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Calcineurin Mediates the Calcium-dependent Inhibition of
Adipocyte Differentiation in 3T3-L1 Cells*
Received for publication, August 2, 2002, and in revised form, September 22, 2002
Published, JBC Papers in Press, September 25, 2002, DOI 10.1074/jbc.M207913200
Joel W. Neal and Neil A. Clipstone‡
From the Department of Microbiology-Immunology, Feinberg School of Medicine, Northwestern University,
Chicago, Illinois 60611
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 differ-
entiation. We found that the specific calcineurin inhib-
itors cyclosporin A and FK506 overcame the antiadipo-
genic effect of calcium ionophore on the differentiation
of 3T3-L1 preadipocytes. This finding suggests that cal-
cineurin is responsible for mediating the previously
documented Ca
2ⴙ
-dependent inhibition of adipogenesis.
We further demonstrate that the expression of a consti-
tutively active calcineurin mutant potently inhibits the
ability of 3T3-L1 cells to undergo adipocyte differentia-
tion by preventing expression of the proadipogenic
transcription factors peroxisome proliferator-activated
receptor
␥
(PPAR
␥
) and CCAAT/enhancer-binding pro-
tein
␣
(C/EBP
␣
). This calcineurin-mediated block in adi-
pocyte differentiation is rescued by ectopic expression
of PPAR
␥
1. Finally, we demonstrate that inhibition of
endogenous calcineurin activity with either FK506 or a
specific calcineurin inhibitory peptide enhances differ-
entiation of 3T3-L1 cells in response to suboptimal adi-
pogenic stimuli, suggesting that endogenous cal-
cineurin activity normally sets a signaling threshold
that antagonizes efficient adipocyte differentiation. Col-
lectively, these data indicate that calcineurin acts as a
Ca
2ⴙ
-dependent molecular switch that negatively regu-
lates commitment to adipocyte differentiation by pre-
venting the expression of critical proadipogenic tran-
scription factors.
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 addi-
tion 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 responsive-
ness, reproduction, vascular remodeling, and the immune re-
sponse (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 dis-
ease 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 differenti-
ation are of considerable scientific interest and have been the
subject of much investigation (1, 4, 5).
Considerable progress in our understanding of adipocyte bi-
ology 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 adipo-
cytes. Adipocyte differentiation is induced in 3T3-L1 preadipo-
cytes by treatment of confluent, growth-arrested cells with the
adipogenic hormones methylisobutylxanthine (Mix),
1
dexam-
ethasone (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
␥
(PPAR
␥
) (1, 4, 5). MDI-treated, growth-arrested 3T3-L1 prea-
dipocytes synchronously enter the cell cycle and initially ex-
press the early transcription factors C/EBP

and C/EBP
␦
(7, 8).
C/EBP

and C/EBP
␦
then elicit the expression of the proadi-
pogenic 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).
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 in-
tracellular calcium concentration ([Ca
2⫹
]
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
[Ca
2⫹
]
i
in human preadipocytes during the later stages of dif-
* 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. This
article must therefore be hereby marked “advertisement” in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
‡ To whom correspondence should be addressed: Dept. of Microbiol-
ogy-Immunology, Northwestern University, 303 E. Chicago Ave., Chi-
cago, IL 60611. Tel.: 312-503-8233; Fax: 312-503-1339; E-mail:
n-clipstone@northwestern.edu.
1
The abbreviations used are: Mix, methylisobutylxanthine; Dex, dex-
amethasone; MDI, methylisobutylxanthine, dexamethasone and insu-
lin; C/EBP, CCAAT/enhancer-binding protein; PPAR
␥
, peroxisome pro-
liferator activated receptor
␥
; NFAT, nuclear factor of activated T cells;
[Ca
2⫹
]
i
, intracellular calcium concentration; CsA, cyclosporin A;
CNmut, constitutively activated calcineurin mutant; MSCV, murine
stem cell virus; IRES, internal ribosomal entry sequence; pEGFP, per-
muted enhanced green fluorescent protein; Ab, antibody; LTR, long
terminal repeat.
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 51, Issue of December 20, pp. 49776–49781, 2002
© 2002 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.
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ferentiation and in Ob1774 cells appear to enhance the expres-
sion 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 [Ca
2⫹
]
i
on the differ-
entiation of 3T3-L1 preadipocytes. Calcineurin, a calcium-de-
pendent 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 Ca
2⫹
-dependent inhibition of
adipocyte differentiation in 3T3-L1 cells. In addition, we dem-
onstrate that inhibition of endogenous calcineurin activity in
3T3-L1 cells enhances the efficiency of adipogenesis in re-
sponse 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.
EXPERIMENTAL PROCEDURES
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
g/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 mMmethylisobutylxanthine, 1
Mdexamethasone, and 10
g/ml insulin, all from Sigma). The cells
were re-fed with growth medium that contained 10
g/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 counter-
stained with Giemsa and visualized by bright field microscopy. Where
indicated, cells were treated additionally with 2
Mionomycin, 5 ng/ml
FK506, 1
g/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-2K
k
cDNA from pMACS Kk.II (Miltenyi Biotec). pMSCV-
PPAR
␥
1 was created by introducing the full-length, cDNA-encoding
murine PPAR
␥
1 (a gift from J. Reddy, Northwestern University) into
pMSCV-H2K. pMSCV-VIVIT-GFP was constructed by inserting an ol-
igonucleotide that encoded a previously described (22) calcineurin-in-
hibitory peptide (MAGPHPVIVITGPHEE) into pEGFP-N3 (Clontech)
and then introducing the resulting VIVIT-GFP fusion sequence into
pMSCV-H2K.
Retrovirus Production and Infection of 3T3-L1 Cells—Retroviral ex-
pression vectors were cotransfected with pVSV-G (Clontech) into the
GP293 pantropic packaging cell line (Clontech) by using Lipo-
fectAMINE Plus (Invitrogen). The medium was replaced after 24 h, and
viral supernatants were harvested at 2 days post-transfection and
stored at ⫺80 °C. For infections, 5 ⫻10
4
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
g/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 in-
fected cells were replated after 3 days and infected with the second
virus as described previously.
Flow Cytometric Analysis—On day 3 after infection, cells were ana-
lyzed for GFP fluorescence or were stained additionally with a phyco-
erythrin-coupled anti-mouse H-2K
k
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
␥
(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 in-
duced. RNA samples (10
g) were separated by using 1.2% agarose, 2.2
Mformaldehyde gel electrophoresis and transferred to Hybond-N mem-
brane (Amersham Biosciences). Immobilized RNA was hybridized with
a
32
P-radiolabeled murine aP2 probe cDNA probe (ATCC) and visual-
ized by exposure to Kodak X-AR film. Membranes were stripped and
reprobed with a glyceraldehyde-3-phosphate dehydrogenase probe as a
control.
RESULTS
Calcineurin Is Required for the Ca
2⫹
-dependent Inhibition of
Adipocyte Differentiation—To investigate the role of cal-
cineurin in the regulation of adipocyte differentiation, we first
examined whether the specific calcineurin inhibitors FK506
and CsA were able to attenuate the previously reported inhib-
itory 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 inhibi-
tory 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 cal-
cineurin is likely to negatively regulate adipocyte
differentiation.
A Calcium-independent, Constitutively Active Form of Cal-
cineurin Inhibits Adipocyte Differentiation in 3T3-L1 Cells—To
FIG.1. The immunosuppressive drugs CsA and FK506 over-
come the antiadipogenic effects of the calcium ionophore, iono-
mycin. 3T3-L1 preadipocytes were induced to undergo adipocyte dif-
ferentiation 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
Mionomycin or vehicle
(ethanol), plus either vehicle (ethanol), 5 ng/ml FK506, or 1
g/ml CsA.
After 10 days, plates of cells were fixed, stained with Oil Red O, and
either directly photographed or counterstained with Giemsa and visu-
alized by bright field microscopy.
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further investigate the effects of calcineurin on adipogenesis,
we used an efficient retroviral gene delivery system to intro-
duce a previously characterized (21) calcium-independent, con-
stitutively 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 mor-
phological 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 inhib-
itory 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-in-
fected cells after treatment with MDI, whereas this transcript
was not detectable in MDI-induced CNmut-expressing cells.
Taken together, these results indicate that sustained cal-
cineurin activity in 3T3-L1 preadipocytes inhibits adipocyte
differentiation.
Sustained Calcineurin Activity Inhibits the Expression of the
Proadipogenic Transcription Factors PPAR
␥
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 un-
derlies the inhibitory effect of calcineurin on adipocyte differ-
entiation, we next examined the effects of CNmut on the ex-
pression 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 differenti-
ation 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 prevent-
ing the expression of the proadipogenic transcription factors
PPAR
␥
and C/EBP
␣
.
Sustained Calcineurin Activity Does Not Affect the Induction
of C⁄EBP

and C⁄EBP
␦
—The expression of PPAR
␥
and
C/EBP
␣
is thought to be regulated during adipocyte differen-
tiation 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 ex-
pression 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
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 retro-
viral vectors. B, flow cytometric analysis of GFP expression after ret-
roviral 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 de-
scribed under “Experimental Procedures.”After 10 days, cells were
stained with Oil Red O. D, Northern blot analysis of aP2 mRNA ex-
pression 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 de-
hydrogenase (GAPDH) as a loading control (lower panel).
FIG.3.Effects of sustained calcineurin activity on the expres-
sion 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 “Ex-
perimental Procedures.”Whole cell extracts were prepared at the indi-
cated time points and analyzed by SDS-PAGE followed by immunoblot-
ting with the indicated Ab: PPAR
␥
(A,upper panel), C/EBP
␣
(A,lower
panel), C/EBP

(B,upper panel), C/EBP
␦
(B,lower panel).
Calcineurin Negatively Regulates Adipocyte Differentiation49778
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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 rela-
tive abundance of the smaller inhibitory liver-enriched inhibi-
tory protein (LIP) isoform of C/EBP

that arises from alterna-
tive translational initiation and is believed to represent a
naturally occurring dominant-negative regulator of C/EBP
family members (26). Thus, it appears that sustained cal-
cineurin 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
␦
.
Ectopic Expression of PPAR
␥
1 Rescues Adipocyte Differenti-
ation in CNmut-expressing 3T3-L1 Cells—We wanted to deter-
mine 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 cal-
cineurin activity might merely nonspecifically perturb 3T3-L1
cellular physiology by creating a cellular environment incom-
patible 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-PPAR
␥
1 or MSCV-H2K as
a control. The MSCV-PPAR
␥
1 retroviral vector directs the ex-
pression of both PPAR
␥
1 and the murine major histocompati-
bility class I molecule, H2K
k
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-H2K
k
mAb. Using this se-
quential infection protocol, we were able to doubly infect ⬎95%
of cells with MSCV-CNmut and either MSCV-H2K or MSCV-
PPAR
␥
1 (Fig. 4B). As expected, cells infected with both MSCV-
CNmut and MSCV-H2K failed to undergo adipocyte differen-
tiation under standard differentiation conditions (Fig. 4C). In
contrast, cells that co-expressed both CNmut and PPAR
␥
1
were found to efficiently undergo the characteristic morpholog-
ical changes associated with adipocyte differentiation and
stained positive for Oil Red O (Fig. 4C). Fluorescent micro-
scopic analysis of these cells revealed that they still expressed
the CNmut-IRES-GFP transgene, ruling out the trivial possi-
bility that PPAR
␥
1 rescued adipogenesis by inhibiting expres-
sion of CNmut (data not shown). Furthermore, we observed
that expression of PPAR
␥
1 overcomes the inhibitory effects of
ionomycin on adipogenesis (Fig. 4D). The efficient rescue of
adipocyte differentiation in both CNmut-expressing and iono-
mycin-treated cells by ectopic expression of PPAR
␥
1 suggests
that calcineurin principally inhibits adipogenesis by prevent-
ing the expression of the proadipogenic transcription factor
PPAR
␥
.
Inhibition of Endogenous Calcineurin Activity Enhances Adi-
pocyte Differentiation—Having demonstrated that the sus-
tained activation of calcineurin either by treatment with cal-
cium 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 adipo-
cyte 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, stim-
ulation 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 differenti-
ated under these suboptimal conditions in the presence of ei-
ther FK506 or CsA, we observed enhanced differentiation with
a significant increase in both the number and the size of these
adipogenic cell clusters (Fig. 5Aand data not shown). To fur-
ther implicate endogenous calcineurin in the regulation of adi-
pocyte differentiation, we took advantage of a previously char-
acterized 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, ex-
pression 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 cal-
cineurin activity acts to antagonize the normal process of adi-
pogenesis and is likely to set a signaling threshold required for
efficient adipocyte differentiation.
DISCUSSION
In the current study, we provide multiple lines of evidence to
indicate that the Ca
2⫹
-calmodulin-regulated phosphatase cal-
cineurin 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
␥
and C/EBP
␣
.
FIG.4.Ectopic expression of PPAR
␥
rescues adipocyte differ-
entiation in CNmut-expressing 3T3-L1 cells. A, schematic repre-
sentation of the MSCV-PPAR
␥
retroviral vector. LTR, long terminal
repeat. B, representative flow cytometric analysis of GFP and H2K
k
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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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 endoge-
nous calcineurin signaling pathway in preadipocyte cells is
likely to play an important role in setting the signaling thresh-
old required for commitment to the terminal phase of adipocyte
differentiation. Given that calcineurin activity is exquisitely
sensitive to changes in [Ca
2⫹
]
i
(27), the role of calcineurin in
the regulation of adipocyte differentiation is likely to be espe-
cially important in the response to environmental cues such as
prostaglandin F
2
␣
that modify the level of intracellular Ca
2⫹
.
Prostaglandin F
2
␣
has been shown previously to inhibit adipo-
genesis by a Ca
2⫹
-dependent mechanism (16). On the basis of
our findings, we propose that calcineurin is an intrinsic nega-
tive regulatory component of the adipogenic signaling pathway
that acts as a Ca
2⫹
-dependent molecular switch to inhibit
adipocyte differentiation in response to exogenous agents that
elevate [Ca
2⫹
]
i
.
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 differenti-
ation, 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 ra-
pamycin indicates that functional FK506-FK506 binding pro-
tein complexes are unable to block adipogenesis. This observa-
tion and our current data argue strongly against a positive role
for calcineurin in adipocyte differentiation, as originally sug-
gested by Ho et al. (23). Instead, our data provide multiple
independent lines of evidence that demonstrate a novel inhib-
itory 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 adi-
pocyte differentiation by preventing the expression of PPAR
␥
and C/EBP
␣
, but not C/EBP

and C/EBP
␦
, suggests a number
of potential mechanisms by which calcineurin may inhibit ad-
ipogenesis. 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 phosphoryl-
ation-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 expres-
sion 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 sig-
naling pathway has been shown to inhibit adipocyte differen-
tiation (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 differ-
entiation 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 differ-
entiation by activating a pathway that directly represses ex-
pression of PPAR
␥
and C/EBP
␣
. Indeed, undifferentiated
3T3-L1 preadipocytes are known to express a number of pro-
teins 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. Inter-
estingly, calcineurin has recently been implicated in up-regu-
lating the expression of GATA-2 in muscle cell precursors,
where it is believed to play a role in the promotion of muscle cell
FIG.5.Inhibition of endogenous calcineurin activity enhances
the adipocyte differentiation of 3T3-L1 preadipocytes in re-
sponse to suboptimal adipogenic stimuli. A, 2-day, postconfluent,
3T3-L1 preadipocytes were incubated in a growth medium that con-
tained 2-fold serial dilutions of 1
MDex and 0.5 mMMix (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 in-
duced 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 in-
duced to undergo adipocyte differentiation by incubation in growth
medium that contained 2-fold serial dilutions of 1
MDex 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.
Calcineurin Negatively Regulates Adipocyte Differentiation49780
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differentiation and hypertrophy (45). Finally, because cal-
cineurin 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 adipogen-
esis (Fig. 5, Band 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 in-
hibits the MDI-induced differentiation of 3T3-L1 cells by pre-
venting 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 delin-
eate the role of calcineurin in the regulation of adipocyte
differentiation.
Collectively, our findings identify calcineurin as a Ca
2⫹
-de-
pendent negative regulator of adipocyte differentiation and
provide evidence that the level of endogenous calcineurin ac-
tivity 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 sug-
gest 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 en-
hanced adipogenesis may explain the increased obesity, hyper-
lipidemia, and type-2 diabetes that have been reported in pa-
tients treated with the immunosuppressive drugs CsA and
FK506 (49 –51). On the basis of our results, further investiga-
tion of the in vivo role of calcineurin in the regulation of obesity
is clearly warranted.
Acknowledgments—We thank Janardan Reddy for the PPAR
␥
1
cDNA and Li Liu for the generation of MSCV-VIVIT-GFP.
REFERENCES
1. Spiegelman, B. M., and Flier, J. S. (1996) Cell 87, 377–389
2. Fruhbeck, G., Gomez-Ambrosi, J., Muruzabal, F. J., and Burrell, M. A. (2001)
Am. J. Physiol. Endocrinol Metab 280, E827–E847
3. Visscher, T. L., and Seidell, J. C. (2001) Annu. Rev. Public Health 22, 355–375
4. Rosen, E. D., and Spiegelman, B. M. (2000) Annu. Rev. Cell Dev. Biol. 16,
145–171
5. Cowherd, R. M., Lyle, R. E., and McGehee, R. E., Jr. (1999) Semin. Cell Dev.
Biol. 10, 3–10
6. Green, H., and Kehinde, O. (1975) Cell 5, 19 –27
7. Wu, Z., Bucher, N. L., and Farmer, S. R. (1996) Mol. Cell. Biol. 16, 4128 –4136
8. Yeh, W. C., Cao, Z., Classon, M., and McKnight, S. L. (1995) Genes Dev. 9,
168 –181
9. Rosen, E. D., Hsu, C. H., Wang, X., Sakai, S., Freeman, M. W., Gonzalez, F. J.,
and Spiegelman, B. M. (2002) Genes Dev. 16, 22–26
10. Tontonoz, P., Hu, E., and Spiegelman, B. M. (1994) Cell 79, 1147–1156
11. Freytag, S. O., Paielli, D. L., and Gilbert, J. D. (1994) Genes Dev. 8, 1654 –1663
12. Morrison, R. F., and Farmer, S. R. (1999) J. Biol. Chem. 274, 17088 –17097
13. Porse, B. T., Pedersen, T. A., Xu, X., Lindberg, B., Wewer, U. M., Friis-Hansen,
L., and Nerlov, C. (2001) Cell 107, 247–258
14. MacDougald, O. A., and Mandrup, S. (2002) Trends Endocrinol. Metab. 13,
5–11
15. Ntambi, J. M., and Takova, T. (1996) Differentiation 60, 151–158
16. Miller, C. W., Casimir, D. A., and Ntambi, J. M. (1996) Endocrinology 137,
5641–5650
17. Shi, H., Halvorsen, Y. D., Ellis, P. N., Wilkison, W. O., and Zemel, M. B. (2000)
Physiol. Genomics 3, 75–82
18. Gaillard, D., Negrel, R., Lagarde, M., and Ailhaud, G. (1989) Biochem. J. 257,
389 –397
19. Vassaux, G., Gaillard, D., Ailhaud, G., and Negrel, R. (1992) J. Biol. Chem.
267, 11092–11097
20. Crabtree, G. R. (2001) J. Biol. Chem. 276, 2313–2316
21. Clipstone, N. A., and Crabtree, G. R. (1993) Ann. N. Y. Acad. Sci. 696, 20 –30
22. Aramburu, J., Yaffe, M. B., Lopez-Rodriguez, C., Cantley, L. C., Hogan, P. G.,
and Rao, A. (1999) Science 285, 2129 –2133
23. Ho, I. C., Kim, J. H., Rooney, J. W., Spiegelman, B. M., and Glimcher, L. H.
(1998) Proc. Natl. Acad. Sci. U. S. A. 95, 15537–15541
24. Lin, F. T., and Lane, M. D. (1992) Genes Dev. 6, 533–544
25. Rosen, E. D., Sarraf, P., Troy, A. E., Bradwin, G., Moore, K., Milstone, D. S.,
Spiegelman, B. M., and Mortensen, R. M. (1999) Mol. Cell 4, 611–617
26. Descombes, P., and Schibler, U. (1991) Cell 67, 569 –579
27. Rusnak, F., and Mertz, P. (2000) Physiol. Rev. 80, 1483–1521
28. Yeh, W. C., Bierer, B. E., and McKnight, S. L. (1995) Proc. Natl. Acad. Sci.
U. S. A. 92, 11086 –11090
29. Bierer, B. E., Somers, P. K., Wandless, T. J., Burakoff, S. J., and Schreiber,
S. L. (1990) Science 250, 556 –559
30. Tang, Q. Q., and Lane, M. D. (1999) Genes Dev. 13, 2231–2241
31. Ron, D., and Habener, J. F. (1992) Genes Dev. 6, 439 –453
32. Hu, E., Kim, J. B., Sarraf, P., and Spiegelman, B. M. (1996) Science 274,
2100 –2103
33. Font de Mora, J., Porras, A., Ahn, N., and Santos, E. (1997) Mol. Cell. Biol. 17,
6068 –6075
34. Sale, E. M., Atkinson, P. G., and Sale, G. J. (1995) EMBO J. 14, 674 –684
35. Bost, F., Caron, L., Marchetti, I., Dani, C., Le Marchand-Brustel, Y., and
Binetruy, B. (2002) Biochem. J. 361, 621–627
36. Engelman, J. A., Berg, A. H., Lewis, R. Y., Lin, A., Lisanti, M. P., and Scherer,
P. E. (1999) J. Biol. Chem. 274, 35630 –35638
37. Reusch, J. E., Colton, L. A., and Klemm, D. J. (2000) Mol. Cell. Biol. 20,
1008 –1020
38. Lee, M. Y., Kong, H. J., and Cheong, J. (2001) Biochem. Biophys. Res. Com-
mun. 281, 1241–1247
39. Zou, Y., Yao, A., Zhu, W., Kudoh, S., Hiroi, Y., Shimoyama, M., Uozumi, H.,
Kohmoto, O., Takahashi, T., Shibasaki, F., Nagai, R., Yazaki, Y., and
Komuro, I. (2001) Circulation 104, 102–108
40. Bito, H., Deisseroth, K., and Tsien, R. W. (1996) Cell 87, 1203–1214
41. Lim, H. W., New, L., Han, J., and Molkentin, J. D. (2001) J. Biol. Chem. 276,
15913–15919
42. Smas, C. M., Chen, L., Zhao, L., Latasa, M. J., and Sul, H. S. (1999) J. Biol.
Chem. 274, 12632–12641
43. Ross, S. E., Hemati, N., Longo, K. A., Bennett, C. N., Lucas, P. C., Erickson,
R. L., and MacDougald, O. A. (2000) Science 289, 950 –953
44. Tong, Q., Dalgin, G., Xu, H., Ting, C. N., Leiden, J. M., and Hotamisligil, G. S.
(2000) Science 290, 134 –138
45. Musaro, A., McCullagh, K. J., Naya, F. J., Olson, E. N., and Rosenthal, N.
(1999) Nature 400, 581–585
46. Clipstone, N. A., and Crabtree, G. R. (1992) Nature 357, 695–697
47. Rao, A., Luo, C., and Hogan, P. G. (1997) Annu. Rev. Immunol. 15, 707–747
48. Crabtree, G. R., and Olson, E. N. (2002) Cell 109 (suppl.),S67–S79
49. Mathieu, R. L., Casez, J. P., Jaeger, P., Montandon, A., Peheim, E., and
Horber, F. F. (1994) Eur. J. Clin. Invest. 24, 195–200
50. Abouljoud, M. S., Levy, M. F., and Klintmalm, G. B. (1995) Transplant. Proc.
27, 1121–1123
51. Mor, E., Facklam, D., Hasse, J., Sheiner, P., Emre, S., Schwartz, M., and
Miller, C. (1995) Transplant. Proc. 27, 1126
2
J. W. Neal and N. A. Clipstone, manuscript in preparation.
Calcineurin Negatively Regulates Adipocyte Differentiation 49781
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Joel W. Neal and Neil A. Clipstone
Differentiation in 3T3-L1 Cells AdipocyteCalcium-dependent Inhibition of
Calcineurin Mediates the
TRANSDUCTION:
MECHANISMS OF SIGNAL
doi: 10.1074/jbc.M207913200 originally published online September 25, 2002
2002, 277:49776-49781.J. Biol. Chem.
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