Induction of Peroxisome Proliferator-Activated Receptor γ during the Conversion of 3T3 Fibroblasts into Adipocytes Is Mediated by C/EBPβ, C/EBPδ, and Glucocorticoids

Abstract

The differentiation of 3T3 preadipocytes into adipocytes is accompanied by a transient induction of C/EBPβ and C/EBPδ expression in response to treatment of the cells with methylisobutylxanthine (MIX) and dexamethasone (DEX), respectively. In this report, we demonstrate that peroxisome proliferator-activated receptor γ (PPARγ) expression in 3T3-L1 preadipocytes is induced by MIX and DEX, suggesting that C/EBPβ and C/EBPδ may be involved in this process. Using a tetracycline-responsive expression system, we have recently shown that the conditional ectopic expression of C/EBPβ in NIH 3T3 fibroblasts (b2 cells) in the presence of DEX activates the synthesis of peroxisome PPARγ mRNA. Subsequent exposure of these cells to PPAR activators stimulates their conversion into adipocytes; however, neither the expression of C/EBPβ nor exposure to DEX alone is capable of inducing PPARγ expression in the β2 cell line. We find that unlike the case for 3T3 preadipocytes, C/EBPδ is not induced by DEX in these 3T3 fibroblasts and therefore is not relaying the effect of this glucocorticoid to the PPARγ gene. To define the role of glucocorticoids in regulating PPARγ expression and the possible involvement of C/EBPδ, we have established an additional set of NIH 3T3 cell lines expressing either C/EBPδ alone (δ23 cells) or C/EBPδ and C/EBPβ together (β/δ39 cells), using the tetracyclineresponsive system. Culture of these cells in tetracycline-deficient medium containing DEX, MIX, insulin, and fetal bovine serum shows that the β/δ39 cells express PPARγ and aP2 mRNAs at levels that are almost equivalent to those observed in fully differentiated 3T3-L1 adipocytes. These levels are approximately threefold higher than their levels of expression in the β2 cells. Despite the fact that these β/δ39 cells produce abundant amounts of C/EBPβ and C/EBPδ (in the absence of tetracycline), they still require glucocorticoids to attain maximum expression of PPARγ mRNA. Furthermore, the induction of PPARγ mRNA by exposure of these cells to DEX occurs in the absence of ongoing protein synthesis. The δ23 cells, on the other hand, are not capable of activating PPARγ gene expression when exposed to the same adipogenic inducers. Finally, attenuation of ectopic C/EBPβ production at various stages during the differentiation process results in a concomitant inhibition of PPARγ and the adipogenic program. These data strongly suggest that the induction of PPARγ gene expression in multipotential mesenchymal stem cells (NIH 3T3 fibroblasts) is dependent on elevated levels of C/EBPβ throughout the differentiation process, as well as an initial exposure to glucocorticoids. C/EBPδ may function by synergizing with C/EBPβ to enhance the level of PPARγ expression.