E2F/p107 and E2F/p130 complexes are regulated by C/EBP alpha in 3T3-L1 adipocytes

We have previously found that loss of C/EBP alpha in hepatocytes of newborn livers leads to increased proliferation, to a reduction in p21 protein lev els and to an induction of S phase-specific E2F/p107 complexes. In this pap er, we investigated C/EBP alpha-dependent regulation of E2F complexes in a well-characterized cell line, 3T3-L1, and in stable transformants that cond itionally express C/EBP alpha, C/EBP alpha and C/EBP beta proteins are indu ced in 3T3-L1 preadipocytes during differentiation with different kinetics and potentially may regulate E2F/Rb family complexes. In pre-differentiated cells, three E2F complexes are observed: cdk2/EPF/p107, E2F/p130 and E2F4, cdk2/E2F/p107 complexes are induced in nuclear extracts of 3T3-L1 cells du ring mitotic expansion, but are not detectable in nuclear extracts at later stages of 3T3-L1 differentiation. The reduction in E2F/p107 complexes is a ssociated with elevation of C/EBP alpha, but is independent of C/EBP beta e xpression, Bacterially expressed, purified His-C/EBP alpha is able to disru pt E2F/p107 complexes that are observed at earlier stages of 3T3-L1 differe ntiation. C/EBP beta, however, does not disrupt E2F/p107 complexes, A short C/EBP alpha peptide with homology to E2F is sufficient to bring about the disruption of E2F/p107 complexes from 3T3-L1 cells in vitro, Induction of C /EBP alpha in stable 3T3-L1 clones revealed that C/EBP alpha causes disrupt ion of p107/E2F complexes in these cells. In contrast, E2F/p130 complexes a re induced in cells expressing C/EBP alpha. Our data suggest that induction of p130/E2F complexes by C/EBP alpha occurs via up-regulation of p21, whic h, in turn, leads to association with and inhibition of, cdk2 kinase activi ty. The reduction in cdk2 kinase activity correlates with alterations of p1 30 phosphorylation and with induction of p130/E2F complexes in 3T3-L1 stabl e clones. Our data suggest two pathways of C/EBP alpha-dependent regulation of E2F/Rb family complexes: disruption of S phase-specific E2F/p107 comple xes and induction of E2F/p130 complexes.