Dominant-negative C/EBP disrupts mitotic clonal expansion and differentiation of 3T3-L1 preadipocytes

Jiang Wen Zhang, Qi Qun Tang, Charles Vinson, M. Daniel Lane

Research output: Contribution to journalArticlepeer-review

Abstract

Hormonal induction of growth-arrested 3T3-L1 preadipocytes rapidly activates expression of CCAAT/enhancer-binding protein (C/EBP) β. Acquisition of DNA-binding activity by C/EBPβ, however, is delayed until the cells synchronously enter the S phase of mitotic clonal expansion (MCE). After MCE, C/EBPβ activates expression of C/EBPα and peroxisome proliferator-activated receptor γ, which then transcriptionally activate genes that give rise to the adipocyte phenotype. A-C/EBP, which possesses a leucine zipper but lacks functional DNA-binding and transactivation domains, forms stable inactive heterodimers with C/EBPβ in vitro. Infection of 3T3-L1 preadipocytes with an adenovirus A-C/EBP expression vector interferes with C/EBPβ function after induction of differentiation. A-C/EBP inhibited events associated with hormone-induced entry of S-phase of the cell cycle, including the turnover of p27/Kip1, a key cyclin-dependent kinase inhibitor, expression of cyclin A and cyclin-dependent kinase 2, DNA replication, MCE, and, subsequently, adipogenesis. Although A-C/EBP blocked cell proliferation associated with MCE, it did not inhibit normal proliferation of 3T3-L1 preadipocytes. Immunofluorescent staining of C/EBPβ revealed that A-C/EBP prevented the normal punctate nuclear staining of centromeres, an indicator of C/EBPβ binding to C/EBP regulatory elements in centromeric satellite DNA. The inhibitory effects of A-C/EBP appear to be due primarily to interference with nuclear import of C/EBPβ caused by obscuring its nuclear localization signal. These findings show that both MCE and adipogenesis are dependent on C/EBPβ.

Original languageEnglish (US)
Pages (from-to)43-47
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume101
Issue number1
DOIs
StatePublished - Jan 2004

Keywords

  • 3T3-L1 adipocytes
  • Adipogenesis
  • Cell cycle
  • Nuclear localization
  • Obesity

ASJC Scopus subject areas

  • Genetics
  • General

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