ABSTRACT
Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (mHMG-CoAS) is a key enzyme in ketogenesis, catalyzing the condensation of acetyl-CoA and acetoacetyl-CoA to generate HMG-CoA, which is eventually converted to ketone bodies. Transcription of the nuclear-encoded gene for mHMG-CoAS is stimulated by peroxisome proliferator-activated receptor (PPAR) alpha, a fatty acid-activated nuclear hormone receptor. Here we show that the mHMG-CoAS protein physically interacts with PPARalpha in vitro, and potentiates PPARalpha-dependent transcriptional activation via the cognate PPAR response element of the mHMG-CoAS gene in vivo. Immunofluorescence of transiently transfected cells demonstrated that in the presence of PPARalpha, mHMG-CoAS is translocated into the nucleus. Binding to PPARalpha, stimulation of PPARalpha activity and nuclear penetration require the integrity of the sequence LXXLL in mHMG-CoAS, a motif known to mediate the interaction between nuclear hormone receptors and coactivators. These findings reveal a novel mechanism of gene regulation whereby the product of a PPARalpha-responsive gene, normally resident in the mitochondria, directly interacts with this nuclear hormone receptor to autoregulate its own nuclear transcription.
Subject(s)
Gene Expression Regulation, Enzymologic , Hydroxymethylglutaryl-CoA Synthase/genetics , Hydroxymethylglutaryl-CoA Synthase/metabolism , Mitochondria/enzymology , Nuclear Proteins/metabolism , Receptors, Cytoplasmic and Nuclear/metabolism , Transcription Factors/metabolism , Amino Acid Sequence , Animals , Biological Transport , COS Cells , Cell Nucleus/metabolism , Molecular Sequence Data , Nuclear Proteins/genetics , Protein Sorting Signals , Receptors, Cytoplasmic and Nuclear/genetics , Transcription Factors/genetics , Transcriptional Activation , TransfectionABSTRACT
Receptor interacting protein 140 (RIP140), a previously identified putative ligand-dependent coactivator of nuclear hormone receptors, was isolated by yeast two-hybrid cloning as a factor that interacts with peroxisome proliferator-activated receptor alpha (PPARalpha). This interaction in yeast required the integrity of the carboxyl-terminal, ligand-dependent activation domain of PPARalpha. However, protein binding studies carried out in vitro showed that full-length RIP140 bound efficiently to PPARalpha in the absence of exogenously added ligand. RIP140 also bound strongly to the liver-X-receptor (LXRalpha) in the absence of an activator for this receptor. In contrast, a strong interaction of RIP140 with the PPARalpha and LXRalpha heterodimerization partner retinoid-X-receptor alpha (RXRalpha) required the presence of its cognate ligand, 9-cis retinoic acid. Transfection analysis in mammalian cells demonstrated that RIP140 antagonized PPARalpha/RXRalpha- and LXRalpha/RXRalpha-mediated signaling. Our findings identify RIP140 as a novel modulator of transcriptional activation mediated by PPARalpha and LXRalpha and indicate that RIP140 can also bind to nuclear hormone receptors in a ligand-independent manner and repress their activity.
Subject(s)
Liver/chemistry , Nuclear Proteins/pharmacology , Receptors, Cytoplasmic and Nuclear/metabolism , Receptors, Retinoic Acid/metabolism , Transcription Factors/metabolism , Transcriptional Activation/drug effects , Adaptor Proteins, Signal Transducing , Cloning, Molecular , Humans , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Nuclear Receptor Interacting Protein 1 , Receptors, Cytoplasmic and Nuclear/genetics , Receptors, Estrogen , Receptors, Retinoic Acid/genetics , Retinoid X Receptors , Signal Transduction/drug effects , Transcription Factors/genetics , Transcription, Genetic , TransfectionSubject(s)
Gene Expression Regulation, Enzymologic , Microbodies/physiology , Receptors, Cytoplasmic and Nuclear/physiology , Receptors, Retinoic Acid/physiology , Receptors, Thyroid Hormone/physiology , Transcription Factors/physiology , Acyl-CoA Oxidase , Animals , DNA-Binding Proteins/physiology , Humans , Microbodies/enzymology , Oxidoreductases/genetics , Rats , Regulatory Sequences, Nucleic Acid , Retinoid X Receptors , Signal Transduction , Transcriptional ActivationABSTRACT
The yeast two-hybrid system was used to isolate novel cellular factors that interact with the mouse peroxisome proliferator-activated receptor alpha (PPARalpha). One of the interacting clones isolated encoded LXRalpha, a recently described human orphan nuclear hormone receptor. LXRalpha bound directly to PPARalpha, as well as to the common heterodimerization partner 9-cis-retinoic acid receptor (RXRalpha). LXRalpha did not form a DNA binding complex with PPARalpha on synthetic hormone response elements composed of direct repeats of the TGACCT consensus half-site or on naturally occurring peroxisome proliferator response elements (PPREs) or LXRalpha response elements. However, LXRalpha inhibited binding of PPARalpha/RXRalpha heterodimers to PPREs, and coexpression of LXRalpha in mammalian cells antagonized peroxisome proliferator signaling mediated by PPARalpha/RXRalpha in vivo. These findings identify a novel partner for PPARalpha and suggest that LXRalpha plays a role in modulating PPAR-signaling pathways in the cell.
Subject(s)
DNA-Binding Proteins/metabolism , Receptors, Cytoplasmic and Nuclear/physiology , Signal Transduction , Transcription Factors/physiology , Tretinoin/pharmacology , Animals , Base Sequence , Binding Sites , Cell Line , Cloning, Molecular , Consensus Sequence , DNA-Binding Proteins/biosynthesis , DNA-Binding Proteins/isolation & purification , Humans , Liver X Receptors , Macromolecular Substances , Mice , Microbodies/drug effects , Microbodies/physiology , Molecular Sequence Data , Oligodeoxyribonucleotides , Orphan Nuclear Receptors , Protein Biosynthesis , Receptors, Cytoplasmic and Nuclear/biosynthesis , Receptors, Cytoplasmic and Nuclear/isolation & purification , Receptors, Retinoic Acid/biosynthesis , Receptors, Retinoic Acid/isolation & purification , Receptors, Retinoic Acid/metabolism , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism , Repetitive Sequences, Nucleic Acid , Retinoid X Receptors , Transcription Factors/biosynthesis , Transcription Factors/isolation & purification , Transcription Factors/metabolism , Transcription, Genetic , Tretinoin/metabolismABSTRACT
Calreticulin is a ubiquitous calcium binding/storage protein found primarily in the endoplasmic reticulum. Calreticulin has been shown to inhibit DNA binding and transcriptional activation by glucocorticoid and androgen hormone receptors by binding to the conserved sequence KXFF(K/R)R, present in the DNA-binding domains of all known members of the steroid/nuclear hormone receptor superfamily. To determine whether calreticulin might be a general regulator of hormone-responsive pathways, we examined its effect on DNA binding in vitro and transcriptional activation in vivo by heterodimers of the peroxisome proliferator-activated receptor (PPAR) and the 9-cis retinoic acid receptor (RXR alpha). We show here that purified calreticulin inhibits the binding of PPAR/RXR alpha heterodimers and of other nuclear hormone receptors, to peroxisome proliferator-responsive DNA elements in vitro. However, overexpression of calreticulin in transiently transfected cultured cells had little or no effect on transactivation mediated by PPAR/RXR alpha. Therefore, while calreticulin inhibits the binding of both nuclear and steroid hormone receptors to cognate response elements in vitro, our findings suggest that calreticulin does not necessarily play an important role in the regulation of all classes of hormone receptors in vivo.
Subject(s)
Calcium-Binding Proteins/pharmacology , DNA/metabolism , Receptors, Cytoplasmic and Nuclear/physiology , Receptors, Retinoic Acid/physiology , Ribonucleoproteins/pharmacology , Transcription Factors/physiology , Transcription, Genetic/drug effects , Amino Acid Sequence , Animals , Base Sequence , Binding Sites , Calreticulin , Macromolecular Substances , Molecular Sequence Data , Rats , Receptors, Cytoplasmic and Nuclear/chemistry , Receptors, Cytoplasmic and Nuclear/genetics , Receptors, Retinoic Acid/chemistry , Receptors, Retinoic Acid/genetics , Retinoid X Receptors , Transcription Factors/chemistry , Transcription Factors/genetics , Transcriptional Activation , TransfectionABSTRACT
Peroxisome proliferator-activated receptors (PPARs) are orphan members of the nuclear hormone receptor superfamily. PPARs bind to cognate response elements through heterodimerization with retinoid X receptors (RXRs). Together PPAR/RXR regulate the transcription of genes for which products are involved in lipid homeostasis, cell growth, and differentiation. PPARs are activated by fatty acids and by nongenotoxic rodent hepatocarcinogens called peroxisome proliferators through as of yet undefined signal transduction pathways. In an effort to elucidate the requirements for PPAR function and the pathways of its activation, we expressed mouse PPAR alpha and human RXR alpha in the yeast Saccharomyces cerevisiae. Mouse PPAR alpha and human RXR alpha had little activity individually in yeast; however, when cosynthesized, they were able to synergistically activate transcription via cognate response elements. Transactivation was independent of exogenously added activators of either receptor but was potentiated by the addition of petroselinic acid, a fatty acid shown to activate PPARs in mammalian cells. Similar experiments were carried out in a mutant yeast strain lacking peroxisomes entirely or in a mutant strain deficient for 3-ketoacyl-CoA thiolase, the final enzyme of the peroxisomal beta-oxidation cascade. The findings showed that constitutive transactivation by PPAR/RXR did not require the complete beta-oxidation pathway or intact peroxisomes but required intact peroxisomes for potentiation by exogenously added petroselinic acid. This study demonstrates that at least part of the mammalian peroxisome proliferator-signaling pathway can be faithfully reconstituted in yeast and that activation of PPAR by at least one particular fatty acid requires the integrity of peroxisomes.
Subject(s)
Fatty Acids/pharmacology , Microbodies/metabolism , Nuclear Proteins/metabolism , Receptors, Cytoplasmic and Nuclear/metabolism , Receptors, Retinoic Acid/metabolism , Saccharomyces cerevisiae/metabolism , Transcription Factors/metabolism , Transcriptional Activation , Animals , Base Sequence , Electrophoresis , Humans , Mice , Microbodies/genetics , Molecular Sequence Data , Nuclear Proteins/genetics , Oleic Acids/metabolism , Rats , Receptors, Cytoplasmic and Nuclear/genetics , Receptors, Retinoic Acid/genetics , Retinoid X Receptors , Saccharomyces cerevisiae/genetics , Signal Transduction/genetics , Signal Transduction/physiology , Transcription Factors/genetics , Transformation, GeneticABSTRACT
The peroxisome proliferator-activated receptor (PPAR) binds cooperatively to cognate peroxisome proliferator-responsive elements (PPRE) in vitro through heterodimerization with retinoid X receptors (RXR). We used the yeast two-hybrid system to determine whether these two nuclear receptors physically interact in vivo. Mouse (m) PPAR and human (h) RXR alpha were synthesized as fusion proteins to either the DNA-binding domain (GBD) or the transactivation domain (GAD) of the yeast GAL4 transcription-activator protein, and were tested for their ability to activate expression of a GAL1::lacZ reporter gene. Strong activation was observed only in yeast transformed with combinations of GBD::mPPAR and GAD::hRXR alpha or with GAD::mPPAR and GBD::hRXR alpha. Homodimeric interaction by mPPAR was not detected. These results provide evidence for the interaction of PPAR and RXR alpha in vivo in the absence of a PPRE target site or exogenously added ligands.
Subject(s)
Nuclear Proteins/metabolism , Receptors, Cytoplasmic and Nuclear/metabolism , Receptors, Retinoic Acid , Saccharomyces cerevisiae Proteins , Transcription Factors/metabolism , Animals , DNA-Binding Proteins/metabolism , Fungal Proteins/metabolism , Humans , Mice , Retinoid X Receptors , Transformation, GeneticABSTRACT
Peroxisome proliferator-activated receptors (PPARs) stimulate the expression of several genes involved in lipid metabolism by binding to specific cis-acting peroxisome proliferator-responsive elements (PPREs) via cooper-ativity with retinoid X receptors. We demonstrate here that hepatocyte nuclear factor-4 (HNF-4), another member of the nuclear hormone receptor superfamily, bound with differing affinities to the PPREs from the genes encoding rat acyl-CoA oxidase and hydratase-dehydrogenase, the first two enzymes of the peroxisomal beta-oxidation pathway. In cotransfection assays, HNF-4 repressed rat PPAR-dependent activation of a reporter gene linked to the acyl-CoA oxidase PPRE, either in the absence or presence of the peroxisome proliferator, Wy-14,643. Rat PPAR-dependent activation of a reporter gene linked to the hydratase-dehydrogenase PPRE was less efficiently repressed by HNF-4 in the absence of Wy-14,643 than was activation from the acyl-CoA oxidase PPRE. However, in the presence of Wy-14,643, HNF-4 functioned cooperatively with PPAR to significantly enhanced induction from the hydratase-dehydrogenase PPRE. These results suggest that the genes encoding the first two enzymes of the peroxisomal beta-oxidation pathway are subject to differential regulation by the interplay of multiple members of the steroid/nuclear hormone receptor superfamily, mitigated in part by the structures of the PPREs and by the presence of activators of PPARs.
Subject(s)
DNA-Binding Proteins , Phosphoproteins , Receptors, Cytoplasmic and Nuclear/genetics , Transcription Factors/genetics , Transcription Factors/metabolism , Transcriptional Activation , Acyl-CoA Oxidase , Animals , Base Sequence , DNA Probes , Hepatocyte Nuclear Factor 4 , Microbodies/drug effects , Molecular Sequence Data , Oxidoreductases/metabolism , Pyrimidines/pharmacology , RatsABSTRACT
Peroxisome proliferators form a family of diverse xenobiotic compounds that includes hypolipidemic agents, herbicides, and plasticizers. These compounds activate transcription of a subset of nuclear genes including those encoding peroxisomal fatty acid beta-oxidation enzymes, whose elevated activities can lead to hepatocarcinogenesis. Induction of the genes encoding fatty acyl-CoA oxidase and hydratase-dehydrogenase, the first and second enzymes of the pathway, is mediated by peroxisome proliferator-activated nuclear receptors (PPARs) that bind to upstream responsive elements (PPREs) through heterodimerization with retinoid X receptors. We demonstrate that the chicken ovalbumin upstream promoter transcription factor 1 (COUP-TF1), another member of the nuclear hormone receptor superfamily, binds to the hydratase-dehydrogenase PPRE in vitro and in vivo and antagonizes PPAR-dependent signaling. These data suggest that members of the COUP-TF family play a role in modulating receptor-mediated activation of peroxisome proliferator-responsive genes.
Subject(s)
DNA-Binding Proteins/metabolism , Ovalbumin/genetics , Receptors, Cell Surface/metabolism , Receptors, Cytoplasmic and Nuclear , Receptors, Retinoic Acid , Transcription Factors/metabolism , Animals , Base Sequence , Binding Sites , COUP Transcription Factor I , Cell Line , Chickens , Cloning, Molecular , DNA-Binding Proteins/biosynthesis , HeLa Cells , Humans , Liver Neoplasms, Experimental , Luciferases/biosynthesis , Luciferases/metabolism , Macromolecular Substances , Molecular Sequence Data , Oligodeoxyribonucleotides , Promoter Regions, Genetic , Protein Biosynthesis , Rats , Receptors, Cell Surface/biosynthesis , Recombinant Proteins/biosynthesis , Recombinant Proteins/metabolism , Retinoid X Receptors , Retinoids/metabolism , Signal Transduction , Transcription Factors/biosynthesis , Transcription, Genetic , Transfection , Tumor Cells, CulturedABSTRACT
A peroxisome proliferator-responsive element is located in the 5'-flanking region of the gene encoding rat hydratase-dehydrogenase, the second enzyme of the peroxisomal beta-oxidation pathway. DNase I footprint analysis with nuclear extracts from proliferator-responsive rat H4IIEC3 cells revealed two protected regions within the 196-base pair peroxisome proliferator-responsive element. Both regions contained multiple copies of a motif related to the consensus steroid hormone receptor binding half-site TGACCT, suggesting that peroxisome proliferator-dependent activation of this gene is mediated via peroxisome proliferator-activated receptors. Region II contains three TGACCT-like motifs in a direct repeat array. An oligonucleotide corresponding to this region was sufficient to confer responsiveness to the peroxisome proliferator ciprofibrate onto a heterologous promoter, as determined by transient transfection assays. Gel retardation assays demonstrated that nuclear factors bound to the hydratase-dehydrogenase oligonucleotide. Mutation of a single G residue within the second repeat motif abolished factor binding and consequently the ability of the element to respond to ciprofibrate, directly demonstrating that factor binding is necessary for peroxisome proliferator responsiveness. These results are discussed in the context of our current understanding of the mechanism of the coordinated transcriptional induction of the genes encoding peroxisomal beta-oxidation enzymes by peroxisome proliferators.
Subject(s)
DNA, Neoplasm/metabolism , DNA-Binding Proteins/metabolism , Enoyl-CoA Hydratase/genetics , Isomerases , Microbodies/enzymology , Multienzyme Complexes/genetics , Racemases and Epimerases/genetics , Receptors, Cell Surface/metabolism , Receptors, Cytoplasmic and Nuclear , Transcription Factors/metabolism , 3-Hydroxyacyl CoA Dehydrogenases/genetics , Animals , Base Sequence , Binding Sites , Cell Nucleus/metabolism , Clofibric Acid/analogs & derivatives , Clofibric Acid/pharmacology , Fibric Acids , Gene Expression Regulation, Enzymologic/drug effects , Hypolipidemic Agents/pharmacology , Liver Neoplasms, Experimental , Luciferases/genetics , Luciferases/metabolism , Molecular Sequence Data , Oligodeoxyribonucleotides , Peroxisomal Bifunctional Enzyme , Rats , Transfection , Tumor Cells, CulturedABSTRACT
The ability of peroxisome proliferator-activated receptors (PPARs) to induce expression of a reporter gene linked to a peroxisome proliferator-responsive element (PPRE) from either the rat enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase gene or acyl-CoA oxidase [acyl-CoA:oxygen 2-oxidoreductase, EC 1.3.3.6] gene was examined by transient transfection assays in COS cells. Mouse and rat PPARs, as well as Xenopus PPAR alpha (xPPAR alpha) could induce expression of a reporter gene linked to the hydratase/dehydrogenase PPRE in the presence of the peroxisome proliferators ciprofibrate or Wy-14,643, whereas xPPAR beta and xPPAR gamma were ineffective. A similar induction of expression of a reporter gene linked to the acyl-CoA oxidase PPRE was observed with all PPARs except xPPAR beta. Extracts from cells transfected with PPAR-encoding genes contained factors that bound to both PPREs. In vitro synthesized PPARs could interact weakly with both PPREs; however, binding of each PPAR to both PPREs was significantly increased by the addition of COS cell nuclear extracts, demonstrating that efficient PPAR/DNA binding requires auxiliary cofactors. One cofactor was identified as the 9-cis-retinoic acid receptor, RXR alpha (retinoid X receptor alpha). Cooperative DNA binding and heteromerization between RXR alpha and each of the PPARs could be seen with both PPREs. Our results demonstrate that PPAR/PPRE binding and cooperativity with RXR alpha (and other cofactors) are obligatory but not necessarily sufficient for peroxisome proliferator-dependent transcription induction and that distinct PPREs can selectively mediate induction by particular PPARs.
