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Molecular Endocrinology 13 (10): 1695-1703
Copyright © 1999 by The Endocrine Society

p120 Acts as a Specific Coactivator for 9-cis-Retinoic Acid Receptor (RXR) on Peroxisome Proliferator-Activated Receptor-{gamma}/RXR Heterodimers

Tsuyoshi Monden, Mikiko Kishi, Takeshi Hosoya, Teturou Satoh, Fredric E. Wondisford, Anthony N. Hollenberg, Masanobu Yamada and Masatomo Mori

First Department of Internal Medicine (T.M., M.K., T.H., T.S., M.Y., M.M.) Gunma University School of Medicine Maebashi 371 Japan
Thyroid Unit (F.E.W., A.N.H.) Department of Medicine Beth Israel Deaconess Medical Center and Harvard Medical School Boston, Massachusetts 02215


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
p120 was originally isolated as a novel nuclear coactivator for thyroid hormone receptor. In this study, we characterized its interaction and transactivation of peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma}) and 9-cis-retinoic acid receptor (RXR) heterodimers. Transient transfection study revealed that p120 enhanced the transcriptional activation of PPAR{gamma}/RXR induced by PPAR{gamma}- or RXR-specific ligands. In the glutathione-S-transferase pull-down assay, while steroid receptor coactivator-1 showed apparent interactions with both RXR and PPAR{gamma}, p120 bound only to RXR in a 9-cis-retinoic acid (RA)-dependent manner and also did not bind to PPAR{gamma} even in the presence of thiazolidinediones. The yeast two-hybrid analysis showed no interaction of p120 with PPAR{gamma} under any conditions, and electophoretic mobility shift assay showed apparent DNA-PPAR{gamma}/RXR/p120 complex formation only in the presence of 9-cis-RA. Furthermore, the yeast three-hybrid assay clearly revealed a significant interaction between p120 and PPAR{gamma} via RXR of PPAR{gamma}/RXR heterodimer only in the presence of 9-cis-RA. These findings indicate that p120 acts as a specific coactivator for the RXR of PPAR{gamma}/RXR heterodimer in a 9-cis-RA-dependent manner.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We have recently cloned p120 using a yeast two-hybrid system and the ligand-binding domain of the thyroid hormone receptor (TR) ß1 as bait (1). The amino acid sequence deduced from its cDNA sequence revealed that p120 consists of 920 amino acids, and part of the sequence was identical to that of a previously identified protein, skeletal muscle abundant protein (SMAP) (2). However, Northern analysis demonstrated that p120 was expressed not only in the skeletal muscle but also in various organs. p120 was shown to interact specifically with the AF-2 domain of TR, and the interacting domains of p120 were localized in the region between amino acids (AA) 186–297. Transient transfection study revealed that p120 enhances the transcriptional activation by TR in a thyroid hormone-dependent manner. Therefore, in this context, p120 is similar to the steroid receptor coactivator-1 (SRC-1). However, subsequent examination showed p120 functions that differed from those of SRC-1: while SRC-1 is a coactivator for most nuclear receptors, including glucocorticoid receptor (GR), progesterone receptor (PR), TR, estrogen receptor (ER), androgen receptor (AR), vitamin D receptor (VitDR), retinoic acid receptor (RAR) and 9-cis-retinoic acid receptor (RXR) (3), p120 acts as a coactivator for AR but not for ER or RAR.

Peroxisome proliferator-activated receptor (PPAR) also is a member of the nuclear receptor superfamily (4). To date, three isoforms of PPARs have been identified (PPAR{alpha}, -{gamma}, and -{delta}), and they exhibit different tissue distribution patterns (5). Furthermore, PPAR{gamma} has two isoforms, PPAR{gamma}1 and {gamma}2, which share a high degree of homology except in the N-terminal region (6). These isoforms also showed different tissue distributions from PPAR{gamma}1, which is expressed ubiquitously, and PPAR{gamma}2, which is expressed predominantly in adipose tissue, where it has been shown to play an important role in adipocyte differentiation (7, 8). The actions of this receptor are known to be regulated by thiazolidinediones, a class of antidiabetic reagent (9), and the fatty acid derivative 15-deoxy-PGJ2 (10, 11), which bind to PPAR{gamma} and promote adipogenesis. Recently, oxidized low-density lipoprotein (12, 13) was reported to be an endogenous ligand of PPAR{gamma} and was suggested to be related to atherosclerosis (14).

When these ligands activate the PPAR{gamma} response gene, PPAR{gamma} heterodimerizes with RXR, a common heterodimer partner with TRs, RAR, and VitDR (15, 16), and this heterodimer binds to the PPAR-response elements (PPREs) in target genes to activate transcription (17, 18). The PPRE has been identified as a direct repeat motif of hexamer half-sites, TGACCT, spaced by one nucleotide (DR1) (19, 20), and has been found in various genes including the peroxisomal ß-oxidation enzymes (21), lipoprotein lipase (19), acyl-CoA oxidase (22), phospoenlpyruvate carboxykinase (23), aP2 (24), UCP-1 (25), and leptin (26). Furthermore, it has been reported that this activation of gene transcription by PPAR/RXR is dependent on the recruitment of coactivators such as SRC-1 (27, 28), PPAR{gamma} coactivator-1 (PGC-1) (29) and PPAR{gamma}-binding protein (PBP) (30). All of these coactivators interact with PPAR{gamma} in either a ligand-dependent or independent manner. In the present study, we examined whether p120 functions as a coactivator for PPAR{gamma} and determined the mechanism by which p120 interacts with PPAR{gamma}/RXR. We demonstrated that p120 acts as a coactivator for PPAR{gamma}/RXR and predominantly interacted with the RXR on PPAR{gamma}/RXR heterodimer complex only in the presence of 9-cis-RA, suggesting that p120 mediates enhancement of PPAR{gamma}/RXR transcriptional activation through a different mechanism from other coac-tivators.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
p120 Is Expressed in Human Fat Tissue
We initially examined the expression of p120 mRNA in human fat tissue. While our previous study showed the ubiquitous expression of the p120 gene in various tissues (1), the expression of p120 in fat tissue was unknown. Therefore, we performed RT-PCR analysis to confirm the expression of p120 in fat tissue. Lane 2 of Fig. 1Go shows the expression of the p120 mRNA in human fat tissue. However, SRC-1 mRNA was not detectable in the fat tissue under the same conditions (Fig. 1Go, lane 4). This result suggests that p120 may play a role in adipocyte differentiation or lipid metabolism along with PPAR{gamma}.



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  		Figure 1. Expression of p120 Gene in Fat Tissue

Lanes 1 and 2 were hybridized with {alpha}32P-labeled p120 cDNA, and lanes 3 and 4 were hybridized with {alpha}32P-labeled SRC-1 cDNA as probes. The following samples were used at each lane as a template: lane 1, p120 cDNA; lanes 2 and 4, cDNA from human fat tissue; lane 3, SRC-1 cDNA. The primers were described in Materials and Methods.

 
p120 Acts as a Coactivator of PPAR{gamma}/RXR in CV-1 Cells
To examine whether p120 acts as a coactivator of PPAR{gamma}/RXR in the presence of specific ligands, we performed transient transfection assays using CV-1 cells transfected with PPAR{gamma}2 and RXR{alpha} expression vector. As shown in the left panel of Fig. 2Go, incubation with 9-cis-retinoic acid (9-cis RA) showed the minimum increase (~2-fold) of the basal activity of the parental vector containing the TK109 promoter, whereas troglitazone (TZ) did not cause any changes. In this system, expression of p120 did not affect the luciferase activity of the thymidine kinase (TK) promoter in either the absence or presence of ligands, suggesting that p120 did not enhance general transcription.



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  		Figure 2. Cotransfected p120 Enhances PPAR{gamma}/RXR-Related Gene Expression Caused by Specific Ligands

CV-1 cells were transfected as described in Materials and Methods with DR1-Luc and mPPAR{gamma}2 and hRXR{alpha} in the presence or absence of p120. After transfection, cells were cultured in the absence or presence of 10 µM TZ or 1 µM 9-cis-RA or 10 µM TZ plus 1 µM 9-cis- RA. As a control, the parental vector TK109-pA3Luc was transfected under the same conditions. The data were quantified as relative luciferase (LUC) activity, where 1 equals the activity of the reporter in the absence of p120 and ligands.

 
In contrast, 1 µM 9-cis-RA and 10 µM TZ caused 8- and 5-fold activation of the TK promoter activity fused to the DR1 element (Fig. 2Go, right panel), respectively, and synergistic activation (19-fold) was observed by adding both ligands simultaneously. Cotransfection of p120 was able to augment each ligand-dependent activation of DR1 reporter expression (28-fold, 9-cis-RA; 14-fold, TZ). The synergistic effect of both ligands was further stimulated by expression of p120 (72-fold). Furthermore, expression of p120 alone enhanced the RXR/PPAR{gamma}-mediated activation of DR1 reporter expression by 3.4-fold (Fig. 2Go). This result suggested the presence of endogenous 9-cis-RA-like activity in this culture system, and an equally likely senario is that p120 is interacting with RXR in a ligand-independent manner under these experimental conditions. These observations demonstrated that p120 functions as a coactivator of PPAR{gamma}/RXR heterodimer in the presence of specific ligands.

p120 Interacts with RXR{alpha} but Not with PPAR{gamma}
To understand the mechanisms by which p120 activates PPAR{gamma}/RXR, we studied the protein-protein interactions among these three molecules using glutathione-S-transferase (GST) pull-down assay and the yeast two-hybrid assay. We previously demonstrated that the first 297 amino acids of p120 and AA 186–297 of p120 containing the LXXLL motif are essential for the interaction with TR in a ligand-dependent manner; therefore, we used these regions to study its interaction with RXR{alpha} or PPAR{gamma}. As shown in Fig. 3Go, the interacting domain of SRC-1, containing three LXXLL motifs, clearly interacted with RXR{alpha} in a 9-cis-RA-dependent manner and with PPAR{gamma} in either the presence or absence of ligand. In contrast, AA 1–297 of p120 interacted with RXR{alpha} only in the presence of 9-cis-RA, and AA 186–297 of p120 showed stronger interaction with RXR{alpha}. Furthermore, no interaction was detected between p120 and PPAR{gamma} in either the presence or absence of TZ (Fig. 3Go). Similar results were observed when AA 1–920 (the whole protein) for AA 1–297 of p120 was used (Fig. 3Go).



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  		Figure 3. p120 Interacts with RXR but Not with PPAR{gamma} in the GST Pull-Down Assay

Similar amounts of Sepharose beads containing the bound GST fusion proteins of p120 derivative from AA 1–920 (full length), AA 1–297, and AA 186–297 and SRC-1 (AA 583–779) were incubated with radiolabeled in vitro translated RXR{alpha} either in the presence or absence of 1 µM 9-cis-RA, and with PPAR{gamma}2 in either the presence or absence of 10 µM TZ. As a control, an identical amount of GST alone was used.

 
To confirm the above results, we next examined whether PPAR{gamma} interacted with p120 using the yeast two-hybrid assay. As shown in Fig. 4AGo, although the ligand-binding domain of PPAR{gamma} including the hinge region strongly interacted with RXR{alpha} in either the presence or absence of TZ, PPAR{gamma} was not able to interact with any derivatives of p120 that covered all coding regions of p120 even in the presence of TZ. In the same yeast system, p120 interacted with RXR in a 9-cis-RA-dependent manner and also this data suggested that AA 186–297 of p120 are important for the interaction with RXR as well as TR (Fig. 4BGo).



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  		Figure 4. PPAR{gamma} Does Not Interact with any Region of p120 in the Yeast Two-Hybrid System

A, GAL4 activation domain-p120 fusion proteins were constructed as described previously (1 ). These constructs were examined for interaction with AA 193–505 of the ligand-binding domain of PPAR{gamma}2 fused to the GAL4 DNA-binding domain in HF7c yeast cells in either the presence or absence of 10 µM TZ. ß-Galactosidase activities of individual transformants were determined. The full-length of RXR{alpha} fused to the GAL4 activation domain was used as a positive control. B, The full-lenghth RXR{alpha} ligated to pGBT9 was cotransformed with GAL4 activation domain-p120 constructs into HF7c yeast cells onto the SD plate in either the presence or absence of 1 M 9-cis-RA. The values shown represent the means ± SE of six separate colonies from three separate transformations.

 
Taken together, these observations suggested that the activation PPAR{gamma}/RXR by p120 may be caused by the direct protein-protein interaction of PPAR{gamma}-RXR but not by PPAR{gamma}-p120.

Detection of p120-RXR{alpha}-PPAR{gamma} Complex on the DR1 DNA Element
To confirm the results obtained in the above in vitro assays, we next tested whether p120 could interact with PPAR{gamma} and RXR-DNA complex using electrophoretic mobility shift assay (EMSA) with the bacterially expressed AA 186–297 of p120, in vitro translated PPAR{gamma}2, RXR{alpha}, and labeled DR1 probe. Our previous study showed that this domain of p120 bound to TR homodimers and TR/RXR heterodimers on the DR4 element in the presence of thyroid hormone. As shown in Fig. 5Go, PPAR{gamma} did not form either monomer or homodimers on the DR1 element even in the presence of p120. On the other hand, RXR{alpha} formed homodimers on the DR1 element, and addition of 9-cis-RA promoted this homodimerization. Furthermore, addition of GST-p120 (AA 186–297) supershifted this complex only in the presence of 9-cis-RA, indicating the formation of the p120/RXR/RXR complex in the presence of 9-cis-RA. As expected, PPAR{gamma} and RXR{alpha} heterodimer complex strongly bound to the DR1 elements, and addition of p120 supershifted PPAR{gamma}/RXR{alpha} heterodimer complex only in the presence of 9 cis-RA or a combination of TZ and 9-cis-RA. While the p120/RXR/RXR complex and the p120/RXR{alpha}/PPAR{gamma} complex were not distinct because these complexes showed the same size on the gel, addition of p120 made the RXR{alpha}/PPAR{gamma} heterodimer faint, and the RXR/RXR homodimers still remained, strongly suggesting that these supershifts were formed mainly by the p120/RXR{alpha}/PPAR{gamma} complex. However, this supershift was not observed after addition of TZ alone, indicating that p120 forms a ternary complex with PPAR{gamma}-RXR{alpha} heterodimer on PPREs, which is 9-cis-RA dependent. Furthermore, the GST proteins alone did not bind to the DR1 probe.



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  		Figure 5. p120 Interacts with PPAR{gamma}/RXR Heterodimer in the Presence of 9-cis-RA on DNA

GST-p120 (AA 186–297) was used in EMSA with in vitro translated PPAR{gamma}2 and RXR{alpha} in the presence of the radiolabeled DR1 element as a probe. Each of the complexes formed was identified. TZ (10 µM) or 9-cis-RA (1 µM) was used in this experiment. The same amount of GST was used as a control.

 
The Yeast Three-Hybrid Assay Demonstrates that 9-cis-RA Promotes the Interaction of p120 with the RXR on PPAR{gamma}/RXR Heterodimers
To examine specific interactions of p120 with the RXR on PPAR{gamma}/RXR heterodimers, we next performed yeast three-hybrid assay between the GAL4 DNA-binding domain-PPAR{gamma} fusion and GAL4 activation domain-p120 fusion in the presence or absence of RXR{alpha} expression (Fig. 6Go). For this system, we fused cDNAs of RXR{alpha}, PPAR{gamma}, and p120 into the pAUR123, a yeast-Escherichia coli shuttle vector (Takara, Berkeley, CA), pGBT9 vector, and pGAD24 vector (CLONTECH Laboratories, Inc., Palo Alto, CA), respectively, all of which were driven by the alcohol dehydrogenase 1 promoter (ADH1) (31). Under the control of ADH1 promoter, the heterologous protein is expressed at a high level in yeast cells during growth on glucose-rich medium (32). We then transformed pGBT9-PPAR{gamma} with the pAUR123-RXR and/or pGAD-P120 into HF7c yeast cells and plated them on plates lacking leucine, tryptophan, and histidine. As shown in the left panel of Fig. 6AGo, in the absence of both p120 and RXR, addition of 9-cis-RA or TZ had no effect on basal ß-galactosidase activity. Cotransfection of RXR alone also showed no effect (second panel). On the other hand, in the presence of p120 (AA 1–297), TZ and 9-cis-RA showed a slight increase in ß-galactosidase activity in the absence of RXR, but these changes were not significant. In contrast, in the presence of RXR and p120, while TZ alone did not cause significant changes in ß-galactosidase activity, 9-cis-RA promoted activity 3.8-fold, and the combination of 9-cis-RA and TZ induced a 6-fold increase in activity (the right panel of Fig. 6AGo). The yeast colonies in the presence of RXR were approximately 3- to 4-fold larger and showed more distinct salmon pink color than those in the absence of RXR on plates containing 9-cis-RA (data not shown). The observation that only yeast cells expressing RXR were able to survive suggested that RXR was necessary to bridge between PPAR{gamma} fused to the GAL4-DNA binding domain to p120 fused to the GAL4-activation domain (Fig. 6BGo). These results clearly demonstrated that p120 interacts with the RXR of PPAR{gamma}/RXR heterodimers, and that 9-cis-RA is required for this interaction.



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  		Figure 6. A Novel Method Using the Yeast Three-Hybrid Assay Demonstrates the 9-cis-RA-Dependent Interaction of p120 with PPAR{gamma}/RXR Heterodimer

A, GAL4 activation domain-p120 (AA 1–297) construct (pGAD-p120) and GAL4 DNA-binding domain-PPAR{gamma}2 (AA 193–505) construct (pGBT9-PPAR{gamma}) were cotransformed to HF7c yeast cells in either the presence or absence of RXR{alpha} protein overexpressed by pAUR123 expression vector. TZ (10 µM) and 9-cis-RA (1 µM) were used in the SD plates lacking histidine, leucine, and tryptophan. ß-Galactosidase activities of individual transformants were determined. B, Schematic diagram of the yeast three-hybrid assay used in this experiment.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The present study demonstrated that p120 interacts with PPAR{gamma}/RXR heterodimers on PPREs in a 9-cis-RA-dependent manner, and p120 functions as a coactivator of PPAR{gamma} and RXR heterodimers. Several other coactivators that interact with various nuclear receptors to enhance transcriptional activity have been reported. Some of these belong to the histone acetyltransferase family (33), while others bind to other histone acetyltransferase proteins such as CBP/p300, which contribute to promoter activation by altering or disrupting the repressive chromatin structure (34, 35). The coactivators of PPARs/RXR, SRC-1, PBP, and PGC-1 have been cloned. It is of interest that these molecules function as coactivators for PPAR{gamma}/RXR by different mechanisms. SRC-1 and PBP interact with both PPAR and RXR in a ligand-dependent manner, and the AF-2 region of PPARs is important for their interactions (28, 30). A more recent study of the crystal structure using x-ray analysis demonstrated that the AF-2 region of PPAR{gamma} formed a pocket-like structure and bound to SRC-1 (36). In fact, in our GST pull-down study the interaction domain of SRC-1 clearly interacted with PPAR{gamma} in a ligand-independent manner (Fig. 3Go). On the other hand, PGC-1 has been reported to interact with PPAR{gamma} in a ligand-independent manner, and the hinge region of PPAR{gamma} is essential for the interaction (29). In contrast to these coactivators, the present results from EMSA and GST pull-down analyses suggested that p120 did not interact with PPAR{gamma} at all but interacted with RXR in a 9-cis-RA-dependent manner. These observations indicated that the transactivation of ligand-induced PPAR{gamma}/RXR by p120 is mediated through the RXR of PPAR{gamma}/RXR heterodimer complex. In addition, the expression of the p120 gene in fat tissue indicated a cooperative relationship between p120 and PPAR{gamma} in adipocyte differentiation.

Although the yeast two-hybrid assay is a powerful technique with which to analyze protein-protein interactions, this system is limited to detection of protein interactions between two proteins. Thus, this system fails to detect ternary complex formation. Since the three-hybrid system is helpful for detection of X/Y/Z complex formation when X does not bind to Z individually but only binds to the complex produced by the combination of X/Y and Y/Z (37), we used this assay to further confirm the interaction of p120 and RXR on RXR/PPAR{gamma} complex. Our results clearly demonstrated the interaction of p120 (X) with the PPAR{gamma} (Z) molecule only in the presence of RXR (Y). Furthermore, this interaction required the presence of 9-cis-RA, and TZ did not affect this protein-protein interaction (Fig. 7Go). Taken together, these results indicated that p120 act as a coactivator of PPAR/RXR through its interaction with the RXR molecule. Furthermore, this yeast three-hybrid assay is a useful strategy for detecting the partners of various heterodimer complexes.



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  		Figure 7. Model of the Interaction between p120 and PPAR{gamma}/RXR Heterodimer

A, In the presence of 9-cis-RA, p120 interacts with RXR directly causing activation of gene expression. B, On the other hand, p120 is not able to interact with PPAR{gamma}/RXR heterodimer immediately in the absence of 9-cis-RA, although TZ binds to the PPAR{gamma}.

 
However, the reason for the indirect activation by p120 in the presence of TZ alone in the CV-1 cells remains unclear. The first possibility is that p120 interacts directly with other transcriptional factors, such as CBP, which can interact with PPAR{gamma} in the presence of TZ (38). This was supported by our previous results, which demonstrated synergistic activation between p120 and CBP in a transfection study using the TRß1 expression vector and TRE-TK Luc reporter vector. The second possibility is that endogenous ligands for RXR, other than 9-cis RA, exist which might cooperate with exogenous TZ in the serum used in the transfection experiments. The third possibility is that not all retinoid is removed in stripped media and that TZ then potentiates the interaction.

SRC-1 is the best characterized nuclear receptor coactivator. Although SRC-1 was originally isolated as a protein interacting with PR, it interacts with most known nuclear receptors, including TR, PPAR, RXR, RAR, ER, AR, VitDR, and GR, and functions as a coactivator for these receptors to enhance transcriptional activation (3). In contrast, functional analysis of p120 revealed enhancement of the transcriptional activation only by TR, PPAR, RXR, and AR, but not by ER or RAR. Although it remains unclear whether p120 interacts with AR, ER, and RAR, the present study demonstrated the importance of AA 186–297 of p120, containing the LXXLL motif, which is a signature sequence for the binding of coactivators to nuclear receptors (39), for the interaction with RXR and TR, but not with PPAR{gamma}. These findings indicated the specificity of nuclear receptor-coactivator interactions and suggested that the LXXLL motif is not sufficient for this interaction. Therefore, it is speculated that the specific recruitment of coactivators by nuclear receptors may allow the flexible regulation by nuclear receptors in vivo. Further studies are required to determine how these coactivators affect each other and regulate ligand-dependent activation of gene transcription by nuclear receptors.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Plasmids and Constructs
The full-length mouse PPAR{gamma}2 cDNA was amplified by PCR using the mouse skeletal muscle cDNA as a template (Marathon cDNA, CLONTECH Laboratories, Inc.). The PCR product was verified as PPAR{gamma}2 cDNA by sequencing and was cloned into the expression vector pKCR2. For the yeast two-hybrid assay, the ligand-binding domain of PPAR{gamma}2 (AA 193–505) was subcloned into the pGBT9 plasmid (CLONTECH Laboratories, Inc.). The human SRC-1 cDNA was kindly provided by Dr. Takeshita (Harvard Medical School, Boston) (40). For the GST pull-down assay, the interacting domain of SRC-1 (AA 583–779) was generated by PCR amplification and inserted into the pGEX-4T-1 plasmid (Amersham Pharmacia Biotech, Piscataway, NJ). The PPRE reporter construct consisted of two copies of the DR1 element upstream of the TK109 promoter in the vector pA3Luc (26). TZ was provided by Sankyo Co., Ltd. (Tokyo, Japan), and 9-cis-RA was purchased from Sigma Chemical Co. (St. Louis, MO). These chemicals were dissolved in dimethyl sulfoxide.

RT-PCR
Human mesenteric fat tissue was obtained at the time of operation at the First Department of Surgery in Gunma University Hospital (courtesy of Dr. Mochiki). Total RNA was extracted using the modified acid-phenol method as described previously (40), and 2 µg of total RNA were reverse transcribed into first-strand cDNA for 2 h at 37 C using Moloney murine leukemia virus reverse transcriptase (Roche Molecular Biochemicals, Indianapolis, IN). p120 cDNA and SRC-1 cDNA were amplified from 3 µl of cDNA (from a total of 20 µl) using the following primer combinations of sense/antisense primers (p120: 5'-tcaaggtggaacctgca-3'/5'-tagcattgtgtatgctga-3': nucleotide 1729 to 2235), (SRC-1: 5'-atggtgccgatgccaatccct-3'/5'-ttcagtcagtagctgctgaag-3': nucleotide 3957 to 4514). PCR was carried out for 30 cycles consisting of denaturation for 1 min at 94 C, annealing for 2 min at 60 C, and extension for 2 min at 72 C, followed by a 15-min final extension at 72 C. Twenty microliters (50 µl total) of PCR products were visualizing by agarose gel electrophoresis. The Southern blot analysis of PCR products was performed. The PCR products were blotted onto Hybond N+ membrane and hybridized with the p120 or SRC-1 cDNA probes labeled with [{alpha}32P]dCTP using a DNA labeling kit (Amersham Pharmacia Biotech).

Transfection
The CV-1 cells were maintained in DMEM containing 10% FCS, 0.25 mg/ml streptomycin, 100 mg/ml penicillin, and 0.25 mg/ml Amphotericin (Life Technologies, Inc., Rockville, MD). The cells were plated in six-well plates 24 h before transfection, and the medium was changed 4 h before transfection. Using the calcium-phosphate method, the cells were transfected with the following amounts of DNA per six wells: 10 µg of pA3 Luc-TK reporter construct containing two DR1 elements upstream; 100 ng of pKCR2-mouse PPAR{gamma}2; 100 ng of pKCR2-human RXR{alpha}; and 3 µg of either pKCR2-p120 or 3 µg of pKCR2. Sixteen hours after transfection, the cells were cultured in the absence or presence of 10 µM TZ or 1 µM 9-cis-RA for 24 h and then harvested for luciferase assays after a further 24-h incubation. Luciferase activities were normalized by protein content. All experiments were performed in triplicate and repeated at least twice. The data shown are pooled results ± SE.

GST Pull-Down Assay
Purified GST and GST fusion protein were bound to glutathione-Sepharose beads. PPAR{gamma}2 and RXR{alpha} labeled with [35S]methionine were synthesized by the TNT-coupled in vitro translation system (Promega Corp., Madison, WI). These labeled proteins were incubated with GST-Sepharose or GST-fusion proteins-Sepharose for 2 h at 4 C in the presence or absence of the appropriate ligands (10 µM TZ or 1 µM 9-cis- RA) in reaction buffer (150 mM NaCl, 20 mM Tris, pH 7.5, 0.3% NP-40, 0.1 mM EDTA, 1 mM dithiothreitol, and 1 mM PMSF). The beads were then recovered by centrifugation. After washing the beads, bound proteins were eluted in Laemmli buffer, boiled for 2 min, and analyzed by SDS-PAGE followed by autoradiography.

Yeast Two-Hybrid and Three-Hybrid Assays
p120 cDNA ligated into the plasmid pGAD24 expressing the GAL4 activating domain, the PPAR{gamma} cDNA encoding AA 193–505 ligated to the plasmid pGBT9 (42) expressing the GAL4-binding protein, or the full-length hRXR{alpha} ligated to pGBT9 in frame, and pAUR123 (43) expressing hRXR{alpha} protein, were cotransformed into yeast HF7c cells, and transformants were plated onto synthetic complete medium plates lacking histidine, leucine, and tryptophan. Liquid assays of ß-galactosidase were carried out as described previously (1).

EMSA
The sequence, 5'-acgtagaagcttgaaatgAGGTAAAAGGTCAg-agtccaagct-3', derived from the mouse leptin promoter region, was used as the DR1 probe (26). The probe was labeled by [{alpha}-32P]dCTP using the PCR method. In vitro translated mPPAR{gamma}2, hRXR{alpha} proteins, and GST or GST-p120 AA187–297 were incubated with 100,000 cpm of the labeled double-stranded oligonucleotide in the presence of 1 µg of poly(dI-dC), 1 mM dithiothreitol, 0.1 µg of salmon sperm DNA, and EMSA binding buffer (20% glycerol, 20 mM HEPES, 50 mM KCl) was added to a final volume of 30 µl. Reaction was performed at room temperature for 20 min, and the samples were analyzed on 5% nondenaturing polyacrylamide gels. Electroporation was performed at 250 V for 90 min.


   ACKNOWLEDGMENTS
 
We thank Dr. Takeshita for the SRC-1 plasmid and Dr. Mochiki for the fat tissue specimen.


   FOOTNOTES
 
Address requests for reprints to: Tsuyoshi Monden, M.D., First Department of Internal Medicine, Gunma University School of Medicine, 3–39-15 Showa-machi, Maebashi, Gunma 371-8511, Japan.

Received for publication February 19, 1999. Revision received June 2, 1999. Accepted for publication June 29, 1999.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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