Dominant-negative C/EBP disrupts mitotic clonal expansion and differentiation of 3T3-L1 preadipocytes.
Hormonal induction of growth-arrested 3T3-L1 preadipocytes rapidly activates expression of CCAAT/enhancer-binding protein (C/EBP) beta. Acquisition of DNA-binding activity by C/EBPbeta, however, is delayed until the cells synchronously enter the S phase of mitotic clonal expansion (MCE). After MCE, C/EBPbeta activates expression of C/EBPalpha and peroxisome proliferator-activated receptor gamma, 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/EBPbeta in vitro. Infection of 3T3-L1 preadipocytes with an adenovirus A-C/EBP expression vector interferes with C/EBPbeta 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/EBPbeta revealed that A-C/EBP prevented the normal punctate nuclear staining of centromeres, an indicator of C/EBPbeta 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/EBPbeta caused by obscuring its nuclear localization signal. These findings show that both MCE and adipogenesis are dependent on C/EBPbeta.
Zhang
Jiang-Wen
JW
Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
Tang
Qi-Qun
QQ
Vinson
Charles
C
Lane
M Daniel
MD
D041721:3T3-L1 Cells; D000256:Adenoviridae / Q000235:genetics; D017667:Adipocytes / Q000166:cytology* / Q000378:metabolism*; D000818:Animals; D022782:CCAAT-Enhancer-Binding Protein-beta / Q000737:chemistry / Q000378:metabolism; D022762:CCAAT-Enhancer-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002453:Cell Cycle; D002454:Cell Differentiation; D002467:Cell Nucleus / Q000378:metabolism; D002503:Centromere / Q000378:metabolism; D005822:Genetic Vectors; D046911:Macromolecular Substances; D051379:Mice; D008938:Mitosis; D019913:Nuclear Localization Signals / Q000235:genetics; D013234:Stem Cells / Q000166:cytology / Q000378:metabolism
D022782:CCAAT-Enhancer-Binding Protein-beta; D022762:CCAAT-Enhancer-Binding Proteins; D046911:Macromolecular Substances; D019913:Nuclear Localization Signals