help button home button Endocrine Society Molecular Endocrinology ENDO 08 Sessions Library
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Molecular Endocrinology, doi:10.1210/me.2006-0520
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
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 HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Li, D.
Right arrow Articles by Wang, D.-M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Li, D.
Right arrow Articles by Wang, D.-M.
Molecular Endocrinology 21 (10): 2320-2333
Copyright © 2007 by The Endocrine Society

Constitutive Coactivator of Peroxisome Proliferator-Activated Receptor (PPAR{gamma}), a Novel Coactivator of PPAR{gamma} that Promotes Adipogenesis

Dechun Li, Qiaohua Kang and Dan-Ming Wang

Division of Pulmonary, Critical Care, and Sleep Medicine, Departments of Internal Medicine (D.L., Q.K., D.-M.W.) and Biochemistry and Molecular Biology (D.L.), Saint Louis University, St. Louis, Missouri 63110-0250

Address all correspondence and requests for reprints to: Dechun Li, M.D., Ph.D., Department of Internal Medicine, Division of Pulmonary, Critical Care, and Sleep Medicine, Saint Louis University, Desloge Towers, Seventh Floor, 3635 Vista Avenue, St. Louis, Missouri 63110-0250. E-mail: dli2{at}slu.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Peroxisome proliferator-activated receptor{gamma} (PPAR{gamma}) plays essential roles in adipogenesis by transcriptionally regulating adipocyte-specific genes through recruitment of coregulators including coactivators and corepressors. However, the precise repertoire of coactivators required for PPAR{gamma} transactivation remains unresolved. In this report, we cloned and characterized a novel PPAR{gamma} interacting protein, constitutive coactivator of PPAR{gamma} (CCPG), which is expressed in multiple adult tissues and throughout embryonic development. CCPG is localized in nucleus and contains four LXXLL motifs, which are characteristic for nuclear receptor coactivators. A delineation of CCPG-PPAR{gamma} interaction by glutathione-S-transferase pull-down and coimmunoprecipitation assays indicated that CCPG interacts with the hinge region of PPAR{gamma} in a ligand-independent manner. However, mutation of four motifs of LXXLL to LXXAA in CCPG does not compromise its interaction with PPAR{gamma}, suggesting LXXLL motif is not required for the interaction. Glutathione-S-transferase pull-down assays showed that CCPG binds to retinoic X receptor-{alpha} and estrogen receptor-{alpha} independent of their ligands, but not to thyroid hormone receptor-ß. CCPG coactivates PPAR{gamma} in PPAR response element reporter assays, and the N terminus (amino acids 1–561) of CCPG acts to significantly augment the transactivation of PPAR{gamma}, whereas the C terminus (amino acids 562–786) represses PPAR{gamma} activity, indicating the N terminus possesses the activation domain. Using an adenoviral-mediated system, we also revealed that overexpression of CCPG promoted differentiation of OP9 preadipocyte into adipocyte, and knockdown of CCPG by RNA interference blocked this process, as examined by Oil Red O staining and Western blots of adipocyte-specific protein, adiponectin, and perilipin. Taken together, our data indicate that CCPG is a bona fide coactivator and promotes adipogenesis in a PPAR{gamma}-dependent manner.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
NUCLEAR RECEPTORS (NRs) are a superfamily of transcription factors (TFs) that regulate the expression of target genes in response to steroid hormones and other ligands. The biological effects of these ligands are manifested in processes ranging from organogenesis during embryonic development, to regulation of metabolic pathways, to stimulating proliferation in reproductive tissues, to the process of carcinogenesis, and in the progression of cardiovascular diseases (1, 2, 3, 4, 5, 6). To date, there are 48 NRs reported, and each plays distinct or interrelated functions (4). However, recent studies have demonstrated that NR functions are modulated by a large group of proteins called "coregulators." These coregulators include coactivators that promote transcription and corepressors that attenuate promoter activity when recruited into the promoter regions of specific genes (1, 2, 6, 7, 8, 9, 10, 11, 12, 13). There are now about 200 identified NR coactivators that interact with those 48 NRs to perform multiple diversified functions in response to specific cell signaling or hormonal molecules in different tissues and cells (1, 10, 14, 15). These coactivators play complex biological roles depending upon the spatiotemporal interaction with specific NR and other transcription-related proteins, such as TFs and their interacting proteins (2, 8, 14, 16, 17, 18, 19).

Although NRs exhibit a similar structure module, their coactivators are highly diversified (http://www.nursa.org) (6, 7, 13, 14, 20). Coactivators exert their function in a variety of ways and can be categorized in three classes based on the mechanisms of action: 1) directly modifying histones in ways that allow the more facile access of other proteins to DNA such as cAMP response element binding protein-binding protein/p300; 2) binding to TFs, recruiting RNA polymerase II, and interacting with the general transcription apparatus such as members of the thyroid hormone receptor-associated protein (TRAP)/vitamin D receptor-interacting protein/Mediator/activator-recruited cofactor complex; and 3) containing ATP-dependent DNA unwinding activities such as Brahma-related gene-1 (21). Moreover, methylase activity, RNA processing, and cell cycle regulator functions are also reported (1, 6, 13, 14, 21, 22). It has been confirmed that coactivators contribute to the transcriptional process mainly through a diverse array of enzymatic activities ranging from protein kinase (23), to ubiquitin-conjugating enzyme (24), to nuclease (25), and to acetyltransferase (6, 26, 27, 28). These functions eventually coordinate to fashion a transcriptionally permissive environment in the promoter region of the specific genes (14).

Among NRs, PPAR{gamma} is a decisive TF in adipogenesis, and its coregulators have been shown to modulate this process in different ways (29, 30). Of PPAR{gamma} coactivators identified to date, it is difficult to address which coactivators are essential for adipogenesis due to their functional redundancy. The dissection of the transcriptional regulation network in adipogenic process necessitates identification of the precise coactivator repertoire for PPAR{gamma}. In this study we have cloned and characterized a novel constitutive coactivator of PPAR[gamma] (CCPG) that we termed "CCPG" because it is widely expressed in variety of adult mouse tissues and embryos throughout developmental stages. CCPG interacts with PPAR{gamma} and retinoid X receptor (RXR){alpha} in a ligand-independent manner and enhances the transactivation of PPAR{gamma}. Additionally, we have further demonstrated that CCPG promotes the adipogenic action of PPAR{gamma} in OP9 preadipocytes. The unique structure and expression pattern of CCPG suggest that it may play fundamental roles in the regulation of certain NR functions by forming a scaffold for other TFs or coregulators in the control of gene expression. Furthermore, CCPG-promoted adipogenesis may have critical roles in obesity, and inhibition of this process by disruption of CCPG-PPAR{gamma} interaction may provide new avenues for prevention and therapy of obesity and related diseases.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cloning, Expression, and Subcellular Localization of CCPG
We previously performed a cDNA microarray (Incyte Genomics, 9400 gene elements) on hypoxia-treated mouse lungs (10% O2 for 4 d) and found that the expressed sequence tag W62706 was increased 2.7-fold among 270 up-regulated genes (data not shown) (31). The nucleotide sequence of expressed sequence tag W62706 was used to search the GenBank database and found to be homologous to the transcript (accession no.: NM_024203) encoding an uncharacterized KIAA1838-like protein. Based on the sequence information, we then cloned the full-length cDNA sequence using rapid amplification of cDNA Ends (RACE) (GenBank accession no.: DQ873694). To investigate the function of the protein encoded by this novel cDNA, we performed yeast two-hybrid screening for its potential interaction partner(s). Using this cDNA as bait, the 17-d mouse embryo cDNA (1.4 x 106clones) library was screened and yielded 13 strong positive colonies under high-stringency selection strategy. One of the positive clones encoded the NR PPAR{gamma}. Further study demonstrated that this protein is constitutively expressed in multiple adult mouse tissues and throughout embryonic development. It also enhanced PPAR{gamma} activity and we named it CCPG. Therefore, efforts have been focused on the investigation of the roles that CCPG plays in the interaction, regulation of PPAR{gamma} transactivation, and adipogenesis. Northern blots showed that the transcript of mouse CCPG was approximately 3.0 kb long, encoding a protein of 786 amino acid residues with four characteristic LXXLL (L for leucine and X for any amino acid) motifs. We have identified its human (GenBank accession no.: DQ873695), mouse (DQ873694), rat (XM_218006), dog (XP_855466), and cattle (XP_602628) homolog either with cDNA cloning (for mouse and human) or by searching current available databases and found they were highly conserved (Fig. 1AGo). An analysis of protein sequence alignment indicates that CCPG is composed of an N-terminal conserved XPG-like (xeroderma pigmentosum group G-like) domain containing one LXXLL motif, a central variable hinge region, and a C-terminal conserved domain containing three LXXLL motifs (Fig. 1BGo). A rabbit polyclonal antibody against mouse CCPG was raised using a synthetic peptide (GILGEDTDYLIYDTC) as antigen, and mouse CCPG was found to be expressed as a approximately 95-kDa molecular mass protein in human embryonic kidney (HEK) 293 cells (Fig. 1CGo). Gene structure analysis indicates that CCPG from mouse and human consists of 11 exons and 10 introns, and is localized at chromosome 17(A1) in mice and 6q26–27 in humans. Northern and Western blots showed that CCPG was expressed in all adult mouse tissues examined with the higher expression in testis, brain, spleen, heart, and fat tissues (Fig. 2Go, A and B). A time-course examination also showed that CCPG was expressed throughout the embryonic developmental stages from embryonic d 5 (E5) through E19, as well as in placenta and uterus of pregnant mouse (E13) (Fig. 2Go, C and D). To determine its subcellular localization, CCPG was fused in frame with green fluorescent protein (GFP) (GFP-CCPG), and when expressed in NIH3T3 cells, it totally localized to the nucleus of the cells (Fig. 2EGo).


Figure 1
View larger version (40K):
[in this window]
[in a new window]

  		Fig. 1. Cloning and Alignment of CCPG Sequences

A, Alignment of human, mouse, rat, dog, and cattle CCPG amino acid sequences. Shaded are conserved amino acids among species. LXXLL motifs are underlined. B, Schematic representation of the structure of mouse CCPG. CCPG consists of an N-terminal XPG-like domain (XLD), a central viable hinge region (CVR), and a C-terminal conserved domain (CCD). Putative and characteristic domains are marked with numbers. C, Western blot analysis of recombinant mouse CCPG with anti-CCPG polyclonal antibody. HEK 293 cells were transfected with plasmid pcDNA3-CCPG (lane 1) and pcDNA3 empty vector (lane 2) or without transfection (lane 3). The arrow indicates the recombinant CCPG expressed as an approximately 95-kDa protein.

 

Figure 2
View larger version (18K):
[in this window]
[in a new window]

  		Fig. 2. Expression Profile and Subcellular Localization of CCPG

CCPG protein (A and C) and mRNA (B and D) expression in adult mouse tissues and embryos from E5 through E19 and pregnant mouse placenta and uterus (E13). CTL represents HEK 293 cell lysate expressing the recombinant mouse CCPG. Protein (100 µg) or 25 µg total RNA isolated from various mouse tissues, embryos (E5–E19), placenta and pregnant mouse uterus (E13) were used. Ethidium bromide (EtBr) staining of 18S/28S RNA was used as loading control. E, CCPG is localized in the nucleus. 3T3 cells were transfected with GFP-fused full-length CCPG. Cells were examined and photographed under a fluorescence microscopy (x400) 24 h after transfection. Scale bar, 10 µm. CTL, Control.

 
Characterization of CCPG-PPAR{gamma} and Other NR Interactions
GST Pull-Down Assays Showed that CCPG Interacts with PPAR{gamma}, RXR{alpha}, and ER{alpha}, But Not TRß.
To examine whether CCPG interacts with NRs, Myc-tagged PPAR{gamma}, RXR{alpha}, ER{alpha}, and TRß were expressed in COS7 cells, and the resulting cell lysates were incubated with purified GST or CCPG-GST fusion protein immobilized on glutathione Sepharose 4B beads in the presence of ligand or vehicle as stated in figure legends. As shown in Fig. 3AGo, CCPG bound to PPAR{gamma}, RXR{alpha} and ER{alpha}, but not TRß in a ligand-independent manner.


Figure 3
View larger version (12K):
[in this window]
[in a new window]

  		Fig. 3. CCPG Interacts with PPAR{gamma} and Other NRs in a Ligand-Independent Manner

A, For GST pull-down assays, COS7 cells were transfected with myc-tagged PPAR{gamma}, RXR{alpha}, ER{alpha}, or TRß. The cell lysates were incubated with 20 µg of purified GST or GST-CCPG protein immobilized on glutathione Sepharose beads in the presence (+) or absence (–) of ligand. For PPAR{gamma}, Tro was used at 10 µM; for RXR{alpha}, 9-cis RA was used at 10 µM; for ER{alpha}, 17ß-estradiol was used at 1 µM; and for TRß, T3 was used at 1 µM; 10% of protein was used as input loading for each pull down, except that 5% was used for RXR{alpha}. HRP-conjugated anti-myc antibodies were used to detect the interactions. Please note that the input proteins are 1/10 for PPAR{gamma}, ER{alpha}, and TRß and 1/15 for RXR{alpha} of the pull-down reactions. B, For Co-IP, HCT-116 cells were cotransfected with Flag-tagged PPAR{gamma} plus GFP or GFP-CCPG and treated with 1.0 µM Tro or 0.1% vehicle (DMSO). Cell lysates were incubated with anti-GFP antibody, and PPAR{gamma} was detected by Western blotting with HRP-conjugated anti-Flag antibody. After stripping, the membrane was reprobed with anti-GFP or anti-GAPDH antibody, respectively. WB, Western blot.

 
CCPG Binds to PPAR{gamma} in a Ligand-Independent Manner.
To further examine whether CCPG interacts with PPAR{gamma} in vivo, COS7 cells were cotransfected with Flag-tagged PPAR{gamma} with either GFP or GFP-tagged CCPG expression vector and then coimmunoprecipitated (Co-IP) with anti-GFP antibody. Western blots showed that CCPG interacted with PPAR{gamma} in a ligand-independent manner (Fig. 3BGo).

CCPG Binds to the D Hinge Region of PPAR{gamma}.
To map the domain in PPAR{gamma} that mediates CCPG-PPAR{gamma} interaction, we generated C-terminal myc-tagged PPAR{gamma}-truncated expression constructs with corresponding domain deletions based on a PPAR{gamma} structure module (Fig. 4AGo) (20). Purified GST or GST-CCPG were incubated with COS7-expressed, myc-tagged PPAR{gamma}-truncated protein. Co-IP with an anti-myc antibody was used to detect the interactions between CCPG and PPAR{gamma} truncates. As shown in Fig. 4BGo, full-length PPAR{gamma} and PPAR{gamma} with deletions of amino acids 1–205 still retained the binding ability to CCPG, but the more proximal PPAR{gamma} activation function 1 domain (amino acids 1–138) or PPAR{gamma} with deletion of amino acids 1–281 had shown no ability to bind CCPG, indicating that the D hinge region of PPAR{gamma} spanning amino acids 205–280 is responsible for its interaction with CCPG.


Figure 4
View larger version (13K):
[in this window]
[in a new window]

  		Fig. 4. Characterization of PPAR{gamma}-CCPG Interaction

A, Diagram of different PPAR{gamma} deletions according to a PPAR{gamma} structure module. B, GST pull-down assays were performed using COS7-expressed, myc-tagged, different PPAR{gamma} deletions and purified GST or GST-CCPG immobilized on glutathione Sepharose beads. The input lanes represent 10% of the total volume of the lysate used for the pull downs for each sample. Interactions were detected by Western blot with an anti-myc antibody. C, Diagram of truncated N-terminal (amino acids 1–561) and C-terminal (amino acids 562–786) CCPG tagged with GFP. D, Co-IPs were performed exactly as in Fig. 3BGo. E, Diagram of GFP-tagged CCPG mutants with a series of mutated LXXLL motifs (LXXLL to LXXAA). F, Co-IPs were performed exactly as in Fig. 3BGo. CCD, C-terminal conserved domain; CVR, central viable hinge region; WB, Western blot; XLD, XPG-like domain.

 
CCPG Utilizes Its Multiple Sites to Interact with PPAR{gamma}.
To determine the regions in CCPG that mediate its interaction with PPAR{gamma}, we truncated CCPG in two parts: GFP-tagged CCPG N terminus (amino acids 1–561) and GFP-tagged C terminus (amino acids 562–786), respectively (Fig. 4CGo). Flag-tagged PPAR{gamma} was coexpressed either with GFP, or GFP-tagged CCPG fragments in COS7 cells. Figure 4DGo showed that addition of GFP antibody resulted in Co-IP of Flag-PPAR{gamma} with both GFP-1–561 and GFP-562–786 of CCPG, but not with GFP alone, indicating that there are at least two regions in CCPG located in N terminus and C terminus interacting with PPAR{gamma}.

The LXXLL Motifs in CCPG Are Not Required for Its Interaction with PPAR{gamma}.
The LXXLL motif has been thought to mediate ligand-dependent recruitment of p160-type of coactivator to NR (32, 33). Notably, CCPG has four characteristic LXXLL motifs: one in the N terminus and three in the C terminus. We subsequently examined whether the presence of LXXLL motifs in CCPG contributes to CCPG-PPAR{gamma} interaction. We generated GFP-tagged CCPG mutants with a series of mutated LXXLL motifs (LXXLL to LXXAA) (Fig. 4EGo) and used Co-IP assay to analyze their interactions with PPAR{gamma}. As shown in Fig. 4FGo, even CCPG mutated with all four LXXLL motifs did not compromise its binding to PPAR{gamma}. Thus, our data support that LXXLL motifs of CCPG are not required for its interaction with PPAR{gamma}, indicating the presence of novel structural motif(s) in CCPG responsible for its interaction with PPAR{gamma}.

CCPG Enhances Transactivation of PPAR{gamma}, and N Terminus of CCPG Possesses Activation Domain
Knowing that CCPG interacts with PPAR{gamma}, we next asked whether CCPG transactivates PPAR{gamma}. CCPG and PPAR{gamma} were coexpressed in HCT-116 cells with a consensus PPAR{gamma}-responsive luciferase reporter construct peroxisome proliferator response element (PPRE)-TK-LUC (34, 35). A previously identified PPAR{gamma} coactivator (PGC)-1{alpha} was used as a positive control (36). As indicated in Fig. 5AGo, CCPG alone did not activate the transcription of PPRE-TK-LUC, while in the presence of PPAR{gamma} and its ligand troglitazone (Tro) (1.0 µM), CCPG significantly increased the transactivation of PPAR{gamma} in HCT-116 cells in a degree similar to PGC-1{alpha}. Interestingly, a moderate synergistic effect on the transactivation of PPAR{gamma} was observed when CCPG was cotransfected with PGC-1{alpha}. However, there was no direct interaction between CCPG and PGC-1{alpha} in Co-IP assay (data not shown).


Figure 5
View larger version (17K):
[in this window]
[in a new window]

  		Fig. 5. CCPG Coactivates PPAR{gamma} and ER{alpha}, and N Terminus of CCPG Possesses an Activation Domain

A, HCT-116 cells were cotransfected with 300 ng of PPRE-TK-LUC, 50 ng of PPAR{gamma}, 50 ng of CCPG, and 25 ng of a previously identified PPAR coactivator PGC-1{alpha} expression plasmids with Tro (1.0 µM) or 0.1% vehicle (DMSO). B, HCT-116 cells were cotransfected with 300 ng of PPRE-TK-LUC, 50 ng of PPAR{gamma}, either 50 ng of CCPG, CCPG 1–561, or CCPG 562–786 expression plasmids with Tro (1.0 µM) or 0.1% vehicle (DMSO). C, HCT-116 cells were cotransfected with 150 ng of PPRE-TK-LUC, 50 ng of PPAR{gamma}, 50 ng of CCPG expression plasmids with increasing amount (5, 25, 100 ng) of CCPG 1–561, or CCPG 562–786 expression plasmid plus Tro (1.0 µM) or 0.1% vehicle (DMSO). Empty pCDNA3.1 plasmid was added to ensure equal DNA amount used in each transfection. D, COS7 cells were cotransfected with the reporter plasmid pG5luc (Promega) containing five copies of the upstream-activated sequence (UAS) linked to luciferase and plasmid expressing GAL4 DBD-fused CCPG or PGC-1{alpha} shown as a positive control. E, HCT-116 cells were cotransfected with 300 ng of a native acyl-CoA oxidase PPRE reporter plasmid, 50 ng of PPAR{gamma}, and 50 ng of CCPG or empty vector plasmids supplemented with Tro (1.0 µM). F, MCF-7 cells were cotransfected with 300 ng of ER reporter pERE-TK-LUC and 25 ng of CCPG or empty vector plasmid in the presence of 17ß-estradiol (100 nM) or vehicle (ethanol). All the firefly luciferase and Renilla luciferase activities were determined 18–24 h after transfection. Data were normalized to Renilla luciferase activity derived from the internal control plasmid phR-TK. Values were expressed relative to activation of empty vector, and each value was derived from at least three independent experiments. Statistical analysis was done vs. the value from PPAR{gamma} alone in the presence of Tro using Student’s t test. *, P < 0.05. DBD, DNA-binding domain; E2, 17ß-estradiol.

 
Given that N-terminal and C-terminal regions of CCPG are able to bind to PPAR{gamma}, we subsequently dissected which region of CCPG mediated PPAR{gamma} transactivation. To avoid the interference of GFP in transactivation reporter assays, we subcloned N-terminal CCPG (amino acids 1–561) and C-terminal CCPG (amino acids 562–786) into pcDNA3.1 expression vector. As shown in Fig. 5BGo, when cotransfected with equal amount of CCPG expression constructs, the N terminus (amino acids 1–561) had slightly higher ability to activate PPAR{gamma}, as compared with full-length CCPG. In contrast, C terminus (amino acids 562–786) had no ability to coactivate PPAR{gamma} and instead showed a suppressive effect on PPAR{gamma} activation. To evaluate whether either of these CCPG truncates suppresses wild-type CCPG activity, full-length CCPG was cotransfected with increasing quantities of either its N terminus (amino acids 1–561) or C terminus (amino acids 562–786) of CCPG expression constructs. Coexpression of CCPG and its N terminus (amino acids 1–561) generated synergistic effects on PPAR{gamma} transactivation in a dose-dependent manner, whereas coexpression of CCPG and its C terminus (562–786) resulted in a dose-dependent inhibition of PPAR{gamma} activation, indicating that the N terminus of CCPG may have an activation domain (Fig. 5CGo). To test whether CCPG bears intrinsic transcriptional activity like many coactivators such as PGC-1{alpha} (37), CCPG was fused to GAL4 DNA binding domain (GAL4 DBD). Transcriptional activity was analyzed through a luciferase reporter containing five GAL binding sites. As shown in Fig. 5DGo, CCPG fused to GAL4 DBD did not activate transcription whereas a strong transcriptional activity was seen with GAL4 DBD-fused PGC-1{alpha}. Thus, CCPG does not possess intrinsic transcriptional activity. Furthermore, cotransfection assay showed that CCPG coactivated PPAR{gamma} transactivation (Fig. 5EGo) when using a native acyl-coenzyme A (CoA) oxidase PPRE luciferase reporter (38). In addition, estrogen response element-reporter assay also demonstrated that CCPG moderately coactivates endogenous ER{alpha} in MCF-7 cells (Fig. 5FGo) in a ligand-dependent manner.

CCPG Promotes Adipogenesis of OP9 Preadipocytes
Given that CCPG augments the transactivation of PPAR{gamma}, we next investigated whether CCPG promotes adipogenesis of OP9 preadipocytes, a new model for adipogenesis (39). We initially examined the expression profiles of CCPG and PPAR{gamma} in differentiating OP9 cells. OP9 preadipocytes were stimulated to enter an adipocyte differentiation process with adipogenic mix cocktail (39) and subjected to time-course sampling. As shown by Northern and Western blots, CCPG mRNA transcription is up-regulated in OP9 cells starting from d 2 and peaking at d 4 during adipogenesis, whereas PPAR{gamma} mRNA transcription reached peak on d 2 and then declined (Fig. 6AGo). The protein changes of CCPG had a similar pattern as its mRNA and PPAR{gamma} were increased until d 6 (Fig. 6BGo). To clarify whether CCPG interacts with endogenous PPAR{gamma} in differentiating OP9 cells, GFP and GFP-tagged CCPG were constructed in an adenovirus expression vector, and the resulting viruses (designated as Ad-GFP or Ad-GFP-CCPG) were used to transduce OP9 preadipocytes subjected to adipocyte differentiation with stimulation of adipogenic mix cocktail. On the adipocyte differentiating d 2, OP9 cells were collected and subjected to Co-IP assay. As shown in Fig. 6CGo, CCPG interacts with endogenous PPAR{gamma} in differentiating OP9 cells. We further investigated whether CCPG promotes the adipogenesis of OP9 preadipocytes. OP9 preadipocytes were transduced with virus Ad-GFP or Ad-GFP-CCPG and then subjected to adipocyte differentiation. To avoid the overwhelming effects of adipogenesis induced by high concentrations of cAMP and insulin (39), which may overshadow the effects of CCPG, low concentration of exogenous PPAR agonists (Tro, 0.5 µM) and other adipogenic agents such as dexamethasone (0.1 µM) and 3-isobutyl-1-methylxanthine (50 µM) were added to OP9 cell culture medium. As shown in Fig. 7GoA, OP9 preadipocytes transduced with Ad-GFP have very few cells (< 5%) and showed morphological differentiation toward adipocytes, whereas OP9 preadipocytes transduced with Ad-GFP-CCPG had a marked morphological differentiation into adipocytes at d 4 (>90%, ascertained by Oil Red O staining for lipid deposition). This result was further validated by immunodetection of adipocyte-specific marker adiponectin and perilipin (Fig. 7BGo). To evaluate the roles of endogenous CCPG in adipogenesis, CCPG transcripts were knocked down by adenovirus-delivered RNA interference (RNAi). OP9 preadipocytes were subjected to CCPG RNAi or control LacZ RNAi and adipogenesis with adipogenic mix cocktail for 4 d. Real-time RT-PCR showed that CCPG mRNA was significantly reduced in CCPG RNAi-treated cells (Fig. 7CGo). Meanwhile, the adipocyte differentiation of OP9 cells was remarkably compromised in CCPG RNAi1 or CCPG RNAi2-treated OP9 cells, but not in LacZ RNAi and CCPG RNAi2 mutation (CCPG RNAi2M)-treated OP9 cells as examined by Oil Red O staining (Fig. 7DGo) and immunodetection of adipocyte-specific marker adiponectin and perilipin (Fig. 7EGo). Taken together, our data indicate that CCPG is a bona fide coactivator and promotes the adipogenic action of PPAR{gamma}.


Figure 6
View larger version (17K):
[in this window]
[in a new window]

  		Fig. 6. CCPG Expression Is Up-Regulated, and CCPG Interacts with Endogenous PPAR{gamma} in Differentiating OP9 Cells during Adipogenesis

A, Total RNAs (10 µg) were isolated on different dates from OP9 cells subjected to adipocyte differentiation induced by adipogenic mix cocktail and hybridized with DIG-labeled CCPG probe. After stripping, membrane was rehybridized with DIG-labeled PPAR{gamma}2 probe. Ethidium bromide (EtBr)-stained 28S RNA was used as loading control. B, Western blot of CCPG and PPAR{gamma} during adipogenesis as in panel A. C, OP9 preadipocytes were subjected to an adipogenic mix cocktail stimulation, simultaneously infected with adenovirus expressing GFP or GFP-CCPG fusion proteins. On differentiation d 2, cells were lysed for Co-IP assay. Anti-GFP monoclonal antibody was used to coimmunopreciptiate GFP and GFP fusion protein, and anti-PPAR{gamma} antibody was used to detect the interaction. WB, Western blot.

 

Figure 7
View larger version (67K):
[in this window]
[in a new window]

  		Fig. 7. CCPG Promotes Adipogenesis of OP9 Preadipocytes

A, OP9 preadipocytes were transduced with an adenovirus expressing either GFP (Ad-GFP) or CCPG fused to GFP (Ad-GFP-CCPG). The resulting cells were subjected to a standard differentiation for 4 d with one tenth concentration of the standard adipogenic mix cocktail in the media. Cells were fixed and stained with Oil Red O for microscopic examination and photographed at x400 magnification. There are many positive cells (red) in OP9 cells treated with Ad-GFP-CCPG. B, Western blots for adiponectin and perilipin protein in OP9 cells treated with either Ad-GFP or AD-GFP-CCPG for 4 d. C, In another set of experiments, total RNAs were extracted from the cells and subjected to real-time RT-PCR analysis. Adenoviral-CCPG RNAi was used to knock down CCPG transcripts, and LacZ RNAi was used as control. *, P < 0.05 (t test) compared with the LacZ RNAi group. D, Oil Red O staining of OP9 cells treated with only adipogenic mix cocktail [Control (CTL)], Ad-LacZ RNAi or Ad-CCPG RNAi1, Ad-CCPG RNAi2, and Ad-CCPG RNAi2 mutation (RNAi2M) for 4 d. E, Western blot analyses for CCPG, adiponectin, perilipin, and GAPDH in OP9 cells treated for 4 d. Scale bars, 30 µm.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The major finding of this study is that we have identified and characterized a novel PPAR{gamma} coactivator CCPG, and explored its role in promoting PPAR{gamma}-directed adipogenesis. The interaction between CCPG and PPAR{gamma} is independent of PPAR{gamma} ligand. However, the ability of CCPG in PPAR{gamma} transactivation is similar to that of PGC-1{alpha} but in a ligand-dependent manner. These data support the notion that CCPG may enhance PPAR{gamma} function by synergizing ligand-stimulated PPAR{gamma} transactivation even though the CCPG-PPAR{gamma} interaction is not influenced by ligand or by recruiting other transcription-related proteins that promote PPAR{gamma} activity. Nevertheless, the exact mechanism of CCPG-mediated PPAR{gamma} coactivation is not clear. Further studies are needed to clarify this issue. Moreover, the ubiquitous expression pattern in adult and embryonic tissues, and unique structural characteristics of CCPG, strongly suggest that CCPG may play fundamental roles not only in the modulation of PPAR{gamma} activity during the processes of adipogenesis, but also in other biological functions related to PPAR{gamma} transactivation.

PPAR{gamma} has an overall NR structure, heterodimerizes with RXR{alpha}, and then binds to specific peroxisome proliferator response elements (PPREs) to initiate gene transcription. This event requires binding of PPAR{gamma} to a ligand and recruitment of transcriptional coactivators (20). Due to structural variations of coactivators, their interactions with PPAR{gamma} are in divergent ways as well. Most coactivators identified to date interact with PPAR{gamma} through its C-terminal AF-2 domain or ligand-binding domain (LBD) in a ligand-dependent or ligand-enhanced manner, and these interactions are largely mediated by the signature motif, LXXLL (33), as seen with coactivators steroid receptor coactivator (SRC)-1/nuclear receptor coactivator 1, cAMP response element binding protein-binding protein/p300, pCAF, and TRAP220, which interact with the LBD of PPAR{gamma} (13, 30, 40, 41). However, other coactivators, such as PGC-1{alpha}, which interacts with PPAR{gamma} through part of PPAR{gamma} DNA-binding and hinge domains (36), and PGC-2, which binds to PPAR{gamma} A/B domain, demonstrate ligand and LXXLL motif independency. Many NR coactivators, such as members of the p160/SRC and PGC-1 family, are highly versatile. They are expressed in a variety of tissues and coactivate a wide spectrum of NRs including PPAR{gamma} (7, 36, 42). For example, PGC-1{alpha} is also a coactivator for estrogen receptor (ER) {alpha}. Multiple sites in PGC-1{alpha} govern its interaction with ER{alpha}, but the presence of an LXXLL motif is required for PGC-1{alpha}’s ligand-dependent binding to ER{alpha} (43). Therefore, whether ligand is required for the NR-coactivator interaction depends mainly upon whether the LBD of NR is involved in the interaction. Interestingly, CCPG has multiple sites in contacting with PPAR{gamma}, and mutation of all four LXXLL motifs of CCPG did not remarkably compromise its interaction with PPAR{gamma}, implying novel structural characteristics present in CCPG. Although CCPG is ubiquitously expressed and interacts with PPAR{gamma} D hinge region, and PGC-1{alpha} interacts with part of PPAR{gamma} DNA binding and D hinge region (36), we did not observe a direct interaction between CCPG and PGC-1{alpha} by Co-IP (data not show). Nonetheless, a synergistic effect was seen in in vitro PPRE luciferase reporter assay when CCPG and PGC-1{alpha} were coexpressed. This effect can be reconciled because both CCPG and PGC-1{alpha} are coactivators for PPAR{gamma}. The sequential assembling of transcription machinery is a complex process involving participation of many TFs, coactivators, and corepressors that play different roles. PGC-1{alpha} normally is expressed in brown fat, heart, kidney, and brain, but not in white fat (36). Brown fat develops earlier than white fat and is mainly involved in control of adaptive thermogenesis (44). Loss of PGC-1{alpha} does not alter brown fat differentiation but severely reduces the induction of thermogenic genes (45). PGC-1{alpha} plays a key role in transcriptional regulation of adaptive thermogenesis (36, 46). The different expression profiles of CCPG and PGC-1{alpha} also reflect their distinct roles in adipogenesis. The exact amino acid determinants responsible for PPAR{gamma}-CCPG interaction and PPAR{gamma}-PGC-1{alpha} interaction deserve an in-depth investigation. We further found that CCPG interacts with RXR{alpha} and ER{alpha}, but not with TRß, suggesting CCPG may act as a selective coactivator for certain NRs. Furthermore, the interaction between CCPG and RXR{alpha} is also intriguing. Because formation of heterodimer with RXR is a prerequisite in PPAR, vitamin D receptor, and thyroid receptor (TR)-directed gene expression, dissection of CCPG and RXR interaction will assess whether CCPG modulates transactivation of these RXR-heterodimerized NRs (29, 47).

Notably, a structure prediction reveals a Xeroderma pigmentosum G-like (XPG like) domain containing a helix-hairpin-helix (HhH)2 motif located in the CCPG N terminus. This characteristic domain is observed in DNA repair enzymes and in DNA polymerases (48). The HhH2 motif is capable of binding to single-stranded DNA (49) and plays a role in segregating achiasmate chromosomes during meiosis (50), probably facilitating formation of an opening transcriptional complex in the promoter region. The highest expression levels of CCPG in testis may be related to this process. We speculate that upon unwinding double-stranded DNA in the promoter region, CCPG may bind to single-stranded DNA with its HhH2 motif and interacts with the hinge region of PPAR{gamma} to accelerate transcription. However, more detailed studies are warranted to answer whether CCPG bears nuclease activity and plays a role in DNA repair and replication.

Adipogenesis is a multistage process and highlights spatiotemporal expression of a set of TFs that initiate the transcription of preadipocyte- or adipocyte-specific genes. This transcriptional cascade process includes expression of key adipogenesis regulators, such as PPARs, CCAAT/enhancer binding protein, and the basic helix-loop-helix family of TFs such as ADD1/SREBP1c (51). These TFs activate genes encoding enzymes involved in lipid storage and transport such as adipocyte-specific fatty acid binding protein (aP2) and perilipin, and genes encoding secreted proteins or adipokines that modulate preadipocyte and adipocyte functions such as adiponectin and leptin. In adipocytes, PPAR{gamma} regulates the expression of numerous genes involved in lipid synthesis, storage, and transportation (29). For example, PPAR{gamma} is essential to activate the promoters of acyl-CoA oxidase and aP2 and many other fat cell-specific genes (52, 53). Coactivators have been shown to play critical roles in NR-directed adipogenesis programs. Direct evidence in the reflection of the importance of coactivators in metabolism and adipogenesis comes from several recent reports (30, 36, 46, 54, 55, 56, 57). PGC-1{alpha} has been shown to determine the fat cell differentiation to brown or white adipose tissues (30, 58, 59, 60). Activation of PGC-1{alpha} can convert white adipocytes into brown fat cells, which changes a cell function to store energy into a thermogenic cell that dissipates energy (36). The other PGC-1 family member, PGC-2, also plays important roles in adipogenesis. Ectopic expression of PGC-2 in preadipocytes containing endogenous PPAR{gamma} causes a dramatic increase in fat cell differentiation at both the morphological and molecular levels. In 3T3-F442A cells, PGC-2 expression induced PPAR{gamma} and aP2 up-regulation. Similar functions of the coactivators also have been reported in TRAP220 and PPAR-interacting protein in PPAR{gamma}-dependent adipogenesis (54, 55). Furthermore, Wang and co-workers have demonstrated that there is a developmental arrest in mice lacking both the p160 family transcriptional coactivators SRC-1 and p300/CBP-interacting protein due to a failure in induction of selective PPAR{gamma} target genes involved in adipogenesis and mitochondrial uncoupling (57). The p300/CBP-interacting protein and SRC-1 double-knockout mice eat more food on both regular chow and a high-fat diet because of decreased blood leptin levels but they are lean and resistant to high-fat diet-induced obesity. All this evidence strongly suggests that coactivators are critical elements in PPAR{gamma} transactivation, in controlling adipocyte-specific gene expression, and in the promotion of preadipocyte differentiation toward adipocytes. PPAR{gamma} is the utmost important TF in the determination of adipogenesis, and its coregulators have been shown to modulate this process through different ways. Further characterization of CCPG-PPAR{gamma} interaction will enrich our knowledge in the understanding of NR-coactivator associations in gene expression regulation during adipogenesis (20, 29, 30, 61). Although 3T3-L1 is a classic model cell line and has been used in many studies for adipogenesis, it bears several limitations such as loss of potential to differentiate into adipocyte at high density or after high passage numbers. Because 3T3-L1 is derived from fibroblast, it is also refractory to transduction by adenovirus. Instead, OP9 cell line is a newly characterized model for adipogenesis study and circumvents these disadvantages (39). After hormonal stimulation, it can be induced to rapidly undergo adipogenesis and demonstrates all adipocytic characteristics. Moreover, OP9 cells are also easily transduced by adenovirus-mediated gene expression system (Li, D., and D. Wang, unpublished data) and undergo marked morphological and molecular changes toward adipocytes. Considering those advantages, we decided to use OP9 cell line in our system.

The CCPG truncation studies have shown that the N terminus of CCPG (amino acids 1–561) possesses an activating function for PPAR{gamma}, and its C terminus acts as an inhibitor of PPAR{gamma} activation, indicating that these two regions are necessary for the execution of integrated CCPG biological activities. By our study, we explored the modulation of CCPG on PPAR{gamma} transactivation and adipogenesis. It is also intriguing to ask whether CCPG interacts with other key TFs involved in adipogenesis such as CCAAT/enhancer binding proteins and ADD1/SREBP1c.

In summary, we report here that CCPG, which not only interacts with PPAR{gamma} but also with RXR{alpha} and ER{alpha}, is a bona fide coactivator for PPAR{gamma} that promotes adipogenesis. Its mechanism of action on PPAR{gamma}, and possibly other NRs, in the modulation of gene expression warrants further investigation. Moreover, CCPG-promoted adipogenesis may have certain roles in obesity, and disruption of CCPG-PPAR{gamma} interaction may provide a new method for the prevention and therapy of obesity and related disorders in the future.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Cells, Plasmid, Antibodies, and Animals
HCT-116, COS7, and NIH3T3 cells were cultured in medium recommended by American Type Culture Collection (ATCC, Manassas, VA). On the day of transfection, the medium was switched to medium containing 10% charcoal/dextran-treated fetal bovine serum (Hyclone Laboratories, Inc., Logan, UT). PPAR reporter plasmid PPRE-TK-LUC, RXR{alpha}, and PPAR{gamma} expression constructs were described previously (34, 35) and were used as PCR templates. PGC-1{alpha} expression plasmid was from Addgene (Cambridge, MA) (36). The CCPG mutant (LXXLL to LXXAA) expression constructs were generated using GeneTailor site-directed mutagenesis kit (Invitrogen, Carlsbad, CA). All other expression constructs were generated by PCR cloning and validated by DNA sequencing. A rabbit anti-CCPG antibody was generated using synthesized peptide GILGEDTDYLIYDTC as antigen (Lampire Biological Laboratories, PA). Adiponectin and perilipin antibodies were from Sigma Chemical Co. (St. Louis, MO). All other antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). All protocols and procedures were approved by the Animal Care and Use Committee of Saint Louis University and followed the NIH Guide for the Care and Use of Laboratory Animals. Mouse tissues were removed and collected from male (8 wk old) or female pregnant C57BL/6 mice (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) at the specified time points after halothane overdose inhalation.

RACE and cDNA Cloning of CCPG
EST W62706 was used to search the GenBank database and homologous to the transcript (accession no.: NM_024203), from which RACE PCRs were performed. Briefly, for 5'-RACE, first-strand cDNA was synthesized by using CCPG gene-specific primer 5'-GAG ACA GCA CAT GGC GTC CAC CAC G-3' and adult mouse lung total RNA as template. An oligo-dT anchor primer 5'-GAC CAC GCG TAT CGA TGT CGA CTT TTT TTT TTT TTT TT(A/C/G)-3' and a nested CCPG gene-specific primer 5'-GGT GAC GCT CTG CCA GCT CGT GG-3' were used to amplify the 5'-end of CCPG cDNA. For 3'-RACE, first-strand cDNA was synthesized by using above oligo-dT anchor primer. The 3'-end of CCPG cDNA was amplified by using the primer 5'-GAC CAC GCG TAT CGA TGT C-3' and the CCPG-specific primer 5'-CGT AGG CAG AAC GCA TTG GGA CTC-3'.

Yeast Two-Hybrid Screening
The yeast two-hybrid screen was performed following the Matchmaker GAL4 Two-Hybrid System 3 kit (CLONTECH Laboratories, Inc.) protocol. Briefly, the CCPG cDNA was inserted into the GAL4 DNA-binding domain of pGBKT7 bait plasmid (pGBKT7-CCPG) and used to screen a mouse 17-d embryo cDNA library constructed in the pACT2 plasmid expressing the GAL4 activation domain. pGBKT7-CCPG was transformed into yeast AH109 and maintained in tryptophan-Dropout medium (AH109-CCPG). The mouse 17-d embryo cDNA library was transformed into yeast AH109-CCPG, and positive colonies appeared in blue on X-{alpha}-Gal-containing medium due to secretion of {alpha}-galactosidase.

Northern and Western Blot Analyses
Total RNA (25 µg) isolated from mouse tissues or embryos was separated on 1% formaldehyde agarose gels and transferred to nitrocellulose membranes. The 1.5-kb digoxigenin (DIG)-labeled CCPG cDNA probe was hybridized with the membranes overnight. The membrane was visualized using a horseradish peroxidase (HRP)-conjugated anti-DIG antibody (Roche, Indianapolis, IN). Western blots were performed according to the methods reported previously (62).

Transient Transfection and Reporter Assay
HCT-116 cells were cotransfected with plasmids as indicated. After 5 h incubation, cells were treated with appropriate medium supplemented with 10% charcoal/dextran-treated fetal bovine serum (Hyclone) and 1.0 µM Tro or 0.1% vehicle [dimethylsulfoxide (DMSO)]. The luciferase activity was measured 16–20 h after transfection, and the transfection efficiency was normalized by dividing the firefly luciferase activity by the Renilla luciferase activity according to the Dual-Luciferase Reporter Assay kit manual (Promega Corp., Madison, WI).

GST Pull-Down Assay
GST or GST-CCPG fusion protein (subcloned into the pGEX-4T-2 vector) was expressed in Escherichia coli. BL21 and purified by glutathione Sepharose 4B affinity chromatography (Amersham Pharmacia Biotech, Piscataway, NJ). COS7 cells were transfected with appropriately tagged expression constructs or control plasmid DNA. Cell lysates were incubated in binding buffer (50 mM HEPES, pH 7.5; 120 mM NaCl; 1 mM EDTA; 1 mM dithiothreitol; 0.5% Nonidet P-40; 10% glycerol) plus a protein inhibitor cocktail (Roche) with approximately 20 µg of purified GST or GST-CCPG fusion protein immobilized on glutathione-conjugated Sepharose 4B beads in the presence of ligand or vehicle (10 µM Tro and 10 µM 9-cis retinoid acid) for at least 3 h, and washed with binding buffer four times. Beads were boiled in protein 1x SDS-PAGE loading buffer, and the supernatants were analyzed by Western blot.

Co-IP
COS7 cells were cotransfected with plasmid vectors expressing Flag-tagged PPAR{gamma} plus plasmid vector expressing GFP or various GFP-tagged proteins. For those ligand treatment assays, 16 h after transfection, cells were treated with 1.0 µM Tro or vehicle (0.1% DMSO) for additional 2 h before harvesting. Cells were then washed with PBS twice and suspended in ice-cold IP binding buffer. For each IP assay, anti-GFP antibody was used to Co-IP GFP or GFP fusion proteins in the presence or absence of NR ligands (10 µM Tro for PPAR{gamma} or 10 µM 9-cis retinoid acid for RXR) for 1 h and then incubated with 50 µl of Protein A/G Plus agarose beads (Santa Cruz) for 3 h at 4 C. Beads were washed with washing buffer four times at 4 C. Coprecipitated proteins were released by boiling the beads in 1x SDS-PAGE loading buffer and analyzed by Western blot.

Real-Time RT-PCR
Total RNAs were isolated using Trizol reagent (Invitrogen) and reverse-transcribed into First-Stranded cDNAs (Roche). Real-time RT-PCR of CCPG expression was performed using the SYBR Green PCR Master Mix and ABI Prism 7700 Sequence Detector (Applied Biosystems, Foster City, CA) with forward primer 5'-GAA GCA CTC ATG TGT ACA CAC CCTG-3' and reverse primer 5'-CCA CTC CTT GAC CAC TGG GCC AG-3'. The value of each sample was normalized to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

Adenovirus Preparation
cDNA encoding GFP or GFP-CCPG was inserted into the pAd/CMV-V5-DEST Gateway adenoviral vector, and the adenoviruses of Ad-GFP and Ad-CCPG were prepared and titrated in 293A cells according to the manufacturer’s instructions (Invitrogen, Grand Island, NY). The adenovirus-delivered small hairpin RNAi was used to knock down target gene expression. Briefly, oligonucleotides representing CCPG or LacZ used as negative control (provided by Invitrogen) were annealed, cloned into pENTR/H1/TO vector (Invitrogen), and then subcloned into adenoviral vector pAd/BLOCK-iT-DEST (Invitrogen) according to the manufacturer’s instructions. The sense strands of the CCPG RNAi and its mutant sequence were as follows (including linkers): CCPG RNAi1 5'-CAC CGC AAA TGG TGA GTT TAA ATC CCG AAG GAT TTA AAC TCA CCA TTT GC-3, and CCPG RNAi2 5'-CAC CGC CCA CAC ATA TGT ACC ATA GCG AAC TAT GGT ACA TAT GTG TGG GC-3'. For the CCPG RNAi control, the mutated sequence from CCPG RNAi2 was 5'-CAC CGC TTA GAG ATA ACA ACC ATA GCG AAC TAT GGT TGT TAT CTC TAA GC-3', which has been shown no significant similarity found in BLAST search.

Induction of Adipogenesis
OP9 cells were cultured and the induction of adipogenesis followed the previously reported protocols (39). Briefly, 2 d after reached confluency (d 0), OP9 preadipocytes were transduced with adenoviruses Ad-GFP or Ad-GFP-CCPG (5 multiplicity of infection/cell) and cultured in MEM-{alpha} plus 10% BSA supplemented with or without adipogenic mix cocktail for further culture for up to 8 d. The culture media were refreshed every 2 d. At the specified time point, the morphological changes of the cells were photographed, and the cells were collected either for real-time RT-PCR or Northern and Western blot analyses. In addition, Oil Red O staining was performed for the accumulated lipids in the adipocytes (30).


   ACKNOWLEDGMENTS
 
We thank Drs. Janardan Reddy and Yijun Zhu, Northwestern University (Chicago, IL), for providing the PPRE-TK LUC, PPAR{gamma}, and RXR expression plasmids and Dr. Perry E. Bickel, Department of Medicine, Washington University (St. Louis, MO), for providing OP9 cells.

These sequence data have been submitted to the GenBank database under accession nos. DQ873694 (mouse) and DQ873695 (human).


   FOOTNOTES
 
This work is supported by National Institutes of Health Grant RO1HL075755 (to D.L.).

Disclosure Statement: The authors have nothing to disclose.

First Published Online June 26, 2007

Abbreviations: CCPG, Constitutive coactivator of PPAR{gamma}; CoA, coenzyme A; Co-IP, coimmunoprecipitation/coimmunoprecipitated; DIG, digoxigenin; DMSO, dimethylsulfonylfluoride; E5, embryonic d 5; ER, estrogen receptor; GAL4 DBD, GAL4 DNA binding domain; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; GST, glutathione-S-transferase; HEK, human embryonic kidney; HhH, helix-hairpin-helix; HRP, horseradish peroxidase; LBD, ligand-binding domain; NR, nuclear receptor; PGC-1: PPAR{gamma} coactivator-1; PPAR, peroxisome proliferator-activated receptor; PPRE, peroxisome proliferator response elements; RACE, rapid amplication of cDNA ends; RNAi, RNA interference; RXR: retinoid X receptor; SRC, steroid receptor coactivator; TF, transcription factor; TR, thyroid receptor; TRAP, thyroid hormone receptor-associated protein; Tro, troglitazone; XPG, xeroderma pigmentosum group G.

Received for publication December 4, 2006. Accepted for publication June 20, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. McKenna NJ, O’Malley BW 2002 Minireview: nuclear receptor coactivators—an update. Endocrinology 143:2461–2465[Abstract/Free Full Text]
  2. Glass CK 2006 Going nuclear in metabolic and cardiovascular disease. J Clin Invest 116:556–560[CrossRef][Medline]
  3. Novac N, Heinzel T 2004 Nuclear receptors: overview and classification. Curr Drug Targets Inflamm Allergy 3:335–346[CrossRef][Medline]
  4. Ruan XZ, Varghese Z, Powis SH, Moorehead JF 2005 Nuclear receptors and their coregulators in kidney. Kidney Int 68:2444–2461[CrossRef][Medline]
  5. Hager GL, Nagaich AK, Johnson TA, Walker DA, John S 2004 Dynamics of nuclear receptor movement and transcription. Biochim Biophys Acta 1677:46–51[Medline]
  6. Aranda A, Pascual A 2001 Nuclear hormone receptors and gene expression. Physiol Rev 81:1269–1304[Abstract/Free Full Text]
  7. McKenna NJ, O’Malley BW 2002 Combinatorial control of gene expression by nuclear receptor and coregulators. Cell 108:465–474[CrossRef][Medline]
  8. Smith CL, O’Malley BW 2004 Coregulator function: a key to understanding tissue specificity of selective receptor modulators. Endocr Rev 25:45–71[Abstract/Free Full Text]
  9. Darimont BD, Wagner RL, Apriletti JW, Stallcup MR, Kushner PJ, Baxter JD, Fletterick RJ, Yamamoto KR 1998 Structure and specificity of nuclear receptor-coactivator interactions. Genes Dev 12:3343–3356[Abstract/Free Full Text]
  10. Xu W 2005 Nuclear receptor coactivators: the key to unlock chromatin. Biochem Cell Biol 83:418–428[CrossRef][Medline]
  11. O’Malley BW 2003 Sequentiality and processivity of nuclear receptor coregulators in regulation of target gene expression. Nucl Recept Signal 1:e010
  12. Lee JW, Lee YC, Na S-Y, Jung DJ, Lee SK 2001 Transcriptional coregulators of the nuclear receptor superfamily: coactivators and corepressors. Cell Mol Life Sci 58:289–297[CrossRef][Medline]
  13. 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]
  14. Lonard DM, O’Malley BW 2006 The expanding cosmos of nuclear receptor coactivators. Cell 125:411–414[CrossRef][Medline]
  15. Horwitz KB, Jackson TA, Bain DL, Richer JK, Takimoto GS, Tung L 1996 Nuclear receptor coactivators and corepressors. Mol Endocrinol 10:1167–1177[Abstract/Free Full Text]
  16. Shibata H, Spencer TE, Onate SA, Jenster G, Tsai SY, Tsai MJ, O’Malley BW 1997 Role of co-activators and co-repressors in the mechanism of steroid/thyroid receptor action. Recent Prog Horm Res 52:141–164[Medline]
  17. Lonard DM, O’Malley BW 2005 Expanding functional diversity of the coactivators. Trends Biochem Sci 30:126–132[CrossRef][Medline]
  18. Jung SY, Malovannaya A, Wei J, O’Malley BW, Qin J 2005 Proteomic analysis of steady-state nuclear hormone receptor coactivator complexes. Mol Endocrinol 19:2451–2465[Abstract/Free Full Text]
  19. Kumar R, Wang RA, Barnes CJ 2004 Coregulators and chromatin remodeling in transcription control. Mol Carcinog 41:221–230[CrossRef][Medline]
  20. Rosen ED, Spiegelman BM 2001 PPAR{gamma}: a nuclear regulator of metabolism, differentiation, and cell growth. J Biol Chem 276:37731–37734[Free Full Text]
  21. Spiegelman BM, Heinrich R 2004 Biological control through regulated transcriptional coactivators. Cell 119:157–167[CrossRef][Medline]
  22. Auboeuf D, Dowhan DH, Kang YK, Larkin K, Lee JW, Berget SM, O’Malley BW 2004 Differential recruitment of nuclear receptor coactivators may determine alternative RNA splice site choice in target genes. Proc Natl Acad Sci USA 101:2270–2274[Abstract/Free Full Text]
  23. Chen D, Riedl T, Washbrook E, Pace PE, Coombes RC, Egly J-M, Ali S 2000 Activation of estrogen receptor a by S118 phophorylation involves a ligand-dependent interaction with TFIIH and participation of CDK7. Mol Cell 6:127–137[CrossRef][Medline]
  24. Verma S, Ismail A, Gao X, Fu G, Li X, O’Malley BW, Nawaz Z 2004 The ubiquitin-conjugating enzyme UBCH7 acts as a coactivator for steroid hormone receptors. Mol Cell Biol 24:8716–8726[Abstract/Free Full Text]
  25. Liu Y, Ando S, Xia X, Yao R, Kim M, Fondell J, Yen PM 2005 p62, A TFIIH subunit, directly interacts with thyroid hormone receptor and enhances T3-mediated transcription. Mol Endocrinol 19:879–884[Abstract/Free Full Text]
  26. Spencer TE, Jenster G, Burcin MM, Allis CD, Zhou J, Mizzen CA, McKenna NJ, Onate SA, Tsai SY, Tsai MJ, O’Malley BW 1997 Steroid receptor coactivator-1 is a histone acetyltransferase. Nature 389:194–198[CrossRef][Medline]
  27. Chen H, Lin RJ, Schiltz RL, Chakravarti D, Nash A, Nagy L, Privalsky ML, Nakatani Y, Evans RM 1997 Nuclear receptor coactivator ACTR is a novel histone acetyltransferase and forms a multimeric activation complex with P/CAF and CBP/p300. Cell 90:569–580[CrossRef][Medline]
  28. Bannister AJ, Kouzarides T 1996 The CBP co-activator is a histone acetyltransferase. Nature 384:641–643[CrossRef][Medline]
  29. Berger J, Moller DE 2002 The mechanisms of action of PPARs. Annu Rev Med 53:409–435[CrossRef][Medline]
  30. Castillo G, Brun RP, Rosenfield JK, Hauser S, Park CW, Troy AE, Wright ME, Spiegelman BM 1999 An adipogenic cofactor bound by the differentiation domain of PPAR{gamma}. EMBO J 18:3676–3687[CrossRef][Medline]
  31. Teng X, Li D, Champion HC, Johns RA 2003 FIZZ1/RELM{alpha}—a novel hypoxia-induced mitogenic factor in lung with vasoconstrictive and angiogenic properties. Circ Res 92:1065–1067[Abstract/Free Full Text]
  32. Heery DM, Kalkhoven E, Hoare S, Parker MG 1997 A signature motif in transcriptional co-activators mediates binding to nuclear receptors. Nature 387:733–736[CrossRef][Medline]
  33. McInerney EM, Rose DW, Flynn SE, Westin S, Mullen TM, Krones A, Inostroza J, Torchia J, Nolte RT, Assa-Munt N, Milburn MV, Glass CK, Rosenfeld MG 1998 Determinants of coactivator LXXLL motif specificity in nuclear receptor transcriptional activation. Genes Dev 12:3357–3368[Abstract/Free Full Text]
  34. Zhu Y, QI C, Calandra C, Rao MS, Reddy JK 1996 Cloning and identification of mouse steroid receptor coactivator-1 (mSRC-1), as a coactivator of peroxisome proliferator-activated receptor {gamma}. Gene Expr 6:185–195[Medline]
  35. 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]
  36. 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]
  37. Puigserver P, Adelmant G, Wu Z, Fan M, Xu J, O’Malley B, Spiegelman BM 1999 Activation of PPAR coactivator-1 through transcription factor docking. Science 286:1368–1371[Abstract/Free Full Text]
  38. Park Y, Freedman BD, Lee EJ, Park S 2003 A dominant negative PPAR{gamma} mutant shows altered cofactor recruitment and inhibits adipogenesis in 3T3-L1 cells. Diabetologia 46:365–377[Medline]
  39. Wolins NE, Quaynor BK, Skinner JR, Tzekov A, Park C, Choi K, Bickel PE 2006 OP9 mouse stromal cells rapidly differentiate into adipocytes: characterization of a useful new model of adipogenesis. J Lipid Res 47:450–460[Abstract/Free Full Text]
  40. Nolte RT, Wisely GB, Westin S, Cobb JE, Lambert MH, Kurokawa R, Rosenfeld MG, Willson TM, Glass CK, Milburn MV 1998 Ligand binding and co-activator assembly of the peroxisome proliferator-activated receptor-{gamma}. Nature 395:137–143[CrossRef][Medline]
  41. 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]
  42. Lin J, Handschin C, Spiegelman BM 2005 Metabolic control through the PGC-1 family of transcription coactivators. Cell Metab 1:361–370[CrossRef][Medline]
  43. Tcherepanova I, Puigserver P, Norris JD, Spiegelman BM, McDonnell DP 2000 Modulation of estrogen receptor-{alpha} transcriptional activity by the coactivator PGC-1. J Biol Chem 275:16302–16308[Abstract/Free Full Text]
  44. Lowell MD, Flier MD 1997 Brown adipose tissue, 3-adrenergic receptors and obesity. Annu Rev Med 48:307–316[CrossRef][Medline]
  45. Uldry M, Yang W, St Pierre J, Lin J, Seale P, Spiegelman BM 2006 Complementary action of the PGC-1 coactivators in mitochondrial biogenesis and brown fat differentiation. Cell Metab 3:333–341[CrossRef][Medline]
  46. Puigserver P, Spiegelman BM 2003 Peroxisome proliferator-activated receptor-{gamma} coactivator 1{alpha} (PGC-1{alpha}): transcriptional coactivator and metabolic regulator. Endocr Rev 24:78–90[Abstract/Free Full Text]
  47. IJpenberg A, Jeannin E, Wahli W, Desvergne B 1997 Polarity and specific sequence requirements of peroxisome proliferator-activated receptor (PPAR)/retinoid X receptor heterodimer binding to DNA. A functional analysis of the malic enzyme gene PPAR response element. J Biol Chem 272:20108–20117[Abstract/Free Full Text]
  48. Sancar A, Lindsey-Boltz LA, Unsal-Kacmaz K, Linn S 2004 Molecular mechanisms of mammalian DNA repair and the DNA damage checkpoints. Annu Rev Biochem 73:39–85[CrossRef][Medline]
  49. Tsodikov OV, Enzlin JH, Scharer OD, Ellenberger T 2005 Crystal structure and DNA binding functions of ERCC1, a subunit of the DNA structure-specific endonuclease XPF-ERCC1. Proc Nat Acad Sci USA 102:11236–11241[Abstract/Free Full Text]
  50. Cui W, Hawley RS 2005 The HhH(2)/NDD domain of the Drosophila Nod chromokinesin-like protein is required for binding to chromosomes in the oocyte nucleus. Genetics 171:1823–1835[Abstract/Free Full Text]
  51. Rosen ED, Walkey CJ, Puigserver P, Spiegelman BM 2000 Transcriptional regulation of adipogenesis. Genes Dev 14:1293–1307[Free Full Text]
  52. Kliewer SA, Umesono K, Mangelsdorf DJ, Evans RM 1992 Retinoid X receptor interacts with nuclear receptors in retinoic acid, thyroid hormone and vitamin D3 signaling. Nature 355:446–449[CrossRef][Medline]
  53. Tontonoz P, Hu E, Spiegelman BM 1994 Stimulation of adipogenesis in fibroblast by PPAR {gamma} 2, a lipid-activated transcription factor. Cell 79:1147–1156[CrossRef][Medline]
  54. Qi C, Surapureddi S, Zhu YJ, Yu S, Kashireddy P, Rao MS, Reddy JK 2003 Transcriptional coactivator PRIP, the peroxisome proliferator-activated receptor {gamma} (PPAR{gamma})-interacting protein, is required for PPAR{gamma}-mediated adipogenesis. J Biol Chem 278:25281–25284[Abstract/Free Full Text]
  55. Ge K, Guermah M, Yuan CX, Ito M, Wallberg AE, Spiegelman BM, Roeder RG 2002 Transcription coactivator TRAP220 is required for PPAR{gamma}2-stimulated adipogenesis. Nature 417:563–567[CrossRef][Medline]
  56. Andersen G, Wegner L, Yanagisawa K, Rose CS, Lin J, Glumer C, Drivsholm T, Borch-Johnsen K, Jorgensen T, Hansen T, Spiegelman BM, Pedersen O 2005 Evidence of an association between genetic variation of the coactivator PGC-1ß and obesity. J Med Genet 42:402–407[Abstract/Free Full Text]
  57. Wang Z, Qi C, Krones A, Woodring P, Zhu X, Reddy JK, Evans RM, Rosenfeld MG, Hunter T 2006 Critical roles of the p160 transcriptional coactivators p/CIP and SRC-1 in energy balance. Cell Metab 3:111–122[CrossRef][Medline]
  58. Puigserver P 2006 Tissue-specific regulation of metabolic pathways through the transcriptional coactivator PGC1-{alpha}. Int J Obesity 29:55–59
  59. Monsalve M, Wu Z, Adelmant G, Puigserver P, Fan M, Spiegelman BM 2000 Direct coupling of transcription and mRNA processing through the thermogenic coactivator PGC-1. Mol Cell 6:307–316[CrossRef][Medline]
  60. Corton JC, Brown-Borg HM 2005 Peroxisome proliferator-activated receptor {gamma} coactivator 1 in caloric restriction and other models of longevity. J Gerontol A Biol Sci Med Sci 60:1494–1509[Abstract/Free Full Text]
  61. Kersten S, Desvergne B, Wahli W 2000 Roles of PPARs in health and disease. Nature 405:421–424[CrossRef][Medline]
  62. Tong Q, Zheng L, Dodd-O J, Langer J, Wang D, Li D 2006 Hypoxia-induced mitogenic factor modulates surfactant protein B and C cxpression in mouse lung. Am J Respir Cell Mol Biol 34:28–38[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Med. Genet.Home page
M Futter, H Diekmann, E Schoenmakers, O Sadiq, K Chatterjee, and D C Rubinsztein
Wild-type but not mutant huntingtin modulates the transcriptional activity of liver X receptors
J. Med. Genet., July 1, 2009; 46(7): 438 - 446.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
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 HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Li, D.
Right arrow Articles by Wang, D.-M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Li, D.
Right arrow Articles by Wang, D.-M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals