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Molecular Endocrinology 16 (4): 707-721
Copyright © 2002 by The Endocrine Society

Selective Intranuclear Redistribution of PPAR Isoforms by RXR{alpha}

Taro E. Akiyama1, Christopher T. Baumann1, Shuichi Sakai2, Gordon L. Hager and Frank J. Gonzalez

Laboratory of Metabolism (T.E.A., S.S., F.J.G.) and Laboratory of Receptor Biology and Gene Expression (C.T.B., G.L.H.), National Cancer Institute, Bethesda, Maryland 20892

Address all correspondence and requests for reprints to: Frank J. Gonzalez, Laboratory of Metabolism, National Institutes of Health, Building 37, Room 3E-24, 9000 Rockville Pike, Bethesda, Maryland 20892. E-mail: fjgonz{at}helix.nih.gov.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The intracellular localization of transcriptionally active green fluorescent protein (GFP) chimeras linked to PPARs for human PPAR{alpha} (GFP-PPARh{alpha}) and mouse PPAR{alpha}, ß, and {gamma}1 (GFP-PPARm{alpha}, GFP-PPARmß, and GFP-PPARm{gamma}, respectively) was examined in the mouse hepatoma cell line, Hepa-1, using fluorescence microscopy. A predominantly nuclear and diffuse distribution of each isoform was found in both the presence and absence of specific ligands for each receptor. GFP-PPARm{alpha}-G (containing a Glu282Gly substitution of PPARm{alpha}) and a phosphorylation mutant, GFP-PPARm{gamma}-A (containing a Ser82Ala substitution of PPARm{gamma}), exhibited altered transcriptional activities, but displayed similar intracellular localization patterns compared with their respective wild-type receptors. Coexpression of nuclear receptor corepressor suppressed, whereas steroid receptor coactivator-1 enhanced the transcriptional activity of each of the GFP-PPAR isoforms, but did not discernibly alter their intracellular distributions, both in the presence and absence of PPAR ligands. Interestingly, coexpression of the obligate heterodimeric partner of PPARs, RXR{alpha}, resulted in an intranuclear redistribution of the GFP-PPARm{gamma} isoform characterized by a reticulated pattern of the green fluorescent label for PPAR{gamma} within the nucleus, but not in nucleoli, and a heightened concentration of the fluorescent label surrounding nucleolar structures and at the nuclear membrane. Conversely, coexpression of yellow fluorescent protein-RXR{alpha} and native PPARm{gamma} resulted in a similar distribution of the yellow fluorescent tag. This localization pattern was not discernibly altered by PPAR{gamma} or RXR{alpha}-specific ligands. These results implicate RXR{alpha} in the nuclear reorganization of PPAR{gamma} and suggest that PPAR{gamma} colocalizes with RXR{alpha} at specific locations within the nucleus independent of added ligand.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE PPARs ARE MEMBERS of the nuclear hormone receptor superfamily. Three major isoforms of PPARs ({alpha}, ß, and {gamma}) have been identified (1, 2, 3, 4, 5). PPAR{alpha} mediates the response in rodents to a diverse group of chemicals called peroxisome proliferators, which include hypolipidemic drugs, plasticizers, synthetic fatty acids, and pesticides (6). Peroxisome proliferators increase the number and size of peroxisomes and cause significant hepatomegaly in susceptible rodent species. PPAR{alpha} target genes encode proteins involved in peroxisomal and mitochondrial fatty acid ß-oxidation (CYP4A1, CYP4A3, bifunctional enzyme, and thiolase), fatty acid transporter, fatty acid synthase, liver fatty acid-binding protein, S14, and apolipoproteins, thus suggesting a role for this receptor in lipid metabolism and transport (6, 7, 8, 9). Chronic administration of peroxisome proliferators also results in liver cancer in rodent species (10, 11). Significant species-specific differences exist in the response to peroxisomal proliferators. For example, hypolipidemic drugs do not induce peroxisome proliferation in human liver (12). Likewise, there is no evidence that hypolipidemic therapeutics induce cancers in human liver (13). The molecular mechanism for such species differences is not presently well understood, but could be related to low hepatic content of PPAR{alpha} in human liver (14).

PPAR{gamma} has been implicated in adipocyte differentiation (15, 16), insulin resistance (17, 18, 19), macrophage foam cell formation (20), and the mediation of high-fat diet-induced adipocyte hypertrophy (21). Ligands for PPAR{gamma} include synthetic insulin-sensitizing thiazolidinedione compounds such as BRL-49653 (rosiglitazone) and troglitazone (used in the treatment of type 2 diabetes mellitus in humans) and natural ligands such as 15-deoxy-{Delta}12,14-PGJ2, 9-and 13-hydroxyoctadecanoic acid, 15-hydroxyeicosatetraenoic acid, and linoleic acid (22, 23, 24, 25).

Compared with PPAR{alpha} and PPAR{gamma}, relatively little is known about the function of PPARß (26). Fatty acids (3) and fibrate drugs such as bezafibrate (27) have been identified as PPARß ligands. More recently, a potent and selective agonist of PPARß, GW501516, was identified. This compound was shown to increase serum high-density lipoprotein cholesterol and lower triglycerides in fasted obese rhesus monkeys (28).

RXRs serve as obligate heterodimeric partners for all three PPAR isoforms in the binding of DNA and activation of transcription of target genes (29, 30). PPAR-RXR{alpha} heterodimers bind to degenerate direct repeats of the hexameric nucleotide sequence, AGGTCA, separated by 1 bp (DR1) called peroxisome proliferator response elements (PPREs) (2). PPREs mediate the response to PPARs and have been identified in the promoters of a number of genes involved in lipid metabolism and adipocyte function (15, 29, 31, 32).

The regulation of target gene expression mediated by nuclear receptors may occur in response to activation of the receptors by ligands (33) or by phosphorylation (34) or by a combination of both events. With regard to the PPAR family of receptors, the Glu282Gly substitution within the ligand binding domain of murine PPAR{alpha} (PPARm{alpha}) results in lowered constitutive activity but full ligand-inducible activity (35). In addition, the phosphorylation status of several amino acids of PPAR{gamma} was shown to affect receptor activity (36, 37). In particular, the phosphorylation of PPARm{gamma} at Ser82 by ERK causes a decrease in both basal and ligand-dependent transcriptional activity (38).

A class of proteins called coregulators, which includes corepressors and coactivators, was shown to repress and enhance, respectively, the activity of genes regulated by nuclear hormone receptors in a ligand-dependent fashion (39). The activity of the PPAR family of proteins was shown to be regulated by coregulators. A direct protein-protein interaction between nuclear receptor corepressor (NCoR) and PPAR{alpha} correlates with a suppression of PPAR{alpha}-dependent transcriptional activation (40). This protein interaction and repression of transcriptional activity can be reversed by PPAR{alpha} agonists (40). In addition to NCoR, the silencing mediator of retinoid and thyroid hormone receptors is a corepressor of PPARs (41). Silencing mediator of retinoid and thyroid hormone receptors and NCoR mediate transcriptional repression through the recruitment of a histone deacetylase complex that modifies chromatin structure and by sequestration of initiation complex factors (39). Identified coactivators of PPARs include p300, which functions as a coactivator for PPAR{alpha} (42). In addition, steroid receptor coactivator 1 (SRC-1) (43) and cAMP response element binding protein-binding protein (CBP/p300) (42) have been shown to markedly enhance ligand-dependent transcription by PPAR{gamma} (39). Binding of the PPAR{gamma} ligand, BRL-49653, to PPAR{gamma}, results in the assembly of a complex which has been shown by x-ray crystallography to include the ligand binding domain of PPAR{gamma}, BRL-49653, and SRC-1 (44). The docking of another recently identified transcriptional activator, called PPAR{gamma} coactivator-1, to PPAR{gamma} permits the binding of SRC-1 and CBP/p300 (45).

In this report, green fluorescent protein (GFP) chimeras were developed for PPAR{alpha}, -ß, and -{gamma} isoforms that are efficiently expressed and responsive to respective ligands in a way that is not significantly different from native PPAR proteins. Using fluorescence microscopy to visualize the GFP tag, each of the GFP-PPAR isoforms was shown to be predominantly localized in the nucleus, in both the presence and absence of ligands. Substitutions of specific amino acids of PPARm{alpha} and PPARm{gamma} that were shown to be important in regulating the transcriptional activity of each isoform produced no discernable changes in the intracellular localizations. Coexpression of the heterodimeric partner of PPARs, RXR{alpha}, produced an intranuclear redistribution of the PPAR{gamma} isoform but not PPAR{alpha} and PPARß, whereas the coexpression of SRC-1 and NCoR was shown to have no significant effect on the localization of any of the GFP-PPAR isoforms.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Expression of GFP-PPAR and GFP-RXR{alpha} Chimeras
GFP-PPAR chimeras and yellow fluorescent protein (YFP)-human RXR{alpha} (hRXR{alpha}) were constructed using the appropriate fluorescent protein vector and the corresponding full-length cDNAs corresponding to each receptor (as described in Materials and Methods). A point mutation (TCT 224 GCT) of PPARm{gamma} cDNA corresponding to a Ser82Ala substitution was introduced into GFP-PPARm{gamma} by PCR-directed mutagenesis (GFP-PPARm{gamma}-A). Likewise, GFP-PPARm{alpha}-G was constructed by a (GAG 224 GGG) mutation of PPARm{alpha} cDNA corresponding to a Glu282Gly substitution.

Introduction of the GFP chimeras into cultured Hepa-1 cells results in the expression of fusion polypeptides of approximately 82 kDa, 80 kDa, 80 kDa, 82 kDa, and 30 kDa for GFP-PPARmß, GFP-PPARm{gamma}, GFP-PPARm{gamma}-A, YFP-hRXR{alpha}, and pEGFP, respectively (Fig. 1AGo, lanes 1, 2, 3, 5, and 6, respectively). These values correspond well with the predicted molecular masses of each protein. GFP-PPARh{alpha}, GFP-PPARm{alpha}, and GFP-PPARm{alpha}-G chimeras each express an approximately 90-kDa protein that is slightly larger than the predicted size (79 kDa) of each of the fusion proteins (Fig. 1A, lanes 7, 8, and 4, respectively). The larger-than-expected size of the PPAR{alpha} isoform has been observed in human primary hepatocytes and in COS-1 cells transfected with an expression vector for PPARh{alpha} (46) and is consistent with the fact that PPAR{alpha} is known to be a phosphorylated protein (47, 48). A number of other immunoreactive proteins were shown to be present in cells transfected with the fusion constructs. As these proteins were evident in all of the protein samples tested, it is likely that they represent nonspecific binding to the GFP antibody.



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  		Figure 1. Western Blot Analysis of GFP-PPARs

A, Cell extracts (50 µg) were subjected to 10% SDS-PAGE gel and Western blotting using an anti-GFP monoclonal antibody (lanes 1–6 and lanes 7 and 8 represent separate experiments). B, Hepa-1 cells were transfected with either GFP-PPARm{gamma} or GFP vector as indicated. Cell extracts (50 µg) and 50 µg total protein from murine white adipose tissue were subjected to 10% SDS-PAGE gel and Western blotting using an anti-PPAR{gamma} monoclonal antibody.

 
Transfection of Hepa-1 cells with GFP-PPARm{gamma} results in the expression of an 80-kDa immunoreactive protein detected using a PPAR{gamma} antibody (Fig. 1BGo). The size of this protein is consistent with the putative GFP-PPARm{gamma} fusion protein that was detected using a GFP monoclonal antibody (Fig. 1AGo). In contrast, no detectable band was observed when Hepa-1 cells were transfected with the vector for GFP (Fig. 1BGo). In total protein derived from white adipose tissue, two distinct immunoreactive proteins (~50 and 56 kDa) were detected using the PPAR{gamma} antibody, consistent with the known sizes of the PPAR{gamma}1 and PPAR{gamma}2 isoforms (49, 50, 51). The level of expression of GFP-PPARm{gamma} protein was approximately 3- to 4-fold higher than endogenous PPAR{gamma} in murine white adipose tissue, thus suggesting that expression of the exogenous chimeric protein is within physiological range of expression of PPAR{gamma}.

Transcriptional Competence of GFP-PPARs and YFP-hRXR{alpha}
GFP-PPAR chimera were introduced into cultured Hepa-1 cells. For each GFP-PPAR chimera, treatment of the cells with PPAR ligands (WY-14,643 for PPAR{alpha}, bezafibrate for PPARß, and BRL-49653 for PPAR{gamma}) resulted in a dose-dependent activation of a cotransfected reporter construct (3XZ-CAT) containing the chloramphenicol acetyl transferase (CAT) reporter gene under the control of three PPREs (52) (Fig. 2Go, A–D). A similar dose-dependent activation of each of the corresponding native PPARs was observed (Fig. 2Go, A–D). GFP-PPARm{alpha}-G exhibits a very low constitutive transcriptional activity relative to the wild-type fusion protein but responds fully to WY-14,643 (Fig. 3AGo). This result corresponds well with the reported transcriptional activities of native PPARm{alpha}-G protein expressed in COS-1 cells in the presence and absence of added peroxisome proliferators (35). The phosphorylation of PPARm{gamma} at Ser82 by ERK and c-Jun N-terminal kinase (JNK) was shown to cause a decrease in both its basal and ligand-dependent transcriptional activity (38). GFP-PPARm{gamma}-A, which has a substitution of Ser82 by the nonphosphorylatable amino acid Ala, was shown to exhibit an elevation in its constitutive transcriptional activity relative to wild-type GFP-PPARm{gamma} receptor while still capable of being activated by BRL-49653 (Fig. 3BGo). This result, which demonstrates that the absence of phosphorylation at Ser82 of PPARm{gamma} leads to elevated basal and inducible transcriptional activity of PPARm{gamma}, confirms an earlier study (38) showing that phosphorylation at Ser82 negatively regulates PPARm{gamma} transcriptional activity.



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  		Figure 2. Dose-Response Curves of GFP-PPARs

Hepa-1 cells transfected with pSV-ß-Gal, 3XZ-CAT, and the indicated plasmids (GFP-PPAR or pSG5-PPAR) were treated with the indicated concentrations of ligand. Ligand-induced activity of GFP-PPARh{alpha} (A), GFP-PPARm{alpha} (B), GFP-PPARmß (C), and GFP-PPARm{gamma} (D) isoforms using the 3XZ-CAT reporter were measured. The activities were normalized to ß-galactosidase activity, and fold inductions are relative to untreated controls. The experiments were performed four times and the indicated values are mean ± SE.

 


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  		Figure 3. Dose-Response of GFP-PPAR Mutants

Hepa-1 cells transfected with pSV-ß-Gal, 3XZ-CAT, and the indicated GFP-PPARs (or pSG5-PPARs) were treated with the indicated concentrations of ligand. Ligand-induced activities of GFP-PPARm{alpha}-G (A) and GFP-PPARm{gamma}-A (B) using the 3XZ-CAT reporter were measured. The activities were normalized to ß-galactosidase activity, and fold inductions are relative to wild-type GFP-PPAR activity in untreated cells. The experiments were performed four times and the indicated values are mean ± SE. *, P < 0.05 (PPAR mutant activity vs. similarly treated wild-type controls).

 
Similar dose-dependent inductions of GFP-PPARm{alpha}, GFP-PPARmß, and GFP-PPARm{gamma} activities were also observed using the reporter, PPRE3-TK-LUC (29), thus suggesting that the effect is not limited to specific regulatory PPREs in a given context (data not shown).

Because RXR{alpha} is an obligate heterodimeric partner of PPAR{alpha}, ß, and {gamma}, the ligand-dependent induction in the activities of each of the GFP-PPAR chimeras suggests that there are sufficient levels of endogenous RXR{alpha} in Hepa-1 cells to mediate this response. To test whether PPAR ligand inducibility could be further enhanced by exogenously expressed RXR{alpha}, GFP-PPARs and YFP-hRXR{alpha} were coexpressed along with 3XZ-CAT. The overall transcription of the 3XZ-CAT reporter was enhanced, and an additive response to both peroxisome proliferators and 9-cis-RA was observed, consistent with the findings of other groups (53, 54, 55) (Fig. 4Go, A–D). Similar results were observed when pSG5-hRXR{alpha} was cotransfected with individual GFP-PPARs (data not shown).



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  		Figure 4. PPAR and RXR{alpha} Cooperatively Transactivate 3XZ-CAT Reporter Gene Expression

Hepa-1 cells were transfected with 3XZ-CAT, pSV-ß-Gal, and the indicated GFP-PPAR [GFP-PPARh{alpha} (A), GFP-PPARm{alpha} (B), GFP-PPARmß (C), and GFP-PPARm{gamma} (D)] alone or in combination with YFP-hRXR{alpha}. The activities were normalized to ß-galactosidase activity, and fold inductions are relative to GFP-PPAR activity in untreated cells. Treatments included no ligand (DMSO vehicle-CTRL), 100 µM WY-14,643 for GFP-PPARh{alpha} and GFP-PPARm{alpha}, 1.0 mM bezafibrate for GFP-mß, and 1.0 µM BRL-49653 for GFP-m{gamma}, and 0.1 µM 9-cis-RA in combination with the above PPAR ligands. The experiments were performed four times and the indicated values are mean ± SE.

 
Taken together, these data suggest that the expressed GFP-PPARh{alpha}, GFP-PPARm{alpha}, GFP-PPARmß, GFP-PPARm{gamma}, GFP-PPARm{alpha}-G, GFP-PPARm{gamma}-A, and YFP-hRXR{alpha} fusion proteins are fully functional in Hepa-1 cells.

Intracellular Distribution of Unliganded and Ligand-Activated Forms of GFP-PPARs
Visualization of GFP-PPARm{alpha} transfected Hepa-1 cells by fluorescence microscopy shows a predominantly nuclear distribution of the fluorescent label with only a slight cytoplasmic component (Fig. 5AGo). GFP-PPARm{alpha} was absent in nucleoli but otherwise appeared to be evenly distributed throughout the nucleus. Exposure of the cells to 100 µM WY-14,643, the dose that conferred maximal transcriptional activity of GFP-PPARm{alpha}, produced no discernible change to the nuclear/cytoplasmic distribution of the GFP-PPARm{alpha} fusion protein (Fig. 5AGo). Similar results were obtained for GFP-PPARh{alpha} (data not shown). GFP-PPARmß and GFP-PPARm{gamma} were also noticeably present in the nucleus, but not in the nucleoli, of all transfected cells viewed. This localization pattern for GFP-PPARmß and GFP-PPARm{gamma} was independent of the addition of ligand (Fig. 5AGo). The predominantly nuclear distribution of each of the GFP-PPAR isoforms was also evident using lower amounts of transfected GFP-PPAR DNA (25 ng) in the absence of ligand (Fig. 5AGo). The localization of the mutants, GFP-PPARm{alpha}-G and GFP-PPARm{gamma}-A, as detected by their fluorescent tags, was indistinguishable from that of their respective wild-type receptors (Fig. 5BGo). This localization pattern was also independent of the addition of PPAR ligands (data not shown).



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  		Figure 5. Intracellular Localization of Uninduced and Induced GFP-PPARs

A, Hepa-1 cells were transfected with either 25 ng or 200 ng of the indicated GFP-PPARs and were treated with DMSO vehicle (-Ligand) or respective ligands for the chimeric receptors (+Ligand: 100 µM WY-14, 643 for GFP-PPARm{alpha}, 1.0 mM bezafibrate for GFP-mß, and 1.0 µM BRL-49653 for GFP-m{gamma}). B, Hepa-1 cells transfected with either GFP-PPARm{alpha}-G or GFP-PPARm{gamma}-A (200 ng) as indicated were treated with DMSO. Cells were visualized by confocal microscopy. Bar, 2 µm.

 
To examine the possibility that endogenous PPAR ligands in the culture media may have contributed to the nuclear localization of each of the PPAR chimeras, transfections were performed in charcoal-stripped media. Under these conditions, GFP-PPARh{alpha}, GFP-PPARm{alpha}, GFP-PPARmß, and GFP-PPARm{gamma} remained predominantly nuclear in distribution (data not shown).

Intranuclear Redistribution of GFP-PPARm{gamma} in Response to Exogenously Expressed RXR{alpha}
Cotransfection of Hepa-1 cells with GFP-PPARm{gamma} and pSG5-hRXR{alpha} results in a redistribution of GFP-PPARm{gamma} into distinct clusters within the nucleus in approximately 75% of transfected cells (Fig. 6Go, A and B). These accumulations of GFP-PPARm{gamma}, which give the appearance of a reticulated pattern in the nucleus, are noticeably absent in nucleoli. A portion of GFP-PPARm{gamma} + pSG5-hRXR{alpha} transfected cells also display a perinuclear distribution of the fluorescent tag at the nuclear membrane and at the periphery of the nucleoli (Fig. 6CGo). Conversely, the coexpression of YFP-hRXR{alpha} and untagged PPARm{gamma} (pSG5-PPARm{gamma}) results in a similar nuclear distribution of the yellow fluorescent tag (Fig. 6DGo). This pattern of distribution was also similar to that observed when YFP-hRXR{alpha} was expressed by itself (data not shown), thus raising the possibility that the redistribution of PPAR{gamma} corresponds to its colocalization with RXR{alpha}. Addition of BRL-49,653, 9-cis-RA, or both (not shown) had no observable effect on the localization of GFP-PPARm{gamma} when cotransfected with pSG5-hRXR{alpha}. Similar results were observed when the phosphorylation mutant, GFP-PPARm{gamma}-A, was cotransfected with pSG5-hRXR{alpha} (data not shown). In contrast, coexpression of hRXR{alpha} produced no significant effect on the localization of GFP-PPARh{alpha}, GFP-PPARm{alpha}, GFP-PPARm{alpha}-G, and GFP-PPARmß, despite altering their transcriptional activities (shown for GFP-PPARm{alpha} and GFP-PPARmß in Fig. 6Go, E and F, respectively).



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  		Figure 6. Influence of RXR{alpha} on PPAR Localization

Hepa-1 cells were cotransfected with GFP-PPARm{gamma} and pSG5 (A), GFP-PPARm{gamma} and pSG5-hRXR{alpha} (B and C), YFP-hRXR{alpha} and pSG5-PPARm{gamma} (D), GFP-PPARm{alpha} and pSG5-hRXR{alpha} (E), and GFP-PPARmß and pSG5-hRXR{alpha} in the presence of DMSO. The intracellular distribution of the fluorescent tags was examined by confocal microscopy. Bar, 2 µm.

 
Effect of NCoR and SRC-1 Coexpression on the Transcriptional Activity and Localization of GFP-PPARs
NCoR and SRC-1 are coregulators that have been shown to repress and enhance, respectively, the activity of PPAR target genes (39). To assess the effect of NCoR and SRC-1 on the transcriptional activity of GFP-PPAR fusion proteins, NCoR (and SRC-1) was coexpressed with each of the GFP-PPAR isoforms. Given the ligand inducibility of each of the GFP-PPAR isoforms in the absence of RXR{alpha} coexpression, it was assumed that there is sufficient endogenous RXR{alpha} in Hepa-1 cells to form functional PPAR/RXR{alpha} heterodimers.

Coexpression of NCoR resulted in a suppression of the constitutive activity of GFP-PPARm{alpha}, which was reversed upon addition of the PPARm{alpha} ligand, WY-14,643, consistent with the findings of earlier studies using kidney 293 cells (40). Coexpression of SRC-1 enhanced both the constitutive and WY-14,643-inducible transcriptional activity of GFP-PPARm{alpha} (Fig. 7AGo). Upon coexpression of NCoR, GFP-PPARmß exhibited decreased constitutive activity, whereas bezafibrate-inducible transcriptional activity was nearly fully restored (Fig. 7BGo). Coexpression of SRC-1 with GFP-PPARmß resulted in an increase in constitutive and ligand-inducible GFP-PPARmß activity (Fig. 7BGo). Coexpression of NCoR with GFP-PPARm{gamma} reduced its constitutive activity, whereas BRL-49653 restored its ligand-inducible activity. Coexpression of SRC-1 enhanced both the constitutive and BRL-49653-inducible transcriptional activity of GFP-PPARm{gamma} (Fig. 7CGo), which is consistent with the findings of others (43).



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  		Figure 7. Effect of NCoR and SRC-1 on Transcriptional Activation Mediated by GFP-PPARs

Hepa-1 cells were transfected with the 3XZ-CAT reporter, pSV-ß-Gal, and, where indicated, the expression vector for NCoR and SRC-1. Transfected cells were treated with vehicle (-) or the respective ligands for each GFP-PPAR isoform (+) in concentrations of 100 µM WY-14,643 for GFP-PPARm{alpha} (A), 1.0 mM bezafibrate for GFP-PPARmß (B), and 1.0 µM BRL-49653 for GFP-PPARm{gamma} (C). The activities were normalized to ß-galactosidase activity, and fold inductions are relative to GFP-PPAR activity in untreated cells. The experiments were performed four times and the indicated values are mean ± SE. Bar, 2 µm.

 
In summary, NCoR and SRC-1 repress and enhance, respectively, the constitutive activity of each of the GFP-PPAR isoforms. Specific PPAR ligands are still capable of inducing transcriptional activity of their respective GFP-PPARs (to varying extents) in the presence of coexpressed NCoR (or SRC-1). The ability of NCoR and SRC-1 to alter the transcriptional activities of each GFP-PPAR isoform did not, however, correlate with changes in the localization of any of the GFP-PPAR isoforms (Fig. 8Go), both in the presence or absence of respective PPAR ligands (not shown).



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  		Figure 8. Influence of NCoR and SRC-1 on PPAR Localization

Hepa-1 cells were cotransfected with the indicated GFP-PPAR and, where indicated, the expression vectors for NCoR and SRC-1. Cells were visualized by confocal microscopy. Bar, 2 µm.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
In this report, GFP chimeras for PPARm{alpha}, PPARmß, and PPARm{gamma} isoforms that are efficiently expressed and responsive to respective ligands were developed. Using fluorescence microscopy to visualize the GFP tag, each of the GFP-PPAR isoforms was shown to be predominantly localized in the nucleus of Hepa-1 cells, in both the presence and absence of added ligands. These results are consistent with the findings of others showing PPAR{alpha} expression in the nucleus of COS-1 cells (of kidney origin) and CV-1 cells (46, 56), and PPAR{gamma} nuclear localization in COS-1 cells (57) and 3T3-L1 preadipocytes (20). However, PPAR{alpha} was also shown to reside constitutively in the cytoplasm of differentiated human macrophages by immunocytochemistry analysis, whereas PPAR{gamma} was localized predominantly in the nucleus (58). In addition, cytoplasmic-to-nuclear translocation of each of the PPARs was demonstrated in the endothelial cell line ECV-304, upon treatment of the cells with 15-deoxy-{Delta}12,14-PGJ2 (59). The general consensus is that members of the nuclear hormone receptor family can be classified into two distinct groups, one that requires ligand binding for cytoplasmic-to-nuclear translocation (e.g. GR) and another that is nuclear, even in the absence of ligand binding (typlified by the ER) (56, 60, 61, 62, 63). It appears that in the case of the PPAR family of receptors, localization is dependent on the context of the cell type. Perhaps, variations in PPAR localization between cell types may reflect differences in the presence of endogenous ligands, nuclear translocation proteins, or other proteins that may interact with and affect PPAR localization.

In Hepa-1 cells, the demonstration that PPARs are nuclear in localization in the presence or absence of ligands suggests that activation of the receptor doesn’t require nuclear translocation as in the case of GR (64) and that other modes of regulation of receptor transcriptional activity (such as phosphorylation) are limited to the nucleus. The fact that PPARh{alpha} and PPARm{alpha} are both localized in the nucleus also suggests that the species-specific differences in the hepatic response to peroxisome proliferators between humans and mice is not likely to be due to a difference in intracellular receptor localization. It has been suggested that PPAR isoforms contain nuclear localization signals (46). A splicing variant form of PPARh{alpha} that lacks part of the hinge region and the entire ligand-binding domain was shown to reside in the cytoplasm and nucleus, depending on cell culture conditions. Interestingly, under cell culture conditions in which the variant PPARh{alpha} resided in the nucleus, it exhibited dominant negative activity on wild-type PPAR{alpha} transactivation function (46). Although the presence of a specific amino acid sequence with homology to known nuclear localization signals (NLS) has yet to be reported, these data suggest that PPARh{alpha} likely contains a constitutive NLS within the E/F domain and may also have an inducible NLS that could influence receptor trafficking (46). Alternatively, PPARs may lack the ability to be exported from the nucleus in cells that exhibit nuclear localization of PPARs. A class of chaperone proteins that bind to nuclear export signals of steroid hormone receptors has been reported (65, 66). These possibilities remain to be examined.

The Glu282Gly substitution in the ligand binding domain of PPARm{alpha}-G reduces the constitutive activity of the GFP fusion receptor, although it is still fully responsive to added peroxisome proliferators. These results are in agreement with previous studies using COS-1 cells (35). Because the mutation occurs within the ligand binding domain of PPAR{alpha}, the low constitutive activity of PPARm{alpha}-G may reflect the presence of endogenous ligands that are insufficient in concentration to activate PPARm{alpha}-G as fully as its corresponding wild-type receptor. Despite the change in transcriptional activity, there were no changes in the nuclear/cytoplasmic localization of the mutant PPARm{alpha} receptor either in the absence or presence of added ligands. Because it was shown that PPAR{alpha}-specific ligands do not alter nuclear/cytoplasmic distribution, the fact that the amino acid substitution in the ligand binding domain of PPAR{alpha} did not result in any changes in distribution was not unexpected. It was also reported that the phosphorylation of PPARm{gamma} at Ser82 by ERK causes a decrease in both basal and ligand-dependent transcriptional activity (38). In agreement with this, GFP-PPARm{gamma}-A, which contains a substitution of Ser82 by the nonphosphorylatable Ala, exhibits enhanced constitutive and ligand-inducible activity. Despite this, PPARm{gamma}-A has a similar nuclear/cytoplasmic distribution to PPARm{gamma}, suggesting that the phosphorylation status of the receptor at this particular amino acid affects transcriptional activity but not receptor localization.

The effects of the PPAR corepressor and coactivator, NCoR and SRC-1, respectively, on the transactivation activity and localization of the individual PPAR isoforms were also examined. The observation that the coexpression of NCoR and SRC-1 repress and enhance, respectively, the constitutive and ligand-inducible activity of each of the PPAR isoforms suggests that they are functional as coregulators with the GFP-fusion receptors. Changes in transactivation activity in the absence of added ligand may reflect the intrinsic histone acetyltransferase activity of the coregulators (45, 67). The lack of a discernible effect on the nuclear/cytoplasmic distribution of any of the GFP-PPAR isoforms, either in the presence or absence of respective PPAR ligands, indicates that the putative roles of these coregulators in the recruitment of coactivator proteins and release of corepressors may be limited to the nucleus, at least in the context of PPARs. Similarly, a GFP-ER{alpha} fusion protein and SRC-1 were shown to exhibit intranuclear colocalization at the same nuclear matrix-bound foci in the presence of 17ß-E2 (68).

The addition of ligands, the phosphorylation status of PPARm{alpha} and PPARm{gamma}, and the coexpression of NCoR and SRC-1 each affected the transcriptional activity of respective receptors while not altering their nuclear distribution. Based on these findings, it appears that the link between receptor transcription and localization is tenuous, at best. This may be indicative of the fact that in the case of the PPAR family of receptors, changes that generally affect receptor function, such as the binding of ligand, phosphorylation, and interactions with coregulators and other transcription factors, may be limited to the nucleus in a way that is not readily apparent by fluorescent microscopy. Alternatively, the fact that clear changes in transcription were not accompanied by corresponding changes in receptor localization may point to inherent weaknesses of using transient transfections to study receptor localization, in general. One of the major concerns with this type of analysis is the degree to which exogenous proteins are overexpressed or exhibit variable expression between cells. For example, overexpression can lead to oversaturation of binding sites. This may result in a diffuse pattern of receptor distribution that masks localization at distinct sites and changes in localization in response to ligand. Alternatively, insoluble protein aggregates may form when the protein is highly expressed in cells. Overexpression may also saturate endogenous chaperone proteins that are involved in mediating receptor trafficking or disturb the complex interactions that are known to exist between coregulators (often multiple) and transcription factors in regulating gene transcription.

Coexpression of the heterodimeric partner of PPARs, RXR{alpha}, produced an intranuclear redistribution of the PPARm{gamma} isoform but not PPARm{alpha} and PPARmß. This was characterized by a transition from a diffuse nuclear distribution to a reticulated pattern of the green fluorescent label of PPARm{gamma} within the nucleus and a heightened concentration of the fluorescent label surrounding nucleolar structures and at the nuclear membrane. Conversely, the coexpression of YFP-hRXR{alpha} and nonfluorescent native PPAR{gamma} (pSG5-PPAR{gamma}) produced a similar nuclear distribution of the yellow fluorescent tag. These observations indicate that RXR{alpha} expression causes an intranuclear redistribution of PPAR{gamma} and suggest that PPAR{gamma} and RXR{alpha} colocalize at specific locations within the nucleus. Biluminescence studies using different fluorescent tags for RXR{alpha} and PPAR{gamma} may provide evidence regarding a possible colocalization. In light of the fact that the coexpression of RXR{alpha} with PPAR{gamma} produced an increase in transcriptional activity, it is tempting to speculate that the fluorescent spots in the nucleus represent active PPAR{gamma}/RXR{alpha} heterodimers associated with DNA or proteins comprising the transcriptional machinery of the cell. However, because the coexpression of RXR{alpha} also increased the transcriptional activities of PPARm{alpha} and PPARmß, which are also known to heterodimerize with RXR{alpha}, it is difficult to make an association between PPARm{gamma} transcriptional activity and localization. Along these lines, the reorganization foci seen in many receptor systems may not be relevant to its transcription. For example, the pure antagonist of ER{alpha}, ICI 182,780, was shown to have similar (although more subtle) effects on ER{alpha} nuclear reorganization as the agonist, 17ß-E2, from a diffuse to a hyperspeckled pattern (68). In this study, only a small subset of ER{alpha} foci were shown to be coincident with the transcriptional enzyme, pol IIo, indicating that most of the ER{alpha} pool is not directly involved in transcription. Similarly, a punctate nuclear distribution of ligand-bound GR was observed in the presence of either agonists (dexamethasone) or antagonists (RU486) (69, 70), suggesting that nuclear clustering of GR is also independent of its transcriptional activity. In contrast to these studies, however, GFP-GR was found in a diffuse nuclear distribution in the presence of RU486 (71). It was also shown by the same group that PR agonists and antagonist (RU486) caused import of both GFP-PRA and GFP-PRB chimeras into the nucleus (72). In support of these studies, a colocalization study using a variety of transcription factors (including the GR, Oct1, and E2F-1) showed that most transactivators do not overlap with sites of transcription or the localization of RNA polymerase II (73). It should be noted, however, that the question of whether clustering of receptor in the nucleus represent sites of active transcription is still a topic of debate. For example, some studies have proposed that nuclear clusters or foci represent sites of active transcription. This is supported by the notion that some receptors adopt a punctate nuclear distribution in the presence of respective ligands but exhibit a more diffuse distribution in response to receptor antagonists, most notably in the case of the AR (56).

A punctate distribution of PPAR{gamma} has been shown by immunofluorescence microscopy in 3T3-L1 preadipocytes that express high levels of PPAR{gamma} and also in human peripheral blood monocytes (20). In addition, PPAR{gamma} localization exhibited a punctate and perinuclear reticular pattern in ECV-304 endothelial cells (59). Thus, the punctate/perinuclear distribution of PPAR{gamma} is not limited to hepatocytes and is also not likely to be an artifact of overexpression of these receptors in vitro. Further, the punctate distribution of PPAR{gamma} in cells expressing high levels of the receptor suggests that the specific effect of RXR{alpha} on the PPAR{gamma} isoform is not due to the lack of endogenously expressed PPAR{gamma}, relative to PPAR{alpha} and PPARß, in hepatocytes.

Taken together, these studies establish the nuclear localization of each of the PPAR isoforms. Evidence is also provided showing that ligand activation of PPARs, the phosphorylation status of particular amino acids that were shown to affect transcriptional activity and receptor function, and the coexpression of the coregulators, NCoR and SRC-1, do not alter the nuclear distribution of the PPARs, despite affecting their transcriptional activity. Further, it was shown that coexpression of RXR{alpha} is associated with a shift in the nuclear distribution of PPAR{gamma} from a diffuse to punctate pattern, a fact that may have interesting implications.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Construction of Plasmids
Full-length cDNAs for PPARh{alpha} (74) and PPARm{alpha} (74) were introduced into the pEGFP-C1 vector (CLONTECH Laboratories, Inc., Palo Alto, CA) using SalI (5'-end) and PspOM1 (3'-end) cloning sites. Full-length cDNAs for PPARmß (3) and PPARm{gamma} (75) were introduced into the pCI-nGFP-C656G vector (71) using SalI (5'-end) and NotI (3'-end) cloning sites. Expression vectors for the native PPARs, pSG5-PPARmß and pSG5-PPARm{gamma}, were provided by Walter Wahli (Universite de Lausanne, Lausanne, Switzerland), whereas pSG5-PPARm{alpha} and pSG5-PPARh{alpha} were previously described (74). pSG5-hRXR{alpha} was provided by Ronald Evans (Howard Hughes Medical Institute, La Jolla, CA). Full-length hRXR{alpha} cDNA (from pSG5-hRXR{alpha}) was introduced into pEYFP-C1 using SmaI (5'-end) and XbaI (3'-end) cloning sites. A (TCT 224 GCT) mutation of PPARm{gamma} cDNA corresponding to a Ser82Ala substitution was introduced into GFP-PPARm{gamma} by PCR-directed mutagenesis (GFP-PPARm{gamma}-A). Likewise, GFP-PPARm{alpha}-G was constructed by a (GAG 224 GGG) mutation of PPARm{alpha} cDNA corresponding to a Glu282Gly substitution. Construction of the reporter 3XZ-CAT in which the CAT gene is under the control of three PPREs of CYP4A6 (52) has been described (74). Likewise, construction of the reporter PPRE3-TK-LUC in which the luciferase gene is under the control of the herpes simplex virus thymidine kinase promoter and three PPREs, has been described (29). pSV-ß-Galactosidase control vector (pSV-ß-Gal) (Promega Corp., Madison, WI) was used to normalize for transfection efficiency. The expression vectors for NCoR (76) and SRC-1 (77) have been described. The sequence of all of the fluorescent protein constructs was confirmed by nucleotide sequencing.

Cell Lines and Cell Culture
Hepa-1 cells were maintained with DMEM (Life Technologies, Inc., Gaithersburg, MD) with 10% FBS (Atlanta Biologicals, Norcross, GA) plus antibiotics (100 U/ml penicillin and streptomycin, 0.5 mg/ml gentamycin; Life Technologies, Inc.) and 2 mM L-glutamine (Life Technologies, Inc.) in a 5% CO2 incubator at 37 C.

Transfections, Reporter Gene Assay, and ß-Galactosidase Assay
Cells (2 x 105) were transfected with 500 ng 3XZ-CAT (or 500 ng PPRE3-TK-Luc), 500 ng pSV-ß-Gal, and 200 ng (unless otherwise indicated) of one of the following plasmids: GFP-PPARh{alpha}, GFP-PPARm{alpha}, GFP-PPARmß, GFP-PPARm{gamma}, GFP-PPARm{alpha}-G, GFP-PPARm{gamma}-A, YFP-hRXR{alpha}, pSG5-hRXR{alpha}, NCoR, SRC-1, or pEGFP by lipofection according to the protocols provided with LipofectAMINE Reagent (Life Technologies, Inc.). After incubation for 18 h at 37 C, the cells were refed culture media containing WY-14,643 (Chemsyn Science Laboratories, Lenexa, KS), bezafibrate (Sigma, St. Louis, MO), BRL-49653 (BIOMOL Research Laboratories, Inc., Plymouth Meeting, PA), 9-cis-RA (Sigma), or dimethylsulfoxide (DMSO) vehicle (Sigma) and further incubated at 37 C for 24 h.

To determine reporter gene activity, cells were washed twice in PBS before collection by scraping in PBS. After three freeze-thaw cycles, the cell extracts were centrifuged at 10,000 x g for 5 min and the supernatant was collected. CAT activities were determined as previously described (78). Luciferase assays were performed using a luminometer (Analytical Luminescent Laboratories, San Diego, CA) to detect light emitted from the mixture of 20 µl of supernatant and 100 µl of Luciferase Assay Substrate (Promega Corp., Madison, WI). 150 µl of cell extract was mixed with 150 µl of 2x ß-galactosidase solution (Promega Corp.) and incubated at 37 C until a faint yellow color was produced. The reaction was stopped with 500 µl 1 M sodium carbonate. ß-Galactosidase values were determined by measuring the optical density at 420 nm. Reporter gene activities were normalized by ß-galactosidase levels.

Western Blot Analysis
Total protein contained in the Hepa-1 cell extracts (or isolated from murine white adipose tissue) was assayed by the BCA protein assay (Pierce Chemical Co., Rockford, IL). Total protein (50 µg) from each sample was separated by a 10% SDS-PAGE gel and transferred to Immobilon-P membranes (Millipore Corp., Bedford, MA) and probed according to the manufacturer using an anti-GFP monoclonal antibody (lot 8362-1, CLONTECH Laboratories, Inc.) or an anti-PPAR{gamma} monoclonal antibody (sc-7273, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) as indicated. Detection of immunoreactive proteins was done by an enhanced chemiluminescence blot detection system (Amersham Pharmacia Biotech, Arlington Heights, IL).

Fluorescence Imaging
Live cell microscopy of GFP-fusion proteins was performed on a Leica TCS SP confocal microscope mounted on a DMIRBE inverted microscope (Leica Corp. Microsystems, Exton, PA). GFP was excited with the 488-nm laser line of an air-cooled Ar laser (20-mW nominal output, Coherent Inc., Santa Clara, CA), and emission was monitored between 505 nm and 590 nm. All images were processed as tiffs on Corel Photo-Paint (Corel Corp., Ottawa, Ontario, Canada) using standard image processing techniques.


   ACKNOWLEDGMENTS
 
Cell images were collected in the National Cancer Institute Fluorescent Imaging Facility with assistance from James McNally. We thank Cem Elbi and Dawn Walker for their assistance in preparation of the manuscript.


   FOOTNOTES
 
1 T.E.A. and C.T.B. contributed equally to this work. Back

2 Present address: Fuji Gotemba Research Laboratories, Chugai Pharmaceutical Co., Ltd., 135-1 Komakado Gotemba, Shizuoka 412-8513, Japan. Back

Abbreviations: CAT, Chloramphenicol transferase; CBP, cAMP response element binding protein-binding protein; DMSO, dimethylsulfoxide; GFP, green fluorescent protein; hRXR{alpha}, human RXR{alpha}; NCoR, nuclear receptor corepressor; NLS, nuclear localization signal; PPARm{alpha},-ß,-{gamma}, mouse PPAR{alpha}, -ß, and -{gamma}; PPRE, peroxisome proliferator response element; SRC, steroid receptor coactivator; YFP, yellow fluorescent protein.

Received for publication January 25, 2001. Accepted for publication December 19, 2001.


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