Human Translocation Liposarcoma-CCAAT/Enhancer Binding Protein (C/EBP) Homologous Protein (TLS-CHOP) Oncoprotein Prevents Adipocyte Differentiation by Directly Interfering with C/EBPbeta Function*

Guillaume AdelmantDagger , Jeff D. Gilbert, and Svend O. Freytag§

From the Departments of Molecular Biology and Radiation Oncology, Henry Ford Health System, Detroit, Michigan 48202-3450

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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/EBPbeta in naïve NIH3T3 cells, and their ability to inhibit C/EBPbeta -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/EBPbeta and inhibit C/EBPbeta 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, PPARgamma 2, was able to rescue TLS-CHOP-inhibited cells, indicating that TLS-CHOP interferes primarily with C/EBPbeta -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/EBPbeta 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.

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 alpha  (C/EBPalpha ),1 a basic-leucine zipper protein, is among the transcription factors that are induced prior to the onset of morphological differentiation. C/EBPalpha 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/EBPalpha 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/EBPalpha 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/EBPbeta (8, 9), and PPARgamma 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/EBPbeta and C/EBPdelta are the earliest events to occur upon treatment with differentiation inducers (8). These factors, in conjunction with adipogenic hormones, induce the expression of PPARgamma 2 (9). The next event, which coincides with cell cycle withdrawal and commitment to the adipocyte differentiation program, is the induction of C/EBPalpha . The presence of C/EBP and PPAR binding sites in the C/EBPalpha promoter suggest that its expression may be regulated by C/EBPbeta and PPARgamma 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/EBPbeta (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/EBPalpha and C/EBPbeta , 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/EBPalpha and C/EBPbeta 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/EBPbeta -driven adipogenesis. Three lines of evidence make C/EBPbeta the likely molecular target of TLS-CHOP in myxoid liposarcoma; (i) C/EBPbeta protein is expressed in myxoid liposarcoma cells whereas C/EBPalpha is not (17)2; (ii) C/EBPbeta is induced early in the adipogenic pathway and is likely to trigger many of the downstream events (8, 9); and (iii) C/EBPbeta is the preferred heterodimerizing partner of TLS-CHOP (13). We demonstrate here that TLS-CHOP completely blocks C/EBPbeta -driven adipogenesis by directly interfering with the normal function of the C/EBPbeta protein. The results support the thesis that a blockade to normal differentiation is important in the development of the cancer phenotype.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Retroviral and Plasmid Constructs-- The parental vector, pLN(phi -phi ), 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/EBPbeta coding sequence was amplified by PCR from pMEX-CRP2 (22) and cloned into the EcoRI restriction site of pLN(phi -phi ) to generate pLN(C/EBPbeta -phi ). pLN(C/EBPbeta -CHOP) and pLN(C/EBPbeta -TLS-CHOP) were constructed by insertion of PCR-amplified human CHOP- and TLS-CHOP-coding sequences into the BamHI site of pLN(C/EBPbeta -phi ), respectively. Human TLS-CHOP-coding sequences were obtained by PCR amplification of a 402/91 myxoid liposarcoma (17) (obtained from P. Aman) lambda 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 pWZLhygroPPARgamma 2, the mouse PPARgamma 2-coding sequence was PCR-amplified from pBluescript-PPARgamma 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 WZLhygroPPARgamma 2 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 PPARgamma 2 were induced to differentiate with 5 µM pioglitazone as described previously (11). To test for C/EBPbeta 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/EBPbeta (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.

PPARgamma 2 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-PPARgamma 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/EBPbeta (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'.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

TLS-CHOP Inhibits C/EBPbeta -driven NIH3T3 Adipogenesis-- To determine whether TLS-CHOP could inhibit C/EBPbeta function in adipogenesis, we developed a system where both the inducer of differentiation (C/EBPbeta ) 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/EBPbeta 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/EBPbeta 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 right-arrow Gly, Lys410 right-arrow Gly, Arg412 right-arrow Asn). These three basic amino acids were selected for mutagenesis because they were previously shown to be critical for C/EBPalpha 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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Fig. 1.   Schematic diagram of retroviral vectors. The solid rectangles represent the Moloney murine leukemia virus long terminal repeats. IRES, internal ribosome entry site of the encephalomyocarditis virus; SV40, SV40 early promoter; Neo, neomycin resistance gene. The precise location of the point mutations in the two TLS-CHOP mutants are indicated at the bottom.

As shown in Fig. 2A, the five cell pools infected with the vectors encoding C/EBPbeta either alone or in conjunction with CHOP, TLS-CHOP, and the TLS-CHOP mutants, express the same amount of the 36-kDa C/EBPbeta (LAP) protein (lanes 2-6). This amount is significantly higher than the endogenous level of C/EBPbeta found in NIH3T3 cells infected with the control vector (lane 1). Importantly, the level of C/EBPbeta protein expressed in the NIH3T3 pools (lanes 2-6) is identical to that observed in 3T3-L1 adipoblasts on day 2 of differentiation (lane 8), whereas its level of expression in cells infected with the control vector (lane 1) is similar to that of undifferentiated 3T3-L1 cells (lane 7). Similarly, the level of CHOP expression in cells stably infected with the C/EBPbeta -CHOP retrovirus (Fig. 2B, lane 3) is comparable to that observed in 3T3-L1 cells grown in low glucose for 2 days (lane 8). Likewise, TLS-CHOP and its two mutants are expressed at a roughly equal level (lanes 4-6), which is slightly greater than that observed in the 402/91 myxoid liposarcoma cell line (lane 9). These results demonstrate that in this model system, the expression of C/EBPbeta , CHOP, and TLS-CHOP closely mimics the normal or pathological conditions occurring in adipose tissue.


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Fig. 2.   Western blot analysis of G418-resistant NIH3T3 pools. A, Western blot showing expression of C/EBPbeta . Lanes 1-6, NIH3T3 pools expressing the six retroviral vectors (phi -phi , C/EBPbeta -phi , C/EBPbeta -CHOP, C/EBPbeta -TLS-CHOP, C/EBPbeta -TLS-CHOP/BR, and C/EBPbeta -TLS-CHOP/LZ, respectively); lane 7, undifferentiated 3T3-L1 cells on day 0 (confluence); lane 8, 3T3-L1 cells on day 2 of the differentiation program. The 36-kDa (LAP) and 20-kDa (LIP) C/EBPbeta proteins are indicated. The 20-kDa C/EBPbeta (LIP) protein is a translation product from an internal methionine codon (27). B, Western blot showing expression of CHOP and TLS-CHOP. Lanes 1-6 as in A; lane 7, 3T3-L1 cells on day 0; lane 8, 3T3-L1 cells on day 2 following addition of adipogenic hormones in low glucose medium; lane 9, 402/91 human myxoid liposarcoma cells. The positions of the 75-kDa TLS-CHOP (and mutants) and 29-kDa CHOP proteins are indicated. Each lane was loaded with an equal amount of nuclear protein.

NIH3T3 cells expressing exogenous C/EBPbeta can be induced to differentiate in a manner very similar to 3T3-L1 cells (8, 9). To determine the adipogenic potential of the various cell pools, confluent cell monolayers were treated with adipogenic hormones for two days and the extent of differentiation was scored at day ten. As expected, the differentiation of NIH3T3 cells was completely dependent on ectopic expression of C/EBPbeta (Fig. 3A, top, compare phi -phi to C/EBPbeta -phi ). Under these conditions, C/EBPbeta was able to convert essentially 100% of the cell monolayer into mature, fat-laden adipocytes. Co-expression of CHOP (C/EBPbeta -CHOP) strongly inhibited C/EBPbeta -driven adipogenesis as only a low percentage of the cells acquired small fat droplets. Similarly, TLS-CHOP abolished C/EBPbeta -driven adipogenesis and proved to be slightly, but consistently, better than CHOP. The extent of differentiation of TLS-CHOPexpressing cells (C/EBPbeta -TLS-CHOP) was essentially identical to that of cells infected with the parental vector (phi -phi ). Importantly, this inhibitory effect of TLS-CHOP was not attributable to its transforming activity as only a minute percentage (<< 1%) of cells co-expressing C/EBPbeta and TLS-CHOP showed signs of morphological transformation (fusiform morphology or foci formation). On the contrary, the vast majority of these cells displayed a morphology which was indistinguishable from that of control cells. Surprisingly, the TLS-CHOP basic region mutant (C/EBPbeta -TLS-CHOP/BR) blocked adipogenic conversion by C/EBPbeta as effectively as CHOP. By contrast, TLS-CHOP bearing point mutations in its heterodimerizing domain (C/EBPbeta -TLS-CHOP/LZ) had no effect on C/EBPbeta -driven differentiation.


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Fig. 3.   Results of differentiation assays. Top, the six NIH3T3 pools were induced to differentiate as described under "Materials and Methods." Monolayers were fixed and stained with Oil Red O, which stains for lipid, after 7 days. Differentiation assays were repeated five times with similar results. Bottom, Western blot analysis demonstrating expression of aP2. Each lane was loaded with an equal amount of cellular protein.

These morphological observations were corroborated by examining the expression of aP2, a late differentiation marker. As shown in Fig. 3B (bottom), expression of aP2 was strongly induced upon differentiation of the C/EBPbeta -phi and C/EBPbeta -TLS-CHOP/LZ cell pools. In contrast, there was little or no induction of aP2 in those pools that failed to differentiate morphologically. Importantly, the failure of the CHOP-, TLS-CHOP-, and TLS-CHOP/BR-expressing cell pools to differentiate can not be attributed to reduced expression of the transcription factor driving the adipogenic program as C/EBPbeta was expressed equally among these cell pools (see Fig. 2A).

Because the leucine zipper domain of TLS-CHOP was required for its ability to inhibit C/EBPbeta -driven adipogenesis, we examined whether this effect of TLS-CHOP correlated with its ability to heterodimerize with C/EBPbeta in situ. Both TLS-CHOP (Fig. 4, lane 4) and its basic region mutant (lane 5) could be co-immunoprecipitated from nuclear extracts with C/EBPbeta -specific antibodies, indicating that these proteins were stably complexed with C/EBPbeta . As expected, mutation of the leucine zipper domain of TLS-CHOP (lane 6) abolished its ability to heterodimerize with C/EBPbeta . Thus, the ability of TLS-CHOP to inhibit C/EBPbeta -driven adipogenesis requires its leucine zipper domain and correlates with its ability to form a stable heterodimer with C/EBPbeta in situ.


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Fig. 4.   Co-immunoprecipitation of C/EBPbeta and the TLS-CHOP proteins. C/EBPbeta complexes were precipitated from crude nuclei with anti-C/EBPbeta antibodies, and immunoprecipitates were analyzed for the various CHOP proteins by Western blotting. Lanes 1-6, NIH3T3 pools expressing the six retroviral vectors (phi -phi , C/EBPbeta -phi , C/EBPbeta -CHOP, C/EBPbeta -TLS-CHOP, C/EBPbeta -TLS-CHOP/BR, and C/EBPbeta -TLS-CHOP/LZ, respectively). The positions of the 75-kDa TLS-CHOP (wild-type and mutants) and 29-kDa CHOP proteins are indicated.

Ectopic Expression of PPARgamma 2 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/EBPbeta function. However, they do not rule out the possibility that TLS-CHOP may block other requisite events which are independent of C/EBPbeta function. To address this possibility, we examined whether ectopic expression of PPARgamma 2, another adipogenic transcription factor (10, 11), could rescue TLS-CHOP-inhibited cells from their differentiation block. NIH3T3 pools expressing phi -phi and C/EBPbeta -TLS-CHOP were infected with a control retrovirus (WZLhygro) or one encoding PPARgamma 2 (WZLhygroPPARgamma 2). Following a brief selection in hygromycin B, cells were pooled and examined for expression of PPARgamma 2 and their ability to differentiate into adipocytes. Immunofluorescence studies demonstrated that ~80% of cells infected with WZLhygro PPARgamma 2 expressed PPARgamma 2 in the nucleus (not shown). By contrast, cells infected with the control virus and uninfected C/EBPbeta -TLS-CHOP cells (see Fig. 8, below right) did not express PPARgamma 2. Results of the differentiation assays demonstrated clearly that PPARgamma 2 was able to rescue TLS-CHOP-inhibited cells from their differentiation block (Fig. 5). Indeed, following treatment with the PPARgamma 2 activator pioglitazone (11), C/EBPbeta -TLS-CHOP cells expressing PPARgamma 2 differentiated as well as PPARgamma 2-expressing control (phi -phi ) cells. The results demonstrate that TLS-CHOP-inhibited cells are capable of adipogenesis, suggesting that TLS-CHOP does not interfere with other requisite C/EBPbeta -independent events.


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Fig. 5.   PPARgamma 2 can rescue TLS-CHOP-inhibited NIH3T3 cells. phi -phi and C/EBPbeta -TLS-CHOP cell pools infected with the parental WZLhygro retroviral vector (no PPARgamma 2) or WZLhygroPPARgamma 2 (PPARgamma 2) were examined for their ability to differentiate in the presence of 5 µM pioglitazone. Cell monolayers were fixed and stained with Oil Red O 8 days later. Differentiation assays were repeated three times with similar results.

TLS-CHOP Blocks C/EBPbeta Function by Preventing Its Binding to Target DNA Sequences-- To gain a better understanding of how TLS-CHOP inhibits C/EBPbeta function, we examined the possibility that TLS-CHOP blocks C/EBPbeta from binding to its DNA target sites. In a first test, the amount of C/EBPbeta 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/EBPbeta resulted in an increase in the amount of complex formed between C/EBPbeta and the aP2 probe (Fig. 6, compare lane 2 to lane 8). The presence of C/EBPbeta in this complex is demonstrated by the fact that an anti-C/EBPbeta antibody could completely "supershift" this complex (lane 11). Although the five cell pools overexpressing C/EBPbeta contain the same amount of C/EBPbeta protein in the nucleus (Fig. 2A), the C/EBPbeta 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/EBPbeta -phi cells (lane 8). In contrast, C/EBPbeta DNA binding activity was unaffected in cells co-expressing TLS-CHOP/LZ (lane 32).


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Fig. 6.   C/EBPbeta DNA binding activity in NIH3T3 pools. Electrophoretic mobility shift assays using the aP2 probe. phi -phi (lanes 1-6), C/EBPbeta -phi (lanes 7-12), C/EBPbeta -CHOP (lanes 13-18), C/EBPbeta -TLS-CHOP (lanes 19-24), C/EBPbeta -TLS-CHOP/BR (lanes 25-30), and C/EBPbeta -TLS-CHOP/LZ (lanes 31-36). No nuclear extract (lanes 1, 7, 13, 19, 25, and 31); nuclear extract with no additions (lanes 2, 8, 14, 20, 26, and 32); addition of nonspecific (Sp1 binding site) competitor DNA (lanes 3, 9, 15, 21, 27, and 33); addition of specific (aP2 binding site) competitor DNA (lanes 4, 10, 16, 22, 28, and 34); addition of anti-C/EBPbeta antibodies (lanes 5, 11, 17, 23, 29, and 35); and addition of anti-CHOP antibodies (lanes 6, 12, 18, 24, 30, and 36). The positions of the C/EBPbeta -C/EBPbeta homodimer, supershifted complexes, and free aP2 probe are indicated.

The C/EBPbeta -CHOP heterodimer was recently shown to bind a subset of high affinity C/EBP sites (AAATGCAATCCCC, SAAB4 site) (16). Thus, the same nuclear extracts were examined for C/EBPbeta DNA binding activity using the SAAB4 probe. The results were essentially the same as for the aP2 probe, except that a relatively minor complex was observed below the C/EBPbeta -C/EBPbeta homodimer complex in cells co-expressing C/EBPbeta and CHOP (not shown). This complex was the C/EBPbeta -CHOP heterodimer, as it is eliminated by both anti-C/EBPbeta and anti-CHOP antibodies. No additional complexes were observed with extracts co-expressing C/EBPbeta and TLS-CHOP (or TLS-CHOP/BR), suggesting that unlike the C/EBPbeta -CHOP heterodimer, the C/EBPbeta -TLS-CHOP heterodimer can not bind to the SAAB4 sequence.

This issue was examined further by measuring C/EBPbeta transactivation activity in the different NIH3T3 pools. For this purpose, each cell pool was transfected with a CAT reporter gene driven by a C/EBP-responsive promoter containing a trimerized aP2 binding site. A modest (average of 4-fold, n = 5), but reproducible, increase in CAT reporter gene activity was observed in cells overexpressing C/EBPbeta (C/EBPbeta -phi ) relative to control (phi -phi ) cells (Fig. 7, compare lanes 1 and 2). Importantly, TLS-CHOP completely abolished C/EBPbeta transactivation activity, as cells co-expressing C/EBPbeta and TLS-CHOP showed the same level of CAT activity as cells expressing the parental vector (compare lane 4 to lane 1). As expected, TLS-CHOP mutated in its leucine zipper had no effect on C/EBPbeta transactivation activity (compare lane 6 to lane 2). CHOP (lane 3) and TLS-CHOP/BR (lane 5) were less effective than wild-type TLS-CHOP (lane 4) at inhibiting C/EBPbeta transactivation activity, demonstrating that these results qualitatively paralleled those of the differentiation assays (Fig. 3).


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Fig. 7.   C/EBPbeta transactivation activity in NIH 3T3 pools. NIH3T3 pools were transfected with p(aP2)3TATA-CAT as described under "Materials and Methods." Twenty-four hours later, cells were harvested and assayed for CAT activity. Lanes 1-6, phi -phi , C/EBPbeta -phi , C/EBPbeta -CHOP, C/EBPbeta -TLS-CHOP, C/EBPbeta -TLS-CHOP/BR, and C/EBPbeta -CHOP/LZ, respectively. An equal amount of each cell extract, after correcting for transfection efficiency, was assayed. The transfection assays were repeated five times with similar results.

TLS-CHOP Blocks Induction of the Adipogenic Transcription Factor PPARgamma 2-- Two indirect lines of evidence suggest that the PPARgamma 2 gene is a downstream target of C/EBPbeta . Conditional expression of C/EBPbeta results in the induction of PPARgamma 2 mRNA (9), and the mouse PPARgamma 2 promoter contains potential C/EBP binding sites (29). Because PPARgamma 2 is an important component of the adipocyte differentiation machinery, we examined whether TLS-CHOP blocked the induction of PPARgamma 2 in C/EBPbeta -expressing cells following treatment with differentiation inducers. Cells expressing C/EBPbeta either alone (C/EBPbeta -phi ) or with TLS-CHOP (C/EBPbeta -TLS-CHOP) were grown to confluence, treated with differentiation inducers, and examined for expression of PPARgamma 2 by immunofluorescence 2 days later. As expected, cells expressing C/EBPbeta alone showed clear evidence of PPARgamma 2 expression in the nucleus (Fig. 8, left). In contrast, cells co-expressing C/EBPbeta 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/EBPbeta -phi cells prior to treatment with differentiation inducers (not shown). Only an occasional C/EBPbeta -TLS-CHOP cell showed weak nuclear expression of PPARgamma 2 following treatment with differentiation inducers (Fig. 8, right). Together, the results demonstrate that TLS-CHOP effectively blocks C/EBPbeta function, induction of PPARgamma 2, and development of the mature adipocyte phenotype.


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Fig. 8.   TLS-CHOP-inhibited cells fail to induce PPARgamma 2. Confluent cell monolayers expressing either C/EBPbeta alone (C/EBPbeta -phi , left) or co-expressing C/EBPbeta and TLS-CHOP (C/EBPbeta -TLS-CHOP, right) were treated with differentiation inducers for 2 days and examined for expression of PPARgamma 2 by immunofluorescence. Cells on the right were intentionally overexposed to show the uniform background immunofluorescence and lack of nuclear staining. Cells were photographed at a magnification of 500×. Differentiation assays were repeated three times with similar results.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Using a differentiation model that closely mimics physiological conditions and where the expression of C/EBPbeta is constitutive and sufficient to drive the adipogenic program, we demonstrate here that TLS-CHOP functions as a potent inhibitor of C/EBPbeta -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/EBPbeta 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/EBPbeta -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 PPARgamma 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/EBPbeta -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/EBPbeta . As shown here, this interaction inhibits C/EBPbeta 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/EBPbeta . 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/EBPalpha 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/EBPalpha is both sufficient (1-3) and necessary (5-7) for adipogenesis in vitro and in vivo. C/EBPbeta and C/EBPdelta , two proteins closely related to C/EBPalpha , and PPARgamma 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/EBPbeta is one of the first events to occur following treatment with adipogenic hormones (8). Expression of C/EBPbeta , in collaboration with C/EBPdelta , generates a second wave of transcriptional activation that leads to the induction of PPARgamma 2 (9). Although it is not known whether the induction of PPARgamma 2 expression is a direct effect of C/EBPbeta , the presence of C/EBP binding sites in the PPARgamma 2 promoter makes it likely that PPARgamma is a downstream effector of C/EBPbeta . The regulation of C/EBPalpha expression is more complex and subject to both positive and negative control. The presence of PPAR and C/EBP binding sites in the C/EBPalpha promoter (12), coupled with the fact that C/EBPbeta (LAP) can transactivate this promoter in transfection assays (our unpublished results), suggests that induction of C/EBPalpha results from a cooperation between C/EBPbeta and PPARgamma 2. Whereas the induction of C/EBPbeta and PPARgamma 2 occurs early (by 24 h) and at least 1 day prior to any signs of morphological differentiation, the induction of C/EBPalpha (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/EBPalpha 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/EBPalpha is the critical event which commits adipoblasts to the differentiation program. However, the fact that C/EBPbeta , but not C/EBPalpha , is expressed in myxoid liposarcoma suggests that TLS-CHOP is blocking differentiation of this lineage at a point prior to induction of C/EBPalpha , making C/EBPbeta the likely target. Given that PPARgamma 2 and C/EBPalpha are likely downstream targets of C/EBPbeta , inhibition of C/EBPbeta 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-RARalpha ), 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.

    ACKNOWLEDGEMENTS

We thank B. Spiegelman for providing the PPARgamma 2 cDNA, D. Lane for the aP2 antibody, and K. Rogulski for comments on the manuscript.

    FOOTNOTES

* 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.

Dagger 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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Materials & Methods
Results
Discussion
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