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J. Biol. Chem., Vol. 277, Issue 51, 49776-49781, December 20, 2002
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From the Department of Microbiology-Immunology, Feinberg School of
Medicine, Northwestern University, Chicago, Illinois 60611
Received for publication, August 2, 2002, and in revised form, September 22, 2002
Recent studies have revealed that the
calcium-dependent serine/threonine phosphatase calcineurin
mediates the effects of intracellular calcium in many different cell
types. In this study we investigated the role of calcineurin in the
regulation of adipocyte differentiation. We found that the specific
calcineurin inhibitors cyclosporin A and FK506 overcame the
antiadipogenic effect of calcium ionophore on the differentiation of
3T3-L1 preadipocytes. This finding suggests that calcineurin is
responsible for mediating the previously documented Ca2+-dependent inhibition of
adipogenesis. We further demonstrate that the expression of a
constitutively active calcineurin mutant potently inhibits the ability
of 3T3-L1 cells to undergo adipocyte differentiation by preventing
expression of the proadipogenic transcription factors peroxisome
proliferator-activated receptor Adipocytes are highly specialized cells that play a key role in
energy homeostasis by regulating the storage and release of energy in
response to changing nutritional needs (1). In addition to their role
in energy balance, adipocytes also perform important endocrine
functions by secreting a variety of factors that regulate such
processes as food intake, insulin responsiveness, reproduction, vascular remodeling, and the immune response (2). Although adipocytes
clearly play an important physiological role, the excessive accumulation of adipose tissue can result in obesity, which is known to
be a significant risk factor for a number of other disease states
including insulin resistance, type-2 diabetes, hypertensions, cardiovascular disease and cancer (3). Obesity can arise from either an
increase in individual adipocyte cell size or from an increase in total
adipocyte cell number as a result of increased de novo
adipocyte differentiation (1). Accordingly, the molecular mechanisms
that govern the regulation of adipocyte growth and differentiation are
of considerable scientific interest and have been the subject of much
investigation (1, 4, 5).
Considerable progress in our understanding of adipocyte biology has
come from the study of the 3T3-L1 preadipocyte cell line (6), which
under the appropriate in vitro culture conditions can be
efficiently induced to undergo terminal differentiation into
morphologically distinct, triglyceride-laden, mature adipocytes. Adipocyte differentiation is induced in 3T3-L1 preadipocytes by treatment of confluent, growth-arrested cells with the adipogenic hormones methylisobutylxanthine
(Mix),1 dexamethasone (Dex),
and insulin, collectively known as MDI. A large body of accumulated
data has revealed that this process of adipocyte differentiation
proceeds via a highly orchestrated and coordinated cascade of
transcription factors, including members of the CCAAT/enhancer-binding
protein (C/EBP) family and peroxisome proliferator-activated receptor
This process of adipocyte differentiation is influenced by a variety of
different extrinsic factors and intracellular signaling pathways (4,
14). Of particular interest to the current study are the effects of
intracellular calcium on adipocyte differentiation. A number of reports
have demonstrated that increases in intracellular calcium concentration
([Ca2+]i) during the early phase of human and
3T3-L1 preadipocyte differentiation act to potently inhibit
adipogenesis (15-17). However, the effects of calcium on this process
may be complex, because increases in [Ca2+]i in
human preadipocytes during the later stages of differentiation and in
Ob1774 cells appear to enhance the expression of certain markers of
mature adipocytes (17-19). In this study we investigated the molecular
mechanism that underlies the inhibitory effect of early increases in
[Ca2+]i on the differentiation of 3T3-L1
preadipocytes. Calcineurin, a calcium-dependent
serine/threonine phosphatase, is known to be a critical downstream
effector of the calcium signal in a wide variety of different cell
types (20). We present evidence to demonstrate that calcineurin
mediates the Ca2+-dependent inhibition of
adipocyte differentiation in 3T3-L1 cells. In addition, we demonstrate
that inhibition of endogenous calcineurin activity in 3T3-L1 cells
enhances the efficiency of adipogenesis in response to suboptimal
adipogenic stimuli. On the basis of our results, we propose that
commitment to the terminal phase of adipocyte differentiation is likely
to be regulated by the level of calcineurin activity in preadipocytes.
Cell Culture and Adipocyte Differentiation--
3T3-L1
preadipocytes (ATCC) were cultured in a growth medium of
Dulbecco's modified Eagle's medium with high glucose (Invitrogen) supplemented with 10% (v/v) fetal calf serum (Hyclone), 100 units/ml penicillin G, and 100 µg/ml streptomycin (Invitrogen). To induce adipocyte differentiation, cells were grown until 2 days postconfluence (day 0) and then treated for 2 days with growth medium plus MDI (0.5 mM methylisobutylxanthine, 1 µM
dexamethasone, and 10 µg/ml insulin, all from Sigma). The cells were
re-fed with growth medium that contained 10 µg/ml insulin at day 2 and every 2 days thereafter with growth medium alone. After 10 days,
cells were fixed with formalin and stained with the lipophilic dye Oil
Red O (Sigma). Stained cells were either photographed or counterstained
with Giemsa and visualized by bright field microscopy. Where indicated, cells were treated additionally with 2 µM ionomycin, 5 ng/ml FK506, 1 µg/ml CsA (all from Calbiochem), or vehicle control (ethanol).
Retroviral Expression Constructs--
The retroviral expression
vector pMSCV-CNmut was generated by insertion of a
XhoI-EcoRI fragment that contained the previously described CNmut (21) into pMSCV-GFP downstream of the viral long
terminal repeat and upstream of the IRES-GFP cassette. The pMSCV-H2K retroviral expression vector was created by replacing GFP in
the pMSCV-GFP retroviral expression vector with a PCR-amplified truncated murine major histocompatibility class I H-2Kk
cDNA from pMACS Kk.II (Miltenyi Biotec). pMSCV-PPAR Retrovirus Production and Infection of 3T3-L1
Cells--
Retroviral expression vectors were cotransfected with
pVSV-G (Clontech) into the GP293 pantropic
packaging cell line (Clontech) by using
LipofectAMINE Plus (Invitrogen). The medium was replaced after 24 h, and viral supernatants were harvested at 2 days post-transfection and stored at Flow Cytometric Analysis--
On day 3 after infection, cells
were analyzed for GFP fluorescence or were stained additionally with a
phycoerythrin-coupled anti-mouse H-2Kk Ab (36-7-5;
BD Biosciences). 10,000 events were analyzed using a FACSCaliber flow
cytometer and CELLQuest software (BD Biosciences).
Immunoblot and Northern Blot Analysis--
Protein extracts
prepared from cells harvested at the indicated times
postdifferentiation were resolved by SDS-PAGE and subjected to
immunoblot analysis with the relevant Ab. All Abs (PPAR Calcineurin Is Required for the
Ca2+-dependent Inhibition of Adipocyte
Differentiation--
To investigate the role of calcineurin in the
regulation of adipocyte differentiation, we first examined whether the
specific calcineurin inhibitors FK506 and CsA were able to attenuate
the previously reported inhibitory effect of calcium ionophore on the
differentiation of the 3T3-L1 preadipocyte cell line (15). As shown in
Fig. 1, 2-day postconfluent plates of
3T3-L1 cells that were treated with MDI efficiently differentiated into
morphologically distinct, fat-laden adipocytes with accumulated
cytoplasmic triglycerides that stained red with Oil Red O. Notably, the
presence of either FK506 or CsA did not affect the ability of MDI to
induce 3T3-L1 cells to undergo adipocyte differentiation. This fact
indicates that calcineurin is not required for MDI-induced adipocyte
differentiation, as was originally proposed by Ho et al.
(23). Consistent with a previous report (15), treatment of 3T3-L1 cells
with the calcium ionophore ionomycin potently blocked their
differentiation. Significantly, we found that this inhibitory effect of
calcium ionophore was abrogated in the presence of either FK506 or CsA
(Fig. 1). These data therefore indicate that calcineurin activity is
required to mediate the inhibitory effects of calcium ionophore on the differentiation of 3T3-L1 preadipocytes into mature adipocytes and
suggest that calcineurin is likely to negatively regulate adipocyte
differentiation.
A Calcium-independent, Constitutively Active Form of
Calcineurin Inhibits Adipocyte Differentiation in 3T3-L1
Cells--
To further investigate the effects of calcineurin on
adipogenesis, we used an efficient retroviral gene delivery system to introduce a previously characterized (21) calcium-independent, constitutively active calcineurin mutant (CNmut) into 3T3-L1 cells. The
cDNA that encoded CNmut was introduced into the MSCV-GFP retroviral vector under the control of the MSCV promoter and upstream of an
IRES-GFP expression cassette, thereby allowing the expression of both
CNmut and GFP from a single bicistronic mRNA (Fig.
2A). Using these vectors, we
were routinely able to generate a high-titer retrovirus capable of
stably infecting >95% of 3T3-L1 cells (Fig. 2B). As shown
in Fig. 2C, 3T3-L1 preadipocytes infected with the control
MSCV-GFP retrovirus and treated with MDI efficiently differentiated into mature Oil Red O-staining adipocytes. In contrast, we found that
cells infected with the MSCV-CNmut retrovirus and stimulated with MDI
failed to undergo the characteristic morphological changes associated
with adipocyte differentiation and did not stain red with Oil Red O. As
expected, we were able to rescue adipogenesis in these CNmut-expressing
cells by treatment with FK506 (data not shown). To confirm the
inhibitory effect of CNmut on adipocyte differentiation, we next
examined the time course of expression of the late adipocyte-specific
marker gene, aP2. As shown in Fig. 2D, expression
of aP2 mRNA was readily detectable in control MSCV-GFP-infected cells after treatment with MDI, whereas this transcript was not detectable in MDI-induced CNmut-expressing cells. Taken together, these
results indicate that sustained calcineurin activity in 3T3-L1
preadipocytes inhibits adipocyte differentiation.
Sustained Calcineurin Activity Inhibits the Expression of the
Proadipogenic Transcription Factors PPAR Sustained Calcineurin Activity Does Not Affect the Induction of
C/EBP Ectopic Expression of PPAR Inhibition of Endogenous Calcineurin Activity Enhances Adipocyte
Differentiation--
Having demonstrated that the sustained activation
of calcineurin either by treatment with calcium ionophore or ectopic
expression of CNmut potently inhibits adipogenesis, we wanted to
investigate the potential role of calcineurin during the normal process
of in vitro adipocyte differentiation. For these
experiments, we took advantage of our observation that treatment of
3T3-L1 preadipocytes with suboptimal adipogenic stimuli (Mix and Dex
without insulin) resulted in only modest adipocyte differentiation that
occurred primarily in isolated patches of cells. As seen in Fig.
5A, stimulation of 3T3-L1
cells with decreasing concentrations of Mix and Dex resulted in a
dose-dependent decrease in adipocyte differentiation. However, when 3T3-L1 cells were differentiated under these suboptimal conditions in the presence of either FK506 or CsA, we observed enhanced
differentiation with a significant increase in both the number and the
size of these adipogenic cell clusters (Fig. 5A and data not
shown). To further implicate endogenous calcineurin in the regulation
of adipocyte differentiation, we took advantage of a previously
characterized specific peptide inhibitor of calcineurin, VIVIT-GFP,
which has been shown to specifically inhibit the ability of calcineurin
to activate NFAT proteins (22). As predicted, expression of VIVIT-GFP
in 3T3-L1 cells was able to overcome the inhibitory effects of
ionomycin on adipocyte differentiation (Fig. 5B). Consistent
with the effect of FK506 (Fig. 5A), we found that inhibition
of endogenous calcineurin activity with VIVIT-GFP dramatically enhanced
adipocyte differentiation in response to suboptimal adipogenic stimuli
(Fig. 5C). Taken together, these results demonstrate that endogenous calcineurin activity acts to antagonize the normal process
of adipogenesis and is likely to set a signaling threshold required for
efficient adipocyte differentiation.
In the current study, we provide multiple lines of evidence to
indicate that the Ca2+-calmodulin-regulated phosphatase
calcineurin acts to negatively regulate adipocyte differentiation. We
demonstrate that the activation of the calcineurin signaling pathway
inhibits adipogenesis by preventing the expression of the proadipogenic
transcription factors PPAR It is important to note that our primary conclusion that calcineurin
acts to negatively regulate adipocyte differentiation conflicts with a
previous study by Ho et al. (23). On the basis of their
identification of an NFAT binding site in a proximal promoter element
of the adipocyte-specific aP2 gene and their observation that CsA
inhibits MDI-induced 3T3-L1 differentiation, these authors proposed a
positive role for the calcineurin/NFAT signaling pathway in the
regulation of adipogenesis. However, a previous study by Yeh et
al. (28) found that neither CsA nor FK506 inhibited the
differentiation of 3T3-L1 cells. In fact, they found that a molar
excess of FK506 was able to overcome the antiadipogenic effects of the
structurally related drug rapamycin. Because both FK506 and rapamycin
are known to mediate their biological activities by binding to a common
intracellular receptor, FK506-binding protein (29), the ability
of FK506 to overcome the antiadipogenic effects of rapamycin indicates
that functional FK506-FK506 binding protein complexes are unable to
block adipogenesis. This observation and our current data argue
strongly against a positive role for calcineurin in adipocyte
differentiation, as originally suggested by Ho et al. (23).
Instead, our data provide multiple independent lines of evidence that
demonstrate a novel inhibitory function for calcineurin in the
regulation of adipocyte differentiation. At present, the reason behind
the discrepancy between our data and those of Ho et al. is
not clear but may be related to either the method of drug delivery or
specific cell culture conditions.
Our finding that sustained calcineurin activity inhibits adipocyte
differentiation by preventing the expression of PPAR Collectively, our findings identify calcineurin as a
Ca2+-dependent negative regulator of adipocyte
differentiation and provide evidence that the level of endogenous
calcineurin activity in preadipocytes plays an important role in
determining the efficiency of adipogenesis. Because it is now clear
that an increase in adipogenesis can contribute to increased adipose
tissue mass and the development of obesity (1), our data suggest a
potential in vivo role for calcineurin in the regulation of
obesity and its associated diseases. Indeed, our observation that
inhibition of endogenous calcineurin activity leads to enhanced
adipogenesis may explain the increased obesity, hyperlipidemia, and
type-2 diabetes that have been reported in patients treated with the
immunosuppressive drugs CsA and FK506 (49-51). On the basis of our
results, further investigation of the in vivo role of
calcineurin in the regulation of obesity is clearly warranted.
We thank Janardan Reddy for the PPAR *
This work was supported in part by a Gramm travel fellowship
award from the Robert H. Lurie Comprehensive Cancer Center of Northwestern University (to J. W. N.) and by National Institutes of
Health Grant R29 GM55292 (to N. A. C.).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, September 25, 2002, DOI 10.1074/jbc.M207913200
2
J. W. Neal and N. A. Clipstone,
manuscript in preparation.
The abbreviations used are:
Mix, methylisobutylxanthine;
Dex, dexamethasone;
MDI, methylisobutylxanthine, dexamethasone and insulin;
C/EBP, CCAAT/enhancer-binding protein;
PPAR
Calcineurin Mediates the
Calcium-dependent Inhibition of Adipocyte Differentiation
in 3T3-L1 Cells*
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(PPAR) and
CCAAT/enhancer-binding protein 
(C/EBP). This calcineurin-mediated block in adipocyte differentiation is rescued by
ectopic expression of PPAR1. Finally, we demonstrate that inhibition
of endogenous calcineurin activity with either FK506 or a specific
calcineurin inhibitory peptide enhances differentiation of 3T3-L1 cells
in response to suboptimal adipogenic stimuli, suggesting that
endogenous calcineurin activity normally sets a signaling threshold
that antagonizes efficient adipocyte differentiation. Collectively,
these data indicate that calcineurin acts as a
Ca2+-dependent molecular switch that negatively
regulates commitment to adipocyte differentiation by preventing the
expression of critical proadipogenic transcription factors.
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DISCUSSION
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(PPAR) (1, 4, 5). MDI-treated, growth-arrested 3T3-L1
preadipocytes synchronously enter the cell cycle and initially express
the early transcription factors C/EBP
and C/EBP
(7, 8). C/EBP
and C/EBP then elicit the expression of the proadipogenic
transcription factor PPAR (7), which in turn induces the expression
of C/EBP (9). Together, PPAR and C/EBP then are believed to
play a dual role in adipogenesis by first inducing withdrawal from the
cell cycle and then directing the expression of adipocyte-specific
genes that ultimately result in the acquisition of the mature adipocyte cell fate (10-13).
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1 was created by introducing the full-length, cDNA-encoding murine PPAR1 (a gift from J. Reddy, Northwestern University) into pMSCV-H2K.
pMSCV-VIVIT-GFP was constructed by inserting an oligonucleotide that
encoded a previously described (22) calcineurin-inhibitory peptide
(MAGPHPVIVITGPHEE) into pEGFP-N3 (Clontech)and
then introducing the resulting VIVIT-GFP fusion sequence into
pMSCV-H2K.
80 °C. For infections, 5 × 104
3T3-L1 cells were plated per well of a 6-well plate. The next day, the
medium was replaced with 2 ml of viral supernatant that contained 8 µg/ml polybrene (Sigma), and plates were centrifuged at 2000 rpm for
1.5 h at room temperature. After removal of the viral supernatant,
cells were expanded in growth medium for subsequent analysis. For
double infections, previously infected cells were replated after 3 days
and infected with the second virus as described previously.
(H-100), C/EBP (14AA), C/EBP (H-7), and C/EBP (C-22)) were purchased from Santa Cruz Biotechnology. For Northern blot analysis, total RNA
was isolated from cells by using Trizol (Invitrogen) on the indicated
day after differentiation was induced. RNA samples (10 µg) were
separated by using 1.2% agarose, 2.2 M formaldehyde gel electrophoresis and transferred to Hybond-N membrane (Amersham Biosciences). Immobilized RNA was hybridized with a
32P-radiolabeled murine aP2 probe cDNA probe (ATCC) and
visualized by exposure to Kodak X-AR film. Membranes were stripped and
reprobed with a glyceraldehyde-3-phosphate dehydrogenase probe as a control.
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Fig. 1.
The immunosuppressive drugs CsA and FK506
overcome the antiadipogenic effects of the calcium ionophore,
ionomycin. 3T3-L1 preadipocytes were induced to undergo
adipocyte differentiation by treatment with MDI as described under
"Experimental Procedures." As indicated, cells were additionally
treated for the first 4 days of differentiation in the presence of 2 µM ionomycin or vehicle (ethanol), plus either vehicle
(ethanol), 5 ng/ml FK506, or 1 µg/ml CsA. After 10 days, plates of
cells were fixed, stained with Oil Red O, and either directly
photographed or counterstained with Giemsa and visualized by bright
field microscopy.
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Fig. 2.
A calcium-independent, constitutively active
form of calcineurin inhibits adipocyte differentiation in 3T3-L1
cells. A, schematic representation of the MSCV-GFP and
MSCV-CNmut retroviral vectors. B, flow cytometric analysis
of GFP expression after retroviral infection of 3T3-L1 preadipocytes
with the MSCV-GFP and MSCV-CNmut retroviruses. The percentage of GFP
expressing 3T3-L1 cells (green) as compared with mock
infected cells (gray) is indicated. C, 3T3-L1
preadipocytes infected with either MSCV-GFP or MSCV-CNmut retroviruses
were induced to undergo differentiation as described under
"Experimental Procedures." After 10 days, cells were stained with
Oil Red O. D, Northern blot analysis of aP2 mRNA
expression in MSCV-GFP- and MSCV-CNmut-infected cells induced to
undergo differentiation for the indicated number of days (upper
panel). The membrane was reprobed by using
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a
loading control (lower panel).
and C/EBP--
The
transcription factors PPAR and C/EBP are known to be both
necessary and sufficient for adipocyte differentiation (10, 11, 24,
25). To investigate the molecular mechanism that underlies the
inhibitory effect of calcineurin on adipocyte differentiation, we next
examined the effects of CNmut on the expression of PPAR and
C/EBP. Thus, cell extracts prepared from 3T3-L1 cells infected with
either MSCV-GFP or MSCV-CNmut that had been induced to undergo adipocyte differentiation by treatment with MDI were analyzed for
expression of PPAR and C/EBP by immunoblot analysis. As shown in
Fig. 3A, the expression of
both PPAR and C/EBP was readily detectable in cells infected with
MSCV-GFP. In contrast, we did not observe any appreciable expression of
either PPAR or C/EBP in CNmut-expressing cells (Fig.
3A). Thus, calcineurin activity appears to inhibit adipocyte
differentiation by preventing the expression of the proadipogenic
transcription factors PPAR and C/EBP.
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Fig. 3.
Effects of sustained calcineurin activity on
the expression of adipogenic transcription factors. 3T3-L1
preadipocytes infected with either MSCV-GFP or MSCV-CNmut retroviruses
were induced to undergo differentiation with MDI as described under
"Experimental Procedures." Whole cell extracts were prepared at the
indicated time points and analyzed by SDS-PAGE followed by
immunoblotting with the indicated Ab: PPAR (A,
upper panel), C/EBP (A, lower
panel), C/EBP (B, upper panel), C/EBP
(B, lower panel).
and C/EBP--
The expression of PPAR and
C/EBP is thought to be regulated during adipocyte differentiation by
the transcription factors C/EBP and C/EBP (7, 8). We therefore
examined whether calcineurin activity blocks the induction of PPAR
and C/EBP by interfering with the expression of C/EBP and
C/EBP. As shown in Fig. 3B, exposure of MSCV-GFP-infected
control cells to differentiation-inducing conditions resulted in the
expression of both C/EBP and C/EBP. We found that ectopic
expression of CNmut did not attenuate the expression of C/EBP and
C/EBP (unlike PPAR and C/EBP), because both were induced in
CNmut-expressing cells with similar kinetics to control cells (Fig.
3B). Moreover, it appeared that calcineurin did not affect
the relative abundance of the smaller inhibitory liver-enriched
inhibitory protein (LIP) isoform of C/EBP that arises from
alternative translational initiation and is believed to represent a
naturally occurring dominant-negative regulator of C/EBP family members
(26). Thus, it appears that sustained calcineurin activity does not
prevent the induction of PPAR and C/EBP by affecting the
expression of the early transcription factors C/EBP and
C/EBP.
1 Rescues Adipocyte Differentiation in
CNmut-expressing 3T3-L1 Cells--
We wanted to determine next whether
the inhibitory effect of CNmut on adipocyte differentiation was
specifically caused by prevention of the expression of PPAR and
C/EBP, or whether sustained calcineurin activity might merely
nonspecifically perturb 3T3-L1 cellular physiology by creating a
cellular environment incompatible with cellular differentiation. To
distinguish between these possibilities, we tested whether ectopic
expression of PPAR was able to bypass the block in adipogenesis and
rescue adipocyte differentiation in CNmut-expressing cells. Thus,
3T3-L1 cells were infected sequentially first with MSCV-CNmut and then
with either MSCV-PPAR1 or MSCV-H2K as a control. The MSCV-PPAR1
retroviral vector directs the expression of both PPAR1 and the
murine major histocompatibility class I molecule, H2Kk from
a single bicistronic mRNA (Fig.
4A). As a result, successful infection with this virus can be monitored readily by
fluorescence-activated cell sorter analysis with a
fluorescence-conjugated anti-H2Kk mAb. Using this
sequential infection protocol, we were able to doubly infect >95% of
cells with MSCV-CNmut and either MSCV-H2K or MSCV-PPAR1 (Fig.
4B). As expected, cells infected with both MSCV-CNmut and
MSCV-H2K failed to undergo adipocyte differentiation under standard
differentiation conditions (Fig. 4C). In contrast, cells
that co-expressed both CNmut and PPAR1 were found to efficiently undergo the characteristic morphological changes associated with adipocyte differentiation and stained positive for Oil Red O (Fig. 4C). Fluorescent microscopic analysis of these cells
revealed that they still expressed the CNmut-IRES-GFP transgene, ruling out the trivial possibility that PPAR1 rescued adipogenesis by inhibiting expression of CNmut (data not shown). Furthermore, we
observed that expression of PPAR1 overcomes the inhibitory effects
of ionomycin on adipogenesis (Fig. 4D). The efficient rescue
of adipocyte differentiation in both CNmut-expressing and ionomycin-treated cells by ectopic expression of PPAR1 suggests that
calcineurin principally inhibits adipogenesis by preventing the
expression of the proadipogenic transcription factor PPAR.
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Fig. 4.
Ectopic expression of PPAR
rescues adipocyte differentiation in CNmut-expressing 3T3-L1
cells. A, schematic representation of the MSCV-PPAR
retroviral vector. LTR, long terminal repeat.
B, representative flow cytometric analysis of GFP and
H2Kk expression after double infection of 3T3-L1
preadipocytes with the MSCV-CNmut and MSCV-H2K retroviruses. Data are
presented as two-color dot plots, and the percentage of cells infected
with both retroviruses is indicated. C, 3T3-L1 preadipocytes
infected with MSCV-CNmut and either MSCV-PPAR or control MSCV-H2K
virus were induced to undergo differentiation as described under
"Experimental Procedures." After 10 days, cells were stained with
Oil Red O. D, 3T3-L1 preadipocytes were infected with either
MSCV-H2K or MSCV-PPAR and then induced to undergo differentiation
with MDI in the presence of ionomycin as described under
"Experimental Procedures."
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Fig. 5.
Inhibition of endogenous calcineurin activity
enhances the adipocyte differentiation of 3T3-L1 preadipocytes in
response to suboptimal adipogenic stimuli. A, 2-day,
postconfluent, 3T3-L1 preadipocytes were incubated in a growth medium
that contained 2-fold serial dilutions of 1 µM Dex and
0.5 mM Mix (1× MD) for 2 days in the presence
or absence of FK506 and thereafter in a growth medium that contained
either FK506 or vehicle control. After 10 days, cells were stained with
Oil Red O. B, 3T3-L1 preadipocytes infected with either
MSCV-VIVIT-GFP or control MSCV-H2K virus were induced to undergo
adipocyte differentiation with MDI in the presence or absence of
ionomycin as described under "Experimental Procedures."
C, 3T3-L1 preadipocytes infected with either MSCV-VIVIT-GFP
or control MSCV-H2K virus were grown for 2 days postconfluence and then
induced to undergo adipocyte differentiation by incubation in growth
medium that contained 2-fold serial dilutions of 1 µM Dex
and 0.5 mM Mix (1× MD) for 2 days and
maintained thereafter in growth medium alone. After 10 days, cells were
stained with Oil Red O.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
and C/EBP. Conversely, we found that
inhibition of endogenous calcineurin activity markedly enhances
adipocyte differentiation in 3T3-L1 cells in response to suboptimal
adipogenic stimuli. Together our findings suggest that the level of
activation of the endogenous calcineurin signaling pathway in
preadipocyte cells is likely to play an important role in setting the
signaling threshold required for commitment to the terminal phase of
adipocyte differentiation. Given that calcineurin activity is
exquisitely sensitive to changes in [Ca2+]i (27),
the role of calcineurin in the regulation of adipocyte differentiation
is likely to be especially important in the response to environmental
cues such as prostaglandin F2
that modify the
level of intracellular Ca2+. Prostaglandin
F2 has been shown previously to inhibit adipogenesis by
a Ca2+-dependent mechanism (16). On the basis
of our findings, we propose that calcineurin is an intrinsic negative
regulatory component of the adipogenic signaling pathway that acts as a
Ca2+-dependent molecular switch to inhibit
adipocyte differentiation in response to exogenous agents that elevate
[Ca2+]i.
and C/EBP,
but not C/EBP and C/EBP, suggests a number of potential mechanisms by which calcineurin may inhibit adipogenesis. First, calcineurin may interfere directly with the activity of C/EBP and
C/EBP. Although C/EBP is thought to be regulated during 3T3-L1
differentiation by a phosphorylation-dependent mechanism (30), no evidence currently exists to suggest that C/EBP is a direct
substrate of calcineurin. In addition, it appears that calcineurin does
not affect the expression of known inhibitors of C/EBP activity such
as LIP (Fig. 3B), an inhibitory C/EBP isoform that arises
by alternative translational initiation (26), or the expression of
CHOP-10 (data not shown), which is believed to represent an
endogenous dominant-negative inhibitor of the C/EBP family (31).
Second, calcineurin may inhibit adipocyte differentiation by affecting
a parallel pathway to C/EBP and C/EBP that is also required for
the efficient expression of PPAR and C/EBP. In this regard,
activation of the mitogen-activated protein kinase signaling
pathway has been shown to inhibit adipocyte differentiation (32, 33).
This effect is controversial, however, because other studies have
suggested a positive role for this pathway in adipogenesis (34, 35). In
addition, the cAMP- response element-binding protein/activating
transcription factor-2 (ATF-2) and p38 kinase signaling pathways have
both been shown to be required for PPAR expression and efficient
adipocyte differentiation in 3T3-L1 cells (36-38). Interestingly,
calcineurin has been shown to affect the activity of each of these
signaling pathways in a number of distinct cell types (39-41),
making each pathway a potential target for the antiadipogenic
activity of calcineurin. Third, calcineurin may inhibit adipocyte
differentiation by activating a pathway that directly represses
expression of PPAR and C/EBP. Indeed, undifferentiated 3T3-L1
preadipocytes are known to express a number of proteins that have been
shown to potently inhibit adipogenesis by preventing the expression of
PPAR. The expression of these proteins, which include PREF-1 (42),
Wnt-10b (43), and the transcription factors GATA-2 and GATA-3 (44),
must be down-regulated to ensure successful adipocyte differentiation. Interestingly, calcineurin has recently been implicated in
up-regulating the expression of GATA-2 in muscle cell precursors, where
it is believed to play a role in the promotion of muscle cell
differentiation and hypertrophy (45). Finally, because calcineurin is
best known for its ability to activate the NFAT family of transcription
factors (20, 46-48), and NFAT proteins are expressed in 3T3-L1 cells (23), it is possible that calcineurin mediates its effects through NFAT. Because the calcineurin- inhibitory peptide VIVIT-GFP has previously been reported to specifically block the ability of calcineurin to activate NFAT proteins (22), the enhancing effect of
VIVIT-GFP on adipogenesis (Fig. 5, B and C)
suggests that NFAT proteins may play a role in the negative regulation
of adipocyte differentiation. This notion is further supported by our
observation that ectopic expression of a constitutively active NFATc1
mutant also inhibits the MDI-induced differentiation of 3T3-L1 cells by
preventing the expression of PPAR and C/EBP, although this may be
secondary to a transforming effect of NFATc1 in these
cells.2 Experiments are
currently under way to further delineate the role of calcineurin in the
regulation of adipocyte differentiation.
ACKNOWLEDGEMENTS
1
cDNA and Li Liu for the generation of MSCV-VIVIT-GFP.
FOOTNOTES
To whom correspondence should be addressed: Dept. of
Microbiology-Immunology, Northwestern University, 303 E. Chicago
Ave., Chicago, IL 60611. Tel.: 312-503-8233; Fax:
312-503-1339; E-mail: n-clipstone@northwestern.edu.
ABBREVIATIONS
, peroxisome proliferator
activated receptor ;
NFAT, nuclear factor of activated T cells;
[Ca2+]i, intracellular calcium concentration;
CsA, cyclosporin A;
CNmut, constitutively activated calcineurin mutant;
MSCV, murine stem cell virus;
IRES, internal ribosomal entry sequence;
pEGFP, permuted enhanced green fluorescent protein;
Ab, antibody;
LTR, long terminal repeat.
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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