TY - JOUR
T1 - Dominant-negative C/EBP disrupts mitotic clonal expansion and differentiation of 3T3-L1 preadipocytes
AU - Zhang, Jiang Wen
AU - Tang, Qi Qun
AU - Vinson, Charles
AU - Lane, M. Daniel
PY - 2004/1
Y1 - 2004/1
N2 - 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.
AB - 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.
KW - 3T3-L1 adipocytes
KW - Adipogenesis
KW - Cell cycle
KW - Nuclear localization
KW - Obesity
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U2 - 10.1073/pnas.0307229101
DO - 10.1073/pnas.0307229101
M3 - Article
C2 - 14688407
AN - SCOPUS:0346458703
SN - 0027-8424
VL - 101
SP - 43
EP - 47
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 1
ER -