From the Departments of Molecular Biology and Radiation Oncology, Henry Ford Health System, Detroit, Michigan 48202-3450
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ABSTRACT |
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Human translocation liposarcoma
(TLS)-CCAAT/enhancer binding protein (C/EBP) homologous protein (CHOP)
is a fusion oncoprotein found specifically in a malignant tumor of
adipose tissue and results from a t(12;16) translocation that fuses the
amino-terminal part of TLS to the entire coding region of CHOP. Being
that CHOP is a member of the C/EBP transcription factor family,
proteins that comprise part of the adipocyte differentiation machinery, we examined whether TLS-CHOP blocked adipocyte differentiation by
directly interfering with C/EBP function. Using a single-step retroviral infection protocol, either wild-type or mutant TLS-CHOP were
co-expressed along with C/EBP in naïve NIH3T3 cells, and their ability to inhibit C/EBP
-driven adipogenesis was determined. TLS-CHOP was extremely effective at blocking adipocyte differentiation when expressed at a level comparable to that observed in human myxoid
liposarcoma. This effect of TLS-CHOP required a functional leucine
zipper domain and correlated with its ability to heterodimerize with
C/EBP
and inhibit C/EBP
DNA binding and transactivation activity
in situ. In contrast, the TLS-CHOP basic region was
dispensable, making it unlikely that the inhibitory effect of TLS-CHOP
is attributable to unscheduled gene expression resulting from
TLS-CHOP's putative transactivation activity. Another adipogenic
transcription factor, PPAR
2, was able to rescue TLS-CHOP-inhibited
cells, indicating that TLS-CHOP interferes primarily with
C/EBP
-driven adipogenesis and not with other requisite events of the
adipocyte differentiation program. Together, the results demonstrate
that TLS-CHOP blocks adipocyte differentiation by directly preventing
C/EBP
from binding to and transactivating its target genes.
Moreover, they provide strong support for the thesis that a blockade to
normal differentiation is an important aspect of the cancer
process.
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INTRODUCTION |
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Adipogenesis is a process in which an undifferentiated mesenchymal cell capable of proliferation matures into a post mitotic, fat-laden adipocyte. This differentiation process results in dramatic changes in gene expression and a spectacular alteration in cell morphology. The early phase of adipocyte differentiation is accompanied by the induction of transcription factors that promote cell cycle withdrawal and activation of cell-specific genes, two key aspects of terminal cell differentiation.
CCAAT/enhancer binding protein (C/EBP
),1 a basic-leucine
zipper protein, is among the transcription factors that are induced prior to the onset of morphological differentiation. C/EBP
was the
first regulatory protein demonstrated to play a central role in
promotion of the adipogenic program. We (1, 2) and others (3-6)
demonstrated that C/EBP
was both necessary and sufficient to promote
adipogenesis in fibroblastic cells such as NIH3T3 and 3T3-L1. These
observations, coupled with the fact that mice deficient in C/EBP
completely lack mature white or brown adipose tissue (7), demonstrate
unequivocally the pivotal role of this transcription factor in
adipogenesis. Following the same approach, several other transcription
factors, including another member of the C/EBP family, C/EBP
(8, 9),
and PPAR
2, a member of the nuclear hormone receptor superfamily (10,
11), proved capable of converting NIH3T3 cells into morphologically and
biochemically differentiated adipocytes. It is now clear that all of
the aforementioned transcription factors play an important and
sequential role in the adipocyte differentiation process. In the 3T3-L1
preadipocyte model, induction of C/EBP
and C/EBP
are the earliest
events to occur upon treatment with differentiation inducers (8). These
factors, in conjunction with adipogenic hormones, induce the expression
of PPAR
2 (9). The next event, which coincides with cell cycle
withdrawal and commitment to the adipocyte differentiation program, is
the induction of C/EBP
. The presence of C/EBP and PPAR binding sites
in the C/EBP
promoter suggest that its expression may be regulated
by C/EBP
and PPAR
2 (12).
In addition to transcription factors that induce and maintain the
mature adipocyte phenotype, there are also factors that negatively
regulate adipogenesis. Interestingly, one of these negative regulators,
CHOP (C/EBP homologous protein), a
stress and DNA damage-induced transcription factor, is a member of the C/EBP family (13-15). Unlike other C/EBPs that can bind
sequence-specific DNA as homodimers, CHOP homodimers cannot bind
sequence-specific DNA due to a nonconsensual sequence in its basic
region. However, CHOP is able to bind weakly to a restricted subset of
high affinity C/EBP binding sites (GCAAT) when complexed as a
heterodimer with other C/EBP proteins, such as C/EBP (16). Although
CHOP participates in the stress signal in response to metabolic
injuries, its downstream effectors are yet to be described.
Interestingly, an oncogenic variant of CHOP, called TLS-CHOP (TLS,
translocation liposarcoma), is
found specifically in myxoid liposarcoma, a malignant tumor of adipose
tissue. TLS-CHOP results from a t(12;16) chromosomal translocation that
fuses the amino-terminal part of TLS to the entire coding sequence of
CHOP (17, 18). Recently, a second chromosomal translocation (EWS-CHOP,
Ewings sarcoma) specific for myxoid liposarcoma
was also found to involve CHOP (19). That two fusion oncoproteins
involving CHOP are consistently (in over 90% of cases) and
specifically associated with myxoid liposarcoma raises the possibility
they are perturbing C/EBP function and causing a blockade in adipocyte
differentiation. Although a previous study demonstrated that induction
of endogenous CHOP by glucose deprivation inhibited 3T3-L1 adipogenesis
(20), the underlying mechanism of this inhibition is unclear.
CHOP-expressing cells failed to induce normal levels of C/EBP and
C/EBP
, two factors which are required to drive the 3T3-L1 adipogenic
program. Thus, it is not possible to discern from that study
whether the failure of CHOP-expressing cells to differentiate was
attributable to inhibition of C/EBP function by CHOP or a lack of
required C/EBP
and C/EBP
expression. Moreover, the effect of
TLS-CHOP, the oncogenic form of CHOP, on adipocyte differentiation
remains unexplored.
In the present study we investigate whether TLS-CHOP can inhibit
C/EBP-driven adipogenesis. Three lines of evidence make C/EBP
the
likely molecular target of TLS-CHOP in myxoid liposarcoma; (i) C/EBP
protein is expressed in myxoid liposarcoma cells whereas C/EBP
is
not (17)2; (ii) C/EBP
is
induced early in the adipogenic pathway and is likely to trigger many
of the downstream events (8, 9); and (iii) C/EBP
is the preferred
heterodimerizing partner of TLS-CHOP (13). We demonstrate here that
TLS-CHOP completely blocks C/EBP
-driven adipogenesis by directly
interfering with the normal function of the C/EBP
protein. The
results support the thesis that a blockade to normal differentiation is
important in the development of the cancer phenotype.
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MATERIALS AND METHODS |
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Retroviral and Plasmid Constructs--
The parental vector,
pLN(-
), was derived from pLXSN (21). The encephalomyocarditis
virus IRES sequence from pWZLneo (2) was introduced into pLXSN as an
EcoRI-BamHI fragment following PCR amplification.
The mouse C/EBP
coding sequence was amplified by PCR from pMEX-CRP2
(22) and cloned into the EcoRI restriction site of
pLN(
-
) to generate pLN(C/EBP
-
). pLN(C/EBP
-CHOP) and pLN(C/EBP
-TLS-CHOP) were constructed by insertion of PCR-amplified human CHOP- and TLS-CHOP-coding sequences into the BamHI
site of pLN(C/EBP
-
), respectively. Human TLS-CHOP-coding
sequences were obtained by PCR amplification of a 402/91 myxoid
liposarcoma (17) (obtained from P. Aman)
ZAP (Stratagene) cDNA
library. The two TLS-CHOP mutant versions were engineered by
PCR-mediated mutagenesis. Mutated regions were verified by DNA
sequencing. TLS-CHOP/BR was found to carry a Glu to Gly mutation at
amino acid 386. To generate pWZLhygroPPAR
2, the mouse
PPAR
2-coding sequence was PCR-amplified from pBluescript-PPAR
2
(10) (obtained from B. Spiegelman) and blunt end-ligated into pWZLhygro
between the BamHI and EcoRI sites. pWZLhygro is
identical to pWZLneo (2) except that it contains the hygromycin
resistance gene in place of the neomycin resistance gene.
p(aP2)3TATA-chloramphenicol acetyltransferase (CAT) was
created by cloning the double-stranded oligonucleotide 5'-AGCTTTTTCTCAACTTTGGAGCTCTTTTCTCAACTTTGGGTACCTTTTCTCAACTTTGG-3' into HindIII/BamHI-digested TATA-CAT (23).
Cell Culture and Gene Transfection--
Retroviruses were
produced by transient transfection of the Bosc 23 ecotropic packaging
cell line (24). Cells (5 × 106, 60-mm diameter dish)
were transfected by the calcium phosphate precipitation method using 10 µg of plasmid DNA. Viral supernatants were harvested 48 h later
and filtered through a 0.45-µm filter syringe. NIH3T3 cells (ATCC)
were cultivated in Dulbecco's modified Eagle's medium supplemented
with 10% calf serum (growth medium). NIH3T3 cells (3 × 105, 60-mm diameter dish) were infected with a similar
titer of the different retroviruses 1 day after plating. Viruses were
left in contact with the cells for 6 h in growth medium containing 4 µg/ml Polybrene. Two days later, infected cells were detached by
trypsinization and seeded into four dishes (100-mm diameter) containing
growth medium supplemented with 500 µg/ml G418 (Life Technologies,
Inc.). After selection for 1 week, G418-resistant cells were pooled and
subcultured for subsequent experiments. NIH3T3 pools infected with
WZLhygro and WZLhygroPPAR2 were treated in exactly the same manner
except cells were selected in growth medium containing 400 µg/ml
hygromycin B for 7 days. To test for differentiation ability, each pool
was seeded at 3 × 105 cells/dish (60-mm diameter) and
treated 3 days later (confluence) with differentiation medium
consisting of Dulbecco's modified Eagle's medium supplemented with
10% fetal calf serum, 1 µM dexamethasone, 0.5 mM methylisobutylxanthine, and 10 µg/ml insulin. After
48 h of treatment, the medium was changed and cells were
maintained thereafter in the same medium but containing only insulin.
Seven days later, cells were either fixed with 3.7% formaldehyde and stained with Oil Red O or processed for whole cell protein analysis. NIH3T3 pools expressing PPAR
2 were induced to differentiate with 5 µM pioglitazone as described previously (11). To test for C/EBP
transactivation, each cell pool (3 × 105,
60-mm diameter dish) was co-transfected with 2.5 µg of a CAT reporter
gene and 0.5 µg of pCMV luciferase using lipofection (Life
Technologies, Inc.). Twenty-four hours later, cells were lysed in
reporter lysis buffer (Promega), and equal amounts of transfection
efficiency-corrected cell extracts were assayed for CAT activity.
Western Blot Analysis and Co-immunoprecipitation--
Nuclear
extracts were prepared from actively dividing cell cultures. Briefly,
cells were washed twice with ice-cold phosphate-buffered saline (PBS)
and collected by centrifugation, and the cell pellet was resuspended in
500 µl of Nonidet P-40 lysis buffer (10 mM Tris, pH 7.4, 6.6 mM NaCl, 3 mM MgCl2, 0.5%
Nonidet P-40, 500 µM phenylmethylsulfonyl fluoride).
After a 15 min incubation on ice, the suspension was homogenized, and
cell debris was removed by centrifugation. The crude nuclei were washed
in 500 µl of the same buffer, collected by microcentrifugation, lysed
in Laemmli sample buffer, and boiled for 10 min. The same protocol was
used to prepare nuclear extracts from 3T3-L1 and 402/91 cells. Proteins were resolved by SDS-polyacrylamide (12%) gel electrophoresis, and
specific proteins were detected by Western blotting using the enhanced
chemiluminescence system (Amersham Corp.). For co-immunoprecipitation studies, nuclei were prepared as described above except the nuclear proteins were extracted from crude nuclei in high salt buffer [20
mM HEPES, pH 7.9, 400 mM NaCl, 1 mM
EDTA, 0.1% (v/v) Triton X-100] for 30 min on ice. The NaCl
concentration was adjusted to 150 mM with the same buffer
lacking NaCl, and proteins were immunoprecipitated for 2 h at room
temperature with 5 µl of anti C/EBP (D298, Santa Cruz
Biotechnology Inc.) and 20 µl of protein A-Sepharose (Pharmacia
Biotech Inc.). Pellets were washed sequentially with PBS containing
0.1% Triton X-100, PBS containing 500 mM NaCl, and PBS.
Immunoprecipitated proteins were boiled for 10 min in Laemmli sample
buffer and processed for Western analysis as described above.
PPAR2 Immunofluorescence--
Cells (105) were
plated on glass chamber slides and allowed to grow to confluence. Cells
were either left untreated or treated with differentiation inducers as
described above. Cells were fixed in 3.7% (v/v) formalin,
permeabilized with methanol, and incubated with a polyclonal rabbit
anti-PPAR
2 antibody (PA3-821, Affinity Bioreagents Inc.) followed by
fluorescein isothiocyanate-conjugated donkey anti-rabbit IgG secondary
antibody (both 1:200 dilution in PBS with 3% bovine serum albumin) for
1 h at 37 °C. Samples were examined and photographed using an
Olympus BX40 fluorescent microscope.
Electrophoretic Mobility Shift Assays--
Nuclear extracts were
prepared exactly as described elsewhere (25). Nuclear extracts prepared
from the different pools were first analyzed by Western blotting to
normalize for LAP expression. Twenty micrograms (20 µg) of nuclear
extract were incubated in C/EBP DNA binding buffer (10 mM
Tris-HCl, pH 7.5, 2 mM dithiothreitol, 50 mM
KCl, 10% (v/v) glycerol, 25 µg/ml poly(dI-dC) (25) on ice for 30 min
in the presence of either 100 ng of unlabeled competitor DNA, 100 ng of
anti-C/EBP (C19, Santa Cruz) or anti-CHOP antibody (R20, Santa Cruz)
prior to the addition of the 32P-labeled DNA probes.
Nucleoprotein complexes were resolved in a 6% nondenaturing
polyacrylamide gel prepared in 0.5× Tris-borate-EDTA. The following
32P-labeled, double-stranded oligonucleotides were used as
probes in DNA-binding reactions: aP2, 5'-agcttgTTTCTCAACTTTGa-3';
SAAB4, 5'-agcttgAAATGCAATCGCCa-3'.
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RESULTS |
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TLS-CHOP Inhibits C/EBP-driven NIH3T3 Adipogenesis--
To
determine whether TLS-CHOP could inhibit C/EBP
function in
adipogenesis, we developed a system where both the inducer of
differentiation (C/EBP
) and the potential inhibitor (TLS-CHOP) could
be co-expressed simultaneously in naive NIH3T3 cells. This was
accomplished through the use of triple gene retroviral vectors in which
the C/EBP
and TLS-CHOP proteins are produced from the same
bicistronic mRNA driven by the proviral long terminal repeat, and
expression of the selectable marker gene (neo) is driven by an internal
(SV40) promoter (Fig. 1). This approach
has several conceptual and technical advantages: (i) unlike the 3T3-L1
system, the differentiation of NIH3T3 cells is completely dependent on the introduction of an exogenous transcription factor(s) making it
possible to express the factor constitutively, (ii) use of the triple
gene vectors eliminates the need to perform sequential infections and
selections thereby avoiding the generation of clonal cell lines which
vary widely in their ability to differentiate, and (iii) co-translation
of the C/EBP
and TLS-CHOP proteins from the same bicistronic
mRNA greatly increases the likelihood that they will be expressed
stoichiometrically within the same cell. To obtain insight into which
structural domains of TLS-CHOP are required for its inhibitory effect,
two mutants were generated. TLS-CHOP/BR contains three site-directed
mutations in its basic region (Arg407
Gly,
Lys410
Gly, Arg412
Asn). These three
basic amino acids were selected for mutagenesis because they were
previously shown to be critical for C/EBP
DNA binding activity (26).
TLS-CHOP/LZ contains five Leu to Gly conversions in the leucine zipper
(positions 427, 434, 441, 452, and 459) and was constructed to
investigate the requirement of the heterodimerization domain. A
retroviral vector encoding the normal CHOP protein was also generated
to serve as a positive control. All six vectors were introduced into
naive NIH3T3 cells by retroviral infection and pooled cell lines
representing several thousand G418-resistant clones were generated
following a brief selection in G418. Cell pools were examined for
protein expression and their ability to undergo adipogenesis.
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Ectopic Expression of PPAR2 Can Rescue TLS-CHOP-inhibited
Cells--
The data presented thus far are consistent with a mechanism
in which TLS-CHOP inhibits C/EBP-driven adipogenesis by directly interfering with C/EBP
function. However, they do not rule out the
possibility that TLS-CHOP may block other requisite events which are
independent of C/EBP
function. To address this possibility, we
examined whether ectopic expression of PPAR
2, another adipogenic transcription factor (10, 11), could rescue TLS-CHOP-inhibited cells
from their differentiation block. NIH3T3 pools expressing
-
and
C/EBP
-TLS-CHOP were infected with a control retrovirus (WZLhygro) or
one encoding PPAR
2 (WZLhygroPPAR
2). Following a brief selection
in hygromycin B, cells were pooled and examined for expression of
PPAR
2 and their ability to differentiate into adipocytes.
Immunofluorescence studies demonstrated that ~80% of cells infected
with WZLhygro PPAR
2 expressed PPAR
2 in the nucleus (not
shown). By contrast, cells infected with the control virus and
uninfected C/EBP
-TLS-CHOP cells (see Fig. 8, below right)
did not express PPAR
2. Results of the differentiation assays
demonstrated clearly that PPAR
2 was able to rescue
TLS-CHOP-inhibited cells from their differentiation block (Fig.
5). Indeed, following treatment with the
PPAR
2 activator pioglitazone (11), C/EBP
-TLS-CHOP cells
expressing PPAR
2 differentiated as well as PPAR
2-expressing control (
-
) cells. The results demonstrate that
TLS-CHOP-inhibited cells are capable of adipogenesis, suggesting that
TLS-CHOP does not interfere with other requisite C/EBP
-independent
events.
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TLS-CHOP Blocks C/EBP Function by Preventing Its Binding to
Target DNA Sequences--
To gain a better understanding of how
TLS-CHOP inhibits C/EBP
function, we examined the possibility that
TLS-CHOP blocks C/EBP
from binding to its DNA target sites. In a
first test, the amount of C/EBP
DNA binding activity in each NIH3T3
pool was determined by the electrophoretic mobility shift assay using the aP2 binding site (28). Ectopic expression of C/EBP
resulted in
an increase in the amount of complex formed between C/EBP
and the
aP2 probe (Fig. 6, compare lane
2 to lane 8). The presence of C/EBP
in this complex
is demonstrated by the fact that an anti-C/EBP
antibody could
completely "supershift" this complex (lane 11). Although
the five cell pools overexpressing C/EBP
contain the same amount of
C/EBP
protein in the nucleus (Fig. 2A), the C/EBP
DNA
binding activity in cells co-expressing CHOP (lane 14),
TLS-CHOP (lane 20), or TLS-CHOP/BR (lane 26) was
significantly reduced relative to C/EBP
-
cells (lane
8). In contrast, C/EBP
DNA binding activity was unaffected in
cells co-expressing TLS-CHOP/LZ (lane 32).
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TLS-CHOP Blocks Induction of the Adipogenic Transcription Factor
PPAR2--
Two indirect lines of evidence suggest that the PPAR
2
gene is a downstream target of C/EBP
. Conditional expression of
C/EBP
results in the induction of PPAR
2 mRNA (9), and the
mouse PPAR
2 promoter contains potential C/EBP binding sites (29). Because PPAR
2 is an important component of the adipocyte
differentiation machinery, we examined whether TLS-CHOP blocked the
induction of PPAR
2 in C/EBP
-expressing cells following treatment
with differentiation inducers. Cells expressing C/EBP
either alone (C/EBP
-
) or with TLS-CHOP (C/EBP
-TLS-CHOP) were grown to
confluence, treated with differentiation inducers, and examined for
expression of PPAR
2 by immunofluorescence 2 days later. As expected,
cells expressing C/EBP
alone showed clear evidence of PPAR
2
expression in the nucleus (Fig. 8,
left). In contrast, cells co-expressing C/EBP
and
TLS-CHOP (right) exhibited only background
immunofluorescence that was evenly distributed over the entire cell.
This uniform background immunofluorescence was similar to that observed
with C/EBP
-
cells prior to treatment with differentiation
inducers (not shown). Only an occasional C/EBP
-TLS-CHOP cell showed
weak nuclear expression of PPAR
2 following treatment with
differentiation inducers (Fig. 8, right). Together, the
results demonstrate that TLS-CHOP effectively blocks C/EBP
function,
induction of PPAR
2, and development of the mature adipocyte
phenotype.
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DISCUSSION |
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Using a differentiation model that closely mimics physiological
conditions and where the expression of C/EBP is constitutive and
sufficient to drive the adipogenic program, we demonstrate here that
TLS-CHOP functions as a potent inhibitor of C/EBP
-driven adipogenesis. Unlike the 3T3-L1 system where a battery of transcription factors, including at least three C/EBP family members, cooperate to
trigger the onset of differentiation, the simple model system described
here allowed us to demonstrate that TLS-CHOP blocks adipocyte
differentiation by directly interfering with C/EBP
function.
Based on the result that three key mutations in the TLS-CHOP basic
region have little effect on its anti-adipogenic activity, and unlike
for CHOP (16),3 there is no
evidence that the C/EBP-TLS-CHOP heterodimer can bind
sequence-specific DNA, we conclude it is unlikely that the inhibitory
effect of TLS-CHOP is attributable to unscheduled gene expression that
prevents adipocyte differentiation. The fact that ectopic expression of
PPAR
2 can rescue TLS-CHOP-inhibited cells strongly supports this
conclusion. Nor can the inhibitory effect of TLS-CHOP be attributed to
its weak oncogenic activity (30), as the vast majority of
C/EBP
-TLS-CHOP-expressing cells showed no signs of morphological
transformation. On the contrary, our results indicate that the ability
of TLS-CHOP to inhibit adipocyte differentiation is mediated largely
through its ability to heterodimerize with and inhibit the function of
C/EBP
. As shown here, this interaction inhibits C/EBP
DNA
binding, resulting in a decrease in its transactivation ability. Our
results with the TLS-CHOP basic region mutant are somewhat at odds with
the results of Ron and colleagues (20) who reported that the basic
region of CHOP was required for its ability to inhibit 3T3-L1
adipogenesis. We believe the most likely explanation for this
discrepancy is that the CHOP basic region mutant used in that study was
poorly expressed relative to wild-type CHOP (see Fig. 3B in
Batchvarova et al. (20)). In contrast, the TLS-CHOP basic
region mutant used here is expressed equally well relative to wild-type
TLS-CHOP and heterodimerizes equally well with C/EBP
. Unfortunately,
because there is no evidence that TLS-CHOP homodimers or heterodimers
can bind sequence-specific DNA, we could not demonstrate that the
introduced mutations had the expected effect on TLS-CHOP's presumed
DNA binding activity. However, given that the three basic amino acids
altered are critical for C/EBP
DNA binding activity it is likely
that if TLS-CHOP is capable of binding sequence-specific DNA, this
mutant of TLS-CHOP would lack this activity.
The finding that TLS-CHOP, the product of a chromosomal translocation
found only in a malignancy of adipose tissue, involves a member of the
C/EBP family makes much sense in light of the pivotal role of the C/EBP
transcription factors in adipocyte differentiation. Results from four
laboratories have shown that C/EBP is both sufficient (1-3) and
necessary (5-7) for adipogenesis in vitro and in
vivo. C/EBP
and C/EBP
, two proteins closely related to C/EBP
, and PPAR
2, a transcription factor belonging to the nuclear hormone receptor superfamily, are also important factors in the adipogenic pathway (8-11). The apparent redundancy of these
transcription factors was initially somewhat puzzling; however, their
respective roles in adipocyte differentiation are becoming clearer. In
the 3T3-L1 model system, induction of C/EBP
is one of the first
events to occur following treatment with adipogenic hormones (8). Expression of C/EBP
, in collaboration with C/EBP
, generates a
second wave of transcriptional activation that leads to the induction
of PPAR
2 (9). Although it is not known whether the induction of
PPAR
2 expression is a direct effect of C/EBP
, the presence of
C/EBP binding sites in the PPAR
2 promoter makes it likely that
PPAR
is a downstream effector of C/EBP
. The regulation of
C/EBP
expression is more complex and subject to both positive and
negative control. The presence of PPAR and C/EBP binding sites in the
C/EBP
promoter (12), coupled with the fact that C/EBP
(LAP) can
transactivate this promoter in transfection assays (our unpublished
results), suggests that induction of C/EBP
results from a
cooperation between C/EBP
and PPAR
2. Whereas the induction of
C/EBP
and PPAR
2 occurs early (by 24 h) and at least 1 day prior to any signs of morphological differentiation, the induction of
C/EBP
(by 48 h) coincides precisely with cell cycle withdrawal and commitment to the adipocyte differentiation program (1, 31). These
observations, coupled with the facts that C/EBP
is known to inhibit
cell growth (1, 2, 4, 32) and is required for adipogenesis both
in vitro and in vivo (5-7), make it likely that
induction of C/EBP
is the critical event which commits adipoblasts
to the differentiation program. However, the fact that C/EBP
, but
not C/EBP
, is expressed in myxoid liposarcoma suggests that TLS-CHOP
is blocking differentiation of this lineage at a point prior to
induction of C/EBP
, making C/EBP
the likely target. Given that
PPAR
2 and C/EBP
are likely downstream targets of C/EBP
,
inhibition of C/EBP
function by TLS-CHOP would prevent their
induction and commitment to the adipocyte differentiation pathway.
Although the studies described here are directly relevant to the
genesis of myxoid liposarcoma, we believe they may have even broader
significance. TLS-CHOP belongs to a growing family of fusion
oncoproteins resulting from chromosomal translocations. That most of
these fusion proteins are specific for a given type of cancer suggests
they are impinging on a mechanism which is specific for that particular
cell lineage (e.g. differentiation machinery, cell-specific
signal transduction pathway) and not some general proliferation
mechanism (cell cycle machinery). Although incomplete differentiation
is a hallmark characteristic of the cancer cell, there is little
direct proof that the inability to differentiate normally is
in fact important in the cancer process. Indeed, because of the well
known reciprocal relationship between proliferation and
differentiation, it was often argued that the inability of a cancer
cell to differentiate completely was simply an indirect consequence of
uncontrolled proliferation. Cancer cells can not irreversibly exit the
cell cycle, which is required for terminal differentiation. This
argument has been very difficult to challenge as it is buttressed by
the fact that many of the genes known to be mutated in human cancer
play a role in the regulation of cell proliferation. The discovery of
oncoproteins such as TLS-CHOP and others (e.g. PML-RAR),
whose molecular targets are integral components of the cell
differentiation machinery, however, significantly weakens this
argument, at least for those cancers that involve them. On the
contrary, the fact that these oncoproteins directly target and inhibit
the cell differentiation machinery argues strongly that a blockade in
differentiation is important, but clearly not sufficient, for
development of the malignant phenotype. Indeed, it is likely that the
other component of the TLS-CHOP fusion oncoprotein (i.e.
TLS) provides an important function required for malignant transformation in vivo, such as a continuous growth signal
to differentiation-arrested cells.
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ACKNOWLEDGEMENTS |
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We thank B. Spiegelman for providing
the PPAR2 cDNA, D. Lane for the aP2 antibody, and K. Rogulski
for comments on the manuscript.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant CA62295.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.
Supported by a fellowship from the French Research Ministry and
from the Association pour la Recherche sur la Cancer (ARC). Present
address: Dept. of Cancer Biology, Dana Farber Cancer Institute, Harvard
Medical School, Boston, MA 02115.
§ To whom correspondence should be addressed: Molecular Biology Research, Henry Ford Health System, One Ford Place, Wing 5D, Detroit, M 48202-3450. Tel.: 313-876-1949; Fax: 313-876-1950.
1 The abbreviations used are: C/EBP, CCAAT/enhancer binding protein; CHOP, C/EBP homologous protein; TLS, translocation liposarcoma; PCR, polymerase chain reaction; CAT, chloramphenicol acetyltransferase.
2 G. Adelmant, J. D. Gilbert, and S. O. Freytag, unpublished observations.
3 G. Adelmant, J. D. Gilbert, and S. O. Freytag, unpublished results.
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REFERENCES |
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