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J. Biol. Chem., Vol. 276, Issue 5, 3175-3182, February 2, 2001

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Peroxisome Proliferator-activated Receptor  (PPAR)-mediated Regulation of Preadipocyte Proliferation and Gene Expression Is Dependent on cAMP Signaling^*

Jacob B. Hansen, Hongbin Zhang, Thomas H. Rasmussen, Rasmus K. Petersen, Esben N. Flindt, and Karsten Kristiansen^§

From the Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense University, DK-5230 Odense M, Denmark

Received for publication, June 25, 2000, and in revised form, November 3, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The peroxisome proliferator-activated receptor  (PPAR) is a key regulator of terminal adipocyte differentiation. PPAR is expressed in preadipocytes, but the importance of this PPAR subtype in adipogenesis has been a matter of debate. Here we present a critical evaluation of the role of PPAR in adipocyte differentiation. We demonstrate that treatment of NIH-3T3 fibroblasts overexpressing PPAR with standard adipogenic inducers led to induction of PPAR2 expression and terminal adipocyte differentiation in a manner that was strictly dependent on simultaneous administration of a PPAR ligand and methylisobutylxanthine (MIX) or other cAMP elevating agents. We further show that ligands and MIX synergistically stimulated PPAR-mediated transactivation. In 3T3-L1 preadipocytes, simultaneous administration of a PPAR-selective ligand and MIX significantly enhanced the early expression of PPAR and ALBP/aP2, but only modestly promoted terminal differentiation as determined by lipid accumulation. Finally, we provide evidence that synergistic activation of PPAR promotes mitotic clonal expansion in 3T3-L1 cells with or without forced expression of PPAR. In conclusion, our results suggest that PPAR may play a role in the proliferation of adipocyte precursor cells, whereas activation of endogenous PPAR in 3T3-L1 cells appears to have only minor impact on the processes leading to terminal adipocyte differentiation.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Adipocyte differentiation proceeds in a cascade-like manner by the sequential action of different classes of transcriptional regulators among which members of the CCAAT/enhancer-binding protein (C/EBP)¹ and the peroxisome proliferator-activated receptor (PPAR) families play crucial roles, and in a complex interdependent manner regulate clonal expansion, withdrawal from the cell cycle, and terminal differentiation (reviewed in Refs. 1-4).

The PPAR family belongs to the superfamily of nuclear hormone receptors and comprises three subtypes, PPAR, PPAR (also designated PPAR, FAAR, or NUC-1) and PPAR, the latter of which exists in two isoforms (5-8). The PPARs are ligand-activated transcription factors that bind as heterodimers with members of the retinoid X receptor (RXR) subfamily to PPAR response elements (PPREs) in the promoters/enhancers of responsive genes. The PPARs are activated by a large variety of fatty acids and fatty acid metabolites, and direct binding of many of these activators to PPARs has been demonstrated (9-12). Synthetic thiazolidinedione insulin-sensitizing antidiabetic drugs have been shown to be high affinity PPAR ligands (13), and selective PPAR ligands have recently been described (14-16). Apart from ligands, the transactivation potential of PPAR and PPAR is regulated by phosphorylation (17-23) and by interaction with different families of coactivators and corepressors (24, 25), and interaction with ligands and cofactors may in part be controlled by phosphorylation (26, 27).

Activators of PPAR promote adipocyte differentiation of preadipocytes and multipotent C3H10T_1/2 cells (13), and ectopic expression of PPAR in fibroblastic NIH-3T3 cells enables ligand-dependent adipocyte differentiation (28). Conclusive evidence for the crucial role of PPAR in adipogenesis was recently reported by the use of mice and mouse cells null for the PPAR gene (29-31). Whereas the role of PPAR in adipocyte differentiation is well documented, the function of PPAR has been a matter of dispute. In 3T3-F442A, 3T3-L1, and Ob1771 preadipocytes, PPAR is expressed at the inception of differentiation, prior to the induction of PPAR expression (28, 32, 33). Ectopic expression of PPAR in NIH-3T3 fibroblasts was shown not to promote adipocyte differentiation (34). However, ectopic expression of PPAR in 3T3-C2 cells has been shown to confer fatty acid-dependent expression of adipocyte marker genes (33). Furthermore, fatty acid treatment of 3T3-C2 cells with forced expression of PPAR was demonstrated to promote both the expression of PPAR and adipose conversion, the latter in a manner depending on the combined treatment with fatty acids and a selective PPAR ligand (35). Recently, it was reported that gonadal adipose tissue mass was reduced in PPAR knockout female mice, suggesting a modulatory function of PPAR in preadipocyte proliferation and/or adipogenesis in vivo (36). However, the role of PPAR in normal preadipocyte gene expression and differentiation, if any, is poorly understood.

In this report we present a critical evaluation of the potential of PPAR as an adipogenic inducer and the effect of activation of endogenous PPAR in adipose conversion of preadipocytes. We provide evidence that PPAR-mediated transactivation is synergistically enhanced by combined treatment with ligand and a cAMP-elevating agent, and that this combination allowed adipocyte differentiation of NIH-3T3 cells with forced expression of PPAR. We show that activation of endogenously or ectopically expressed PPAR in 3T3-L1 and NIH-3T3 cells promoted mitotic clonal expansion. However, even though synergistic activation of endogenous PPAR in preadipocytes enhanced the expression of the key adipogenic regulator PPAR, terminal differentiation was only modestly promoted, indicating that PPAR activation is not a decisive factor in terminal differentiation of adipocytes, but rather plays a role in the expansion of the pool of precursor cells.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Plasmids and Transient Transfections-- The retroviral expression vector pBabe-mPPAR (gift from B. M. Spiegelman) has been described (34). The 3xPPRE-TK-Luc reporter plasmid was a gift from R. M. Evans (37). The expression vectors used were pSG5-PPAR (gift from P. A. Grimaldi) (33), pCMX-RXR (gift from R. M. Evans) (38), and pSV--galactosidase (Promega). NIH-3T3 cells were transiently transfected by the calcium phosphate method in six-well plates. Fifty percent confluent cells per well were transfected with reporter plasmid (3 µg), PPAR and RXR expression vectors (1 µg of each), and pSV--galactosidase (0.5 µg). Empty expression vectors were added to a total of 5.5 µg of DNA/well. Following transfection, the cells were incubated for 24 h with medium containing 10% resin-charcoal-stripped bovine serum supplemented as indicated with 2-bromopalmitate, L-165041 (kindly provided by Merck Research Laboratories) (20) and/or MIX (Sigma). The total concentration of vehicle was kept constant by the addition of Me₂SO (to 0.1%) and/or potassium hydroxide (1% 0.1 N potassium hydroxide). Cells were analyzed for luciferase and -galactosidase activities by standard procedures.

Retrovirus Production and Transduction-- Phoenix cells were transfected with viral DNA at 50% confluence. Forty-eight h after transfection, virus supernatant was harvested and filtered. Fifty percent confluent NIH-3T3 or 3T3-L1 cells were transduced with virus supernatant diluted with one volume of fresh growth medium (Dulbecco's modified Eagle's medium (DMEM) containing 10% bovine serum) in the presence of 6 µg/ml Polybrene. The following day, the cells were split and subjected to puromycin (Sigma) selection (2 µg/ml). Approximately 5 days later, the selected clones were pooled and replated for differentiation.

Cell Culture and Differentiation-- Selected NIH-3T3 cells were grown to confluence in DMEM containing 10% bovine serum and puromycin. At confluence, puromycin was withdrawn and cells were left for 2 days in DMEM with 10% bovine serum. Two-day postconfluent cells (designated day 0) were induced to differentiate with DMEM containing 10% fetal bovine serum (FBS), dexamethasone (1 µM) (Sigma), and insulin (1 µg/ml) (Roche Molecular Biochemicals). MIX (0.5 mM) and PPAR ligands were added as indicated. After 48 h, the cells were refed with DMEM containing 10% FBS supplemented with 1 µg/ml insulin and ligand (when added). From day 4, medium consisted of DMEM with 10% FBS and ligand (when added) and cells were refed every other day. Cells not treated with PPAR ligands or MIX received similar volumes of vehicle (0.05% Me₂SO and 1% 0.1 N potassium hydroxide, respectively). Ligands used for NIH-PPAR and NIH-vector cells were 2-bromopalmitate, L-165041, and BRL49653 (kindly provided by Novo Nordisk). Untransduced 3T3-L1 cells were grown in DMEM containing 10% bovine serum. Two-day postconfluent 3T3-L1 cells (designated day 0) were induced to differentiate by exposure for 4 days to DMEM with 10% FBS and insulin (5 µg/ml) and the combinations of MIX and PPAR ligands described in the legend to Fig. 6. Cells not treated with PPAR ligands and MIX received similar volumes of vehicle (0.05% Me₂SO and 1% 0.1 N potassium hydroxide, respectively). From day 4 to day 8, cells were exposed to DMEM with 10% FBS. Medium was replenished on days 2, 4, and 6. Oil Red O staining was performed as described (39). Selected 3T3-L1 cells were grown as described for the transduced NIH-3T3 cells and treated with inducers as described in the legend to Fig. 7.

Whole Cell Extracts and Western Blot Analysis-- Whole cell extracts, electrophoresis, blotting, visualization, and stripping of membranes were performed as described (39). Primary antibodies used were mouse anti-human PPAR and rabbit anti-human TBP (both from Santa Cruz Biotechnology), rabbit anti-mouse ALBP/aP2 (kindly provided by D. A. Bernlohr), and rabbit anti-mouse PPAR (the generation of this antibody will be described elsewhere). The secondary antibodies were horseradish peroxidase-conjugated anti-rabbit and anti-mouse antibodies (Dako).

RNA Purification and Multiplex Reverse Transcription-Polymerase Chain Reaction (RT-PCR)-- RNA purification, reverse transcription and multiplex RT-PCR were performed as described (39). Primers used were (upstream and downstream): mouse ALBP/aP2, 5'-GAACCTGGAAGCTTGTCTTCG, 5'-ACCAGCTTGTCACCATCTCG (325 bp); human -actin, 5'-AATGTCACGCACGATTTCCC, 5'-GACATGGAGAAAATCTGGCA (395 bp); mouse PPAR, 5'-ATGGAACAGCCACAGGAGGAG, 5'-GACATTCCATGTTGAGGCTGC (220 bp); mouse PPAR (pan), 5'-GAGCTGACCCAATGGTTGCTG, 5'-GCTTCAATCGGATGGTTCTTC (254 bp). Primer sets for mouse C/EBP, glycerol-3-phosphate dehydrogenase (GPDH), PPAR1, PPAR2, and TBP has been described (39). The number of cycles performed in individual reactions is described in the figure legends. All reactions contained the TBP or the -actin primer sets as internal standards together with up to two additional primer sets. A representative result for -actin and/or TBP is shown in all figures.

Fluorometric Determination of Cell Numbers and Bromodeoxyuridine (BrdUrd) Labeling-- Relative cell numbers were determined by fluorometry using Hoechst 33258-staining of sonicated cells. Briefly, selected 3T3-L1 and NIH-3T3 cells treated for the indicated periods of time were harvested by trypsinization and resuspended and frozen in high salt buffer (10 mM Tris, 10 mM EDTA, 2 M NaCl) (pH 7.4). Before measurement, the cells were thawed, sonicated and diluted in high salt buffer (with only 2 mM EDTA) containing Hoechst 33258 (0.1 µg/ml). Samples were measured using a DyNA Quant^TM 200 (Hoefer). All samples were diluted to be in the linear range upon measurement. BrdUrd incorporations were carried out using the 5-Bromo-2'-deoxy-uridine Labeling and Detection Kit I according to the instructions of the manufacturer (Roche Molecular Biochemicals).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Ligand-dependent Adipocyte Differentiation of PPAR-expressing NIH-3T3 Cells-- Previous work has established PPAR as a major regulator of adipocyte differentiation (28). Forced expression of PPAR was found not to support adipocyte differentiation of NIH-3T3 fibroblasts (34), whereas forced expression of PPAR in 3T3-C2 cells was reported to promote adipocyte differentiation in a manner depending on the sequential administration of fatty acids and a PPAR-selective ligand (35). Accordingly, we decided to re-investigate the differentiation potential of PPAR in NIH-3T3 cells using retrovirus-mediated expression together with a standard differentiation protocol including the combined treatment with dexamethasone, MIX, and insulin (in this study referred to as DMI treatment). Expression of PPAR in the selected NIH-3T3 cells was verified by RT-PCR and Western blotting. Cells transduced with the empty virus (NIH-vector cells) expressed moderate and low levels of endogenous PPAR mRNA and protein, respectively, and the cells transduced with virus encoding PPAR exhibited a significant increase in the levels of PPAR mRNA and protein (data not shown).

Initially, we employed 2-bromopalmitate as ligand in the treatment of NIH-PPAR cells. DMI treatment alone or in combination with 2-bromopalmitate promoted no accumulation of lipid in NIH-vector cells as determined by Oil Red O staining (Fig. 1, middle row of dishes). This shows that no differentiation of the NIH-3T3 cells occurred in the absence of ectopic expression of PPARs when treated with the DMI differentiation protocol in combination with 2-bromopalmitate or other ligands (Fig. 1 and see below). However, we observed a strong ligand-dependent lipid accumulation in NIH-PPAR cells in response to treatment with DMI and 2-bromopalmitate (Fig. 1, upper row of dishes). Thus, forced expression of PPAR promotes adipocyte differentiation of NIH-3T3 cells.

View larger version (29K): [in this window] [in a new window]   Fig. 1.   Adipose conversion of PPAR-expressing NIH-3T3 cells. Two-day postconfluent NIH-PPAR and NIH-vector cells were treated with dexamethasone and insulin in the absence or presence of ligands and MIX. The upper two rows of dishes were treated in the presence of MIX (+MIX), whereas the lower row of dishes was treated in the absence of MIX (MIX). Ligands used were 2-bromopalmitate (BrPal), L-165041, and BRL49653. Cells were stained with Oil Red O on day 8. Shown are whole stained dishes.

To molecularly characterize the adipose conversion of PPAR-expressing NIH-3T3 cells, we performed a time-course analysis of gene expression by multiplex RT-PCR and Western blotting. In accordance with the lipid-accumulating phenotype of the NIH-PPAR cells treated with DMI and 2-bromopalmitate, induction of PPAR2 and GPDH mRNAs was observed (Fig. 2A). At the protein level, this was accompanied by induction of PPAR2 and adipocyte lipid-binding protein (ALBP)/aP2 (Fig. 2B). It is noteworthy that no PPAR1 mRNA was detectable, implying that only the PPAR2 promoter was active in the NIH-PPAR adipocytes (Fig. 2A). C/EBP mRNA is induced to only very low levels in the differentiated NIH-PPAR cells, an observation consistent with previous findings (40, 41). A slight induction of PPAR2 mRNA was observed in NIH-PPAR cells treated with DMI in the absence of ligand, but protein accumulation was below the detection limit as determined by Western blotting. In NIH-vector cells treated with DMI and 2-bromopalmitate (or other ligands), expression of adipocyte marker genes was very low, consistent with the lack of differentiation (data not shown and see Fig. 3).

View larger version (34K): [in this window] [in a new window]   Fig. 2.   Expression of adipocyte markers during 2-bromopalmitate-induced differentiation of NIH-PPAR cells. Cells were induced to differentiate with DMI in the presence or absence of 2-bromopalmitate (BrPal). A, RNA was isolated on the indicated days of the differentiation program, and the expression of PPAR2 (25 cycles), PPAR1 (25 cycles), C/EBP (25 cycles), GPDH (22 cycles), and TBP (22 or 25 cycles) was analyzed by multiplex RT-PCR. B, whole cell extracts were prepared on the indicated days, and the expression of PPAR, ALBP/aP2, and TBP was analyzed by Western blot analysis. DMSO, Me2SO.

View larger version (68K): [in this window] [in a new window]   Fig. 3.   Expression of adipocyte markers during differentiation of NIH-PPAR cells induced by a selective PPAR ligand. NIH-vector and NIH-PPAR cells were induced to differentiate with DMI in the absence or presence of ligands. RNA was isolated on the indicated days of the differentiation program, and the expression of PPAR2 (22 cycles), PPAR1 (22 cycles) (not detectable and not shown), C/EBP (25 cycles), ALBP/aP2 (17 cycles), GPDH (18 cycles), -actin (17 or 18 cycles), and TBP (22 or 25 cycles) was analyzed by multiplex RT-PCR. Ligands used were L-165041 and BRL49653. DMSO, Me2SO.

A PPAR-selective Ligand Supports Adipose Conversion of NIH-PPAR Cells-- The ligand used above to demonstrate adipose conversion of NIH-PPAR cells was 2-bromopalmitate, an activator of all three PPAR subtypes (34). Although 2-bromopalmitate did not promote the differentiation of NIH-vector cells (see Fig. 1), it has been shown to cause adipose conversion of NIH-3T3 cells ectopically expressing PPAR (34). Therefore, to determine to what extent the adipogenic effect of 2-bromopalmitate on NIH-PPAR cells was mediated by the activation of ectopically expressed PPAR relative to endogenous PPAR, we next employed a ligand selective for PPAR. L-165041 is a nonthiazolidinedione agonist of PPARs with selectivity toward PPAR (15). We used L-165041 at a concentration shown to activate only the PPAR subtype in COS-1 cells (15). The majority of NIH-PPAR cells treated with DMI and 0.5 µM L-165041 underwent adipocyte differentiation (Fig. 1, upper row of dishes). In a separate experiment, we demonstrated that treatment with DMI and 0.1 µM L165041 also induced significant adipose conversion of NIH-PPAR cells (data not shown). Even though 0.5 µM L-165041 is a more potent PPAR activator than 30 µM 2-bromopalmitate (see Fig. 5A), 2-bromopalmitate apparently was a more potent inducer of terminal differentiation as visualized by Oil Red O staining (Fig. 1, upper row of dishes). This most likely relates to the ability of 2-bromopalmitate to activate both PPAR and PPAR, whereby 2-bromopalmitate first activates the ectopically expressed PPAR, which in turn promotes the expression of PPAR. PPAR is then also activated by 2-bromopalmitate, thereby strongly promoting terminal differentiation. As a further control, we treated NIH-PPAR cells with the PPAR-selective ligand BRL49653. Interestingly, when BRL49653 was added at a concentration sufficient to activate only PPAR (0.5 µM) (13, 42, 43), a small number of NIH-PPAR cells differentiated. However, the percentage of cells that accumulated lipid was markedly lower than that of cells treated with 0.5 µM L-165041 (Fig. 1, upper row of dishes). Increased adipocyte differentiation of NIH-PPAR cells treated with BRL49653 may relate to the observation that NIH-PPAR cells treated with DMI (in the absence of ligands) express higher levels of PPAR2 than similarly treated NIH-vector cells (Fig. 3). Therefore, the moderate BRL49653-induced differentiation may be initiated by the activation of the endogenous PPAR expressed in NIH-PPAR cells following the treatment with DMI. NIH-vector cells did not differentiate when treated with DMI in combination with either L-165041 or BRL49653 (Fig. 1, middle row of dishes). Consistent with the morphological differentiation, PPAR2, ALBP/aP2, and GPDH mRNAs were robustly induced in NIH-PPAR cells treated with L-165041 or BRL49653 (Fig. 3).

The expression of PPAR2, C/EBP, ALBP/aP2, and GPDH mRNAs was significantly higher at day 3 in NIH-PPAR cells treated with L-165041 compared with treatment with BRL49653 (Fig. 3). Despite the lower number of differentiated cells in response to BRL49653 compared with L-165041, these markers were expressed at equal levels at day 8 (Fig. 3). This might be due to the observation that PPAR is a much more powerful regulator of adipocyte marker genes than PPAR (34). As mentioned above, in the BRL49653-induced differentiation of NIH-PPAR cells, the function of the ectopically expressed PPAR is most likely to assist in the induction of the endogenous PPAR2 gene, after which the simultaneous presence of low levels of PPAR2 and BRL49653 allows the ligand-activated PPAR2 to promote the expression of adipocyte-specific genes and terminal differentiation. This then implies that the NIH-PPAR cells that differentiate in response to BRL49653 (on a per cell basis) express higher levels of adipocyte marker genes than NIH-PPAR cells differentiated with L-165041, in which the expression of PPAR2 is high, but not maximally activated due to the absence of exogenously added PPAR ligand. Consistent with the lack of differentiation, the adipocyte marker transcripts are induced to much lower levels in NIH-vector cells irrespectively of the ligand used (Fig. 3).

PPAR-mediated Adipose Conversion Is Dependent on Increased cAMP Levels-- A notable difference between our differentiation protocol supporting adipocyte differentiation of NIH-PPAR cells and that used in a previous study (34) is our use of MIX. To test the importance of MIX in the differentiation of NIH-PPAR cells, these were induced to differentiate in medium containing dexamethasone, insulin, and ligand (2-bromopalmitate, L-165041, or BRL49653) in the presence or absence of MIX.

Adipose conversion of NIH-PPAR cells was completely suppressed in the absence of MIX, irrespectively of the ligand used (Fig. 1, compare upper and lower rows of dishes). Thus, MIX appears crucial for adipocyte differentiation of NIH-PPAR cells. Replacement of MIX with either forskolin (100 µM) or 8-(4-chlorophenylthio)-cAMP (100 µM) was compatible with adipose conversion of NIH-PPAR (but not NIH-vector) cells (data not shown). This confirms that an increased level of cAMP was the important additional differentiation stimulus required for the NIH-PPAR cells to differentiate. To further characterize the effects of MIX on NIH-PPAR cells, we compared the expression of selected genes following stimulation with dexamethasone, insulin, and L-165041 (0.5 µM) in the absence or presence of MIX. PPAR2, ALBP/aP2, and GPDH mRNAs were barely detectable in the absence of MIX, whereas they were robustly induced when MIX was present (Fig. 4A). Similarly, Western blotting revealed that PPAR2 protein was induced only in the presence of MIX (Fig. 4B). Similar results were obtained when L-165041 was replaced with 2-bromopalmitate (see Fig. 1 and data not shown).

View larger version (47K): [in this window] [in a new window]   Fig. 4.   Effect of MIX on the expression of adipocyte markers during differentiation of PPAR-expressing NIH-3T3 cells. Cells were differentiated using dexamethasone, insulin and L-165041 (0.5 µM) in the absence (MIX) or presence (+MIX) of MIX. A, RNA was isolated at the indicated days. Expression of PPAR2, PPAR1, ALBP/aP2, GPDH, -actin, and TBP was analyzed by multiplex RT-PCR as described in the legend to Fig. 3. B, whole cell extracts were prepared at the indicated days. Expression of PPAR and TBP was analyzed by Western blot analysis. DMSO, Me2SO.

cAMP Signaling Potentiates Ligand-dependent PPAR-mediated Transactivation in NIH-3T3 Cells-- Given the striking dependence of MIX for adipose conversion of PPAR-expressing NIH-3T3 cells (see Fig. 1), we decided to examine whether increased levels of cAMP modulate PPAR-mediated transactivation. NIH-3T3 cells were transiently transfected with expression plasmids encoding full-length PPAR and RXR together with a PPRE reporter plasmid. The cells were then treated with ligand (2-bromopalmitate or L-165041) in the presence or absence of MIX. Addition of MIX nearly doubled the ligand-dependent transcriptional activity of PPAR using either 2-bromopalmitate or L-165041 as ligands (Fig. 5A). To analyze whether a similar effect was detectable at the level of endogenous PPAR, NIH-3T3 cells were transfected with the PPRE reporter plasmid alone followed by treatment with ligand and/or MIX. Whereas ligands and MIX only marginally activated the reporter when added one at a time, the simultaneous presence of ligands and MIX resulted in a dramatic synergistic activation (Fig. 5B). Furthermore, similar effects as those described in Fig. 5 were observed when MIX was replaced with forskolin (data not shown). These results demonstrate that the transcriptional activity of PPAR is regulated synergistically by ligand and cAMP-dependent signaling. Furthermore, this synergy is observed using either ectopically expressed or endogenous PPAR. These results could at least in part explain the requirement of cAMP-elevating agents for the differentiation of NIH-PPAR cells described above.

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Transactivation by ectopically expressed and endogenous PPAR in NIH-3T3 cells. A, NIH-3T3 cells were transiently transfected with expression vectors encoding PPAR, RXR, and -galactosidase, together with a PPRE luciferase reporter plasmid. B, NIH-3T3 cells were transiently transfected with a PPRE luciferase reporter plasmid and an expression vector encoding -galactosidase. In both A and B, the cells were exposed to either 2-bromopalmitate (BrPal) (30 µM) or L-165041 (0.5 µM) in the absence or presence of MIX (0.5 mM). Twenty-four hours later, cells were lysed and assayed for luciferase and -galactosidase activities. Luciferase values were normalized to -galactosidase activities. The normalized value obtained from cells treated with vehicle solvents only was set as 1. Each transfection was performed in triplicate and repeated at least three times. Due to the cotransfected PPAR and RXR expression vectors, the absolute transactivation in column 1 in A was 19-fold higher than column 1 in B.

A PPAR-selective Ligand Modulates the Expression of PPAR and Adipose Conversion in 3T3-L1 Cells in a MIX-dependent Manner-- As shown by others in 3T3-C2 cells (35) and above in NIH-3T3 cells, ectopic overexpression of PPAR is able to support adipose conversion of non-preadipocyte fibroblasts. Whereas the strong adipogenic effect of PPAR-selective ligands on preadipocyte cell lines is well established (13, 43), the effects of selective PPAR ligands on preadipocyte gene expression and differentiation are poorly described. As we observed a synergistic effect of ligands and cAMP-elevating agents on transactivation mediated by endogenous PPAR/RXR (see Fig. 5B), we decided to analyze the effects of ligands and/or MIX in 3T3-L1 preadipocytes. Confluent cells were treated for 4 days with ligands selective for either PPAR or PPAR (L-165041 and BRL49653, respectively) in the absence or presence of MIX. On day 4, the expression of PPAR and ALBP/aP2 mRNAs was measured. MIX or L-165041 alone only marginally induced the expression of PPAR, whereas BRL49653 by itself was more potent (Fig. 6, A and B). The combined treatment with MIX and L-165041 led to a significant 2-fold increase in PPAR expression. Simultaneous administration of MIX and BRL49653 moderately blunted the response obtained by BRL49653 alone (Fig. 6, A and B). The synergy between MIX and L-165041 observed on the expression of PPAR also applied to the expression of ALBP/aP2 (Fig. 6, A and C). As for PPAR expression, addition of MIX moderately reduced the BRL49653-induced expression of ALBP/aP2 (Fig. 6, A and C). The induction of ALBP/aP2 by BRL49653 was much more pronounced than that obtained with L-165041/MIX. This may in part relate to the higher affinity of PPAR compared with PPAR for the PPREs in the ALBP/aP2 enhancer (34).

View larger version (22K): [in this window] [in a new window]   Fig. 6.   Effects of a PPAR-selective ligand and MIX on adipocyte-specific gene expression and differentiation in 3T3-L1 preadipocytes. A, confluent 3T3-L1 preadipocytes were treated for 4 days with L-165041 (0.5 µM) or BRL49653 (0.5 µM) in the absence or presence of MIX (0.5 mM). RNA was harvested on day 4, and the expression of PPAR (20 cycles), TBP (20 cycles), ALBP/aP2 (18 cycles), and -actin (18 cycles) was analyzed by multiplex RT-PCR. B, quantification of PPAR expression in three independent experiments (± S.E.) similar to that shown in A. In each experiment the normalized expression of PPAR in cells treated with BRL49653 and MIX is set as 1. C, quantification of ALBP/aP2 expression in three independent experiments (± S.E.) similar to that shown in A. In each experiment the normalized expression of ALBP/aP2 in cells treated with BRL49653 and MIX is set as 1. D, 3T3-L1 cells treated for 4 days as described in A were cultured for an additional 4 days in the presence of only DMEM containing 10% FBS. On day 8, the cells were stained with Oil Red O to visualize lipid accumulation. DMSO, Me2SO.

To address whether the synergistic activation of PPAR and ALBP/aP2 expression by MIX and L-165041 in 3T3-L1 preadipocytes also resulted in increased terminal differentiation, cells cultured in parallel to those used in Fig. 6A were maintained for an additional 4 days (in the absence of MIX and ligands), followed by staining with Oil Red O to visualize accumulated lipid (Fig. 6D). As for the expression of PPAR and ALBP/aP2, MIX or L-165041 alone only slightly promoted lipid accumulation. Combined treatment with MIX and L-165041 resulted in an accumulation of lipid that significantly exceeded that observed with either compound alone, although the total number of differentiated cells still was moderate (Fig. 6D). Consistent with the expression of ALBP/aP2, BRL49653 was a very potent inducer of adipogenesis, with MIX moderately attenuating the BRL49653-induced differentiation (Fig. 6D). The observation that BRL49653 was much more adipogenic than the combined treatment with L-165041 and MIX, even though the expression of PPAR mRNA was approximately equal (see Fig. 6B), is most likely related to a much higher activity of PPAR in BRL49653- compared with L-165041/MIX-treated cells. The observation that MIX markedly increased the effects of L-165041, whereas it decreased the effects of BRL49653 strongly suggest that L-165041 was not working through activation of PPAR in these cells. The synergistic effects observed with MIX and L-165041 described above were specific to MIX as replacement of MIX with dexamethasone revealed no effects on either gene expression (PPAR and ALBP/aP2) or differentiation exceeding simple additivity (data not shown). These results demonstrate that activation of endogenous PPAR by ligand and a cAMP-elevating agent in preadipocytes is able to moderately promote adipocyte-specific gene expression and differentiation, the latter possibly indirectly via induction of PPAR expression.

Activation of PPAR Promotes Mitotic Clonal Expansion-- Following stimulation with adipogenic inducers, density-arrested preadipocytes reenter the cell cycle and undergo clonal expansion (reviewed in Refs. 1 and 2). As PPAR has been linked to cell proliferation in the colon (44), we decided to analyze the effects of selective activation of PPAR on clonal expansion in 3T3-L1 cells transduced with either PPAR or control virus. Cell numbers were determined by measuring total DNA on days 0 and 4, before and after completion of the phase of clonal expansion (39). The number of 3T3-L1-vector and 3T3-L1-PPAR cells did not differ significantly at confluence (day 0) (Fig. 7A). In plates with 3T3-L1-vector cells, treatment for 4 days with either Me₂SO or L-165041 did not increase cell numbers (Fig. 7A). In 3T3-L1-PPAR plates, treatment with L-165041 increased cell numbers by a moderate 25% relative to Me₂SO-treated plates. MIX moderately increased cell numbers for both 3T3-L1-vector and 3T3-L1-PPAR cells (Fig. 7A). The combined treatment of 3T3-L1-vector cells with MIX and L-165041 led to a 20% increase in cell numbers compared with cells treated with only MIX, demonstrating that full activation of endogenous PPAR by both MIX and a PPAR ligand moderately promotes clonal expansion. 3T3-L1-PPAR cells treated with both MIX and L-165041 responded by a 70% increase in cell numbers compared with cells treated with only MIX (Fig. 7A). These data suggest that the magnitude of the proliferative response obtained by activation of PPAR is correlated with the level of PPAR expression. In agreement with this, we observed that treatment of NIH-vector and NIH-PPAR cells with DMI in combination with L-165041 resulted in 15% and 50%, respectively, increases in cell numbers on day 4 compared with cells treated with only DMI (data not shown). To confirm that the increased cell numbers were due to entry of the density-arrested preadipocytes into the cell cycle, we measured incorporation of BrdUrd in 3T3-L1-PPAR cells. We labeled the cells between 12 and 24 h after stimulation with the indicated inducers, the interval during which cells normally enter the first round of clonal expansion (39). After BrdUrd labeling, the cells were fixed and processed. Compared with vehicle, treatment with L-165041 or MIX alone did not significantly increase the number of cells reentering the cell cycle (Fig. 7B). Combined treatment with MIX and L-165041 resulted in a dramatic increase in BrdUrd-incorporating cells, with more than 50% of the cells staining positive (Fig. 7B). These results strongly suggest that the increase in cell numbers by activation of PPAR is due to a significant increase in the number of density-arrested preadipocytes reentering the cell cycle. Taken together, these data support a model in which activation of PPAR early during adipose conversion is able to promote the expansion of the pool of cells undergoing differentiation.

View larger version (32K): [in this window] [in a new window]   Fig. 7.   Effect of activation of PPAR on mitotic clonal expansion. A, 3T3-L1-vector and 3T3-L1-PPAR cells were induced to differentiate with or without MIX in the absence or presence of L-165041 (0.5 µM). On day 4, plates were harvested in triplicate and the amount of genomic DNA was measured by fluorometry. Similar results were obtained in three independent experiments. Shown is a quantification of DNA content in a representative experiment (± S.E.). B, 3T3-L1-PPAR cells were treated as indicated and labeled with BrdUrd (BrdU) in the period between 12 and 24 h after the start of the treatment. Cells incorporating BrdUrd in this 12-h period were detected by incubating fixed cells with monoclonal anti-BrdUrd antibody followed by incubation with fluorescein isothiocyanate (FITC)-conjugated anti-mouse antibody (upper panel). The total number of cells was visualized by counterstaining with Hoechst (lower panel). DMSO, Me2SO.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Here we show that ectopic expression of PPAR in NIH-3T3 fibroblasts supports ligand-dependent adipocyte differentiation. PPAR-dependent differentiation was observed using both a nonselective (2-bromopalmitate) and a selective (L-165041) PPAR ligand. PPAR-induced differentiation of NIH-3T3 cells was accompanied by expression of adipocyte-specific genes, among these PPAR2. Moreover, we observed that activation of either endogenous or ectopically expressed PPAR in 3T3-L1 and NIH-3T3 cells promoted mitotic clonal expansion to a degree that paralleled the level of PPAR expression. Although the molecular mechanism governing this proliferative response is unknown at present, our findings suggest a potential role of PPAR in modulating clonal expansion and preadipocyte proliferation. It was recently reported that forced expression of PPAR in 3T3-C2 cells allowed post-confluent cell proliferation in response to treatment with fatty acids (45). No effect was observed in 3T3-C2 cells upon activation of endogenous PPAR. Surprisingly, the cell number at confluence was reduced by 40% in cells overexpressing PPAR compared with control cells. This reduction in cell number at confluence was completely independent of the presence of fatty acids, but dependent on an intact PPAR AF-2 function (45). Therefore, whether the apparent proliferative response of fatty acids in 3T3-C2-PPAR cells reflects a rescue of the reduced cell density at confluence or a true induction of proliferation is unclear. As seen in Fig. 7A, the cell numbers at confluence in our experiments did not vary significantly between vector- and PPAR-transduced cells, thereby avoiding this problem. Moreover, our data in both 3T3-L1 and NIH-3T3 cells were obtained using a selective PPAR ligand, thereby eliminating the risk of non-PPAR-related metabolic effects of treatment with high concentrations of fatty acids.

Efficient adipocyte differentiation of NIH-PPAR cells required the combined treatment with dexamethasone, MIX, insulin, and ligand, and omission of either MIX or ligand dramatically compromised differentiation. Furthermore, in the well characterized 3T3-L1 preadipocyte cell line, a PPAR-selective ligand enhanced the expression of PPAR and ALBP/aP2 early in the differentiation program in a manner dependent on the presence of MIX.

Our results demonstrating PPAR-dependent adipose conversion of NIH-3T3 cells and a positive effect of a PPAR selective ligand on ALBP/aP2 expression in 3T3-L1 cells are in apparent contrast to previous reports (15, 34). However, in neither of these studies were MIX or other cAMP-elevating agents included in the differentiation protocols; thus, these apparently contradictory findings may readily be explained by the requirement for a cAMP-elevating agent to elicit these PPAR-dependent processes.

Previously, MIX was shown to transiently induce C/EBP expression during 3T3-L1 differentiation (46, 47). In NIH-PPAR cells, MIX only very slightly and transiently increases the level of C/EBP protein (data not shown). It is, however, possible that the slight effect of MIX on C/EBP expression contributes to the improved differentiation of NIH-PPAR cells, but it should be emphasized that treatment with MIX in the absence of forced expression of PPAR did not induce any adipose conversion (see Fig. 1, middle row of dishes).

The findings reported in this paper suggest that cAMP signaling, and hence most likely protein kinase A-dependent processes play an important role in PPAR-mediated transactivation. Protein kinase A may, in analogy with its effects on other nuclear hormone receptors, influence heterodimerization (48-50), DNA binding (51), or transactivation (52, 53). Recent reports show that increased cAMP levels diminish or abolish interaction between nuclear hormone receptors and corepressors (26, 54). PPAR and PPAR have been shown to interact with the SMRT (26, 55) and the N-CoR corepressors (56, 57), and we have shown that N-CoR and SMRT interact strongly with PPAR and repress transactivation by PPAR in a ligand-independent manner.² Whether cAMP-dependent enhancement of PPAR-mediated transactivation proceeds via protein kinase A-dependent phosphorylation of PPAR, dissociation of corepressor complexes, recruitment of coactivators, or by other mechanism(s) is currently under investigation. PPAR has recently been associated with various biological functions, including the regulation of cholesterol and lipid metabolism, epidermal differentiation and proliferation, oligodendrocyte differentiation, embryo implantation, and the development of colorectal cancer (36, 44, 58-62). Whether cAMP signaling participates in these PPAR-regulated events is not known at present.

In conclusion, we have demonstrated that ligands and cAMP-elevating agents synergistically enhance PPAR-mediated transactivation, and that such synergistic activation allows adipocyte differentiation of NIH-3T3 fibroblast with forced expression of PPAR. Activation of PPAR by a selective ligand (in combination with MIX) resulted in a significant increase in cell number, suggesting that PPAR activation may play a role in the expansion of the pool of adipocyte precursor cells. The recent finding that PPAR knockout mice have reduced gonadal adipose tissue stores would be in keeping with this notion (36). Synergistic activation of endogenous PPAR in 3T3-L1 preadipocytes enhanced expression of PPAR, but only modestly promoted terminal differentiation. Taken together, our results suggest that PPAR may play a role in regulating preadipocyte proliferation and gene expression, whereas the impact of PPAR on terminal differentiation of preadipocytes at most is modest.

	ACKNOWLEDGEMENTS

We thank Drs. Ronald M. Evans, Bruce M. Spiegelman, and Paul A. Grimaldi for kind gifts of plasmids and Drs. David A. Bernlohr, and Irina Kratchmarova for kind gifts of antibodies. We thank Novo Nordisk and Merck Research Laboratories for providing ligands.

	FOOTNOTES

^* This work was conducted within the Center for Experimental BioInformatics and supported by the Danish Biotechnology Program, the Danish Natural Science Research Council, the Danish Cancer Society, and the Novo Nordisk Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address: Dept. of Environmental Medicine, Inst. of Community Health, University of Southern Denmark, Odense University, DK-5230 Odense M, Denmark.

^§ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, University of Southern Denmark, Odense University, Campusvej 55, DK-5230 Odense M, Denmark. Tel.: 45-65502408; Fax: 45-65502467; E-mail: kak@bmb.sdu.dk.

Published, JBC Papers in Press, November 7, 2000, DOI 10.1074/jbc.M005567200

² A.-M. Krogsdam, C. A. F. Nielsen, T. Helledie, S. Neve, D. Holst, B. Thomsen, C. Bendixen, S. Mandrup, and K. Kristiansen, manuscript in preparation.

	ABBREVIATIONS

The abbreviations used are: C/EBP, CCAAT/enhancer-binding protein; ALBP/aP2, adipocyte lipid-binding protein; bp, base pair(s); BrdUrd, bromodeoxyuridine; DMEM, Dulbecco's modified Eagle's medium; DMI, dexamethasone, methylisobutylxanthine, and insulin; FBS, fetal bovine serum; GPDH, glycerol-3-phosphate dehydrogenase; MIX, methylisobutylxanthine; PPAR, peroxisome proliferator-activated receptor; PPRE, peroxisome proliferator-activated receptor response element; RT-PCR, reverse transcription-polymerase chain reaction; RXR, retinoid X receptor; TBP, TATA-binding protein.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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