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

Upon exposure to adipogenesis-inducing hormones, confluent 3T3-L1 preadipocytes express C/EBP beta (CCAAT/enhancer binding protein beta). Early induced C/EBP beta is inactive but, after a lag period, acquires its DNA-binding capability by sequential phosphorylation. During this period, preadipocytes pass the G(1)/S checkpoint synchronously. Thr(188) of C/EBP beta is phosphorylated initially to prime the factor for subsequent phosphorylation at Ser(184) or Thr(179) by GSK3 beta, which translocates into the nuclei during the G(1)/S transition. Many events take place during the G(1)/S transition, including reduction in p27(Kip1) protein levels, retinoblastoma (Rb) phosphorylation, GSK3 beta nuclear translocation, and C/EBP beta binding to target promoters. During hypoxia, hypoxia-inducible factor-1 alpha (HIF-1 alpha) is stabilized, thus maintaining expression of p27(Kip1), which inhibits Rb phosphorylation. Even under normoxic conditions, constitutive expression of p27(Kip1) blocks the nuclear translocation of GSK3 beta and DNA binding capability of C/EBP beta. Hypoxia also blocks nuclear translocation of GSK3 beta and DNA binding capability of C/EBP beta in HIF-1 alpha knockdown 3T3-L1 cells that fail to induce p27(Kip1). Nonetheless, under hypoxia, these cells can block Rb phosphorylation and the G(1)/S transition. Altogether, these findings suggest that hypoxia prevents the nuclear translocation of GSK3 beta and the DNA binding capability of C/EBP beta by blocking the G(1)/S transition through HIF-1 alpha-dependent induction of p27(Kip1) and an HIF-1 alpha/p27-independent mechanism.