CCAAT/enhancer-binding protein β is required for mitotic clonal expansion during adipogenesis

doi:10.1073/pnas.0337434100

CCAAT/enhancer-binding protein β is required for mitotic clonal expansion during adipogenesis

Departments of ^*Biological Chemistry and ^†Pediatrics (Division of Endocrinology), Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205

Contributed by M. Daniel Lane

Abstract

Hormonal induction of growth-arrested 3T3-L1 preadipocytes triggers a signaling cascade that culminates in adipogenesis. CCAAT/enhancer-binding protein (C/EBP)β is expressed immediately but gains DNA-binding activity only after a long lag as the cells synchronously begin mitotic clonal expansion (MCE). After MCE, a process required for adipogenesis, C/EBPβ activates expression of C/EBPα and peroxisome proliferator-activated receptor γ, which then transcriptionally activate genes that produce the adipocyte phenotype. When mouse embryo fibroblasts (MEFs) are subjected to the same differentiation protocol, a subset of the MEFs undergoes a similar program of events. Similar to 3T3-L1 preadipocytes, the MEFs reenter the cell cycle (as indicated by the synchronous expression of cyclin A) and undergo MCE as evidenced by the incorporation of BrdUrd into DNA and the formation of mitotic foci of cells that undergo adipogenesis. C/EBPβ is expressed immediately after induction but exhibits delayed acquisition of DNA-binding activity followed by expression of adipocyte markers and the accumulation of cytoplasmic triglyceride. MEFs from C/EBPβ(−/−) mice, however, neither undergo MCE nor differentiate into adipocytes. Forced expression of C/EBPβ (LAP) but not dominant-negative C/EBPβ (LIP) in C/EBPβ(−/−) MEFs restores MCE, expression of adipocyte markers, and the capacity to form mitotic foci of cells that undergo adipogenesis. These findings demonstrate that expression of C/EBPβ is a prerequisite for MCE in the adipocyte-differentiation program.

3T3-L1 preadipocyte‖mouse embryo fibroblasts‖C/EBPα‖PPARγ‖ cell cycle

Footnotes

↵ ‡ To whom correspondence should be addressed at: Department of Biological Chemistry, 512 Wood Basic Science Building, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205. E-mail: qtang1{at}jhem.jhmi.edu.
↵ § Although PPARγ1 is expressed both by cells infected with the control/empty virus or the C/EBPβ (LAP)-containing virus, only cells infected with the latter virus express PPARγ2. This is likely because of the presence of tandem C/EBP regulatory elements in the proximal promoter of the PPARγ2 gene, through which C/EBPβ (LAP) can transactivate the gene (10), whereas the proximal promoter of the PPARγ1 gene does not possess these elements in its proximal promoter (22).
Abbreviations:

MCE,

mitotic clonal expansion;

C/EBP,

CCAAT/enhancer-binding protein;

PPARγ,

peroxisome proliferator-activated receptor γ;

MEF,

mouse embryo fibroblast;

wt,

wild type;

aP2,

adipocyte P2 protein