Terminal differentiation of cultured 3T3-L1 fibroblasts to the adipogenic phenotype is potently stimulated by dexamethasone (DEX) and methylisobutylxanthine (MIX). Previous studies have shown that these hormones induce the expression of genes encoding two members of the CCAAT/enhancer binding protein (C/EBP) family of transcription factors. In the absence of new protein synthesis DEX activates the gene encoding C/EBPδ. Likewise, MIX is a direct inducer of C/EBPβ gene expression. Optimal conditions for differentiation entail a 2-day period wherein confluent fibroblasts are exposed to DEX and MIX, followed by removal of the hormones and subsequent culture in the presence of insulin and fetal bovine serum. During the early phase of differentiation, high levels of C/EBPδ and C/EBPβ accumulate. These transcription factors diminish during the terminal phase of differentiation and come to be replaced by a third member of the C/EBP family, C/EBPα. Conclusive evidence has already shown that C/EBPα regulates terminal adipocyte differentiation, turning on the battery of fat-specific genes required for the synthesis, uptake, and storage of long chain fatty acids. Here we provide evidence that C/EBPδ and C/EBPβ play early catalytic roles in the differentiation pathway, relaying the effects of the hormonal stimulants DEX and MIX in a cascade-like fashion, leading to the activation of the gene encoding C/EBPα. Conditions facilitating the precocious expression of either C/EBPδ or C/EBPβ were observed to accelerate adipogenesis and, in the case of C/EBPβ, relieve dependence on the early hormonal stimulants. Likewise, conditions that prevented the expression of functional C/EBPβ effectively blocked terminal differentiation. Finally, we have discovered that ectopic expression of C/EBPβ in multipotential NIH-3T3 cells results in their conversion into committed adipoblasts capable, upon hormonal stimulation, of synchronous and uniform differentiation into fat- laden adipocytes.
ASJC Scopus subject areas
- Developmental Biology