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
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ABSTRACT |
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The peroxisome proliferator-activated receptor
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 Activators of PPAR In this report we present a critical evaluation of the potential of
PPAR Plasmids and Transient Transfections--
The retroviral
expression vector pBabe-mPPAR 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 At confluence, puromycin was withdrawn and cells were left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 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 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
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 QuantTM 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).
Ligand-dependent Adipocyte Differentiation of
PPAR
Initially, we employed 2-bromopalmitate as ligand in the treatment of
NIH-PPAR
To molecularly characterize the adipose conversion of
PPAR A PPAR
The expression of PPAR PPAR
Adipose conversion of NIH-PPAR cAMP Signaling Potentiates Ligand-dependent
PPAR A PPAR
To address whether the synergistic activation of PPAR Activation of PPAR Here we show that ectopic expression of PPAR Efficient adipocyte differentiation of NIH-PPAR Our results demonstrating PPAR Previously, MIX was shown to transiently induce C/EBP 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 In conclusion, we have demonstrated that ligands and cAMP-elevating
agents synergistically enhance PPAR (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 PPAR
2 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
, 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).
promote adipocyte differentiation of
preadipocytes and multipotent C3H10T1/2 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.
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
(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.
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.
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).
-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), PPAR
1,
PPAR
2, 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.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-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).
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.
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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.
-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 PPAR
2 and
GPDH mRNAs was observed (Fig.
2A). At the protein level,
this was accompanied by induction of PPAR
2 and adipocyte
lipid-binding protein (ALBP)/aP2 (Fig. 2B). It is noteworthy
that no PPAR
1 mRNA was detectable, implying that only the
PPAR
2 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 PPAR
2 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).
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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 PPAR
2 (25 cycles),
PPAR
1 (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.
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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 PPAR
2 (22 cycles),
PPAR
1 (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.
-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 PPAR
2 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, PPAR
2, ALBP/aP2, and GPDH
mRNAs were robustly induced in NIH-PPAR
cells treated with
L-165041 or BRL49653 (Fig. 3).
2, 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 PPAR
2 gene, after which the simultaneous presence of low
levels of PPAR
2 and BRL49653 allows the ligand-activated PPAR
2 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 PPAR
2 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).
-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.
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. PPAR
2, 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 PPAR
2 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).
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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 PPAR
2, PPAR
1, 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.
-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.
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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.
-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).
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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.
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.
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 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
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 PPAR
2. 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.
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.
-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.
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).
-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.
-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.
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