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

Abstract

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/EBPβ interaction. Cell cycle arrest in G1 by l-mimosine inhibited differentiation-induced C/EBPβ-DNA-binding activity and Rb phosphorylation. However, cell cycle arrest after the G1/S transition by aphidicolin or nocodazole did not prevent C/EBPβ-DNA-binding activity or Rb phosphorylation. Furthermore, hypophosphorylated Rb and C/EBPβ coimmunoprecipitated, whereas phosphorylated Rb and C/EBPβ did not. Electrophoretic mobility shift assays demonstrated that recombinant hypophosphorylated Rb decreased C/EBPβ-DNA-binding activity and that Rb overexpression inhibited C/EBPβ-induced transcriptional activation of a C/EBPα-promoter-luciferase reporter gene. We conclude that C/EBPβ-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.

ETO/MTG8 Is an Inhibitor of C/EBPβ Activity and a Regulator of Early Adipogenesis

“…We believe that this is the first assessment of these early time points of differentiation by using this method. However, the data agree well with previously published time courses assessed by gel shift analysis with nuclear extracts from 3T3-L1 preadipocytes (8), which has demonstrated strong binding of C/EBP␤ to the C/EBP␣ promoter within 12 h of differentiation. Subsequent analysis of C/EBP␤ binding to the C/EBP␣ promoter in cells constitutively expressing ETO revealed that C/EBP␤ binding activity was significantly reduced in these cells compared to mock-transfected cells (Fig.…”

Prevention of CCAAT/Enhancer-binding Protein β DNA Binding by Hypoxia during Adipogenesis

“…Normal embryonic fibroblasts from lung buds differentiate into adipocytes in response to hormonal induction; however, embryonic fibroblasts deficient in Rb do not, indicating that Rb is essential for adipogenesis (47). The interaction between Rb and C/EBP␤ occurred only in differentiating cells, and interaction with Rb appeared to inhibit the binding of C/EBP␤ to cognate DNA sequences in vitro (48). Forced expression of C/EBP␤ inhibited the proliferation of wild type MEFs but not of MEFs lacking all three Rb family proteins, suggesting that C/EBP␤ represses genes required for cell cycle progression through interaction with Rb proteins (49).…”

The retinoblastoma gene is involved in multiple aspects of stem cell biology

“…Adipocyte differentiation is a well-defined process that can be recapitulated with murine 3T3 mesenchymal-like stem cells as a model system (Sparks et al, 1986;Cole et al, 2004). The differentiation process can be divided into three steps: (1) growth arrest; (2) cell commitment and reversible differentiation; (3) terminal differentiation (Wang and Scott, 1993).…”