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J. Biol. Chem., Vol. 277, Issue 6, 4062-4068, February 8, 2002
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From the Department of Biological Sciences, Louisiana State
University, Baton Rouge, Louisiana 70803
Received for publication, September 4, 2001, and in revised form, November 29, 2001
Interferon- PPAR PPAR Interferon- The ubiquitin-proteasome pathway is essential for the degradation of
short lived proteins, the levels of which are regulated constitutively
or in response to changes in the cellular environment (22, 23).
Transcription factors and tumor suppressors are among the proteins
regulated by the ubiquitin-proteasome pathway, and included in this
group are members of the nuclear hormone receptor superfamily (24, 25).
Ligand-dependent down-regulation by the
ubiquitin-proteasome system has been demonstrated for several members
of the nuclear hormone receptor family, including the estrogen (26,
27), progesterone (28), thyroid hormone (29), and aryl hydrocarbon
receptors (30).
Substrates of the ubiquitin-proteasome system are targeted to the
proteasome after covalent attachment of multiple ubiquitin molecules.
Ubiquitin, a 76 amino acid protein, is initially activated by E1, the
ubiquitin-activating enzyme. Activated ubiquitin is then transferred to
a ubiquitin-conjugating enzyme (E2), which generally shuttles ubiquitin
to ubiquitin ligase (E3). E3 is bound to the targeted substrate and
catalyzes the covalent attachment of ubiquitin to the substrate. Once
the first ubiquitin is transferred to the substrate, a
polyubiquitination chain is generated via a series of isopeptide
linkages. The multiubiquitinated substrate protein is then degraded by
the 26 S proteasome in an ATP-dependent manner (31).
Our recent studies (13) have shown that acute IFN Materials--
Dulbecco's modified Eagle's medium (DMEM),
OptiMEM, and fetal bovine serum were purchased from Invitrogen.
Calf serum was purchased from Sigma. Murine IFN Constructs--
The pSVSport plasmids encoding wild-type PPAR Cell Culture--
Murine 3T3-L1 preadipocytes were plated and
grown to 2-days postconfluence in DMEM with 10% calf serum. The medium
was changed every 48 h. Cells were induced to differentiate by
changing the medium to DMEM containing 10% fetal bovine serum and 0.5 mM 3-isobutyl-methylxanthine, 1 µM
dexamethasone, and 1.7 µM insulin (MDI). After 48 h,
this medium was replaced with DMEM supplemented with 10% fetal bovine serum, and the cells were maintained in this medium until used for
experimentation. NIH 3T3 cells were grown in DMEM with 10% calf serum.
Preparation of Whole Cell Extracts--
Cell monolayers were
rinsed with phosphate-buffered saline (PBS) and harvested in a lysis
buffer containing 10 mM Tris-Cl, pH 7.4, 150 mM
NaCl, 1 mM EGTA, 1 mM EDTA, 1% Triton X-100,
0.5% Nonidet P-40, 1 µM phenylmethylsulfonyl fluoride, 1 µM pepstatin, 50 trypsin inhibitory milliunits of
aprotinin, 10 µM leupeptin, and 2 mM sodium
vanadate. Samples were extracted on ice for 30 min prior to
centrifugation at 10,000 × g for 15 min. The resulting supernatants were analyzed for protein content by BCA analysis (Pierce)
according to the manufacturer's instructions and stored at
Preparation of Nuclear/Cytosolic Extracts--
Cell monolayers
were rinsed with PBS and harvested in a nuclear homogenization buffer
(NHB) containing 20 mM Tris-Cl, pH 7.4, 10 mM
NaCl and 3 mM MgCl2. Nonidet P-40 was added to
a final concentration of 0.15%, and the cells were homogenized with 16 strokes in a Dounce homogenizer. The resulting homogenate was
centrifuged at 1500 rpm for 5 min, and the supernatant was saved as
cytosolic extract. The nuclear pellet was twice resuspended in 0.5 volume of a nuclear homogenization buffer and centrifuged as before. The nuclear pellet was then resuspended in an extraction buffer containing 20 mM HEPES pH 7.9, 420 mM NaCl, 0.2 mM EDTA and 25% glycerol. Nuclei were extracted for 30 min
on ice followed by incubation with 200 units of DNase I at room
temperature for 15 min. Finally, the sample was centrifuged at 15,000 rpm for 10 min at 4 °C. The resulting nuclear extract and the
previously obtained cytosolic extract were analyzed for protein content
by BCA analysis (Pierce) according to the manufacturer's instructions and stored at Gel Electrophoresis and Immunoblotting--
Proteins were
separated in 12% polyacrylamide (National Diagnostics) gels containing
SDS according to Laemmli (32) and transferred to nitrocellulose
(Bio-Rad) in 25 mM Tris, 192 mM glycine, and 20% methanol. Following transfer, the membrane was blocked in 4% milk
overnight at 4 °C. The immunoblots were visualized with horseradish
peroxidase-conjugated secondary antibodies (Sigma) and enhanced
chemiluminescence (Pierce).
Transient Transfection and Luciferase Assay--
NIH 3T3 cells
were grown to 60-70% confluence and transiently transfected with
either wild-type PPAR Ubiquitin Conjugation Assay--
NIH 3T3 cells were transfected
with 2 µg of PPAR
3T3-L1 adipocytes were serum-deprived overnight in OptiMEM, followed by
incubation with 10 µM MG132 for 2 h. At the end of 2 h, IFN PPAR Basal and IFN IFN IFN The Role of Ser112 Phosphorylation in the Decay of
PPAR Cellular Location of PPAR The novel observations in this study include the increased
ubiquitin conjugation of PPAR In light of our current findings and the studies cited above (13, 34,
39-41), we have formulated a model for the degradation of
PPAR Our results demonstrate that both PPAR The rapid reduction in PPAR The phosphorylation of PPAR We also investigated the cellular location of the IFN Recent studies (12, 54) have shown that reduced PPAR *
This work was supported by National Institutes of Health
Grant R01DK52968-02 and a research grant from the American Diabetes Association (to J. M. S.).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.
Published, JBC Papers in Press, November 30, 2001, DOI 10.1074/jbc.M108473200
The abbreviations used are:
PPAR
Interferon-
-mediated Activation and
Ubiquitin-Proteasome-dependent Degradation of PPAR in
Adipocytes*
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(IFN) treatment of
adipocytes results in a down-regulation of the peroxisome
proliferator-activated receptor (PPAR). The decrease in
PPAR expression is mediated by inhibition of PPAR
synthesis and increased degradation of PPAR. In this study, we
demonstrate that both PPAR1 and PPAR2 are targeted to the
proteasome under basal conditions and that PPAR1 is more labile than
PPAR2. The IFN-induced increase in PPAR turnover is blocked by
proteasome inhibition and is accompanied by an increase in
PPAR-polyubiquitin conjugates. In addition, IFN treatment results
in the transcriptional activation of PPAR. Similar to ligand-dependent activation of PPAR, IFN-induced
activation was greater in the phosphorylation-deficient S112A
form of PPAR when compared with wild-type PPAR. Moreover, the
inhibition of ERKs 1 and 2 with a MEK inhibitor, U1026, lead to an
inhibition in the decay of PPAR proteins, indicating that serine
phosphorylation influences the degradation of PPAR in fat cells. Our
results also demonstrate that the proteasome-dependent
degradation of PPAR does not require nuclear export. Taken together,
these results indicate that PPAR is targeted to the
ubiquitin-proteasome pathway for degradation under basal conditions and
that IFN leads to an increased targeting of PPAR to the
ubiquitin-proteasome system in a process that is affected by
ERK-regulated serine phosphorylation of PPAR proteins.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 is a member of
the nuclear hormone receptor family, a group of transcription factors
that are activated by small lipophilic ligands (1). PPAR exists as
two isoforms, PPAR1 and PPAR2, which are produced by a
combination of different promoters and alternative splicing (2). There
is also a PPAR3 gene that codes for a protein that is
identical to PPAR1 (3). PPAR1 is predominantly expressed in fat
cells but occurs in low levels in multiple tissues. PPAR2 has an
N-terminal extension of 30 amino acids and is very highly expressed in
adipocytes (4, 5). Deletion of the PPAR gene in mice
results in placental dysfunction and embryonic lethality (6, 7).
has been implicated in the regulation of systemic insulin
sensitivity. This was first demonstrated when PPAR was shown to be a
functional receptor for the synthetic antidiabetic
thiazolidinediones (TZDs) (8).
Thiazolidinediones are specific high affinity ligands for PPAR and
the order of their receptor binding affinities in vitro
mirrors their antihyperglycemic activity in vivo (9). Direct
evidence for the association between PPAR and insulin sensitivity
comes from genetic studies showing that mutations in the ligand-binding
domain of PPAR are associated with severe insulin resistance.
Although not obese, these patients developed type 2 diabetes as well as
early onset hypertension (10). Also, insulin has been shown to acutely
regulate the expression of PPAR in human adipocytes (11), and mice
that only express one copy of the PPAR gene have been
shown to be more sensitive to insulin (12). We have recently
demonstrated that IFN results in a substantial loss of PPAR
expression by regulating two cellular events: 1) targeting PPAR to
the proteasome for degradation, and 2) inhibiting the synthesis of
PPAR (13). Moreover, prolonged IFN treatment of 3T3-L1 adipocytes
also results in the development of insulin resistance (13) and
supports the hypothesis that PPAR is involved in conferring insulin sensitivity.
(IFN) is a cytokine that is primarily known for its
roles in immunological responses but has also been shown to affect fat
metabolism and adipocyte gene expression. In adipocytes, IFN
treatment results in a decrease of lipoprotein lipase (LPL) activity
and increased lipolysis (14). In 3T3-F442 adipocytes, exposure to
IFN results in a decreased expression of lipoprotein lipase and
fatty acid synthase. Also, in various rodent preadipocyte cell lines,
IFN inhibits the differentiation of preadipocytes (15-17). Acute
IFN treatment of cultured and native rat adipocytes results in a
dose- and time-dependent activation of STATs 1 and 3 (18).
Moreover, there are studies (19-21) linking IFN and insulin
resistance in humans. IFN has been implicated in the development of
insulin resistance during viral infections (20), and IFN therapy of
cancer patients has been associated with the development of
hyperglycemia (21).
treatment of
3T3-L1 adipocytes results in a repression of PPAR transcription that
is independent of new protein synthesis. Yet, we also demonstrated that
the half-life of PPAR proteins was shorter following IFN treatment. In the current investigation, we observed that proteasomal inhibitors attenuate the TZD- and IFN-induced decrease in PPAR expression. Moreover, we demonstrate that IFN treatment is
associated with an increase in the formation of polyubiquitin-PPAR
conjugates in 3T3-L1 adipocytes. Together, these data indicate that
IFN signaling results in the increased targeting of PPAR to the
ubiquitin-proteasome system in adipocytes. In addition, we have
shown that like TZDs, IFN increases the transcriptional activity of
PPAR. Also, the IFN-induced activation of a
phosphorylation-deficient mutant of PPAR2 (S112A) is substantially
greater than the IFN activation of wild-type PPAR2. Our results
suggest that phosphorylation of PPAR2 at Ser112
contributes to the targeting of PPAR to the ubiquitin-proteasome pathway. Finally, these studies indicate that the IFN-mediated ubiquitin-proteasome-dependent degradation of PPAR
occurs in the nucleus.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
was purchased from
Roche Molecular Biochemicals. PPAR monoclonal (E-8, no. sc-7273) and
polyclonal (H-100, no. sc-7196) antibodies, Mdm2 monoclonal (SMP14, no.
sc-965) antibody, and a STAT 5A polyclonal (L-20, no. sc-1081) antibody were purchased from Santa Cruz Biotechnology. Monoclonal anti-ubiquitin (no. 13-1600) was purchased from Zymed Laboratories
Inc. The proteasome inhibitors epoxomicin, lactacystin, and MG132
(N-carbobenzoxyl-Leu-Leu-Leucinal) were purchased from Boston
Biochemicals. A luciferase assay system, pSV-
-galactosidase control
vector, and a -galactosidase enzyme assay kit were purchased from
Promega. FuGENE 6 was purchased from Roche Molecular Biochemicals.
Darglitazone was kindly provided by Pfizer.
and the S112A PPAR mutant as well as DR-1 luciferase were the
generous gift of Dr. Bruce Spiegelman (Dana Farber Cancer Institute).
The HA-ubiquitin plasmid and leptomycin B (LMB) were kindly provided
by Dr. Dirk Bohmann (European Molecular Biology Laboratories) and
Dr. Minoru Yoshida (The University of Tokyo), respectively.
80 °C.
80 °C.
2 or PPAR2 S112A. To measure PPAR
activity, the cells were cotransfected with DR-1 luciferase and
pSV--galactosidase to normalize for transfection efficiency. FuGENE
6 was used according to the manufacturer's instructions and a FuGENE 6 to DNA ratio of 3:2 was used in the transfections. Transient
transfections were carried out in OptiMEM for 8 h. After 8 h,
the media were replaced with DMEM supplemented with 10% calf serum,
and the cells were incubated overnight. Twenty-four hours after
transfection, the cells were treated with IFN (100 units/ml) or
darglitazone (TZD) (2.5 µM), and the cells were harvested 6 h later. Cell lysates were prepared and analyzed for luciferase activity and -galactosidase activity according to the
manufacturer's instructions (Promega). PPAR transcriptional
activity was reported as the ratio of luciferase activity (relative
light units) to -galactosidase activity.
alone or in combination with 4 µg of
HA-ubiquitin per 100 mm plate using FuGENE 6 as described above. After
24 h, the cells were treated with 10 µM MG132 for
2 h prior to the addition of IFN (100 units/ml). The cells were
harvested after 15- and 30-min incubations and lysed on ice in PBS, pH
7.0, containing 1% Triton X-100, 10 mM N-ethylmaleimide, 1 mM phenylmethylsulfonyl fluoride, 1 µM
pepstatin and 10 µM leupeptin. Immunoprecipitations were
performed by incubation with a polyclonal anti-PPAR followed by
incubation with protein A-Sepharose (RepliGen). PPAR-ubiquitin
complexes were detected by Western blotting with an anti-HA antibody.
(100 units/ml) was added, and the cells were harvested after 15- and 30-min incubations and lysed on ice in PBS containing 1%
Triton X-100, 10 mM N-ethylmaleimide, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 µM pepstatin, and 10 µM leupeptin.
Immunoprecipitations were performed with a polyclonal anti-PPAR, and
PPAR-ubiquitin complexes were detected by Western blotting using
both anti-PPAR (monoclonal) and anti-ubiquitin antibodies.
Stability in Vivo--
Experiments using 3T3-L1
adipocytes were carried out in the presence or absence of cycloheximide
(5 µM) to examine the effect of IFN on the half-life
of PPAR proteins. The half-lives of PPAR1 and PPAR2 were
calculated based on first order decay after quantitation of Western
blot data using Un-Scan-It software (Silk Scientific, Inc). IFN was
added at 100 units/ml and darglitazone was added at 2.5 µM, where indicated. The adipocytes were incubated with
one of three proteasome inhibitors (5 µM lactacystin, 100 nM epoxomicin, or 10 µM MG132) in experiments
designed to assay proteasome targeting of PPAR. In these
experiments, the cells were preincubated with the proteasome inhibitor
for 15-30 min prior to adding the ligand or cycloheximide. A MAPK/ERK
kinase (MEK) inhibitor, U0126 (5 µM), was used to assay
involvement of ERK1/2 in the turnover of PPAR, and the cells were
preincubated with U0126 for 30-45 min. Leptomycin B (10 nM) was added as an inhibitor of
CRM-1-dependent nuclear export (33). Cells were pretreated
with leptomycin B for 0.5-1 h prior to the addition of ligand or
cycloheximide. Vehicle control additions were performed with either
Me2SO (for proteasome inhibitors, TZDs, and U0126) or ethanol (for leptomycin B).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-mediated Targeting of PPAR to the
Proteasome--
We have previously shown that treatment of 3T3-L1
adipocytes with IFN leads to a decrease in the half-life of both
PPAR proteins (13). Recent studies by Spiegelman and co-workers (34) have shown that TZDs target PPAR for proteasome-mediated
degradation. These results suggest that targeting to the proteasome is
an important regulatory event in the control of PPAR expression.
Therefore, we examined PPAR expression in the presence of three
distinct proteasome inhibitors. As shown in Fig.
1, treatment of 3T3-L1 adipocytes with
either epoxomicin, lactacystin, or MG132 resulted in an increase in the
levels of PPAR proteins under basal conditions or in the presence of
IFN or TZD. Lactacystin and epoxomicin are highly specific
proteasome inhibitors and confirm that the observed effects on
degradation are due to proteasomal targeting (35, 36). As shown in Fig.
1, under steady-state conditions, IFN treatment of 3T3-L1 adipocytes
leads to a substantial loss of PPAR when compared with control
levels. The decrease in PPAR after IFN treatment is slightly
greater than the decrease associated with the presence of synthetic
ligand (TZD). Inhibition of the proteasome substantially reduces the
IFN-induced decrease in PPAR expression. These results indicate
that the loss of PPAR following IFN treatment is mediated by the
targeting of PPAR to the 26 S proteasome. Interestingly, PPAR
levels in both IFN- and TZD-treated adipocytes in the presence of
proteasome inhibitors are less than the control levels under the same
conditions. This result is consistent with studies that demonstrate
that both IFN and TZDs can also down-regulate PPAR at the
mRNA level (13, 37).
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Fig. 1.
PPAR is targeted to
the proteasome under basal conditions and after IFN
or TZD treatment. Whole cell extracts were prepared from
fully differentiated 3T3-L1 adipocytes that were untreated or treated
with 100 units/ml IFN or 2.5 µM TZD. Proteasome
activity was inhibited with epoxomicin (100 nM),
lactacystin (5 µM), or MG132 (10 µM).
Steady-state levels of PPAR were measured after 6 h. One
hundred micrograms of each extract was separated by SDS-PAGE,
transferred to nitrocellulose, and subjected to Western blot analysis.
The molecular mass of each protein is indicated to the left of the blot
in kDa. The detection system was horseradish
peroxidase-conjugated secondary antibodies (Sigma) and ECL (Pierce).
This was a representative experiment independently performed three
times.
-mediated Ubiquitin-PPAR
Conjugation--
Ubiquitin-proteasome-dependent degradation of
a substrate requires two separate steps. First, the substrate is
targeted to the proteasome via covalent tagging of the substrate with a
polyubiquitin chain. The polyubiquitin-conjugated substrate is then
recognized by the 26 S proteasome (22). These polyubiquitin-substrate
conjugates are short lived, high molecular mass intermediates of the
ubiquitin-proteasome pathway. Because IFN affects PPAR decay and
this effect can be modulated by proteasome inhibitors, we hypothesized
that there would be an increase in polyubiquitin-PPAR conjugates
after IFN treatment. To test this theory, we examined the formation
of endogenous PPAR-ubiquitin adducts in 3T3-L1 adipocytes. PPAR
proteins were immunoprecipitated from whole cell extracts that had been
incubated in the presence or absence of IFN for the times indicated
in Fig. 2. The immunoprecipitations were
analyzed by immunoblotting using either an anti-PPAR antibody (Fig.
2A) or an anti-ubiquitin (Fig. 2B) antibody. As
shown in Fig. 2, PPAR was detected in high molecular mass forms that
are present under basal conditions and with increased intensity after
IFN treatment. We also ectopically expressed octameric HA-tagged
ubiquitin and PPAR2 in NIH 3T3 cells and observed ubiquitin
conjugation of PPAR under basal conditions and a significant
increase in PPAR ubiquitin conjugation following IFN treatment
(data not shown).
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Fig. 2.
IFN treatment is
associated with an increase in PPAR-ubiquitin
conjugates in 3T3-L1 adipocytes. Fully differentiated 3T3-L1
adipocytes were treated with IFN (100 units/ml) for 15 and 30 min
after preincubation with MG132 (20 µM) for 2 h.
Control samples were incubated for the same time period without the
addition of IFN. Whole cell extracts were harvested and
immunoprecipitations were performed as described under "Experimental
Procedures" using anti-PPAR. Western analysis was performed using
either anti-PPAR (left) or anti-ubiquitin
(right). HC represents IgG heavy chain.
-mediated Activation of PPAR--
Based on our previous
studies showing that IFN treatment of cultured adipocytes has the
dual effect of suppressing PPAR transcription and increasing PPAR
turnover (13), we hypothesized that IFN treatment may also decrease
the transcriptional activity of PPAR. To test this prediction, we
assayed the transcriptional activity of PPAR in NIH 3T3 cells using
a luciferase reporter (DR1 luciferase) construct containing three
PPAR response elements. This construct has previously been used to
measure PPAR activity (34, 38). In this experiment, we also examined
the effect of IFN on the transcriptional activity of the
phosphorylation-deficient PPAR2 S112A mutant. Numerous studies have
shown that this mutant is more transcriptionally active and that
phosphorylation at this site is associated with reduced PPAR
activity (39-41). To measure PPAR activity, NIH 3T3 cells were
transiently cotransfected with DR1 luciferase and PPAR2 or PPAR2
S112A in pSVSport vectors in the presence and absence of IFN or TZD.
As shown in Fig. 3, IFN treatment
activates PPAR2 to the same extent as the
ligand-dependent activation associated with TZD treatment.
In addition, activity of the PPAR2 S112A was greater than wild-type
PPAR2, and the transcriptional activity of the mutant was also
significantly induced by IFN treatment. However, the mutant was more
potently activated by TZD treatment.
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Fig. 3.
IFN mediates
transcriptional activation of both wild-type PPAR
and PPAR S112A. NIH 3T3 cells were
cotransfected with pDR1-luciferase and either wild-type PPAR or
PPAR S112A. The cells were also transfected with
pSV--galactosidase to correct for variability in transfection
efficiency. After 24 h, the cells were incubated with IFN (100 units/ml) or darglitazone (TZD, 2.5 µM) and
harvested 6 h later. PPAR transcriptional activity was
determined by calculating the ratio of luciferase activity (relative
light units, RLU) to -galactosidase activity. The
experiment was independently performed in duplicate.
--
Because IFN and TZDs both activate PPAR and target
it for degradation, we hypothesized that regulators of PPAR
activation could also contribute to PPAR degradation. Therefore, we
examined the contribution of PPAR Ser112 phosphorylation
on PPAR degradation because phosphorylation at this site has
profound effects on PPAR activation. Fully differentiated 3T3-L1
adipocytes were pretreated with the MEK inhibitor, U0126, prior to the
addition of IFN or a vehicle control. Turnover of PPAR was then
measured in the presence or absence of cycloheximide. As shown in Fig.
4A, the turnover of both
PPAR1 and 2 was prolonged in the presence of the MEK inhibitor
(control + MEK I). We also observed that inhibition of
ERK1/2 activity abrogates the IFN-mediated decrease in the half-life
of PPAR (Fig. 4A, IFN + MEK I). The results
in Fig. 4A clearly demonstrate that the presence of the MEK
inhibitor suppresses the decay of PPAR proteins in adipocytes under
control and IFN-treated conditions. We also examined the effect of
IFN and/or MEK I on PPAR levels in the absence of cycloheximide.
The results in Fig. 4B confirm that ERKs 1 and 2 and play a
role in degradation of PPAR proteins under basal as well as
IFN-mediated conditions. In Fig. 4, A and B,
the expression of STAT 5A is shown as a loading control. The results in
Fig. 4A also indicate that the decay of PPAR is much
quicker than the decay of PPAR2. Therefore, we performed an
additional decay experiment to compare the decay of 1 and 2.
Fully differentiated 3T3-L1 adipocytes were treated with cycloheximide,
and whole cell extracts were isolated at various times over a 6 h
period. Fig. 5A shows the
decay of PPAR proteins under basal conditions. The 1 and 2
half-lives were calculated to be 58 min and 1.45 h, respectively.
The bottom panel of Fig. 5A represents an
enlarged display of four of the time points from the top
panel. As shown in this panel, we were also able to resolve the
two bands of PPAR1, which represent the
Ser112-phosphorylated (upper band) and
unphosphorylated forms of the protein. The decay experiment in Fig.
5A clearly demonstrates that PPAR1 is more labile than
2. In addition, the unphosphorylated 1 disappears quicker than
the phosphorylated form of 1. This pattern was also observed in the
presence of IFN. (Fig. 5B).
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Fig. 4.
Inhibition of ERK1/2 prolongs the half-life
of PPAR proteins in adipocytes. PPAR
expression was measured in the presence of 5 µM
cycloheximide (CH) (A) or under steady-state
conditions (B) under control or IFN (100 units/ml)-treated conditions. Where indicated, the 3T3-L1 adipocytes
were pretreated for 45 min with the MEK inhibitor (MEK I), U0126 (5 µM). B, the cells were harvested after a 2-h
incubation in the presence or absence of IFN. One hundred micrograms
of each extract was separated by SDS-PAGE, transferred to
nitrocellulose, and subjected to Western blot analysis. Samples were
processed, and results were visualized as described in the Fig. 1
legend. The molecular mass of each protein is indicated to the left of
the blots in kilodaltons. This was a representative experiment
independently performed three times.
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Fig. 5.
PPAR 1 is more labile
than PPAR2, and the higher mobility form of
PPAR1 decays after the lower mobility
form. A, whole cell extracts were prepared from fully
differentiated 3T3-L1 adipocytes after incubation in the presence of 5 µM cycloheximide for the indicated time points.
Incubations were carried out in the absence (A) or presence
(B) of IFN (100 units/ml). The lower panel
(A) is an enlargement of the indicated time points from the
upper panel. One hundred micrograms of each extract was
separated by SDS-PAGE, transferred to nitrocellulose, and subjected to
Western blot analysis. Samples were processed, and results were
visualized as described in the Fig. 1 legend. This was a representative
experiment independently performed three times.
Degradation--
The majority of
PPAR proteins are found in the nucleus, and this raises the
possibility that the nuclear, rather than cytosolic, ubiquitin-proteasome components may mediate the degradation of PPAR.
To address this question, we treated 3T3-L1 adipocytes with IFN
alone or in the presence of either MG132 or leptomycin B (Fig.
6). LMB acts as an irreversible inhibitor
of the CRM-1-dependent nuclear export pathway via the
modification of Cys529 of CRM-1 (33) and has been used to
determine whether nuclear export is required for the degradation of
nuclear proteins (42-44). We examined the decay of PPAR proteins
following IFN treatment in the presence of either MG132 or LMB. The
results in Fig. 6 indicate that MG132 prolongs the half-life of PPAR
proteins, and the presence of LMB has no effect on PPAR decay. To
confirm LMB activity, we assayed the cellular location of Mdm2 in
3T3-L1 adipocytes in the absence or presence of LMB. Mdm2 has been
characterized as an ubiquitin ligase (E3) that shuttles between the
nucleus and cytoplasm and is required for the degradation of p53 (22). Although p53 expression is down-regulated during differentiation of
3T3-L1 adipocytes, Mdm2 expression is maintained in fully
differentiated 3T3-L1 adipocytes (45). Fig. 6B demonstrates
that Mdm2 accumulates in the nucleus in the presence of LMB, indicating
the effectiveness of LMB in these experiments. These results
demonstrate that CRM-1-dependent nuclear export is not
required for the degradation of PPAR following IFN treatment and
strongly suggests that PPAR is degraded in the nucleus.
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Fig. 6.
PPAR degradation in
adipocytes does not depend on nuclear export. A, fully
differentiated 3T3-L1 adipocytes were incubated in the presence of
cycloheximide (5 µM) and harvested at the indicated time
points. The adipocytes were treated with IFN alone or in the
presence of MG132 (20 µM) or leptomycin B (10 nM) as indicated. B, fully differentiated 3T3-L1
adipocytes were harvested after a 4-h incubation in the presence of
ethanol (LMB) or leptomycin B (+LMB, 10 nM). Cytosolic and nuclear extracts were obtained as
described under "Experimental Procedures." One hundred micrograms
of each extract were separated by SDS-PAGE, transferred to
nitrocellulose, and subjected to Western blot analysis. Samples were
processed, and results were visualized as described in the Fig. 1
legend. This was a representative experiment independently performed
two times.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
following IFN treatment, the
activation of PPAR transcriptional activity by IFN, evidence that
PPAR1 is substantially more labile than PPAR2, evidence that
serine phosphorylation of PPAR contributes to the turnover of
PPAR proteins in adipocytes, and evidence that PPAR proteins are
degraded by the nuclear ubiquitin-proteasome system. These results and recent findings by Spiegelman and co-workers (34) indicate that ubiquitin-proteasome-mediated degradation of PPAR is an important contributor to the cellular levels of PPAR proteins. Moreover, the
cellular levels of PPAR appear to be important because transgenic mice that express half the normal amount of PPAR have been shown to
be more insulin sensitive (12).
proteins in adipocytes. This model, illustrated in Fig. 7, suggests that activation of PPAR by
IFN, TZDs, or endogenous ligands is followed by
ubiquitin-proteasome-mediated degradation. This model also suggests
that serine phosphorylation contributes to PPAR degradation. The
validity of this model is addressed in the following paragraphs.
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Fig. 7.
Proposed model for degradation of
PPAR . PPAR activation is mediated by
ligand binding or exposure to IFN. Phosphorylation of PPAR
influences the IFN and ligand-dependent degradation of
PPAR.
1 and PPAR2 are targeted to
proteasome under basal conditions and following IFN treatment of
adipocytes. We have also observed ubiquitin conjugation of PPAR
under basal conditions and demonstrated a substantial increase in
ubiquitin conjugation of PPAR after IFN exposure. The increase in
PPAR-ubiquitin conjugates occurred within 15 min of IFN treatment and precedes the decrease in PPAR observed in experiments measuring PPAR degradation. Our results demonstrating that proteasome
inhibitors reduce the effect of IFN on PPAR expression and the
results demonstrating the appearance of PPAR-polyubiquitin
conjugates indicate that IFN treatment in adipocytes results in the
rapid degradation of PPAR via the ubiquitin-proteasome pathway.
mRNA and protein levels following
IFN treatment (13) led us to predict that IFN treatment would
suppress PPAR activity in adipocytes. Surprisingly, IFN treatment
of 3T3-L1 adipocytes was associated with the transcriptional activation
of PPAR2. Although unexpected, this result is consistent with the
idea that nuclear hormone receptor turnover occurs concomitantly with
transcriptional activation of these transcription factors (24).
Ligand-dependent activation and subsequent degradation has
been demonstrated for several other nuclear hormone receptors (26-30),
and the paradigm of activation followed by
ubiquitin-proteasome-dependent degradation has been extended to
proteins such as protein kinase C (46). Although IFN has not been
shown to be a ligand for PPAR, the activation of PPAR is a
ligand-dependent process (47), and a recent study has
demonstrated that PPAR2 degradation is associated with the
TZD-induced activation of PPAR2 (34). Our data demonstrating that
IFN treatment results in both the activation of PPAR2 and the
ubiquitin-proteasome-mediated degradation of PPAR
suggest that IFN-mediated signaling in adipocytes may be associated with the binding of an endogenous ligand and the activation and subsequent degradation of PPAR. Moreover, IFN-induced
PPAR2 transcriptional activation is enhanced in the
phosphorylation-deficient S112A mutant of PPAR2. This result is
consistent with previous findings showing that the mutation of
Ser112 to alanine in PPAR (Ser82 in
PPAR1) is associated with increased transcriptional activity (39,
40, 48).
by MAPKs has been described in various
studies (39-41, 48, 49). Although neither IFN nor TZDs directly
activate ERKs 1 and 2 in adipocytes, we found that inhibition of these
MAPKs resulted in an inhibition of PPAR decay. Therefore, the
mechanism(s) by which MAPKs influence PPAR degradation is not clear.
However, phosphorylation plays an important role in targeting many
substrates for ubiquitination and can either inhibit or increase the
targeting of substrates to the ubiquitin-proteasome system (22, 23). In
our experiments, we observed that both PPAR1 and PPAR2 migrate as
a doublet on gels that have been run for 24-30 h (refer to Fig. 5).
This doublet is easily distinguishable for PPAR1. We confirmed that
the slower migrating form corresponds to serine-phosphorylated
PPAR1, and the faster migrating form represents the unphosphorylated
PPAR1 proteins (data not shown), as has been previously described
(34). The results in Fig. 5 demonstrate that the faster migrating form
of PPAR1 disappears prior to the phosphorylated form of the protein.
The observed difference in the decay of these two forms of PPAR1
suggest that phosphorylation of PPAR proteins may serve as a
ubiquitin-proteasome targeting signal in which PPAR is converted to
the phosphorylated form prior to degradation by the
ubiquitin-proteasome pathway. This hypothesis is also consistent with
the increased activation the S112A mutant, and we predict that the
ubiquitin-conjugating machinery may not recognize the
phosphorylation-deficient PPAR as well as the wild-type protein. We
hypothesize that this may contribute to the increased activation
observed with the S112A mutant. This model is also supported by our
data demonstrating that inhibition of PPAR serine phosphorylation
with the MEK inhibitor prolongs the half-life of PPAR proteins. All
of these results support the hypothesis that serine phosphorylation of
PPAR may influence its targeting to the ubiquitin-proteasome system.
However, recent work from the Spiegelman laboratory (34) has shown that both the wild-type and the S112A form of PPAR2 are degraded after ligand activation, but they did not determine whether the half-lives of
these forms of the protein were different. Nonetheless, because the
phosphorylation-deficient mutant can be degraded, it seems unlikely
that serine phosphorylation is the only means by which PPAR proteins
are targeted to the ubiquitin-proteasome system. Interestingly, the
MAPK-regulated serine phosphorylation of the progesterone receptor has
been shown act as a targeting signal for the degradation of this
protein (28, 50).
-mediated
ubiquitin-proteasome-dependent degradation of PPAR.
PPAR proteins are predominantly localized in the nucleus, and recent studies have demonstrated that the nuclear ubiquitin-proteasome is
active in the degradation of selected substrates (42, 51, 52). Our
results demonstrate that the IFN-mediated degradation of PPAR
does not require CRM1-dependent nuclear export, indicating that IFN-induced PPAR degradation likely occurs in the nucleus. In the absence of serum deprivation, we observe active ERKs 1 and 2 in
the nucleus of 3T3-L1 adipocytes (data not shown) and hypothesize that
the presence of these kinases influences the nuclear decay of PPAR
proteins. Finally, the observation that PPAR1 is substantially more
labile than PPAR2 suggests that recognition of PPAR proteins by
the ubiquitin-proteasome system in adipocytes is influenced by the
30-amino acid N-terminal extension found in PPAR2. However,
examination of the N-terminal residues of both forms of PPAR reveals
that neither region contains the characteristic residues involved in
the N-end rule targeting to the ubiquitin-proteasome system (53).
Moreover, neither form contains a lysine residue necessary for
ubiquitin conjugation (22). However, this study does not address the
mechanisms underlying the differences in the half-lives of PPAR1 and
PPAR2.
expression in
mice (PPAR+/
) is associated with resistance to weight
gain along with protection from the insulin resistance that typically
accompanies weight gain. In addition, genetic evidence indicates that
decreased PPAR activity may protect against insulin resistance in
humans (55). Conversely, PPAR is required for the formation of fat
cells, and a lack of adipose cells is associated with insulin
resistance and hyperglycemia (56). These studies suggest that a careful balance between PPAR expression and activity levels must be
maintained to avoid development of diseases such as type II diabetes
and obesity. The current study, along with a previous study showing that ligand activation of PPAR leads to
ubiquitin-proteasome-dependent degradation of PPAR (34),
suggests that the ubiquitin-proteasome pathway plays an important role
in the regulation of PPAR expression in adipocytes.
FOOTNOTES
To whom correspondence should be addressed: Louisiana State
University, Dept. of Biological Sciences, 508 Life Sciences Bldg., Baton Rouge, LA 70803. Tel.: 225-578-1749; Fax: 225-578-2597; E-mail:
jsteph1@lsu.edu
ABBREVIATIONS
, peroxisome
proliferator-activated receptor ;
TZD, thiazolidinedione;
IFN, interferon-;
STAT, signal transducer and activator of transcription;
HA, hemagglutinin;
DMEM, Dulbecco's modified Eagle's medium;
PBS, phosphate-buffered saline;
MAPK, mitogen-activated protein kinase;
ERK, extracellular signal-regulated kinase;
MEK, MAPK/ERK kinase;
LMB, leptomycin B.
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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 J. P. DeLany, Z. E. Floyd, S. Zvonic, A. Smith, A. Gravois, E. Reiners, X. Wu, G. Kilroy, M. Lefevre, and J. M. Gimble Proteomic Analysis of Primary Cultures of Human Adipose-derived Stem Cells: Modulation by Adipogenesis Mol. Cell. Proteomics, June 1, 2005; 4(6): 731 - 740. [Abstract] [Full Text] [PDF]
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K. M. Fuenzalida, M. C. Aguilera, D. G. Piderit, P. C. Ramos, D. Contador, V. Quinones, A. Rigotti, F. C. Bronfman, and M. Bronfman Peroxisome Proliferator-activated Receptor {gamma} Is a Novel Target of the Nerve Growth Factor Signaling Pathway in PC12 Cells J. Biol. Chem., March 11, 2005; 280(10): 9604 - 9609. [Abstract] [Full Text] [PDF]
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 X. Zhang, M. C. Rodriguez-Galan, J. J. Subleski, J. R. Ortaldo, D. L. Hodge, J.-M. Wang, O. Shimozato, D. A. Reynolds, and H. A. Young Peroxisome proliferator-activated receptor-{gamma} and its ligands attenuate biologic functions of human natural killer cells Blood, November 15, 2004; 104(10): 3276 - 3284. [Abstract] [Full Text] [PDF]
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K. E. Davis, M. Moldes, and S. R. Farmer The Forkhead Transcription Factor FoxC2 Inhibits White Adipocyte Differentiation J. Biol. Chem., October 8, 2004; 279(41): 42453 - 42461. [Abstract] [Full Text] [PDF]
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D. A. Culver, B. P. Barna, B. Raychaudhuri, T. L. Bonfield, S. Abraham, A. Malur, C. F. Farver, M. S. Kavuru, and M. J. Thomassen Peroxisome Proliferator-Activated Receptor {gamma} Activity Is Deficient in Alveolar Macrophages in Pulmonary Sarcoidosis Am. J. Respir. Cell Mol. Biol., January 1, 2004; 30(1): 1 - 5. [Abstract] [Full Text] [PDF]
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