A Truncated Human Peroxisome Proliferator-Activated Receptor {alpha} Splice Variant with Dominant Negative Activity

Philippe Gervois, Inés Pineda Torra, Giulia Chinetti, Thilo Grötzinger, Guillaume Dubois, Jean-Charles Fruchart, Jamila Fruchart-Najib, Eran Leitersdorf1 and Bart Staels

U.325 INSERM Département d’Athérosclérose Institut Pasteur de Lille 59019 Lille, France
Faculté de Pharmacie Université de Lille II 59006 Lille, France


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The peroxisome proliferator-activated receptor {alpha} (PPAR{alpha}) plays a key role in lipid and lipoprotein metabolism. However, important inter- and intraspecies differences exist in the response to PPAR{alpha} activators. This incited us to screen for PPAR{alpha} variants with different signaling functions. In the present study, using a RT-PCR approach a variant human PPAR{alpha} mRNA species was identified, which lacks the entire exon 6 due to alternative splicing. This deletion leads to the introduction of a premature stop codon, resulting in the formation of a truncated PPAR{alpha} protein (PPAR{alpha}tr) lacking part of the hinge region and the entire ligand-binding domain. RNase protection analysis demonstrated that PPAR{alpha}tr mRNA is expressed in several human tissues and cells, representing between 20–50% of total PPAR{alpha} mRNA. By contrast, PPAR{alpha}tr mRNA could not be detected in rodent tissues. Western blot analysis using PPAR{alpha}-specific antibodies demonstrated the presence of an immunoreactive protein migrating at the size of in vitro produced PPAR{alpha}tr protein both in human hepatoma HepG2 cells and in human hepatocytes. Both in the presence or absence of 9-cis-retinoic acid receptor, PPAR{alpha}tr did not bind to DNA in gel shift assays. Immunocytochemical analysis of transfected CV-1 cells indicated that, whereas transfected PPAR{alpha}wt was mainly nuclear localized, the majority of PPAR{alpha}tr resided in the cytoplasm, with presence in the nucleus depending on cell culture conditions. Whereas a chimeric PPAR{alpha}tr protein containing a nuclear localization signal cloned at its N-terminal localized into the nucleus and exhibited strong negative activity on PPAR{alpha}wt transactivation function, PPAR{alpha}tr interfered with PPAR{alpha}wt transactivation function only under culture conditions inducing its nuclear localization. Cotransfection of the coactivator CREB-binding protein relieved the transcriptional repression of PPAR{alpha}wt by PPAR{alpha}tr, suggesting that the dominant negative effect of PPAR{alpha}tr might occur through competition for essential coactivators. In addition, PPAR{alpha}tr interfered with transcriptional activity of other nuclear receptors such as PPAR{gamma}, hepatic nuclear factor-4, and glucocorticoid receptor-{alpha}, which share CREB-binding protein/p300 as a coactivator. Thus, we have identified a human PPAR{alpha} splice variant that may negatively interfere with PPAR{alpha}wt function. Factors regulating either the ratio of PPAR{alpha}wt vs. PPAR{alpha}tr mRNA or the nuclear entry of PPAR{alpha}tr protein should therefore lead to altered signaling via the PPAR{alpha} and, possibly also, other nuclear receptor pathways.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Peroxisome proliferator (PP)-activated receptors (PPARs) are ligand-activated transcription factors belonging to the superfamily of nuclear receptors (1, 2). After activation, PPARs heterodimerize with the 9-cis-retinoic acid receptor (RXR) as a preferential partner. The PPAR/RXR complex subsequently binds to DNA on specific response elements termed peroxisome proliferator response elements (PPREs), located in regulatory regions of target genes, thereby modulating their transcriptional activity. PPREs consist of the juxtaposition of two derivatives of a canonical hexamer sequence PuGGTCA spaced by one or two nucleotides and commonly called direct repeat (DR) 1 or 2. Three distinct PPARs, {alpha}, ß, and {gamma}, have been described in several species such as Xenopus (3), rat (4), mouse (5, 6, 7, 8, 9, 10), hamster (11), and human (12, 13, 14, 15, 16, 17), each encoded by different genes and exhibiting distinct tissue distribution patterns.

Mouse PPAR{alpha} was the first identified PPAR family member, and its expression is principally detected in tissues exhibiting high rates of ß-oxidation, i.e. in liver, kidney, heart, and muscle (5). In rodents, PPAR{alpha} mediates a pleiotropic response to a wide variety of compounds, termed peroxisome proliferators (PPs), leading to peroxisome proliferation, hepatomegaly, and possibly hepatocarcinogenesis (see Ref. 18 for review). PPs include various xenobiotics such as plasticizers, herbicides, some dietary compounds (fatty acids), pharmacological drugs (e.g. fibrates), and eicosanoids, some of which have been shown to be PPAR{alpha} ligands (19, 20, 21, 22, 23, 24). The implication of PPAR{alpha} in peroxisome proliferation in mice has been unequivocally demonstrated in PPAR{alpha} knockout mice that become resistant to PPs (25). Interestingly, PPAR{alpha} also mediates the hypolipidemic effect of PPs by regulating the transcription of key genes in lipoprotein metabolism. For instance, the hypolipidemic fibrates act by repressing the transcription of the apo A-I and apo A-II (26), apo A-IV (27), apo C-III (28, 29), hepatic lipase (30), and LCAT (lecithin-cholesterol acyltransferase) (31) genes in rodents, an effect that is mediated by PPAR{alpha} (32). Moreover, fibrates induce the transcription of the lipoprotein lipase gene in rat liver (33). PPAR{alpha} also plays an important role in intracellular lipid metabolism by regulating gene expression of peroxisomal ß-oxidation enzymes such as acyl-coenzyme A (CoA) oxidase (34, 35), multifunctional enzyme (36), and 3-ketoacyl-CoA thiolase (37) in rodents. Moreover, PPREs have been identified in the 5'-upstream region of several extraperoxisomal genes such as microsomal CYP4A6 (38) and 3-hydroxy-3-methylglutaryl-CoA synthase, a key mitochondrial enzyme in ketogenesis (39). In man also, PPAR{alpha} mediates fibrate action on lipoprotein metabolism since functional PPREs have been identified in the regulatory sequences of genes implicated in lipid transport such as apo A-I (40), apo A-II (41), apo C-III (28), and lipoprotein lipase (42). Thus, PPAR{alpha} can be considered as a major regulator of intra- and extracellular lipid metabolism.

Although most of the PPAR{alpha} functions seem to be conserved, some important differences in response exist. Although treatment with fibrates as well as with other PPs leads in rodents to a marked peroxisome proliferation and hepatomegaly (43, 44), such a response has never been shown to occur in man (45, 46, 47). Whereas in the majority of fibrate-treated patients plasma high-density lipoprotein-cholesterol concentrations increase, a significant fraction of patients respond to fibrates with no change or even with a decrease in their plasma high-density lipoprotein-cholesterol levels (48, 49). Such different responses to fibrates among species and within a species among individuals may be linked to variations in the PPAR{alpha} signaling pathways, possibly due to the presence of different PPAR{alpha} variants.

In this report, we therefore searched for the existence of different PPAR{alpha} variants in human tissues. Using a RT-PCR approach, we identified, in addition to the full-length transcript, a transcript lacking exon 6 due to alternative splicing. This splicing leads to the generation of a premature stop codon, giving rise to a C-terminal truncated protein (hPPAR{alpha}tr), which lacks part of the hinge region and the entire ligand-binding domain (LBD). We furthermore demonstrate that the variant transcript is expressed in various cell lines and tissues of human origin but not in rodents. Furthermore, results from Western blot experiments indicate both in vitro and in vivo expression of hPPAR{alpha}tr protein. Moreover, we show that hPPAR{alpha}tr has a repressive activity on the hPPAR{alpha}wt transactivation function, only when it translocates to nucleus. Together, these data indicate that hPPAR{alpha}tr, if produced in sufficient amounts in vivo, may play a regulatory role on hPPAR{alpha}wt signaling function.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The hPPAR{alpha} Gene Gives Rise to Two Distinct Transcripts
To determine the existence of different hPPAR{alpha} variants, RT-PCR experiments were performed using a set of specific primers covering the entire hPPAR{alpha}-coding region (Fig. 1AGo). These primers were designed to hybridize to the extremities of putative exons in the hPPAR{alpha} gene deduced from sequence comparison of the mouse, rat, and human cDNAs and the mouse gene of PPAR{alpha} (5, 4, 13, 14, 50). RT-PCR analysis was first performed on RNA extracted from HepG2 cells. For each primer pair tested, a product of the expected size was detected (Fig. 1BGo). Surprisingly, using primer pairs 35/83, 45/83, and 15/73, a second, approximately 200-bp shorter product was obtained in the same PCR reaction (Fig. 1BGo). However, whenever primer combinations including a putative exon 6 primer were used, only one fragment of a size corresponding to the hPPAR{alpha}wt transcript was obtained. These observations suggest the existence of two transcripts for hPPAR{alpha}, the shorter one lacking at least one portion of approximately 200 bp around the putative exon 6 region. To determine the identity of the shorter transcript, the shorter PCR fragment was isolated, cloned, and sequenced (Fig. 2AGo). Sequence analysis revealed that the hPPAR{alpha} variant lacked 203 bp between bp 632–834 (numbering based on the nucleotide sequence described in Ref. 14), resulting in a frame shift introducing a premature stop codon 3' of the deletion (Fig. 2AGo). The shorter transcript should result in the production of a truncated form of hPPAR{alpha} containing the first 170 N-terminal amino acids of the hPPAR{alpha}wt protein and 4 different C-terminal amino acids (Fig. 2BGo), including the ligand-independent transactivation and DNA-binding domains and part of the hinge region. To determine whether this deleted transcription variant was generated by alternative splicing of exon 6, the intron-exon boundaries of the hPPAR{alpha} gene were determined by direct sequencing of a hPPAR{alpha} gene containing BAC clone. Sequence comparison showed that the deleted fragment localized exactly at the boundaries of exon 6, indicating that it is generated by an alternative splicing event skipping exon 6 (Fig. 2CGo). Therefore, the hPPAR{alpha} gene gives rise in HepG2 cells to two transcripts of distinct size, which are generated by alternative splicing.



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Figure 1. The hPPAR{alpha} Gene Gives Rise to Two Distinct Transcripts

A, Schematic representation of the hPPAR{alpha} coding sequence and position of the primers (arrows) used for RT-PCR analysis. Numbers below arrows indicate the first nucleotide of the 5'-termini of primers. Numbers above the schematic coding sequence represent the number of the first 5'-nucleotide of corresponding primers. B, RT-PCR analysis on RNA isolated from HepG2 cells. Total RNA was extracted, reverse transcribed, and amplified using the indicated pairs of primers as described in Materials and Methods. Control corresponds to PCR reaction without RT product.

 


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Figure 2. The hPPAR{alpha}wt and hPPAR{alpha}tr Transcripts are Generated by an Alternative Splicing Event Skipping Exon 6

A, Sequence alignment of hPPAR{alpha}wt and hPPAR{alpha}tr around the exon 6 region. Sequence of the hPPAR{alpha}tr contains an in-frame TGA (boldface). B, Schematic representation of the splicing events Swt and Str generating hPPAR{alpha}wt and hPPAR{alpha}tr transcripts, respectively. The variant transcript lacks exon 6 resulting in a frame shift generating a premature TGA termination codon. Predicted protein structure of hPPAR{alpha}wt and hPPAR{alpha}tr is represented at the bottom of the figure (DBD, DNA-binding domain; LBD, ligand-binding domain). Numbers above the schematic protein structure correspond to amino acids delimiting PPAR{alpha} domains. C, Intron-exon boundaries of the hPPAR{alpha} and mPPAR{alpha} exon 6.

 
The Variant hPPAR{alpha} Transcript Is Widely Expressed in Human Cell Lines and Tissues, but Not in Rodents
RT-PCR analysis was performed next to test whether the alternative splicing underlying formation of the hPPAR{alpha} variant transcript occurs in different human cell lines and tissues. RT-PCR analysis was therefore performed using the 35 sense primer and either the 73 or 83 antisense primers that cover the alternatively spliced region. Two PCR products of the expected size for wt and variant transcripts were detected in all cells analyzed, indicating a wide distribution of both messages in different human transformed cell lines (Fig. 3AGo). Furthermore, both wt and variant transcripts were detected in human tissues such as liver and adipose tissue (Fig. 3BGo). Therefore, both hPPAR{alpha} transcripts appear to be widely distributed in different human tissues and cells.



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Figure 3. The PPAR{alpha}tr Transcript Is Widely Expressed in Human Cell Lines and Tissues

Expression of the PPAR{alpha}wt and PPAR{alpha}tr transcripts was analyzed by RT-PCR analysis as described in Materials and Methods. Primer pairs used are indicated at the bottom of each panel. A, Expression of the PPAR{alpha}wt and PPAR{alpha}tr transcript in various human cell lines. B, Expression of the PPAR{alpha}wt and PPAR{alpha}tr transcript in two different human liver and adipose tissue (Ad. tissue) samples. C, Expression of the PPAR{alpha}wt transcript in rat and mouse liver and adipose tissue (Ad. tissue).

 
We next tested whether the variant transcript is also expressed in liver and in adipose tissue of two rodent species, namely rats and mice. RT-PCR was carried out on RNA isolated from these tissues using the conserved primer pair 35/73 (5, 4, 13, 14) and resulted in detection of only one fragment of predicted size of 922 bp for the PPAR{alpha}wt transcript (Fig. 3CGo). Thus, the PPAR{alpha} variant transcript appears to be specifically expressed in man but not in rodents.

To quantify the relative level of variant and wt hPPAR{alpha} mRNA, a RNase protection assay was developed that allows simultaneous quantification of both mRNA species. Using a riboprobe that includes exon 6 and part of exon 7 of hPPAR{alpha}, protected fragments corresponding to wt and variant hPPAR{alpha} mRNA were detected in human liver and adipose tissue (Fig. 4Go). When liver samples from different subjects were compared, absolute mRNA levels of hPPAR{alpha}wt and hPPAR{alpha}tr varied from 2.42 to 6.97 relative absorbance units (R.A.U.) and from 1.57 to 2.67 R.A.U., respectively. In adipose tissue, the hPPAR{alpha}wt transcript level ranged from 1.78 to 2.00 R.A.U. whereas the hPPAR{alpha}tr varied from 1.18 to 1.60 R.A.U. Moreover, in liver the wt-variant mRNA ratio also varied among individuals from 1:1 to 4:1, whereas it was almost constant and near 1:1 in each adipose tissue sample analyzed. Interestingly, whereas hPPAR{alpha}wt levels in liver varied substantially between individuals, hPPAR{alpha}tr levels appeared to be more constant and similar in liver and in adipose tissue. These results indicate that both transcripts are widely expressed in human cell lines and tissues and that the wt-variant PPAR{alpha} mRNA ratio seems to vary substantially among individuals in human liver. On the contrary, expression level of each PPAR{alpha} transcript appears fairly constant among individuals in adipose tissue.



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Figure 4. hPPAR{alpha}wt and hPPAR{alpha}tr mRNA Levels Vary between Individuals and Tissues

RNase protection assay was used to assess the expression of hPPAR{alpha}wt and hPPAR{alpha}tr mRNAs in human liver and adipose tissue from different patients. Expression of 36B4 was determined and used as internal control. Quantification of hPPAR{alpha}wt and hPPAR{alpha}tr transcripts was performed as described in Materials and Methods. R.A.U., Relative absorbance units.

 
hPPAR{alpha}tr Protein Can Be Produced in Vitro and in Vivo
To determine whether the alternatively spliced hPPAR{alpha} mRNA could give rise to the production of a protein, in vitro and in vivo translation experiments were performed next. To detect effective synthesis of both hPPAR{alpha}wt and hPPAR{alpha}tr protein, we performed Western blot analysis. An anti-hPPAR{alpha} antibody was generated using as antigen a peptide that covers a part of the A/B domain with 95% identity between mouse and human PPAR{alpha}. This polyclonal anti-PPAR{alpha} antibody recognizes specifically the PPAR{alpha} subtype (51). A specific signal was observed for both PPAR{alpha} forms from in vitro translated plasmids (Fig. 5AGo). Cos-1 cells transfected with either pSG5hPPAR{alpha}wt or pSG5hPPAR{alpha}tr produced proteins of the expected size, respectively (Fig. 5AGo). Although the size of the detected signal in SDS-PAGE was larger than theoretically calculated, the PPARs synthesized either with the in vitro translation rabbit reticulocyte system or in vivo in Cos-1 cells were of similar size. Furthermore, when whole-cell protein extracts from human hepatoma HepG2 cells and from human primary hepatocytes were analyzed for PPAR{alpha} protein expression, specific bands were detected, the size of which was comparable to those using in vitro translated hPPAR{alpha}wt and hPPAR{alpha}tr protein (Fig. 5Go, B and D). To ensure that the shorter protein detected in HepG2 cells and in human primary hepatocytes corresponds to hPPAR{alpha}tr, the membranes were reprobed using an antibody raised against the C-terminal extremity of hPPAR{alpha}, which is lacking in hPPAR{alpha}tr. As expected, a specific band was observed for hPPAR{alpha}wt, whereas no signal was detected for hPPAR{alpha}tr (Fig. 5Go, C and E), strongly suggesting that hPPAR{alpha}tr protein may be produced in both HepG2 cells and human primary hepatocytes. These results indicate that the hPPAR{alpha}tr transcript may lead to the in vivo expression of a corresponding protein containing the ligand-independent transactivation and the DNA-binding domains, but lacking the ligand-dependent transactivation domain.



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Figure 5. hPPAR{alpha}tr Protein Is Produced in Vitro and in Vivo

A, Western blot analysis of in vitro translated protein using the TNT-coupled reticulocyte lysate system and of total protein extract from Cos-1 cells transfected with either pSG5hPPAR{alpha}wt or pSG5hPPAR{alpha}tr. B, Total protein extract from HepG2 cells. Immunodetection was performed using PPAR{alpha} rabbit polyclonal antibody developed against an N-terminal PPAR{alpha} portion. C, Immunoblot used in panel B was stripped and reprobed using a PPAR{alpha} rabbit polyclonal antibody raised against the C-terminal part of hPPAR{alpha}. D, Total protein extract from human primary hepatocytes (HH). Immunodetection was performed using PPAR{alpha} rabbit polyclonal antibody developed against a N-terminal PPAR{alpha} portion. E, Immunoblot used in panel D was stripped and reprobed using a PPAR{alpha} rabbit polyclonal antibody raised against the C-terminal part of hPPAR{alpha}. Protein extracts were prepared as described in Materials and Methods. Total protein extract was separated by SDS-PAGE and analyzed by immunoblot assay.

 
The hPPAR{alpha}tr Isoform Does Not Bind to a PPRE
Since hPPAR{alpha}tr still contains the DNA-binding domain, we next sought to determine whether it could bind a PPRE-containing DNA fragment. Therefore, gel retardation assays were performed using in vitro translated hPPAR{alpha}wt and hPPAR{alpha}tr and the apo A-II PPRE-containing oligonucleotide as probe (41). mRXR{alpha}, hPPAR{alpha}wt, and hPPAR{alpha}tr alone did not bind to the labeled apoA-II PPRE site (Fig. 6Go). However, in the presence of mRXR{alpha}, hPPAR{alpha}wt formed a DNA-protein complex. This binding was specific since excess amounts of cold probe could compete for PPAR/RXR binding. By contrast, even in the presence of mRXR{alpha}, hPPAR{alpha}tr did not bind to DNA (Fig. 6Go). The same results were obtained using different PPREs of the ACO or the apo A-I genes (data not shown). These results demonstrate that hPPAR{alpha}tr protein is not able to interact with DNA either as dimer with RXR or even as monomer. However, it cannot be excluded that hPPAR{alpha}tr heterodimerizes with mRXR{alpha} in solution, leading to the formation of an inactive protein complex. To test this mechanism, hPPAR{alpha}wt was incubated with increasing amounts of hPPAR{alpha}tr in the presence of mRXR{alpha}. The hPPAR{alpha}wt/mRXR{alpha} complex could be detected, but signal intensity did not vary for a constant amount of hPPAR{alpha}wt even when large amounts of hPPAR{alpha}tr were added (data not shown). These results indicate that the hPPAR{alpha}tr form does not bind to DNA and is unable to heterodimerize efficiently with mRXR{alpha} in vitro.



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Figure 6. hPPAR{alpha}tr Does Not Bind to a PPRE

Gel retardation assays were performed on end-labeled apo A-II J site oligonucleotide (which contains the apo A-II PPRE) in the presence of unprogrammed reticulocyte lysate (unprog lys), in vitro translated hPPAR{alpha}wt, hPPAR{alpha}tr, or mRXR{alpha}, in the presence (+) or the absence (-) of 100-fold molar excess of unlabeled apo A-II J site (competition) oligonucleotide as described in Materials and Methods.

 
hPPAR{alpha}tr Affects Transcriptional Activity of hPPAR{alpha}wt
Given the structure of the hPPAR{alpha}tr protein (without transactivation domain), it is tempting to speculate that this isoform may interfere with wild-type function in a dominant negative manner. To test the transactivation activity of both hPPAR{alpha}tr and hPPAR{alpha}wt and possible mutual interference, transient cotransfection experiments were performed in HepG2 cells using a apoA-II PPRE containing luciferase vector as a reporter (J3-TK-luc). Basal level of luciferase activity was unchanged by cotransfection with hPPAR{alpha}tr whereas hPPAR{alpha}wt activated transcription of the J3-TK-luc (Fig. 7Go). When increasing amounts of the hPPAR{alpha}tr were cotransfected with constant amounts of hPPAR{alpha}wt, a slight decrease of J3-TK promoter activation by hPPAR{alpha}wt was observed in the presence of high amounts of hPPAR{alpha}tr (Fig. 7Go). Surprisingly, the repressive activity of hPPAR{alpha}tr on hPPAR{alpha}wt transactivation function was influenced by cell culture conditions using two different batches of FCS (see FCS1 and FCS2 in Fig. 7Go). Indeed, whereas neither batch of FCS tested influenced basal or hPPAR{alpha}wt-stimulated reporter activity, hPPAR{alpha}tr-repressive activity on hPPAR{alpha}wt was more pronounced depending on the FCS tested (Fig. 7Go). These results indicate that the hPPAR{alpha}tr isoform is unable to transactivate a luciferase reporter construct driven by the PPRE-containing thymidine kinase (TK) promoter and that it can efficiently repress hPPAR{alpha}wt activity depending on the presence of certain serum factors.



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Figure 7. hPPAR{alpha}tr Affects Transcriptional Activity of hPPAR{alpha}wt

HepG2 cells were transfected with 1 µg Luc reporter construct driven by the heterologous TK promoter containing three copies of apo A-II PPRE (J3). Increasing amounts (1x, 5x) of pSG5hPPAR{alpha}tr were transfected in the presence or in the absence of pSG5hPPAR{alpha}wt (200 ng). FCS1 and FCS2 correspond to two different batches of FCS. Cotransfection with pSG5 empty vector allows determination of the basal level of expression of the J3-TK-Luc reporter vector, which was set at 1. Empty vector plasmid was added to obtain equal amounts of DNA per well.

 
Nuclear Localized hPPAR{alpha}tr Is a Potent Repressor of Nuclear Receptor Activity
To determine whether the repressive activity of hPPAR{alpha}tr on hPPAR{alpha}wt was linked to its subcellular localization, immunocytochemistry studies were performed next. When CV-1 cells were transfected with the pSG5hPPAR{alpha}wt vector, hPPAR{alpha}wt was predominantly detected in the nucleus with only minor staining in the cytoplasm (Fig. 8BGo). Signals were specific since pSG5-transfected cells did not give a significant fluorescence signal (Fig. 8AGo). In pSG5hPPAR{alpha}tr-transfected cells, the fluorescence signal was predominantly detected in the cytoplasm when cells were cultured in FCS1 (Fig. 8CGo), which yielded low repressive activity. By contrast, a stronger signal was observed in the nucleus when cells were cultured in FCS2 (Fig. 8DGo) in which hPPAR{alpha}tr behaved as a strong repressor. These differences in subcellular localization were confirmed by subcellular fractionation followed by immunoblotting analysis (data not shown). These results indicate that hPPAR{alpha}wt is predominantly localized in the nucleus and that the extent of the repressive effect of hPPAR{alpha}tr is correlated with nuclear localization.



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Figure 8. Serum Factors Induce Nuclear Localization of hPPAR{alpha}tr

Subcellular localization of the hPPAR{alpha}wt and the hPPAR{alpha}tr protein by immunocytochemistry. A, CV-1 cells transfected with pSG5 in FCS1; B, CV-1 cells transfected with pSG5hPPAR{alpha}wt in FCS1; C, CV-1 cells transfected with pSG5hPPAR{alpha}tr in FCS1; D, CV-1 cells transfected with pSG5hPPAR{alpha}tr in FCS2; E, CV-1 cells transfected with pSG5-NLS in FCS1; F, CV-1 cells transfected with pSG5-NLShPPAR{alpha}tr in FCS1.

 
To demonstrate unequivocally that the subcellular localization influenced hPPAR{alpha}tr-repressive activity, the SV40 large T antigen nuclear localization signal (NLS) (52) was inserted 5' of the hPPAR{alpha}tr cDNA to produce a chimeric NLShPPAR{alpha}tr protein that should be able to translocate into the nucleus. To determine the subcellular localization of this protein, CV-1 cells were transfected with pSG5-NLShPPAR{alpha}tr and subjected to immunocytochemistry analysis. A strong signal was detected inside the nucleus, indicating a massive translocation of NLShPPAR{alpha}tr into the nucleus (Fig. 8FGo). The signal was specific since transfection with pSG5-NLS resulted in undetectable fluorescence intensity (Fig. 8EGo). Thus, the NLS system seems to be able to efficiently direct hPPAR{alpha}tr into the nucleus. Next, we analyzed the transcriptional activity of this NLS-hPPAR{alpha}tr by transient cotransfection assays in HepG2 cells (cultured in FCS1-containing medium) using a Luc reporter construct that contains the apo A-II PPRE (J site) in three copies in front of the heterologous TK promoter (J3-TK-Luc). The J3-TK-Luc vector was not activated by NLS-hPPAR{alpha}tr either in the presence or in the absence of Wy 14,643 (Fig. 9AGo). Cotransfection of hPPAR{alpha}wt resulted in a stronger induction of Luc activity, which was repressed when NLS-hPPAR{alpha}tr was cotransfected at a 1:1 ratio. Western blot analysis cell extracts of pSG5hPPAR{alpha}wt- and pSG5hPPAR{alpha}tr-cotransfected cells at a 1:1 ratio demonstrated that both proteins were at approximately equimolar levels (Fig. 9AGo, inset). Interestingly, this transcriptional activation was repressed by increasing amounts of NLS-hPPAR{alpha}tr (Fig. 9AGo). The repressive effect of NLS-hPPAR{alpha}tr was similar both in the absence and in the presence of Wy 14,643. To ensure that the repressive effect of NLS-hPPAR{alpha}tr was not due to a saturation effect of the protein import pathway caused by high levels of NLS peptide, the pSG5-NLS expression vector was cotransfected in increasing amounts against a constant amount of the hPPAR{alpha}wt. As shown in the right part of Fig. 9AGo, the induction of Luc activity by hPPAR{alpha}wt was unaffected, indicating that there was no effect of the NLS peptide itself on hPPAR{alpha}wt-induced Luc activity. Consequently, these experiments confirm that nuclear localized hPPAR{alpha}tr has a repressive activity on hPPAR{alpha}wt transactivation function, independent of the presence of the NLS peptide. Given the fact that the nuclear translocation of hPPAR{alpha}tr has to be induced, we can consider that it acts as a negative modulator on hPPAR{alpha}wt function. Finally, to define the specificity of the negative effect of hPPAR{alpha}tr, we assessed its ability to alter the transactivation of other nuclear receptor family members, namely PPAR{gamma}, hepatic nuclear factor-4 (HNF-4), related orphan receptor-{alpha} (ROR{alpha}), and glucocorticoid receptor-{alpha} (GR{alpha}). HepG2 cells were transfected with the appropriate response element-driven reporter plasmids in the presence of expression vectors expressing either PPAR{gamma}, HNF-4, ROR{alpha}, and GR{alpha} in the presence of hPPAR{alpha}tr (Fig. 9BGo). Cotransfection of hPPAR{alpha}tr almost completely repressed hPPAR{alpha}wt and HNF-4, whereas PPAR{gamma} and GR{alpha} activity was less affected and ROR{alpha} activity was only marginally repressed. These experiments indicate that hPPAR{alpha}tr, if synthetized in significant amounts in vivo, interferes with several nuclear receptor pathways, albeit to a different extent, and suggest that one mechanism through which hPPAR{alpha}tr exerts its inhibitory effect may be by sequestration of a common coactivator necessary for nuclear receptor transcriptional activity.



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Figure 9. Nuclear Localized hPPAR{alpha}tr Is a Potent Repressor of Nuclear Receptor Activity

A, HepG2 cells, cultured in FCS1-containing medium, were cotransfected with 1 µg of Luc reporter construct driven by the heterologous TK promoter containing three copies of the apo A-II PPRE (J3). Activity of the reporter plasmid J3-TK-Luc was set at 1. Increasing amounts (1x, 5x, 10x) of pSG5-NLShPPAR{alpha}tr were transfected in the presence or in the absence of pSG5hPPAR{alpha}wt (200 ng). Proteins extracted from HepG2 cells transfected at a 1:1 ratio of pSG5hPPAR{alpha}wt:pSG5-NLShPPAR{alpha}tr were used for immunoblot analysis (inset). Increasing amounts of pSG5-NLS (1x, 5x, 10x) were added to constant amounts of pSG5hPPAR{alpha}wt (right of the figure). Cells were treated with either Wy 14,643 (1 µM) or vehicle (dimethylsulfoxide), and luciferase activity was measured as described in Materials and Methods. B, HepG2 cells, cultured in FCS2-containing medium, were cotransfected with 1 µg of J3-TK-Luc, and 200 ng of pSG5, pSG5hPPAR{alpha}, pSG5hPPAR{gamma}, or pSG5HNF-4. pSG5ROR{alpha} or GR{alpha} (200 ng) was transfected in the presence of 1 µg of RORE-TK-Luc and MMTV-TK-Luc, respectively. Increasing amounts (1x, 5x) of pSG5hPPAR{alpha}tr were cotransfected as indicated. hPPAR{alpha}, PPAR{gamma}, and GR{alpha} were activated using Wy 14,643 (1 µM), BRL 49653 (0.1 µM) and dexamethasone (1 µM), respectively. Empty vector plasmid was added to obtain equal amounts of DNA per well. Activity of the reporter plasmid J3-TK-Luc, RORE-TK-Luc, and MMTV-TK-Luc was set at 1.

 
Dominant Negative Effect of hPPAR{alpha}wt May Occur through Titration of the Coactivator CREB-Binding Protein (CBP)
Since it was reported that CBP or p300 is a coactivator of PPAR{alpha}, PPAR{gamma}, HNF-4, and GR{alpha}, as well as ROR{alpha} (53, 54, 55, 56, 57), we hypothesized that titration of CBP could constitute a mechanism by which hPPAR{alpha}tr might exert its inhibitory effect. To test this possibility, HepG2 cells, cultured in FCS2-containing medium, were transfected with increasing amounts of CBP expression vector in the presence of hPPAR{alpha}wt alone or both hPPAR{alpha}wt and hPPAR{alpha}tr (Fig. 10Go). As expected, transcriptional activity of hPPAR{alpha}wt was markedly enhanced by increasing amounts of CBP. Interestingly, transcriptional repression of hPPAR{alpha}wt activity by hPPAR{alpha}tr was restored by CBP cotransfection to levels attained by hPPAR{alpha}wt in the absence of hPPAR{alpha}tr. These data indicate that hPPAR{alpha}tr may exert its dominant negative activity through a mechanism involving the titration of common coactivator, such as CBP.



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Figure 10. Dominant Negative Effect of hPPAR{alpha}wt May Occur through Titration of the Coactivator CBP

HepG2 cells, cultured in FCS2-containing medium, were transfected with 1 µg of J3-TK-Luc, 200 ng of pSG5hPPAR{alpha}wt, and 200 ng of pSG5hPPAR{alpha}tr. Increasing amounts of pRSVCBP (0.2 µg, 0.4 µg, 1 µg) were added as indicated. Empty vector plasmid was added to obtain equal amounts of DNA per well. Activity of the reporter plasmid J3-TK-Luc was set at 1.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
PPAR{alpha} plays an important role in lipid and lipoprotein metabolism by regulating a number of genes implicated in ß-oxidation and plasma lipid transport (18). It has been well documented that treatment with hypolipidemic drugs results in distinct effects depending on species (humans vs. rodents) (18). Furthermore, among patients, differential responses to these drugs have been reported as well (48, 49). In an attempt to determine whether these phenomena could be linked to differences in expression level or to the existence of variant forms of hPPAR{alpha}, we performed expression analysis of PPAR{alpha} in humans. In this report we identified a PPAR{alpha}-truncated protein (hPPAR{alpha}tr) encoded by a mRNA derived from an alternative splicing event, results that extend observations published while this work was in progress (58). We furthermore show that the variant transcript appears to be present in all human cells and tissues expressing hPPAR{alpha}wt, but not in rodent liver. The hPPAR{alpha}tr transcripts might result in both in vitro and in vivo production of a protein as shown by immunoblot analysis of extracts from transfected cells or from HepG2 cells and human primary hepatocytes. Moreover, immunocytochemistry studies of transfected cells revealed that whereas transfected hPPAR{alpha}wt is constitutively imported into the nucleus, hPPAR{alpha}tr cellular localization is influenced by cell culture conditions. Furthermore, transient transfection studies with an NLS-containing hPPAR{alpha}tr expression vector showed that this variant can alter hPPAR{alpha}wt transactivation capacities once translocated in the nucleus. Finally, cotransfection experiments with CBP suggested cofactor competition as a potential mechanism of hPPAR{alpha}tr dominant negative activity.

PPAR{alpha} function may be regulated at the transcriptional, the posttranscriptional, and at the protein level. Transcriptional regulation of PPAR{alpha} expression has been shown to occur in rodents. In rat, PPAR{alpha} expression is regulated by hormones, such as glucocorticoids and insulin, or by physiological stimuli such as stress (59, 60, 61). However, very little is known about the regulation of PPAR{alpha} gene expression in man. In the present study, we analyzed the variation of hPPAR{alpha}wt expression in human liver, the principal site of PPAR{alpha} expression, among several individuals. Our results indicate that hPPAR{alpha}wt mRNA levels in the liver vary substantially (from 1 to 3) among individuals. These data suggest that PPAR{alpha} is strongly regulated at the gene level in human liver by genetic and/or environmental factors. By contrast, in adipose tissue, the level of expression of hPPAR{alpha} mRNA appears to be lower and more constant among the individuals analyzed. It will be interesting to determine whether transcriptional regulation of PPAR{alpha} also occurs through the use of alternative promoters, as has been described for another member of the PPAR family, PPAR{gamma}, which results both in mouse and man (9, 62, 63) in the production of two distinct proteins, PPAR{gamma}1 and PPAR{gamma}2, with distinct activation capacities due to differences only in their N-terminal amino acid sequence (64).

Our results furthermore demonstrate that the hPPAR{alpha} variant, identified in this and a previous study (58), is generated by a posttranscriptional mechanism involving alternative splicing resulting in the skipping of exon 6. Generation of isoforms by alternative splicing has already been reported for other nuclear receptors such as the estrogen receptor (65), the glucocorticoid receptor (GR) (66), the thyroid hormone receptor (67), or the vitamin D receptor (68). Alternative splicing of these receptors results in the production of isoforms with repressive activity on the wild-type (wt) receptor. Therefore, the use of alternative splicing may constitute a general mechanism to diversify and to adapt receptor action upon physiological changes.

Alternative splicing could regulate wt receptor activity both at the gene expression level and at the protein level. Our results obtained from the quantitative RNase protection assay demonstrate that the ratio of wt to variant PPAR{alpha} mRNA varies substantially in liver among individuals. The variation in this ratio appears, however, to be mainly due to variation in hPPAR{alpha}wt mRNA levels. Nevertheless, factors that influence this splicing event may interfere with the PPAR{alpha}-signaling pathway by posttranscriptional regulation of the RNA level of hPPAR{alpha}wt. By contrast, in adipose tissue, wt-variant mRNA levels appear to be fairly constant and close to 1:1. Therefore, high levels of hPPAR{alpha}tr mRNA may result in lesser activity of PPAR{alpha} and its ligands, such as fibrates, in this tissue.

In addition, if present in significant amounts in vivo, hPPAR{alpha}tr may interfere with PPAR{alpha}-signaling pathways at the protein level. Immunoblot experiments revealed that both wt and truncated PPAR{alpha} proteins can be produced in vivo by HepG2 cells and by human primary hepatocytes. Moreover, transient expression assays suggest that hPPAR{alpha}tr may display a repressive effect on hPPAR{alpha}wt transactivation function. As shown by immunocytochemistry experiments, modulation of this effect might be explained by the amount of hPPAR{alpha}tr that translocates into the nucleus. Furthermore, when hPPAR{alpha}tr protein is fused to a NLS peptide, nuclear translocation of hPPAR{alpha}tr is induced, and its repressive effect on hPPAR{alpha}wt transactivation function is strongly enhanced. This repressive activity of nuclear hPPAR{alpha}tr could be due to several mechanisms, such as competition for DNA binding, sequestering of RXR by the formation of inactive heterodimers, or titration of nuclear factors necessary for transcription activation (69). The first two mechanisms can be excluded since, as shown by gel retardation assays, hPPAR{alpha}tr does not bind to a PPRE sequence nor does it inhibit DNA binding of the PPAR{alpha}wt/RXR{alpha} complex even when added at a 4-fold excess. These data are in line with recently published findings that demonstrate that the C-terminal part of PPAR{alpha} is required for heterodimerization and DNA binding (70). The third mechanism of inhibitory effect on hPPAR{alpha}wt transactivation might be due to the titration of a cofactor binding to the N-terminal half of the protein, thereby limiting the amounts of common coactivators available for transactivation by hPPAR{alpha}wt and other nuclear receptors. Such a mechanism is probable since cotransfection of CBP could completely revert the inhibition of hPPAR{alpha}wt transcriptional activity by hPPAR{alpha}tr. Furthermore, hPPAR{alpha}tr was also found to exert transcriptional repressive activity on nuclear receptors such as PPAR{gamma}, HNF-4, GR{alpha}, and ROR{alpha}, which all share CBP/p300 as a common coactivator (53, 54, 55, 56, 57). Interestingly, hPPAR{alpha}tr interfered only marginally with ROR{alpha} transcription activity, suggesting that a selectivity of hPPAR{alpha}tr toward certain nuclear receptors exists. The hPPAR{alpha}tr protein structure consists of the A/B and C domains. Similarly, an estrogen receptor isoform with identical structure has been previously described also being derived from alternative splicing and interfering with wt receptor function (71). Although most cofactors thus far identified interact with the LBD, the existence of cofactors binding to the A/B domain has been also demonstrated, such as for thyroid hormone receptor (72). Thus, the expression level of hPPAR{alpha}tr, as well as the selectivity of interaction with cofactors such as CBP, will determine the specificity of hPPAR{alpha}tr action on transcription signaling by hPPAR{alpha}wt and other transcription factors. However, hPPAR{alpha}tr dominant negative action was more effective on hPPAR{alpha}wt, suggesting that hPPAR{alpha}tr may also compete specifically for cofactors other than CBP alone binding the N terminus of hPPAR{alpha}.

To exert its repressive activity, hPPAR{alpha}tr needs to be transported into the nucleus. Transport of transcription factors and nuclear receptors into the nucleus is an essential step in target gene transcription regulation. In eukaryotes, the nuclear membrane constitutes a barrier and thereby offers a way to regulate gene expression. Transport of nuclear proteins across the nuclear pore complex (73) is a regulated process, but the mechanisms by which proteins enter the nucleus are not yet fully understood. To date, the best studied mechanism is the NLS-mediated system. Many nuclear proteins carry NLS. Given the fact that hPPAR{alpha}tr was observed both in the cytoplasm and in the nucleus, with higher intensity of hPPAR{alpha}wt in the nucleus, the constitutive NLS of hPPAR{alpha}wt is probably comprised in the E/F domain. However, immunocytochemistry experiments, showing that the cellular distribution of hPPAR{alpha}tr is influenced by cell culture conditions, suggest the existence of an inducible NLS in hPPAR{alpha}tr that could influence receptor trafficking. Such a regulated nuclear entry has been demonstrated for a number of proteins, including GR (74), for which two NLS appear to mediate regulated nuclear import. Certain proteins, such as mitogen-activated protein kinase kinase, are able to translocate rapidly into the nucleus upon mitogenic stimulation and thus to regulate gene expression through phosphorylation of transcription factors (75, 76). It is therefore possible that the nuclear translocation of hPPAR{alpha}tr may constitute a specific and controlled mechanism that modulates its activity. Several mechanisms of induced nuclear translocation may be suggested. First, protein phosphorylation constitutes a mechanism by which a cryptic NLS may be unmasked. Such mechanism has been shown to occur for the Xenopus nuclear factor Xnf7 (77) implicated in gene regulation during development. Second, PPAR{alpha} might show high affinity for a cytoplasmic retention protein. Such cytoplasmic retention occurs for NF-KB, which is sequestered in the cytoplasm by its inhibitor IK-B. Under activation by a variety of cytokines and mitogenic factors (e.g. interleukin-1, tumor necrosis factor-{alpha}), the NFK-B/IK-B complex dissociates and NFK-B is imported into the nucleus (78). Finally, hPPAR{alpha}tr might be translocated to the nucleus as a part of a cell cycle-dependent process as shown for viral Jun (79). In this respect, it is interesting to note that, upon transfection with hPPAR{alpha}tr, a small number of cells consistently stained more pronouncedly in the nucleus. It will therefore be of interest to determine whether nuclear import of hPPAR{alpha}tr is subject to regulation, for instance during different stages of the cell cycle.

In conclusion, we have described a PPAR{alpha} variant transcript that is expressed in human cell lines and tissues, but not in rodents. This transcript may give rise to the production of a truncated receptor form of hPPAR{alpha} in human liver cells. In addition, upon nuclear translocation, hPPAR{alpha}tr becomes a potent inhibitor of hPPAR{alpha}wt as well as other transcription factors via a mechanism implicating CBP sequestration. The generation of PPAR{alpha} variant transcript by alternative splicing may regulate PPAR{alpha} signaling both at the mRNA and at the protein level. Regulation of variant transcript generation, together with its powerful inducible repressive effect on hPPAR{alpha}wt function upon induction of nuclear translocation, might represent a new approach for modulation of nuclear receptor function. It will be of interest to determine whether hPPAR{alpha}tr also contributes to the species-specific differential response to PPAR{alpha} activators. Moreover, differences in the level of expression of hPPAR{alpha}tr might be implicated in the heterogeneity in response to fibrates among different patients. Further studies are warranted to determine the physiological role of hPPAR{alpha}tr protein in humans.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Tissue and Cell RNA Extraction and RT-PCR Analysis
Total cellular RNA from tissues and cells was prepared by the guanidinium thiocyanate/phenol-chloroform method (80). For analysis of hPPAR{alpha}wt expression by RT-PCR, total RNA was reverse transcribed using random hexamer primers (Amersham Pharmacia Biotech, Buckinghamshire, UK) and Superscript reverse transcriptase (Life Technologies, Inc., Paisley, UK), and the resulting cDNA product was subsequently PCR amplified (35 cycles of 1 min at 94 C, 1 min at 55 C, 1 min at 72 C) using the following primers: oligo 15, 5'-GAA GTT CAA GAT CAA AGT GCC AGC-3'; oligo 35, 5'-TCT GAA GAG TTC CTG CAA GAA ATG G-3'; oligo 45, 5'-ACA CGC TTT CAC CAG CTT CGA CCC-3'; oligo 65, 5'-GAC GAA TGC CAA GAT CTG AGA AAG C-3'; oligo 33, 5'-GTG ATG ACC GAG CCA TCT GAG C-3'; oligo 63, 5'-TGT ATT GTT ACT GGC CTT TCC TGA GAG G-3'; oligo 73, 5'-AGC ATC CCG TCT TTG TTC ATC-3'; oligo 83, 5'-CGT CTC CTT TGT AGT GCT GTC AGC-3'. Each primer corresponds to a region well conserved between the mouse and human PPAR{alpha}-coding sequence (5, 13, 14). The resulting products were separated alongside suitable molecular markers on a 1.5% agarose gel and visualized by ethidium bromide staining.

Cloning and Construction of Recombinant Plasmids
The hPPAR{alpha}wt cDNA containing the entire open reading frame of wt hPPAR{alpha} was excised from the pCMX vector (14) by NotI digestion and blunt ending followed by NruI digestion, and was subsequently cloned into the blunt-ended BamHI site of the pSG5 expression vector (Stratagene, La Jolla, CA), giving pSG5hPPAR{alpha}wt. The hPPAR{alpha}tr-containing pSG5 expression vector was constructed by insertion of the shorter fragment, lacking exon 6, produced by RT-PCR amplification using the 35/73 pair of primers (Fig. 1Go). The PCR fragment was digested with MluI and SphI and inserted into the MluI and SphI sites of the pSG5hPPAR{alpha}wt vector resulting in the pSG5hPPAR{alpha}tr construct. To increase translation efficiency of both hPPAR{alpha}wt and hPPAR{alpha}tr, a Kozak consensus sequence (81) was inserted at the 5'-ATG of both pSG5hPPAR{alpha}wt and pSG5hPPAR{alpha}tr constructs using a PCR method. Briefly, the sense primer 5'-C CAT GGA TCC ACC ATG GTG GAC ACG GAA AGC-3', which includes a BamHI site upstream from the Kozak consensus sequence, and the antisense primer 73 were used to amplify a fragment of 1026 bp, which was subsequently digested with BamHI and MluI. After agarose gel purification, this fragment was inserted into the BamHI and MluI sites of the pSG5hPPAR{alpha}wt or pSG5hPPAR{alpha}tr plasmids. To introduce a nuclear localization signal (NLS) into hPPAR{alpha}tr, a cDNA encoding the SV40 large T antigen NLS (52) was inserted in frame 5' of the hPPAR{alpha}tr cDNA. The Kozak consensus containing pSG5hPPAR{alpha}tr was digested with NcoI, blunt ended, and then BglII digested. The resulting fragment was inserted into pSG5-NLS linearized by BamHI digestion followed by blunt ending and BglII digestion yielding pSG5-NLShPPAR{alpha}tr.

Determination of Intron-Exon Boundaries and DNA Sequencing
The BAC clone containing the hPPAR{alpha} gene was a kind gift from B. Wilkison (Glaxo Wellcome Inc., Research Triangle Park, NC). Sequencing reactions were performed using the Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin Elmer Corp., Foster City, CA) and an automatic ABI prism 377 sequencer (Perkin Elmer Corp.). 5'- And 3'-intron-exon boundaries of exon 6 were sequenced using the oligonucleotides 5'-GAC CCG GGC TTT GAC CTT GTT G-3' and 5'-GAC GAA TGC CAA GAT CTG AGA AAG C-3', respectively, as primers.

RNase Protection Assay
The hPPAR{alpha} antisense probe used was designed to distinguish between the wt and the variant transcripts. A pBSSKhPPAR{alpha} clone containing the full-length cDNA sequence was SmaI and SacI digested, and the resulting fragment (which spans a region located between bp 800–983 according to nucleotide numbering used in Ref. 14 was isolated and subcloned into a pBSKS vector (Stratagene). The resulting hPPAR{alpha} riboprobe covers 54 bp of vector sequence plus 152 bp that are common to both hPPAR{alpha}wt and hPPAR{alpha}tr and 36 bp of exon 6 that are only present in the wt transcript. A 36B4 riboprobe was used as an internal control (82). Therefore, the 36B4 cDNA was PstI/Sau3AI digested and subcloned in the PstI/BamHI sites of the pBSKS vector. The construct was further linearized with XhoI. This control probe contains 140 bp of vector sequence and 170 bp of 36B4 sequence. Identity and orientation of both clones were confirmed by sequencing analysis. Both PPAR{alpha} and 36B4 riboprobes were subsequently produced by in vitro transcription of the respective XhoI-linearized pBSKS plasmid in the presence of [32P]-CTP (800 Ci/mmol) using T3 RNA Polymerase and the RNA transcription Kit (Stratagene). Different molar ratios of [32P]CTP to cold CTP were used (1:166 for 36B4 and 1:0 for hPPAR{alpha}) to synthesize each riboprobe, which allowed signal comparison of the corresponding mRNA on the same gel. The RNase protection assay was carried out using the HybSpeed RNase protection Kit (Ambion, Inc. Austin, TX). Twenty micrograms of total RNA were hybridized simultaneously to hPPAR{alpha} (6 x 104 cpm) and 36B4 (103 cpm) antisense probes. RNA samples were electrophoresed on a 5% denaturing polyacrylamide gel and visualized by autoradiography. Protected fragments representing hPPAR{alpha}wt, hPPAR{alpha}tr, and 36B4 mRNA were quantified on a GS525 Phosphorimager (Bio-Rad Laboratories, Inc. Hercules, CA). The radioactivity was corrected for differences in the radiolabeled nucleotide content between hPPAR{alpha}wt and hPPAR{alpha}tr and subsequently normalized to the internal 36B4 control.

Cell Culture and Transfections
Human hepatoma Hep G2 and Cos-1 cells were obtained from the European Collection of Animal Cell Culture (Porton Down, Salisbury, UK). Cells were grown in DMEM, supplemented with 2 mM glutamine and 10% (vol/vol) FCS, in a 5% CO2 humidified atmosphere at 37 C. Medium was changed every other day. Stimuli were dissolved in dimethylsulfoxide. Control cells received vehicle only. All transfections were performed with a mixture of plasmids, which contained in addition to the reporter (1 µg) and expression vector (0.2–2 µg), 1 µg of Cytomegalovirus-driven ß-galactosidase expression vector as control for transfection efficiency. All samples were complemented to an equal amount of plasmid DNA using empty pSG5 vector. Cells were transfected at 50–60% confluence in 60-mm dishes by the calcium phosphate coprecipitation procedure. After a 4-h incubation period, cells were washed with PBS and then refed with fresh medium and treated with Wy 14,643 (Chemsyn, Lenexa, KS), vehicle, or dexamethasone (Sigma, St. Louis, MO) as indicated. Cells were harvested after 24 h incubation. The luciferase activity in cell extracts was determined using a luciferase assay system (Promega Corp., Madison, WI) following the supplier’s instruction. Transfection experiments were performed in triplicate and repeated at least three times.

Protein Extract Preparation
Total cellular extracts were made from 5–10 x 106 cells. Cells were washed twice with ice-cold PBS, scraped off in 5 ml ice-cold PBS, and collected by centrifugation for 5 min at 800 rpm at 4 C. The pellet was resuspended in 100 µl ice-cold lysis buffer (1% Nonidet P-40, 0.5% sodium desoxycholate, 0.1% SDS in PBS), and protease inhibitors were freshly added (5 µg/ml leupeptin, 5 µg/ml pepstatin, 5 mg/ml EDTA-Na2, 1 mM benzamidine, 5 µg/ml aprotinin, and 0.5 mM phenylmethylsulfonyl fluoride). Cells were immediately transferred into 1.5-ml tubes, vigorously but quickly (10 sec) vortexed, and allowed to swell on ice for 15 min. The cell extract was then obtained by centrifugation (5 min at 13000 rpm at 4 C), and the supernatant was transferred into new tubes, aliquoted, and stored at -80 C. The samples were continuously kept at 4 C during the entire extraction procedure.

SDS-PAGE and Western Blotting
Electrophoresis of samples (100 µg of total protein) and prestained mol wt markers was performed on 10% SDS-polyacrylamide gels (Minigel system, Bio-Rad Laboratories, Inc.) under reducing conditions (sample buffer containing 10 mM dithiothreitol). Proteins were electrophoretically blotted onto a nitrocellulose membrane. Nonspecific binding sites were blocked with 10% skim milk powder in TNT buffer (20 mM Tris, 55 mM NaCl, 0.1% Tween 20), overnight at 4 C. The membrane was probed with a PPAR{alpha} rabbit polyclonal antibody developed against either a N-terminal PPAR{alpha} peptide (amino acids 10–56) or a C-terminal PPAR{alpha} peptide and diluted in 5% skim milk-TNT, for 4 h at room temperature. After washes, membranes were incubated with peroxidase-conjugated antirabbit antibody (Diagnostics Pasteur, Marnes- La-Coquette, France) diluted 1:4000 and visualized using a chemiluminescent system (ECL, Amersham Pharmacia Biotech, Buckinghamshire, UK).

Immunocytochemistry
Cells were cultured on microscopy coverslips of 14 mm diameter (sterilized with ethanol) under standard conditions. Cells were washed with a solution of 0.01 M Tris, 0.5 M NaCl (pH 7.4) and then fixed with 3% paraformaldehyde, 2% sucrose in PBS, pH 7.5, for 15 min at room temperature. After three washes in Tris-NaCl, cells were incubated with 0.1 M lysine for 4 h at room temperature to avoid nonspecific fluorescent signals. Cells were permeabilized using methanol-acetone (1:1 vol/vol) for 5 min at room temperature. The nonspecific staining was blocked by incubation in Tris-NaCl, 0.5% ovalbumin (TNO), and 1% of normal goat serum for 30 min at room temperature. The preparations were incubated overnight at 4 C with primary antibody (N-terminal PPAR{alpha} antibody) diluted in TNO and followed by three washes with TNO (3 x 30 min) and by incubation with secondary fluorescein isothiocyanate-conjugated antirabbit IgG antibody (dil.1:100 in TNO) for 2 h at room temperature. After successive washes in TNO, Tris-NaCl, and distilled water, the preparations were assembled on microscope slides for analysis with a Leitz DMR fluorescence microscope (Leica Corp., Nussloch, Germany).

In Vitro Translation and Gel Retardation Assays
pSG5hPPAR{alpha}wt, pSG5hPPAR{alpha}tr and pSG5mRXR{alpha} were in vitro transcribed with T7 polymerase and translated using the rabbit reticulocyte lysate sytem (Promega Corp.). For gel retardation assay, a synthetic double stranded oligonucleotide, which spans nucleotides -737 to -715 of the human apo A-II gene upstream regulatory sequences and contains a PPRE, was end labeled and used as probe (41). The PPAR and RXR proteins were incubated in a total volume of 20 µl for 15 min on ice with 2.5 µg of poly-dI-dC and 1 µg of herring sperm DNA in TM buffer (10 mM Tris-HCl, pH 7.9, 40 mM KCl, 10% glycerol, 0.05% Nonidet P-40, and 1 mM dithiothreitol). The 32P-radiolabeled oligonucleotide was then added, and the mixture was incubated 20 min on ice. For competition experiments, 100-fold molar excess of cold probe was added just before the labeled oligonucleotide. The complexes were resolved on 5% polyacrylamide gels in 0.25x TBE buffer (90 mM Tris-borate, 2.5 mM EDTA, pH 8.3) at 4 C. Gels were dried and exposed overnight at -70 C to x-ray film (XOMAT-AR, Eastman Kodak, Rochester, NY).


    ACKNOWLEDGMENTS
 
Helpful discussions with Gérard Torpier, Ngoc Vu-Dac, and Piet De Vos are gratefully acknowledged. We thank Odile Vidal, Lluis Fajas, and Isabelle Saves for expert technical assistance. The BAC clone, hepatoma cell line RNA, and human liver samples were kind gifts from B. Wilkison, H. Will/S.F. Chang, and U. Beisiegel/V. Kosykh, respectively. We thank R. Mukherjee for providing a hPPAR{alpha} cDNA clone. The CBP clone is the kind gift of D. Hum. M. Dauça is acknowledged for providing anti-PPAR{alpha} antibody raised against the C-terminal part of PPAR{alpha}. We are grateful to J. J. Berthelon for providing BRL 49653.


    FOOTNOTES
 
Address requests for reprints to: Dr. Bart Staels, U.325 INSERM, Département d’Athérosclérose, Institut Pasteur, 1 Rue Calmette, 59019 Lille, France.

This research was sponsored by grants from INSERM, ARCOL (Comité Français de Coordination des Recherches sur l’Athérosclérose et le Cholestérol), Biomed 2 Concerted action (Grant PL963324), Fondation pour la Recherche Médicale, the Région Nord-Pas de Calais, and the European Community (Grant ERBFMBICT983214).

1 On sabbatical leave from the Center for Research, Prevention and Treatment of Atherosclerosis, Department of Medicine, Hadassah University Hospital, Jerusalem, Israel. Back

Received for publication July 13, 1998. Revision received April 22, 1999. Accepted for publication May 26, 1999.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Tsai M-J, O’Malley B 1994 Molecular mechanisms of action of steroid/thyroid receptor superfamily members. Annu Rev Biochem 63:451–486[CrossRef][Medline]
  2. Mangelsdorf DJ, Thummel C, Beato M, Herrlich P, Schutz G, Umesono K, Blumberg B, Kastner P, Mark E, Chambon P, Evans RM 1995 The nuclear receptor superfamily: the second decade. Cell 83:835–839[Medline]
  3. Dreyer C, Krey G, Keller H, Givel F, Helftenbein G, Wahli W 1992 Control of the peroxisomal ß-oxidation pathway by a novel family of nuclear hormone receptors. Cell 68:879–887[Medline]
  4. Göttlicher M, Widmark E, Li Q, Gustafsson JA 1992 Fatty acids activate chimera of the clofibric acid-activated receptor and the glucocorticoid receptor. Proc Natl Acad Sci USA 89:4653–4657[Abstract]
  5. Isseman I, Green S 1990 Activation of a member of the steroid hormone receptor superfamily by peroxisome proliferators. Nature 347:645–650[CrossRef][Medline]
  6. Chen F, Law SN, O’Malley BW 1993 Identification of two mPPAR related receptors and evidence for the existence of five subfamily members. Biochem Biophys Res Commun 196:671–677[CrossRef][Medline]
  7. Zhu Y, Alvares K, Huang Q, Rao MS, Reddy JK 1993 Cloning of a new member of the peroxisome proliferator activated receptor gene family from mouse liver. J Biol Chem 268:26817–26820[Abstract/Free Full Text]
  8. Kliewer SA, Forman BM, Blumberg B, Ong ES, Borgmeyer U, Mangelsdorf DJ, Umesono K, Evans RM 1994 Differential expression and activation of a family of murine peroxisome proliferator-activated receptors. Proc Natl Acad Sci USA 91:7355–7359[Abstract]
  9. Tontonoz P, Hu E, Graves RA, Budavari AI, Spiegelman BM 1994 mPPAR{gamma}2: tissue-specific regulator of an adipocyte enhancer. Genes Dev 8:1224–1234[Abstract]
  10. Amri E-Z, Bonino F, Ailhaud G, Abumrad NA, Grimaldi PA 1995 Cloning of a protein that mediates transcriptional effects of fatty acids in preadipocytes. J Biol Chem 270:2367–2371[Abstract/Free Full Text]
  11. Aperlo C, Pognonec P, Saladin R, Auwerx J, Boulukos K 1995 Isolation and characterization of the hamster peroxisomal proliferator activated receptor hPPAR{gamma}, a member of the nuclear hormone receptor superfamily. Gene 162:297–302[CrossRef][Medline]
  12. Schmidt A, Endo N, Rutledge SJ, Vogel R, Shinar D, Rodan GA 1992 Identification of a new member of the steroid hormone receptor superfamily that is activated by a peroxisome proliferator and fatty acids. Mol Endocrinol 6:1634–1641[Abstract]
  13. Sher T, Yi HF, McBride W, Gonzalez FJ 1993 cDNA cloning, chromosomal mapping, and functional characterization of the human peroxisome proliferator activated receptor. Biochemistry 32:5598–5604[Medline]
  14. Mukherjee R, Jow L, Noonan D, McDonnell DP 1994 Human and rat peroxisome proliferator activated receptors (PPARs) demonstrate similar tissue distribution but different responsiveness to PPAR activators. J Steroid Biochem Mol Biol 51:157–166[CrossRef][Medline]
  15. Greene ME, Blumberg B, McBride OW, Yi HF, Kronquist K, Kwan K, Hsieh L, Greene G, Nimer SD 1995 Isolation of the human peroxisome proliferator activated receptor gamma cDNA: expression in hematopoietic cells and chromosomal mapping. Gene Expression 4:281–299[Medline]
  16. Elbrecht A, Chen Y, Cullinan CA, Hayes N, Leibowitz MD, Moller DE, Berger J 1996 Molecular cloning, expression and characterization of human peroxisome proliferator activated receptors {gamma}1 and {gamma}2. Biochem Biophys Res Commun 224:431–437[CrossRef][Medline]
  17. Lambe KG, Tugwood JD 1996 A human peroxisome-proliferator activated receptor-g is activated by inducers of adipogenesis, including thiazolidinedione drugs. Eur J Biochem 239:1–7[Abstract]
  18. Schoonjans K, Staels B, Auwerx J 1996 The peroxisome proliferator activated receptors (PPARs) and their effects on lipid metabolism and adipocyte differentiation. Biochim Biophys Acta 1302:93–109[Medline]
  19. Forman BM, Tontonoz P, Chen J, Brun RP, Spiegelman BM, Evans RM 1995 15-Deoxy-{Delta}12,14 prostaglandin J2 is a ligand for the adipocyte determination factor PPAR{gamma}. Cell 83:803–812[Medline]
  20. Kliewer SA, Lenhard JM, Willson TM, Patel I, Morris DC, Lehman JM 1995 A prostaglandin J2 metabolite binds peroxisome proliferator-activated receptor {gamma} and promotes adipocyte differentiation. Cell 83:813–819[Medline]
  21. Devchand PR, Keller H, Peters JM, Vazquez M, Gonzalez FJ, Wahli W 1996 The PPAR{alpha}-leukotriene B4 pathway to inflammation control. Nature 384:39–43[CrossRef][Medline]
  22. Forman BM, Chen J, Evans RM 1997 Hypolipidemic drugs, polyunsaturated fatty acids, and eicosanoids are ligands for peroxisome proliferator-activated receptors {alpha} and {delta}. Proc Natl Acad Sci USA 94:4312–4317[Abstract/Free Full Text]
  23. Kliewer SA, Sundseth SS, Jones SA, Brown PJ, Wisely GB, Koble CS, Devchand P, Wahli W, Willson TM, Lenhard JM, JML 1997 Fatty acids and eicosanoids regulate gene expression through direct interactions with peroxisome proliferator-activated receptors {alpha} and {gamma}. Proc Natl Acad Sci USA 94:4318–4323[Abstract/Free Full Text]
  24. Lehmann JM, Lenhard JM, Oliver BB, Ringold GM, Kliewer SA 1997 Peroxisome proliferator-activated receptors {alpha} and {gamma} are activated by indomethacin and other non-steroidal anti-inflammatory drugs. J Biol Chem 272:3406–3410[Abstract/Free Full Text]
  25. Lee SST, Pineau T, Drago J, Lee EJ, Owens JW, Kroetz DL, Fernandez-Salguero PM, Westphal H, Gonzalez FJ 1995 Targeted disruption of the {alpha} isoform of the peroxisome proliferator-activated receptor gene in mice results in abolishment of the pleiotropic effects of peroxisome proliferators. Mol Cell Biol 15:3012–3022[Abstract]
  26. Staels B, Van Tol A, Andreu T, Auwerx J 1992 Fibrates influence the expression of genes involved in lipoprotein metabolism in a tissue-selective manner in the rat. Arterioscler Thromb Vasc Biol 12:286–294[Abstract]
  27. Staels B, van Tol A, Verhoeven G, Auwerx J 1990 Apolipoprotein A-IV messenger ribonucleic acid abundance is regulated in a tissue-specific manner. Endocrinology 126:2153–2163[Abstract]
  28. Hertz R, Bishara-Shieban J, Bar-Tana J 1995 Mode of action of peroxisome proliferators as hypolipidemic drugs, suppression of apolipoprotein C-III. J Biol Chem 270:13470–13475[Abstract/Free Full Text]
  29. Staels B, Vu-Dac N, Kosykh V, Saladin R, Fruchart JC, Dallongeville J, Auwerx J 1995 Fibrates down-regulate apolipoprotein C-III expression independent of induction of peroxisomal acyl co-enzyme A oxidase. J Clin Invest 95:705–712[Medline]
  30. Staels B, Peinado-Onsurbe J, Auwerx J 1992 Down-regulation of hepatic lipase gene expression and activity by fenofibrate. Biochim Biophys Acta 1123:227–230[Medline]
  31. Staels B, van Tol A, Skretting G, Auwerx J 1992 Lecithin:cholesterol acyltransferase gene expression is regulated in a tissue-selective manner by fibrates. J Lipid Res 33:727–735[Abstract]
  32. Peters JM, Hennuyer N, Staels B, Fruchart JC, Fievet C, Gonzalez FJ, Auwerx J 1997 Alterations in lipoprotein metabolism in PPAR{alpha}-deficient mice. J Biol Chem 272:27307–27312[Abstract/Free Full Text]
  33. Staels B, Auwerx J 1992 Perturbation of developmental gene expression in rat liver by fibric acid derivatives: lipoprotein lipase and {alpha}-fetoprotein as models. Development 115:1035–1043[Abstract/Free Full Text]
  34. Osumi T, Wen JK, Hashimoto T 1991 Two cis-acting regulatory elements in the peroxisome proliferator-responsive element enhancer region of rat acyl-CoA oxidase gene. Biochem Biophys Res Commun 175:866–871[Medline]
  35. Tugwood JD, Isseman I, Anderson RG, Bundell KR, McPheat WL, Green S 1992 The mouse peroxisome proliferator activated receptor recognizes a response element in the 5' flanking sequence of the rat acyl CoA oxidase gene. EMBO J 11:433–439[Abstract]
  36. Bardot O, Aldridge TC, Latruffe N, Green S 1993 PPAR-RXR heterodimer activates a peroxisome proliferator response element upstream of the bifunctional enzyme gene. Biochem Biophys Res Commun 192:37–45[CrossRef][Medline]
  37. Hijikata M, Ishii N, Kagamiyama H, Osumi T, Hashimoto T 1990 Rat peroxisomal 3-ketoacyl-CoA thiolase gene: occurrence of two closely related but differentially regulated genes. J Biol Chem 265:4600–4606[Abstract/Free Full Text]
  38. Muerhoff AS, Griffin KJ, Johnson EF 1992 The peroxisome proliferator activated receptor mediates the induction of CYP4A6, a cytochrome P450 fatty acid {omega}-hydroxylase by clofibric acid. J Biol Chem 267:19051–19053[Abstract/Free Full Text]
  39. Rodriguez JC, Gil-Gomez G, Hegardt FG, Haro D 1994 Peroxisome proliferator activated receptor mediates induction of the mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase gene by fatty acids. J Biol Chem 269:18767–18772[Abstract/Free Full Text]
  40. Vu-Dac N, Schoonjans K, Laine B, Fruchart JC, Auwerx J, Staels B 1994 Negative regulation of the human apolipoprotein A-I promoter by fibrates can be attenuated by the interaction of the peroxisome proliferator-activated receptor with its response element. J Biol Chem 269:31012–31018[Abstract/Free Full Text]
  41. Vu-Dac N, Schoonjans K, Kosykh V, Dallongeville J, Fruchart JC, Staels B, Auwerx J 1995 Fibrates increase human apolipoprotein A-II expression through activation of the peroxisome proliferator-activated receptor. J Clin Invest 96:741–750[Medline]
  42. Schoonjans K, Peinado-Onsurbe J, Lefebvre AM, Heyman R, Briggs M, Cayet D, Deeb S, Staels B, Auwerx J 1996 PPAR{alpha} and PPAR{gamma} activators direct a tissue-specific transcriptional response via a PPRE in the lipoprotein lipase gene. EMBO J 15:5336–5348[Abstract]
  43. Svoboda DJ, Azarnoff DL, Hignite CE 1966 Response of hepatic microbodies to a hypolipidemic agent, ethylchlorophenoxyisobutyrate (CPIB). J Cell Biol 30:442–450[Free Full Text]
  44. Moody DE, Reddy JK 1978 Hepatic peroxisome (microbody) proliferation in rats fed plasticizers and related compounds. Toxicol Appl Pharmacol 45:487–504
  45. De La Iglesia FA, Lewis JE, Buchanan RA, Marcus EL, McMahon G 1982 Light and electron microscopy of liver in hyperlipoproteinemic patients under long-term gemfibrozil treatment. Atherosclerosis 43:19–37[Medline]
  46. Blumcke S, Schwartzkopff W, Lobeck H, Edmondson NA, Prentice DE, Blane GF 1983 Influence of fenofibrate on cellular and subcellular liver structure in hyperlipidemic patients. Atherosclerosis 46:105–116[Medline]
  47. Hanefeld M, Kemmer C, Kadner E 1983 Relationship between morphological changes and lipid-lowering action of p-chlorphenoxyisobutyric acid (CPIB) on hepatic mitochondria and peroxisomes in man. Atherosclerosis 46:239–246[Medline]
  48. Chandler HA, Batchelor AJ 1994 Ciprofibrate and lipid profile. Lancet 344:128–129[CrossRef]
  49. McLeod AJ, Warren RJ, Armitage M 1994 Ciprofibrate and lipid profile. Lancet 344:955
  50. Gearing KL, Crickmore A, Gustafsson JA 1994 Structure of the mouse peroxisome proliferator activated receptor {alpha} gene. Biochem Biophys Res Commun 199:255–263[CrossRef][Medline]
  51. Chinetti G, Griglio S, Antonucci M, Pineda Torra I, Delerive P, Majd Z, Fruchart J-C, Chapman J, Najib J, Staels B 1998 Activation of proliferator-activated receptors {alpha} and {gamma} induces apoptosis of human monocyte-derived macrophages. J Biol Chem 273:25573–25581[Abstract/Free Full Text]
  52. Kalderon D, Roberts BL, Richardson WD, Smith AE 1984 A short amino acid sequence able to specify nuclear location. Cell 39:499–509[Medline]
  53. Dowell P, Ishmael JE, Avram D, Peterson VJ, Nevrivy DJ, Leid M 1997 p300 functions as a coactivator for the peroxisome proliferator-activated receptor {alpha}. J Biol Chem 272:33435–33443[Abstract/Free Full Text]
  54. Mizukami J, Taniguchi T 1997 The antidiabetic agent thiazolidinedione stimulates the interaction between PPAR{gamma} and CBP. Biochem Biophys Res Commun 240:61–64[CrossRef][Medline]
  55. Yoshida E, Aratani S, Itou H, Miyagishi M, Takiguchi M, Osumu T, Murakami K, Fukamizu A 1997 Functional association between CBP and HNF4 in transactivation. Biochem Biophys Res Commun 241:664–669[CrossRef][Medline]
  56. Sheppard KA, Phelps KM, Williams AJ, Thanos D, Glass CK, Rosenfeld MG, Gerritsen ME, Collins T 1998 Nuclear integration of glucocorticoid receptor and nuclear factor-kappaB signaling by CREB-binding protein and steroid receptor coactivator-1. J Biol Chem 273:29291–29294[Abstract/Free Full Text]
  57. Lau P, Bailey P, Dowhan DH, Muscat GE 1999 Exogenous expression of a dominant negative RORalpha1 vector in muscle impairs differentiation: RORalpha1 directly interacts with p300 and myoD. Nucleic Acids Res 27:411–420[Abstract/Free Full Text]
  58. Palmer CNA, Hsu M-H, Griffin KJ, Raucy JL, Johnson EF 1998 Peroxisome proliferator activated receptor-{alpha} expression in human liver. Mol Pharmacol 53:14–22[Abstract/Free Full Text]
  59. Lemberger T, Staels B, Saladin R, Desvergne B, Auwerx J, Wahli W 1994 Regulation of the peroxisome proliferator-activated receptor {alpha} gene by glucocorticoids. J Biol Chem 269:24527–24530[Abstract/Free Full Text]
  60. Steineger HH, Sorensen HN, Tugwood JD, Skrede S, Spydevold O, Gautvik KM 1994 Dexamethasone and insulin demonstrate marked and opposite regulation of the steady-state mRNA level of the peroxisomal proliferator-activated receptor (PPAR) in hepatic cells. Hormonal modulation of fatty acid-induced transcription. Eur J Biochem 225:967–974[Abstract]
  61. Lemberger T, Saladin R, Vazquez M, Assimacopoulos F, Staels B, Desvergne B, Wahli W, Auwerx J 1996 Expression of the peroxisome proliferator-activated receptor {alpha} gene is stimulated by stress and follows a diurnal rhythm. J Biol Chem 271:1764–1769[Abstract/Free Full Text]
  62. Zhu Y, Qi C, Korenberg JR, Chen X-N, Noya D, Rao MS, Reddy JK 1995 Structural organization of mouse peroxisome proliferator activated receptor {gamma} (mPPAR{gamma}) gene: alternative promoter use and different splicing yield two mPPAR{gamma} isoforms. Proc Natl Acad Sci USA 92:7921–7925[Abstract]
  63. Fajas L, Auboeuf D, Raspe E, Schoonjans K, Lefebvre AM, Saladin R, Najib J, Laville M, Fruchart JC, Deeb S, Vidal-Puig A, Flier J, Briggs MR, Staels B, Vidal H, Auwerx J 1997 Organization, promoter analysis and expression of the human PPAR{gamma} gene. J Biol Chem 272:18779–18789[Abstract/Free Full Text]
  64. Werman A, Hollenberg A, Solanes G, Bjorbaek C, Vidal-Puig AJ, Flier JS 1997 Ligand-independent activation domain in the N terminus of peroxisome proliferator-activated receptor {gamma}. J Biol Chem 272:20230–20235[Abstract/Free Full Text]
  65. Fuqua SAW, Fitzgerald SD, Craig Allred D, Elledge RM, Nawaz Z, McDonnell DP, O’Malley BW, Greene GL, McGuire WL 1992 Inhibition of estrogen receptor action by a naturally occurring variant in human breast tumor. Cancer Res 52:483–486[Abstract]
  66. Oakley RH, Sar M, Cidlowski JA 1996 The human glucocorticoid receptor ß isoform. Expression, biochemical properties and putative function. J Biol Chem 271:9550–9559[Abstract/Free Full Text]
  67. Koenig DJ, Lazar MA, Hodin RA, Brent GA, Larsen R, Chin WW, Moore DD 1989 Inhibition of thyroid hormone action by a non hormone binding c-erbA protein generated by alternative mRNA splicing. Nature 337:659–661[CrossRef][Medline]
  68. Ebihara K, Masuhiro Y, Kitamoto T, Suzawa M, Uematsu Y, Yoshizawa T, Ono T, Harada H, Matsuda K, Hasegawa T, Masushige S, Kato S 1996 Intron retention generates a novel isoform of the murine vitamin D receptor that acts in a dominant negative way on the vitamin D signaling pathway. Mol Cell Biol 16:3393–3400[Abstract]
  69. Yen PM, Chin WW 1994 Molecular mechanisms of dominant negative activity by nuclear hormone receptors. Mol Endocrinol 8:1450–1454[Medline]
  70. IJpenberg A, Jeannin E, Wahli W, Desvergne B 1997 Polarity and specific sequence requirements of peroxisome proliferator-activated receptor (PPAR)/retinoid X receptor heterodimer binding to DNA. J Biol Chem 272:20108–20117[Abstract/Free Full Text]
  71. Satoshi I, Hoshino SJ, Miyoshi H, Akishita M, Hosoi T, Orimo H, Ouchi Y 1996 Identification of a novel isoform of estrogen receptor, a potential inhibitor of estrogen action, in vascular smooth muscle cells. Biochem Biophys Res Commun 219:766–772[CrossRef][Medline]
  72. Hollenberg AN, Monden T, Madura JP, Lee K, Wondisford FE 1996 Function of nuclear co-repressor protein on thyroid hormone response elements is regulated by the receptor A/B domain. J Biol Chem 271:28516–28520[Abstract/Free Full Text]
  73. Feldherr CM, Kallenbach E, Schultz N 1984 Movement of a karyophilic protein through the nuclear pores of oocytes. J Cell Biol 99:2216–2222[Abstract]
  74. Picard D, Yamamoto KR 1987 Two signals mediate hormone-dependent nuclear localization of the glucocorticoid receptor. EMBO J 6:3333–3340[Abstract]
  75. Seger R, Krebs EG 1995 The MAPK signalling cascade. FASEB J 9:726–735[Abstract/Free Full Text]
  76. Jaaro H, Rubinfeld H, Hanoch T, Seger R 1997 Nuclear translocation of mitogen-activated protein kinase kinase (MEK1) in response to mitogenic stimulation. Proc Natl Acad Sci USA 94:3742–3747[Abstract/Free Full Text]
  77. Miller M, Reddy BA, Kloc M, Li XX, Dreyer C, Etkin LD 1991 The nuclear-cytoplasmic distribution of the Xenopus nuclear factor, xnf7, coincides with its state of phosphorylation during early development. Development 113:569–575[Abstract]
  78. Baeurle PA, Baltimore D 1988 IK-B: a specific inhibitor of NFK-B transcription factor. Science 242:540–546[Medline]
  79. Chida K, Vogt PK 1992 Nuclear translocation of viral jun but not cellular jun is cell cycle dependent. Proc Natl Acad Sci USA 89:4290–4294[Abstract]
  80. Chomczynski P, Sacchi N 1987 Single step method for RNA isolation by acid guanidinium-thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159[CrossRef][Medline]
  81. Kozak M 1986 Point mutation define a sequence flanking the AUG initiation codon that modulates translation by eukaryotic ribosomes. Cell 44:283–292[Medline]
  82. Masiakowski P, Breathnach R, Bloch J, Gannon F, Krust A, Chambon P 1982 Cloning of cDNA sequences of hormone-regulated genes from MCF-7 human breast cancer cell line. Nucleic Acids Res 10:7895–7903[Abstract]