CG&D
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cell Growth & Differentiation

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
Services
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
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Saladin, R.
Right arrow Articles by Briggs, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Saladin, R.
Right arrow Articles by Briggs, M.
Cell Growth & Differentiation Vol. 10, 43-48, January 1999
© 1999 American Association for Cancer Research

Differential Regulation of Peroxisome Proliferator Activated Receptor {gamma}1 (PPAR{gamma}1) and PPAR{gamma}2 Messenger RNA Expression in the Early Stages of Adipogenesis1

Régis Saladin2,, 3, Lluis Fajas2, Sharon Dana, Yuan-Di Halvorsen, Johan Auwerx and Michael Briggs4

Ligand Pharmaceuticals, Inc., San Diego, California 92121-3016 [R. S., S. D., M. B.]; Institut National de la Santé et de la Recherche Médicale U325 and Département d’Athérosclérose, Institut Pasteur de Lille, 59019 Lille, France [L. F., J. A.]; and Zen-Bio, Inc., Research Triangle Park, North Carolina 27709 [Y-D. H.]


    Abstract
 TOP
 Abstract
 Introduction
 Results and Discussion
 Materials and Methods
 References
 
Adipocyte differentiation is driven by the expression and activation of three transcription factor families: the differentially expressed CAAT/enhancer binding proteins (C/EBPs) {alpha}, ß, and {delta}; the helix-loop-helix adipocyte differentiation and determination factor-1; and peroxisome proliferator activated receptor {gamma} (PPAR{gamma}), expressed as two isoforms, PPAR{gamma}1 and the adipocyte-specific PPAR{gamma}2. Overexpression of PPAR{gamma} can induce adipocyte differentiation; therefore, we analyzed the expression of the two PPAR{gamma} isoforms during early stages of differentiation to determine whether one was preferentially induced as an early determining event. Surprisingly, in the first 24 h, a 3–6-fold increase of PPAR{gamma}2 mRNA was observed, whereas PPAR{gamma}1 mRNA remained unchanged. PPAR{gamma}1 was induced 1 day later. Overexpression of C/EBPß has also been shown to induce adipocyte differentiation. A C/EBP site was identified only in the human PPAR{gamma}2 promoter. Its deletion blunted the response of PPAR{gamma}2 promoter to cotransfected C/EBPß or methylisobutylxanthine treatment. We hypothesize that PPAR{gamma}2 initiates adipocyte differentiation.


    Introduction
 TOP
 Abstract
 Introduction
 Results and Discussion
 Materials and Methods
 References
 
Obesity is a major risk factor for several pathologies, such as type 2 diabetes, hypertension, and hyperlipidemia, and it is a major contributor to mortality and morbidity in industrialized countries (1) . Obesity is characterized by a positive energy balance, which leads to abnormally elevated storage of triglycerides in adipocytes, resulting in adipocyte hypertrophy and hyperplasia (2) . Adipocytes are derived from pluripotent stem cells, from which myocytes and chondrocytes also originate (3) . The capacity of these precursor cells to differentiate is gradually reduced with age; however, they are still able to be recruited to adipocytes, even in the elderly (4) . Therefore, an in vitro system suitable for studying adipogenesis is desirable. The mouse 3T3-L1 fibroblast cell line has been the most studied cell lines for this purpose because it can be differentiated into adipocytes by hormonal treatment (5) . To date, no human preadipocyte transformed cell line is available, but human primary preadipocytes can be cultured in vitro and induced to differentiate into adipocytes (6) .5

Over the last several years, the involvement of several transcription factors in adipocyte differentiation has been demonstrated (for review see Ref. 7 ). C/EBP{alpha},6 C/EBPß, and C/EBP{delta}, members of the C/EBP family of transcription factors (for review see Ref. 8 ), are expressed at distinct times during adipogenesis. C/EBPß and C/EBP{delta} are expressed very early and appear to be involved in the phase of mitotic clonal expansion that occurs just before the differentiation process begins (9) . C/EBPß and C/EBP{delta} activate the expression of the gene encoding C/EBP{alpha} (10) . C/EBP{alpha} is expressed just after the clonal expansion phase and is able to drive the program of adipocyte differentiation (11 , 12) . Adipocyte differentiation and determination factor-1/sterol regulatory element-binding protein 1, a member of the helix-loop-helix transcription factor that is expressed in the early stages of differentiation, is capable of inducing fat accumulation and adipocyte differentiation when overexpressed in fibroblasts (13) . In addition, PPAR{gamma}, a member of the PPAR subfamily of nuclear receptors, is expressed early in the adipocyte differentiation process and, when activated by a ligand, promotes adipocyte differentiation (11 , 14, 15, 16) . Both the mouse and the human PPAR{gamma} are expressed as two isoforms, PPAR{gamma}1 and PPAR{gamma}2 (15 , 17, 18, 19, 20) . PPAR{gamma}2 is predominantly expressed in adipose tissue in both mouse (15) and human (18, 19, 20) . Moreover, PPAR{gamma} genes have recently been shown to possess transcription activation capacities through the NH2-terminal region, this effect being ligand independent (21) . PPAR{gamma}2 was {approx}10 times more active than PPAR{gamma}1 in ligand-independent transcriptional activation, and this effect required INS (21) . This data suggests that PPAR{gamma}1 and PPAR{gamma}2 may have different functions, with PPAR{gamma}1 being used when ligand is abundant and PPAR{gamma}2 being important under conditions of low ligand concentration, such as might occur in early adipocyte differentiation.

PPAR{gamma} appears to have multiple roles in adipocyte differentiation. In NIH-3T3 cells, ectopic expression of PPAR{gamma} accompanied by the addition of one of its ligands, pioglitazone, can halt the division of exponentially growing cells and induce adipocyte differentiation (22) . In addition to its effect on cell cycle withdrawal, PPAR{gamma} also regulates a number of adipose tissue-specific genes by binding as a heterodimer with RXR to PPREs (reviewed in Ref. 23 ). ARE6 and ARE7, two sequence elements identified in the promoter of the gene coding for the lipid binding protein aP2, are PPREs that mediate the transcriptional response to PPAR{gamma} and are capable of interacting with both isoforms (15) .

Although PPAR{gamma}1 is the most abundant isoform in preadipocytes, PPAR{gamma}2 is the adipocyte-specific form. Because PPAR{gamma}1 has been shown to be less transcriptionally active in some systems and PPAR{gamma}2 was a more potent ligand-independent activator, it was of interest to determine whether PPAR{gamma}2 was induced early in differentiation when ligand supply is thought to be low, thereby providing the necessary impetus to move the process forward. We set out to examine the regulation of the two isoforms during the differentiation process and specifically to attempt to identify the trigger for PPAR{gamma}2 expression in the mature adipocyte.

Our results demonstrate that PPAR{gamma}1 and PPAR{gamma}2 have distinctly different patterns of expression and that only PPAR{gamma}2 is directly up-regulated during the initiation of adipocyte differentiation by C/EBPß or inducers of C/EBPß expression or activity such as IBMX, suggesting a key role for PPAR{gamma}2 in driving the initiation of adipogenesis.


    Results and Discussion
 TOP
 Abstract
 Introduction
 Results and Discussion
 Materials and Methods
 References
 
Induction of PPAR{gamma} mRNAs during Adipocyte Differentiation.
As a first step to elucidating the regulation of the PPAR{gamma} isoforms, a time course experiment was performed. Mouse 3T3-L1 preadipocytes were cultured in DMEM containing 10% calf serum until 2 days postconfluency. Cells were then subjected to the differentiation protocol described in "Materials and Methods." Mouse PPAR{gamma} mRNA was assayed by an RNase protection assay in parallel with the 28S rRNA. PPAR{gamma}2 mRNA was first detectable 3 h after addition of the hormone mixture and was induced 6-fold compared to the first detected value by 24 h after induction of differentiation (Fig. 1A)Citation . Meanwhile, mRNA for PPAR{gamma}1 was initially slightly down-regulated in this period (Fig. 1A)Citation . After the first 24 h, PPAR{gamma}2 mRNA continued to increase, and after 8 days of differentiation, an increase of {approx}30-fold was observed (Fig. 1A)Citation . After the first quiescent 24 h, PPAR{gamma}1 mRNA became induced during the next 3 days, reaching a plateau with levels {approx}7-fold higher than the initial levels (Fig. 1A)Citation .



View larger version (22K):
[in this window]
[in a new window]
 
Fig. 1. Kinetic of expression of PPAR{gamma}1 and PPAR{gamma}2 mRNA during adipogenesis. A, 3T3-L1 preadipocytes, 2 days postconfluent, were treated with 10 µg/ml INS, 500 µM IBMX, and 1 µM DEX. Cells were harvested at the indicated times, and total mRNA was prepared as described in "Materials and Methods." PPAR{gamma}1 and PPAR{gamma}2 mRNAs and 28S rRNA were detected by RNase protection assay using 20 µg of total mRNA. The values obtained for PPAR{gamma}s were normalized to those for 28S rRNA. Data points, average values of four different experiments. B, confluent human primary preadipocytes were treated as described in A, except that 1 µM BRL49653 was added. Cells were harvested at the indicated times, and total mRNA was prepared as described in "Materials and Methods." PPAR{gamma}1, PPAR{gamma}2, and ß-microglobulin mRNA levels were quantified by competitive RT-PCR using 50 ng of RNA equivalent of cDNA. The values obtained for PPAR{gamma}s were normalized to those for ß-microglobulin. Data points, average values of two different experiments.

 
A similar experiment was next performed with human primary preadipocytes. Cells were cultured in DMEM/F10 (1:1) containing 10% FBS to confluency. The preadipocytes were then induced to differentiate as described in "Materials and Methods." Because of the low amount of mRNA obtained from these cells, PPAR{gamma} mRNA was assayed by competitive RT-PCR in parallel with the mRNA for the ß-microglobulin as a normalization control. In the first 24 h, PPAR{gamma}2 was induced by 3–4-fold, whereas levels of PPAR{gamma}1 mRNA remained stable (Fig. 1B)Citation . Thereafter, both isoforms were induced. Strikingly, a 70-fold induction was observed in PPAR{gamma}2 expression, whereas PPAR{gamma}1 was up-regulated by 6-fold (Fig. 1B)Citation .

These results clearly demonstrate that one of the earliest events of both human and mouse adipocyte differentiation is the selective induction of PPAR{gamma}2 mRNA over PPAR{gamma}1, which extends results previously described in rodent cell lines (15) . PPAR{gamma}2 mRNA was detected in human preadipocytes, whereas it could not be detected in the mouse 3T3-L1 preadipocyte cell line. This is probably due to the higher sensitivity of the competitive RT-PCR versus the RNase protection assay used for the detection of mouse PPAR{gamma} mRNA. Nevertheless, the trace amount of PPAR{gamma}2 mRNA detected in human preadipocytes is at a level insufficent for adipocyte differentiation. Interestingly, previous studies in 3T3L1 cells suggested that RXR{alpha} is induced a few hours after differentiation is initiated (24) . This coinduction of both partners of the heterodimer PPAR{gamma}2/RXR{alpha} suggests that they have a key role in the commitment of the cells to adipogenesis. Furthermore, because the relative levels of retinoic acid receptor, another heterodimeric partner of RXR, decrease upon adipocyte differentiation (25) , a switch in RXR’s partners could result in differential recruitment of corepressors and coactivators to DR-1-type PPREs (26) . The results of this study clearly show that induction of PPAR{gamma}2 transcription appears very early after the initiation of differentiation. Considering the importance of PPAR{gamma} in adipocyte differentiation and the fact that obesity is associated with increased expression of PPAR{gamma}2 and not PPAR{gamma}1 in adipose tissue (20) , we propose that up-regulation of PPAR{gamma}2 transcription may be a determining factor in the development of obesity through hyperplastic mechanisms.

PPAR{gamma}1 and PPAR{gamma}2 Promoter Activation Confirms Differential Regulation.
To address the question of transcriptional regulation of the PPAR{gamma} isoforms versus mRNA stability, transfection assays using luciferase reporter constructs driven by the promoter for each PPAR{gamma} human isoform were performed. Because the human primary preadipocytes are difficult to transfect and because a similar induction was seen with both cell types, transfection assays were carried out in mouse 3T3-L1 cells. Briefly, 3T3-L1 preadipocytes were grown in regular medium 2 days postconfluency and then transfected for 3 h using Superfect reagent (Qiagen). Medium was then changed to the differentiation conditions. Thus, promoter assays were carried out in cells primed for differentiation. Luciferase activity was assayed 24 and 48 h after transfection. In the presence of 500 µM IBMX, 1 µM DEX, and 10 µg/ml INS, the human PPAR{gamma}2 promoter was up-regulated by 10-fold within the first 24 h (Fig. 2A)Citation , consistent with the mRNA data. Maximum induction of the PPAR{gamma}2 promoter activity was obtained when the combination IBMX/DEX was present (data not shown). This large induction of PPAR{gamma}2 persisted for 48 h (Fig. 2B)Citation . The human PPAR{gamma}1 promoter showed a different regulation. In presence of the hormone mixture, the human PPAR{gamma}1 promoter was down-regulated by 30% (Fig. 2A)Citation , consistent with the slight decrease obtained at the mRNA level. This inhibitory effect on PPAR{gamma}1 promoter activity was caused by IBMX because it was the only compound in the hormone mixture capable of repressing PPAR{gamma}1 promoter by itself (70% of control; data not shown). After 48 h of transfection, this down-regulation was no longer observed (Fig. 2B)Citation . Indeed, as a result of the combined effect of INS and DEX (data not shown), PPAR{gamma}1 promoter activity increased relative to control conditions, and the absolute level of expression was higher than that of PPAR{gamma}2 promoter (Fig. 2B)Citation .



View larger version (15K):
[in this window]
[in a new window]
 
Fig. 2. Human PPAR{gamma}1 and PPAR{gamma}2 promoter activity in the early stage of adipocyte differentiation. 3T3-L1 preadipocytes 2 days postconfluency were transiently transfected with 2 µg of the indicated reporter plasmids and 1 µg of ß-galactosidase plasmid as internal control. Cells were treated with 10 µg/ml INS, 500 µM IBMX, and 1 µM DEX or vehicle (CON). Luciferase activity was normalized to ß-galactosidase activity. Over the time of transfection, for unknown reasons, luciferase activity declined, which explains the difference in scales in A versus B.

 
The results of the transfection study complement the mRNA analysis and demonstrate that PPAR{gamma}2 mRNA levels and promoter activity are strongly induced very early during adipocyte differentiation, further supporting the hypothesis that PPAR{gamma}2 is the key player in the initiation of adipogenesis. Human PPAR{gamma}1 expression appears to be more constitutive, being initially stable and then gradually increasing during the first 4 days of differentiation. The hormone cocktail showed a dramatic effect on PPAR{gamma}2 promoter activity but only a minor effect on the PPAR{gamma}1 promoter activity. However, both PPAR{gamma}1 and PPAR{gamma}2 mRNA levels are induced throughout adipogenesis with PPAR{gamma}2, preceding PPAR{gamma}1 by about 24 h. The increased level of PPAR{gamma}1 mRNA seen after the first 24 h is not driven by PPAR{gamma} and likely arises from other transcriptional regulatory pathways. Analysis of the 5' flanking sequence of both genes did not reveal any consensus PPREs. Moreover, cotransfection of a vector expressing PPAR{gamma} did not induce expression from the human PPAR{gamma} promoter when ligand was added (data not shown).

C/EBPß Increases PPAR{gamma}2 Promoter Activity.
C/EBPß expression has been shown to be induced by IBMX in 30A5 preadipocytes (27) and 3T3-L1 cells (28) . Ectopic expression of C/EBPß in NIH-3T3 cells was reported to induce PPAR{gamma} gene expression (29) . Ectopically expressed C/EBPß, however, required the addition of DEX to the culture medium to fully initiate the adipogenic program (29) . In view of the strong preferential induction of the human PPAR{gamma}2 promoter by combination treatment with IBMX/DEX, it was investigated whether the up-regulation of the human PPAR{gamma}2 promoter during adipocyte differentiation could be mediated by C/EBPß. Cotransfection experiments performed in growing 3T3-L1 cells showed that C/EBPß induced the activity of PPAR{gamma}2 promoter (Fig. 3)Citation . In contrast, no effect was observed on the human PPAR{gamma}1 promoter (Fig. 3)Citation . Interestingly, cotransfection of a expression vector for C/EBP{alpha}, not normally expressed early in differentiation but still able to bind the cognate C/EBP site, also resulted in the induction of only the human PPAR{gamma}2 promoter (Fig. 3)Citation . Sequence analysis confirmed the presence of a putative C/EBP site in the human PPAR{gamma}2 promoter, whereas no homology was found in the PPAR{gamma}1 promoter. To demonstrate that the effects of C/EBPß were mediated by this sequence element, the putative C/EBPß site was next deleted within the PPAR{gamma}2 promoter/reporter construct to generate the construct PPAR-h{gamma}2p{Delta}C/EBP. As shown in Fig. 3Citation , when the PPAR-h{gamma}2p{Delta}C/EBP construct was examined in transfection experiments, no induction of promoter activity was observed in response to either C/EBPß or C/EBP{alpha}. While this work was in progress and consistent with our observation on the human promoter, Clarke et al. (30) reported that deletion of a similar C/EBP site in the mouse PPAR{gamma}2 promoter reduced the inductive effects of C/EBPß on the mouse PPAR{gamma}2 promoter (30) . The identification of a C/EBP site within the mouse (30) and human PPAR{gamma}2 promoter provides a molecular basis for the observation that ectopic expression of C/EBPß resulted in an induction of PPAR{gamma} mRNA (29) . In addition, we extended the observation to show that C/EBPß induction of PPAR{gamma} is specific to PPAR{gamma}2 and not PPAR{gamma}1 mRNA, thus supporting our hypothesis that PPAR{gamma}2 drives the initiation of differentiation. C/EBP{alpha} and C/EBPß have different kinetics of expression during adipogenesis (28) . The fact that both these factors were able to induce the human PPAR{gamma}2 promoter indicated that the C/EBP family was responsible for the expression of PPAR{gamma}2 throughout the process of adipocyte differentiation. C/EBPß (and possibly C/EBP{delta}) drove the early induction of PPAR{gamma}2 when adipogenesis was initiated. Because C/EBPß expression declined as differentiation progressed, the increased expression of C/EBP{alpha} would be able to maintain the expression of PPAR{gamma}2.



View larger version (20K):
[in this window]
[in a new window]
 
Fig. 3. C/EBPß induces the human PPAR{gamma}2 promoter. Growing 3T3-L1 preadipocytes were transiently transfected with 2 µg of the indicated reporter plasmids, 200 ng C/EBPß or C/EBP{alpha}, and 1 µg of ß-galactosidase plasmid as internal control. After 24 h of transfection, cells were lysed, and luciferase activity was determined and normalized to ß-galactosidase activity.

 
To unequivocally implicate C/EBPß as the effector of the hormone mixture treatment on the human PPAR{gamma}2 promoter, transfection experiments were performed using both the human wild-type PPAR{gamma}2 promoter and the C/EBP binding site mutant construct. When IBMX and DEX were added in the medium, as expected, a increase in human PPAR{gamma}2 promoter activity was observed (Fig. 4)Citation . This induction was blunted when the PPAR{gamma}2 promoter construct that carried the mutation in the C/EBP site was used (Fig. 4)Citation . No significant induction in response to drug treatment was observed for the mutant human PPAR{gamma}2 promoter activity, thus ruling out the possibility of a cryptic glucocorticoid response element or other dominant regulatory site in the promoter. These data clearly demonstrated that endogenous C/EBPß mediated the effect of IBMX on the human PPAR{gamma}2 promoter through the C/EBP site. C/EBP{delta} is expressed in early differentiation. It has been shown that C/EBP{delta} gene expression could be induced by DEX (27 , 28) . The fact that DEX was required for maximal activation of the human PPAR{gamma}2 promoter indicated a supporting a role for C/EBP{delta} in early maximal activation of PPAR{gamma}2 expression.



View larger version (13K):
[in this window]
[in a new window]
 
Fig. 4. C/EBPß mediates the effect of IBMX and DEX on the human PPAR{gamma}2 promoter. Growing 3T3-L1 preadipocytes were transiently transfected with 2 µg of the indicated reporter plasmids and 1 µg of ß-galactosidase plasmid as internal control. Cells were treated for 24 h with 500 µM IBMX and 1 µM DEX (IBMX + DEX) or vehicle (Control). Luciferase activity was normalized to ß-galactosidase activity.

 
In conclusion, this study demonstrates that PPAR{gamma}1 and PPAR{gamma}2 are differentially regulated during adipocyte differentiation in human preadipocytes and in the mouse 3T3L1 preadipocyte cell line. The early induction observed for PPAR{gamma}2 relative to PPAR{gamma}1 suggests a key role for the PPAR{gamma}2 isoform in driving early events in adipogenesis. This is consistent with the fact that PPAR{gamma}2 isoform has greater ligand-independent transcriptional activity and is induced in the preadipocyte before the adipocyte differentiation and determination factor-1/sterol regulatory element-binding protein 1 pathway, which has been proposed to be important for ligand production (31) . This work firmly establishes the hypothesis that PPAR{gamma}2 is the important isoform in very early stages of adipogenesis and suggests that isoform activity differences may have profound biological impact. For example, if PPAR{gamma}2 can be prevented from being induced, would some or most adipocyte differentiation be inhibited? This work sets the stage for a more comprehensive analysis of the roles of PPAR{gamma}1 and PPAR{gamma}2 in adipogenesis and obesity. Reagents to selectively inhibit the expression or activity of either isoform have thus far been difficult to prepare and are the subject of ongoing work to elucidate a functional difference between these isoforms.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Results and Discussion
 Materials and Methods
 References
 
Cell Culture and Differentiation.
3T3-L1 preadipocytes (American Type Culture Collection) were cultured in growth medium containing DMEM and 10% bovine calf serum until they reached confluency. The medium was then changed, and the cells grown for 2 additional days. Adipocyte differentiation was then induced by replacing the growth medium by DMEM supplemented with 10% FBS, 500 µM IBMX (Sigma Chemical Co., St. Louis, MO), 1 µM DEX (Sigma), and 10 µg/ml INS (Sigma). Three days later, medium was replaced with DMEM supplemented with 10% FBS and 10 µg/ml INS, and cell were cultured for 2 additional days. Thereafter, the cells were refed at 48-h intervals with DMEM supplemented with 10% FBS only. The human primary preadipocytes were isolated by approved protocols from excised s.c. adipose tissue of a 37-year-old healthy female undergoing abdominal plastic surgery. The preadipocytes were prepared and cultured based on the procedures reported by Petruschke and Hauner (32) . Adipocyte differentiation was induced by replacing the medium with DMEM/Ham’s nutrient broth F-10 (1:1, v/v) supplemented with 10% FBS, 500 µM IBMX (Sigma), 1 µM DEX (Sigma), 10 µg/ml INS (Sigma), and 1 µM BRL49653.

RNase Protection Assay.
Total mRNA from 3T3-L1 cells and human primary preadipocytes and adipocytes was isolated by the acid, guanidium thiocyanate, phenol, and chloroform method (33) . A partial cDNA containing nucleotides 4–273 of mouse PPAR{gamma} was subcloned into the pCRII vector (Invitrogen). The pTRI-RNA-28S plasmid (Ambion) was used as a control. HindIII-linearized plasmids were used to make 32P-labeled antisense riboprobes using the T7 RNA polymerase and Maxiscript in vitro transcription kit (Ambion). RNase protection assay was performed using the RPAII kit (Ambion). Band intensities were quantitated on a PhosphoImager (Molecular Dynamics).

Competitive RT-PCR Assay.
Each RNA sample was reverse transcribed with oligo(dT) primers using the Superscript Preamplification System for First Strand cDNA Synthesis (Life Technologies, Inc., Gaithersburg, MD) including a DNaseI pretreatment. Competitive PCR was performed on 50 ng of RNA equivalent of cDNA per 50 µl reaction in the presence of varying concentrations of competitor plasmid and using AmpliTaq Gold and the manufacturer’s standard buffer (Perkin-Elmer/Roche Molecular Systems, Inc., Branchburg, NJ). Cycling was as described (34) , with an additional first step of 10 min at 95°C to activate the AmpliTaq Gold. Amplified products were quantitated by agarose gel electrophoresis in the presence of EtBr, followed by analysis with Gel Pro Analyzer software (Media Cybernetics, Silver Spring, MD). Competitor plasmids have been described (34 , 35) . All PPAR values were normalized to ß-microglobulin values to adjust for differences in input RNA and efficiency of cDNA synthesis. PPAR{gamma}1 values were obtained by subtracting PPAR{gamma}2 from total PPAR{gamma}.

Transfection.
To analyze the regulation of the human PPAR{gamma} promoters, the PPAR{gamma}1 and PPAR{gamma}2 promoters were each subcloned into the reporter vector pGL3 (Promega). Briefly, a 3Kb SacI/XhoI (PPAR{gamma}1) and a 1-kb SmaI/KpnI (PPAR{gamma}2) fragments were inserted in their respective sites in pGL3, resulting in pGL3-h{gamma}1p and pGL3-h{gamma}2p constructs (19) . pGL3-h{gamma}1p contains 3000 bp of 5' promoter sequence, and pGL3-h{gamma}2p contains 1000 bp. The pGL3-h{gamma}2p{Delta}C/EBP was constructed by deleting the CCAATT sequence located at the position -56 of the PPAR{gamma}2 promoter by splicing overlapping ends-PCR. 3T3-L1 transfections were carried out in 12-well plates using Superfect reagent (Qiagen). Luciferase and ß-galactosidase assays were carried out as described (36) .


    Acknowledgments
 
We thank Jim Fraser, Kay Klausing, Ira Schulman, Ranjan Mukherjee, and Rich Heyman for critical comments on the manuscript; Lawrence Hamann for synthesizing BRL49653; and Peter Johnson for providing the C/EBPs expression vector plasmids. The excellent technical assistance of Patricia Hoener for the competitive RT-PCR is also acknowledged.


    Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by grants from Institut National de la Santé et de la Recherche Médicale, ARC, Institut Pasteur de Lille, Institut de Chimie Pharmaceutique, and Ligand Pharmaceuticals, Inc. L. F. was supported by a fellowship from the Janssen Research Foundation, and J. A. is a research director with Centre National de la Recherche Scientifique. Back

2 The first two authors contributed equally to this work. Back

3 To whom requests for reprints should be addressed, at Ligand Pharmaceuticals, Inc., 10255 Science Center Drive, San Diego, CA 92121. Phone: (619) 550-7652; Fax: (619) 550-7876; E-mail: rsaladin{at}ligand.com Back

4 Present address: SmithKline Beecham Pharmaceuticals, King of Prussia, PA 19406. Back

5 Y-D. Halvorsen and R. Saladin, unpublished data. Back

6 The abbreviations used are: C/EBP, CAAT/enhancer binding protein; PPAR{gamma}, peroxisome proliferator activated receptor {gamma}; RXR, retinoid X receptor; PPRE, PPAR response element; IBMX, 1-methyl-3-isobutylxanthine; FBS, fetal bovine serum; RT-PCR, reverse transcriptase-PCR; DEX, dexamethasone; INS, insulin. Back

Received for publication 8/11/98. Revision received 11/12/98. Accepted for publication 11/23/98.


    References
 TOP
 Abstract
 Introduction
 Results and Discussion
 Materials and Methods
 References
 

  1. Martin L. F., Hunter S. M., Lauve R. M., O’Leary J. P. Severe obesity: expensive to society, frustrating to treat, but important to confront. Southern Med. J., 88: 895-902, 1995.[Medline]
  2. Hirsch J. Adipose cellularity in relation to human adiposity Stollerman G. H. eds. . Advances in Internal Medicine, 17: 289-300, Year Book Medical Publishers Chicago 1971.[Medline]
  3. Cornelius P., MacDouglad O. A., Lane M. D. Regulation of adipocyte development. Annu. Rev. Nutr., 14: 99-129, 1994.[Medline]
  4. Prins J. B., O’Rahilly S. Regulation of adipose cell number in man. Clin. Sci., 92: 3-11, 1997.[Medline]
  5. Reed B. C., Lane M. D. Expression of insulin receptors during preadipocyte differentiation. Adv. Enzyme. Regul., 18: 97-117, 1980.[Medline]
  6. Deslex S., Negrel R., Vannier C., Etienne J., Ailhaud G. Differentiation of human adipocyte precursors in a chemically defined serum-free medium. Int. J. Obesity, 11: 19-27, 1986.
  7. Fajas, L., Fruchart, J. C., and Auwerx, J. Transcriptional control of adipogenesis. Curr. Opin. Cell Biol., in press, 1999.
  8. Mandrup S., Lane M. D. Regulating adipogenesis. J. Biol. Chem., 272: 5367-5370, 1997.[Free Full Text]
  9. MacDouglad O. A., Lane M. D. Adipocyte differentiation. When precursors are also regulators. Curr. Biol., 5: 618-621, 1995.[Medline]
  10. Yeh W. C., Cao Z., Classon M., McKnight L. M. Cascade regulation of terminal adipocyte differentiation by three members of the C/EBP family of leucine zipper proteins. Genes Dev., 9: 168-181, 1995.[Abstract/Free Full Text]
  11. Freytag S. O., Paielli D. L., Gilbert J. D. Ectopic expression of the CCAAT/enhancer-binding protein {alpha} promotes the adipogenic program in a variety of mouse fibroblastic cells. Genes Dev., 8: 1654-1663, 1994.[Abstract/Free Full Text]
  12. Shao D., Lazar M. A. Peroxisome proliferator activated receptor {gamma}, CCAAT/enhancer-binding protein {alpha}, and cell cycle status regulate the commitment to adipocyte differentiation. J. Biol. Chem., 272: 21473-21478, 1997.[Abstract/Free Full Text]
  13. Kim J., Spiegelman B. M. ADD1/SREBP1 promotes adipocyte differentiation and gene expression linked to fatty acid metabolism. Genes Dev., 10: 1096-1107, 1996.[Abstract/Free Full Text]
  14. Brun R. P., Tontonoz P., Forman B. M., Ellis R., Chen J., Evans R. M., Spiegelman B. M. Differential activation of adipogenesis by multiple PPAR isoforms. Genes Dev., 10: 974-984, 1996.[Abstract/Free Full Text]
  15. Tontonoz P., Hu E., Graves R. A., Budavari A. I., Spiegelman B. M. mPPAR{gamma}2: tissue-specific regulator of an adipocyte enhancer. Genes Dev., 8: 1224-1234, 1994.[Abstract/Free Full Text]
  16. Tontonoz P., Hu E., Spiegelman B. M. Regulation of adipocyte gene expression and differentiation by peroxisome proliferator activated receptor {gamma}. Cell, 79: 1147-1156, 1994.[Medline]
  17. Zhu Y., Alvares K., Huang Q., Rao M. S., Reddy J. K. Cloning of a new member of the peroxisome proliferator-activated receptor gene family from mouse liver. J. Biol. Chem., 268: 26817-26820, 1993.[Abstract/Free Full Text]
  18. Mukherjee R., Jow L., Croston G. E., Paterniti J. R., Jr. Identification, characterization, and tissue distribution of human peroxisome proliferator-activated receptor (PPAR) isoforms PPAR{gamma}2 versus PPAR{gamma}1, and activation with retinoid X receptor agonists, and antagonists. J. Biol. Chem., 272: 8071-8076, 1997.[Abstract/Free Full Text]
  19. Fajas L., Auboeuf D., Raspé E., Schoonjans K., Lefebvre A. M., Saladin R., Najib J., Laville M., Fruchart J. C., Deeb S., Vidal-Puig A., Flier J., Briggs M. R., Staels B., Vidal H., Auwerx J. The organization, promoter analysis, and expression of the human PPAR{gamma} gene. J. Biol. Chem., 272: 18779-18789, 1997.[Abstract/Free Full Text]
  20. Vidal-Puig A. J., Considine R. V., Jimenez-Linan M., Werman A., Pories W. J., Caro J. F., Flier J. S. Peroxisome proliferator-activated receptor gene expression in human tissues. Effects of obesity, weight loss, and regulation by insulin and glucocorticoids. J. Clin. Invest., 99: 2416-2422, 1997.[Medline]
  21. Werman A., Hollenberg A., Solanes G., Bjørbaek C., Vidal-Puig A. J., Flier J. S. Ligand-independent activation domain in the N terminus of peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}). J. Biol. Chem., 272: 20230-20235, 1997.[Abstract/Free Full Text]
  22. Altiok S., Xu M., Spiegelman B. M. PPAR{gamma} induces cell cycle withdrawal: inhibition of E2F/DP DNA-binding activity via down-regulation of PP2A. Genes Dev., 11: 1987-1998, 1997.[Abstract/Free Full Text]
  23. Schoonjans K., Staels B., Auwerx J. Role of the peroxisome proliferator-activated receptor (PPAR) in mediating the effects of fibrates and fatty acids on gene expression. J. Lipid Res., 37: 907-925, 1996.[Abstract]
  24. Chawla A., Lazar M. A. Peroxisome proliferator and retinoid signaling pathways co-regulate preadipocyte phenotype and survival. Proc. Natl. Acad. Sci. USA, 91: 1786-1790, 1994.[Abstract/Free Full Text]
  25. Xue J. C., Schwarz E. J., Chawla A., Lazar M. A. Distinct stages in adipogenesis revealed by retinoid inhibition of differentiation after induction of PPAR{gamma}. Mol. Cell. Biol., 16: 1567-1575, 1996.[Abstract/Free Full Text]
  26. DiRenzo J., Sàderstràm M., Kurokawa R., Ogliastro M. H., Ricote M., Ingrey S., Hörlein A., Rosenfeld M. G., Glass C. K. Peroxisome proliferator-activated receptor and retinoic acid receptors differentially control the interactions of retinoid X receptor heterodimers with ligands, coactivators and corepressors. Mol. Cell. Biol., 17: 2166-2176, 1997.[Abstract/Free Full Text]
  27. Tae H. J., Zhang S., Kim K. H. cAMP activation of CAAT enhancer-binding protein-ß gene expression and promoter I of acetyl-CoA carboxylase. J. Biol. Chem., 270: 21487-21494, 1995.[Abstract/Free Full Text]
  28. Cao Z., Umek R., McKnight S. L. Regulated expression of three C/EBP isoforms during adipose conversion of 3T3–L1 cells. Genes Dev., 5: 1538-1542, 1991.[Abstract/Free Full Text]
  29. Wu Z., Xie Y., Bucher N. L. R., Farmer S. R. Conditional ectopic expression of C/EBPß in NIH-3T3 cells induces PPAR{gamma} and stimulates adipogenesis. Genes Dev., 9: 2350-2363, 1995.[Abstract/Free Full Text]
  30. Clarke S. L., Robinson C. E., Gimble J. M. CAAT/enhancer binding proteins directly modulate transcription from the peroxisome proliferator-activated receptor {gamma}2 promoter. Biochem. Biophys. Res. Commun., 240: 99-103, 1997.[Medline]
  31. Kim J. B., Wright H. M., Wright M., Spiegelman B. M. AAD1/SREBP-1 activates PPAR{gamma} through the production of endogenous ligand. Proc. Natl. Acad. Sci. USA, 95: 4333-4337, 1998.[Abstract/Free Full Text]
  32. Petruschke T. H., Hauner H. Tumor necrosis factor-{alpha} prevents the differentiation of human adipocyte precursor cells and causes delipidation of newly developed fat cells. J. Clin. Endocrinol. Metab., 76: 742-747, 1993.[Medline]
  33. Saladin R., De Vos P., Guerre-Millo M., Leturque A., Girard J., Staels B., Auwerx J. Transient increase in obese gene expression after food intake or insulin administration. Nature (Lond.), 377: 527-529, 1995.[Medline]
  34. Laville M., Auboeuf D., Khalfallah Y., Vega N., Riou J. P., Vidal H. Acute regulation by insulin of phosphatidylinositol-3-kinase, Rad, Glut 4, and lipoprotein lipase mRNA levels in human muscle. J. Clin. Invest., 98: 43-49, 1996.[Medline]
  35. Auboeuf D., Rieusset J., Fajas L., Vallier P., Frering V., Riou J. P., Staels B., Auwerx J., Laville M., Vidal H. Tissue distribution and quantification of the expression of PPARs and LXRa in humans: no alterations in adipose tissue of obese and NIDDM patients. Diabetes, 46: 1319-1327, 1997.[Abstract/Free Full Text]
  36. McDonnell D. P., Vegeto E., Gleeson M. A. G. Nuclear hormone receptors as targets for new drug discovery. Bio/Technology, 11: 1256-1261, 1993.[Medline]



This article has been cited by other articles:


Home page
DiabetesHome page
M. Park, J.-W. Yi, E.-M. Kim, I.-J. Yoon, E.-H. Lee, H.-Y. Lee, K.-Y. Ji, K.-H. Lee, J.-H. Jang, S.-S. Oh, et al.
Triggering Receptor Expressed on Myeloid Cells 2 (TREM2) Promotes Adipogenesis and Diet-Induced Obesity
Diabetes, January 1, 2015; 64(1): 117 - 127.
[Abstract] [Full Text] [PDF]


Home page
J Biol ChemHome page
C.-Y. Ou, T.-C. Chen, J. V. Lee, J.-C. Wang, and M. R. Stallcup
Coregulator Cell Cycle and Apoptosis Regulator 1 (CCAR1) Positively Regulates Adipocyte Differentiation through the Glucocorticoid Signaling Pathway
J. Biol. Chem., June 13, 2014; 289(24): 17078 - 17086.
[Abstract] [Full Text] [PDF]


Home page
ReproductionHome page
P J Williams, N Marten, V Wilson, J C Litten-Brown, A M Corson, L Clarke, M E Symonds, and A Mostyn
Influence of birth weight on gene regulators of lipid metabolism and utilization in subcutaneous adipose tissue and skeletal muscle of neonatal pigs
Reproduction, September 1, 2009; 138(3): 609 - 617.
[Abstract] [Full Text] [PDF]


Home page
J EndocrinolHome page
L. Zhang, C. Paddon, M. D Lewis, F. Grennan-Jones, and M. Ludgate
Gs{alpha} signalling suppresses PPAR{gamma}2 generation and inhibits 3T3L1 adipogenesis
J. Endocrinol., August 1, 2009; 202(2): 207 - 215.
[Abstract] [Full Text] [PDF]


Home page
J Biol ChemHome page
P. Bai, S. M. Houten, A. Huber, V. Schreiber, M. Watanabe, B. Kiss, G. de Murcia, J. Auwerx, and J. M.-d. Murcia
Peroxisome Proliferator-activated Receptor (PPAR)-2 Controls Adipocyte Differentiation and Adipose Tissue Function through the Regulation of the Activity of the Retinoid X Receptor/PPAR{gamma} Heterodimer
J. Biol. Chem., December 28, 2007; 282(52): 37738 - 37746.
[Abstract] [Full Text] [PDF]


Home page
RNAHome page
Y. Xu, S.-H. Mirmalek-Sani, F. Lin, J. Zhang, and R. O.C. Oreffo
Adipocyte differentiation induced using nonspecific siRNA controls in cultured human mesenchymal stem cells
RNA, August 1, 2007; 13(8): 1179 - 1183.
[Abstract] [Full Text] [PDF]


Home page
Proc. Natl. Acad. Sci. USAHome page
J.-F. Louet, A. Coste, L. Amazit, M. Tannour-Louet, R.-C. Wu, S. Y. Tsai, M.-J. Tsai, J. Auwerx, and B. W. O'Malley
Oncogenic steroid receptor coactivator-3 is a key regulator of the white adipogenic program
PNAS, November 21, 2006; 103(47): 17868 - 17873.
[Abstract] [Full Text] [PDF]


Home page
Am. J. Physiol. Cell Physiol.Home page
A. G. Smith and G. E. O. Muscat
Orphan nuclear receptors: therapeutic opportunities in skeletal muscle
Am J Physiol Cell Physiol, August 1, 2006; 291(2): C203 - C217.
[Abstract] [Full Text] [PDF]


Home page
Am. J. Physiol. Endocrinol. Metab.Home page
U. Kintscher and R. E. Law
PPAR{gamma}-mediated insulin sensitization: the importance of fat versus muscle
Am J Physiol Endocrinol Metab, January 6, 2005; 288(2): E287 - E291.
[Abstract] [Full Text] [PDF]


Home page
J. Lipid Res.Home page
M. Grohmann, M. Sabin, J. Holly, J. Shield, E. Crowne, and C. Stewart
Characterization of differentiated subcutaneous and visceral adipose tissue from children: the influences of TNF-{alpha} and IGF-I
J. Lipid Res., January 1, 2005; 46(1): 93 - 103.
[Abstract] [Full Text] [PDF]


Home page
Mol. Cell. Biol.Home page
N. Salma, H. Xiao, E. Mueller, and A. N. Imbalzano
Temporal Recruitment of Transcription Factors and SWI/SNF Chromatin-Remodeling Enzymes during Adipogenic Induction of the Peroxisome Proliferator-Activated Receptor {gamma} Nuclear Hormone Receptor
Mol. Cell. Biol., June 1, 2004; 24(11): 4651 - 4663.
[Abstract] [Full Text] [PDF]


Home page
Proc. Natl. Acad. Sci. USAHome page
H. Koutnikova, T.-A. Cock, M. Watanabe, S. M. Houten, M.-F. Champy, A. Dierich, and J. Auwerx
Compensation by the muscle limits the metabolic consequences of lipodystrophy in PPAR{gamma} hypomorphic mice
PNAS, November 25, 2003; 100(24): 14457 - 14462.
[Abstract] [Full Text] [PDF]


Home page
J Biol ChemHome page
L. Choy and R. Derynck
Transforming Growth Factor-{beta} Inhibits Adipocyte Differentiation by Smad3 Interacting with CCAAT/Enhancer-binding Protein (C/EBP) and Repressing C/EBP Transactivation Function
J. Biol. Chem., March 14, 2003; 278(11): 9609 - 9619.
[Abstract] [Full Text] [PDF]


Home page
DiabetesHome page
C. P. Sewter, F. Blows, A. Vidal-Puig, and S. O'Rahilly
Regional Differences in the Response of Human Pre-Adipocytes to PPAR{gamma} and RXR{alpha} Agonists
Diabetes, March 1, 2002; 51(3): 718 - 723.
[Abstract] [Full Text] [PDF]


Home page
J Biol ChemHome page
G. Elberg, J. M. Gimble, and S. Y. Tsai
Modulation of the Murine Peroxisome Proliferator-activated Receptor {gamma}2 Promoter Activity by CCAAT/Enhancer-binding Proteins
J. Biol. Chem., September 8, 2000; 275(36): 27815 - 27822.
[Abstract] [Full Text] [PDF]


Home page
J Biol ChemHome page
K. Bordji, J.-P. Grillasca, J.-N. Gouze, J. Magdalou, H. Schohn, J.-M. Keller, A. Bianchi, M. Dauca, P. Netter, and B. Terlain
Evidence for the Presence of Peroxisome Proliferator-activated Receptor (PPAR) {alpha} and {gamma} and Retinoid Z Receptor in Cartilage: PPAR{gamma} ACTIVATION MODULATES THE EFFECTS OF INTERLEUKIN-1{beta} ON RAT CHONDROCYTES
J. Biol. Chem., April 21, 2000; 275(16): 12243 - 12250.
[Abstract] [Full Text] [PDF]


Home page
Mol. Cell. Biol.Home page
L. Fajas, K. Schoonjans, L. Gelman, J. B. Kim, J. Najib, G. Martin, J.-C. Fruchart, M. Briggs, B. M. Spiegelman, and J. Auwerx
Regulation of Peroxisome Proliferator-Activated Receptor {gamma} Expression by Adipocyte Differentiation and Determination Factor 1/Sterol Regulatory Element Binding Protein 1: Implications for Adipocyte Differentiation and Metabolism
Mol. Cell. Biol., August 1, 1999; 19(8): 5495 - 5503.
[Abstract] [Full Text] [PDF]


Home page
J Biol ChemHome page
G. Elberg, J. M. Gimble, and S. Y. Tsai
Modulation of the Murine Peroxisome Proliferator-Activated Receptor{gamma}2 Promoter Activity by CCAAT/Enhancer Binding Proteins
J. Biol. Chem., June 20, 2000; (2000) 3593200.
[Abstract]


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
Services
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
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Saladin, R.
Right arrow Articles by Briggs, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Saladin, R.
Right arrow Articles by Briggs, M.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cell Growth & Differentiation