Mechanisms Mediating the Regulation of Peroxisomal Fatty Acid Beta-Oxidation by PPARα.

In mammalian cells, two cellular organelles, mitochondria and peroxisomes, share the ability to degrade fatty acid chains. Although each organelle harbors its own fatty acid ß-oxidation pathway, a distinct mitochondrial system feeds the oxidative phosphorylation pathway for ATP synthesis. At the same time, the peroxisomal ß-oxidation pathway participates in cellular thermogenesis. A scientific milestone in 1965 helped discover the hepatomegaly effect in rat liver by clofibrate, subsequently identified as a peroxisome proliferator in rodents and an activator of the peroxisomal fatty acid ß-oxidation pathway. These peroxisome proliferators were later identified as activating ligands of Peroxisome Proliferator-Activated Receptor α (PPARα), cloned in 1990. The ligand-activated heterodimer PPARα/RXRα recognizes a DNA sequence, called PPRE (Peroxisome Proliferator Response Element), corresponding to two half-consensus hexanucleotide motifs, AGGTCA, separated by one nucleotide. Accordingly, the assembled complex containing PPRE/PPARα/RXRα/ligands/Coregulators controls the expression of the genes involved in liver peroxisomal fatty acid ß-oxidation. This review mobilizes a considerable number of findings that discuss miscellaneous axes, covering the detailed expression pattern of PPARα in species and tissues, the lessons from several PPARα KO mouse models and the modulation of PPARα function by dietary micronutrients.

Unique microRNA alterations in hepatocellular carcinomas arising either spontaneously or due to chronic exposure to Ginkgo biloba extract (GBE) in B6C3F1/N mice.

Ginkgo biloba extract (GBE) is used in traditional Chinese medicine as a herbal supplement for improving memory. Exposure of B6C3F1/N mice to GBE in a 2-year National Toxicology Program (NTP) bioassay resulted in a dose-dependent increase in hepatocellular carcinomas (HCC). To identify key microRNAs that modulate GBE-induced hepatocarcinogenesis, we compared the global miRNA expression profiles in GBE-exposed HCC (GBE-HCC) and spontaneous HCC (SPNT-HCC) with age-matched vehicle control normal livers (CNTL) from B6C3F1/N mice. The number of differentially altered miRNAs in GBE-HCC and SPNT-HCC was 74 (52 up and 22 down) and 33 (15 up and 18 down), respectively. Among the uniquely differentially altered miRNAs in GBE-HCC, miR-31 and one of its predicted targets, Cdk1 were selected for functional validation. A potential miRNA response element (MRE) in the 3'-untranslated regions (3'-UTR) of Cdk1 mRNA was revealed by in silico analysis and confirmed by luciferase assays. In mouse hepatoma cell line HEPA-1 cells, we demonstrated an inverse correlation between miR-31 and CDK1 protein levels, but no change in Cdk1 mRNA levels, suggesting a post-transcriptional effect. Additionally, a set of miRNAs (miRs-411, 300, 127, 134, 409-3p, and 433-3p) that were altered in the GBE-HCCs were also altered in non-tumor liver samples from the 90-day GBE-exposed group compared to the vehicle control group, suggesting that some of these miRNAs could serve as potential biomarkers for GBE exposure or hepatocellular carcinogenesis. These data increase our understanding of miRNA-mediated epigenetic regulation of GBE-mediated hepatocellular carcinogenesis in B6C3F1/N mice.

Epigenetic modification of histone 3 lysine 27: mediator subunit MED25 is required for the dissociation of polycomb repressive complex 2 from the promoter of cytochrome P450 2C9.

The Mediator complex is vital for the transcriptional regulation of eukaryotic genes. Mediator binds to nuclear receptors at target response elements and recruits chromatin-modifying enzymes and RNA polymerase II. Here, we examine the involvement of Mediator subunit MED25 in the epigenetic regulation of human cytochrome P450 2C9 (CYP2C9). MED25 is recruited to the CYP2C9 promoter through association with liver-enriched HNF4α, and we show that MED25 influences the H3K27 status of the HNF4α binding region. This region was enriched for the activating marker H3K27ac and histone acetyltransferase CREBBP after MED25 overexpression but was trimethylated when MED25 expression was silenced. The epigenetic regulator Polycomb repressive complex (PRC2), which represses expression by methylating H3K27, plays an important role in target gene regulation. Silencing MED25 correlated with increased association of PRC2 not only with the promoter region chromatin but with HNF4α itself. We confirmed the involvement of MED25 for fully functional preinitiation complex recruitment and transcriptional output in vitro. Formaldehyde-assisted isolation of regulatory elements (FAIRE) revealed chromatin conformation changes that were reliant on MED25, indicating that MED25 induced a permissive chromatin state that reflected increases in CYP2C9 mRNA. For the first time, we showed evidence that a functionally relevant human gene is transcriptionally regulated by HNF4α via MED25 and PRC2. CYP2C9 is important for the metabolism of many exogenous chemicals including pharmaceutical drugs as well as endogenous substrates. Thus, MED25 is important for regulating the epigenetic landscape resulting in transcriptional activation of a highly inducible gene, CYP2C9.

Regulation of human CYP2C9 expression by electrophilic stress involves activator protein 1 activation and DNA looping.

Cytochrome P450 (CYP)2C9 and CYP2C19 are important human enzymes that metabolize therapeutic drugs, environmental chemicals, and physiologically important endogenous compounds. Initial studies using primary human hepatocytes showed induction of both the CYP2C9 and CYP2C19 genes by tert-butylhydroquinone (tBHQ). As a pro-oxidant, tBHQ regulates the expression of cytoprotective genes by activation of redox-sensing transcription factors, such as the nuclear factor E2-related factor 2 (Nrf2) and members of the activator protein 1 (AP-1) family of proteins. The promoter region of CYP2C9 contains two putative AP-1 sites (TGAGTCA) at positions -2201 and -1930, which are also highly conserved in CYP2C19. The CYP2C9 promoter is activated by ectopic expression of cFos and JunD, whereas Nrf2 had no effect. Using specific kinase inhibitors for mitogen-activated protein kinase, we showed that extracellular signal-regulated kinase and Jun N-terminal kinase are essential for tBHQ-induced expression of CYP2C9. Electrophoretic mobility shift assays demonstrate that cFos distinctly interacts with the distal AP-1 site and JunD with the proximal site. Because cFos regulates target genes as heterodimers with Jun proteins, we hypothesized that DNA looping might be required to bring the distal and proximal AP-1 sites together to activate the CYP2C9 promoter. Chromosome conformation capture analyses confirmed the formation of a DNA loop in the CYP2C9 promoter, possibly allowing interaction between cFos at the distal site and JunD at the proximal site to activate CYP2C9 transcription in response to electrophiles. These results indicate that oxidative stress generated by exposure to electrophilic xenobiotics and metabolites induces the expression of CYP2C9 and CYP2C19 in human hepatocytes.

Hepatic steatosis and peroxisomal fatty acid beta-oxidation.

Three subhepatocellular compartments concur for fatty acids degradation including ω-oxidation in endoplasmic reticulum and ß-oxidation in both mitochondria and peroxisomes. Deficits affecting the peroxisomal physiology may be associated with multiple metabolic disturbances. Nowadays, a growing body of evidence underlines the key role of peroxisomal ß-oxidation in the sensing of lipid metabolism through the production/degradation of some essential metabolites. Lessons from several mice models strengthen the link between fatty acid ß-oxidation in peroxisomes and the nuclear hormone receptor Peroxisome Proliferator-Activated Receptor (PPAR)-α with an additional level of coregualtor complexity, which couples regulation of body energetic balance and hepatic caloric flux to functional peroxisome status. Here, we review key determinants of disrupted peroxisomal ß-oxidation pathway, which in liver promotes hepatic steatosis and hepatocarcinogenesis.

Human CYP2C8 is post-transcriptionally regulated by microRNAs 103 and 107 in human liver.

The CYP2C genes are extensively regulated at the transcriptional stage. The present study shows for the first time that CYP2Cs are also regulated post-transcriptionally by microRNAs (miRNAs). By using online search engines, we found potential miRNA response elements (MREs) in the 3'-untranslated region (3'-UTR) of the CYP2C mRNAs. Among these were a MRE for the miRNAs miR-103 and miR-107 in the 3'-UTR of human CYP2C8. CYP2C8 protein levels (measured through immunoblot analyses) did not correlate with CYP2C8 mRNA levels (measured through quantitative polymerase chain reaction analyses) in human liver samples. The translation efficiency (protein/mRNA ratio) for CYP2C8 was inversely correlated with the expression of miR-103 and miR-107. When three copies of the putative MRE from CYP2C8 were inserted downstream from a luciferase expression reporter, transfection with precursors for miR-103 or miR-107 decreased luciferase activity in primary hepatocytes, whereas transfection with antisense oligonucleotides (AsOs) for miR-103/miR-107 increased luciferase activity. As expected, there was no effect of the precursors or AsOs when three copies of the putative MRE were inserted in the reverse orientation. When precursors for miR-103/miR-107 were transfected into primary human hepatocytes, CYP2C8 protein levels were decreased, whereas AsOs increased CYP2C8 protein levels. Neither precursors nor AsOs affected CYP2C8 mRNA levels, which indicated that the effect was post-transcriptional. Putative MRE motifs were also found in the 3'-UTRs of CYP2C9 and CYP2C19, which suggested that the same miRNAs could regulate translation of other members of the CYP2C family, although to a lesser degree than CYP2C8. These results clearly show that CYP2Cs are regulated post-transcriptionally by miR-103 and miR-107.

NCOA6 differentially regulates the expression of the CYP2C9 and CYP3A4 genes.

CYP2Cs and CYP3A4 sub families of enzymes of the Cytochrome P450 super family metabolize clinically prescribed therapeutics. Constitutive and induced expressions of these enzymes are under the control of HNF4α and rifampicin activated PXR. In the present study, we show a mechanism for ligand dependent synergistic cross talk between PXR and HNF4α. Two-hybrid screening identified NCOA6 as a HNF4α interacting protein. NCOA6 was also found to interact with PXR through the first LXXLL motif in GST pull down and mammalian two hybrid assays. NCOA6 enhances the synergistic activation of CYP2C9 and CYP3A4 promoter activity by PXR and HNF4α in the presence of rifampicin. However silencing NCOA6 abrogated the synergistic activation and induction of CYP2C9 by PXR-HNF4α but not of CYP3A4. ChIP analysis revealed that NCOA6 could bridge HNF4α and PXR binding sites of the CYP2C9 promoter. Our results indicate that NCOA6 is responsible for the synergistic activation of CYP2C9 by HNF4α and PXR and NCOA6 differentially regulates CYP2C9 and CYP3A4 gene expression though both the genes are regulated by the same nuclear receptors.

Med25 is required for RNA polymerase II recruitment to specific promoters, thus regulating xenobiotic and lipid metabolism in human liver.

Hepatocyte nuclear factor 4α (HNF4α) controls the expression of many critical metabolic pathways, and the Mediator complex occupies a central role in recruiting RNA polymerase II (Pol II) to these gene promoters. An impaired transcriptional HNF4α network in human liver is responsible for many pathological conditions, such as altered drug metabolism, fatty liver, and diabetes. Here, we report that Med25, an associated member of the Mediator complex, is required for the association of HNF4α with Mediator, its several cofactors, and RNA Pol II. Further, increases and decreases in endogenous Med25 levels are reflected in the composition of the transcriptional complex, Pol II recruitment, and the expression of HNF4α-bound target genes. A novel feature of Med25 is that it imparts "selectivity." Med25 affects only a significant subset of HNF4α target genes that selectively regulate drug and lipid metabolism. These results define a role for Med25 and the Mediator complex in the regulation of xenobiotic metabolism and lipid homeostasis.

Hepatocyte nuclear factor 4{alpha} regulates rifampicin-mediated induction of CYP2C genes in primary cultures of human hepatocytes.

CYP2C enzymes are expressed constitutively and comprise approximately 20% of the total cytochrome P450 in human liver. However, the factors influencing the transcriptional regulation of the CYP2C subfamily have only been addressed recently. In the present study, we used primary cultures of human hepatocytes to investigate the role of HNF4alpha in the pregnane X receptor (PXR)/rifampicin-mediated up-regulation of CYP2C8, CYP2C9, and CYP2C19 gene expression. We first identified new proximal cis-acting HNF4alpha sites in the proximal CYP2C8 promoter [at -181 base pairs (bp) from the translation start site] and the CYP2C9 promoter (at -211 bp). Both sites bound HNF4alpha in gel shift assays. Thus, these and recent studies identified a total of three HNF4alpha sites in the CYP2C9 promoter and two in the CYP2C8 promoter. Mutational studies showed that the HNF4alpha sites are needed for up-regulation of the CYP2C8 and CYP2C9 promoters by rifampicin. Furthermore, silencing of HNF4alpha abolished transactivation of the CYP2C8 and CYP2C9 promoters by rifampicin. Constitutive promoter activity was also decreased. Quantitative polymerase chain reaction analysis demonstrated that silencing HNF4alpha reduced the constitutive expression of CYP2C8 (53%), CYP2C9 (55%), and CYP2C19 (43%) mRNAs and significantly decreased the magnitude of the rifampicin-mediated induction of CYP2C8 (6.6- versus 2.7-fold), CYP2C9 (3- versus 1.5-fold), and CYP2C19 (1.8- versus 1.1-fold). These results provide clear evidence that HNF4alpha contributes to the constitutive expression of the human CYP2C genes and is also important for up-regulation by the PXR agonist rifampicin.

Hepatocyte-specific deletion of SIRT1 alters fatty acid metabolism and results in hepatic steatosis and inflammation.

Hepatic metabolic derangements are key components in the development of fatty liver, insulin resistance, and atherosclerosis. SIRT1, a NAD+-dependent protein deacetylase, is an important regulator of energy homeostasis in response to nutrient availability. Here we demonstrate that hepatic SIRT1 regulates lipid homeostasis by positively regulating peroxisome proliferators-activated receptor alpha (PPARalpha), a nuclear receptor that mediates the adaptive response to fasting and starvation. Hepatocyte-specific deletion of SIRT1 impairs PPARalpha signaling and decreases fatty acid beta-oxidation, whereas overexpression of SIRT1 induces the expression of PPARalpha targets. SIRT1 interacts with PPARalpha and is required to activate PPARalpha coactivator PGC-1alpha. When challenged with a high-fat diet, liver-specific SIRT1 knockout mice develop hepatic steatosis, hepatic inflammation, and endoplasmic reticulum stress. Taken together, our data indicate that SIRT1 plays a vital role in the regulation of hepatic lipid homeostasis and that pharmacological activation of SIRT1 may be important for the prevention of obesity-associated metabolic diseases.

Nuclear receptor coactivator 6 mediates the synergistic activation of human cytochrome P-450 2C9 by the constitutive androstane receptor and hepatic nuclear factor-4alpha.

Nuclear receptor coactivator 6 (NCOA6) also known as PRIP/RAP250/ASC-2 anchors a steady-state complex of cofactors and function as a transcriptional coactivator for certain nuclear receptors. This is the first study to identify NCOA6 as a hepatic nuclear factor 4alpha (HNF4alpha)-interacting protein. CYP2C9 is an important enzyme that metabolizes both commonly used therapeutic drugs and important endogenous compounds. We have shown previously that constitutive androstane receptor (CAR) (a xenobiotic-sensing receptor) up-regulates the CYP2C9 promoter through binding to a distal site, whereas HNF4alpha transcriptionally up-regulates CYP2C9 via proximal sites. We demonstrate ligand-enhanced synergistic cross-talk between CAR and HNF4alpha. We identify NCOA6 as crucial to the underlying mechanism of this cross-talk. NCOA6 was identified as an HNF4alpha-interacting protein in this study using a yeast two-hybrid screen and GST pull-down assays. Furthermore, we identified NCOA6, CAR, and other coactivators as part of a mega complex of cofactors associated with HNF4alpha in HepG2 cells. Although the interaction of NCOA6 with CAR is specifically through the first LXXLL motif of NCOA6, both LXXLL motifs are involved in its interaction with HNF4alpha. Silencing of NCOA6 abrogated the synergistic activation of the CYP2C9 promoter and the synergistic induction of the CYP2C9 gene by CAR-HNF4alpha. Chromatin immunoprecipitation analysis revealed that NCOA6 can pull down both the proximal HNF4alpha and distal CAR binding sites of the CYP2C9 promoter and provides the basis for the recruitment of other cofactors. We conclude that the coactivator NCOA6 mediates the mechanism of the synergistic activation of the CYP2C9 gene by CAR and HNF4alpha.

Pigment epithelium-derived factor is an angiogenesis and lipid regulator that activates peroxisome proliferator-activated receptor alpha.

Pigment epithelium-derived factor (PEDF) is an endogenous antiangiogenic protein that also possesses antitumor activity. The mechanisms by which PEDF exerts its actions remains poorly understood. We sought to understand the role of PEDF in hepatocellular carcinoma (HCC), a hypervascular malignancy that has been shown to upregulate enzymes involved in fatty acid synthesis. PEDF expression occurs in two HCC cell lines and is oxygen dependent. Migration studies confirm PEDF's role as an endogenous inhibitor of angiogenesis in HCC cells. Loss of PEDF in an animal model leads to hepatocyte lipid accumulation, proliferation, and cellular atypia. To investigate potential interactions with transcription factors that are involved in fatty acid metabolism and cellular proliferation, we examined PEDF's interaction with PPARalpha in vitro and its functional activity through transactivation assays. We show that PEDF binds to PPARalpha but minimally to PPARgamma. In the presence of the ligand, ciprofibrate, PEDF binding to PPARalpha decreases whereas the presence of troglitazone does not alter PEDF interactions with PPARgamma. Transfection of the PEDF gene in the presence of the PPARalpha/RXR heterodimer demonstrates transcriptional activation of PPARalpha by PEDF. These data show that PEDF regulates lipid metabolism through activation of the transcription factor PPARalpha.

Transcriptional regulation of Cidea, mitochondrial cell death-inducing DNA fragmentation factor alpha-like effector A, in mouse liver by peroxisome proliferator-activated receptor alpha and gamma.

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.

PRIC320, a transcription coactivator, isolated from peroxisome proliferator-binding protein complex.

Ciprofibrate, a potent peroxisome proliferator, induces pleiotropic responses in liver by activating peroxisome proliferator-activated receptor alpha (PPARalpha), a nuclear receptor. Transcriptional regulation by liganded nuclear receptors involves the participation of coregulators that form multiprotein complexes possibly to achieve cell and gene specific transcription. SDS-PAGE and matrix-assisted laser desorption/ionization reflection time-of-flight mass spectrometric analyses of ciprofibrate-binding proteins from liver nuclear extracts obtained using ciprofibrate-Sepharose affinity matrix resulted in the identification of a new high molecular weight nuclear receptor coactivator, which we designated PRIC320. The full-length human cDNA encoding this protein has an open-reading frame that codes for a 320kDa protein containing 2882 amino acids. PRIC320 contains five LXXLL signature motifs that mediate interaction with nuclear receptors. PRIC320 binds avidly to nuclear receptors PPARalpha, CAR, ERalpha, and RXR, but only minimally with PPARgamma. PRIC320 also interacts with transcription cofactors CBP, PRIP, and PBP. Immunoprecipitation-immunoblotting as well as cellular localization studies confirmed the interaction between PPARalpha and PRIC320. PRIC320 acts as a transcription coactivator by stimulating PPARalpha-mediated transcription. We conclude that ciprofibrate, a PPARalpha ligand, binds a multiprotein complex and PRIC320 cloned from this complex functions as a nuclear receptor coactivator.

P53 mediates amosite asbestos-induced alveolar epithelial cell mitochondria-regulated apoptosis.

Asbestos causes pulmonary toxicity in part by generating reactive oxygen species that cause DNA damage. We previously showed that the mitochondria-regulated (intrinsic) death pathway mediates alveolar epithelial cell (AEC) DNA damage and apoptosis. Because p53 regulates the DNA damage response in part by inducing intrinsic cell death, we determined whether p53-dependent transcriptional activity mediates asbestos-induced AEC mitochondrial dysfunction and apoptosis. We show that inhibitors of p53-dependent transcriptional activation (pifithrin and type 16-E6 protein) block asbestos-induced AEC mitochondrial membrane potential change (DeltaPsim), caspase 9 activation, and apoptosis. We demonstrate that asbestos activates p53 promoter activity, mRNA levels, protein expression, and Bax and p53 mitochondrial translocation. Further, pifithrin, E6, phytic acid, or rho(0)-A549 cells (cells incapable of mitochondrial reactive oxygen species production) block asbestos-induced p53 activation. Finally, we show that asbestos augments p53 expression in cells at the bronchoalveolar duct junctions of rat lungs and that phytic acid prevents this. These data suggest that p53-dependent transcription pathways mediate asbestos-induced AEC mitochondria-regulated apoptosis. This suggests an important interactive effect between p53 and the mitochondria in the pathogenesis of asbestos-induced pulmonary toxicity that may have broader implications for our understanding of pulmonary fibrosis and lung cancer.

Transcription coactivator peroxisome proliferator-activated receptor-binding protein/mediator 1 deficiency abrogates acetaminophen hepatotoxicity.

Peroxisome proliferator-activated receptor-binding protein (PBP), also known as thyroid hormone receptor-associated protein 220/vitamin D receptor-interacting protein 205/mediator 1, an anchor for multisubunit mediator transcription complex, functions as a transcription coactivator for nuclear receptors. Disruption of the PBP gene results in embryonic lethality around embryonic day 11.5 by affecting placental and multiorgan development. Here, we report that targeted deletion of PBP in liver parenchymal cells (PBP(Liv-/-)) results in the abrogation of hypertrophic and hyperplastic influences in liver mediated by constitutive androstane receptor (CAR) ligands phenobarbital (PB) and 1,4-bis-[2-(3,5-dichloropyridyloxy)]benzene, and of acetaminophen-induced hepatotoxicity. CAR interacts with the two nuclear receptor-interacting LXXLL (L, leucine; X, any amino acid) motifs in PBP in a ligand-dependent manner. We also show that PBP interacts with the C-terminal portion of CAR, suggesting that PBP is involved in the regulation of CAR function. Although the full-length PBP only minimally increased CAR transcriptional activity, a truncated form of PBP (amino acids 487-735) functioned as a dominant negative repressor, establishing that PBP functions as a coactivator for CAR. A reduction in CAR mRNA and protein level observed in PBP(Liv-/-) mouse liver suggests that PBP may regulate hepatic CAR expression. PBP-deficient hepatocytes in liver failed to reveal PB-dependent translocation of CAR to the nucleus. Adenoviral reconstitution of PBP in PBP(Liv-/-) mouse livers restored PB-mediated nuclear translocation of CAR as well as inducibility of CYP1A2, CYP2B10, CYP3A11, and CYP7A1 expression. We conclude that transcription coactivator PBP/TRAP220/MED1 is involved in the regulation of hepatic CAR function and that PBP deficiency in liver abrogates acetaminophen hepatotoxicity.

Transcription coactivator PBP, the peroxisome proliferator-activated receptor (PPAR)-binding protein, is required for PPARalpha-regulated gene expression in liver.

Nuclear receptor coactivator PBP (peroxisome proliferator-activated receptor (PPAR)-binding protein) functions as a coactivator for PPARs and other nuclear receptors. PBP serves as an anchor for TRAP (thyroid hormone receptor-associated proteins)/mediator multisubunit cofactor transcription complex. Disruption of the PBP/TRAP220 gene results in embryonic lethality around embryonic day 11.5 by affecting placental, cardiac, hepatic, and bone marrow development. Because PPAR isoforms alpha, gamma, and beta/delta function as important regulators of lipid homeostasis in mammals, it becomes important to assess the requirement of coactivator PBP in the regulation of PPAR functions in vivo. Sustained activation of PPARalpha by structurally diverse classes of chemicals of biological importance, designated peroxisome proliferators, leads to proliferation of peroxisomes in liver, induction of PPARalpha target genes including those involved in fatty acid oxidation, and the eventual development of liver tumors. Here, we show that targeted deletion of PBP in liver parenchymal cells, using the Cre-loxP system, results in the near abrogation of PPARalpha ligand-induced peroxisome proliferation and liver cell proliferation, as well as the induction of PPARalpha-regulated genes in PBP-deficient liver cells. In contrast, scattered PBP(+/+) hepatocytes in these livers showed DNA synthesis and were markedly hypertrophic with peroxisome proliferation in response to PPARalpha ligands. Chromatin immunoprecipitation data suggest that in PBP conditional null livers, there appears to be reduced association of cofactors, especially of CBP and TRAP150, to the mouse enoyl-CoA hydratase/l-3-hydroxyacyl-CoA dehydrogenase gene promoter. These observations suggest that PBP is required for the stabilization of multiprotein cofactor complexes. In essence, the absence of PBP in hepatocytes in vivo appears to mimic the absence of PPARalpha, indicating that coactivator PBP is essential for PPARalpha-regulated gene expression in liver parenchymal cells.

Transcriptional coactivator PRIP, the peroxisome proliferator-activated receptor gamma (PPARgamma)-interacting protein, is required for PPARgamma-mediated adipogenesis.

Nuclear receptor coactivator PRIP (peroxisome proliferators-activated receptor (PPARgamma)-interacting protein) appears to serve as a linker between cAMP response element-binding protein-binding protein (CBP/p300)anchored and PBP (PPARgamma-binding protein)-anchored coactivator complexes involved in the transcriptional activity of nuclear receptors. Disruption of PRIP and PBP genes results in embryonic lethality between embryonic day 11.5 and 12.5 (postcoitum), indicating that PRIP and PBP are essential and nonredundant coactivators. Both PRIP and PBP were initially identified as PPARgamma coactivators, suggesting a role for these molecules in PPARgamma-induced adipogenesis. PBP-/- mouse embryonic fibroblasts fail to exhibit PPARgamma-stimulated adipogenesis indicating that PBP is a downstream regulator of PPARgamma-mediated adipogenesis. We now show that PRIP-/- mouse embryonic fibroblasts are also refractory to PPARgamma-stimulated adipogenesis and fail to express adipogenic marker aP2, a PPARgamma-responsive gene. Chromatin immunoprecipitation assays reveal reduced association in PRIP-/- cells of PIMT (PRIP-binding protein) and PBP with aP2 gene promoter, suggesting that PRIP is required for the linking of CBP/p300-anchored cofactor complex with PBP-anchored mediator complex. These data indicate that PRIP, like PBP, is a downstream regulator of PPARgamma-mediated adipogenesis and that both these coactivators are required for the successful completion of adipogenic program.

PAT5A: a partial agonist of peroxisome proliferator-activated receptor gamma is a potent antidiabetic thiazolidinedione yet weakly adipogenic.

PAT5A [5-[4-[N-(2-pyridyl)-(2S)-pyrrolidine-2-methoxyl]phenylmethylene[thiazolidine-2,4-dione, malic acid salt]], a chemically distinct unsaturated thiazolidinedione, activates peroxisome proliferator-activated receptor gamma (PPARgamma) submaximally in vitro with the binding affinity approximately 10 times less than that of rosiglitazone, a highly potent thiazolidinedione. PAT5A reduces plasma glucose level and improves insulin sensitivity in insulin resistant db/db mice, similar to that of rosiglitazone, while exerting a relatively weak adipogenic effect. In contrast to rosiglitazone, PAT5A inhibits cholesterol and fatty acid biosynthesis suggesting that PAT5A possesses a unique receptor-independent non-PPAR related property. PAT5A induces qualitatively similar but quantitatively different protease digestion patterns and interacts with PPARgamma differently than rosiglitazone. PAT5A shows differential cofactor recruitment and gene activation than that of rosiglitazone. Thus, the partial agonism of PAT5A to PPARgamma together with its receptor independent effects may contribute to its antidiabetic potency similar to rosiglitazone in vivo despite reduced affinity for PPARgamma. These biological effects suggest that PAT5A is a PPARgamma modulator that activates some (insulin sensitization), but not all (adipogenesis), PPARgamma-signaling pathways.

Novel prostaglandin D(2)-derived activators of peroxisome proliferator-activated receptor-gamma are formed in macrophage cell cultures.

Incubation of RAW 264.7 murine macrophages with 9,15-dihydroxy-11-oxo-, (5Z,9alpha,13E,15(S))-Prosta-5,13-dien-1-oic acid [prostaglandin D(2) (PGD(2))] induced formation of considerable peroxisome proliferator-activated receptor-gamma (PPARgamma) activity [Nature 391 (1998) 79]. Because PGD(2) itself is a poor PPARgamma ligand, we incubated RAW 264.7 macrophage cultures with prostaglandin D(2) for 24 h and studied the ability of the metabolites formed to activate PPARgamma. PGD(2) products were extracted and fractionated by reverse phase high-performance liquid chromatography. Chemical identification was achieved by UV spectroscopy, gas-liquid chromatography/mass spectrometry and chemical syntheses of reference compounds. PGD(2) was converted to eight products, six of which were identified. Ligand-induced interaction of PPARgamma with steroid receptor coactivator-1 was determined by glutathione-S-transferase pull-down assays and PPARgamma activation was investigated by transient transfection of RAW 264.7 macrophages. In addition to the previously known ligand 11-oxo-(5Z,9,12E,14Z)-Prosta-5,9,12,14-tetraen-1-oic acid (15-deoxy-delta(12,14)-PGJ(2)), a novel PPARgamma ligand and activator viz. 9-hydroxy-11-oxo-, (5Z,9alpha,12E,14Z)-Prosta-5,12,14-trien-1-oic acid (15-deoxy-delta(12,14)-PGD(2)) was identified. The biological significance of these results is currently under investigation.