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Differential Regulation of Peroxisome Proliferator Activated Receptor 1 (PPAR1) and PPAR2 Messenger RNA Expression in the Early Stages of Adipogenesis¹

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) , ß, and ; the helix-loop-helix adipocyte differentiation and determination factor-1; and peroxisome proliferator activated receptor (PPAR), expressed as two isoforms, PPAR1 and the adipocyte-specific PPAR2. Overexpression of PPAR can induce adipocyte differentiation; therefore, we analyzed the expression of the two PPAR 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 PPAR2 mRNA was observed, whereas PPAR1 mRNA remained unchanged. PPAR1 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 PPAR2 promoter. Its deletion blunted the response of PPAR2 promoter to cotransfected C/EBPß or methylisobutylxanthine treatment. We hypothesize that PPAR2 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,⁶ C/EBPß, and C/EBP, 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 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 activate the expression of the gene encoding C/EBP (10) . C/EBP 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, 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 are expressed as two isoforms, PPAR1 and PPAR2 (15 , 17, 18, 19, 20) . PPAR2 is predominantly expressed in adipose tissue in both mouse (15) and human (18, 19, 20) . Moreover, PPAR genes have recently been shown to possess transcription activation capacities through the NH₂-terminal region, this effect being ligand independent (21) . PPAR2 was 10 times more active than PPAR1 in ligand-independent transcriptional activation, and this effect required INS (21) . This data suggests that PPAR1 and PPAR2 may have different functions, with PPAR1 being used when ligand is abundant and PPAR2 being important under conditions of low ligand concentration, such as might occur in early adipocyte differentiation.

PPAR appears to have multiple roles in adipocyte differentiation. In NIH-3T3 cells, ectopic expression of PPAR 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 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 and are capable of interacting with both isoforms (15) .

Although PPAR1 is the most abundant isoform in preadipocytes, PPAR2 is the adipocyte-specific form. Because PPAR1 has been shown to be less transcriptionally active in some systems and PPAR2 was a more potent ligand-independent activator, it was of interest to determine whether PPAR2 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 PPAR2 expression in the mature adipocyte.

Our results demonstrate that PPAR1 and PPAR2 have distinctly different patterns of expression and that only PPAR2 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 PPAR2 in driving the initiation of adipogenesis.

	Results and Discussion

TOP Abstract Introduction Results and Discussion Materials and Methods References

 
Induction of PPAR mRNAs during Adipocyte Differentiation.
As a first step to elucidating the regulation of the PPAR 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 mRNA was assayed by an RNase protection assay in parallel with the 28S rRNA. PPAR2 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) . Meanwhile, mRNA for PPAR1 was initially slightly down-regulated in this period (Fig. 1A) . After the first 24 h, PPAR2 mRNA continued to increase, and after 8 days of differentiation, an increase of 30-fold was observed (Fig. 1A) . After the first quiescent 24 h, PPAR1 mRNA became induced during the next 3 days, reaching a plateau with levels 7-fold higher than the initial levels (Fig. 1A) .

View larger version (22K): [in this window] [in a new window]   Fig. 1. Kinetic of expression of PPAR1 and PPAR2 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." PPAR1 and PPAR2 mRNAs and 28S rRNA were detected by RNase protection assay using 20 µg of total mRNA. The values obtained for PPARs 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." PPAR1, PPAR2, and ß-microglobulin mRNA levels were quantified by competitive RT-PCR using 50 ng of RNA equivalent of cDNA. The values obtained for PPARs were normalized to those for ß-microglobulin. Data points, average values of two different experiments.

These results clearly demonstrate that one of the earliest events of both human and mouse adipocyte differentiation is the selective induction of PPAR2 mRNA over PPAR1, which extends results previously described in rodent cell lines (15) . PPAR2 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 mRNA. Nevertheless, the trace amount of PPAR2 mRNA detected in human preadipocytes is at a level insufficent for adipocyte differentiation. Interestingly, previous studies in 3T3L1 cells suggested that RXR is induced a few hours after differentiation is initiated (24) . This coinduction of both partners of the heterodimer PPAR2/RXR 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 PPAR2 transcription appears very early after the initiation of differentiation. Considering the importance of PPAR in adipocyte differentiation and the fact that obesity is associated with increased expression of PPAR2 and not PPAR1 in adipose tissue (20) , we propose that up-regulation of PPAR2 transcription may be a determining factor in the development of obesity through hyperplastic mechanisms.

PPAR1 and PPAR2 Promoter Activation Confirms Differential Regulation.
To address the question of transcriptional regulation of the PPAR isoforms versus mRNA stability, transfection assays using luciferase reporter constructs driven by the promoter for each PPAR 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 PPAR2 promoter was up-regulated by 10-fold within the first 24 h (Fig. 2A) , consistent with the mRNA data. Maximum induction of the PPAR2 promoter activity was obtained when the combination IBMX/DEX was present (data not shown). This large induction of PPAR2 persisted for 48 h (Fig. 2B) . The human PPAR1 promoter showed a different regulation. In presence of the hormone mixture, the human PPAR1 promoter was down-regulated by 30% (Fig. 2A) , consistent with the slight decrease obtained at the mRNA level. This inhibitory effect on PPAR1 promoter activity was caused by IBMX because it was the only compound in the hormone mixture capable of repressing PPAR1 promoter by itself (70% of control; data not shown). After 48 h of transfection, this down-regulation was no longer observed (Fig. 2B) . Indeed, as a result of the combined effect of INS and DEX (data not shown), PPAR1 promoter activity increased relative to control conditions, and the absolute level of expression was higher than that of PPAR2 promoter (Fig. 2B) .

View larger version (15K): [in this window] [in a new window]   Fig. 2. Human PPAR1 and PPAR2 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.

C/EBPß Increases PPAR2 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 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 PPAR2 promoter by combination treatment with IBMX/DEX, it was investigated whether the up-regulation of the human PPAR2 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 PPAR2 promoter (Fig. 3) . In contrast, no effect was observed on the human PPAR1 promoter (Fig. 3) . Interestingly, cotransfection of a expression vector for C/EBP, 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 PPAR2 promoter (Fig. 3) . Sequence analysis confirmed the presence of a putative C/EBP site in the human PPAR2 promoter, whereas no homology was found in the PPAR1 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 PPAR2 promoter/reporter construct to generate the construct PPAR-h2pC/EBP. As shown in Fig. 3 , when the PPAR-h2pC/EBP construct was examined in transfection experiments, no induction of promoter activity was observed in response to either C/EBPß or C/EBP. 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 PPAR2 promoter reduced the inductive effects of C/EBPß on the mouse PPAR2 promoter (30) . The identification of a C/EBP site within the mouse (30) and human PPAR2 promoter provides a molecular basis for the observation that ectopic expression of C/EBPß resulted in an induction of PPAR mRNA (29) . In addition, we extended the observation to show that C/EBPß induction of PPAR is specific to PPAR2 and not PPAR1 mRNA, thus supporting our hypothesis that PPAR2 drives the initiation of differentiation. C/EBP and C/EBPß have different kinetics of expression during adipogenesis (28) . The fact that both these factors were able to induce the human PPAR2 promoter indicated that the C/EBP family was responsible for the expression of PPAR2 throughout the process of adipocyte differentiation. C/EBPß (and possibly C/EBP) drove the early induction of PPAR2 when adipogenesis was initiated. Because C/EBPß expression declined as differentiation progressed, the increased expression of C/EBP would be able to maintain the expression of PPAR2.

View larger version (20K): [in this window] [in a new window]   Fig. 3. C/EBPß induces the human PPAR2 promoter. Growing 3T3-L1 preadipocytes were transiently transfected with 2 µg of the indicated reporter plasmids, 200 ng C/EBPß or C/EBP, 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.

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 PPAR2 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.

	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 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. PPAR1 values were obtained by subtracting PPAR2 from total PPAR.

Transfection.
To analyze the regulation of the human PPAR promoters, the PPAR1 and PPAR2 promoters were each subcloned into the reporter vector pGL3 (Promega). Briefly, a 3Kb SacI/XhoI (PPAR1) and a 1-kb SmaI/KpnI (PPAR2) fragments were inserted in their respective sites in pGL3, resulting in pGL3-h1p and pGL3-h2p constructs (19) . pGL3-h1p contains 3000 bp of 5' promoter sequence, and pGL3-h2p contains 1000 bp. The pGL3-h2pC/EBP was constructed by deleting the CCAATT sequence located at the position -56 of the PPAR2 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.

² The first two authors contributed equally to this work.

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

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

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

⁶ The abbreviations used are: C/EBP, CAAT/enhancer binding protein; PPAR, peroxisome proliferator activated receptor ; 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.

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

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TOP Abstract Introduction Results and Discussion Materials and Methods References

 

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