©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification of a Peroxisome Proliferator-responsive Element Upstream of the Human Peroxisomal Fatty Acyl Coenzyme A Oxidase Gene (*)

(Received for publication, August 14, 1995; and in revised form, November 14, 1995)

Usha Varanasi Ruiyin Chu Qin Huang Raquel Castellon Anjana V. Yeldandi Janardan K. Reddy (§)

From the Department of Pathology, Northwestern University Medical School, Chicago, Illinois 60611

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Peroxisome proliferators cause a rapid and coordinated transcriptional activation of genes encoding the enzymes of the peroxisomal beta-oxidation pathway in rats and mice. Cis-acting peroxisome proliferator responsive elements (PPREs) have been identified in the 5`-flanking region of H(2)O(2)-producing rat acyl-CoA oxidase (ACOX) gene and in other genes inducible by peroxisome proliferators. To gain more insight into the purported nonresponsiveness of human liver cells to peroxisome proliferator-induced increases in peroxisome volume density and in the activity of the beta-oxidation enzyme system, we have previously cloned the human ACOX gene, the first and rate-limiting enzyme of the peroxisomal beta-oxidation system. We now present information on a regulatory element for the peroxisome proliferator-activated receptor (PPAR)/retinoid X receptor (RXR) heterodimers. The PPRE, consists of AGGTCA C TGGTCA, which is a direct repeat of hexamer half-sites interspaced by a single nucleotide (DR1 motif). It is located at -1918 to -1906 base pairs upstream of the transcription initiation site of this human ACOX gene. This PPRE specifically binds to baculovirus-expressed recombinant rat PPARalpha/RXRalpha heterodimers. In transient transfection experiments, the maximum induction of luciferase expression by ciprofibrate and/or 9-cis-retinoic acid is dependent upon cotransfection of expression plasmids for PPARalpha and RXRalpha. The functionality of this human ACOX promoter was further demonstrated by linking it to a beta-galactosidase reporter gene or to a rat urate oxidase cDNA and establishing stably transfected African green monkey kidney (CV1) cell lines expressing reporter protein. The human ACOX promoter has been found to be responsive to peroxisome proliferators in CV1 cells stably expressing PPARalpha, whereas only a basal level of promoter activity is detected in stably transfected cells lacking PPARalpha. The presence of a PPRE in the promoter of this human peroxisomal ACOX gene and its responsiveness to peroxisome proliferators suggests that factors other than the PPRE in the 5`-flanking sequence of the human ACOX gene may account for differences, if any, in the pleiotropic responses of humans to peroxisome proliferators.


INTRODUCTION

Peroxisomes are cellular organelles that are present in virtually all eukaryotic cells(1) . At present, >50 proteins have been identified in peroxisomes, and more than half of these play a role in lipid metabolism(2) . Of particular interest is that these organelles contain H(2)O(2)-producing flavin oxidases together with catalase, which decomposes H(2)O(2)(3) . Peroxisomes in liver parenchymal cells can be stimulated to proliferate by the administration of certain nonmutagenic chemicals designated as peroxisome proliferators(4, 5) . These form a broad group of compounds of industrial, pharmaceutical, and agricultural value; they include certain phthalate ester plasticizers, industrial solvents, herbicides, leukotriene D(4) antagonists, the adrenal steroid dehydroepiandrosterone, and amphipathic carboxylates such as the hypolipidemic drugs clofibrate and ciprofibrate(2, 5) . When peroxisome proliferators, with ostensibly dissimilar structures and pharmacokinetic properties, are administered to rodents and certain nonrodent species including primates, they cause profound proliferation of peroxisomes in hepatic parenchymal cells and marked increases in the activities of the enzymes required for peroxisomal beta-oxidation of fatty acids, namely fatty acyl-CoA oxidase (ACOX), (^1)enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase bi-(tri)functional enzyme (PBE), and 3-ketoacyl-CoA thiolase(2, 6) . The increased activities of these enzymes are related to the rapid and coordinated transcriptional activation of the nuclear genes encoding these enzymes(7) .

Peroxisome proliferators owe their importance, in part, to the fact that they induce hepatocellular carcinomas in rats and mice on continued exposure(8, 9) . Since these hepatocarcinogens neither bind covalently to DNA nor produce somatic mutations directly or after metabolic activation, it has been postulated that any genetic alterations responsible for carcinogenesis that may eventually occur during chronic exposure to these nongenotoxic carcinogens should be attributable to the tissue-specific pleiotropic responses induced by such agents(9) . Support for a mechanistic relationship between peroxisome proliferation and hepatocarcinogencity is provided, in part, by a close concordance with the magnitude of hepatic peroxisome proliferation and liver tumor development in rats and mice(9, 10) . In liver cells with massive increases in peroxisomal volume density caused by peroxisome proliferators, there is differential transcriptional regulation of genes encoding catalase and of H(2)O(2)- producing peroxisomal ACOX, the first and rate-limiting enzyme of the beta-oxidation pathway(7, 11, 12) . In these livers catalase activity is increased approx2-fold, whereas the ACOX level increases by >20-fold, thus leading to excess production of H(2)O(2) and possibly other reactive oxygen intermediates(7, 12) . Corroborative evidence for the hypothesis that sustained overproduction of intracellular levels of H(2)O(2) in livers of rats and mice with persistent maximal increase in peroxisome volume density leads to neoplastic transformation has been derived from a variety of observations, including the recent finding that African green monkey kidney (CV1) cells stably overexpressing rat peroxisomal ACOX when exposed to a fatty acid substrate formed transformed foci, grew efficiently in soft agar, and developed adenocarcinomas when injected into nude mice(13, 14) .

The postulated link between induction of peroxisome proliferation vis à vis ACOX and hepatocarcinogenicity implies that species that do not respond to peroxisome proliferators, i.e. fail to exhibit significant degree of hepatic peroxisome proliferative response, are less likely to develop liver tumors on chronic exposure(9, 13) . The available data on human hepatocytes in vivo and in vitro suggest that compounds that are peroxisome proliferators in rats and mice have little, if any, effect on human liver cells(15, 16) . Although additional studies are nonetheless required to establish unequivocally the nonresponsiveness of human hepatocytes, the apparent disparity between the effects of peroxisome proliferators in rodent and human hepatocytes may be due to a number of modulating or confounding factors. These include the dose administered, pharmacokinetics, bioavailability, affinity of the agents at the target site, distribution of peroxisome proliferator-activated receptor (PPAR) isoforms in the target (responsive) cells, and the nature of the cis-acting peroxisome proliferator-responsive element (PPRE) in the 5`-flanking region of target genes(2) . Transactivation of peroxisome proliferator-responsive genes is mediated through ligand-activated receptors, collectively referred to as PPARs; these receptors are members of the steroid/thyroid hormone receptor superfamily(17, 18) . Three PPAR isoforms (PPARalpha, PPARbeta, and PPAR) have been isolated both from the mouse and Xenopus(19, 20, 21, 22) . These PPARs have been shown to be activated by a wide array of peroxisome proliferators, as well as, natural and synthetic fatty acids(23) . The discovery of PPAR isoforms has facilitated the identification of PPRE in the upstream regions of the rat ACOX gene(17, 24) . This PPRE is composed of two direct AGG(A/T)CA repeats separated by a single nucleotide(24) . Similar PPREs have been identified in other genes known to be activated by peroxisome proliferators, such as the rat PBE gene(25, 26) , the rabbit CYP4A6 fatty acid -hydroxylase gene(27) , the rat CYP4A1 -hydroxylase gene(28) , the rat malic enzyme gene(29) , and the rat mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase gene(30) . The PPREs characterized to date consist of direct repeats (DR) of AGGTCA related sequences, the consensus half-site recognition sequence for the TR/RAR/RXR receptor family(31, 32) . Like TR, RAR, and VDR, which strongly heterodimerize with RXR to enhance binding to their respective response elements, PPAR also heterodimerizes with RXR to enhance the transactivation of the PPREs of rat ACOX and PBE genes(23, 33) . The relative spacing and orientation of the TGACCT/AGGTCA half-site motifs determine the selective binding of PPAR/RXR to these response elements (34) . The DNA binding of the PPARalpha/RXRalpha heterodimers occurs preferentially when the spacing between the direct repeats was one base pair (DR1) as demonstrated with the PPRE of rat ACOX gene(23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35) . Thus, the convergence of the peroxisome proliferator/retinoid signaling pathways resulting in PPAR/RXR heterodimers to interact with PPREs appears to be a crucial mechanism in the transcriptional activation of ACOX of the rat peroxisomal beta-oxidation enzyme system and by inference in hepatocarcinogenesis by peroxisome proliferators.

To gain more insight into the purported nonresponsiveness of human liver cells to peroxisome proliferator-induced early pleiotropic responses, such as peroxisome proliferation and increase in the activity of beta-oxidation enzyme system, we have undertaken studies to examine the transcriptional regulation of the human ACOX gene. Analysis of the regulation of the human ACOX gene is an important step in our attempts to understand the implications of peroxisome proliferator-induced pleiotropic responses to human health. We previously cloned the human ACOX gene (36) and its corresponding cDNA (37) and now demonstrate the presence of a PPRE in the promoter of this gene. The human ACOX promoter is constitutively active in CV1 cells. This regulatory element of human ACOX gene is a DR1, which interacts specifically with PPARalpha/RXRalpha and is responsive to peroxisome proliferators in CV1 cells stably expressing PPARalpha. These observations indicate that factors other than the PPRE in the 5`-flanking region of the ACOX gene may account for possible differences in the peroxisome proliferator-induced responses between human and rodent.


EXPERIMENTAL PROCEDURES

Plasmid Constructions

The pGL2-basic vector (Promega), which contains the luciferase gene as a reporter, was used for the generation of all constructs for promoter analysis. The isolation of human ACOX gene and subcloning of the 7.8-kb SacI-SacI clone (pSKACOX) containing 5`-flanking region of this ACOX gene (Fig. 1) into pBluescript SK vector (Stratagene) were described previously(36) . The pHACOXLUC7 plasmid, which contains 294 bp of the minimal promoter of ACOX gene, was constructed by the application of polymerase chain reaction, using a pair of oligonucleotide primers, UV1 (5`-cggaagcttCGCGACGACCAGCTGGC, corresponding to coordinates +53 to +37 of the 5`-flanking region of human ACOX gene; transcription initiation site numbered as +1; lower case letters represent linkers) and UV2 (5`-ccgaagcttCCTTTCCGAGGATCAGCTC, -241 to -223) and pSKACOX as template. Both primers have HindIII linkers, which facilitated subcloning the product into the HindIII site of pGL2-basic vector. pHACOXLUC2 contains a 1.6-kb fragment, which was isolated by digesting the pSKACOX plasmid (Fig. 1A, b) with BamHI and cloning this fragment into the BglII site of the pGL2-basic vector. pHACOXLUC4 has the same 1.6-kb BamHI fragment mentioned above linked to the minimal promoter (pHACOXLUC7) using the BglII site. pHACOXLUC6 was made by amplifying a 200-bp fragment encompassing the PPRE from pSKACOX using a pair of oligonucleotide primers, UV3 (5`- cggagatctTCTCTCCCACAGATCAAA, -1829 to -1812) with a BglII linker and UV4 (5`-ccggtcgacAAATCGAGAAGTAGATTC, -2015 to -1998) with a SacI linker. This fragment was linked to the minimal promoter element (-241 to +53). The BglII and SacI sites of the construct pHACOXLUC7 were used to subclone this fragment. pHACOXLUC3 is the same as pHAOX-LUC previously described (36) . pHACOXLUC1 has the most upstream 3.7-kb SacI/BamHI fragment of pSKACOX inserted into the SacI and BglII sites of the construct pHACOXLUC7 in the pGL2-basic vector. pHACOXLUC5 was made by the application of polymerase chain reaction using a pair of oligonucleotide primers, UV1 and UV5 (5`-ggcagatctTACGTTGACGTGAGGTCGG, -2165 to -2147). UV1 has a HindIII linker, and UV5 has a BglII linker to facilitate subcloning in the HindIII and BglII sites of pGL2basic. Rat PPARalpha and rat RXRalpha were subcloned into pSG5 expression vectors. The promoterless beta-galactosidase vector, pNASSbeta(Clontech) was used to insert the human ACOX promoter corresponding to pHACOXLUC1 (Fig. 1A, a) into the XhoI/EcoRI cloning sites of the beta-galactosidase vector (Fig. 2a). This plasmid is designated pbetaHACOX. In order to construct the human ACOX promoter driving urate oxidase (UOX), the beta-galactosidase cDNA was replaced by full-length rat UOX cDNA using the NotI cloning site (Fig. 2b). UOX is used here as a novel indicator gene because of its unique ultrastructural appearance and its ability to target to the peroxisome. This plasmid is designated pHACOXUOX. The rat ACOX promoter cloned into the HindIII/BglII sites of the pGL2-basic vector was used as a positive control(38) . pCH110 (Pharmacia Biotech Inc.) was used as the reporter plasmid expressing the beta-galactosidase gene to normalize for transfection efficiency.


Figure 1: Ciprofibrate-mediated induction of the HACOX promoter requires an upstream cis-acting site. A, diagram of the promoter constructs linked to the luciferase reporter gene, pGL2-basic. A restriction map of the 7.8-kb SacI fragment of the HACOX gene (pSKACOX), which includes the transcription start site as well as the first and second exons is shown at the top. The luciferase reporter constructs are numbered pHACOXLUC1-7 (a-g). Their position with respect to pSKACOX is indicated. pHACOXLUC7 represents the 294-bp minimal promoter generated by polymerase chain reaction as described under ``Experimental Procedures.'' B, the HACOX promoter requires the sequence of the 200-bp fragment between -2015 and -1812 for responsiveness to ciprofibrate. All of the constructs were transfected into H4IIEC3 cells in the presence of Me(2)SO alone (black bar) or 0.5 mM ciprofibrate (gray bar). -Fold induction is the ratio of the value obtained from ciprofibrate-treated cells to those obtained from untreated cells. Values are the means of three experiments normalized to a beta-galactosidase transfection control.




Figure 2: Constructs used in the generation of the stable cell lines CVPHACOX, CVHACOX and CVHACOXUOX. a, this construct designated pbetaHACOX, contains the promoter fragment corresponding to the pHACOXLUC1 plasmid linked to the beta-galactosidase cDNA, which was used in the generation of both the CVPHACOX and the CVHACOX cell lines. b, this construct, designated pHACOXUOX, contains the same ACOX promoter fragment mentioned above linked to the full-length rat UOX cDNA. c, this represents the PPARalpha cDNA under the transcriptional control of the cytomegalovirus promoter. A stably expressing PPARalpha cell line, termed PPARalpha-CV1, was used in the generation of CVPHACOX cell line.



Cell Culture and Transfections

Rat hepatoma H4IIEC3 cells (ATCC CRL 1600) were cultured as monolayers in Dulbecco's modified minimal essential medium. CV1 cells were cultured in minimal essential medium. To both culture media, 10% fetal bovine serum, 1% fungizone, and 100 units of penicillin per ml and 100 µg of streptomycin per ml were added. H4IIEC3 cells, at an 80% confluence in 10-cm culture dishes, were transiently transfected typically with 5 µg of luciferase reporter construct using a modification of the calcium phosphate procedure(39) . Transient transfection experiments with CV1 cells involved cotransfection with RXRalpha and PPARalpha expression plasmids (2 µg each) or control vector pSG5 (Stratagene) along with 0.5 µg of beta-galactosidase vector, pCH110 (Pharmacia). The calcium phosphate DNA precipitate was removed 24 h after transfection, and the cells were refed with the appropriate medium. Experiments with ligands included either vehicle alone (Me(2)SO, or ethanol) or ligand (1.5 mM ciprofibrate in Me(2)SO, or 0.1 µM 9-cis retinoic acid in ethanol). Cells were harvested 48 h after refeeding, washed with phosphate-buffered saline, and incubated for 5 min in 500 µl of lysis buffer containing 100 mM K(2)HPO(4) and 1 mM dithiotreitol and lysed by freeze/thaw (3 times). The supernatant (100 µl) was assayed for luciferase activity with D-luciferin as substrate in a monolight luminometer(20) . To account for transfection efficiency, luciferase values were normalized to beta-galactosidase activity from the cotransfected plasmid pCH110.

Stable Transfection of CV1 Cells with beta-Galactosidase or UOX Genes Under the Transcriptional Control of the Human ACOX Promoter

To generate stable transfectants containing the human pbetaHACOX transgene, a CV1 cell line stably expressing rat PPARalpha (PPARalpha-CV1) was used (Fig. 2c). The PPARalpha cDNA was initially cloned into the pCEP4 vector (Invitrogen) (Fig. 2c). Following transfection, it was selected for over a period of 3 weeks using hygromycin, after which resistant colonies were visible on the plates. The PPARalpha-CV1 cells were subsequently co-transfected with pbetaHACOX (Fig. 2a) and a neomycin-resistant reporter plasmid at a ratio of 20:1. Following transfection, subclones were isolated within 3-4 weeks using G418 for selection and cultured separately. One such cell line designated CVPHACOX was used to analyze the effect of a fatty acid (elaidic acid; C(18)), two structurally diverse peroxisome proliferators (ciprofibrate and Wy-14,643) and the plasticizer, DEHP on the inducibility of beta-galactosidase. In addition, we transfected regular CV1 cells with pbetaHACOX or pHACOXUOX plasmids to generate stable transfectants to assess for induction.

Treatment with Peroxisome Proliferators

For dose-response studies, the medium was removed from the wells and replaced with fresh selecting medium containing Wy-14,643, ciprofibrate, elaidic acid, or DEHP at various concentrations. Cells without the drug were incubated in the presence of Me(2)SO alone. Cells were incubated for 24 h at 37 °C in a 5% CO(2) atmosphere. Duplicate plates were also set up for determination of cell viability using the Cell Titer 96 proliferation kit (Promega) according to manufacturer's instructions. Following transfection, each well was washed twice with phosphate-buffered saline followed by the addition of 100 µl of fresh selecting medium. Cell viability was determined by the addition of 15 µl of tetrazolium dye solution to each well and incubating for at least 24 h. Plates were read at 570 nm with an enzyme-linked immunosorbent assay plate reader using a reference filter at 630 nm to correct for cell debris. The proportion of live cells in each well (percent of control) was calculated by dividing its absorbance by the mean absorbance of the untreated wells. The viability factor for each dose was calculated as the inverse of the proportion of live cells multiplied by 100. Data points represent the mean of two individual experiments, each containing quadruplicate wells for every dose tested. Duplicate cultures were used for beta-galactosidase assay as described below. For time-course experiments, cells were set up as described above and treated with Wy-14,643 (1.5 mM), ciprofibrate (1.5 mM), elaidic acid (1.0 mM), or DEHP (1.5 mM) for 0, 1, 2, 4, 6, 8, 10, 12, 18, 24, 36, and 48 h. Replicate plates were used for cell viability assay as described above.

beta-Galactosidase Assay

Cells were washed twice with phosphate-buffered saline after they had been incubated with the appropriate drug for the specified length of time. 75 µl of reporter lysis buffer (Promega) was added to each well followed by incubation at 37 °C for 4 h. To this, 75 µl of assay 2 times buffer (120 mM Na(2)HPO(4), 80 mM NaH(2)HPO(4), 1.33 mg/ml O-nitrophenyl beta-D-galactopyranoside, and 100 mM beta-mercaptoethanol) was added. Plates were incubated for 24 h at 37 °C and read at 407 nm with an enzyme-linked immunosorbent assay plate reader using a reference filter at 630 nm to correct for cell debris. beta-Galactosidase activity for each well was calculated by dividing its absorbance by the mean value of the untreated wells and multiplying this value by the cell viability correction factor for that particular dose. Data points represent the mean of two individual experiments each containing quadruplicate wells for the doses tested. To visualize the presence of beta-galactosidase in the cells, they were washed twice with phosphate-buffered saline and fixed for 15 min in 1% glutaraldehyde. After fixation, they were washed 4 times with phosphate-buffered saline and stained with an X-gal solution, which contained 400 µg/ml X-gal, 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, and 1 mM magnesium chloride diluted in phosphate-buffered saline. The cells were allowed to stand at 37 °C for 1-4 h, washed with phosphate-buffered saline, and examined using a light microscope.

Mobility Shift Assays

Recombinant rat RXRalpha and PPARalpha were expressed using the baculovirus insect cell system and purified from Sf9 insect cells(26) . The nucleotide sequences of the synthetic oligonucleotides comprising the human ACOX PPRE and its mutant derivatives are as follows:

These double-stranded oligonucleotides were end-labeled using [P]dCTP, dATP, dTTP, and dGTP with Klenow DNA polymerase. The unincorporated nucleotides were removed by a Sephadex G-50 spin column. The purified probes were diluted to 20,000 cpm/µl for binding assays. Purified receptor proteins were incubated with 5 µl of the radiolabeled double-stranded oligonucleotide probe at room temperature for 20 min as described previously(26) . The respective double-stranded oligonucleotides were also used for cloning into a pTKLUC reporter plasmid to assess their activity following transfection into CV1 cells(26) . Transfections typically contained 1 µg of a reporter gene construct, 0.2 µg of rPPARalpha expression plasmid, 0.2 µg of rRXRalpha expression plasmid, and 0.5 µg of a bacterial beta-galactosidase expression vector pCMVbeta (Invitrogen) as an internal control. Cells were incubated in the presence of 1.5 mM ciprofibrate or 0.5% dimethyl sulfoxide, as required. Following a 40-h incubation, cells were processed to assess luciferase activity; the activity obtained for individual transfections was expressed relative to the beta-galactosidase activity obtained for the same preparation of lysate(26) .

Miscellaneous

DNA sequencing was performed using Sequenase (U. S. Biochemical Corp.). RNA extraction and Northern blotting were done using standard procedures(39) . Oligonucleotides were synthesized by the Biotechnology Facility (Northwestern University, Chicago). Polymerase chain reaction procedures employed reagents from Perkin Elmer Cetus and were run under routine conditions: 94 °C for 1 min, 55 °C for 1 min, and 72 °C for 1.5-2 min for a total of 35 cycles. UOX assay and electron microscopy were performed as described previously(40, 41) . One unit of UOX activity is defined as the activity that converts uric acid to allantoin at the rate of 1 µmol/min(40) .


RESULTS

Analysis of the Human ACOX Promoter in H4IIEC3 Cells

Treatment of H4IIEC3 rat hepatoma cells with ciprofibrate or other peroxisome proliferators results in the transcriptional activation of the genes encoding the beta-oxidation enzymes(7, 38) . Therefore, we used H4IIEC3 cells for transfection analysis. Luciferase expression of the promoter and 5`-flanking sequences of the human ACOX constructs exceeded by at least 100-1000-fold activity observed for a promoterless luciferase construct in H4IIEC3 in the presence of ciprofibrate. Owing to the low transfection efficiency of H4IIEC3 cells, all transfections were carried out in duplicate with the promoterless pGL2-basic as a negative control and the rat ACOX construct as a positive control. pCH110, the beta-galactosidase vector, was used in each case to normalize for transfection efficiency and protein concentration. A restriction map of the pSKACOX plasmid containing the 7.8-kb SacI promoter fragment is shown in Fig. 1A. The minimal promoter of human ACOX cloned in a luciferase reporter was designated pHACOXLUC7 (-241 to +53) (Fig. 1A, g). It had a fairly high basal luciferase activity, which exceeded that of the negative control by 100-1000-fold, but the inducibility with ciprofibrate was not marked (only about 1.3 times higher than control; Fig. 1B, g). The other three constructs, which also exhibited high basal luciferase activity, were pHACOXLUC2 (-1767 to -228), pHACOXLUC3 (-1402 to +53) and pHACOXLUC4 (-1767 to +53). Construct pHACOXLUC2 lacks the minimal promoter, whereas both pHACOXLUC3 and pHACOXLUC4 are linked to the minimal promoter (Fig. 1A, b, c, and d). For all of these three constructs, the relative induction using 0.5 mM ciprofibrate ranged from 1.2 to 1.5-fold, which is very similar to the level of inducible activity obtained using ciprofibrate with the minimal promoter alone (Fig. 1B, b, c, d, and g). pHACOXLUC3 has greater activity in H4IIEC3 cells when compared with HepG2 cells as reported previously(36) . The observation that H4IIEC3 cells exhibit greater activity than HepG2 cells holds true for all the constructs used in the transfection analysis (data not shown).

The plasmid pHACOXLUC1, which includes the native minimal promoter linked in tandem to the 3.7-kb 5`-most SacI-BamHI fragment (Fig. 1A, a) revealed a comparatively higher level of induction (approx 4.0-fold) with ciprofibrate than that observed using the minimal promoter alone (Fig. 1B; a and g). The highest inducible luciferase activity, 5-fold over the minimal, was obtained with pHACOXLUC5 (-2165 to +53). This construct encompasses the minimal, the 1.6-kb BamHI fragment, and 200 bp further upstream (Fig. 1A). To delineate this region further, a 200-bp fragment immediately upstream of the second BamHI site (-2015 to -1812) was linked to the minimal (-241 to +53) (Fig. 1A, f). The level of activity obtained for this construct, pHACOXLUC6, was 3.5-fold over that obtained using the minimal alone (Fig. 1B, f, and g). Since this 200-bp region between -2015 and -1812 is able to confer inducibility on the otherwise minimally responsive minimal human ACOX promoter, this region most probably contains a positively acting PPRE that functions independent of the other regions of this promoter. Also the other two constructs, pHACOXLUC1 and pHACOXLUC5 (Fig. 1, a and e), which include this 200-bp fragment, have a higher level of induction than pHACOXLUCs 2, 3 and 4 that lack this region. The level of inducible luciferase activity that was obtained for the HACOXLUC constructs was similar to that observed for the inducible rat ACOX in our system (data not shown).

Cotransfection with PPARalpha and RXRalpha

In order to determine the influence, if any, of PPARalpha and RXRalpha on the level of expression, the two constructs pHACOXLUCs 6 and 7 (Fig. 1A, f and g) were cotransfected with rat PPARalpha and rat RXRalpha expression plasmids into CV1 cells (Fig. 3). pHACOXLUC6 contains the minimal promoter linked to the 200-bp fragment encompassing the PPRE. pHACOXLUC7 represents the minimal promoter alone. The empty expression vector pSG5 was transfected individually for each of the constructs as a control. For each construct, there were four separate transfections in the following order: vehicle alone, 1.5 mM ciprofibrate, 0.1 µM 9-cis-retinoic acid, and lastly, both ciprofibrate and 9-cis-retinoic acid (Fig. 3). pHACOXLUC6, which contains the minimal promoter linked to the 200-bp fragment (Fig. 1A, f) showed an augmented response to ciprofibrate and 9-cis-retinoic acid when compared with pHACOXLUC7 (Fig. 3). There is a greater level of induction with ciprofibrate in contrast to 9 cis-retinoic acid. A synergistic induction was noted when both activators are used together.


Figure 3: PPARalpha and RXRalpha transactivate reporter expression cooperatively for the HACOX promoter. CV1 cells were transfected with constructs pHACOXLUCs 6 or 7 reporters, which are the two constructs from Fig. 1, together with PPARalpha and RXRalpha expression vectors. pSG5 plasmid DNA was used as a control. Treatments included no ligand (Me(2)SO alone), ciprofibrate alone, 9-cis-retinoic acid and both ciprofibrate and 9-cis-retinoic acid. The luciferase expression, depicted as -fold induction, is calculated in relative light units.



Generation of the CVPHACOX, CVHACOX, and CVHACOXUOX Cell Lines

The PPARalpha cDNA under the transcriptional control of the CMV promoter (Fig. 2c) was initially transfected into CV1 cells. Colonies resistant to hygromycin were selected within a period of 3-4 weeks and cultured separately. One such cell line expressing PPARalpha was termed PPARalpha-CV1. This particular cell line was subsequently cotransfected with pbetaHACOX (Fig. 2a) and a neomycin-resistant reporter plasmid. The pbetaHACOX plasmid contains the human ACOX promoter fragment corresponding to pHACOXLUC1 (Fig. 1A, a) linked to the beta-galactosidase cDNA. Transient transfection analysis into CV1 cells stably expressing rat PPARalpha revealed that the human ACOX promoter is active (Fig. 4A). Stable transfectants of the same beta-galactosidase construct in the PPARalpha-CV1 cells were generated using G418 as a selection marker. Several clones were isolated and subcultured separately. One such clone was termed CVPHACOX. The presence of a 2.7-kb PPARalpha transcript was detected on Northern blot analysis from the CVPHACOX cell line, but no signal was detected when an equal amount of total RNA extracted from untransfected CV1 cells was used as a control (data not shown). This cell line shows >98% of the cells expressing beta-galactosidase as visualized by the histochemical staining procedure (Fig. 4B). These cells when cultured in the presence of ciprofibrate reveal a perceptible increase in the intensity of the beta-galactosidase staining (Fig. 4C).


Figure 4: Transient and stable transfection with the beta-galactosidase expression vector (pbetaHACOX plasmid) in PPARalpha-CV1 cells. A, transient expression of beta-galactosiadse under the influence of human ACOX promoter. B and C represent CVPHACOX cells that were generated by stable transfection with pbetaHACOX. This cell line, CVPHACOX, had approximately 98% of the cells expressing beta-galactosidase as visualized by the histochemical staining (B, stably transfected control; C, stably transfected treated with ciprofibrate).



The pbetaHACOX and pHACOXUOX plasmids were also transfected individually into regular CV1 cells (i.e. those not expressing PPARalpha), and stable transfectants were generated using G418 as a selection marker. The cell line expressing beta-galactosidase was termed CVHACOX, and that of rat UOX was designated CVHACOXUOX. No distinct differences in the beta-galactosidase staining intensities were noted between control and ciprofibrate-treated CVHACOX cells (data not shown). Further evidence that the human ACOX promoter is constitutively active in CV1 cells is derived from the demonstration that this promoter is capable of driving the expression of UOX, which can be visualized as crystalloid cores in the stably transfected CV1 cells. In these stably transfected CV1 cells, UOX containing crystalloid core-like structures are detected within single-membrane limited structures, indicating that human ACOX promoter is active (Fig. 5, A and B). The identity of these UOX containing recombinant organelles as peroxisomes was confirmed by the demonstration of catalase (data not shown). As expected, UOX, which is a liver-specific protein, is not detectable in untransfected CV1 cells, which are derived from monkey kidney (Fig. 5C).


Figure 5: Stable transfection using the pHACOXUOX plasmid, which contains the full-length rat urate oxidase cDNA under the transcriptional control of the HACOX promoter. Stable transfectants express the typical crystalloid-core like structure characteristic of UOX. A, peroxisomes indicated by arrowheads in CV1 cells stably expressing UOX under the transcriptional control of HACOX display dense cores. B, higher magnification of these in the peroxisomes of stably transfected cells show crystalloid cores characteristic of UOX. C, regular CV1 cells that lack the core-like structures as these cells normally do not express UOX.



Responsiveness of Human ACOX Promoter to Structurally Diverse Peroxisome Proliferators

To characterize the responsiveness of the CVPHACOX cell line to peroxisome proliferators, dose-response experiments were performed using ciprofibrate, Wy-14,643, elaidic acid, and the plasticizer DEHP. The cells were seeded in 96-well plates and incubated for 24 h in the presence of an inducer. The negative controls for this experiment were cells cultured in the presence of vehicle. For all compounds tested, there was a dose-dependent increase in the percentage of beta-galactosidase activity over the control. Of the four compounds, ciprofibrate displayed the highest potency in terms of induction. There was an approximately 400% increase in beta-galactosidase activity over the control at a concentration of 0.75 mM (Fig. 6). At a 0.75 mM concentration, elaidic acid and the plasticizer showed an approx250 and 300% increase over the control (Fig. 6). The highest level of induction was obtained for Wy-14,643 which is almost 700% over the control at a 2.5 mM concentration. The proportion of viable cells in each well (percent of control) was maintained as constant and was calculated by dividing its absorbance by the mean absorbance of the untreated wells. Treatment of the CVHACOX cell line (lacking PPARalpha) with Wy-14,643 showed no increase in beta-galactosidase activity when compared with untreated cells (Fig. 6). This was confirmed by further experiments using the CVHACOXUOX cell line, which also revealed a minimal difference in the UOX activity between treated and untreated cells (data not shown). The time course of induction was ascertained using 1.5 mM DEHP, 1.5 mM Wy-14,643, 1.0 mM elaidic acid, and 1.5 mM ciprofibrate as illustrated in Fig. 7. At these dose levels, 80-85% of cells were viable. Within 2 h of administration, there is a significant increase in the beta-galactosidase activity for all the agents when compared with controls (Fig. 7). Again, the highest level of activity is obtained for Wy-14,643, which is approx600% over the control following 8-10 h of treatment with the drug (Fig. 7). Transcriptional activity of human ACOX appears to be maximal between 12-24 h following treatment with all compounds in this sytem, but it tapers off by 48 h of treatment (Fig. 7). The range of concentrations of drugs used in our system (0.01 to 3 mM) is somewhat higher than published values from other studies(34) . However, in our experimental system, the effect of the drugs is being assayed on viable cells, which is a different approach from methods used in previous studies.


Figure 6: Dose-response of the CVPHACOX cell line. Ciprofibrate, Wy-14,643, dioctyl phthalate (DEHP), and elaidic acid were used for this experiment. Concentrations of the drugs ranged from 0.01 to 3 mM. beta-galactosidase activity was assessed as described under ``Experimental Procedures.'' Data points represent the mean of two individual experiments, each containing quadruplicate wells for the doses tested. Percent beta-galactosidase activity represents -fold induction over the control (untreated) cells.




Figure 7: Time-course of response of the CVPHACOX cell line. DEHP (1.5 mM), ciprofibrate (1.5 mM), Wy-14,643 (1.5 mM), and elaidic acid (1 mM) were added to the medium, and cells were harvested at 0, 2, 4, 6, 8, 10, 12, 18, 24, 36, and 48 h following treatment to assess the reporter activity. Data points represent the mean of two individual experiments, each containing quadruplicate wells for the doses tested. Percent beta-galactosidase activity represents -fold induction over the control (untreated) cells.



Human ACOX Gene Contains a PPRE

Previously, we reported the nucleotide sequence of the 5`-flanking region of human ACOX gene 505 bp upstream of the putative transcription initiation site(36) . Although this -505-bp region is highly G+C-rich and contains multiple GC boxes similar to those present in the genes encoding rat ACOX, PBE, and thiolase(42) , it contained no identifiable PPRE. We then sequenced upto -2500 bp of the 5`-flanking region of the human ACOX (Fig. 8). A putative PPRE with the sequence AGGTCA C TGGTCA was identified whose coordinates are -1918 to -1906 (Fig. 8). The region in which it was identified is included in the 200-bp fragment of the plasmid pHACOXLUC6 spanning, 2015 to -1812 and in plasmids pHACOXLUCs 1 and 5 (Fig. 1A, a, e, and f). This site matches the perfect DR1 motif with the sequence AGGTCA C AGGTCA (Fig. 8). It is of interest to note that both the PPREs of human and rat ACOX genes have DR1 motifs. Also worth noting is that the location of the PPRE of human ACOX gene is much farther upstream (-1918 to -1906 bp), when compared with its rat counterpart, which is located between -570 and -558 bp(24) . Two putative CACCC boxes are depicted (Fig. 8), and these are presumably important for hormone-mediated responsiveness of several genes having responsive elements far upstream of their transcription start sites (25, 28) .


Figure 8: Sequence of the human ACOX promoter. The -2015 to -1725 region of the human ACOX promoter is shown. The numbering of bp is with respect to the transcription start site = +1. The potential PPRE (boldfaced and underlined) is very similar to the cognate response elements recognized by the nuclear hormone receptor superfamily and is composed of AGGTCACTGGCTA (-1918/-1906). Two putative CACCC boxes (italicized and underlined) are presumed to be important for hormone-mediated responsiveness of several genes having response elements further upstream of their transcription start sites such as the one seen with human ACOX.



Cooperative Binding of PPARalpha and RXRalpha to the PPRE of Human ACOX

The half-site motifs of the PPRE of human ACOX are separated by one nucleotide. Mobility shift experiments were performed to determine whether PPARalpha and RXRalpha are capable of interacting with this PPRE. Rat PPARalpha and rat RXRalpha were expressed using a baculovirus-insect cell system. Recombinant PPARalpha and RXRalpha purified from insect cells were used in an electrophoretic mobility shift assay with a P-labeled double-stranded oligonucleotide probe corresponding to the PPRE sequence (between coordinates -1918 and -1906 bp) of human ACOX. Migration of the probe was not retarded with PPARalpha alone (Fig. 9, lane 1), but it was strongly retarded when using a combination of PPARalpha and RXRalpha receptor proteins (Fig. 9, lanes 2-8). Relatively weak binding was observed when RXRalpha alone was used (Fig. 9, lane 9). The combination experiments were performed either with a 1-5-fold increase in the amount of RXRalpha added to an equal amount of PPARalpha (proceeding from lane 2 to lane 5) or 1-5-fold increase in the amount of PPARalpha added to the reaction mixture containing an equal amount of RXRalpha (i.e. lane 8 to lane 5). Maximum binding is observed in lane 5 (Fig. 9), where equal amounts of PPARalpha and RXRalpha are included. Competition assays using a 25-fold (lane 11) and 100-fold (lane 12) molar excess of unlabeled oligonucleotide results in a vast decrease in the amount of complex formed. Nonspecific competition using a 25-fold molar excess of an unlabeled, unrelated oligonucleotide does not compete out the complex (Fig. 9, lane 10).


Figure 9: Specific binding of RXRalpha and PPARalpha with the PPRE of the HACOX promoter. A P-labeled DNA fragment containing the human ACOX PPRE (WT) was incubated with PPARalpha and RXRalpha purified from insect Sf9 cells. Lanes 1-5 have the same amount of PPAR (50 ng). Lanes 2-5 proceeding from left to right have a 10-50 ng increase in RXRalpha. In the reverse direction, lanes 9-5 have the same amount of RXR (50 ng), while increasing PPAR from 10 to 50 ng in lanes 8-5. Lanes 10-12 used same amount of receptor proteins as lane 5. Lane 10 represents nonspecific competition using a 25-fold molar excess of an unrelated, unlabeled oligonucleotide. Lanes 11 and 12 represent specific competition using a 25- and 100-fold molar excess of unlabled human ACOX PPRE DNA.



Binding of PPARalpha and RXRalpha to Mutant HACOX PPRE

Four mutant oligonucleotides were made from the wild-type HACOX PPRE, which is AGGTCA C TGGTCA. Mutant 1 (M1) has the second and third Gs of both the hexameric direct repeats mutated to Cs. Mutant 2 (M2) has the fifth nucleotide of each motif changed from a C to a G. Mutant 3 (M3) has the C in between the two motifs converted to a T, and mutant 4 (M4) has the two Gs of the second half site motif converted to Cs. Recombinant RXR alpha and PPARalpha purified from insect cells were used in the mobility shift assay as described above. Migration of the wild-type (WT) probe was strongly retarded (Fig. 10A, lane 1). Binding of RXRalpha and PPARalpha to M1 is essentially abolished (Fig. 10A, lane 2), indicating that the two Gs of both motifs are crucial for binding. Some binding activity is restored when M2 is used as the probe (Fig. 10A, lane 3), implying that the fifth nucleotide is perhaps not as crucial for binding. Conversion of the C, which is in between the two motifs to a T has little impact on the binding of heterodimer as can be visualized by the strong band similar to the native PPRE WT (Fig. 10A, lane 4). Nonetheless, conversion of the two Gs of the second hexameric repeat to Cs also interfered with the binding (Fig. 10A, lane 5). Transfection of the wild-type and mutant double-stranded oligonucleotides using a pTKLUC reporter plasmid into CV1 cells revealed that both the WT and M3 exhibited similar increases in luciferase activity (Fig. 10B, lanes 1 and 4), whereas M1, M2, and M4 mutants showed negligible activity (Fig. 10B, lanes 2, 3, 5, and 6), suggesting that receptor binding is necessary for the activation of HACOX PPRE.


Figure 10: Mutation of HACOX PPRE influences receptor binding and peroxisome proliferator responsiveness. A, electropheretic mobility shift assay using P-labeled WT (lane 1), M1 (lane 2), M2 (lane 3), M3 (lane 4), M4 (lane 5) HACOX PPRE probes. B, transfection of CV1 cells was carried out with rat PPARalpha and rat RXRalpha expression vector and TK reporter constructs containing WT (lane 1), M1 (lane 2), M2 (lane 3), M3 (lane 4), M4 (lane 5) HACOX PPREs, respectively. Lane 6 is the pTKLUC control plasmid. After incubation with DNA for 16 h, the cells were washed and either solvent (Me(2)SO, open bars) or 1.5 mM of ciprofibrate was added to the fresh medium for an additional incubation of 48 h. The bar graphs represent the mean values plus standard deviation obtained from three independent transfections and show the activity normalized to that obtained with WT PPRE. The pCMVbeta (Clontech) plasmid was used as an internal control.




DISCUSSION

Elucidation of the mechanism(s) by which peroxisome proliferators modulate gene expression necessitates, in part, the identification of promoter elements and transcription factors that are responsible for mediating the biological responses to these nongenotoxic agents. Induction of peroxisomal ACOX is the most widely used marker to assess the magnitude of peroxisome proliferator-induced pleiotropic responses. Different species appear to exhibit varying degrees of increase in ACOX activity and peroxisome volume density in response to peroxisome proliferators(13, 16) . Evidence indicates that human hepatocytes show either minimal or no increases in peroxisome volume density when exposed to peroxisome proliferators(15, 16) . Since PPAR isoform(s) as well as enzymes of peroxisomal beta-oxidation system are present in the human liver (43) and thus cannot account for the nonresponsiveness of human liver cells to peroxisome proliferator-induced early pleiotropic responses, it is essential to determine whether PPREs are present and functional in human ACOX gene and other human genes involved in lipid metabolism. In this paper, we show that the 5` region of the human peroxisomal ACOX gene contains a PPRE and that it appears essential for the response of this gene to peroxisome proliferators. The PPRE of human ACOX gene is a direct repeat of the consensus AGGTCA half-site interspaced by one nucleotide, the so-called DR1. Several genes, which are transactivated by PPARalpha and RXRalpha heterodimers and are involved in the function of peroxisomes, mitochondria, and cytosol are comprised of DR1 motifs. Thus, the presence of a functionally active PPRE in human ACOX gene as demonstrated in this report, and the existence of a functional PPAR in human liver(43) , suggests that malfunctioning of these two components of the transcriptional machinery cannot account for the reported nonresponsiveness, or weak responsiveness, of human liver to peroxisome proliferators.

Recent evidence demonstrates that peroxisome proliferators, like other lipophilic molecules such as steroids, retinoic acid, thyroid hormone, and vitamin D(3) function by interacting with ligand-activable transcription factors that comprise the steroid/nuclear receptor superfamily(17, 18, 21) . VDR, TR, and RAR form heterodimers with RXR on bipartite hormone-response elements composed of two direct repeats of the consensus sequence 5`-AGGTCA-3` separated by 3-5 bp(31, 32) . The hormone response elements for VDR, TR, and RAR vary from one another only in the number of base pairs (3-5 bp) separating the hexameric direct repeats (DR3-DR5). To date, almost all of the PPREs identified in rat and rabbit peroxisomal and nonperoxisomal enzyme genes that are inducible by peroxisome proliferators have a DR1 motif that binds PPAR/RXR heterodimers with greater affinity. The PPAR/RXR heterodimers also exhibit greater potential for transactivation than either PPAR or RXR alone. As demonstrated in the present study, the PPRE of human ACOX gene is also a DR1 and appears essentially similar to the PPRE of rat ACOX gene in its ability to bind PPAR/RXR heterodimers (Fig. 9). Our mobility shift analysis indicates that rat RXRalpha/PPARalpha heterdimers are capable of binding to the PPRE of human ACOX cooperatively, indicating that they (i.e. their counterparts in human) possibly heterodimerize in order to transactivate the human ACOX PPRE (Fig. 9). Also, our mobility shift analysis with the mutant PPREs has demonstrated that the second and third nucleotides of both the half-site motifs are crucial for binding and transcriptional activity (Fig. 10). It has been previously reported that the highest stimulation for the rat ACOX reporter plasmid was observed by cotransfection with PPARalpha and RXRalpha in the presence of their respective ligands(23) . Transfection experiments have clearly indicated that both ciprofibrate and 9-cis-retinoic acid contribute toward gene activation for rat ACOX(23) . This synergistic induction has been reported for other systems as well(18, 28, 29) . Our results also indicate that ciprofibrate and 9-cis-retinoic acid act synergistically in transactivating the human ACOX promoter constructs that include the PPRE if cotransfected with PPARalpha and RXRalpha. Recently, it has been demonstrated that the DR1 motif alone is not sufficient to constitute a PPRE in the case of gene encoding rabbit CYP4A6, and that sequences immediately 5` of the DR1 are required for the PPARalpha/RXRalpha heterodimer binding to the PPRE of CYP4A6 gene(44) . Apparently, the minimal sequence that preserves strong binding with PPARalpha/RXRalpha heterodimers for the CYP4A6 gene includes six nucleotides upstream of the DR1 motif(44) . The role of sequences 5` and 3` of the human ACOX PPRE in promoting the binding of the PPAR/RXR heterodimers to DR1 and in the transcriptional efficiency of this gene remains unclear.

Transient transfection analysis of the pbetaHACOX promoter fragment into the CV1 cell line stably expressing PPARalpha reveals a number of blue cells staining positively for beta-galactosidase activity. Transient expression assays have provided extensive information on the peroxisome proliferator-induced regulations on receptor-mediated transactivations, but the development of a stably transfected mammalian cell line will facilitate studies on the ligand/human PPRE/PPAR-mediated transactivation of a reporter gene. Toward this goal, we have developed a CV1 cell line, in which the human ACOX promoter is driving the beta-galactosidase, and CMV promoter is driving the rat PPARalpha. This cell line, designated CVPHACOX, reveals a 700% increase in activity of the reporter gene using Wy-14,643 when compared with untreated controls. In contrast, both the CVHACOX and CVHACOXUOX cell lines, both of which lack stably transfected PPARalpha gene, demonstrate a very minimal increase in the activity of beta-galactosidase and UOX, respectively, upon treatment with Wy-14,643. The CVPHACOX cell line stably expressing PPARalpha, has provided new data regarding the inducibility of the human ACOX promoter with structurally different peroxisome proliferators, namely ciprofibrate, Wy-14,643, DEHP, and a fatty acid. Although published studies on the responsiveness of human liver cells to peroxisome proliferators have provided negative or inconclusive results(15, 16) , our transfection analysis data both from the transient luciferase, as well as stable beta-galactosidase and UOX experiments, indicate that the human ACOX promoter is indeed constitutively active and inducible. Thus the presence of a functionally active PPRE in human ACOX as shown here, and a functional PPAR in human liver(43) , suggest that the reported nonresponsiveness of human liver cells to peroxisome proliferators may be due to other factors such as the bioavailability of the ligand, interference with PPAR/RXR heterodimerization and its binding to PPRE and possibly a myriad of complex interactions involved in the transcriptional activation/repression of genes in vivo. It is of interest to note that mice, in which the PPARalpha gene has been disrupted by homologous recombination, failed to display the peroxisome proliferator-induced early pleiotropic responses, implying that PPARalpha plays a crucial role in mediating the effects of peroxisome proliferators(45) . Nonetheless, many other DR1 binding proteins are present in liver that may antagonize PPAR signaling; these include COUP-TF1, ARP-1, and HNF-4(27, 44, 46, 47) . Also, since direct binding of peroxisome proliferators to PPAR has yet to be demonstrated, it remains uncertain whether peroxisome proliferators activate PPARalpha directly. Additional stringently controlled studies are nevertheless desirable to unequivocally establish the nonresponsiveness of human hepatocytes to peroxisome proliferators. The transcriptional controls of genes involved in lipid metabolism appear extremely complex, and the net effect of peroxisome proliferators may depend upon the heterodimerization and binding or displacement of nuclear factors that may have positive or negative transcriptional influence. It is worth noting that hNUC1, a PPAR isoform isolated from an osteogenic sarcoma (48) , acts as repressor of human PPARalpha-mediated transcriptional activation of rat ACOX gene(49) . The tissue distribution of hNUC1 is not known, and it remains to be ascertained whether hNUC is a dominant isoform in human liver when compared with rat liver and that the relative lack of peroxisome proliferation in human liver is due to the inability of peroxisome proliferators to overcome hNUC1 repression.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant R37 GM23750, The Veteran's Administration Merit Review, and the Joseph L. Mayberry Sr. Endowment Fund. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Pathology, Northwestern University Medical School, 303 E. Chicago Ave., Chicago, IL 60611. Tel.: 312-503-8144; Fax: 312-503-8240; jkreddy{at}merle.acns.nwu.edu.

(^1)
The abbreviations used are: ACOX, peroxisomal acyl-CoA oxidase; PPAR, peroxisome proliferator-activated receptor; PPRE, peroxisome proliferator-response element; RXR, retinoid X receptor; RAR, retinoic acid receptor; TR, thyroid hormone receptor; VDR, vitamin D receptor; PBE, enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase bi-(tri)functional enzyme; bp, base pair(s); kb, kilobase pair(s); DEHP, di(2-ethylhexyl) phthalate; UOX, urate oxidase; CMV, cytomegalovirus; X-gal, 5-bromo-4-chloro-3-indoyl beta-D-galactoside.


ACKNOWLEDGEMENTS

We thank Dr. Frank Gonzalez (National Institutes of Health, Bethesda, MD) for providing the plasmid containing the rat PPARalpha and Drs. Takashi Osumi and Takashi Hashimoto (Shinshu University, Matsumoto, Japan) for providing the rat ACOX promoter. We also thank Dr. Bernard Mirkin (Children's Memorial Hospital, Northwestern University Medical School, Chicago, IL) for the use of equipment to perform beta-galactosidase assays. We also thank Dr. Jie Pan for assistance in preparing the electron micrographs and Yulian Lin and Kirthi Reddy in generating the pHACOXUOX stable transfectants.


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