is a key enzyme that is responsible for the production of glucose in the liver during fasting or in type 2 diabetes mellitus (T2DM). During fasting or in T2DM, peroxisome proliferator-activated receptor α (PPARα) is activated, which may contribute to increased hepatic glucose output. However, the mechanism by which PPARα up-regulates hepatic G6Pase gene expression in these states is not well understood. We evaluated the mechanism by which PPARα up-regulates hepatic G6Pase gene expression in fasting and T2DM states. In PPARα-null mice, both hepatic G6Pase and phosphoenolpyruvate carboxykinase levels were not increased in the fasting state. Moreover, treatment of primary cultured hepatocytes with Wy14,643 or fenofibrate increased the G6Pase mRNA level. In addition, we have localized and characterized a PPAR-responsive element in the promoter region of the G6Pase gene. Chromatin immunoprecipitation (ChIP) assay revealed that PPARα binding to the putative PPAR-responsive element of the G6Pase promoter was increased in fasted wild-type mice and db/db mice. These results indicate that PPARα is responsible for glucose production through the up-regulation of hepatic G6Pase gene expression during fasting or T2DM animal models.

Peroxisome Proliferator-Activated Receptor-γ Increases Adiponectin Secretion via Transcriptional Repression of Endoplasmic Reticulum Chaperone Protein ERp44

Adiponectin, an adipocyte-derived hormone, is a versatile player involved in the regulation of energy homeostasis, cardiovascular disease, and diabetes. Within adipocytes, adiponectin is retained in the lumen of the endoplasmic reticulum (ER) by binding to the thiol protein ER resident protein 44 kDa (ERp44), which is apparently regulated by the activation of nuclear receptor peroxisome proliferator-activated receptor (PPAR)-gamma. However, the precise role of ERp44 in adiponectin secretion remains elusive. In the present study, we investigated the functional correlation between ERp44 and adiponectin in a pig model. The transcription of porcine ERp44 was regulated by PPARgamma, which was consistent with the finding of putative peroxisome proliferator response element sites within ERp44 promoter. Using chromatin immunoprecipitation and luciferase reporter assays, we demonstrated that the transcription of porcine ERp44 is repressed through binding of PPARgamma to a peroxisome proliferator response element site located between positions -981 and -1004 in its 5'-flanking region. In human embryonic kidney 293 cells stably transfected with cDNA encoding porcine adiponectin, the secretion of adiponectin was significantly up-regulated and the ERp44 mRNA was down-regulated observably, by either the treatment of PPARgamma agonist rosiglitazone or the overexpression of PPARgamma in these cells. Taken together, our results indicated that PPARgamma is an essential regulatory factor for the transcriptional activity of ERp44, which in turn controls the secretion of adiponectin.

Mouse OCTN2 is directly regulated by peroxisome proliferator-activated receptor α (PPARα) via a PPRE located in the first intron

Recent studies provided strong evidence to suggest that organic cation transporter 2 (OCTN2) is a direct target gene of peroxisome proliferator-activated receptor α (PPARα). However, subsequent studies failed to demonstrate a functional peroxisome proliferator response element (PPRE) in the promoter region of the OCTN2 gene. In the present study we hypothesized that the OCTN2 gene is transcriptionally induced by PPARα via a functional PPRE located in the first intron. In silico-analysis of the first intron of mouse OCTN2 revealed 11 putative PPRE with high similarity to the consensus PPRE. In addition, reporter gene assays using a mouse OCTN2 intron reporter construct containing a cluster of three partially overlapping PPRE (PPREint-1-8-10) revealed a marked response to exogenous mouse PPARα/RXRα and subsequent stimulation with PPARα agonist WY-14,643. Introduction of a selective mutation in either PPRE8 or PPRE10 in the PPREint-1-8-10 reporter constructs caused a substantial loss of the responsiveness to PPARα activation, but a selective mutation in PPRE1 resulted in a complete loss of responsiveness to PPARα activation. Moreover, gel shift assays revealed binding of PPARα/RXRα heterodimer to the PPRE1 of mouse OCTN2 first intron. In conclusion, the present study shows that mouse OCTN2 is a direct target gene of PPARα and that transcriptional upregulation of OCTN2 by PPARα is likely mediated via PPRE1 in its first intron.

Cloning and expression pattern of peroxisomal β-oxidation genes palmitoyl-CoA oxidase, multifunctional protein and 3-ketoacyl-CoA thiolase in mussel Mytilus galloprovincialis and thicklip grey mullet Chelon labrosus

Due to the ability to respond after exposure to organic toxic compounds, peroxisome proliferation is used as biomarker of exposure to organic pollutants in mussels and in fish. Mussels are worldwide studied as sentinels of pollution in marine environments while mullets such as the thicklip grey mullet Chelon labrosus have been proposed as appropriate sentinel species since they inhabit highly polluted environments. In order to study genes of the inducible peroxisomal β-oxidation pathway in mussels Mytilus galloprovincialis and in C. labrosus, genes coding for the three enzymes in the inducible peroxisomal β-oxidation pathway, palmitoyl-CoA oxidase (AOX1), multifunctional protein (MFP1 in mullet and MFP2 in mussels), and 3-ketoacyl-CoA thiolase (THIO), were cloned. Additionally, a fragment of the peroxisomal Δ 2, Δ 4 dienoyl-CoA reductase 2 (DECR) necessary for the β-oxidation of unsaturated fatty acids was cloned in mullets. The whole open reading frame of aox1 sequenced in both mussels and mullets revealed high homology with known aox1 sequences, with highly conserved important domains such as the FAD binding motif or the typical peroxisomal targeting signal (PTS1). A thorough in silico analysis of the gene and genome databases allowed to identify in fish and molluscs sequence homologs of all the enzymes necessary for 2 of the 3 different paralog peroxisomal β-oxidation pathways described in metazoans (AOX1, AOX3, MFP1, MFP2, THIO and sterol carrier protein X). Only the enzyme necessary for the oxidation of branched chain fatty acids, AOX2, described in mammalian, avian and amphibian species, seems to be lacking from the genomes of fish and molluscs. In order to study the expression and regulation capacity of peroxisomal β-oxidation genes, aox1 and thio expression was determined in different tissues of mature and immature mullets and mussels collected in January and June, both genes being expressed higher in the digestive gland of mussels collected in June compared to January. Finally, in silico studies of the promoter regions in the piscine genomes available in the Ensembl genome repository, allowed the identification of putative peroxisome proliferator response elements that could explain the possible cellular and molecular mechanisms leading to peroxisome proliferation in fish. Further studies are needed to decipher molecular mechanisms of peroxisome proliferation in aquatic organisms under exposure to peroxisome proliferator xenobiotics.

Effect of troglitazone on CYP1A1 induction

Several peroxisome proliferators enhance CYP1A1 activity, but the mechanisms involved in this enhancement remain unknown. In this study, we examined the effect of troglitazone, a peroxisome proliferator-activated receptor-γ (PPAR-γ) agonist, on CYP1A1 gene expression and explored the mechanisms involved in these effects. Troglitazone increased gene expression of CYP1A1 mRNA and also increased CYP1A1-specific 7-ethoxyresorufin- O-deethylase (EROD) activity in a dose-dependent manner. Moreover, concomitant treatment with troglitazone and GW9662, a PPAR antagonist, markedly reduced the troglitazone-inducible EROD activity. Luciferase reporter assays using Hepa-1c1c7 cells showed a significant transactivation by troglitazone with a reporter plasmid containing a region from −1395 to +7 of the CYP1A1 gene. We found that a putative peroxisome proliferator-response element (PPRE) between −521 and −500 is located in the CYP1A1 gene promoter. Their inactivation by deletion mutagenesis suppressed the inductive effect of troglitazone on CYP1A1 promoter activation. Electrophoretic mobility shift assay revealed that troglitazone induced the activation of the PPAR-γ to a form capable of binding specifically to the PPRE sequence of the CYP1A1 gene promoter. Furthermore, troglitazone increased the formation of the benzo[ a]pyrene (BaP)–DNA adduct. Overall, our results suggest that troglitazone induces CYP1A1 enzyme activity and gene expression through PPAR-γ activation, and may be involved in carcinogenesis.

The acyl-CoA thioesterase I is regulated by PPARα and HNF4α via a distal response element in the promoter

The cytosolic acyl-coenzyme A thioesterase I (Acot1) is an enzyme that hydrolyzes long-chain acyl-CoAs of C(12)-C(20)-CoA in chain length to the free fatty acid and CoA. Acot1 was shown previously to be strongly upregulated at the mRNA and protein level in rodents by fibrates. In this study, we show that Acot1 mRNA levels were increased by 90-fold in liver by treatment with Wy-14,643 and that Acot1 mRNA was also increased by 15-fold in the liver of hepatocyte nuclear factor 4alpha (HNF4alpha) knockout animals. Our study identified a direct repeat 1 (DR1) located in the Acot1 gene promoter in mouse, which binds the peroxisome proliferator-activated receptor alpha (PPARalpha) and HNF4alpha. Chromatin immunoprecipitation (ChIP) assay showed that the identified DR1 bound PPARalpha/retinoid X receptor alpha (RXRalpha) and HNF4alpha, whereas the binding in ChIP was abrogated in the PPARalpha and HNF4alpha knockout mouse models. Reporter gene assays showed activation of the Acot1 promoter in cells by the PPARalpha agonist Wy-14,643 after cotransfection with PPARalpha/RXRalpha. However, transfection with a plasmid containing HNF4alpha also resulted in an increase in promoter activity. Together, these data show that Acot1 is under regulation by an interplay between HNF4alpha and PPARalpha.

Transcriptional Regulation of Cidea, Mitochondrial Cell Death-inducing DNA Fragmentation Factor α-Like Effector A, in Mouse Liver by Peroxisome Proliferator-activated Receptor α and γ

Cidea (cell death-inducing DNA fragmentation factor alpha-like effector A), a member of a novel family of proapoptotic proteins, is expressed abundantly in the brown adipose tissue of the mouse. Although Cidea mRNA is not detectable in the mouse liver, we now show that peroxisome proliferator-activated receptor (PPAR) alpha ligands Wy-14,643 and ciprofibrate increase the Cidea mRNA level in a PPARalpha-dependent manner, whereas Cidea induction in liver by PPARgamma overexpression is PPARalpha independent. Increase in Cidea mRNA content in liver did not alter the expression of uncoupling protein 1 (Ucp1) gene, which regulates thermogenesis, lipolysis, and conservation of energy. Although Cidea is considered to be a proapoptotic factor, Cidea induction in liver did not result in increased apoptosis. To elucidate the mechanism by which PPARalpha and PPARgamma regulate Cidea gene expression in the liver, we analyzed the promoter region of the Cidea gene. Three putative peroxisome proliferator response elements (PPREs) are found in the Cidea gene promoter. Transactivation, gel-shift, and chromatin immunoprecipitation assays indicated that the proximal PPRE in Cidea gene (Cidea-PPRE1 at -680/-668) is functional for both PPARalpha and -gamma. We conclude that Cidea is a novel target gene for both PPARalpha and -gamma in the liver where these two transcription factors utilize the same PPRE region for dual regulation. The induction of Cidea in liver with these PPARalpha and -gamma agonists suggests a possible role for Cidea in energy metabolism and a less likely role in hepatocyte apoptosis.

Differential regulation of the cynomolgus, human, and rat acyl-CoA oxidase promoters by PPARα

Peroxisome proliferator-activated receptor α (PPARα) is a member of the nuclear receptor family of transcription factors and is recognized as the molecular target of the hypolipidemic fibrate drugs. Fibrates promote lipid catabolism by inducing genes involved in fatty acid β-oxidation. In rodents this is accompanied by peroxisome proliferation, and after chronic dosing hepatocarcinoma, whereas epidemiological studies suggest these adverse events are lacking in humans. Rodents such as rats and mice appear particularly sensitive to PPARα-induced peroxisome proliferation while humans are resistant. These findings question the utility of rodent models for safety monitoring of experimental PPARα agonists and highlight the need for additional preclinical models that display greater physiological relevance for human response. Thus we have focused on elucidating the molecular mechanism of species-dependent peroxisome proliferation by understanding the PPARα-dependent regulation of the acyl-CoA oxidase (AOX) promoter, the rate-limiting step of peroxisomal β-oxidation. We have chosen the cynomolgus monkey as a model that is modestly responsive to peroxisome proliferation and functionally characterized it against the highly responsive rat and non-responsive human species. We report the identification of a putative peroxisome proliferator response element (PPRE) within the 2.3 kb proximal promoter of the cynomolgus monkey AOX gene. Characterization of these promoters using a series of constitutively active, PPARα constructs demonstrate that the PPREs within the proximal cynomolgus and human AOX promoters are non-responsive to PPARα whereas the rat PPRE is highly responsive. These findings were verified in vivo using a small molecule PPARα agonist. Taken together, we demonstrate concordant regulation of the AOX promoter by PPARα in cynomolgus monkeys and humans and suggest that this model is superior to rodent models with respect to preclinical evaluation of PPARα agonists.

Evidence for a new human CYP1A1 regulation pathway involving PPAR-α and 2 PPRE sites

Cytochrome P450 1A1 catalyzes the degradation of endobiotics (estradiol, fatty acids, and so on) and the bioactivation of numerous environmental procarcinogens, such as arylamines and polycyclic aromatic hydrocarbons, that are found in food. Several peroxisome proliferators and arachidonic acid derivatives enhance cytochrome P450 1A1 activity, but the mechanisms involved remain unknown. The aim of this work was to study the role of peroxisome proliferator-activated receptors in cytochrome P450 1A1 gene induction. The role of peroxisome proliferator-activated receptor transcription factors in cytochrome P450 1A1 induction was assessed by means of enzymatic activities, quantitative real-time polymerase chain reaction, gene reporter assays, mutagenesis, and electrophoretic mobility shift assay. We show that peroxisome proliferator-activated receptor-alpha agonists (WY-14643, bezafibrate, clofibrate, and phthalate) induce human cytochrome P450 1A1 gene expression, whereas 2,4-thiazolidinedione, a specific peroxisome proliferator-activated receptor-gamma agonist, represses it. The induction of cytochrome P450 1A1 transcripts by WY-14643 was associated with a marked increase of ethoxyresorufin O -deethylase activity (10-fold at 200 mumol/L). Transfection of peroxisome proliferator-activated receptor-alpha complementary DNA enhanced cytochrome P450 1A1 messenger RNA induction by WY-14643, although WY-14643 failed to activate xenobiotic responsive element sequences. Two peroxisome proliferator response element sites were located at positions -931/-919 and -531/-519 of the cytochrome P450 1A1 promoter. Their inactivation by directed mutagenesis suppressed the inductive effect of WY-14643 on cytochrome P450 1A1 promoter activation. Electrophoretic mobility shift assay and chromatin immunoprecipitation assay experiments showed that the 2 cytochrome P450 1A1 peroxisome proliferator response element sites bind the peroxisome proliferator-activated receptor-alpha/retinoid X receptor-alpha heterodimer. We describe here a new cytochrome P450 1A1 induction pathway involving peroxisome proliferator-activated receptor-alpha and 2 peroxisome proliferator response element sites, indicating that peroxisome proliferator-activated receptor-alpha ligands, which are common environmental compounds, may be involved in carcinogenesis.

Human aquaporin adipose (AQPap) gene. Genomic structure, promoter analysis and functional mutation.

Aquaporin adipose (AQPap), which we identified from human adipose tissue, is a glycerol channel in adipocyte [Kishida et al. (2000) J. Biol. Chem. 275, 20896-20902]. In the current study, we determined the genomic structure of the human AQPap gene, and identified three AQPap-like genes that resembled (approximately 95%) AQPap, with little expression in human tissues. The AQPap promoter contained a putative peroxisome proliferator response element (PPRE) at -46 to -62, and a putative insulin response element (IRE) at -542/-536. Deletion of the PPRE abolished the pioglitazone-mediated induction of AQPap promoter activity in 3T3-L1 adipocytes. Deletion and single base pair substitution analysis of the IRE abolished the insulin-mediated suppression of the human AQPap gene. Analysis of AQPap sequence in human subjects revealed three missense mutations (R12C, V59L and G264V), and two silent mutations (A103A and G250G). The cRNA injection of the missense mutants into Xenopus oocytes revealed the absence of the activity to transport glycerol and water in the AQPap-G264V protein. In the subject homozygous for AQPap-G264V, exercise-induced increase in plasma glycerol was not observed in spite of the increased plasma noradrenaline. We suggest that AQPap is responsible for the increase of plasma glycerol during exercise in humans.

Studies on regulation of the peroxisomal beta-oxidation at the 3-ketothiolase step. Dissection of the rat liver thiolase B gene promoter.

The peroxisomal 3-oxoacyl-CoA thiolase (thiolase) is the last enzyme involved in the beta-oxidation of fatty acids. The enzyme cleaves long chain fatty acyl-CoA to generate acetyl-CoA and shortened acyl-CoA. The enzyme is nuclear encoded, synthesized in the cytoplasm and transported into peroxisomes. The thiolase B gene is inducible by the peroxisome proliferator compounds, like other genes involved in beta-oxidation of fatty acids in peroxisomes. The importance of studying thiolase is that it generates acetyl-CoA which is the precursor for the synthesis of molecules like cholesterol and fatty acids. The structural and functional analysis of thiolase at molecular level may add to the knowledge of fatty acid metabolism and further the obesity phenomenon. It is known that several genes mediate lipid homeostasis in target organs like liver, adipose tissue and are regulated by peroxisome proliferator activated receptors (PPAR alpha and PPAR gamma). To elucidate the mechanism of induction of rat liver thiolase B gene, an upstream 2.8 kb fragment containing promoter element has been subcloned and partially sequenced. The sequence analysis revealed a putative PPRE (Peroxisome Proliferator Response Element) of AGACCT T TGAACC sequence at -681 to -668 [Kliever et al. (1992) Nature 358:771-774]. By transient expression of a luciferase reporter gene in HeLa cells, we conclude that the identified PPRE could be functional in induction of thiolase B gene, but other sequences of genes might be involved.

The Peroxisome Proliferator-activated Receptor α (PPARα) Regulates Bile Acid Biosynthesis

Fibrates are a group of hypolipidemic agents that efficiently lower serum triglyceride levels by affecting the expression of many genes involved in lipid metabolism. These effects are exerted via the peroxisome proliferator-activated receptor alpha (PPARalpha). In addition, fibrates also lower serum cholesterol levels, suggesting a possible link between the PPARalpha and cholesterol metabolism. Bile acid formation represents an important pathway for elimination of cholesterol, and the sterol 12alpha-hydroxylase is a branch-point enzyme in the bile acid biosynthetic pathway, which determines the ratio of cholic acid to chenodeoxycholic acid. Treatment of mice for 1 week with the peroxisome proliferator WY-14,643 or fasting for 24 h both induced the sterol 12alpha-hydroxylase mRNA in liver. Using the PPARalpha knockout mouse model, we show that the induction by both treatments was dependent on the PPARalpha. A reporter plasmid containing a putative peroxisome proliferator-response element (PPRE) identified in the rat sterol 12alpha-hydroxylase promoter region was activated by treatment with WY-14,643 in HepG2 cells, being dependent on co-transfection with a PPARalpha expression plasmid. The rat 12alpha-hydroxylase PPRE bound in vitro translated PPARalpha and retinoid X receptor alpha, albeit weakly, in electrophoretic mobility shift assay. Treatment of wild-type mice with WY-14,643 for 1 week resulted in an increased relative amount of cholic acid, an effect that was abolished in the PPARalpha null mice, verifying the functionality of the PPRE in vivo.