Considerable insight into the molecular mechanisms governing adipocyte differentiation has been gained from studies using the murine 3T3-L1 preadipocyte cell line. These cells differentiate into mature adipocytes within five days of hormonal stimulation. Following induction of differentiation, these quiescent cells re-enter the cell cycle undergoing a mitotic clonal expansion phase and again withdraw from cell cycle prior to completing terminal differentiation into mature adipocytes. Through interactions with the E2F transcription factors, the retinoblastoma proteins(pRb,p107 and p130) have been shown to be critical in regulating cell cycle progression, and recently it has been demonstrated that a switch in E2F binding complexes from p130/E2F to p107/E2F occurs during the clonal expansion phase of adipogenesis after which the switch reverts back to the p130/E2F state. TNFα has been shown to inhibit adipocyte differentiation and given its production by adipocytes may serve to feedback inhibit expansion of fat stores. We therefore wanted to determine if TNFα interfered with p130/p107 regulation and clonal expansion during 3T3-L1 adipogenesis. Differentiation in the presence of TNFα (10 ug/ml) resulted in a significant reduction in the percentage of mature adipocytes as assessed by cell counting and Oil Red O staining. TNFα treatment did not prevent the switch from p130/E2F to p107/E2F as determined by Western blot nor did it block entry into the clonal expansion phase as assessed by BrdU proliferation assays. However TNFα did prevent the switch from reverting back to the predominant p130/E2F following Day 1 when the cells normally withdraw from the cell cycle. p107 protein and mRNA levels absent at Day 0 dramaticaly increased at Day 1 and remained elevated through Day 4 of differentiation while p130 protein levels which were high at Day 0 decreased dramatically and remained low. Interestingly, morphologic evidence of apoptosis appeared early in the course of treatment with TNFα coincident with the p130/p107 alterations and was confirmed by the TUNEL method. These results suggest that the inhibition of differentiation by TNFα is due in part to loss of cells by apoptosis. Furthermore, TNFα-induced pertubations in p130/p107 regulation during the mitotic clonal expansion phase may lead to apoptosis and therefore represent an important mechanism in the TNFα inhibition of 3T3-L1 adipogenesis.
