Department of Nutrition, University of North Carolina School of Public Health, Chapel Hill, North Carolina 27599
Submitted 18 June 2003 ; accepted in final form 9 October 2003
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ABSTRACT |
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cell cycle regulation; cellular proliferation; differentiation
Progression of the cell cycle from G1 to S phase is regulated by the phosphorylation status of the retinoblastoma protein Rb. In G1, hypophosphorylated Rb binds and sequesters transcription factors that are required for the expression of genes necessary for DNA synthesis (16, 17). Rb phosphorylation at the G1/S boundary results in its dissociation from these transcription factors and triggers cell cycle progression. Rb not only binds transcription factors involved in cell cycle progression, it also interacts with transcription factors involved in differentiation (7, 14, 15). Rb has been shown to bind to members of the CCAAT/enhancer binding protein (C/EBP) family of transcription factors (24).
Transcription factors in the C/EBP family function in a cascadelike manner to activate adipogenesis (1, 29). The MDI protocol induces the synthesis of C/EBP and C/EBP
, which prime the differentiation pathway (29). C/EBP
and C/EBP
activate the synthesis of two critical adipogenic transcription factors, peroxisome proliferator-activated receptor-
and C/EBP
, which are required for the expression of adipocyte genes. Whereas C/EBP
and C/EBP
are synthesized and translocated to the nucleus within 4 h after the induction of differentiation, they do not bind to DNA for another 8 h (24). This delay in DNA-binding activity is not well characterized.
Previous studies (19) have suggested that C/EBP-DNA-binding activity may be linked to cell cycle progression during differentiation. Preadipocytes induced to differentiate in the presence of a G1 cell cycle inhibitor, expressed C/EBP
but had decreased C/EBP
-DNA-binding activity and decreased C/EBP
-mediated transactivation of a reporter gene (19). In this report, we show that C/EBP
acquires the ability to bind to DNA in a cell cycle-dependent manner (after the G1/S transition). Importantly, we demonstrate that although hypophosphorylated Rb associates with C/EBP
, phosphorylated Rb does not. We also show that Rb inhibits C/EBP
-DNA-binding activity in vitro and overexpression of Rb prevents C/EBP
-mediated transactivation of a C/EBP
-promoter-reporter gene. These studies suggest that Rb is a critical regulator of C/EBP
-DNA binding activity during the early stages of 3T3-L1 adipogenesis.
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EXPERIMENTAL PROCEDURES |
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L-Mimosine, aphidicolin, and nocodazole were obtained from Cal-Biochem (San Diego, CA). Aprotinin, leupeptin, antipain, benzamidine, chymostatin, and pepstatin-A were purchased from Sigma-Aldrich (St. Louis, MO). C/EBP mouse monoclonal antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The Rb monoclonal antibody was purchased from PharMingen (San Diego, CA). The C/EBP
rabbit polyclonal antibody was generously provided by Dr. M. Daniel Lane (Johns Hopkins University). Recombinant full-length Rb and a truncated Rb protein, p56Rb (amino acids 379928), were purchased from QED Bioscience (San Diego, CA). The recombinant truncated Rb p25Rb (amino acids 773928) was purchased from Upstate (Charlottesville, VA).
Methods
Cell culture. 3T3-L1 preadipocytes were cultured in Dulbecco's modified Eagle medium (DMEM) containing 10% calf serum (CS) until confluence and then maintained for 48 h (day 0). Preadipocytes were then maintained in CS (time 0) or were induced to differentiate with 0.5 mM 3-isobutyl-1-methylxanthine, 1 µM dexamethasone, and 1 µg/ml insulin in DMEM containing 10% fetal bovine serum for 48 h (MDI protocol). Where indicated, cell cycle inhibitors (5 µM aphidicolin, 500 µM mimosine, or 10 µM nocodazole) were added at the time of induction.
Immunoblot analysis. 3T3-L1 preadipocyte whole cell lysates were prepared as previously described (13). Protein concentrations were determined and protein (50 µg) was subjected to SDS-PAGE and transferred to Immobilon-P membranes (Millipore). The membranes were incubated with antisera as indicated, followed by incubation with a peroxidase-conjugated secondary antibody. Proteins were visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech).
Nuclear extract preparation. Nuclear extracts were prepared from 3T3-L1 preadipocytes as described previously (6, 23). Cells were washed with PBS and scraped in a hypotonic lysis buffer containing 20 mM Tris, pH 7.5, 10 mM NaCl, 3 mM MgCl2, 1 mM dithiothreitol (DTT), and 2 µl/ml of protease inhibitor cocktails PIC I and PIC II. PIC I contains (in mg/ml) 1 leupeptin, 1 antipain, and 10 benzamidine in 1 aprotinin in distilled water. PIC II contains 1 mg/ml chymostatin and 1 mg/ml pepstatin-A in dimethyl sulfoxide. Nonidet P-40 (NP-40) was added to a final concentration of 1%. The samples were Dounce homogenized and centrifuged at 2,200 g for 5 min at 4°C. The crude nuclear pellet was resuspended in hypotonic lysis buffer and centrifuged at 2,200 g for 5 min at 4°C. The pellet was resuspended in a nuclei storage buffer containing 40% glycerol, 1 M Tris pH 8.0, 3 mM MgCl2, 1 mM DTT, and 2 µl/ml PIC I and PIC II and was centrifuged at 6,500 g for 10 s at 4°C. The pellet was resuspended in NUN buffer (0.3 M NaCl, 1 M urea, 1% NP-40, 25 mM HEPES, pH 7.9, 1 mM DTT, and 2 µl/ml PIC I and PIC II), incubated on ice for 30 min, and centrifuged at 14,000 g for 10 min at 4°C. Supernatants were collected and glycerol was added to a final concentration of 10%.
EMSA. A [32P]-labeled double-stranded oligonucleotide (20 bp), corresponding to the C/EBP site in the C/EBP promoter was used for EMSA. The labeled probe and 10 µg of nuclear protein were incubated on ice for 15 min and then subjected to nondenaturing PAGE. Protein-DNA complexes were visualized by autoradiography. For gel supershift experiments, 2.5 µg of the indicated antibody were added to the nuclear protein 15 min before the addition of the labeled probe. All recombinant Rb proteins were resuspended in a solution containing 20 mM sodium phosphate, 200 mM NaCl, 1 mM EDTA, and 10% glycerol, pH 7.5. In competition experiments, 1 µg of either p56Rb or p25Rb was added to 12 µg of nuclear protein 15 min before the addition of labeled probe. In dose-dependent competition experiments, 0.050.5 µg of purified, full-length recombinant Rb was added to the nuclear protein. Protein concentration was equalized with BSA, and the samples contained equal amounts of Rb diluent.
Immunoprecipitation. 3T3-L1 preadipocytes were washed twice in ice-cold PBS containing 1 mM orthovanadate, scraped in ice-cold gentle lysis buffer (25 mM Tris·HCl, pH 7.5, 1% NP-40, 10% glycerol, 50 mM NaF, 10 mM NaH2PO4, 137 mM NaCl, 2 mM Na3VO4, 1 mM PMSF, and 10 µg/ml aprotinin), sonicated, and then centrifuged at 6,000 g at 4°C for 20 min. Supernatants were removed, and protein concentrations were determined. Antibodies (10 µg/ml) to either C/EBP or Rb were added to protein lysates (800 µg) and allowed to incubate at 4°C for 1 h. Agarose beads (protein A/G PLUS-agarose beads) were mixed with the immunoprecipitates for 1 h at 4°C. Immunoprecipitates were recovered by centrifugation at 2,500 g and washed three times with ice-cold lysis buffer. Immunoprecipitated proteins were dissolved in 2x Laemmli buffer, heated at 95°C for 5 min, subjected to SDS-PAGE, and transferred to Immobilon-P membranes (Millipore). Membranes were incubated with the indicated primary antibodies and visualized by enhanced chemiluminescence.
Transfection. 3T3-L1 preadipocytes were transiently cotransfected on day 0 by calcium phosphate co-precipitation with a C/EBP promoter-luciferase construct alone (24) or with either a cytomegalovirus (CMV)-C/EBP
expression vector (generously provided by Dr. M. Daniel Lane, Johns Hopkins University) and/or a CMV-Rb or a Rb mutant vector with a deletion corresponding to amino acid 389580 (Rbmut) (generously provided by Dr. Jonathan M. Horowitz, North Carolina State University) (27). Cells were then maintained in DMEM containing 10% CS for 24 h. Differentiation was induced as described previously. Cell lysates were prepared 24 h after induction and assayed for luciferase activity, which was normalized to cells transfected with the C/EBP
-promoter-luciferase construct alone and treated with MDI.
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RESULTS |
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To determine the temporal pattern of C/EBP-DNA-binding activity in relation to the G1/S transition of the cell cycle, day 0 preadipocytes were maintained in CS (time 0) or were treated with the MDI protocol. Nuclear extracts were prepared at 6-h intervals (for 24 h) and subjected to EMSA with the use of an oligonucleotide corresponding to the C/EBP binding site of the C/EBP
promoter. C/EBP
-DNA-binding activity increased dramatically 12 h after the induction of differentiation (Fig. 1B). Nuclear extracts incubated with a C/EBP
-specific antibody supershifted the protein-DNA complex, thereby confirming that C/EBP
was the predominant C/EBP isoform binding to the oligonucleotide at these time points (Fig. 1B). Because both Rb phosphorylation and C/EBP
-DNA-binding activity occur
12 h after induction of differentiation, we wanted to determine whether these two events link the cell cycle to the priming of differentiation during adipogenesis.
Cell cycle inhibition affects C/EBP-DNA-binding activity. Because C/EBP
is maximally expressed 4 h after the induction of differentiation but does not bind to DNA for another 8 h, we wanted to determine whether the delay in C/EBP
-DNA-binding activity is related to cell cycle progression. Day 0 preadipocytes were maintained in 10% CS or were induced to differentiate with the MDI protocol in the absence or presence of various cell cycle inhibitors. Twenty-four hours after induction, nuclear extracts were prepared and 10 µg of nuclear protein were subjected to EMSA, as described under EXPERIMENTAL PROCEDURES (Fig. 2A). Whole cell lysates were also prepared 24 h after treatment and subjected to immunoblot analysis using either an Rb or C/EBP
-specific antibody (Fig. 2B, top and bottom, respectively). The cell cycle inhibitor L-mimosine (a late G1 cell cycle inhibitor) inhibited C/EBP
-DNA binding activity without affecting C/EBP
expression (Fig. 2, A and B). As expected, L-mimosine treatment prevented the phosphorylation of Rb (Fig. 2B). In contrast, preadipocytes induced to differentiate in the presence of either aphidicolin (an agent that prevents DNA synthesis without preventing Rb phosphorylation) or nocodazole (an agent that arrests the cell cycle at the G2/M transition) had similar levels of C/EBP
expression and C/EBP
-DNA-binding activity as preadipocytes treated with MDI alone (Fig. 2, A and B). Rb was hyperphosphorylated in preadipocytes treated with either aphidicolin or nocodazole because both agents arrest the cell cycle after the G1/S transition (Fig. 2B). Cell cycle arrest by the various inhibitors was confirmed by fluorescence-activated cell sorting analysis (data not shown).
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Rb and C/EBP interact in a cell cycle-dependent manner. Before its hyperphosphorylation at the G1/S transition, Rb binds and sequesters transcription factors required for DNA synthesis. Rb has also been shown to interact with members of the C/EBP family, including C/EBP
(24). Because the DNA-binding activity of C/EBP
correlates with the hyperphosphorylation of Rb (Figs. 1 and 2), we wanted to determine whether C/EBP
and Rb interact in a cell cycle-dependent manner. Preadipocytes are in G1 6 h after MDI induction and have progressed beyond the G1/S transition point by 18 h (18). Day 0 3T3-L1 preadipocytes were either maintained in CS (time 0) or induced to differentiate by the MDI protocol for 6 or 18 h. Coimmunoprecipitation assays were conducted with the use of a C/EBP
-specific antibody and immunoblotted by using either an Rb or a C/EBP
antibody (Fig. 3). Immunoprecipitation reactions yielded only hypophosphorylated Rb (compare the migration pattern in the whole cell lysates with that in the immunoprecipitates). Immunoprecipitated C/EBP
associates with Rb strongly at 0 and 6 h, when Rb is hypophosphorylated, but only weakly at 18 h (Fig. 3), when most of the Rb is hyperphosphorylated. These studies suggest that the Rb-C/EBP
interaction depends on the phosphorylation state of Rb. The pattern of C/EBP
expression in whole cell lysates and immunoprecipitates was similar (Fig. 3).
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Rb decreases C/EBP-DNA-binding activity. Because Rb and C/EBP
interact at time points when Rb is hypophosphorylated and C/EBP
-DNA-binding activity is low, we wanted to determine whether unphosphorylated Rb alters C/EBP
-DNA-binding activity. Two-day postconfluent 3T3-L1 preadipocytes were maintained in 10% CS (0 h) or were treated with MDI for 18 h; nuclear extracts were prepared as described in EXPERIMENTAL PROCEDURES. C/EBP
-DNA-binding activity is maximal at 12 h and continues to be high 18 h after induction (Fig. 4B). Nuclear extracts from 0 and 18 h were subjected to EMSA. Recombinant, full-length unphosphorylated Rb protein (0, 0.05, 0.1, 0.25, and 0.5 µg) was incubated with 18 h nuclear extracts and subjected to EMSA (Fig. 4B, lanes 2 and 58). Full-length unphosphorylated Rb decreased C/EBP
-DNA-binding activity. Because previous studies have demonstrated that C/EBP
can bind to the two pocket domains (PDA and PDB) of Rb (Fig. 4A) (3), we examined the effect of truncated Rb proteins (p56Rb or p25Rb) on C/EBP
-DNA-binding activity in 18 h nuclear extracts (Fig. 4B, lanes 3 and 4). The addition of p56Rb decreased C/EBP
-DNA-binding activity, whereas p25Rb did not (Fig. 4B). These findings suggest that unphosphorylated Rb can inhibit the DNA-binding activity of C/EBP
even after the G1/S transition.
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Overexpression of Rb inhibits C/EBP-mediated transcription. Because association with Rb inhibits the DNA-binding activity of C/EBP
in vitro, we wanted to determine whether the overexpression of Rb would inhibit C/EBP
-mediated transcriptional activation. The C/EBP
-promoter contains a C/EBP binding site that mediates transactivation by members of the C/EBP family. To determine whether Rb can alter C/EBP
-mediated transcriptional activation, 3T3-L1 preadipocytes were mock transfected or were transfected with the C/EBP
-promoter-luciferase reporter transgene alone or with a CMV-Rb, a Rbmut (deletion of the PDA domain) (27), and/or a CMV-LAP expression vector by calcium-phosphate coprecipitation. Transfected preadipocytes were then induced to differentiate with MDI for 24 h. Luciferase activity was assayed 24 h after MDI induction (results are presented as means ± SD, n = 4). Cells cotransfected with a C/EBP
promoter-luciferase reporter gene and a CMV-LAP expression vector resulted in approximately a threefold increase in luciferase activity compared with cells transfected with the C/EBP
-luciferase reporter gene alone (Fig. 5). Cotransfection of the C/EBP
-luciferase reporter gene and a CMV-Rb expression vector resulted in a slight inhibition of luciferase activity compared with the level in cells transfected with a C/EBP
-luciferase reporter gene alone. Overexpression of Rb inhibited the C/EBP
-mediated transcriptional activation of the C/EBP
promoter to a level similar to cells transfected with the C/EBP
-luciferase reporter gene alone (Fig. 5). Transfection of an Rb expression vector (Rbmut) lacking the PDA domain (a C/EBP-binding site) did not inhibit C/EBP
-mediated transcriptional activation of the C/EBP
promoter. Taken together, these findings suggest that Rb regulates the transcriptional activity of C/EBP
by directly interacting with it and preventing DNA binding.
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DISCUSSION |
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A critical component of 3T3-L1 adipogenesis is the temporal expression and activation of C/EBP and C/EBP
. Although C/EBP
is at maximal levels 4 h after induction, it does not bind to DNA for another 8 h (Fig. 1) (24). Several mechanisms have been proposed in the regulation of C/EBP
activity, including conformational change, interaction with dimerization partners, and phosphorylation (20, 25, 28, 29). In this report, we demonstrate that C/EBP
-DNA-binding activity is also regulated by the cell cycle (Figs. 1 and 2). Our previous studies have shown that C/EBP
-DNA-binding activity is inhibited in the presence of the calpain inhibitor ALLN, which arrests cells in G1 (19). To determine whether calpain activity or cell cycle progression mediates C/EBP
-DNA-binding activity, we employed another known G1 cell cycle inhibitor. Inhibition of the cell cycle in G1 by L-mimosine also blocked C/EBP
-DNA-binding activity even after 24 h (Fig. 2). In contrast, inhibition of the cell cycle after the G1/S transition by either aphidicolin (cell cycle arrest in S phase) or nocodazole (arrest at G2/M) did not prevent C/EBP
-DNA-binding activity. Because all of these studies were conducted 24 h after MDI induction, our findings suggests that C/EBP
-DNA-binding activity is cell cycle-dependent, not time dependent, during the early stages of adipogenesis.
Linking C/EBP-DNA-binding activity to the cell cycle further suggests a connection between the initiation of differentiation and cell cycle progression. Our studies demonstrate a cell cycle-dependent interaction between C/EBP
and Rb (Fig. 3). Rb is a critical regulator of cell cycle progression at the G1/S transition. Previous studies have shown that Rb and C/EBP family members are able to interact in in vitro experiments (4). While these studies demonstrate an interaction between Rb and C/EBP isoforms, that interaction was not investigated in the context of cell cycle progression. Our study is the first to examine the interaction between Rb and C/EBP
as a function of the cell cycle. We propose a mechanism for cell cycle-mediated C/EBP
-DNA-binding activity during 3T3-L1 adipogenesis. Specifically, our studies show that hypophosphorylated Rb (6 h after induction) binds to C/EBP
, whereas hyperphosphorylated Rb (18 h) does not (Fig. 3). During time points when Rb and C/EBP
interact, the DNA-binding activity of C/EBP
is minimal (Figs. 1 and 3). Conversely, maximal C/EBP
-DNA-binding activity occurs when Rb and C/EBP
no longer associate (Figs. 1 and 3). Previous studies (4) have shown that C/EBP
is able to bind to Rb by interacting with the pocket domains PDA and PDB. Our studies demonstrate that recombinant Rb proteins that contain the pocket domains (either full-length or p56Rb) reduce C/EBP
-DNA-binding activity in 18 h nuclear extracts, whereas p25Rb, which lacks both C/EBP
-DNA-binding domains (PDA and PDB), does not affect C/EBP
-DNA-binding activity (Fig. 4). Because hypophosphorylated Rb interacts with many different proteins with varying affinities (14), relatively high concentrations of recombinant Rb proteins were used to alter C/EBP
-DNA-binding activity in competition assays. The addition of full-length recombinant hypophosphorylated Rb reduced C/EBP
-DNA-binding activity when the concentration of exogenous Rb was
4% of the total protein in the assay (Fig. 4). Furthermore, we show that overexpression of Rb inhibited the C/EBP
-induced transactivation of the C/EBP
-promoter-luciferase reporter (Fig. 5). Collectively, our findings suggest that the hypophosphorylated Rb-C/EBP
interaction couples cell cycle progression with the initiation of differentiation.
In conclusion, this study demonstrates that hypophosphorylated Rb is a negative regulator of C/EBP-DNA binding activity during 3T3-L1 preadipocyte differentiation. These studies add to the growing body of evidence that demonstrate a requirement for mitotic clonal expansion during 3T3-L1 adipogenesis. Our findings also provide evidence that clonal expansion participates in the temporal regulation of transcriptional effects required for differentiation (C/EBP
-DNA-binding activity). These studies provide a link between mitotic clonal expansion and initiation of differentiation. Although this study proposes a mechanism for the delay in C/EBP
-DNA-binding activity, further studies are required to determine the purpose of this delay.
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ACKNOWLEDGMENTS |
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This work was supported in part by National Institutes of Health Nutrition Training Grant DK-07686 (to K. A. Cole).
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FOOTNOTES |
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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.
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