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INTRODUCTION |
The nuclear factor of activated T cells
(NFAT)1 family of
transcription factors is composed of four calcium-responsive family members (NFATc1 (NFAT2/NFATc), NFATc2 (NFAT1/NFATp), NFATc3
(NFAT4/NFATx), and NFATc4 (NFAT3)) and is best known for its role in
the regulation of the T cell immune response (1-3). The principal
function of NFAT proteins in T cells is to couple stimulation of the T
cell antigen receptor to changes in the expression of a number of
cytokine and other immunologically important genes. NFAT proteins are
regulated primarily at the level of their subcellular localization
through the actions of the calcium/calmodulin-dependent
serine/threonine phosphatase, calcineurin (1-3). In resting cells,
NFAT family members are normally located in the cytoplasm in a
hyperphosphorylated latent form. However, following an increase in the
intracellular calcium concentration, activated calcineurin directly
dephosphorylates NFAT proteins, inducing their rapid nuclear import and
increasing their intrinsic DNA binding activity. Once located in the
nucleus, NFAT proteins are then free to bind to their target promoter
elements and activate the transcription of specific NFAT target genes, either alone or in combination with other nuclear partners. This calcineurin-mediated activation pathway is strongly opposed by a number
of specific NFAT kinases, which act to directly rephosphorylate NFAT
proteins, thereby directly antagonizing NFAT activity by inhibiting
their DNA binding activity and promoting their rapid export back into
the cytoplasm (4-10). Consequently, NFAT-dependent transcription is highly dynamic and exquisitely sensitive to changes in
the intracellular calcium concentration (11, 12). This aspect of NFAT
regulation is likely to be significant, since both the extent and
duration of NFAT activity have recently been shown to influence the
qualitative pattern of NFAT-dependent gene expression induced during T cell activation (13).
Whereas the calcineurin/NFAT signaling pathway is certainly best known
for its role in the regulation of the immune response, it has become
increasingly apparent that this pathway also plays an important role in
the regulation of a wide variety of cellular responses in a number of
other tissues (2, 14). Thus, important roles for distinct NFAT proteins
have been demonstrated in the development and function of the
cardiovascular system, including regulation of cardiac valve
morphogenesis, cardiac hypertrophy, early patterning of the
vasculature, and angiogenesis in mature blood vessels (15-19). In
addition, NFAT proteins have also been shown to be involved in the
regulation of chondrocyte growth and differentiation (20) as well as to
play important roles in skeletal muscle, where they have been shown to
differentially regulate myogenesis, muscle fiber type gene expression,
and myocyte hypertrophy (21-24). These findings indicate that the
calcineurin/NFAT signaling pathway is likely to play a much broader
role in the regulation of cell growth and development than previously appreciated.
Previous studies have revealed the expression of NFAT proteins in the
murine 3T3-L1 preadipocyte cell line, suggesting a potential role for
these proteins in the regulation of adipocyte differentiation and
function (25). The 3T3-L1 preadipocyte cell line is a well established
in vitro model of adipocyte differentiation that has been
extensively used to investigate the molecular processes that control
adipocyte growth and development (26, 27). When confluent, growth-arrested 3T3-L1 cells are exposed to the adipogenic hormones methylisobutylxanthine, dexamethasone, and insulin (collectively known
as MDI), they synchronously enter the cell cycle and undergo a defined
genetic program of terminal differentiation. This process requires the
ordered expression of a number of transcription factors, including
members of the CCAAT/enhancer-binding protein (C/EBP) family and the
peroxisome proliferator-activated receptor
(PPAR
) before
permanently exiting the cell cycle and giving rise to mature morphologically distinct adipocytes containing large cytoplasmic triglyceride depots (26, 27).
The current study was prompted by our recent demonstration that
calcineurin is a critical effector of a calcium-dependent signaling pathway that acts to inhibit adipocyte differentiation (28).
Given this role of calcineurin and the known expression of NFAT
proteins in preadipocytes (25), we wished to examine the potential role
of the NFAT signaling pathway in the regulation of adipocyte
differentiation. To address this question, we took advantage of a
previously characterized constitutively active NFATc1 mutant (caNFATc1)
that is known to constitutively localize to the nucleus, bind DNA with
high affinity, and activate endogenous chromatin-embedded NFAT target
genes (29). We have used this constitutively active mutant to examine
the effects of sustained NFATc1 activation on the adipocyte
differentiation of 3T3-L1 cells. Remarkably, we find that whereas
ectopic expression of this NFATc1 mutant in 3T3-L1 cells is able to
potently inhibit their ability to differentiate into mature adipocytes,
it is also sufficient to induce these immortalized cells to acquire the
well established hallmarks of cellular transformation (30). Hence, we
find that cells expressing caNFATc1 (a) lose
contact-mediated growth inhibition, (b) exhibit reduced
serum growth requirements, (c) are protected from growth
factor deprivation-induced apoptosis, (d) gain growth factor
autonomy and continue to proliferate in the absence of serum as a
result of an NFATc1-induced autocrine regulatory growth loop,
(e) undergo anchorage-independent cell growth in semisolid media, and (f) form tumors in athymic nude mice. Taken
together, these results indicate that sustained NFATc1 activity is able to subvert the mechanisms that regulate the normal cell growth and
differentiation of 3T3-L1 cells and is sufficient to induce these
immortalized cells to adopt a transformed cell phenotype, thereby
establishing the oncogenic potential of the NFATc1 transcription factor.
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EXPERIMENTAL PROCEDURES |
Cell Culture and Adipocyte Differentiation--
3T3-L1
preadipocytes (ATCC) were cultured in growth medium: Dulbecco's
modified Eagle's medium (DMEM) containing 4.5 g/liter glucose
(Invitrogen) supplemented with 10% (v/v) fetal calf serum (FCS;
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), 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 refed with growth medium containing 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) as previously
described (28). Stained cells were either photographed directly or
counterstained with Giemsa and visualized by bright field microscopy.
Production of Recombinant Retroviruses and Infection of 3T3-L1
Cells--
The retroviral expression vectors pMSCV-GFP and
pMSCV-caNFATc1 have been previously described (29). Recombinant
retroviruses were produced by co-transfecting either the pMSCV-GFP or
pMSCV-caNFATc1 proviral vectors together with pVSV-G
(Clontech), encoding the vesicular stomatitis
virus-glycoprotein, into the GP293 pantrophic packaging cell line
(Clontech) using LipofectAMINE Plus (Invitrogen). Medium was replaced after 24 h, and viral supernatants were
harvested 2 days post-transfection and stored at
80 °C. For
infections, 5 × 104 3T3-L1 cells were plated per well
of a six-well plate. The next day, medium was replaced with 2 ml of
viral supernatant containing 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 and typically used within 5-7 days of
infection. To ensure reproducibility, each experiment was repeated
using cells derived from independent viral infections and independently
derived retroviral stocks. Flow cytometric analysis of green
fluorescent protein routinely revealed that greater than 95% of cells
were virally infected.
Immunoblot and Northern Blot Analysis--
Protein extracts
prepared from cells harvested at the indicated times following
induction of differentiation or withdrawal of serum were resolved by
SDS-PAGE and subjected to immunoblot analysis with the relevant
antibody (Ab). The Abs PPAR
(H-100), C/EBP
(14AA), C/EBP
(H-7), C/EBP
(C-22), cyclin D (H-295), c-Myc (N-262), and actin
(C-2) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz,
CA); Rb (G3-245) was purchased from BD Pharmingen. For Northern blot
analysis, total RNA was isolated from cells using Trizol (Invitrogen)
on the indicated day after the induction of differentiation. RNA
samples (10 µg) were separated 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 Eastman Kodak Co. X-AR film. Membranes were
stripped and reprobed with a glyceraldehyde-3-phosphate dehydrogenase
probe as a loading control.
Focus Formation and Methylene Blue Staining--
For
focus-forming studies, 3T3-L1 cells infected with either the control
MSCV-GFP or the MSCV-caNFATc1 viruses were diluted 1:30 with uninfected
wild type 3T3-L1 cells. These were plated at a final density of 1 × 104 cells/well of a six-well plate, and the growth
medium was changed every 2 days. After 2 weeks, the cells were
visualized by both phase-contrast microscopy and fluorescence
microscopy for detection of GFP expression. Subsequently, the plates
were rinsed in phosphate-buffered saline and fixed with methanol and
then stained with 0.4% methylene blue in 0.5 M sodium
acetate to visualize foci.
Cell Proliferation Studies--
3T3-L1 cells infected with
either the control MSCV-GFP or the MSCV-caNFATc1 virus were plated in
triplicate. After 24 h, the cells were rinsed three times with
DMEM and cultured in DMEM supplemented with either 10, 0.5, or 0%
(v/v) FCS. At the indicated times after the medium change, cells were
trypsinized and enumerated with a Coulter particle counter set to
record events between 7 and 25 µm.
Cell Cycle Analysis--
On the indicated day following medium
exchange, cells were trypsinized, washed once with growth medium and
twice with sample buffer (Dulbecco's phosphate-buffered saline
containing 1 g/liter glucose), and fixed in ice-cold 70% ethanol. At
least 24 h later, 5 × 105 fixed cells were
stained for 30 min in 0.5 ml of sample buffer containing 50 µg/ml
propidium iodide and 100 units/ml RNase A. Analysis of cellular DNA
content was determined by collecting 10,000 events using a FACScaliber
flow cytometer and CELLQuest software (BD Biosciences).
Conditioned Medium Experiments--
3T3-L1 cells infected with
either the control MSCV-GFP or the MSCV-caNFATc1 virus were grown until
they had achieved 80% confluence and then washed three times with DMEM
and cultured in DMEM lacking serum for a further 2 days. The medium
from each cell population was then collected and centrifuged at
2000 × g for 10 min to yield a debris-free supernatant
of serum-free conditioned medium. Where indicated, the conditioned
medium was heated to 94 °C for 20 min prior to use. To determine the
effects of serum-free conditioned medium on cell growth and survival,
wild type 3T3-L1 cells at 20% confluence were washed three times in
DMEM and then cultured in a 1:1 mix of the appropriate conditioned
medium and an equal volume of fresh DMEM. For adipocyte differentiation
assays, serum-free conditioned medium was combined with an equal volume
of DMEM containing 20% FCS, which was then used to induce adipocyte
differentiation as described above.
Methylcellulose Growth and Nude Mouse Injections--
To assay
cells for growth in methylcellulose medium, 60-mm Petri dishes were
coated with 1% agarose to resist cell adhesion. Cells were
trypsinized, and 1 × 105 cells were resuspended in 8 ml of growth medium containing 1.8% methylcellulose and allowed to
grow in the Petri dishes for 4 weeks, after which representative
colonies were photographed, and colonies were counted. For analysis of
tumorigenic potential in vivo, cells that had been infected
with either the control MSCV-GFP or the MSCV-caNFATc1 virus were
trypsinized, washed, and resuspended in phosphate-buffered saline at a
concentration of 2.5 × 106 cells/ml. Athymic
nu/nu mice (Harlan) were injected subcutaneously in each
flank with 1 × 106 cells, and tumor volume was
determined after 16 days using a previously described formula (31).
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RESULTS |
caNFATc1 Inhibits Adipocyte Differentiation in 3T3-L1
Cells--
In a previous study, we identified calcineurin as a
critical intrinsic component of a
Ca2+-dependent signaling pathway involved in
the inhibition of adipocyte differentiation (28). Since NFAT proteins
are known to be directly activated by calcineurin and are also known to
be expressed in 3T3-L1 cells (25), we initially examined whether
increased NFAT activity could affect adipogenesis. For this experiment,
we took advantage of our previously characterized caNFATc1 mutant that is known to be constitutively localized to the nucleus, able to bind
DNA with high affinity, and capable of activating endogenous gene
expression (29). To facilitate efficient gene transfer, the caNFATc1
cDNA was introduced into the MSCV-GFP retroviral vector under the
transcriptional control of the MSCV-long terminal repeat and
upstream of an internal ribosome entry site-GFP expression cassette, thereby allowing the expression of both caNFATc1 and GFP from
a single bicistronic mRNA. We have previously shown that we are
able to generate high titer virus capable of routinely infecting >95%
of 3T3-L1 cells by pseudotyping our recombinant retroviruses with the
vesicular stomatitis virus glycoprotein-G (28). This high level of
infection obviates the process of isolating and expanding clonal cell
lines and allows analysis of bulk populations of cells immediately
after infection.
To determine the effects of sustained NFATc1 signaling on adipocyte
differentiation, 3T3-L1 preadipocyte cells were infected with either a
retrovirus encoding caNFATc1 (MSCV-caNFATc1) or the control MSCV-GFP
retrovirus and then induced to differentiate using a well established
adipocyte differentiation protocol that involves exposure to the MDI
adipogenic mixture. As shown in Fig. 1A, cells infected with the
control MSCV-GFP virus and treated with MDI readily differentiated into
morphologically distinct fat-laden adipocytes, as assessed by staining
with the neutral lipid-specific dye, Oil Red O. In contrast, 3T3-L1
cells expressing caNFATc1 failed to differentiate into mature
adipocytes, based upon both morphological criteria and a lack of
specific staining with Oil Red O. This lack of differentiation was
confirmed by analyzing cells for their expression of both the
adipocyte-specific marker gene aP2 and the critical proadipogenic
transcription factors PPAR
and C/EBP
. Whereas control cells
expressed aP2, PPAR
, and C/EBP
following treatment with MDI, the
expression of these gene products was undetectable in MDI-treated,
caNFATc1-expressing cells (Fig. 1, B-D). However, ectopic
expression of caNFATc1 did not completely disrupt all responses of
3T3-L1 cells to the MDI adipogenic stimuli. Treatment of
caNFATc1-expressing cells with MDI induced expression of C/EBP
and
C/EBP
, two early transcription factors in the adipogenic cascade,
with similar kinetics to those observed in control cells (Fig. 1,
E and F). Taken together, these data suggest that
sustained activation of the NFATc1 signaling pathway inhibits the
differentiation of 3T3-L1 cells into mature adipocytes by preventing
the expression of the critical proadipogenic transcription factors
PPAR
and C/EBP
.

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Fig. 1.
A constitutively active form of NFATc1
inhibits adipocyte differentiation in 3T3-L1 cells. A,
3T3-L1 preadipocytes infected with either MSCV-GFP (control)
or MSCV-caNFATc1 retroviruses were either left untreated or induced to
undergo adipocyte differentiation by treatment with MDI as described
under "Experimental Procedures." 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.
B, Northern blot analysis of aP2 mRNA expression in
control and caNFATc1-expressing cells induced to undergo
differentiation for the indicated number of days. The membrane was
reprobed using glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) as a loading control. C-F, effects of
sustained NFATc1 activity on the expression of the adipogenic
transcription factors PPAR , C/EBP , C/EBP , and C/EBP . Whole
cell extracts were prepared from control and caNFATc1-expressing cells
on the indicated days following the induction of differentiation and
analyzed by SDS-PAGE followed by immunoblotting with the indicated
Ab.
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Sustained NFATc1 Activity Induces Morphological Changes and Loss of
Contact-dependent Growth Inhibition in 3T3-L1
Cells--
When we initially analyzed 3T3-L1 cells expressing
caNFATc1, we noticed that they exhibited a very different cellular
morphology compared with either control 3T3-L1 cells or cells infected
with the control MSCV-GFP virus. Whereas control 3T3-L1 cells and cells infected with control MSCV-GFP virus were relatively uniform in size
and shape and represented a typical fibroblast morphology, cells
expressing caNFATc1 adopted a wider range of morphologies, including
many tracts of highly refractile spindle-like cells interspersed with
occasional large, flat cells (Fig.
2A). Moreover, unlike control
cells that stopped growing once they had reached confluence, we found
that cells expressing caNFATc1 overgrew the monolayer and continued to
proliferate beyond confluence (Fig. 2B). In fact, visual
inspection of caNFATc1-expressing cells under the microscope revealed
that they accumulated in dense mutilayers of cells growing on top of
one another. These observations suggested that expression of caNFATc1
had caused these 3T3-L1 cells to lose contact-mediated growth
inhibition, one of the known hallmarks of cellular transformation (30).
To more clearly visualize this phenotype, we performed a focus-forming
assay in which a small number of either MSCV-GFP- or
MSCV-caNFATc1-infected cells were mixed together with an excess of
uninfected wild type 3T3-L1 cells and then grown in culture for 10-14
days and analyzed for the formation of transformed foci by either
methylene blue staining or direct microscopy. Whereas cells infected
with the control MSCV-GFP virus did not give rise to any foci as
detected by methylene blue staining, we found that caNFATc1-expressing
cells formed numerous foci (Fig. 2C). Because caNFATc1 and
GFP are expressed from the same bicistronic mRNA in the
MSCV-caNFATc1 retroviral vector, both caNFATc1-expressing cells and
cells infected with the control retrovirus can be readily discriminated
from wild type uninfected 3T3-L1 cells on the basis of their GFP
expression. This allowed us to identify cells infected with either the
MSCV-caNFATc1 or control MSCV-GFP retrovirus, which in turn allowed us
to evaluate their cellular and colony morphologies. As a result of this
analysis, we found that all of the caNFATc1-expressing cells in the
mixed cultures formed readily detectable foci, whereas cells infected with the control MSCV-GFP virus were morphologically indistinguishable from their uninfected neighboring cells (Fig. 2D). Together,
these results suggest that expression of caNFATc1 in 3T3-L1 cells
promotes the loss of contact-mediated growth inhibition and induces
these cells to form foci in culture.

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Fig. 2.
Sustained NFATc1 activity induces
morphological changes and loss of contact-dependent growth
inhibition in 3T3-L1 cells. A, phase microscopy
demonstrates morphological changes in 3T3-L1 cells expressing caNFATc1
as compared with control cells. B, effects of caNFATc1 on
cell proliferation in medium containing 10% (v/v) FCS. Cells infected
with either the control or caNFATc1 retroviruses were plated in
triplicate in medium containing 10% (v/v) FCS, and the cell number was
determined daily with a Coulter particle counter. Cell number is
expressed as thousands of cells/cm2. The S.D. values are
indicated. C, caNFATc1-expressing cells form foci in
culture. Cells infected with either the control or caNFATc1
retroviruses were mixed 1:30 with uninfected 3T3-L1 cells, grown 10 days postconfluence, fixed with methanol, and then stained with
methylene blue. D, representative fields from C
were visualized by phase microscopy (upper panel) and by
fluorescence microscopy for GFP expression (lower
panel).
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Sustained NFATc1 Activity in 3T3-L1 Cells Promotes Cell Cycle
Progression in the Presence of Reduced Serum
Concentrations--
Having demonstrated that ectopic expression of
caNFATc1 promotes the proliferation of 3T3-L1 cells in the presence of
10% FCS, we next investigated whether caNFATc1 could affect cell
proliferation under reduced serum conditions. As shown in Fig.
3, control 3T3-L1 cells cultured in
medium containing only 0.5% FCS fail to proliferate (Fig.
3A) and accumulate in the G1 phase of the cell
cycle after a 24-h period (Fig. 3B). In contrast, we found
that caNFATc1-expressing cells steadily continue to proliferate under
these reduced serum conditions (Fig. 3A) and maintain a cell
cycle profile typical of actively dividing cells (Fig.
3B).

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Fig. 3.
Sustained NFATc1 activity in 3T3-L1 cells
promotes cell cycle progression in the presence of reduced serum
concentrations. A, effects of caNFATc1 on cell
proliferation in the presence of reduced serum concentrations. Cells
infected with either the control or caNFATc1 retroviruses were plated
in triplicate in medium containing 0.5% (v/v) FCS, and the cell number
was determined daily with a Coulter particle counter. Cell number is
expressed as thousands of cells/cm2. The S.D. values are
indicated. B, cell cycle analysis of control and
caNFATc1-expressing cells 1 day after changing to medium containing
0.5% serum. Cells were fixed and stained with propidium iodide
(PI) and then analyzed by flow cytometric analysis. The
percentage of cells in each stage of the cell cycle (G1, S,
G2/M) is indicated. C, effects of caNFATc1 on
the expression of cyclin D, Rb, and c-Myc. Whole cell extracts were
prepared from cells infected with either the control or caNFATc1
retroviruses on the indicated days following transfer to medium
containing 0.5% serum and analyzed by SDS-PAGE followed by
immunoblotting with the indicated Ab. Cyclins D1 and D2 and the
phosphorylated form of Rb (pRb) are indicated by
arrowheads.
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Since cells expressing caNFATc1 appeared to overcome the G1
cell cycle arrest induced by low serum conditions, we next examined the
effects of caNFATc1 on the expression of the cell cycle-related genes
cyclin D, retinoblastoma protein (Rb), and c-myc,
which are all known to play a role in cell cycle progression. Within 24 h of shifting to reduced serum conditions, the expression of the D-type cyclin proteins, which is known to be dependent
upon the presence of mitogenic growth factors (32), was significantly diminished in control cells. In contrast, the levels of cyclin D1 and
D2 were maintained at a high level in caNFATc1-expressing cells, even
after 48 h in the presence of reduced serum (Fig. 3C).
Since cyclin D-dependent protein kinases are
known to contribute toward cell cycle progression by directly
phosphorylating and inhibiting the growth-inhibitory activity of the Rb
cell cycle regulatory protein (33), we next examined the effects of
caNFATc1 on the relative distribution of phosphorylated Rb polypeptide species. In resting cells, Rb is found in a hypophosphorylated active
form (Rb). Following mitogenic stimulation and the activation of
cyclin-dependent protein kinases, Rb becomes
hyperphosphorylated and consequently inactive (pRb), as detected by a
more slowly migrating form on SDS-PAGE. As shown in Fig. 3C,
shifting control cells to low serum conditions results in the
disappearance of the hyperphosphorylated Rb forms. Conversely, these
inactive Rb isoforms are still readily detectable in
caNFATc1-expressing cells maintained in the presence of 0.5% FCS for
more than 48 h. Finally, we examined expression of the c-Myc
oncoprotein expression in both control and caNFATc1-expressing cells.
Interestingly, caNFATc1-expressing cells consistently exhibited high
levels of c-Myc protein even under these reduced serum conditions (Fig.
3C). Taken together, these results indicate that ectopic
expression of caNFATc1 in 3T3-L1 cells is able to reduce serum growth
requirements by promoting cell cycle progression even under conditions
of limiting serum growth factors.
Sustained NFATc1 Activity Protects Cells from Growth Factor
Withdrawal-induced Apoptosis--
We next tested the effects of
caNFATc1 on the cellular response to complete serum withdrawal, which
is known to induce normal fibroblasts to undergo apoptosis. As
determined by phase microscopy and direct cell counts, transfer of
control MSCV-GFP retrovirally infected cells from growth medium
containing 10% FCS into serum-free medium resulted in a rapid and
pronounced decrease in the number of viable cells (Fig.
4, A and B). In
contrast, we observed that cells infected with the MSCV-caNFATc1
retrovirus not only maintained viability but also continued to
proliferate after a brief lag phase despite the complete absence of
serum. As indicated by flow cytometric analysis of the cellular DNA
profile of control MSCV-GFP-infected cells, complete serum withdrawal
resulted in the accumulation of a significant proportion of cells
exhibiting a sub-G1 DNA content that is known to be
indicative of cells undergoing apoptosis. Conversely, we found that
cells infected with the MSCV-caNFATc1 virus maintained a typical cell
cycle DNA profile with little evidence of apoptosis (Fig.
4C). These data suggest that expression of caNFATc1 is able
to protect 3T3-L1 cells from undergoing apoptosis in response to growth
factor withdrawal.

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Fig. 4.
Sustained NFATc1 activity protects cells from
growth factor withdrawal-induced apoptosis. A, phase
microscopy of control and caNFATc1-expressing cells on the indicated
day following complete withdrawal of serum. B, cells
infected with either the control or caNFATc1 retroviruses were plated
in triplicate in medium lacking serum, and their number was determined
at daily intervals with a Coulter particle counter. The data are
presented as the percentage change in cell number compared with the
number of cells present at day 0, which was arbitrarily set to 100%.
The S.D. values are indicated. C, effects of caNFATc1 on
apoptosis induced by serum growth factor withdrawal. Cells infected
with either the control or caNFATc1 retroviruses were plated in
triplicate in medium lacking serum and were analyzed by PI staining
followed by flow cytometric analysis at daily intervals. The data are
presented as percentage of apoptotic cells, as determined by the
percentage of cells in the sub-G1 peak compared with the
number of cells in the cell cycle (left panel).
Representative profiles from day 4 are also shown (right
panels).
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Sustained NFATc1 Activity in 3T3-L1 Cells Induces the Production of
One or More Autocrine Heat-labile Prosurvival/Antiadipogenic
Factors--
In the previous experiments, we observed that
caNFATc1-expressing cells were able to proliferate even in the complete
absence of exogenously derived serum growth factors (see Fig. 4,
A and B). The underlying mechanism for this
effect could be the result of an intrinsic cell-autonomous effect of
caNFATc1 on the expression of genes involved in the regulation of cell
cycle progression. Alternatively, the effect could be
non-cell-autonomous and result from the caNFATc1-dependent
production of one or more prosurvival/promitogenic factors that act in
an autocrine fashion to promote cell survival and proliferation. In
order to gain initial insights into the underlying molecular mechanism,
we first assayed serum-free conditioned medium from either control or
caNFATc1-expressing cells for the presence of survival/mitogenic
factors. Thus, serum-free conditioned medium collected from either
control or caNFATc1-expressing cells were tested for their effects on
the survival of wild type 3T3-L1 cells cultured in medium lacking
serum. As shown in Fig. 5A,
serum-free conditioned medium isolated from control MSCV-GFP-infected
cells did not support the survival of wild type 3T3-L1 cells, which appeared to rapidly succumb to apoptosis. In contrast, serum-free conditioned medium isolated from caNFATc1-expressing cells promoted both the survival and proliferation of wild type 3T3-L1 cells (Fig.
5A). Further investigation revealed that the activity of the
survival/mitogenic factor present in the serum-free conditioned medium
isolated from caNFATc1-expressing cells was sensitive to heat
treatment, potentially suggesting the involvement of a heat-labile polypeptide.

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Fig. 5.
Enforced expression of caNFATc1 in 3T3-L1
cells induces the production of an autocrine heat-labile
promitogenic/antiadipogenic factor(s). A, phase
microscopy of wild type 3T3-L1 cells incubated for either 0 or 4 days
with serum-free conditioned medium (CM) isolated from either
control or caNFATc1-expressing cells. Where indicated, the conditioned
medium was first heated to 94 °C for 20 min. B, Oil Red O
staining of wild type 3T3-L1 preadipocytes induced to undergo
adipocyte differentiation by treatment with MDI together with
conditioned medium from either control or caNFATc1-expressing
cells. Where indicated, the conditioned medium was heat-treated at
94 °C for 20 min.
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Since mitogenic growth factors have previously been shown to inhibit
adipocyte differentiation (34, 35), we next investigated whether the
production of a caNFATc1-induced autocrine factor might also explain
the observed blockade of adipocyte differentiation in
caNFATc1-expressing cells (Fig. 1). Thus, we examined the ability of
normal wild type 3T3-L1 cells to undergo MDI-induced adipocyte differentiation in the presence of conditioned medium isolated from
either control or caNFATc1-expressing cells. Whereas the conditioned
medium isolated from control cells did not affect the ability of 3T3-L1
cells to efficiently differentiate into mature adipocytes, as
determined by staining with Oil Red O, we found that the conditioned
medium isolated from caNFATc1-expressing cells potently inhibited
adipogenesis (Fig. 5B). As with the mitogenic activity
described above, the antiadipogenic activity present in conditioned
medium from caNFATc1-expressing cells was found to be heat-labile (Fig.
5B). Taken together, these data indicate that caNFATc1
induces the production of one or more heat-labile factors that act in
an autocrine fashion to promote both cell survival and proliferation of
3T3-L1 cells as well as potently inhibiting their ability to undergo
terminal differentiation into mature adipocytes.
Sustained NFATc1 Activity in 3T3-L1 Cells Promotes
Anchorage-independent Cell Growth and the Formation of Tumors in
Athymic Nude Mice--
Collectively, our data indicate that sustained
NFATc1 activity causes 3T3-L1 cells to adopt many of the well
established hallmarks of transformed cells. To further analyze the
transforming potential of caNFATc1, we next tested the ability of
caNFATc1 to induce anchorage-independent cell growth, another well
established property of transformed cells. Thus, either control or
caNFATc1-expressing cells were plated in semisolid methylcellulose
medium and monitored for their ability to form colonies in the absence
of a solid substratum. We found that control cells remained in
suspension as single cells and never formed colonies, whereas the
caNFATc1-expressing cells readily formed large colonies (Fig.
6, A and B).

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Fig. 6.
Enforced expression of caNFATc1 in 3T3-L1
cells promotes both anchorage-independent cell growth and the formation
of tumors in athymic nude mice. A, phase microscopy of
either control or caNFATc1-expressing 3T3-L1 cells grown in semisolid
methylcellulose medium. B, quantitation of colonies growing
in each dish of methylcellulose medium from 1 × 105
control or caNFATc1 input cells. ND, none detected.
C, tumor formation in nude mice following subcutaneous flank
injection with 1 × 106 control or caNFATc1-expressing
cells. Tumors are indicated by white
arrowheads.
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As a final examination of the transforming potential of caNFATc1,
we tested the ability of caNFATc1 expression to promote the formation
of tumors in athymic nude mice, the sine qua non of cellular
transformation. Thus, athymic nude mice were injected in each flank
with 106 3T3-L1 cells infected with either control MSCV-GFP
virus or the MSCV-caNFATc1 virus, and the animals were monitored for
tumor formation. Whereas all of the mice injected with
caNFATc1-expressing cells displayed large bilateral flank tumors (Fig.
6C; mean tumor volume = 700 ± 170 mm3), none of the mice injected with control cells
exhibited detectable tumors. These results indicate that ectopic
expression of caNFATc1 is able to promote anchorage-independent cell
growth and is sufficient to cause cells to form tumors in nude mice.
Taken together with our other data, these results clearly highlight the
oncogenic potential of the NFATc1 transcription factor.
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DISCUSSION |
In the current study, we provide evidence that deregulated NFATc1
activity is able to subvert the mechanisms that regulate the normal
cell growth and differentiation of 3T3-L1 preadipocytes and is
sufficient to induce these immortalized cells to acquire the well
established hallmarks of cellular transformation (30). Thus, we find
that cells expressing caNFATc1 lose contact-mediated growth inhibition,
exhibit reduced serum growth requirements, and are fully protected from
apoptosis following growth factor deprivation. Furthermore, we observe
that caNFATc1-expressing cells acquire full growth factor
autonomy and are able to continue to proliferate in the complete
absence of exogenously added serum growth factors. In fact, we provide
evidence that this growth factor independence is caused by the
NFATc1-dependent production of one or more autocrine
factors that are capable of promoting both the growth and survival of
wild type 3T3-L1 cells as well as inhibiting their differentiation into
mature adipocytes. Finally, we demonstrate that, unlike control cells,
cells expressing caNFATc1 establish colonies in semisolid medium and
form tumors when injected into athymic nude mice. Collectively, these
results indicate that sustained NFATc1 activity in 3T3-L1 preadipocytes
induces an autocrine regulatory growth loop and is sufficient to induce
the immortalized 3T3-L1 preadipocyte cell line to adopt a transformed
cell phenotype, thereby establishing the oncogenic potential of the
NFATc1 transcription factor in vitro and raising the
possibility that deregulated NFATc1 activity may play a causative role
in tumorigenic progression in vivo.
Significant insight into the potential molecular mechanisms underlying
the transforming effects of caNFATc1 was gained by our observation that
enforced expression of caNFATc1 is sufficient to promote growth factor
autonomy, allowing cells to grow and survive in the complete absence of
exogenously added serum growth factors. We provide evidence that this
independence from exogenous growth factors is mediated by a
non-cell-autonomous mechanism, in which caNFATc1 induces the expression
of a soluble prosurvival/promitogenic factor(s) that is capable of
acting in an autocrine fashion to promote cell growth and survival.
Thus, we find that whereas serum-free conditioned medium isolated from
control cells is unable to support the serum-free survival and
proliferation of wild type 3T3-L1 cells, conditioned medium isolated
from caNFATc1-expressing cells contains a mitogenic factor(s) capable
of promoting both the survival and proliferation of normal uninfected
3T3-L1 cells. In addition to its effects on cell survival and
proliferation, we also find that conditioned medium isolated from
caNFATc1-expressing cells is able to potently inhibit adipocyte
differentiation. This antiadipogenic activity of the
caNFATc1-conditioned medium is probably related to its mitogenic
activity, since mitogenic growth factors are known to inhibit adipocyte
differentiation by preventing preadipocytes from permanently exiting
the cell cycle (34, 35). Further analysis revealed that both the
mitogenic and antiadipogenic activities present in caNFATc1-conditioned
medium are sensitive to heat treatment, suggesting the involvement of a
polypeptide factor(s). Based upon these observations, we propose that
the growth factor autonomy and antiadipogenic activity induced by
caNFATc1 is most likely caused by a direct transcriptional effect
of NFATc1 on the expression of an endogenous factor(s) capable of
acting in an autocrine fashion to promote both cell proliferation and
the inhibition of adipocyte differentiation. Remarkably, the ability of
caNFATc1 to induce an autoregulatory growth loop in 3T3-L1 cells has a
striking parallel with the known function of NFAT proteins in the
regulation of the T cell immune response. There, NFAT proteins are
known to induce the expression of the primary T cell growth factor,
interleukin-2, and a component of its high affinity receptor, the
interleukin-2 receptor
-chain gene (1, 36, 37), thereby establishing an interleukin-2-dependent autocrine growth loop that is
known to promote T cell clonal expansion (38). Although we cannot completely rule out a cell autonomous role for NFATc1, our data certainly favor a mechanism in which sustained NFATc1 activity promotes
cell cycle progression by inducing the expression of an autocrine
growth factor(s). Since the autocrine production of mitogenic
growth factors is a common mechanism employed by tumor cells to escape
the normal regulatory constraints that restrict the proliferation and
survival of normal nontransformed cells (39), we believe that this
NFATc1-induced autocrine regulatory growth loop induced in 3T3-L1 cells
is likely to play an important role in the transforming effects of
caNFATc1.
Based upon our observation that sustained NFATc1 activity is
sufficient to induce the transformation of the immortalized 3T3-L1 cell
line, it is tempting to speculate that increased and prolonged NFATc1
activity may directly contribute toward tumor progression in certain
cell lineages in vivo. At present, there is no evidence that
the well known mechanisms of proto-oncogene activation, such as
gain-of-function mutations, chromosomal translocations, and gene
amplification, are involved in deregulating the activity of any NFAT
family member. However, one potential mechanism by which NFATc1
activity could be sustained in vivo is by the continuous presence of an external stimuli that is capable of directly stimulating signal transduction pathways leading to the prolonged induction of
NFATc1 activity. In this regard, it is interesting to note that
signaling through the insulin-like growth factor (IGF)-1 receptor is
known to enhance both the expression and activity of NFATc1 in a number
of different cell types, including preadipocytes (23, 24, 40, 41). This
observation is of particular interest, since both IGF-1 and IGF-2 are
known to play an important role in the etiology and progression of a
wide variety of different tumors (42). In fact, IGF-1 and IGF-2 are
found to be overexpressed in more than 90% of liposarcomas (43),
suggesting a potential mechanism by which IGF-1/IGF-2 autocrine
signaling may lead to sustained NFATc1 activity. However, whether
IGF-1/IGF-2-induced NFATc1 activity contributes toward the development
of liposarcomas and other tumors of the adipose lineage remains to be
seen. In addition to tyrosine kinase growth factor receptors such as
the IGF-1 receptor, several other signaling pathways, including those regulated by G-protein-coupled receptors, integrins, and the receptors for the Wnt family of proteins, are known to induce NFAT activity in a
wide variety of different cell types (44-47). Since excessive signaling through each of these different pathways is known to influence various aspects of the tumorigenic phenotype (48-51), it
will be interesting to determine whether any of these effects are
mediated through the actions of NFATc1.
Finally, although our results are the first to directly demonstrate the
oncogenic potential of an NFAT family member, other recent studies have
implicated NFAT proteins in the regulation of other distinct aspects of
the cancer phenotype. Based upon their observation of uncontrolled
proliferation of abnormal extraarticular cartilage cells in NFATc2-null
animals, Glimcher and colleagues (20) have proposed a potential tumor
suppressor role for NFATc2 in the chondrocyte lineage. When isolated
and placed in culture, these NFATc2-deficient cells were found to
proliferate rapidly, lose their ability to undergo contact-mediated
growth inhibition, exhibit an increase in aneuploidy, and form tumors
in nude mice. Support for an inhibitory role of NFATc2 in the
regulation of cellular growth control is provided by the recent
observation that this NFAT family member acts to repress the expression
of the key cell cycle regulatory kinase, cyclin-dependent
kinase-4, via the direct recruitment of transcriptional co-repressors
(52). Hence, it appears that NFATc1 and NFATc2 are likely to play
opposing roles in the tumorigenic process, with NFATc1 exhibiting the
properties of an oncogene and NFATc2 appearing to function as a tumor
suppressor. The opposing effects of these two NFAT family members on
the transformation process reflect their respective roles in the
regulation of the immune response, since deficiency in NFATc1 results
in a defect in immune cell function and T cell proliferation (53, 54), as would be expected for a positively acting transcription factor, whereas a deficiency in NFATc2 has been shown to lead to T and B cell
hyperproliferation (55, 56). Importantly, the role of NFAT family
members in the regulation of the tumorigenic phenotype may not be
restricted to the early stages of cell growth control, since a number
of recent studies have suggested roles for NFAT proteins in
angiogenesis and metastasis, processes that occur during the later
stages of tumorigenesis. NFAT proteins have been shown to play a role
in the regulation of vascular endothelial growth factor-mediated
angiogenesis via direct regulation of cyclooxygenase-2 gene expression
(19), an enzyme that is known to play a pivotal role in
neovascularization (57). This activity would be predicted to play an
important role in tumor progression, since successful tumor
colonization in vivo is known to require the recruitment of
an adequate blood supply in order to provide the tumor with oxygen and
essential nutrients (30). More recently, a role for NFAT proteins in
the regulation of tumor cell invasion has also been proposed (45). In
this case, NFAT proteins have been shown to be expressed in an active
form in human breast carcinoma cell lines, and the ability of these
cells to undergo integrin-mediated invasion of an extracellular matrix
barrier has been shown to depend on the activity of the
calcineurin/NFAT signaling pathway. Based upon these multiple lines of
evidence, it appears that the NFAT family of transcription factors is
likely to contribute to many distinct aspects of the tumorigenic
phenotype, including the initial dysregulation of cellular growth
control, the recruitment of an adequate blood supply, and the
regulation of tumor metastasis. Further investigation into the emerging
role of NFAT family members in the regulation of the tumorigenic
process is clearly warranted.