The Peroxisome Proliferator Response Element of the Gene Encoding the Peroxisomal β-Oxidation Enzyme Enoyl-CoA Hydratase/3-Hydroxyacyl-CoA Dehydrogenase Is a Target for Constitutive Androstane Receptor β/9-cis-Retinoic Acid Receptor-mediated Transactivation*

The genes encoding the first two enzymes of the peroxisomal β-oxidation pathway, acyl-CoA oxidase (AOx) and enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase (HD), contain upstreamcis-acting regulatory regions termed peroxisome proliferator response elements (PPRE). Transcription of these genes is mediated through the binding of peroxisome proliferator-activated receptor α (PPARα), which binds to a PPRE as a heterodimer with the 9-cis-retinoic acid receptor (RXRα). Here we demonstrate that the HD-PPRE is also a target for the constitutive androstane receptor β (CARβ). In vitro binding analysis showed that CARβ bound the HD-PPRE, but not the AOx-PPRE, as a heterodimer with RXRα. Binding of CARβ/RXRα to the HD-PPRE occurred via determinants that overlap partially with those required for PPARα/RXRα binding. In vivo, CARβ/RXRα activated transcription from an HD-PPRE luciferase reporter construct. Interestingly, CARβ was shown to also modulate PPARα/RXRα-mediated transactivation in a response element-specific manner. In the presence of the peroxisome proliferator, Wy-14,643, CARβ had no effect on PPARα/RXRα-mediated transactivation from the HD-PPRE but antagonized transactivation from the AOx-PPRE in both the presence and the absence of proliferator. Our results illustrate that transcription of the AOx and HD genes is differentially regulated by CARβ and that the HD gene is a specific target for regulation by CARβ. Overall, this study proposes a novel role for CARβ in the regulation of peroxisomal β-oxidation.

The peroxisome proliferator-activated receptors (PPAR) 1 are members of the nuclear hormone receptor superfamily (1). Functioning as ligand-activated transcription factors, PPARs regulate the expression of genes involved in lipid metabolism, adipogenesis, inflammation, and glucose metabolism (2). PPARs respond to a broad class of structurally diverse ligands, including xenobiotic chemicals called peroxisome proliferators and both naturally occurring and synthetic fatty acids (3)(4)(5)(6)(7). Upon binding of ligand, PPARs heterodimerize with the 9-cisretinoic acid receptor, RXR␣, and bind to specific cis-acting DNA response elements termed peroxisome proliferator response elements (PPRE). PPREs consist of direct repeats of the consensus hexameric motif TGACC(T/C) separated by one base pair (DR1) and have been found upstream of a number of target genes, including those encoding the first two enzymes of the peroxisomal ␤-oxidation pathway, acyl-CoA oxidase (AOx) and enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase (HD) (8,9). In addition to PPARs, several other nuclear hormone receptors have been shown to bind PPREs and differentially modulate PPAR function. These include chicken ovalbumin upstream promoter-transcription factor (10), hepatocyte nuclear factor-4 (11), thyroid hormone receptor ␣ (12), retinoid Z receptor ␣ (13), and RevErb␣ (14). Accordingly, transcriptional regulation from PPREs is a net aggregate response generated in part by the availability of PPARs and other factors that bind PPREs, the complexity of the response elements themselves, and the interplay of PPARs with other nuclear hormone receptors and cofactors, including corepressors and coactivators. This complex interaction of various factors and elements ensures that the correct transcriptional response to extra-and intracellular stimuli will be elicited from appropriate target genes.
The nuclear hormone receptor constitutive androstane receptor ␤ (CAR␤) (NR1I4 -Nuclear Receptor Nomenclature Committee, 1999) (15) heterodimerizes with RXR␣ to activate a subset of retinoic acid response elements (15)(16)(17) consisting of DRs related to the half-site consensus motif and separated by 2 or 5 base pairs. CAR␤ response elements include the retinoic acid receptor ␤2 response element (␤-RARE) (15)(16)(17) and the phenobarbital response element module of the CYP2B gene (18,19). Interestingly, CAR␤/RXR␣ possesses ligand-independent transcriptional activation activity from both the ␤-RARE and phenobarbital response element module in the absence of retinoids or any other exogenously added ligand (15). CAR␤ is therefore among a growing number of orphan receptors, such as hepatocyte nuclear factor-4 (20 -22), steroidogenic factor 1 (23), nerve growth factor 1B (24 -26), and OR-1 (27) that are capable of activating transcription in the absence of added ligand. Recently, Forman and co-workers (17) showed that unlike the classical steroid/nuclear hormone receptors that are activated by their cognate ligands, CAR␤ binds the steroid androstane metabolites androstanol and androstenol to antagonize its ligand-independent transcriptional activation, suggesting that CAR␤ could form part of a novel class of liganddeactivated receptors (28).
Considering the similarities between CAR␤ and PPAR␣ with respect to the compositions of their response elements, their strong expression in liver, and their heterodimerization with RXR␣ to modulate transcription, we investigated the potential of CAR␤ to regulate expression from the PPREs of genes involved in peroxisomal ␤-oxidation.

EXPERIMENTAL PROCEDURES
Plasmids-The in vivo and in vitro expression plasmids for rat PPAR␣ and human RXR␣ and the luciferase reporter constructs pCPSluc, pAOx(X2)luc, and pHD(X3)luc have been described (9,10,29,30). The plasmid pM2(X3)luc, which contains the HD-PPRE with a mutation in its second TGACCT-like half-site, has been described (13). The CAR␤/SG5 expression plasmid was constructed by excision of the cDNA for mouse CAR␤ as a SalI-NotI fragment from the plasmid T7lacHisMyc/mCAR␤ (15) (a kind gift of D. D. Moore, Massachusetts General Hospital, Boston, MA), followed by its cloning as a blunt fragment into the SmaI site of pGEM-7Zf(ϩ) (Promega), removal as an EcoRI/BamHI fragment, and insertion into the EcoRI/BglII site of the eukaryotic expression vector pSG5 (Invitrogen). The integrity of the CAR␤/SG5 construct was confirmed by sequencing. The ␤RARE(X2)-TK-luc reporter plasmid was constructed by synthesizing oligonucleotides containing two copies of the ␤-RARE (bold) (31)(32)(33) and overhangs for MluI and BglII (lowercase) 5Ј-cgcgtAAGGGTTCACCGAAAGT-TCACTCGCATAAGGGTTCACCGAAAGTTCACTCGCATA and its complement, 5Ј-gatctATGCGAGTGAACTTTCGGTGAACCCTTATG-CGAGTGAACTTTCGGTGAACCCTTA. The oligonucleotides were phosphorylated with T4 polynucleotide kinase, annealed, and cloned into the pGL2 control vector (Promega) via MluI and BglII sites. The SV40 promoter was excised by digestion with BglII and HindIII and replaced with a 170-base pair fragment of the TK promoter, which was excised from the plasmid TK-luc-pGL2 (34) by digestion with BglII and HindIII. The integrity of the construct was verified by sequencing.
Electrophoretic Mobility Shift Analysis-To examine the DNA-binding dynamics of PPAR␣, RXR␣, and CAR␤ on the AOx-and HD-PPREs, electrophoretic mobility shift analysis (EMSA) was performed with in vitro synthesized proteins and radiolabeled AOx-and HD-PPRE double-stranded oligonucleotides. PPAR␣, RXR␣, and CAR␤ were synthesized in vitro using the TNT-coupled transcription/translation reticulocyte lysate system (Promega) according to the manufacturer's protocol. Reactions were carried out with L-[ 35 S]methionine and analyzed on 15% polyacrylamide gels. Parallel reactions were performed with unlabeled methionine to yield translation products for use in EMSA. Oligonucleotides corresponding to the AOx-PPRE (5Ј-gatCCTTTCCCGAACGT-GACCTTTGTCCTGGTCCCCTTTTGCT and its complement) or the HD-PPRE (5Ј-gatCCTCTCCTTTGACCTATTGAACTATTACCTAC-ATTTGA and its complement) were annealed and end-labeled with the Klenow fragment of DNA polymerase I and [␣-32 P]dATP. Bold sequences indicate TGACCT-like motifs. Combinations of PPAR␣-, RXR␣-, or CAR␤-programmed reticulocyte lysate (as indicated) were incubated at 25°C for 5 min in reaction buffer containing 6 mM HEPES, pH 7.9, 120 mM NaCl, 0.4 mM MgCl 2 , 0.1 mM EDTA, 7% (v/v) glycerol, 4 g of bovine serum albumin, 4 g of nonspecific competitor DNA (poly(dI-dC) and sonicated salmon sperm DNA, 1:1 weight ratio), and 0.2 mM dithiothreitol. 2 pmol of labeled AOx-or HD-PPRE probe was added to the reactions (total volume, 15 l), and the incubation continued for 25 min. The total amount of reticulocyte lysate in each reaction was kept constant by the inclusion of unprogrammed lysate. Reaction mixtures were analyzed by electrophoresis at 4°C on prerun 3.5% polyacrylamide gels (30:1 acrylamide/N,NЈ-methylenebisacrylamide weight ratio) with 22 mM Tris base/22 mM boric acid/1 mM EDTA as running buffer, followed by autoradiography.

Transient Transfections and Measurement of Luciferase Activity-
Transfections were performed in African green monkey kidney BSC40 cells at 60 -80% confluence using the calcium phosphate method (10). Monolayer cultures in 10-cm plates were transfected with 5 g of luciferase reporter plasmid (pAOx(X2)luc, pHD(X3)luc, or pM2(X3)luc), 2 g each of PPAR␣ and RXR␣ expression plasmids, and 0.5-4 g of CAR␤ expression plasmid as indicated in figure legends. 2 g of the ␤-galactosidase expression vector pCH110 (Amersham Pharmacia Biotech) was included in transfections as an internal reference. Effector plasmid dosage was kept constant by addition of appropriate amounts of the corresponding empty vectors. Total DNA was normalized to 20 g by addition of salmon sperm DNA. Transfection medium without phenol red and containing 5% (v/v) charcoal-stripped fetal bovine serum was supplemented with either 0.1 mM Wy-14,643 (Chemsyn, Lenexa, KS), 5 and 10 M 5␣-androstan-3␣-ol (Steraloids, Newport, RI) or an equivalent volume of dimethyl sulfoxide. Cells were harvested 48 h post-transfection, and relative luciferase activity was determined as described previously (29). Transfections were carried out in duplicate, and all values presented represent the averages of at least three independent experiments.

CAR␤ Binds to the HD-PPRE as a Heterodimer with RXR␣-
Both the AOx-and HD-PPREs contain arrays of the consensus hexameric binding motif TGACCT that could potentially serve as binding sites for CAR␤. To illustrate the binding capability of CAR␤ to these response elements, we performed EMSA with radiolabeled PPRE probes and in vitro translated receptors. As previously shown (13,14), PPAR␣ and RXR␣ bound as a heterodimer to the HD-PPRE (Fig. 1A). CAR␤ did not bind alone to the HD-PPRE but was capable of binding as a heterodimer with RXR␣, generating a complex with slightly faster mobility than that of PPAR␣/RXR␣ (Fig. 1A). Coincubation of PPAR␣, RXR␣ and CAR␤ with the HD-PPRE produced two distinct complexes corresponding to PPAR␣/RXR␣ and CAR␤/RXR␣ heterodimers (Fig. 1A). No higher order ternary complex was observed, suggesting the lack of cooperativity of all three receptors in binding to the HD-PPRE. In contrast to the results obtained with the HD-PPRE, binding of CAR␤ as a heterodimer with RXR␣ on the AOx-PPRE was not observed (Fig. 1B). Binding of CAR␤/RXR␣ to the HD-PPRE was specific, because the radiolabeled complex was refractory to competition by nonspecific unlabeled oligonucleotide but was effectively competed out by the addition of unlabeled HD-PPRE oligonucleotide (Fig. 2).
CAR␤/RXR␣ Recognizes Hexameric Half-Sites II and III of the HD-PPRE-The HD-PPRE consists of four consensus hexameric TGACCT half-sites (I-IV) in an arrangement of two DR1 elements separated by two base pairs, thereby forming an internal DR2 element (Fig. 3). Oligonucleotide probes harboring mutations in each of the four half-sites were used in binding studies to determine which half-sites are responsible for CAR␤/RXR␣ binding. PPAR␣/RXR␣ requires the integrity of half-sites III and IV (the 3Ј DR1 element) of the HD-PPRE for binding (Refs.13, 14, and 30 and data not presented). Disruption of half-sites II and III (comprising the DR2 element) abrogated CAR␤/RXR␣ binding, whereas half-sites I and IV were dispensable for CAR␤/RXR␣ binding (Fig. 3). These results show that CAR␤/RXR␣ and PPAR␣/RXR␣ overlap in their binding to the HD-PPRE at half-site III.
CAR␤ Competes with PPAR␣ for Binding to the HD-PPRE-To determine the influence of CAR␤ on PPAR␣/RXR␣ binding to the HD-PPRE, EMSA was performed using radiolabeled HD-PPRE, constant amounts of PPAR␣ and RXR␣, and increasing amounts of CAR␤. Increasing the amount of CAR␤ reduced the binding of PPAR␣/RXR␣ on the HD-PPRE and led to a concomitant increase in CAR␤/RXR␣ binding (Fig. 4). This result suggests a competition between CAR␤ and PPAR␣ for limiting amounts of the heterodimerization partner, RXR␣. Moreover, the data are suggestive of a dynamic balance be-tween CAR␤/RXR␣ and PPAR␣/RXR␣ for binding to the HD-PPRE.
CAR␤ Differentially Affects PPAR␣/RXR␣-mediated Transactivation from the HD-and AOx-PPREs-To investigate the effects of CAR␤ on transcriptional regulation mediated by PPAR␣/RXR␣, we carried out transient transfection assays using luciferase reporter plasmids containing the HD-PPRE (pHD(X3)luc) and AOx-PPRE (pAOx(X2)luc), along with expression plasmids for PPAR␣, RXR␣, and CAR␤. Cotransfection of PPAR␣ and RXR␣ expression plasmids with the HD-PPRE reporter plasmid led to an ϳ6-fold induction in the levels of transcription over basal levels in the absence of the peroxisome proliferator, Wy-14,643, and to an ϳ20-fold induction in the levels of transcription in the presence of proliferator (Fig.  5A). Increasing amounts of the expression plasmid for CAR␤ did not significantly affect PPAR␣/RXR␣-mediated transcription in the presence of proliferator and modestly reduced transcription levels in its absence. Interestingly, increasing amounts of CAR␤ expression plasmid antagonized PPAR␣/ RXR␣-mediated transactivation from the AOx-PPRE in a dosedependent manner both in the presence and the absence of Wy-14,643 (Fig. 5B). These data illustrate that CAR␤ differentially affects PPAR␣/RXR␣-mediated transcription from the HD-and AOx-PPREs.
CAR␤/RXR␣ Potentiates Transactivation from the HD-PPRE-Because we observed that CAR␤/RXR␣ heterodimers could bind the HD-PPRE in vitro, we sought to determine whether the HD-PPRE is a specific target for CAR␤/RXR␣mediated transcription in vivo. Transient transfections of BSC40 cells were carried out with the pHD(X3)luc reporter plasmid in the absence or presence of expression plasmid for RXR␣ and with varying amounts of CAR␤ expression plasmid. We observed that CAR␤ in the presence of coexpressed RXR␣ could potentiate transcription from the HD-PPRE in a dose-dependent manner, and with equal competence in both the absence and presence of the peroxisome proliferator, Wy-14,643 (Fig. 6A). In the presence of exogenously expressed RXR␣, transcription from the HD-PPRE was induced ϳ12-fold over basal levels by 4 g of CAR␤ expression plasmid. This potentiation of transcription by CAR␤/RXR␣ was abrogated when a reporter plasmid harboring a mutation at half-site II of the HD-PPRE (pM2(X3)luc) was used in transfection (Fig. 6B), in agreement with in vitro results demonstrating that the integ-rity of half-site II of the HD-PPRE is required for CAR␤/RXR␣ binding (Fig. 3B).
The CAR␤ Ligand 5␣-Androstan-3␣-ol Reduces Transactivation from the HD-PPRE by CAR␤/RXR␣-The steroid androstanol metabolite, 5␣-androstan-3␣-ol, has recently been shown to serve as a ligand for the CAR␤ receptor (17). Interestingly, this ligand acts to reduce transcriptional activation from a ␤-RARE by the CAR␤ receptor (17). We investigated whether 5␣-androstan-3␣-ol would also inhibit transactivation from the HD-PPRE by CAR␤/RXR␣. In transient transfections performed in the presence of expression plasmids for CAR␤ and RXR␣, addition of 5␣-androstan-3␣-ol led to an ϳ50% reduction in transcriptional activity of luciferase reporter construct containing two copies of ␤-RARE (p␤RARE(X2)-TK-luc) and to an ϳ30% reduction in transcriptional activity of a reporter construct containing three copies of the HD-PPRE (pHD(X3)luc) (Fig. 7). These results suggest that in BSC40 cells, the CAR␤ ligand retains moderate transcriptional inhibitory effects, with a reporter plasmid containing two copies of the ␤RARE showing slightly greater transcriptional inhibition than a reporter containing three copies of the HD-PPRE. Lysate volumes were kept constant by the addition of unprogrammed lysate as appropriate. Radiolabeled HD-PPRE was added to the binding reactions following a 5-min preincubation, and reactions were kept at 25°C for an additional 25 min before electrophoresis.

FIG. 5. Transactivation by PPAR␣/RXR␣ from the HD-and
AOx-PPREs is differentially modulated by CAR␤. BSC40 monolayer cells were transfected with 5 g of the luciferase reporter pHD(X3)luc (A) or pAOx(X2)luc (B) and expression plasmids for PPAR␣ (2 g), RXR␣ (2 g) and CAR␤ (0.5-4 g) in the absence or presence of 0.1 mM Wy-14, 643. Plasmid dosage was normalized by the addition of empty expression vector. Cells were harvested 48 h post-transfection, and luciferase activity was quantitated. Transfections were carried out in duplicate, and the values reported represent the average of three independent experiments. Values from independent experiments did not vary by more than 15%.

DISCUSSION
Because of the abundant expression of the nuclear hormone receptor CAR␤ in liver and its functional interaction with RXR␣, we investigated whether CAR␤ had a role in the peroxisomal ␤-oxidation of fatty acids through regulation of transcription of the HD and AOx genes via their PPREs. We have demonstrated that the HD-PPRE is a target for CAR␤/RXR␣ heterodimers and that CAR␤/RXR␣ stimulates transcription from the HD-PPRE. This increase in transcriptional activity is approximately the same in either the absence or the presence of the peroxisome proliferator, Wy-14,643, affirming the ligandindependent transcriptional activation activity that has previously been demonstrated for CAR␤ (16).
The presence of CAR␤ does not affect transactivation from the HD-PPRE by PPAR␣/RXR␣. In contrast, CAR␤ serves to antagonize PPAR␣/RXR␣-mediated transcriptional induction from the AOx-PPRE. We attribute the effects of CAR␤ on transcription from the HD-and AOx-PPREs in the presence of PPAR␣ to the availability of RXR␣ required for heterodimerization with both CAR␤ and PPAR␣. Because CAR␤/RXR␣ heterodimers form a complex on the HD-PPRE, we suggest that CAR␤ sequesters RXR␣ away from PPAR␣ to form a heterodimeric CAR␤/RXR␣ complex that is less transcriptionally robust than the PPAR␣/RXR␣ complex on the HD-PPRE. In the case of the AOx-PPRE, which does not bind CAR␤/RXR␣, sequestration of RXR␣ away from PPAR␣ by CAR␤ would result in decreased transcriptional activity, likely as a result of the formation of nonbinding, transcriptionally inactive CAR␤/ RXR␣ heterodimers, concomitant with a reduction in the number of transcriptionally active PPAR␣/RXR␣ heterodimers.
The involvement of cofactors such as SRC-1, p300, N-CoR, and SMRT-1 in transcriptional regulation by nuclear hormone receptors is well established (35)(36)(37)(38). The binding of the ligand 5␣-androstan-3␣-ol to CAR␤ has been suggested to result in the dissociation of SRC-1 from CAR␤, thereby bringing about transcriptional deactivation (17,39). Addition of 5␣-androstan-3␣-ol in transient transfections did result in transcriptional deactivation by CAR␤/RXR␣ from both the ␤-RARE and the HD-PPRE; however, the extent of deactivation was less than what has previously been reported. The observed decrease in deactivation could stem from endogenous levels of PPAR␣ and RXR␣ in BSC40 cells, which may overcome androstanol deactivation by promoting transcriptional induction from the HD-PPRE via PPAR␣/RXR␣ heterodimers. In effect, in transient transfections expressing PPAR␣, RXR␣, and CAR␤, addition of androstanol did not affect the level of transcriptional activation from the HD-PPRE (data not presented). PPAR␣/RXR␣ heterodimers bind weakly to ␤-RARE (40), and therefore endogenous cellular levels of PPAR␣ and RXR␣ would probably be precluded from potentiating transcription from the ␤-RARE, allowing for the observed deactivation by CAR␤ in the presence of androstanol. Surprisingly, we observed only a moderate reduction of ligand-independent activity by CAR␤/RXR␣ on the ␤-RARE as compared with previously published results (17). These differences could be attributed to inherent differences in FIG. 6. CAR␤/RXR␣ potentiates transcription from the HD-PPRE. A, transfections of BSC40 cells were carried out with 5 g of the reporter plasmid pHD(X3)luc in the absence or presence of RXR␣ expression plasmid (2 g) and varying amounts of CAR␤ expression plasmid (0.5-4 g) and in the absence or presence of the peroxisome proliferator, Wy-14, 643 (0.1 mM). Transfections were carried out in duplicate and represent the average of three independent experiments. Values from independent transfections did not vary by more than 15%. B, half-site II of the HD-PPRE is required for transactivation by CAR␤/ RXR␣. BSC40 cells were transfected with 5 g of the reporter plasmid pM2(X3)luc, which harbors a mutation in the second TGACCT-like half-site of the HD-PPRE, and with expression plasmids for RXR␣ (2 g) and CAR␤ (2 g or 4 g), as indicated. The values shown represent the averages of three independent transfections carried out in duplicate. Values from independent transfections did not vary by more than 15%.
FIG. 7. The CAR␤ ligand, 5␣-androstan-3␣-ol, reduces transactivation from the HD-PPRE by CAR␤/RXR␣. Transient transfections were performed in BSC40 cells with expression plasmids for RXR␣ (2 g) and CAR␤ (4 g) and with 5 g of the luciferase reporter plasmids pHD(X3)luc or p␤RARE(X2)-TK-luc, in the absence or presence of 5␣androstan-3␣-ol (5 and 10 M). The values reported represent the averages of three independent transfections done in duplicate. Values from independent transfections did not vary more than 15%. the cell lines used, including the relative levels of endogenous nuclear hormone receptors, corepressors and coactivators. These and other cell-specific factors could influence the observed differences in ligand-dependent deactivation.
The HD-PPRE is a complex response element composed of four hexameric DRs arranged as two tandem DR1 arrays separated by two nucleotides, thereby forming a DR2. This complex arrangement permits the interaction of a variety of nuclear hormone receptors on the HD-PPRE (9 -11, 13, 14), leading to a complex model of transcriptional regulation from this response element. CAR␤/RXR␣ heterodimers bind to halfsites on the HD-PPRE that partially overlap with half-sites recognized by PPAR␣/RXR␣ heterodimers. Half-sites II and III of the HD-PPRE, which form a DR2, are required for CAR␤/ RXR␣ binding, whereas PPAR␣/RXR␣ binds to sites III and IV. CAR␤/RXR␣-mediated transactivation is eliminated when half-site II or III is mutated. It has been reported that half-site III of the HD-PPRE is occupied by RXR␣, whereas the more distal half-site (IV) is occupied by PPAR␣ (41). Considering this and the fact that CAR␤/RXR␣ heterodimers have been shown to bind ␤-RARE with DR2 spacing (15), it is not surprising that CAR␤ would likely occupy half-site II, which matches perfectly the consensus hexameric half-site sequence. Furthermore, the intricate combinations of various nuclear hormone receptors that can modulate the transcriptional response from the HD-PPRE converge on the second hexameric half-site (13,14). Nevertheless, the limitations of our in vitro assays do not allow us to distinguish which half-site is specifically occupied by CAR␤ when bound as a heterodimer with RXR␣ on the HD-PPRE.
Our findings suggest that CAR␤ can modulate the transcriptional response of the genes coding for two enzymes of the peroxisomal ␤-oxidation pathway, AOx and HD. The peroxisomal ␤-oxidation pathway is involved preferentially in the metabolism of long and very long chain fatty acyl-CoAs. AOx, the first enzyme in the pathway, is rate-limiting. We have shown that CAR␤ decreases transcriptional activation of the AOx gene while increasing transcriptional activation of the HD gene, which encodes the second enzyme in the peroxisomal ␤-oxidation pathway. This differential regulation of the genes encoding the first two enzymes of the peroxisomal ␤-oxidation pathway may represent an adaptive cellular response that primes the pathway to respond rapidly to cellular oxidative demands under physiological conditions where repression of the transcription of the AOx gene is relieved. The convergence of the CAR␤, PPAR␣ and RXR␣ signaling pathways underscores the complex and dynamic processes by which various metabolic cues are integrated to elicit the correct transcriptional response leading to control of peroxisomal ␤-oxidation.
In summary, we demonstrate that CAR␤/RXR␣ heterodimers play a role in fatty acid homeostasis by regulating the transcription of the genes encoding the first two enzymes of the peroxisomal ␤-oxidation pathway, acyl-CoA oxidase and enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase. We also show that the PPRE of the HD gene is a target for CAR␤/ RXR␣ heterodimers. An understanding of the various transcription factors that control peroxisomal ␤-oxidation could contribute to the development of pharmacological agents that specifically target this pathway as a way to treat disorders of lipid metabolism and homeostasis.