When induced to differentiate, growth-arrested 3T3-L1 preadipocytes synchronously reenter the cell cycle and undergo mitotic clonal expansion (MCE) followed by expression of genes that produce the adipocyte phenotype. The preadipocytes traverse the G1͞S checkpoint synchronously as evidenced by the expression͞ activation of cdk2-cyclin-E͞A, turnover of p27͞kip1, hyperphosphorylation of Rb, translocation of cyclin D1 from nuclei to cytoplasm and GSK-3 from cytoplasm to nuclei, and incorporation of [ 3 H]thymidine into DNA. As the cells cross the G1͞S checkpoint, C͞EBP acquires DNA-binding activity, initiating a cascade of transcriptional activation that culminates in the expression of adipocyte proteins. The mitogen-activated protein kinase͞extra-cellular signal-regulated kinase kinase (MEK) inhibitor PD98059 delays, but does not block, MCE and differentiation, the extent of the delay causing a comparable delay in the expression of cell-cycle markers, MCE, and adipogenesis. The more potent and specific MEK inhibitor UO126 and the cyclin-dependent kinase inhibitor roscovitine, which inhibit the cell cycle at different points, block MCE, expression of cell cycle and adipocyte markers, as well as adipogenesis. These results show that MCE is a prerequisite for differentiation of 3T3-L1 preadipocytes into adipocytes.cell cycle ͉ adipogenesis ͉ 3T3-L1 preadipocyte ͉ C͞EBP␣ ͉ PPAR␥
The increase of adipose tissue mass associated with obesity is due in part to an increase in the number of adipocytes. This hyperplasia results from recruitment of pluripotent stem cells present in the vascular stroma of adipose tissue. A model cell culture system has been developed that recapitulates this process both ex vivo and in vivo. After treatment of pluripotent C3H10T1͞2 stem cells with bone morphogenic protein 4 (BMP4) during proliferation followed by differentiation inducers at growth arrest, the cells synchronously enter S phase and undergo mitotic clonal expansion, a hallmark of preadipocyte differentiation. Upon exiting the cell cycle, these cells express adipocyte markers and acquire adipocyte characteristics at high frequency. C3H10T1͞2 cells treated with BMP4 in cell culture and implanted s.c. into athymic mice develop into tissue indistinguishable from adipose tissue in normal fat depots. We interpret the findings as evidence that BMP4 is capable of triggering commitment of pluripotent C3H10T1͞2 stem cells to the adipocyte lineage.adipose tissue ͉ differentiation ͉ bone morphogenic protein 4 ͉ mitotic clonal expansion ͉ development
Hormonal induction of growth-arrested 3T3-L1 preadipocytes triggers a signaling cascade that culminates in adipogenesis. CCAAT͞ enhancer-binding protein (C͞EBP) 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, C͞EBP activates expression of C͞EBP␣ and peroxisome proliferator-activated receptor ␥, 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. C͞EBP is expressed immediately after induction but exhibits delayed acquisition of DNAbinding activity followed by expression of adipocyte markers and the accumulation of cytoplasmic triglyceride. MEFs from C͞EBP(؊͞؊) mice, however, neither undergo MCE nor differentiate into adipocytes. Forced expression of C͞EBP (LAP) but not dominant-negative C͞EBP (LIP) in C͞EBP(؊͞؊) 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 C͞EBP is a prerequisite for MCE in the adipocyte-differentiation program.3T3-L1 preadipocyte ͉ mouse embryo fibroblasts ͉ C͞EBP␣ ͉ PPAR␥ ͉ cell cycle A fter induction of differentiation, postconfluent, growtharrested 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 (1-4). Recent evidence (5, 6) has established that MCE is required for the progression of the differentiation program. Immediately (within 2-4 h) after induction CCAAT͞ enhancer-binding protein (C͞EBP) 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 C͞EBP acquire DNA-binding activity (7). Acquisition of binding activity occurs as the cells synchronously reenter the cell cycle, traverse the G 1 -S checkpoint, and begin MCE (7). Coincident with the acquisition of DNA-binding activity, C͞EBP binds to centromeres through consensus C͞EBP-binding sites in centromeric satellite DNA (7). After acquiring DNA-binding activity C͞EBP activates transcription of the C͞EBP␣ and peroxisome proliferator-activated receptor ␥ (PPAR␥) genes mediated by C͞EBP regulatory elements in their promoters (8-10). Together, C͞EBP␣ and PPAR␥ coordinately activate the transcription of genes that give rise to the adipocyte phenotype (1, 3, 4). Since both C͞EBP␣ and PPAR␥ are antimitotic (11)(12)(13)(14), the timing of this gain of function by C͞EBP is critical, because premature e...
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