A Role for C/EBPbeta in Regulating Peroxisome Proliferator-activated Receptor gamma  Activity during Adipogenesis in 3T3-L1 Preadipocytes*

Jonathan K. Hamm, Bae Hang Park, and Stephen R. FarmerDagger

From the Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 2118

Received for publication, January 29, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The differentiation of 3T3-L1 preadipocytes is regulated in part by a cascade of transcriptional events involving activation of the CCAAT/enhancer-binding proteins (C/EBPs) and peroxisome proliferator-activated receptor gamma  (PPARgamma ) by dexamethasone (DEX), 3-isobutyl-1-methylxanthine (MIX), and insulin. In this study, we demonstrate that exposure of 3T3-L1 preadipocytes to DEX and insulin fails to induce adipogenesis as indicated by a lack of C/EBPalpha , PPARgamma 2, and adipose protein 2/fatty acid-binding protein expression; however, PPARgamma 1 is expressed. Treatment of these MIX-deficient cells with a PPARgamma ligand, troglitazone, induces C/EBPalpha expression and rescues the block in adipogenesis. In this regard, we also show that induction of C/EBPalpha gene expression by troglitazone in C3H10T1/2 cells ectopically expressing PPARgamma occurs in the absence of ongoing protein synthesis, suggesting a direct transactivation of the C/EBPalpha gene by PPARgamma . Furthermore, ectopic expression of a dominant negative isoform of C/EBPbeta (liver-enriched transcriptional inhibitory protein (LIP)) inhibits the induction of C/EBPalpha , PPARgamma 2, and adipose protein 2/fatty acid-binding protein by DEX, MIX, and insulin in 3T3-L1 cells without affecting the induction of PPARgamma 1 by DEX. Exposure of LIP-expressing preadipocytes to troglitazone along with DEX, MIX, and insulin induces differentiation into adipocytes. Additionally, we show that sustained expression of C/EBPalpha in these LIP-expressing adipocytes requires constant exposure to troglitazone. Taken together, these observations suggest that inhibition of C/EBPbeta activity not only blocks C/EBPalpha and PPARgamma 2 expression, but it also renders the preadipocytes dependent on an exogenous PPARgamma ligand for their differentiation into adipocytes. We propose, therefore, an additional role for C/EBPbeta in regulating PPARgamma activity during adipogenesis, and we suggest an alternative means of inducing preadipocyte differentiation that relies on the dexamethasone-associated induction of PPARgamma 1 expression.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The differentiation of preadipocytes into mature fat cells is regulated by a cascade of transcription factors that interact in a complex fashion to control expression of several hundred adipogenic genes (1, 2). Many different nuclear factors have been shown to influence the adipogenic process, but two families of factors in particular have received the most attention as follows: the CCAAT enhancer-binding proteins (C/EBPs)1 and the peroxisome proliferator-activated receptors (PPAR) family of nuclear hormone receptors. Three members of the C/EBP family, alpha , beta , and delta , have been shown to play important roles in regulating adipose tissue development in mice and preadipocyte differentiation in vitro (3). In contrast, only the gamma  form of the PPAR family is considered to regulate adipogenesis in vitro and in vivo (4). The temporal pattern of expression of these important adipogenic factors, and control of their activity during adipogenesis, is dependent on a variety of biological effectors and other transcription factors (5-7).

The C/EBPs belong to a larger family of basic leucine zipper (bZIP) transcription factors, which have a C-terminal leucine zipper domain for dimerization and a basic domain for binding to DNA. There are at least six members of this family, alpha , beta , delta , gamma , epsilon , and zeta , that can both homodimerize and heterodimerize with each other and bind to the same C/EBP regulatory element in the promoters/enhancers of many different genes. In addition, each family member can give rise to several isoforms by a process of selective use of translational start sites within each mRNA or by proteolysis of a larger precursor protein (8). The C/EBPbeta mRNA, for instance, gives rise to at least four isoforms corresponding to the following peptides, 38, 34, 30, and 20 kDa. The 34-kDa protein is often referred to as LAP (liver-enriched transcriptional activator protein) since it has been shown to be a potent transactivator of liver gene expression (9). The 20-kDa polypeptide, however, can inhibit hepatic gene expression and is, therefore, referred to as LIP (liver-enriched transcriptional inhibitory protein) (9). This LIP isoform of C/EBPbeta corresponds to the C-terminal portion of the LAP protein that lacks the transactivation domain but contains the basic leucine zipper region. Consequently, LIP can act as a potent dominant negative repressor of C/EBPbeta activity. In fact, ectopic expression of LIP, resulting in a LAP/LIP ratio of ~1, blocks adipogenesis in preadipocytes in culture (10).

PPARgamma exists as two protein isoforms, gamma 1 and gamma 2, that are generated by alternative splicing of at least three different mRNAs, which are transcribed from the same gene (11, 12). PPARgamma 1 and gamma 2 share almost all the same exon sequences, except gamma 2 contains an additional 30 amino acids at the N terminus. Both isoforms of PPARgamma form an obligate heterodimer with the retinoid X receptor to bind to regulatory elements within the promoters/enhancers of many genes associated with lipid metabolism. Activation of PPARgamma ·RXR complexes requires association with a series of ligands that include RXR ligands such as 9-cis-retinoic acid as well as ligands for PPARgamma (13). The latter includes polyunsaturated fatty acids and their derivatives as well as the thiazolidinedione family of insulin sensitizers such as troglitazone (14-16). Transcription from the PPARgamma gene has been detected in many tissues in which the gamma 1 isoform is the predominant transcript (17). In contrast, transcription from the PPARgamma 2 promoter is highly adipose tissue-selective giving rise to abundant production of the PPARgamma 2 polypeptide in addition to the more ubiquitous PPARgamma 1 isoform (18). Ectopic expression of PPARgamma 2 or -gamma 1 in non-adipogenic fibroblasts under appropriate hormonal conditions results in potent induction of adipocyte differentiation (19).

Various mouse cell lines have been used to delineate the many different processes involved in regulating adipogenesis. Most notable are 3T3-L1 preadipocytes, which can be induced to differentiate into mature fat cells following exposure to a mixture of hormonal inducers including dexamethasone (DEX), isobutylmethylxanthine (MIX), insulin, and FBS. MIX and DEX induce expression of C/EBPbeta and C/EBPdelta , respectively, which in turn activate C/EBPalpha and PPARgamma expression (10, 20, 21). PPARgamma and C/EBPalpha are then capable of cross-activating each other as well as governing expression of the mature adipocytic phenotype (22, 23). The normal differentiation of preadipocytes in culture does not require addition of an exogenous PPARgamma ligand. In contrast, non-adipogenic fibroblasts that ectopically express a C/EBP or PPARgamma require exposure to a potent PPARgamma ligand to undergo conversion into adipocytes (24, 25). Preadipocytes have likely acquired the ability to produce an appropriate ligand of PPARgamma . The molecular mechanisms that regulate production of such molecules are not known. Earlier studies by others (26) have suggested a role for the sterol regulatory element-binding proteins (SREBPs).

We demonstrate that attenuation of C/EBPbeta activity by omitting MIX from the culture medium or ectopically expressing a dominant negative form of C/EBPbeta (LIP) renders 3T3-L1 preadipocytes dependent on an exogenous PPARgamma ligand for their differentiation into adipocytes. These studies have also uncovered an alternative pathway of adipogenesis, which involves a glucocorticoid-associated induction of PPARgamma 1 in the absence of C/EBPbeta activity. Furthermore, activation of PPARgamma 1 in the LIP-expressing cells with troglitazone directly activates C/EBPalpha gene expression.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Dexamethasone, 3-isobutyl-1-methylxanthine, insulin, puromycin, aprotinin, leupeptin, and digitonin were purchased from Sigma. Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum were supplied by Life Technologies, Inc. [alpha -32P]dCTP and [gamma -32P[ATP were purchased from PerkinElmer Life Sciences. Klenow fragment of DNA polymerase I was obtained from Promega (Madison, WI). Troglitazone was from Parke-Davis/Warner Lambert.

Antibodies-- We used a monoclonal anti-PPARgamma antibody and polyclonal antibodies against the C/EBPs, alpha , beta , and delta  (Santa Cruz Biotechnology, Santa Cruz, CA), and polyclonal anti-aP2 serum (kindly provided by Dr. David Bernlohr, University of Minnesota, MN).

Plasmids and Stable Cell Lines-- The BOSC 23 packaging cells (27), pBabe-Puro and pBabe-PPARgamma -Puro retroviral expression vectors (19), were kind gifts of Dr. Bruce Spiegelman (Dana Farber Cancer Institute, Harvard Medical School). The pBabe vector expressing either the LAP or LIP isoforms of C/EBPbeta were constructed by subcloning corresponding PCR products of the C/EBPbeta cDNA (20) into the BamHI and EcoRI sites of the pBabe-puro vector. The LAP PCR fragments were generated using the following primers: cbeta -1 (5'-CGCGGATCCCCACCATGGAAGTGGCCAACTT) and cbeta -3 (5'-CCGGAATTCGCATCAAGTCCCGAAACCCGGT), and the LIP PCR fragments were generated using cbeta -2 (5'-CGCGGATCCCCACCATGGCGGCCGGCTT) and cbeta -3 primers. Transfection of BOSC 23 packaging cells and subsequent infection of target cells were performed as described by others (19, 27). Infected target cells were selected for 6-10 days in medium containing 2.0 µg/ml puromycin.

Cell Culture-- Murine 3T3-L1 preadipocytes were cultured, maintained, and differentiated as described previously (28, 29). Briefly, cells were plated and grown for 2 days post-confluence in DMEM supplemented with 10% calf serum. Differentiation was then induced (Day 0) by changing the medium to DMEM containing 10% FBS, 0.5 mM 3-isobutyl-1-methylxanthine, 1 µM dexamethasone, and 1.67 µM insulin. After 48 h, cells were maintained in DMEM containing 10% FBS. 3T3-L1 cells expressing either C/EBPbeta LIP or control vector and 10T1/2 cells expressing PPARgamma were differentiated by the same protocol for 3T3-L1 cells, except growth medium was DMEM containing 10% FBS and 2.0 µg of puromycin, and the cells were differentiated and maintained in the presence or absence of 10 µM troglitazone, except as noted.

Oil Red O Staining-- Oil Red O staining was performed following the procedure described previously (29). The cells were then photographed using phase contrast microscopy.

Preparation of Whole Cell Extracts-- At the indicated times, cultured cells grown in 10-cm dishes were rinsed with phosphate-buffered saline (140 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, 8.1 mM Na2HPO4, pH 7.4) and then harvested in 1 ml of ice-cold buffer containing 50 mM Tris (pH 7.4), 100 mM NaCl, 1% sodium deoxycholate, 4% Nonidet P-40, 0.4% SDS, 5 µM aprotinin, and 50 µM leupeptin. Lysates were vortexed for 1 min and centrifuged for 15 min at full speed (13,000 rpm) in a microcentrifuge. Pellets were discarded and supernatants stored at -80 °C. Protein content of supernatants was determined using the BCA kit (Amersham Pharmacia Biotech).

Gel Electrophoresis and Immunoblotting-- Proteins were separated in SDS-polyacrylamide (acrylamide from American BioAnalytical) gels as described previously (23) and transferred to polyvinylidene difluoride membrane (Bio-Rad) in 25 mM Tris, 192 mM glycine. After transfer, the membrane was blocked with 4% nonfat dry milk in PBST for 1 h at room temperature. After incubation with the primary antibodies specified above, horseradish peroxidase-conjugated secondary antibodies (Sigma) and an enhanced chemiluminescent (ECL) substrate kit (PerkinElmer Life Sciences) were used for detection.

RNA Analysis-- Total RNA was harvested according to the procedure of Chomczynski and Sacchi (30). Cells were lysed in buffer containing M guanidinium isothiocyanate. Lysates were extracted with acid phenol/chloroform, and RNA was precipitated in 50% isopropyl alcohol overnight at -20 °C. Northern blot analysis was performed on 20 µg of each sample RNA as described. cDNA probes for C/EBPalpha and PPARgamma were labeled using Klenow fragment of DNA polymerase I and [alpha -32P[dCTP by random priming.

Preparation of Nuclear Protein Extracts-- Nuclear protein extracts were prepared essentially as described. Cells were washed twice with ice-cold phosphate-buffered saline and then lysed in nuclear lysis buffer (10 mM Tris (pH 7.6), 10 mM NaCl, 3 mM MgCl2, 0.5% Nonidet P-40). Samples were spun at low speed in a clinical centrifuge. Supernatants were discarded, and nuclei were lysed in nuclear extraction buffer (20 mM HEPES (pH 7.9), 350 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA (pH 8.0), 25% glycerol). Nuclear extracts were incubated on ice for 15 min and centrifuged at full speed (13,000 rpm) at 4 °C. The resulting supernatants were stored at -80 °C. Protein concentrations were determined using the BCA protein assay kit (Amersham Pharmacia Biotech).

Electrophoretic Mobility Shift Assay-- DNA binding assays were performed as described previously (31). Ten micrograms of nuclear extract was incubated with 3 µg of poly(dI-dC), 2 µl of carrier mix (50 mM MgCl2, 340 mM KCl), and delta buffer (0.1 mM EDTA, 40 mM KCl, 25 mM HEPES (pH 7.6), 8% Ficoll, 1 mM dithiothreitol) at 4 °C for 15 min. Double-stranded oligonucleotides corresponding to a C/EBP-binding site (5'-gatccGCGTTGCGCCACGATG-3' and 5'-CATCGTGGCGCAACGCggatc-3') were end-labeled with [gamma -32P[ATP using T4 polynucleotide kinase according to the manufacturer (New England Biolabs). For supershift experiments, antibody was added, and samples were incubated at room temperature for 1.5 h. Reactions were mixed with labeled probes on ice for 30 min and resolved on nondenaturing 6% polyacrylamide (39.5:0.5 acrylamide/bisacrylamide) gels at 200 V for 2-2.5 h at 4 °C in TBE buffer (80 mM Tris borate, 2 mM EDTA (pH 8.0)). Gels were vacuum-dried for 1 h before exposure to Biomax MR-1 autoradiography film (Eastman Kodak Co.). Antibodies used for C/EBP supershifts were the same as described above. Goat anti-rabbit IgG antibodies (Sigma) were used in negative control experiments.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

To understand the roles of various inducers and the C/EBPs in regulating adipogenesis, we generated 3T3-L1 cell lines expressing vector DNA alone (designated L1-V cells) or the dominant negative C/EBPbeta , LIP (designated L1-LIP cells). To determine the effect of the different inducers on expression of PPARgamma and C/EBPalpha , we stimulated L1-V cells to differentiate by exposure to different combinations of insulin, dexamethasone (DEX), and isobutylmethylxanthine (MIX) in the presence or absence of 10 µM troglitazone. Cells were maintained according to the procedure described under "Experimental Procedures," and total protein was harvested 4 days after induction. Fig. 1, lane 7, shows the abundant expression of PPARgamma and C/EBPalpha by 4 days of differentiation following exposure to DEX, MIX, insulin, and FBS. The presence of troglitazone appears to attenuate expression of these transcription factors (lane 8). Omission of DEX and/or MIX from the culture medium significantly attenuates differentiation of these preadipocytes into mature fat cells as indicated by the barely detectable expression of C/EBPalpha and aP2 in each case (lanes 1, 3, and 5). Interestingly, PPARgamma 1 is abundantly expressed in cells exposed to either DEX or MIX alone but not to the same extent as that when the two inducers are used together (compare lane 7 with lanes 3 and 5). PPARgamma 2, however, appears to be more highly expressed in cells exposed to DEX compared with those exposed to MIX (compare lane 5 with lane 3). Activation of PPARgamma 1 in these differentiation-compromised cells (cells deprived of DEX or MIX) by exposure to troglitazone resulted in an extensive induction of C/EBPalpha and aP2 (lanes 4 and 6) and a corresponding conversion of the preadipocytes into morphologically distinct adipocytes (data not shown).


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Fig. 1.   Effect of different effectors on PPARgamma and C/EBPalpha expression. 3T3-L1 cells transfected with vector DNA alone were induced to differentiate in media containing insulin and FBS in the presence or absence of dexamethasone (D), isobutylmethylxanthine (M), or 10 µM troglitazone (Trog). Four days later, whole cell extracts were prepared, and 100 µg of each sample was subjected to Western blot analysis for PPARgamma , C/EBPalpha , and aP2 expression. I, 1.67 µM insulin; D, 1 µM dexamethasone; M, 0.5 mM isobutylmethylxanthine; CRM, cross-reacting material.

Earlier studies by us and others (10, 21, 29) have shown that one of the mechanisms by which DEX and MIX induce adipogenesis is to enhance expression of C/EBPbeta and C/EBPdelta during the initial few hours of adipogenesis in 3T3-L1 cells, which in turn activate expression of PPARgamma and C/EBPalpha . Fig. 1 suggests that these effectors may in fact be regulating two separate pathways in which DEX alone can induce PPARgamma 1 expression, but MIX is required along with DEX for C/EBPalpha expression. Consequently, since MIX has been shown to regulate C/EBPbeta (20), we questioned whether the induction of C/EBPalpha by troglitazone in cells deprived of MIX was due to a corresponding troglitazone-associated induction of C/EBPbeta . In the experiment presented in Fig. 2, 3T3-L1 cells were exposed to insulin and DEX in the presence or absence of MIX or troglitazone, and total protein extracts harvested at the indicated times were subjected to Western blot analysis. At 4 h post-induction, C/EBPbeta is expressed in cells treated with the complete set of inducers (lanes 3 and 4), whereas very little C/EBPbeta is produced in the absence of MIX (Fig. 2, lanes 1 and 2). Addition of troglitazone has no significant effect on C/EBPbeta expression in the presence or absence of MIX (compare lane 1 with lane 2, and lane 3 with lane 4). This figure also shows that expression of PPARgamma 1 occurs much earlier (at 24 h) in cells exposed to mixture lacking MIX than cells cultured in the complete mixture (Fig. 1, compare lanes 5 and 7). By 72 h after treatment, PPARgamma 1 is abundantly expressed in both populations of cells (minus or plus MIX); however, PPARgamma 2 and C/EBPalpha are expressed to any significant extent only in cells exposed to DEX and MIX (compare lanes 9 and 11). The presence of troglitazone resulted in an extensive induction of C/EBPalpha and PPARgamma 2 in MIX-deprived cells but also enhanced expression of C/EBPalpha in cells exposed to MIX.


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Fig. 2.   Time course of protein expression during adipogenesis with and without MIX and troglitazone. At the indicated times following induction of differentiation of L1-vector cells in the presence or absence of isobutylmethylxanthine (ID versus DIM) and troglitazone (Trog), total protein extracts were collected and analyzed by Western blot analysis for expression of PPARgamma , C/EBPalpha , and C/EBPbeta .

The induction of C/EBPalpha by troglitazone suggests that PPARgamma may be capable of directly transactivating the C/EBPalpha gene. To test this idea, we ectopically expressed PPARgamma 2 in C3H10T1/2 mesenchymal stem cells to create a cell line (10T-Pgamma ) whose differentiation into adipocytes was dependent on an exogenous PPARgamma ligand. The Northern blot in Fig. 3 shows that exposure of these cells to 10 µM troglitazone results in the induction of C/EBPalpha mRNA expression that also occurs in the absence of ongoing protein synthesis (+ cycloheximide). These data are consistent with the notion that PPARgamma is interacting directly with the C/EBPalpha gene to enhance C/EBPalpha mRNA production.


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Fig. 3.   PPARgamma induces C/EBPalpha mRNA expression in the absence of ongoing protein synthesis. 10T1/2 cells ectopically expressing PPARgamma 2 were exposed to DMEM containing 1 µM DEX, 0.5 mM MIX, 1.67 µM insulin, and 10% FBS for 48 h. Cells were maintained in 10% FBS for an additional 24 h and then treated with or without cycloheximide (CHX, 5 µg/ml), in the presence or absence of 5 µM troglitazone (Trog). Total RNA was harvested at the indicated times post-treatment and analyzed by Northern blot for C/EBPalpha and PPARgamma mRNA expression.

To determine if the selective effect of omitting MIX on C/EBPalpha compared with PPARgamma 1 expression was primarily due to its role in regulating C/EBPbeta , we generated a 3T3-L1 cell line ectopically expressing a dominant negative isoform of C/EBPbeta (designated L1-LIP cells). Fig. 4A shows abundant expression of LIP (3rd lane) in proliferating 3T3-L1 preadipocytes compared with undetectable levels of this polypeptide in the vector cells (1st lane). Also shown is the ectopic expression of the LAP (34 kDa) isoform of C/EBPbeta in a corresponding L1-LAP cell line (2nd lane). As observed previously by McKnight and co-workers (10), ectopic expression of LIP completely blocks the ability of DEX, MIX, insulin, and FBS to induce the differentiation of 3T3-L1 preadipocytes to differentiate into fat cells. The data shown in Fig. 4B confirm this result, but they also show that troglitazone can reverse this inhibitory action of LIP as judged by the accumulation of Oil Red O-positive lipid droplets following exposure of the L1-LIP cells to troglitazone.


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Fig. 4.   Ectopic expression of a dominant negative isoform of C/EBPbeta (LIP) inhibits adipogenesis in 3T3-L1 preadipocytes. A, total proteins harvested from proliferating preadipocytes, which ectopically express either vector (V), LAP, or LIP were subjected to Western blot analysis using an anti-C/EBPbeta antibody. CRM, cross-reacting material. B, confluent L1-vector and L1-LIP cells were induced to differentiate in the presence or absence of troglitazone (Trog). At day 7, cells were fixed and stained for neutral lipids with Oil Red O.

To gain insight into the effect of LIP and troglitazone on the differentiation of preadipocytes, L1-LIP cells were induced to differentiate as in Fig. 1, where MIX and/or DEX were omitted from the culture medium. Extracts of total protein were then harvested at day 4 and subjected to Western blot analysis. Fig. 5 shows that expression of LIP greatly attenuates adipogenesis under all hormonal conditions as indicated by a lack of C/EBPalpha and aP2 expression (lanes 1, 3, 5, and 7). DEX is capable of enhancing PPARgamma 1 expression above the low basal levels produced in the presence of insulin and FBS, but it has no positive effect on PPARgamma 2 expression (compare lane 1 and 3). Addition of troglitazone along with DEX induces adipogenesis as indicated by the expression of C/EBPalpha , PPARgamma 2, and aP2. It is worth noting that DEX has a similar effect on gene expression in the presence or absence of LIP (compare Fig. 5, lanes 3 and 4 with Fig. 1, lanes 3 and 4). In contrast, exposure of LIP cells to MIX and insulin only slightly enhances PPARgamma 1 with no PPARgamma 2 expression, and when troglitazone is added under these conditions it does not induce adipogenesis (i.e. minimal aP2 and C/EBPalpha expression). This pattern of gene expression differs significantly from that observed in the absence of LIP. Specifically, MIX induces both PPARgamma 1 and -2 in the L1-vector cells, and consequently, exposure of these cells to troglitazone promotes adipogenesis (compare Fig. 1, lanes 5 and 6, with Fig. 4, lanes 5 and 6). Taken together, these data are consistent with a model in which DEX is capable of priming the preadipocytes to be responsive to troglitazone even in the absence of C/EBPbeta ; this likely involves induction of PPARgamma 1 expression. MIX, however, is only capable of a similar priming process if C/EBPbeta is actively expressed in the absence of LIP. These data also strongly suggest that inhibiting C/EBPbeta activity blocks production of an endogenous activator of PPARgamma , which renders the 3T3-L1 preadipocytes dependent on an exogenous PPARgamma ligand for their differentiation into adipocytes.


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Fig. 5.   Troglitazone induces adipogenic gene expression in L1-LIP cells exposed to dexamethasone. 3T3-L1 preadipocytes expressing LIP were induced to differentiate by exposure to the indicated combinations of the adipogenic inducers, DEX (D), MIX (M), and insulin (I) in the presence or absence of 10 µM troglitazone (Trog). At 4 days post-induction, whole cell proteins were extracted and analyzed by Western blot for expression of PPARgamma , C/EBPalpha , and aP2.

To gain more insight into the ligand dependence of these LIP-expressing preadipocytes, we analyzed the temporal pattern of gene expression following exposure to troglitazone as well as determining the optimum dose of troglitazone required to induce adipogenesis. In the experiment shown in Fig. 6, confluent L1-LIP cells were exposed to DEX, MIX, and insulin in the presence or absence of troglitazone, and total cellular proteins were subjected to Western blot analysis. The combination of DEX, MIX, and insulin is capable of initiating the early phase of adipogenesis in these LIP-expressing cells as indicated by induction of C/EBPbeta as well as PPARgamma 1 (compare lane 2 and 4 with lane 1). Exposure of these cells to troglitazone appears to have no significant effect on this pattern of gene expression during the first 2 days. After this time, however, troglitazone is essential for the induction of C/EBPalpha and aP2 expression. Taken together, the studies shown above demonstrate that culturing LIP cells in troglitazone for 6 days, along with an initial priming with DEX, MIX, and insulin, results in their conversion into adipocytes based on accumulation of lipid droplets in >95% of the cells (Fig. 4B) and the abundant expression of PPARgamma 2, C/EBPalpha , and aP2 (Fig. 6). To establish the troglitazone dose dependence of LIP cells, both L1-LIP and L1-V cells were exposed to differentiation medium containing DEX, MIX, insulin, and increasing concentrations of troglitazone. Total protein samples were harvested 6 days later and subjected to Western blot analysis of the indicated proteins. Fig. 7 demonstrates that expression of both C/EBPalpha and PPARgamma 2 increased substantially with increasing doses of troglitazone. Expression of aP2 also seemed proportionate to troglitazone concentration, correlative to the number of cells accumulating lipid droplets (data not shown). LIP expression was unaffected by the PPARgamma ligand.


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Fig. 6.   Time course of adipogenic gene expression following exposure of L1-LIP cells to troglitazone. L1-LIP cells were induced to differentiate with DEX, MIX, insulin, and FBS in the presence or absence of 10 µM troglitazone (Trog). Total protein extracts were harvested on the indicated days after induction. Western blot analysis was performed as described using antibodies for the indicated proteins.


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Fig. 7.   Troglitazone dose-dependent rescue of adipogenic gene expression in L1-LIP cells. L1-LIP cells and L1-V cells (L1) were induced to differentiate as in Fig. 6 in the presence of varying concentrations of troglitazone (Trog). Six days after induction, whole cell proteins were harvested and subjected to Western blot analysis for expression of the indicated proteins. L1-V controls (0 and 10 µM troglitazone) are included.

The LIP polypeptide retains the C-terminal basic leucine zipper region of the full-length C/EBPbeta protein, and therefore, it can dimerize with other C/EBPbeta isoforms and bind to DNA. It was important, therefore, to determine what effect LIP and/or troglitazone may have on the DNA binding activity of the different C/EBPs during adipogenesis. Consequently, L1-V and L1-LIP cells were treated to differentiate in the presence or absence of troglitazone, and nuclear proteins were harvested at day 1 and day 6. The electrophoretic mobility shift assay presented in Fig. 8 shows binding of nuclear protein complexes to an oligonucleotide corresponding to the C/EBP regulatory element within the promoter of the C/EBPalpha gene (31). The profile and intensity of binding observed at day 1 is very similar in the LIP and vector cells, with one exception. There is a faster migrating species in the LIP cells that likely corresponds to LIP-LIP homodimers (Fig. 8A). Troglitazone has no significant effect on the overall binding activity in either cell line. Fig. 8A also shows that the complexes present in L1-V samples at day 6 migrate with a slightly larger mass than the day 1 complexes, which is probably due to the presence of C/EBPalpha . This same shift in migration is observed in the LIP samples following exposure to troglitazone for 6 days. To examine the composition of these DNA-protein complexes, a series of supershift assays were performed using antibodies corresponding to C/EBPalpha , C/EBPbeta , and C/EBPdelta . Fig. 8B, lane 4, demonstrates that a proportion of the complexes expressed at day 6 in L1-V cells consist of C/EBPalpha homodimers. As expected, there is a significant increase in C/EBPalpha binding activity in LIP cells following exposure to troglitazone for 6 days (Fig. 8B, compare lane 12 with lane 8). In fact, the C/EBPalpha binding activity is slightly higher in the LIP cells plus troglitazone compared with that expressed in the vector cells. Furthermore, ectopic expression of LIP has not affected the ability of C/EBPalpha to bind to the C/EBP regulatory element. This figure also shows the existence of LIP-LIP homodimers binding to the C/EBP oligonucleotide since the faster migrating complexes present in the LIP cells can be supershifted selectively with an anti-C/EBPbeta antibody (lanes 6 and 10). C/EBPdelta is minimally expressed at 6 days under all conditions since there is no detectable supershift with an anti-C/EBPdelta antibody.


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Fig. 8.   Changes in C/EBP DNA binding activity during adipogenesis in L1-V and L1-LIP cells. L1-V and L1-LIP cells were treated to differentiate in the presence or absence of 10 µM troglitazone (Trog). A, at 1 and 6 days after induction (d1 and d6, respectively), nuclear proteins were harvested and subjected to electrophoretic mobility shift assay, as described under "Experimental Procedures." B, supershift analysis of C/EBP DNA binding activity in L1-V and L1-LIP cells induced in the presence or absence of troglitazone. Day 6 nuclear protein samples from L1-V and L1-LIP cells induced to differentiate in the standard inducers, with or without troglitazone, were analyzed by supershift analysis using antibodies to C/EBPbeta (Cbeta ), C/EBPdelta (Cdelta ), C/EBPalpha (Calpha ), or IgG (-).

Our observation that LIP cells require an exogenous PPARgamma ligand such as troglitazone for their complete conversion into adipocytes suggests that they are not capable of producing the endogenous ligand(s) or activators. Alternatively, it is possible that they express PPARgamma 1 at levels below a threshold required for activation by the endogenous activator(s). To determine the reason for the ligand dependence of the LIP cells, we induced PPARgamma and C/EBPalpha to fully differentiated levels by exposing LIP cells to DEX, MIX, insulin, and 10 µM troglitazone for 6 days. We then questioned whether these LIP adipocytes still require the exogenous PPARgamma ligand to maintain normal adipocyte gene expression. This was achieved by withdrawing troglitazone from half the cultures at day 6 and measuring expression of PPARgamma , C/EBPalpha , and aP2 in these and a control set of cultures that were maintained in troglitazone for the entire experiment. The Western blot in Fig. 9 shows abundant levels of PPARgamma , C/EBPalpha , and aP2 following 7 days of exposure of LIP cells to troglitazone. Withdrawal of the exogenous ligand at day 6, however, results in an extensive dedifferentiation as indicated by a drop in expression of C/EBPalpha and aP2 to virtually undetectable levels by day 10 (4 days of withdrawal). Interestingly, the abundance of both PPARgamma 1 and -gamma 2 remains constant throughout this period even in the absence of troglitazone. Notably, expression of LIP does not increase when troglitazone is withdrawn; on the contrary, it appears to decrease (Fig. 9).


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Fig. 9.   L1-LIP cells require exposure to troglitazone throughout the differentiation process in order to maintain adipogenic gene expression. L1-LIP cells were induced to differentiate and maintained in the presence of 10 µM troglitazone (Trog). Six days after induction, troglitazone was either withdrawn (WD) from the media or maintained at a concentration of 10 µM (+). Total protein extracts were harvested at 1 (day 7, d7), 2 (day 8, d8), 3 (day 9, d9), and 4 (day 10, d10) days later. Samples were subjected to Western blot analysis for PPARgamma , C/EBPalpha , aP2, and LIP expression.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The differentiation of 3T3-L1 cells into mature adipocytes requires their exposure to a mixture of hormonal inducers including DEX, MIX, insulin, and FBS. These effectors have been shown to activate a cascade of transcriptional events that culminate in expression of the mature adipocytic phenotype. Most notably, they facilitate the induction of C/EBPbeta and C/EBPdelta , which together activate expression of PPARgamma and C/EBPalpha (10, 20, 21, 29). The data presented in this study suggest an additional role for C/EBPbeta , and the effectors that control its expression, in regulating the production of PPARgamma ligands. These studies further show that adipogenesis can be induced in 3T3-L1 preadipocytes in the absence of C/EBPbeta by exposing the cells to an exogenous PPARgamma ligand. This alternative mechanism appears to depend on the ability of insulin and DEX to induce PPARgamma 1 expression, which is then capable of inducing C/EBPalpha and PPARgamma 2 expression following exposure to troglitazone. Induction of C/EBPalpha gene expression in the absence of an exogenous PPARgamma ligand depends on MIX and/or C/EBPbeta expression. It appears, therefore, that expression of C/EBPalpha during adipogenesis can be regulated by at least two independent pathways. One pathway involves a MIX-associated induction of C/EBPbeta , which transactivates a C/EBP regulatory element within the promoter of the C/EBPalpha gene (32). The other mechanism can occur in the absence of C/EBPbeta due to a DEX-associated induction of PPARgamma 1, which is also capable of transactivating the C/EBPalpha gene in the presence of troglitazone.

Previous studies have shown that an important role for MIX and DEX is to induce expression of C/EBPbeta and C/EBPdelta , respectively, which in turn activate C/EBPalpha and PPARgamma 2 expression through C/EBP regulatory elements in the promoters of the corresponding genes (10, 31-34). Our data are consistent with these observations since inhibition of C/EBPbeta activity by either omitting MIX or expressing LIP blocks both C/EBPalpha and PPARgamma 2 expression. Of interest is the observation that DEX can induce PPARgamma 1 expression in the absence of C/EBPbeta activity, which may be due to a DEX-associated induction of C/EBPdelta . Other studies, however, have shown that ectopic expression of C/EBPdelta alone in 3T3 fibroblasts does not induce C/EBPalpha , PPARgamma 1, or -gamma 2 expression (10, 29). Despite the abundant expression of PPARgamma 1 in the C/EBPbeta -deficient preadipocytes, they are incapable of expressing the adipogenic program unless exposed to an exogenous PPARgamma ligand (i.e. troglitazone) along with the normal mixture of hormonal inducers. This observation suggests that C/EBPbeta may play a role in regulating processes that lead to production of PPARgamma ligands/activators. Further support for this notion are the data in Fig. 9 showing a continuing requirement of the LIP cells for troglitazone to maintain adipogenic gene expression even after they have completely converted into adipocytes by a 6 -day exposure to the PPARgamma ligand.

Mechanisms by Which C/EBPbeta May Regulate PPARgamma Activity-- The most likely determinant of PPARgamma activity is the availability of ligands within the preadipocyte. Even though the natural cellular ligand for PPARgamma has not been identified, evidence suggests that derivatives of polyunsaturated fatty acids are potent activators of PPARgamma both in in vitro assays as well as in vivo (13, 16) Mechanisms that control the cellular production of polyunsaturated fatty acids or their derivatives may play an important role in regulating adipogenesis. In this regard, studies have shown that ADD1/SREBP-1, a transcription factor that is linked to processes controlling fatty acid production, appears to be involved in the production of endogenous PPARgamma ligands (26). In fact, ADD1/SREBP-1 is induced early during adipogenesis, and its ectopic expression in non-adipogenic cells can enhance fat cell formation by directly activating the PPARgamma 2 gene as well as stimulating production of PPARgamma ligands (26, 35, 36). It is conceivable, therefore, that a role for C/EBPbeta in regulating PPARgamma activity may involve induction and/or activation of ADD1/SREBP-1.

PPARgamma Ligand-dependent Induction of C/EBPalpha Expression-- Several investigations have demonstrated that activation of PPARgamma in a variety of different fibroblast lines results in expression of many adipogenic genes including C/EBPalpha (19, 22-25, 37). Similarly, ectopic expression of C/EBPalpha in non-adipogenic cells can induce PPARgamma expression (22, 23). In fact, Spiegelman and co-workers (22) have suggested that cross-regulation between C/EBPalpha and PPARgamma is important in maintaining the differentiated state. The molecular mechanisms involved in such a cross-regulatory process are not known. It is very likely that C/EBPalpha directly transactivates PPARgamma 2 gene expression through the C/EBP regulatory elements within the PPARgamma 2 promoter (33, 34). The data presented in Fig. 3 suggest that PPARgamma may also be capable of a similar direct transactivation of the C/EBPalpha gene based on the observation that activation of PPARgamma by troglitazone in 10T1/2 cells induces C/EBPalpha mRNA expression in the absence of ongoing protein synthesis. For most PPARgamma target genes, PPARgamma initiates transcription by binding to cognate PPAR regulatory elements at DR-1 sites within the promoter/enhancer regions of the genes (18, 38). It seems likely that similar DR-1 sites exist within the C/EBPalpha gene. Analysis of sequences in the 5'-flanking region of the C/EBPalpha gene have identified DR-1 elements that bind strongly to particular COUP-TF proteins, but very weakly to PPARgamma .2 We are presently determining whether this or any other elements facilitate the PPARgamma -dependent induction of C/EBPalpha gene expression.

What Are the Roles of C/EBPbeta and/or C/EBPdelta in Regulating Adipogenesis?-- Several studies performed in a variety of cultured cell systems have led to a model for the transcriptional control of adipogenesis, which involves the sequential activation of C/EBPs and PPARgamma . The function of C/EBPbeta and C/EBPdelta in this process is to induce expression of both PPARgamma 2 and C/EBPalpha . Investigations using mice lacking C/EBPbeta and/or C/EBPdelta suggest an alternative role for these C/EBPs in regulating adipose tissue formation and function in the animal (39). Specifically, C/EBPbeta (-/-)·C/EBPdelta (-/-) mice express defects in lipid accumulation despite normal expression of C/EBPalpha and PPARgamma . However, primary embryonic fibroblasts derived from these knock-out animals have lost the potential to undergo adipogenesis and, in so doing, do not express PPARgamma or C/EBPalpha in response to DEX, MIX, insulin, and FBS. Taken together, these observations suggest a role for C/EBPbeta and/or C/EBPdelta in regulating PPARgamma and C/EBPalpha gene expression in cultured cells (cell lines or mouse embryonic fibroblasts) exposed to a restricted set of inducers. In preadipocytes in adipose tissue, however, PPARgamma and C/EBPalpha may be activated by an alternative mechanism due to the presence of effectors not present in the culture system. As mentioned above, there appears to be a role for C/EBPbeta and C/EBPdelta in facilitating the formation and function of adipose tissue in vivo that is independent of C/EBPalpha and PPARgamma expression, which may involve production of PPARgamma ligands.

In summary, we propose an alternative model for the transcriptional control of adipogenesis (Fig. 10) that incorporates the conclusions drawn from these studies with those already presented by others (10, 21, 22). In this model, C/EBPbeta and C/EBPdelta regulate production of PPARgamma ligands as well as PPARgamma 2 and C/EBPalpha expression. Additionally, we suggest that physiological effectors can induce expression of PPARgamma 1 in the absence of C/EBPbeta and C/EBPdelta as part of a default pathway. This event can then initiate a cascade of transcription factor expression, commencing with C/EBPalpha , which in turn induces expression of the entire adipogenic program providing the preadipocyte is exposed to PPARgamma ligands. Further dissection of the transcriptional events that regulate production of PPARgamma ligands should provide a greater understanding of the processes controlling adipogenesis.


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Fig. 10.   Transcriptional control of PPARgamma ligand production is a central component of the signaling cascade that regulates adipogenesis. Initiation of adipogenesis involves induction of C/EBPbeta , C/EBPdelta , and PPARgamma 1 in response to exposure of preadipocytes to a variety of physiological effectors including insulin, glucocorticoids, and agonists that elevate cAMP. C/EBPbeta and C/EBPdelta activate expression of C/EBPalpha and PPARgamma 2 as well as stimulate a pathway that leads to production of PPARgamma ligands. Ligand-activated forms of PPARgamma 1 and PPARgamma 2 can directly induce expression of C/EBPalpha to establish a positive feedback loop in which C/EBPalpha maintains expression of the PPARs. The synergistic activity of C/EBPalpha and PPARgamma ensures expression of the entire adipogenic gene program.


    ACKNOWLEDGEMENTS

We thank Drs. Bruce Spiegelman, David Bernlohr, and Steve McKnight for their generosity in providing reagents. We also thank Dr. Marthe Moldes for critical reading of the manuscript and valuable suggestions.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant DK51586.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 To whom correspondence should be addressed: Dept. of Biochemistry, Boston University School of Medicine, 715 Albany St., Boston, MA 02118. Tel.: 617-638-4186; Fax: 617-638-5339; E-mail: farmer@biochem.bumc.bu.edu.

Published, JBC Papers in Press, February 27, 2001, DOI 10.1074/jbc.M100797200

2 Y. Xie, J. Hamm, and S. Farmer, unpublished data.

    ABBREVIATIONS

The abbreviations used are: C/EBP, CCAAT/enhancer-binding protein; DMEM, Dulbecco's modified Eagle's medium; DEX, dexamethasone; MIX, 3-isobutyl-1-methylxanthine; FBS, fetal bovine serum; PPAR, peroxisome proliferator-activated receptor; aP2, adipose protein 2/fatty acid-binding protein; LIP, liver-enriched transcriptional inhibitory protein; LAP, liver-enriched transcriptional activator protein; PCR, polymerase chain reaction; SREBP, sterol regulatory element-binding protein.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Morrison, R. F., and Farmer, S. R. (1999) J. Cell. Biochem. 75 Suppl. 32, 59-67[CrossRef]
2. Rosen, E. D., Walkey, C. J., Puigserver, P., and Spiegelman, B. M. (2000) Genes Dev. 14, 1293-1307[Free Full Text]
3. Darlington, G. J., Ross, S. E., and MacDougald, O. A. (1998) J. Biol. Chem. 273, 30057-30060[Free Full Text]
4. Wu, Z., Puigserver, P., and Spiegelman, B. M. (1999) Curr. Opin. Cell Biol. 11, 689-694[CrossRef][Medline] [Order article via Infotrieve]
5. Gregoire, F. M., Smas, C. M., and Sul, H. S. (1998) Physiol. Rev. 78, 783-809[Abstract/Free Full Text]
6. Rosen, E. D., and Spiegelman, B. M. (2000) Annu. Rev. Cell Dev. Biol. 16, 145-171[CrossRef][Medline] [Order article via Infotrieve]
7. Morrison, R. F., and Farmer, S. R. (2000) J. Nutr. 130, 3116-3121
8. Welm, A. L., Timchenko, N. A., and Darlington, G. J. (1999) Mol. Cell. Biol. 19, 1695-1704[Abstract/Free Full Text]
9. Descombes, P., and Schibler, U. (1991) Cell 67, 569-579[Medline] [Order article via Infotrieve]
10. Yeh, W. C., Cao, Z., Classon, M., and McKnight, S. L. (1995) Genes Dev. 9, 168-181[Abstract]
11. Zhu, Y., Qi, C., Korenberg, J. R., Chen, X.-N., Noya, D., Rao, M. S., and Reddy, J. K. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 7921-7925[Abstract]
12. Fajas, L., Fruchart, J. C., and Auwerx, J. (1998) FEBS Lett. 438, 55-60[CrossRef][Medline] [Order article via Infotrieve]
13. Kliewer, S., Sundseth, S., Jones, S., Brown, P., Wisely, G., Koble, C., Devchand, P., Wahli, W., Willson, T., Lenhard, J., and Lehmann, J. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 4318-4323[Abstract/Free Full Text]
14. Forman, B. M., Tontonoz, P., Chen, J., Brun, R. P., Spiegelman, B. M., and Evans, R. M. (1995) Cell 83, 803-812[Medline] [Order article via Infotrieve]
15. Kliewer, S. A., Lenhard, J. M., Wilson, T. M., Patel, I., Morris, D. C., and Lehmann, J. M. (1995) Cell 83, 813-819[Medline] [Order article via Infotrieve]
16. Forman, B. M., Chen, J., and Evans, R. M. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 4312-4317[Abstract/Free Full Text]
17. Braissant, O., Foufelle, F., Scotto, C., Dauca, M., and Wahli, W. (1996) Endocrinology 137, 354-366[Abstract]
18. Tontonoz, P., Hu, E., Graves, R. A., Budavari, A. I., and Spiegelman, B. M. (1994) Genes Dev. 8, 1224-1234[Abstract]
19. Tontonoz, P., Hu, E., and Spiegelman, B. M. (1994) Cell 79, 1147-1156[Medline] [Order article via Infotrieve]
20. Cao, Z., Umek, R. M., and McKnight, S. L. (1991) Genes Dev. 5, 1538-1552[Abstract]
21. Wu, Z., Xie, Y., Bucher, N. L. R., and Farmer, S. R. (1995) Genes Dev. 9, 2350-2363[Abstract]
22. Wu, Z., Rosen, E. D., Brun, R., Hauser, S., Adelmont, G., Troy, A. E., McKeon, C., Darlington, G. J., and Spiegelman, B. M. (1999) Mol. Cell 3, 151-158[Medline] [Order article via Infotrieve]
23. El-Jack, A. K., Hamm, J. K., Pilch, P. F., and Farmer, S. R. (1999) J. Biol. Chem. 274, 7946-7951[Abstract/Free Full Text]
24. Brun, R. P., Tontonoz, P., Forman, B. M., Ellis, R., Chen, J., Evans, R. M., and Spiegelman, B. M. (1996) Genes Dev. 10, 974-984[Abstract]
25. Wu, Z., Xie, Y., Morrison, R. F., Bucher, N. L. R., and Farmer, S. R. (1998) J. Clin. Invest. 101, 22-32[Abstract/Free Full Text]
26. Kim, J. B., Wright, H. M., Wright, M., and Spiegelman, B. M. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 4333-4337[Abstract/Free Full Text]
27. Pear, W. S., Nolan, G. P., Scott, M. L., and Baltimore, D. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 8392-8396[Abstract/Free Full Text]
28. Student, A. K., Hsu, R. Y., and Lane, M. D. (1980) J. Biol. Chem. 255, 4745-4750[Abstract/Free Full Text]
29. Wu, Z., Bucher, N. L. R., and Farmer, S. R. (1996) Mol. Cell. Biol. 16, 4128-4136[Abstract]
30. Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem. 162, 156-159[CrossRef][Medline] [Order article via Infotrieve]
31. Rana, B., Xie, Y., Mischoulon, D., Bucher, N. L. R., and Farmer, S. R. (1995) J. Biol. Chem. 270, 18123-18132[Abstract/Free Full Text]
32. Tang, Q. Q., Jiang, M. S., and Lane, M. D. (1999) Mol. Cell. Biol. 19, 4855-4865[Abstract/Free Full Text]
33. Clarke, S. L., Robinson, C. E., and Gimble, J. M. (1997) Biochem. Biophys. Res. Commun. 240, 99-103[CrossRef][Medline] [Order article via Infotrieve]
34. Elberg, G., Gimble, J., M., and Tsai, S. Y. (2000) J. Biol. Chem. 275, 27815-27822[Abstract/Free Full Text]
35. Kim, B. J., and Spiegelman, B. M. (1996) Genes Dev. 10, 1096-1107[Abstract]
36. Fajas, L., Schoonjans, K., Gelman, L., Kim, J. B., Najib, J., Martin, G., Fruchart, J. C., Briggs, M., Spiegelman, B. M., and Auwerx, J. (1999) Mol. Cell. Biol. 19, 5495-5503[Abstract/Free Full Text]
37. Hu, E., Tontonoz, P., and Spiegelman, B. M. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 9856-9860[Abstract]
38. Juge-Aubry, C., Pernin, A., Favez, T., Burger, A. G., Wahli, W., Meier, C. A., and Desvergne, B. (1997) J. Biol. Chem. 272, 25252-25259[Abstract/Free Full Text]
39. Tanaka, T., Yoshida, N., Kishimoto, T., and Akira, S. (1997) EMBO J. 16, 7432-7443[Abstract/Free Full Text]


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