Rb regulates C/EBP -DNA-binding activity during 3T3-L1 adipogenesis

Cole, Kathryn A., Anne W. Harmon, Joyce B. Harp, and Yashomati M. Patel. Rb regulates C/EBP -DNA-binding activity during 3T3-L1 adipogenesis. Am J Physiol Cell Physiol 286: C349–C354, 2004. First published October 22, 2003; 10.1152/ajpcell.00255.2003.—Two pathways are initiated upon 3T3-L1 preadipocyte differentiation: the reentry of cells into the cell cycle and the initiation of a cascade of transcriptional events that “prime” the cell for differentiation. The “priming” event involves the synthesis of members of the CCAAT/enhancer binding protein (C/EBP) family of transcription factors. However, the relationship between these two pathways is unknown. Here we report that in the 3T3-L1 preadipocytes induced to differentiate, cell cycle progression and the initiation of differentiation are linked by a cell cycle-dependent Rb-C/EBP interaction. Cell cycle arrest in G1 by L-mimosine inhibited differentiation-induced C/EBP -DNA-binding activity and Rb phosphorylation. However, cell cycle arrest after the G1/S transition by aphidicolin or nocodazole did not prevent C/EBP -DNA-binding activity or Rb phosphorylation. Furthermore, hypophosphorylated Rb and C/EBP coimmunoprecipitated, whereas phosphorylated Rb and C/EBP did not. Electrophoretic mobility shift assays demonstrated that recombinant hypophosphorylated Rb decreased C/EBP -DNA-binding activity and that Rb overexpression inhibited C/EBP -induced transcriptional activation of a C/EBP -promoter-luciferase reporter gene. We conclude that C/EBP -DNA-binding activity is regulated by its interaction with hypophosphorylated Rb, thereby linking the progression of the cell cycle to the initiation of differentiation during 3T3-L1 adipogenesis.