Cascade regulation of terminal adipocyte differentiation by three members of the C/EBP family of leucine zipper proteins.

Abstract

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/EBP delta. Likewise, MIX is a direct inducer of C/EBP beta 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/EBP delta and C/EBP beta 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/EBP alpha. Conclusive evidence has already shown that C/EBP alpha 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/EBP delta and C/EBP beta 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/EBP alpha. Conditions facilitating the precocious expression of either C/EBP delta or C/EBP beta were observed to accelerate adipogenesis and, in the case of C/EBP beta, relieve dependence on the early hormonal stimulants. Likewise, conditions that prevented the expression of functional C/EBP beta effectively blocked terminal differentiation. Finally, we have discovered that ectopic expression of C/EBP beta 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.