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Proc Natl Acad Sci U S A. Feb 4, 2003; 100(3): 850–855.
Published online Jan 13, 2003. doi:  10.1073/pnas.0337434100
PMCID: PMC298690
Biochemistry

CCAAT/enhancer-binding protein β is required for mitotic clonal expansion during adipogenesis

Abstract

Hormonal induction of growth-arrested 3T3-L1 preadipocytes triggers a signaling cascade that culminates in adipogenesis. CCAAT/enhancer-binding protein (C/EBP)β is expressed immediately but gains DNA-binding activity only after a long lag as the cells synchronously begin mitotic clonal expansion (MCE). After MCE, a process required for adipogenesis, C/EBPβ activates expression of C/EBPα and peroxisome proliferator-activated receptor γ, which then transcriptionally activate genes that produce the adipocyte phenotype. When mouse embryo fibroblasts (MEFs) are subjected to the same differentiation protocol, a subset of the MEFs undergoes a similar program of events. Similar to 3T3-L1 preadipocytes, the MEFs reenter the cell cycle (as indicated by the synchronous expression of cyclin A) and undergo MCE as evidenced by the incorporation of BrdUrd into DNA and the formation of mitotic foci of cells that undergo adipogenesis. C/EBPβ is expressed immediately after induction but exhibits delayed acquisition of DNA-binding activity followed by expression of adipocyte markers and the accumulation of cytoplasmic triglyceride. MEFs from C/EBPβ(−/−) mice, however, neither undergo MCE nor differentiate into adipocytes. Forced expression of C/EBPβ (LAP) but not dominant-negative C/EBPβ (LIP) in C/EBPβ(−/−) MEFs restores MCE, expression of adipocyte markers, and the capacity to form mitotic foci of cells that undergo adipogenesis. These findings demonstrate that expression of C/EBPβ is a prerequisite for MCE in the adipocyte-differentiation program.

Keywords: 3T3-L1 preadipocyte‖mouse embryo fibroblasts‖C/EBPα‖PPARγ‖ cell cycle

After induction of differentiation, postconfluent, growth-arrested 3T3-L1 preadipocytes synchronously reenter the cell cycle, undergo several rounds of mitotic clonal expansion (MCE), and then express genes that produce the adipocyte phenotype (14). Recent evidence (5, 6) has established that MCE is required for the progression of the differentiation program. Immediately (within 2–4 h) after induction CCAAT/enhancer-binding protein (C/EBP)β is expressed but unable to bind DNA and thus cannot activate the regulatory genes responsible for terminal differentiation. Only after a long lag period (10–12 h) does C/EBPβ acquire DNA-binding activity (7). Acquisition of binding activity occurs as the cells synchronously reenter the cell cycle, traverse the G1-S checkpoint, and begin MCE (7). Coincident with the acquisition of DNA-binding activity, C/EBPβ binds to centromeres through consensus C/EBP-binding sites in centromeric satellite DNA (7). After acquiring DNA-binding activity C/EBPβ activates transcription of the C/EBPα and peroxisome proliferator-activated receptor γ (PPARγ) genes mediated by C/EBP regulatory elements in their promoters (810). Together, C/EBPα and PPARγ coordinately activate the transcription of genes that give rise to the adipocyte phenotype (1, 3, 4). Since both C/EBPα and PPARγ are antimitotic (1114), the timing of this gain of function by C/EBPβ is critical, because premature expression of C/EBPα and PPARγ would prevent MCE. Thus, delayed acquisition of DNA-binding function by C/EBPβ seems to forestall expression of C/EBPα and PPARγ.

Evidence that C/EBPβ plays a vital role in adipogenesis was reported first by Tanaka et al. (15), who found that disruption of the C/EBPβ and C/EBPδ genes in mice caused impaired development of adipose tissue. In view of this finding and the dependence of adipogenesis on MCE (6), the possibility was considered that C/EBPβ may play a role in or even be required for MCE. A strong precedent exists for participation of C/EBPβ in the clonal expansion of hepatocytes after partial hepatectomy (16), which has many features in common with the MCE of adipocyte differentiation. C/EBP family members play important roles in both of these processes (6, 17, 18). After partial hepatectomy, growth-arrested liver cells reenter the cell cycle and proliferate to restore liver mass and function. During this clonal-expansion process, the expression of C/EBPβ (transcriptional activator of the C/EBPα gene) is followed by expression of C/EBPα. The dependence of hepatocyte proliferation on C/EBPβ is supported by the finding that partial hepatectomy in mice carrying a targeted disruption of the C/EBPβ gene results in impaired liver regeneration (19). Furthermore, a mutation in C/EBPβ that renders it inactive prevents hepatocyte proliferation both in vivo and ex vivo (16).

The present study investigates the role of C/EBPβ in the MCE of adipogenesis by mouse embryo fibroblasts (MEFs). When subjected to the same differentiation protocol as 3T3-L1 preadipocytes, a subset of MEFs undergoes MCE and adipogenesis. We demonstrate that the expression of C/EBPβ is required for MCE in MEFs induced to differentiate into adipocytes.

Materials and Methods

Cell Culture and Differentiation MEFs.

To induce differentiation, 2-day postconfluent MEFs (designated day 0) were fed DMEM containing 10% FBS, 1 μg/ml insulin, 1 μM dexamethasone, and 0.5 mM 3-isobutyl-1-methyl-xanthine until day 2. Cells then were fed DMEM supplemented with 10% FBS/1 μg/ml insulin for 2 days, after which they were fed every other day with DMEM containing 10% FBS. MEFs, prepared from wild-type (wt) and C/EBPβ(−/−) mice, were generously provided by Peter Johnson (National Cancer Institute, National Institutes of Health). The MEFs were maintained in DMEM containing 10% FBS, grown to 2-day postconfluence, and then subjected to differentiation as described above. Day-3 cells were stained by hematoxylin (Vector Laboratories), and day-8 cells were stained with oil red O.

C/EBPβ Adenoviral Expression Vectors and Infection.

The adenoviral expression vectors (Adeno-X, CLONTECH) encoding C/EBPβ (LAP), dominant-negative C/EBPβ (LIP), and Lac Z were provided by Hiroshi Sakaue and Masato Kasuga (Kobe University, Kobe, Japan). Growth-arrested confluent C/EBPβ(−/−) MEFs were infected with the adenoviral vectors at a multiplicity of infection of 10 1 h before induction of differentiation by using the standard 3T3-L1-differentiation protocol; 24 h later, whole-cell lysates were prepared and the expression of LAP or LIP was assessed by immunoblotting. On day 6 whole-cell lysates were prepared and immunoblotted with antibodies to PPARγ, C/EBPα, and 422/adipocyte P2 protein (aP2), and on day 8 cells were fixed, and cytoplasmic triglyceride was stained with oil red O.

Immunoblotting.

At various time points, cell monolayers (6-cm dishes) were washed once with cold PBS, pH 7.4, and then scraped into lysis buffer (1% SDS/60 mM Tris[center dot]Cl, pH 6.8). Lysates were heated at 100°C for 10 min and clarified by centrifugation, and equal amounts of protein were separated by SDS/PAGE. Proteins were transferred to poly(vinylidene difluoride) membranes and immunoblotted with antibodies to C/EBPα, 422/aP2, and PPARγ [antibodies to C/EBPα or 422/aP2 were as described (7, 20), and the antibody to PPARγ was provided by Mitchell Lazar (University of Pennsylvania, Philadelphia)].

Immunofluorescence Microscopy and BrdUrd Labeling.

MEFs were plated onto glass coverslips in 3.5-cm dishes, grown to 2-day postconfluence, and then induced to differentiate as described above. At 8 and 20 h after induction, cell monolayers were washed with cold PBS and fixed for 20 min with 4% formaldehyde at room temperature. Cells were permeabilized for 30 min with 0.075% Triton X-100 in 2 mg/ml BSA and blocked with 2 mg/ml BSA for 1–2 h at room temperature. Cells then were incubated with the primary antibody (C/EBPβ antibody, 1:200 dilution) in 2 mg/ml BSA for 1–2 h and incubated for 1 h with FITC-labeled secondary antibody in the same buffer. After each step cells were washed with PBS three times. Antifade solution (Molecular Probes) was added to the monolayers, and coverslips were mounted on slides.

For BrdUrd labeling, wt C/EBPβ(+/+) or C/EBPβ(−/−) MEFs were plated on coverslips and maintained in DMEM containing 10% FBS until 2 days after confluence was achieved. Cells were induced to differentiate with standard differentiation protocol and 18 h after induction (during S phase) were pulse-labeled for 2 h with 30 μg/ml BrdUrd and then shifted to normal medium. On day 3 coverslips were fixed in 70% ethanol for 30 min and incubated in 100% methanol for 10 min at room temperature. Fixed cells on coverslips then were treated for 30 min with 1.5 M HCl, blocked with 0.5% Tween 20 in PBS for 5 min, incubated with anti-BrdUrd primary antibody (1:500) in the same buffer for 2 h at room temperature, and then incubated with FITC-conjugated secondary antibody (1:200) with 0.1 μg/ml 4′,6-diamidino-2-phenylindole (DAPI) for 1 h at room temperature. After each step cells were washed with PBS three times. Antifade solution (Molecular Probes) was added to the monolayers, and coverslips were mounted on slides for examination with a confocal microscope.

Oil Red O Staining.

Cells were washed three times with PBS and then fixed for 2 min with 3.7% formaldehyde. Oil red O (0.5% in isopropanol) was diluted with water (3:2), filtered through a 0.45-μm filter, and incubated with the fixed cells for 1 h at room temperature. Cells were washed with water, and the stained fat droplets in the cells were visualized by light microscopy and photographed.

Results

MCE and Adipogenesis by MEFs.

When subjected to the same differentiation protocol used to induce adipogenesis with 3T3-L1 preadipocytes, a subset of wt MEFs undergoes MCE and differentiation into adipocytes. Thus, 8 days after induction ≈15% of MEFs exhibit cytoplasmic triglyceride accumulation as visualized by oil red O staining (Fig. (Fig.1A1A Right). Presumably, the subset of MEFs that underwent adipogenesis originated from pluripotent MEFs that already had undergone commitment to the adipose lineage. During differentiation, MEFs express adipocyte markers, e.g., C/EBPα, PPARγ, and 422/aP2, typical of differentiated 3T3-L1 adipocytes (Fig. (Fig.11B).

Figure 1
Effect of the differentiation-induction protocol on adipogenesis and adipocyte markers in wt C/EBPβ(+/+) and C/EBPβ(−/−) MEFs. wt C/EBPβ(+/+) ...

Interestingly, MEFs that differentiated were found only in foci of tightly packed cells that closely resembled differentiated 3T3-L1 adipocytes [Fig. 2A, C/EBPβ(+/+)]. Compared with the undifferentiated cells in the regions surrounding the foci, differentiated MEFs within foci exhibited higher cell density, increased cell number (Fig. (Fig.22B), and incorporation of BrdUrd into DNA [Fig. 2C, C/EBPβ(+/+)]. These characteristics and the fact that the cells in the mitotic foci express cytoplasmic triglyceride indicate that the cells that have differentiated are those that have undergone MCE. This is particularly evident in the foci shown in Fig. Fig.22A [C/EBPβ(+/+)] that were stained with hematoxylin early in the differentiation program, i.e., 3 days after induction, before full expression of cytoplasmic triglyceride had obscured the nuclei. As illustrated in Fig. Fig.2A2A Inset, cells within mitotic foci were destined to become adipocytes, because many had begun already to accumulate minute cytoplasmic triglyceride-laden vesicles.

Figure 2
Effect of differentiation induction on the formation of mitotic foci, cell number, and BrdUrd labeling of C/EBPβ by wt and C/EBPβ(−/−) MEFs. wt C/EBPβ(+/+) ...

Previous investigations showed that C/EBPβ is expressed rapidly (within 2–4 h) after the induction of differentiation by 3T3-L1 preadipocytes. However, acquisition of DNA-binding activity by C/EBPβ (measured in vitro with nuclear extracts) in 3T3-L1 preadipocytes is delayed for 12–16 h (7). It also was shown ex vivo that, after acquiring DNA-binding activity, C/EBPβ associates with centromeres. This association was found to be due to the binding of C/EBPβ to consensus C/EBP-binding sites in centromeric satellite DNA (7). This phenomenon also occurs with similar kinetics in wt MEFs that have been induced to differentiate. As shown in Fig. Fig.33A, expression of C/EBPβ (both LAP and LIP) occurs within 4 h after induction of differentiation and continues at this level for the next 24 h. By 8 h C/EBPβ exhibits diffuse nuclear immunostaining (Fig. (Fig.33B), indicative of its presence in nuclei. However, at this point C/EBPβ has not acquired DNA-binding activity yet as shown by the lack of punctate staining, indicating that it has not yet associated with centromeres. Similar to 3T3-L1 preadipocytes (6, 7), MEFs traverse the G1-S checkpoint at 14–16 h as demonstrated by the expression of cyclin A (Fig. (Fig.33A) before C/EBPβ acquires DNA-binding activity as shown by its association with centromeres (Fig. (Fig.33B). Verification that C/EBPβ associates with centromeres at this point is indicated by the coincidence of punctate staining of C/EBPβ and 4′,6-diamidino-2-phenylindole, which stains centromeric satellite DNA (Fig. (Fig.33B; see ref. 7). These findings show that after induction of differentiation, C/EBPβ in MEFs exhibits the same pattern of delayed acquisition of DNA-binding activity and centromeric localization as observed with 3T3-L1 preadipocytes (7).

Figure 3
Expression of C/EBPβ and cyclin A and association of C/EBPβ with centromeres in MEFs after induction of differentiation. (A) At the times indicated after induction, cell lysates were prepared, and 50 μg of protein ...

Effect of C/EBPβ Deficiency on MCE and Adipogenesis.

The effect of C/EBPβ deficiency on MCE and subsequent differentiation was investigated by using MEFs isolated from C/EBPβ(−/−) mouse embryos. Unlike wt C/EBPβ(+/+) MEFs, C/EBPβ(−/−) MEFs neither form mitotic foci nor give rise to cells with adipocyte characteristics, e.g., the accumulation of cytoplasmic triglyceride (Fig. (Fig.1A1A Left). Careful inspection of the C/EBPβ(−/−) cell monolayers revealed no “densely packed” cells, i.e., mitotic foci, indicating that MCE had not occurred. Quantification of foci in 10 randomly selected microscopic fields revealed an average of 12 ± 1 foci per 1.5 × 1.5-cm field of wt C/EBPβ(+/+) MEFs and <0.5 ± 0.3 foci per field of C/EBPβ(−/−) MEFs. Moreover, quantification of cell number showed that, similar to 3T3-L1 preadipocytes (7), wt C/EBPβ(+/+) MEFs undergo an increase in cell number when induced to differentiate, with the total cell number per culture dish increasing 40–45% by day 3 (Fig. (Fig.22B). In contrast, the cell number of C/EBPβ(−/−) MEFs remained unchanged (Fig. (Fig.22B). The magnitude of the increase in cell number of wt MEFs is consistent with the fact that only a fraction (i.e., 10–15%) of wt C/EBPβ(+/+) MEFs differentiate into triglyceride-laden adipocytes (Fig. (Fig.11A). This 3- to 4-fold increase in cell number is consistent with the results of BrdUrd pulse-labeling experiments conducted 18 h after induction during S phase when expression of cyclin A is maximal (Fig. (Fig.33A). Labeling of C/EBPβ(+/+) MEFs with BrdUrd gave rise to a large fraction of cells (within mitotic foci) labeled with BrdUrd (Fig. (Fig.2C2C Left). Little BrdUrd labeling was detected in C/EBPβ(−/−) MEFs (Fig. (Fig.2C2C Right) or in the regions surrounding foci of wt C/EBPβ(+/+) MEFs (Fig. (Fig.2C2C Left). Quantification of 10 randomly selected fields for BrdUrd labeling revealed that virtually all foci of wt C/EBPβ(+/+) MEFs and 90 ± 5% of the cells within those foci exhibited BrdUrd labeling. Fewer than 5% of C/EBPβ(−/−) MEFs exhibited BrdUrd labeling. Taken together, these results indicate that C/EBPβ is required for MCE.

Rescue of MCE and Adipogenesis in C/EBPβ(−/−) MEFs by Forced Expression of C/EBPβ.

To verify the requirement of C/EBPβ per se for MCE, C/EBPβ-deficient MEFs were infected with adenoviral expression vectors for the “active” C/EBPβ (LAP) or dominant-negative C/EBPβ (LIP) isoforms. As illustrated in Fig. Fig.44A, both C/EBPβ (LAP) and C/EBPβ (LIP) were expressed in C/EBPβ(−/−) MEFs infected with expression vectors for these isoforms. Expression of C/EBPβ (LAP) restored both MCE and adipogenesis in a subset (10–15%) of C/EBPβ(−/−) MEFs. These MEFs produced foci of tightly packed cells, accumulated adipocyte markers (PPARγ,§ C/EBPα, and 422/aP2 protein; Fig. Fig.44B), and expressed cytoplasmic triacylglycerol (Fig. (Fig.44C). The frequency of differentiation of MEFs infected with the C/EBPβ (LAP) expression vector (Fig. (Fig.44C, LAP) was comparable to that of wt C/EBPβ(+/+) MEFs (see Fig. Fig.1A1A Top). In contrast, forced expression of dominant-negative C/EBPβ (LIP) did not lead to acquisition of these functions (Fig. (Fig.44C). These results indicate that expression of the LAP isoform of C/EBPβ in C/EBPβ(−/−) MEFs is sufficient to rescue both MCE and adipogenesis.

Figure 4
Forced expression of C/EBPβ (LAP) in C/EBPβ(−/−) MEFs restores both MCE and the capacity for differentiation. C/EBPβ(−/−) MEFs were maintained in DMEM containing ...

Discussion

C/EBPβ seems to serve dual functions in the 3T3-L1 adipocyte-differentiation program. C/EBPβ acts as transcriptional activator of the C/EBPα and PPARγ genes, both of which possess C/EBP regulatory elements in their proximal promoters (810). Once expressed, C/EBPα and PPARγ act as pleiotropic transcriptional activators for a large group of adipocyte genes, the products of which give rise to the terminally differentiated phenotype (14). In this capacity C/EBPβ functions late in the differentiation program. The present investigation shows that C/EBPβ also functions earlier in the differentiation program, i.e., during MCE.

When wt MEFs are subjected to the differentiation protocol, a subset of cells produce mitotic foci of high cell density (Figs. (Figs.11A and and22A) that contain a large proportion of S-phase cells (as evidenced by the expression of cyclin A; Fig. Fig.33A) and exhibit extensive BrdUrd labeling (Fig. (Fig.22C). These features indicate that the cells in the foci undergo MCE. Most of the MEFs in these foci begin to exhibit minute cytoplasmic triacylglyceride-containing vesicles (Fig. (Fig.2A2A Inset) just as MCE shuts down. Because the onset of adipogenesis occurs in MEFs that have undergone proliferation, it is evident that these cells are destined to become the fully differentiated adipocytes with the much larger triglyceride-laden vesicles observed in foci 5–6 days later, after terminal differentiation has been completed (Fig. (Fig.11A).

In contrast, when MEFs from C/EBPβ-deficient mice are subjected to the differentiation protocol, they do not produce mitotic foci (Figs. (Figs.11A and and22A), undergo labeling with BrdUrd (Fig. (Fig.22C), express adipocyte markers (Fig. (Fig.11B), or accumulate cytoplasmic triglyceride (Figs. (Figs.11A and and22A). These results are consistent with our previous finding (6) that MCE is required for the differentiation of 3T3-L1 preadipocytes into adipocytes. Importantly, MCE and adipogenesis by C/EBPβ(−/−) MEFs can be rescued by the forced expression of C/EBPβ (LAP) (Fig. (Fig.44 B and C). It should be noted also that, similar to 3T3-L1 preadipocytes, wt MEFs exhibit similar kinetics of expression of C/EBPβ and cyclin A (Fig. (Fig.33A) and delayed acquisition of DNA-binding activity (Fig. (Fig.33B) when induced to differentiate. Taken together these findings indicate that C/EBPβ plays a vital role in both MCE and adipogenesis.

Recent findings (J. Zhang, C. R. Vinson, and M.D.L., results not shown) with dominant-negative A-zip C/EBP, a construct containing the C/EBPβ leucine zipper but lacking basic DNA-binding and transactivation domains (21), support this conclusion. Thus, forced expression of A-zip C/EBP in wt 3T3-L1 preadipocytes prevents translocation of C/EBPβ to the nucleus and thereby blocks MCE and differentiation. Although A-zip C/EBP can form inactive heterodimers with C/EBPβ and C/EBPα, only inactivation of C/EBPβ is relevant for MCE, because C/EBPβ is expressed and activated before MCE and the expression of C/EBPα.

Acknowledgments

We thank Peter Johnson, National Cancer Institute, National Institutes of Health (NIH), for providing the wt and C/EBPβ(−/−) MEFs and Drs. Hiroshi Sakaue and Masato Kasuga (Kobe University) for supplying the C/EBPβ adenoviral vectors. This research was supported by NIH Research Grant DK38418. Q.-Q.T. was supported by NIH K01 Award DK61355, and T.C.O. was supported by NIH National Research Service Award DK61840.

Abbreviations

MCE
mitotic clonal expansion
C/EBP
CCAAT/enhancer-binding protein
PPARγ
peroxisome proliferator-activated receptor γ
MEF
mouse embryo fibroblast
wt
wild type
aP2
adipocyte P2 protein

Footnotes

§Although PPARγ1 is expressed both by cells infected with the control/empty virus or the C/EBPβ (LAP)-containing virus, only cells infected with the latter virus express PPARγ2. This is likely because of the presence of tandem C/EBP regulatory elements in the proximal promoter of the PPARγ2 gene, through which C/EBPβ (LAP) can transactivate the gene (10), whereas the proximal promoter of the PPARγ1 gene does not possess these elements in its proximal promoter (22).

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