CONDITIONAL ECTOPIC EXPRESSION OF C EBP-BETA IN NIH-3T3 CELLS INDUCESPPAR-GAMMA AND STIMULATES ADIPOGENESIS/

Activation of adipogenesis in 3T3 preadipocytes by exposure to the adi pogenic inducers dexamethasone, methylisobutylxanthine, insulin, and f etal bovine serum is accompanied by a transient burst of C/EBP beta pr otein expression that precedes the induction of the fat gene program. In this study we have investigated the role of C/EBP beta in initiatin g the adipogenic program by overexpressing C/EBP beta in multipotentia l NIH-3T3 fibroblasts. Conditional ectopic expression of C/EBP beta wa s accomplished by using an artificial transcriptional regulatory syste m based on the Escherichia coli tetracycline repressor to generate a s table cell line, beta 2, that expresses C/EBP beta mRNA and protein in a tightly controlled tetracycline dose-dependent manner. Induction of C/EBP beta DNA-binding activity in NIH-3T3 beta 2 cells exposed to de xamethasone in the presence of insulin and fetal bovine serum activate s the expression of an adipocyte-specific nuclear hormone receptor, PP AR gamma, that stimulates the conversion of these fibroblasts into com mitted preadipocytes. Either ectopic expression of C/EBP beta or treat ment with dexamethasone alone is incapable of inducing PPAR gamma expr ession, but when present together, they have a synergistic effect on t he adipogenic program. Exposure of these stimulated cells to a PPAR ac tivator 5,8,11,14-eicosatetraynoic acid (ETYA) results in the accumula tion of fat droplets and expression of the adipocyte-enriched genes aP 2 and glycerol phosphate dehydrogenase (GPD). The number of beta 2 cel ls that can differentiate into adipocytes is related to the concentrat ion of tetracycline and, therefore, the amount of the exogenous C/EBP beta protein expressed. C/EBP beta can induce PPAR gamma mRNA in the a bsence of ETYA; however, expression of aP2 mRNA and maximum fat deposi tion is dependent on the PPAR activator. Our results suggest that enha nced expression of C/EBP beta converts multipotential mesenchymal prec ursor cells into preadipocytes that respond to adipogenic inducers, in cluding dexamethasone and PPAR activators to differentiate into adipoc ytes.