CASCADE REGULATION OF TERMINAL ADIPOCYTE DIFFERENTIATION BY 3 MEMBERSOF THE C EBP FAMILY OF LEUCINE-ZIPPER PROTEINS/

Terminal differentiation of cultured 3T3-L1 fibroblasts to the adipoge nic phenotype is potently stimulated by dexamethasone (DEX) and methyl isobutylxanthine (MIX). Previous studies have shown that these hormone s induce the expression of genes encoding two members of the CCAAT/enh ancer 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 expressio n. Optimal conditions for differentiation entail a 2-day period wherei n confluent fibroblasts are exposed to DEX and MIX, followed by remova l of the hormones and subsequent culture in the presence of insulin an d fetal bovine serum. During the early phase of differentiation, high levels of C/EBP delta and C/EBP beta accumulate. These transcription f actors diminish during the terminal phase of differentiation and come to be replaced by a third member of the C/EBP family, C/EBP alpha. Con clusive evidence has already shown that C/EBP alpha regulates terminal adipocyte differentiation, turning on the battery of fat-specific gen es 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 eff ects of the hormonal stimulants DEX and MIX in a cascade-like fashion, leading to the activation of the gene encoding C/EBP alpha. Condition s facilitating the precocious expression of either C/EBP delta or C/EB P beta were observed to accelerate adipogenesis and, in the case of C/ EBP beta, relieve dependence on the early hormonal stimulants. Likewis e, conditions that prevented the expression of functional C/EBP beta e ffectively blocked terminal differentiation. Finally, we have discover ed that ectopic expression of C/EBP beta in multipotential NIH-3T3 cel ls results in their conversion into committed adipoblasts capable, upo n hormonal stimulation, of synchronous and uniform differentiation int o fat-laden adipocytes.