help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology, doi:10.1210/en.2004-0011
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
145/6/2886    most recent
Author Manuscript (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lim, H. J.
Right arrow Articles by Han, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lim, H. J.
Right arrow Articles by Han, K.
Endocrinology Vol. 145, No. 6 2886-2895
Copyright © 2004 by The Endocrine Society

Transcriptional Cofactors Exhibit Differential Preference toward Peroxisome Proliferator-Activated Receptors {alpha} and {delta} in Uterine Cells

Hyunjung J. Lim, Irene Moon and Kyuyong Han

Departments of Obstetrics & Gynecology (H.J.L., I.M., K.H.) and Cell Biology & Physiology (H.J.L.), Washington University School of Medicine, St. Louis, Missouri 63110

Address all correspondence and requests for reprints to: Dr. H. Jade Lim, Department of Obstetrics & Gynecology, Washington University School of Medicine, Campus Box 8064, 4566 Scott Avenue, St. Louis, Missouri 63110. E-mail: limj{at}wustl.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We previously showed that peroxisome proliferator-activated receptor {delta} (PPAR{delta}) is crucial for embryo implantation as a receptor for cyclooxygenase-2-derived prostacyclin in mice. PPARs belong to the nuclear receptor superfamily. They form heterodimer with a retinoid X receptor, recruit transcriptional cofactors, and bind to a specific recognition element for regulation of target genes. Although cofactors are generally shared by various nuclear receptors, some are involved in cell-specific events. The objective of this investigation was to examine interactions of transcriptional cofactors with PPAR{delta} in uterine cells for its effectiveness in regulating gene expression. We chose two uterine cellular systems: periimplantation mouse uterus and AN3CA human uterine cell line. As examined by in situ hybridization, steroid receptor coactivator (SRC)-2, SRC-3, PPAR-interacting protein, receptor-interacting protein 140 (RIP140), nuclear receptor corepressor (N-CoR), and silencing mediator for retinoid and thyroid hormone receptor (SMRT) exhibit overlapping expression with that of PPAR{delta} in the periimplantation mouse uterus. Glutathione-S-transferase (GST) pull-down assays show that PPAR{delta} physically interacts with SRC 1–3, RIP140, PPAR-binding protein, N-CoR, and SMRT in the absence of ligands, suggesting their potent interactions with PPAR{delta}. Transient transfection assays in AN3CA cells show that among members of the SRC family, only SRC-2 serves as a true coactivator for PPAR{delta}, whereas all SRC members could enhance PPAR{alpha}-induced transcriptional activation. Interestingly, N-CoR and SMRT potently repress PPAR{delta}-induced transcriptional activation but fail to repress PPAR{alpha} activity. RIP140 is effective in repressing basal and PPAR-induced transcriptional activation. Collectively, the results suggest that gene regulation by PPAR{delta} in the uterine cells uniquely responds to SRC-2, N-CoR, SMRT, or RIP140, and these interactions may be operative during implantation when these cofactors are abundantly expressed.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
LIGAND-DEPENDENT TRANSCRIPTION factors such as nuclear hormone receptors modulate transcription by directly binding to sequence-specific DNA response elements in promoters of the target genes, resulting in activation or repression of transcription (1, 2). Although biological functions and profiles of activating ligands are extremely diverse among the members of nuclear receptors, gene regulation by ligand-activated nuclear receptors converges on the usage of transcriptional cofactors (3, 4). Thus, many cofactors are known for their promiscuity toward various nuclear receptors. On the other hand, certain cofactors exhibit tissue-specific expression patterns and function in a specific cellular context (4, 5, 6, 7, 8, 9, 10). Because a combination of different cofactors contributes to proper functioning of a nuclear receptor, it is necessary to examine the status of various transcriptional cofactors in a given cellular context followed by a study of physical interactions between the nuclear receptor of interest and available cofactors to generate insightful information.

The peroxisome proliferator-activated receptor (PPAR) family of transcription factors (PPAR{alpha}, PPAR{gamma}, and PPAR{delta}) belongs to the nuclear hormone superfamily (11). Transcriptional activation of target genes by PPAR depends on ligand-induced heterodimerization with a retinoid X receptor (RXR) and recruitment of several cofactors (4, 12). Identification of several PPAR cofactors predicts complex network of cofactors that are shared by various nuclear hormone receptors. Among these, PPAR-binding protein (PBP/PPARBP), PPAR-interacting protein/nuclear receptor-activating protein 250 (PRIP/RAP250), receptor-interacting protein 140 (RIP140), and PPAR{gamma} coactivator-1 (PGC-1) were identified in yeast two-hybrid screening systems using either PPAR{alpha} or PPAR{gamma} as a bait (13, 14, 15, 16, 17, 18). They are also capable of increasing transcriptional activity of other nuclear receptors (15, 16, 18). Likewise, other cofactors identified as interactors of other nuclear receptors, including members of steroid hormone receptor coactivators (SRC) family, silencing mediator for retinoid and thyroid hormone receptor (SMRT), and nuclear receptor corepressor (N-CoR), were shown to interact with PPAR{alpha} or PPAR{gamma} in vitro and influence their transcriptional activation (4, 19). As for PPAR{delta}, interactions with N-CoR, SMRT, SHARP [SMRT and histone deacetylases (HDACs)-associated repressor protein], and HDACs under certain in vitro or in vivo settings have been demonstrated by two groups (20, 21). A recent report also showed that PPAR{delta} physically interacts with SRC-1 (22). However, direct interaction of other transcriptional cofactors with PPAR{delta} remains to be investigated.

The uterus consists of three major cell types: epithelium, stroma, and myometrium. Proliferation and differentiation of these cells before implantation is regulated by ovarian steroid hormones. Implantation in mice occurs around midnight of d 4. After the attachment of the blastocyst onto uterine epithelium, stromal cells proliferate and differentiate into decidual cells to support the developing conceptus under progesterone (23). During these dynamic hormonal changes, PPAR{delta} is not expressed in the preimplantation mouse uterus (d 1–4 morning) but is dramatically induced in the stromal cells surrounding the implanting embryo at the time of attachment reaction (d 4 midnight onward). The expression of PPAR{delta} persists through decidualization process, whereas PPAR{alpha} and PPAR{gamma} are undetectable or expressed at very low levels during the periimplantation period (24). This observation suggests that the expression of PPAR{delta} in the uterus demarcates the onset of implantation process. Similar expression of PPAR{delta} during implantation has also been observed in rats (25). We have previously shown that PPAR{delta} plays an important role in blastocyst implantation as the receptor for cyclooxygenase-2-derived PGI2 (24). However, no information is available regarding cofactor usage or downstream target genes of PPAR{delta} during implantation, and thus the mechanism by which this nuclear receptor contributes to PGI2 signaling during the crucial process of embryo implantation remains elusive. PPAR{delta} is widely expressed in many tissues (11, 26), and PPAR{delta}-deficient mice exhibit genetic background-dependent phenotypes ranging from early developmental defects leading to midgestational lethality to altered fatty acid metabolism and variable subfertility in surviving adult mice (reviewed in Ref. 27). Mounting evidence now suggests that PPAR{delta}, aside from embryo implantation (24), regulates a variety of cellular processes including apoptosis, colorectal cancer, vascular functions, fat metabolism, and atherogenic inflammation (28, 29, 30, 31, 32, 33, 34). Therefore, scrutinizing the downstream signaling mechanism of PPAR{delta} in a specific cellular setting will help identify generality and specificity of the signaling pathways exerted by this nuclear receptor.

In the present investigation, we sought to determine cofactor interactions with PPAR{delta} in the uterine context, using both mouse and human systems. We first examined which cofactors are available to interact with PPAR{delta} during implantation using the periimplantation mouse uterus. We then confirmed direct physical interactions between cofactors and PPAR{delta} by glutathione-S-transferase (GST) pull-down assays. In vivo interactions were investigated using reporter gene assays in transient transfection experiments. The results show that eight transcriptional cofactors studied herein exhibit cell type-specific expression patterns in the periimplantation mouse uterus, showing that some are available to interact with PPAR{delta} during implantation, and that PPAR{delta} and PPAR{alpha} exhibit distinct cofactor preferences in uterine cells, with PPAR{delta} preferentially interacting with SRC-2, N-CoR, and SMRT for gene regulation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice
Adult CD-1 mice were housed in the Animal Care Facility at the Washington University School of Medicine according to National Institutes of Health and institutional guidelines for laboratory animals. Protocols for animal work were approved by the Washington University Institutional Committee on Animal Care. Female mice were mated with fertile males of the same strain to induce pregnancy. The morning of finding a vaginal plug was designated d 1 of pregnancy. Implantation sites on d 5 were visualized by iv injection (0.1 ml/mouse) of Chicago blue dye solution (1% in saline) (35).

Materials
Carbaprostacyclin (cPGI) and 9-cis-retinoic acid (9cRA) were purchased from Cayman Chemical and Biomol, respectively. The cDNAs for human PPAR (hPPAR) {alpha}, hPPAR{delta}, and activator of thyroid and retinoic acid receptor (ACTR) (SRC-3) in mammalian expression vectors and the PPREx3-tk-luciferase construct were kindly provided by Dr. R. Evans (The Salk Institute, La Jolla, CA). Dr. J. Reddy provided mouse cDNAs for PBP and PRIP, and Dr. M. Stallcup provided mouse glucocorticoid receptor-interacting protein 1 (GRIP1) (SRC-2) cDNA (University of Southern California, Los Angeles, CA). Mouse cDNA for RIP140 was a generous gift from Dr. L. Wei (University of Minnesota Medical School, Minneapolis, MN), and mouse cDNAs for RXRs from Dr. P. Chambon (Institute de Genetique et de Biologie Moleculaire et Cellulaire, Illkirch Cedex, France), respectively. Human SRC-1 was provided by Dr. M. Tsai (Baylor College of Medicine, Houston, TX). RAP250 (PRIP) cDNA was a generous gift from Dr. P. Antonson (Karolinska Institutet, Stockholm, Sweden). When necessary, partial cDNAs of cofactors were subcloned into riboprobe vectors to generate 35S-labeled probes for in situ hybridization. SRC-3 riboprobe vector was generated by RT-PCR. Antipeptide polyclonal antibodies for PPAR{delta}, SRC-1, GRIP1 (SRC-2), SRC-3, RIP140, N-CoR, and thyroid hormone receptor-associated protein 220 (TRAP220) (PBP) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

In situ hybridization
In situ hybridization was performed as described previously (36). Uteri from pregnant mice were cut into 4- to 6-mm pieces and flash frozen in Histo-Freeze (Fisher Scientific, Pittsburgh, PA). Frozen sections (12 µm) were mounted onto poly-L-lysine-coated slides and fixed in cold 4% paraformaldehyde in PBS. The sections were prehybridized and hybridized at 45 C for 4 h in 50% formamide hybridization buffer containing the 35S-labeled antisense cRNA probe (specific activities ~2 x 109 dpm/ml). After hybridization and washing, the sections were incubated with ribonuclease A (20 µg/ml) at 37 C for 20 min. Ribonuclease A-resistant hybrids were detected by autoradiography using Kodak NTB-2 liquid emulsion (Eastman Kodak Co., Rochester, NY). Sections hybridized with the corresponding sense probe served as negative controls. Slides were poststained with hematoxylin and eosin. In situ hybridization of each gene was repeated at least three times.

Western blotting
Protein extracts from mouse uteri or cell lines were prepared in solubilization buffer [50 mM Tris-Cl (pH 7.5), 150 mM NaCl, 1% Nonidet P-40 (NP-40), 10% glycerol, 1 mM EGTA] containing an aliquot of Complete protease inhibitor cocktail (Roche, Indianapolis, IN). Briefly, mouse uteri were collected in the solubilization buffer and homogenized with a Polytron power homogenizer. Cultured cells were harvested in the same buffer and sonicated. Lysates were centrifuged at 12,000 x g, and supernatants were subjected to Bradford assays for quantitation. To examine expression of putative PPAR{delta} cofactors, 100 µg of protein extracts were loaded onto 7.5% SDS-PAGE gels. After transferring onto nitrocellulose membranes, the membranes were subjected to Western blotting with a cofactor-specific antibody as indicated.

GST binding assays
Full-length SRC-1, SRC-2, SRC-3, RIP140, N-CoR, SMRT, and PBP cDNAs were transcribed and translated using the TNT reticulocyte lysate kit (Promega, Madison, WI) in the presence of [35S]methionine. The construct encoding the GST-PPAR{delta} fusion protein was generated by cloning full-length hPPAR{delta} in frame into the pGEX-5X-3 vector (Amersham Pharmacia Biotech Inc., Alameda, CA). GST and GST-PPAR{delta} fusion proteins were expressed in Escherichia coli BL21(DE3)pLysS (Novagen, Madison, WI) by induction with 0.1 mM isopropyl ß-D-thiogalactoside at 37 C. Proteins were isolated by cell lysis and purified using glutathione-Sepharose 4B (Amersham Pharmacia Biotech Inc.). Purified GST and GST-PPAR{delta} fusion proteins were incubated with 2 µl volume of in vitro-translated cofactors in 500 µl of binding buffer [20 mM Tris-Cl (pH 7.9), 180 mM KCl, 0.05% NP-40, 0.2 mM EDTA, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride] with constant rotation at 4 C for 2 h. Unbound proteins were removed by three washes with washing buffer [20 mM Tris-Cl (pH 7.9), 180 mM KCl, 0.1% NP-40, 0.2 mM EDTA, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride]. Bound proteins were eluted from the beads by boiling in SDS-PAGE sample buffer and resolved on SDS-PAGE gels. [35S]Methionine-labeled proteins were visualized by autoradiography. Representative gels were stained with Gelcode Blue Stain Reagent (Pierce, Rockford, IL) before being subjected to autoradiography to ensure that equal amounts of GST fusion proteins were included in each reaction (data not shown).

Cell culture, transfection, and luciferase assays
AN3CA human uterine carcinoma cells were grown in DMEM (Life Technologies, Inc., Gaithersburg, MD) supplemented with 10% FBS (Sigma, St. Louis, MO), penicillin (100 U/ml)-streptomycin (100 µg/ml) in a 5% CO2 atmosphere. Cells were split into six-well plates a day before transfection. Cells were transfected with a mixture containing FuGENE6 transfection reagent (Roche), 0.6 µg/ml PPRE3-tk-luciferase, 0.2 µg/ml ß-galactosidase, 0.6 µg/ml empty, hPPAR{alpha}, or hPPAR{delta} expression vector under the control of cytomegalovirus promoter, with or without the addition of 0.6 µg/ml cofactor in Opti-MEM I (Life Technologies, Inc.) for 5 h. All transfection reactions were normalized to a total of 2.0 µg/ml plasmid DNA with pCDNA3. The transfection mixture was replaced with complete media containing vehicle (dimethylsulfoxide) or 1 µM cPGI and/or 5 µM 9cRA. After 24 h, cells were harvested in lysis buffer [0.05% Tris/2-[N-morpholino]ethanesulfonic acid (pH 7.8), 1% Triton X-100]. Relative light units from luciferase activity were determined using LUMIstar Galaxy (BMG Labtechnologies, Durham, NC) and normalized to the ß-galactosidase activity. Data are presented as fold activation relative to vehicle-treated cells in the same group and represent the mean from at least four independent transfection experiments. A statistical significance was examined by a Student’s t test (two tails) using Microsoft Excel program. (See Figs. 5Go and 6Go; all error bars represent SD from the mean.)



View larger version (20K):
[in this window]
[in a new window]
  		FIG. 5. SRC family of coactivators differentially influences transcriptional activation by PPAR{alpha} and PPAR{delta} in the AN3CA human uterine adenocarcinoma cells. AN3CA cells were transfected as with the indicated receptor and the reporter construct with or without a cofactor for 5 h. Cells were then treated with vehicle (dimethylsulfoxide) or ligand(s) for 24 h. Transcriptional response by PPAR{alpha} or PPAR{delta} was measured by luciferase activity of the rat acyl coenzyme A PPREs (PPREx3-tk-luc) and was normalized to the internal controls of ß-galactosidase expression. Data are presented as fold activation relative to vehicle-treated cells in the same group and represent the mean from at least four independent transfection experiments. See Materials and Methods for details. Error bars represent SDs and statistical significance was examined by a Student’s t test. *, P < 0.01.

 


View larger version (25K):
[in this window]
[in a new window]
  		FIG. 6. Transcriptional corepressors exhibit preferential repression of PPAR{delta}-induced transcriptional activation in the AN3CA human uterine adenocarcinoma cells. See Fig. 5Go legend.

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Transcriptional cofactors exhibit spatiotemporal expression patterns in the periimplantation mouse uterus
The objective of these experiments was to determine putative cofactors that are available to interact with PPAR{delta} for its role as a PGI2 receptor during implantation. We chose to study expression of coactivators SRC-1, SRC-2, SRC-3, PBP, and PRIP on uterine sections from d 1, 4, 5, or 8 of pregnancy by in situ hybridization. These four time points were chosen because they exemplify changing cellular environment under steroid hormones before and after the attachment reaction (23). All of these factors interact with either PPAR{alpha} or PPAR{gamma} in in vitro binding experiments or in transient transfection experiments, but only SRC-1, SMRT, and N-CoR have been shown to interact with PPAR{delta} (4, 17, 20, 21, 22, 37).

SRC family.
The SRC family (p160 family) of cofactors forms a protein complex containing histone acetyltransferase activity. Three members have been cloned in mice as SRC-1, SRC-2 (GRIP1/transcription initiation factor 1), and SRC-3 [p/CIP/RAC3/ACTR/amplified in breast cancer 1 (AIB1)] (10). All three members are capable of interacting with PPAR{alpha} or PPAR{gamma} in vitro (4, 19). These SRC family members exhibit diverse expression patterns and functions (3, 10). Indeed, these three members exhibit unique spatiotemporal expression patterns distinct from one another in the mouse uterus. As shown in Fig. 1AGo, SRC-1 is weakly expressed in the uterine epithelium on d 1 of pregnancy but is expressed mainly in the stroma on d 4, suggesting this gene may be under the regulation of steroid hormones. On d 5, the mRNA is expressed in the stromal cells in a diffused pattern but not at the vicinity of implanting blastocyst. On d 8, the expression is down-regulated. In contrast, SRC-2 mRNA is highly abundant in the periimplantation mouse uterus. On d 1, SRC-2 is expressed mainly in the epithelium, and at low levels in the myometrium and stroma. On d 4, epithelial expression is sustained at high levels and there is patchy expression in the stroma. On d 5, SRC-2 is highly expressed in the stroma, epithelium, and myometrium, and on d 8, the maternal deciduum as well as developing embryo shows high levels of expression. In contrast, SRC-3 is not detected in the preimplantation uterus (d 1 and 4 of pregnancy). In the postimplantation uterus, SRC-3 mRNA is weakly expressed in the stroma around the implanting blastocyst on d 5 and in the deciduum on d 8. Overall, SRC-2 and SRC-3 exhibit overlapping expression with PPAR{delta}.



View larger version (138K):
[in this window]
[in a new window]
  		FIG. 1. In situ hybridization of SRC-1, SRC-2, SRC-3, PRIP, and PBP mRNAs in the pregnant mouse uterus. Gene expression in the periimplantation mouse uterus was assessed by in situ hybridization of paraformaldehyde-fixed frozen sections with 35S-labeled cRNA probes. Dark-field photomicrographs of representative uterine sections are shown at x40, except for d 8 uterine sections, which are shown at x20. le, Luminal epithelium; s, stroma; myo, myometrium; bl, blastocyst; em, embryo.

 
PRIP and PBP.
PRIP (RAP250) was identified as a PPAR{gamma} and PPAR{alpha} cofactor independently (17, 18). PRIP is shown to interact with PPAR{alpha}, RAR{alpha}, RXR{alpha}, estrogen receptor (ER), and thyroid hormone receptor (TR) (17). As shown in Fig. 1BGo, PRIP mRNA is weakly expressed in the epithelium on d 1, but predominantly in the subepithelial stroma on d 4 of pregnancy. On d 5, PRIP mRNA exhibits concentrated expression around the implanting blastocyst, which is reminiscent of PPAR{delta} expression. On d 8, PRIP mRNA is expressed weakly in the deciduum and embryo.

PBP was first identified in a yeast two-hybrid screening system as a PPAR{gamma} cofactor and is also called vitamin D receptor-interacting protein 205 (DRIP205) or thyroid hormone receptor-associated protein 220 (TRAP220) (13, 38, 39). PBP interacts with PPAR{alpha} and PPAR{gamma} in a ligand-enhanced manner and is widely expressed in various mouse tissues including reproductive organs (13). In situ hybridization reveals that PBP is expressed in the mouse uterus. On d 1 of pregnancy, PBP mRNA is weakly detected in the stroma and longitudinal muscle layer (Fig. 1BGo). This pattern of expression is intensified on d 4 and 5. The signal is the most intense in submyometrial stroma on d 4. On d 8, PBP is weakly expressed in the decidua and the myometrium in a diffused pattern.

Corepressors RIP140, N-CoR, and SMRT.
RIP140 is originally identified as a cofactor of PPAR{alpha} or TR2 (14, 16). This factor was shown to actively compete with SRC-1 in transcriptional assays using the PPAR response element (PPRE) reporter system (16), implying its role as a true corepressor in PPAR/RXR signaling. As the first known corepressor that directly down-regulates PPAR/RXR activity, we examined its expression in the periimplantation mouse uterus. As shown in Fig. 2Go, RIP140 mRNA is abundantly expressed in the epithelium on d 1, both in the epithelium and stroma on d 4 of pregnancy. Notably, RIP140 mRNA shows focal expression in the antimesometrial stroma around the implanting blastocyst on d 5 when the blastocyst makes the initial contact with the uterine epithelium. On d 8 of pregnancy, RIP140 mRNA shows a condensed expression in the maternal deciduum surrounding the embryo with a weak diffused expression in the entire deciduum.



View larger version (146K):
[in this window]
[in a new window]
  		FIG. 2. In situ hybridization of RIP140, N-CoR, and SMRT mRNAs in the pregnant mouse uterus. Gene expression in the periimplantation mouse uterus was assessed by in situ hybridization of paraformaldehyde-fixed frozen sections with 35S-labeled cRNA probes. Dark-field photomicrographs of representative uterine sections are shown at x40, except for d 8 uterine sections, which are shown at x20. le, Luminal epithelium; s, stroma; myo, myometrium; bl, blastocyst; em, embryo.

 
N-CoR is initially cloned as a transcriptional corepressor of TR and RXR (40), but it has been shown to interact with all three members of PPAR with differential affinity (20, 21, 41). In the mouse uterus, N-CoR is expressed at low levels in the epithelium on d 1 of pregnancy, and abundantly in the subepithelial stroma on d 4 of pregnancy. On d 5, N-CoR mRNA is expressed in the stroma closer to the implanting blastocyst. On d 8, the mRNA is highly expressed in the overall deciduum as well as in the embryo.

SMRT is also a transcriptional corepressor that interacts with a wide range of nuclear receptors including PPARs (42, 43, 44). SMRT mRNA is expressed also at low levels in the uterine epithelium on d 1 of pregnancy and mainly in the stroma on d 4. Notably, the message is highly concentrated on the stromal cells surrounding the implanting blastocyst d 5. On d 8, this corepressor also shows abundant expression in the decidua (Fig. 2Go). Thus, all of these corepressors exhibit focal or diffused expression in the stroma around the implanting blastocyst at the time of implantation, overlapping with the PPAR{delta} expression (24).

Collectively, these in situ hybridization results show that coactivators SRC-2, SRC-3, and PRIP, and corepressors RIP140, N-CoR, and SMRT are abundant in the periimplantation mouse uterus and exhibit cell type-specific expression patterns that overlap PPAR{delta} expression around the time of implantation. These cofactors, therefore, are available to be recruited to uterine PPAR{delta}/RXR complex to regulate downstream target genes during implantation.

Western blot analyses of transcriptional cofactors in the mouse uterus and in the AN3CA human uterine adenocarcinoma cells
We next determined protein expression of the above cofactors in the uterine tissues collected from d 5–8 pregnant mice by Western blot analysis. PPAR{delta} is expressed in the uterine stroma during the early phase of implantation (d 5–6) and in the maternal deciduum and developing embryo on d 7–8 of pregnancy (24). As shown in Fig. 3Go, all three members of SRC family, RIP140, N-CoR, and PBP show steady-state levels of protein expression in d 5–8 of postimplantation mouse uterus (left panel). The results confirm that these cofactors are available to physically interact with the uterine PPAR{delta} during implantation.



View larger version (76K):
[in this window]
[in a new window]
  		FIG. 3. Western blot analysis of transcriptional cofactors in the mouse uterus and cell lines. Left panel, Total uterine extracts were collected from mice at d 5, 6, 7, or 8 of pregnancy. About 100 µg of total proteins were loaded onto 7.5% SDS-PAGE gels. Gels were transferred on nitrocellulose papers and subjected to Western blot analyses using specific antibodies indicated. Right panel, Total cellular lysates of NIH3T3 mouse fibroblasts and AN3CA human uterine adenocarcinoma cells were used in Western blot analyses. For N-CoR, two distinct bands are detected in NIH3T3 cells, seemingly representing RIP13, a shorter form of N-CoR (~150 kDa), and the full-length N-CoR (~270 kDa). No, No transfection; P{delta}, PPAR{delta}-transfected cells.

 
Our previous work showed that uterine adenocarcinoma cell line AN3CA supports PPAR{delta}-mediated transcriptional activation on a PPRE by PPAR{delta}-selective agonists, suggesting this uterine cell line is capable of supporting PPAR{delta}-mediated transcriptional activation (24). To examine which cofactors are endogenously expressed to support transcriptional activation by PPAR{delta}, we analyzed basal expression of transcriptional cofactors in this cell line. We performed same experiments with NIH3T3 mouse fibroblast cell line for comparison. Figure 3Go (right panel) shows that SRC-2, SRC-3, RIP140, N-CoR, and PBP are endogenously expressed in AN3CA, but SRC-1 and SMRT proteins are not detected with the antibodies we used. In NIH3T3 cells, similar profile of cofactor expression is noted with an exception of SRC-1 and N-CoR. SRC-1 is endogenously expressed in these cells, whereas N-CoR is expressed at a very low level. Two distinct bands are detected in NIH3T3 cells by anti-N-CoR antibody, a band of approximately 150 kDa apparently representing RIP13, a smaller variant of N-CoR (45), along with very low levels of full-length N-CoR. Endogenous PPAR{delta} is not detected by Western blotting in both of these cell lines, and transfection of full-length PPAR{delta} expression construct directed high levels of PPAR{delta} expression. Transfection of the PPAR{delta} plasmid did not alter endogenous expression of transcriptional cofactors examined.

In vitro interaction between transcriptional cofactors and PPAR{delta}
The results so far showed that the transcriptional cofactors exhibit spatiotemporal expression patterns in the mouse uterus and that they are available to interact with PPAR{delta}. With the exception of SRC-1, N-CoR, and SMRT, it was not determined whether PPAR{delta} also can directly interact with other coactivators (20, 21, 22). Thus, we performed GST pull-down assays using GST-PPAR{delta} fusion protein and 35S-labeled in vitro-translated cofactors. As shown in Fig. 4Go, all three members of SRC family, PBP, and corepressors RIP140, N-CoR, and SMRT show interaction with PPAR{delta}. This interaction is specific to PPAR{delta} because no band was observed with the GST control protein. Specific interactions with the GST-PPAR{delta} are not enhanced by the addition of cPGI and/or 9cRA (a RXR ligand) (data not shown). The results show that PPAR{delta} is capable of interacting with a wide range of transcriptional cofactors in vitro for transcriptional regulation of target genes.



View larger version (76K):
[in this window]
[in a new window]
  		FIG. 4. In vitro interaction of various cofactors with PPAR{delta}. [35S]Methionine-labeled full-length cofactors generated by in vitro transcription-translation were incubated with GST-hPPAR{delta} or with GST in the binding buffer in the presence or in the absence of cPGI and 9cRA. After extensive washing, the bound proteins were eluted from the beads by boiling in SDS-PAGE sample buffer and resolved on SDS-PAGE gels. Interactions between cofactors and GST-PPAR{delta} were visualized by autoradiography. Addition of ligands (cPGI and/or 9cRA) did not alter band intensity (data not shown).

 
Differential effects of coactivators on the transcriptional response mediated by PPAR{alpha} and PPAR{delta}
As mentioned, the human uterine adenocarcinoma cell line AN3CA is a suitable system to monitor transcriptional regulation by PPAR{delta}. We previously showed that addition of cPGI (a ligand for both PPAR{alpha} and PPAR{delta}) and 9cRA in PPAR{delta}-transfected AN3CA cells induces more than 20-fold induction of transcriptional activation of the PPRE-luciferase reporter gene (24). This response was more pronounced with PPAR{delta} than with PPAR{alpha}, suggesting that this uterine cell line provides a suitable cellular environment for PPAR{delta} with respect to gene regulation. In this study, we sought to determine effects of transcriptional coactivators and corepressors on PPAR{delta}-induced transcriptional activation on the uterine context in comparison with PPAR{alpha}.

First, we examined whether addition of a member of SRC family transcription coactivators alters the transcriptional regulation by ligand-activated PPAR{delta}. Western blotting showed that, whereas SRC-1 was undetectable, both SRC-2 and SRC-3 were expressed in AN3CA cells (Fig. 3Go). As shown in Fig. 5Go, in a natural cellular context of AN3CA cells, activation of the luciferase reporter gene by ligands is indeed higher in PPAR{delta}-transfected AN3CA cells than in PPAR{alpha}-transfected cells (Fig. 5Go, open bars), consistent with our previous finding (24). Synergistic activation by combination of cPGI and 9cRA is also noted. The addition of SRC-1 has a significant effect on the transcriptional activation by PPAR{alpha} in all ligand groups, but not by PPAR{delta} (Fig. 5Go, solid bars). In contrast, SRC-2 enhances transcriptional activation by PPAR{delta} in the presence of 9cRA and cPGI + 9cRA by about 4-fold, and about 2-fold of transcriptional activity by PPAR{alpha} in all ligand groups (hatched bars). SRC-3 apparently plays a role as a coactivator with PPAR{alpha}, as fold activation of the luciferase activity doubled with cotransfection with this cofactor in all ligand groups. However, SRC-3 reduces luciferase activity by approximately 30% in the case of PPAR{delta}, suggesting that overexpression of SRC-3 above endogenous levels may lead to a slight repression of the reporter activation by PPAR{delta} (Fig. 5Go, dotted bars; *, P < 0.01). Interestingly, SRC-1 and SRC-2 suppress PPAR{delta} activity in the presence of cPGI, a PPAR{delta} ligand. This effect is not observed in 9cRA-treated group, suggesting that cofactor interaction by PPAR{delta} is affected by differential ligand usage. Collectively, these results show that members of SRC family of transcriptional coactivators contribute differentially to gene activation exerted by PPAR{alpha} and PPAR{delta} in AN3CA cells, and SRC-2 is a preferential cofactor for PPAR{delta} transactivation in AN3CA cells.

Differential effects of corepressors on the transcriptional response mediated by PPAR{alpha} and PPAR{delta}
We then studied effects of three transcriptional corepressors on PPAR{alpha}- or PPAR{delta}-induced activation of the reporter gene. Western blotting showed that AN3CA cells endogenously express RIP140 and N-CoR (Fig. 3Go). We could not detect SMRT with the antibody we used (data not shown). As shown in Fig. 6Go, cotransfection of RIP140 strongly represses basal and ligand-induced transactivation of PPRE reporter construct by PPAR{alpha} and PPAR{delta} (solid bars), showing that this cofactor is a potent repressor for PPAR signaling. We tested the 1/10th amount of RIP140 plasmid in similar transfection experiments, and the tendency of repression was still observed albeit at a lower extent (data not shown). Cotransfection of either N-CoR or SMRT exhibits strong repressive effects on PPAR{delta}-induced transcriptional activation, whereas N-CoR significantly enhances PPAR{alpha} transactivation in all ligand groups (Fig. 6Go, hatched and dotted bars; *, P < 0.01). SMRT marginally represses PPAR{alpha} transactivation. This result is consistent with previous reports that PPAR{delta} preferentially interacts with these corepressors (20, 21) and shows that both N-CoR and SMRT are more effective corepressors for PPAR{delta} than PPAR{alpha} in the regulation of target genes.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Transcriptional regulation by nuclear hormone receptors uses an intertwined network of transcriptional cofactors and basal transcriptional machinery. Therefore, investigating the in vivo context, i.e. which cofactors are actually available to modulate transcriptional regulation by certain ligand-receptor complex, gives meaningful information as to how a ligand-receptor complex modulates expression of target genes during a specific cellular event.

Our previous observations of expression of PPAR{delta} in an implantation-specific manner and of cyclooxygenase-2-derived PGI2 serving as a ligand for this receptor provide strong evidence that PGI2-PPAR{delta} signaling is a crucial component of early implantation events, such as the attachment reaction by the blastocyst, decidualization, and angiogenesis (24, 46). In the present investigation, we sought to determine putative PPAR{delta} cofactors in uterine cellular environments using two systems with proven PPAR{delta} activity. First, PPAR{delta} in the periimplantation mouse uterus serves as a PGI2 receptor for embryo implantation, possibly by regulating angiogenesis and decidualization (24, 46). Secondly, in the AN3CA human uterine adenocarcinoma cell line, PPAR{delta} is shown to be more active as a cPGI receptor than PPAR{alpha} in a natural cellular environment without addition of cofactors (24).

In the periimplantation mouse uterus, SRC-1, SRC-2, SRC-3, PRIP, PBP, RIP140, N-CoR, and SMRT are expressed differentially during the early phase of implantation (d 5 of pregnancy). Among these, SRC-2, SRC-3, PRIP, and three corepressors RIP140, N-CoR, and SMRT exhibit expression patterns that overlap that of PPAR{delta} (Figs. 1Go and 2Go). Especially on d 5 of pregnancy when PPAR{delta} is initially induced in the uterine stromal cells, these cofactors are expressed in the same cell type as well. The potent interaction of these cofactors with PPAR{delta} observed by GST pull-down assays did not require the presence of any ligand (Fig. 4Go). In contrast, PPAR{gamma} has been shown to interact all three members of SRC family in the presence of 15-deoxy-{Delta}12,14-PGJ2, a ligand for this isoform (47). This suggests that PPAR isoforms exhibit distinct ligand requirements for cofactor binding.

Our transfection experiments in AN3CA cells using a common ligand for PPAR{alpha} and PPAR{delta} clearly reveal that PPAR{alpha} and PPAR{delta} exhibit differential preference toward transcriptional cofactors. Although SRC-2 is the only member which significantly enhances PPAR{delta}-induced transcriptional activation (Fig. 5Go), all three members of SRC family augment transactivation by PPAR{alpha} to different extents. Because SRC-2 is one of the most abundant cofactors in the periimplantation mouse uterus, it is notable that SRC-2-deficient female mice exhibit subfertility due to placental hypoplasia possibly stemming from earlier uterine defects (9). This suggests that the interaction between PPAR{delta} and SRC-2 may be operative during the process of implantation.

As for corepressors, N-CoR, SMRT, and RIP140 all potently repressed transactivation by PPAR{delta} (Fig. 6Go), whereas RIP140 was the only corepressor antagonizing reporter activation by PPAR{alpha}. The strong repression by RIP140 on PPAR-induced transactivation observed herein is consistent with previous reports that RIP140 antagonizes PPAR-mediated transactivation (37) and SRC-1 coactivation on PPAR{gamma}/RXR signaling (16). Other studies have shown that RIP140 is a strong repressor for transcription induced by PPAR{alpha}, liver X receptor {alpha} (LXR{alpha}), glucocorticoid receptor (GR), RAR, and RXR (37, 48, 49). Overall, our results suggest that PPAR{delta} exhibits high affinity toward transcriptional corepressors with respect to target gene regulation. Indeed, two studies reported similar findings on the corepressor usage by PPAR{delta} (20, 21). These studies showed that PPAR{delta} associates with corepressors including N-CoR, SMRT, SHARP, or HDCA. PPAR{delta} was also shown to be a potent transcriptional repressor itself, down-regulating gene induction by PPAR{alpha} or PPAR{gamma} (21). Given the abundant expression of RIP140, N-CoR, and SMRT in the implantation sites and strong interactions of PPAR{delta} with these cofactors, it is likely that PPAR{delta} is involved in gene repression that is a prerequisite for successful implantation. Further in-depth investigation is required to elucidate the mechanism of target gene regulation by PPAR{delta} during this process.

Functional classification and physiological and biochemical characteristics of cofactors studied in the present investigation are presented in Table 1Go.
Cointegrators CBP [cAMP response element binding protein-binding protein (CBP)] and p300 were not included in our study, because of well known ubiquitous nature of cointegrators (50). Although we chose two uterine cellular systems to find putative cofactors for PPAR{delta} during implantation, these factors are widely expressed and involved in various cellular processes. Except for PGC-1 and SMRT, gene targeted mouse models have been developed for these cofactors. Among these, gene targeted mice for CBP, p300, PBP, PRIP, and N-CoR all exhibit embryonic lethality associated with early developmental events. This is suggestive of fundamental roles of transcriptional cofactors in cell survival and development. Among mouse models that survive to adulthood, RIP140-deficient mice have been well characterized with respect to female infertility. Female mice devoid of RIP140 are infertile due to defective ovulation and their phenotype of infertility is not associated with any uterine defects (8, 51). Although SRC-1-deficient mice do not have fertility-related phenotypes (5), one study have shown that SRC-3-deficient female mice are subfertile with decreased ovulation and reduced litter size (7). For cofactors that show embryonic lethality after gene targeting, tissue-specific ablation to avoid early embryonic lethality will help in elucidating their involvement in implantation and PPAR{delta}-induced gene regulation.


View this table:
[in this window]
[in a new window]
  		TABLE 1. Summary of transcriptional cofactors shown to interact with PPARs in vitro

 
In conclusion, the present investigation offers several new observations: first, transcriptional cofactors SRC 1–3, PRIP, PBP, RIP140, N-CoR, and SMRT exhibit cell type-specific expression during implantation and some of them show overlapping expression patterns with PPAR{delta}; second, gene regulation by PPAR{delta} in AN3CA cells is affected by the presence of specific cofactors such as SRC-2, RIP140, N-CoR, and SMRT; and third, transactivation by PPAR{alpha} and PPAR{delta} is differentially influenced by cofactors in the uterine contexts. The question still remains as to which genes are targeted by PPAR{delta} during implantation. This is currently pursued in our laboratory and the information herein will be helpful to optimize the cellular environment for studying regulation of potential target genes by PPAR{delta} in the uterus.


   Acknowledgments
 
We thank Ms. S. Jeong for technical assistance and Drs. S. K. Dey and H. Song for critical reading of the manuscript.


   Footnotes
 
This work was supported by National Institutes of Health Grant HD40810 (to H.L.).

Abbreviations: ACTR, Activator of thyroid and retinoic acid receptor; CBP, cAMP response element binding protein-binding protein; cPGI, carbaprostacyclin; 9cRA, 9-cis-retinoic acid; ER, estrogen receptor; GRIP, glucocorticoid receptor-interacting protein; GST, glutathione-S-transferase; HDAC, histone deacetylase; hPPAR, human PPAR; N-CoR, nuclear receptor corepressor; NP-40, Nonidet P-40; PBP, PPAR-binding protein; PGC, PPAR{gamma} coactivator; PGI2, prostacyclin; PPAR, peroxisome proliferator-activated receptor; PPRE, PPAR response element; PRIP, PPAR-interacting protein; RAP, receptor-activating protein; RIP140, receptor-interacting protein 140; RXR, retinoid X receptor; SHARP, SMRT and HDAC-associated repressor protein; SMRT, silencing mediator for retinoid and TR; SRC, steroid receptor coactivator; TR, thyroid hormone receptor.

Received January 6, 2004.

Accepted for publication February 24, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. 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]
  2. Tugwood JD, Issemann 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[Medline]
  3. McKenna NJ, Lanz RB, Malley BW 1999 Nuclear receptor coregulators: cellular and molecular biology. Endocr Rev 20:321–344[Abstract/Free Full Text]
  4. Robyr D, Wolffe AP, Wahli W 2000 Nuclear hormone receptor coregulators in action: diversity for shared tasks. Mol Endocrinol 14:329–347[Free Full Text]
  5. Xu J, Qiu Y, DeMayo FJ, Tsai SY, Tsai MJ, Malley BW 1998 Partial hormone resistance in mice with disruption of the steroid receptor coactivator-1 (SRC-1) gene. Science 279:1922–1925[Abstract/Free Full Text]
  6. Wang Z, Rose DW, Hermanson O, Liu F, Herman T, Wu W, Szeto D, Gleiberman A, Krones A, Pratt K, Rosenfeld R, Glass CK, Rosenfeld MG 2000 Regulation of somatic growth by the p160 coactivator p/CIP. Proc Natl Acad Sci USA 97:13549–13554[Abstract/Free Full Text]
  7. Xu J, Liao L, Ning G, Yoshida K, Deng C, Malley BW 2000 The steroid receptor coactivator SRC-3 (p/CIP/RAC3/AIB1/ACTR/TRAM-1) is required for normal growth, puberty, female reproductive function, and mammary gland development. Proc Natl Acad Sci USA 97:6379–6384[Abstract/Free Full Text]
  8. White R, Leonardsson G, Rosewell I, Ann J, Milligan S, Parker M 2000 The nuclear receptor co-repressor nrip1 (RIP140) is essential for female fertility. Nat Med 6:1368–1374[CrossRef][Medline]
  9. Gehin M, Mark M, Dennefeld C, Dierich A, Gronemeyer H, Chambon P 2002 The function of TIF2/GRIP1 in mouse reproduction is distinct from those of SRC-1 and p/CIP. Mol Cell Biol 22:5923–5937[Abstract/Free Full Text]
  10. Xu J, Li Q 2003 Review of the in vivo functions of the p160 steroid receptor coactivator family. Mol Endocrinol 17:1681–1692[Abstract/Free Full Text]
  11. 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/Free Full Text]
  12. Kliewer SA, Umesono K, Noonan DJ, Heyman RA, Evans RM 1992 Convergence of 9-cis retinoic acid and peroxisome proliferator signalling pathways through heterodimer formation of their receptors. Nature 358:771–774[CrossRef][Medline]
  13. Zhu Y, Qi C, Jain S, Rao MS, Reddy JK 1997 Isolation and characterization of PBP, a protein that interacts with peroxisome proliferator-activated receptor. J Biol Chem 272:25500–25506[Abstract/Free Full Text]
  14. Lee CH, Chinpaisal C, Wei LN 1998 Cloning and characterization of mouse RIP140, a corepressor for nuclear orphan receptor TR2. Mol Cell Biol 18:6745–6755[Abstract/Free Full Text]
  15. Puigserver P, Wu Z, Park CW, Graves R, Wright M, Spiegelman BM 1998 A cold-inducible coactivator of nuclear receptors linked to adaptive thermogenesis. Cell 92:829–839[CrossRef][Medline]
  16. Treuter E, Albrektsen T, Johansson L, Leers J, Gustafsson JA 1998 A regulatory role for RIP140 in nuclear receptor activation. Mol Endocrinol 12:864–881[Abstract/Free Full Text]
  17. Zhu Y, Kan L, Qi C, Kanwar YS, Yeldandi AV, Rao MS, Reddy JK 2000 Isolation and characterization of peroxisome proliferator-activated receptor (PPAR) interacting protein (PRIP) as a coactivator for PPAR. J Biol Chem 275:13510–13516[Abstract/Free Full Text]
  18. Caira F, Antonson P, Pelto H, Treuter E, Gustafsson JA 2000 Cloning and characterization of RAP250, a novel nuclear receptor coactivator. J Biol Chem 275:5308–5317[Abstract/Free Full Text]
  19. DiRenzo J, Soderstrom M, Kurokawa R, Ogliastro MH, Ricote M, Ingrey S, Horlein A, Rosenfeld MG, Glass CK 1997 Peroxisome proliferator-activated receptors and retinoic acid receptors differentially control the interactions of retinoid X receptor heterodimers with ligands, coactivators, and corepressors. Mol Cell Biol 17:2166–2176[Abstract]
  20. Krogsdam AM, Nielsen CA, Neve S, Holst D, Helledie T, Thomsen B, Bendixen C, Mandrup S, Kristiansen K 2002 Nuclear receptor corepressor-dependent repression of peroxisome-proliferator-activated receptor {delta}-mediated transactivation. Biochem J 363:157–165[CrossRef][Medline]
  21. Shi Y, Hon M, Evans RM 2002 The peroxisome proliferator-activated receptor {delta}, an integrator of transcriptional repression and nuclear receptor signaling. Proc Natl Acad Sci USA 99:2613–2618[Abstract/Free Full Text]
  22. Shaw N, Elholm M, Noy N 2003 Retinoic acid is a high affinity selective ligand for the peroxisome proliferator-activated receptor ß/{delta}. J Biol Chem 278:41589–41592[Abstract/Free Full Text]
  23. Dey SK 1996 Implantation. In: Adashi EY, Rock JA, Rosenwaks Z, eds. Reproductive endocrinology, surgery and technology. New York: Lippincott-Raven; 421–434
  24. Lim H, Gupta RA, Ma WG, Paria BC, Moller DE, Morrow JD, DuBois RN, Trzaskos JM, Dey SK 1999 Cyclo-oxygenase-2-derived prostacyclin mediates embryo implantation in the mouse via PPAR{delta}. Genes Dev 13:1561–1574[Abstract/Free Full Text]
  25. Ding NZ, Teng CB, Ma H, Ni H, Ma XH, Xu LB, Yang ZM 2003 Peroxisome proliferator-activated receptor {delta} expression and regulation in mouse uterus during embryo implantation and decidualization. Mol Reprod Dev 66:218–224[CrossRef][Medline]
  26. Braissant O, Wahli W 1998 Differential expression of peroxisome proliferator-activated receptor-{alpha}, -ß, and -{gamma} during rat embryonic development. Endocrinology 139:2748–2754[Abstract/Free Full Text]
  27. Lim H, Dey SK 2002 A novel pathway of prostacyclin signaling-hanging out with nuclear receptors. Endocrinology 143:3207–3210[Abstract/Free Full Text]
  28. He TC, Chan TA, Vogelstein B, Kinzler KW 1999 PPAR{delta} is an APC-regulated target of nonsteroidal anti-inflammatory drugs. Cell 99:335–345[CrossRef][Medline]
  29. Gupta RA, Tan J, Krause WF, Geraci MW, Willson TM, Dey SK, DuBois RN 2000 Prostacyclin-mediated activation of peroxisome proliferator-activated receptor delta in colorectal cancer. Proc Natl Acad Sci USA 97:13275–13280[Abstract/Free Full Text]
  30. Zhang J, Fu M, Zhu X, Xiao Y, Mou Y, Zheng H, Akinbami MA, Wang Q, Chen YE 2002 Peroxisome proliferator-activated receptor {delta} is upregulated during vascular lesion formation and promotes post-confluent cell proliferation in vascular smooth muscle cells. J Biol Chem 277:11505–11512[Abstract/Free Full Text]
  31. Hatae T, Wada M, Yokoyama C, Shimonishi M, Tanabe T 2001 Prostacyclin-dependent apoptosis mediated by PPAR{delta}. J Biol Chem 276:46260–46267[Abstract/Free Full Text]
  32. Tan NS, Michalik L, Noy N, Yasmin R, Pacot C, Heim M, Fluhmann B, Desvergne B, Wahli W 2001 Critical roles of PPAR ß/{delta} in keratinocyte response to inflammation. Genes Dev 15:3263–3277[Abstract/Free Full Text]
  33. Wang YX, Lee CH, Tiep S, Yu RT, Ham J, Kang H, Evans RM 2003 Peroxisome-proliferator-activated receptor {delta} activates fat metabolism to prevent obesity. Cell 113:159–170[CrossRef][Medline]
  34. Lee CH, Chawla A, Urbiztondo N, Liao D, Boisvert WA, Evans RM 2003 Transcriptional repression of atherogenic inflammation: modulation by PPAR{delta}. Science 302:453–457[Abstract/Free Full Text]
  35. Psychoyos A 1973 Endocrine control of egg implantation. In: Greep RO, Astwood EG, Geiger SR, eds. Handbook of physiology. Washington, DC: American Physiology Society; 187–215
  36. Chakraborty I, Das SK, Wang J, Dey SK 1996 Developmental expression of the cyclo-oxygenase-1 and cyclo-oxygenase-2 genes in the peri-implantation mouse uterus and their differential regulation by the blastocyst and ovarian steroids. J Mol Endocrinol 16:107–122[Abstract]
  37. Miyata KS, McCaw SE, Meertens LM, Patel HV, Rachubinski RA, Capone JP 1998 Receptor-interacting protein 140 interacts with and inhibits transactivation by, peroxisome proliferator-activated receptor alpha and liver-X-receptor {alpha}. Mol Cell Endocrinol 146:69–76[CrossRef][Medline]
  38. Yuan CX, Ito M, Fondell JD, Fu ZY, Roeder RG 1998 The TRAP220 component of a thyroid hormone receptor-associated protein (TRAP) coactivator complex interacts directly with nuclear receptors in a ligand-dependent fashion. Proc Natl Acad Sci USA 95:7939–7944[Abstract/Free Full Text]
  39. Rachez C, Lemon BD, Suldan Z, Bromleigh V, Gamble M, Naar AM, Erdjument B, Tempst P, Freedman LP 1999 Ligand-dependent transcription activation by nuclear receptors requires the DRIP complex. Nature 398:824–828[CrossRef][Medline]
  40. Horlein A, Naar AM, Heinzel T, Torchia J, Gloss B, Kurokawa R, Ryan A, Kamei Y, Soderstrom M, Glass CK, Rosenfeld MG 1995 Ligand-independent repression by the thyroid hormone receptor mediated by a nuclear receptor co-repressor. Nature 377:397–404[CrossRef][Medline]
  41. Dowell P, Ishmael JE, Avram D, Peterson VJ, Nevrivy DJ, Leid M 1999 Identification of nuclear receptor corepressor as a peroxisome proliferator-activated receptor {alpha} interacting protein. J Biol Chem 274:15901–15907[Abstract/Free Full Text]
  42. Chen JD, Evans RM 1995 A transcriptional co-repressor that interacts with nuclear hormone receptors. Nature 377:454–457[CrossRef][Medline]
  43. Gurnell M, Wentworth JM, Agostini M, Adams M, Collingwood TN, Provenzano C, Browne PO, Rajanayagam O, Burris TP, Schwabe JW, Lazar MA, Chatterjee VK 2000 A dominant-negative peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) mutant is a constitutive repressor and inhibits PPAR{gamma}-mediated adipogenesis. J Biol Chem 275:5754–5759[Abstract/Free Full Text]
  44. Xu HE, Stanley TB, Montana VG, Lambert MH, Shearer BG, Cobb JE, McKee DD, Galardi CM, Plunket KD, Nolte RT, Parks DJ, Moore JT, Kliewer SA, Willson TM, Stimmel JB 2002 Structural basis for antagonist-mediated recruitment of nuclear co-repressors by PPAR{alpha}. Nature 415:813–817[Medline]
  45. Seol W, Mahon MJ, Lee YK, Moore DD 1996 Two receptor interacting domains in the nuclear hormone receptor corepressor RIP13/N-CoR. Mol Endocrinol 10:1646–1655[Abstract]
  46. Matsumoto H, Ma WG, Daikoku T, Zhao X, Paria BC, Das SK, Trzaskos JM, Dey SK 2002 Cyclooxygenase-2 differentially directs uterine angiogenesis during implantation in mice. J Biol Chem 277:29260–29267[Abstract/Free Full Text]
  47. Kodera Y, Takeyama K, Murayama A, Suzawa M, Masuhiro Y, Kato S 2000 Ligand type-specific interactions of peroxisome proliferator-activated receptor gamma with transcriptional coactivators. J Biol Chem 275:33201–33204[Abstract/Free Full Text]
  48. Subramaniam N, Treuter E, Okret S 1999 Receptor interacting protein RIP140 inhibits both positive and negative gene regulation by glucocorticoids. J Biol Chem 274:18121–18127[Abstract/Free Full Text]
  49. Farooqui M, Franco PJ, Thompson J, Kagechika H, Chandraratna RA, Banaszak L, Wei LN 2003 Effects of retinoid ligands on RIP140: molecular interaction with retinoid receptors and biological activity. Biochemistry 42:971–979[CrossRef][Medline]
  50. Yao TP, Oh SP, Fuchs M, Zhou ND, Ch n, Newsome D, Bronson RT, Li E, Livingston DM, Eckner R 1998 Gene dosage-dependent embryonic development and proliferation defects in mice lacking the transcriptional integrator p300. Cell 93:361–372[CrossRef][Medline]
  51. Leonardsson G, Jacobs MA, White R, Jeffery R, Poulsom R, Milligan S, Parker M 2002 Embryo transfer experiments and ovarian transplantation identify the ovary as the only site in which nuclear receptor interacting protein 1/RIP140 action is crucial for female fertility. Endocrinology 143:700–707[Abstract/Free Full Text]
  52. Ito M, Yuan CX, Okano HJ, Darnell RB, Roeder RG 2000 Involvement of the TRAP220 component of the TRAP/SMCC coactivator complex in embryonic development and thyroid hormone action. Mol Cell 5:683–693[CrossRef][Medline]
  53. Kuang SQ, Liao L, Zhang H, Pereira FA, Yuan Y, DeMayo FJ, Ko L, Xu J 2002 Deletion of the cancer-amplified coactivator AIB3 results in defective placentation and embryonic lethality. J Biol Chem 277:45356–45360[Abstract/Free Full Text]
  54. Antonson P, Schuster GU, Wang L, Rozell B, Holter E, Flodby P, Treuter E, Holmgren L, Gustafsson JA 2003 Inactivation of the nuclear receptor coactivator RAP250 in mice results in placental vascular dysfunction. Mol Cell Biol 23:1260–1268[Abstract/Free Full Text]
  55. Zhu YJ, Crawford SE, Stellmach V, Dwivedi RS, Rao MS, Gonzalez FJ, Qi C, Reddy JK 2003 Coactivator PRIP, the peroxisome proliferator-activated receptor-interacting protein, is a modulator of placental, cardiac, hepatic, and embryonic development. J Biol Chem 278:1986–1990[Abstract/Free Full Text]
  56. Cavailles V, Dauvois S, Horset F, Lopez G, Hoare S, Kushner PJ, Parker MG 1995 Nuclear factor RIP140 modulates transcriptional activation by the estrogen receptor. EMBO J 14:3741–3751[Medline]
  57. Jepsen K, Hermanson O, Onami TM, Gleiberman AS, Lunyak V, McEvilly RJ, Kurokawa R, Kumar V, Liu F, Seto E, Hedrick SM, Mandel G, Glass CK, Rose DW, Rosenfeld MG 2000 Combinatorial roles of the nuclear receptor corepressor in transcription and development. Cell 102:753–763[CrossRef][Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
145/6/2886    most recent
Author Manuscript (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal