Abstract
During 3T3-L1 adipocyte differentiation, growth-arrested, postconfluent preadipocytes are required to reenter the cell cycle and proceed through a mitotic clonal expansion phase prior to terminal differentiation. The retinoblastoma proteins (pRB, p107, and p130) are thought to be critical in controlling cell cycle progression by binding to and regulating the activity of the E2F transcription factors. We show here that p130/p107 protein levels, p107 mRNA levels, and E2F DNA binding complexes are regulated during 3T3-L1 adipogenesis. The predominant E2F binding complex in day 0 preadipocytes was p130-E2F with no detectable free E2F or p107. On Day 1, during mitotic clonal expansion, there was a distinct switch to free E2F and p107-E2F complexes associated with increased p107 mRNA and protein along with decreased p130 protein levels. Following differentiation, the day 0 pattern is reestablished. The switch is not just a consequence of reentry into the cell cycle, in that p107 protein levels are both detectable and unchanged in dividing, serum-restricted, or serum restimulated preconfluent cells. Interestingly, hormonal stimulation of 3T3-C2 cells, a related nondifferentiating cell line, also induces a mitotic clonal expansion phase that is associated with the p130:p107 switch in a pattern very similar to 3T3-L1 cells, suggesting the block in differentiation observed in 3T3-C2 cells occurs after clonal expansion. Combined, these findings suggest that the regulatory mechanisms of the p130:p107 switch are not specific to differentiation but may play a key role in regulating the mitotic clonal expansion necessary for adipocyte differentiation in 3T3-L1 cells.
Highlights
Obesity is a major health problem in the United States and is a risk factor for many serious illnesses such as heart disease, arthritis, diabetes, and others
Following hormonal induction of differentiation with IDX1, these growth-arrested confluent cells proceed through the G1/S boundary, reentering the cell cycle, and undergo several rounds of mitotic clonal expansion that is essential to completing terminal differentiation into mature adipocytes [4]
Following the burst in proliferation observed in day 1, the cells begin to withdraw from the cell cycle and by day 3 of differentiation were essentially quiescent again and arrested at G0 (Fig. 1C), a time when late markers of adipocyte differentiation are beginning to be expressed
Summary
Obesity is a major health problem in the United States and is a risk factor for many serious illnesses such as heart disease, arthritis, diabetes, and others. On Day 1, during mitotic clonal expansion, there was a distinct switch to free E2F and p107-E2F complexes associated with increased p107 mRNA and protein along with decreased p130 protein levels.
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