Wu Z, Bucher N L, Farmer S R
Department of Biochemistry, Boston University School of Medicine, Massachusetts 02118, USA.
Mol Cell Biol. 1996 Aug;16(8):4128-36. doi: 10.1128/MCB.16.8.4128.
The differentiation of 3T3 preadipocytes into adipocytes is accompanied by a transient induction of C/EBPbeta and C/EBPdelta 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 gamma (PPARgamma) expression in 3T3-L1 preadipocytes is induced by MIX and DEX, suggesting that C/EBPbeta and C/EBPdelta may be involved in this process. Using a tetracycline-responsive expression system, we have recently shown that the conditional ectopic expression of C/EBPbeta in NIH 3T3 fibroblasts (beta2 cells) in the presence of DEX activates the synthesis of peroxisome PPARgamma mRNA. Subsequent exposure of these cells to PPAR activators stimulates their conversion into adipocytes; however, neither the expression of C/EBPbeta nor exposure to DEX alone is capable of inducing PPARgamma expression in the beta2 cell line. We find that unlike the case for 3T3 preadipocytes, C/EBPdelta is not induced by DEX in these 3T3 fibroblasts and therefore is not relaying the effect of this glucocorticoid to the PPARgamma gene. To define the role of glucocorticoids in regulating PPARgamma expression and the possible involvement of C/EBPdelta, we have established an additional set of NIH 3T3 cell lines expressing either C/EBPdelta alone (delta23 cells) or C/EBPdelta and C/EBPbeta together (beta/delta39 cells), using the tetracycline-responsive system. Culture of these cells in tetracycline-deficient medium containing DEX, MIX, insulin, and fetal bovine serum shows that the beta/delta39 cells express PPARgamma 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 beta2 cells. Despite the fact that these beta/delta39 cells produce abundant amounts of C/EBPbeta and C/EBPdelta (in the absence of tetracycline), they still require glucocorticoids to attain maximum expression of PPARgamma mRNA. Furthermore, the induction of PPARgamma mRNA by exposure of these cells to DEX occurs in the absence of ongoing protein synthesis. The delta23 cells, on the other hand, are not capable of activating PPARgamma gene expression when exposed to the same adipogenic inducers. Finally, attenuation of ectopic C/EBPbeta production at various stages during the differentiation process results in a concomitant inhibition of PPARgamma and the adipogenic program. These data strongly suggest that the induction of PPARgamma gene expression in multipotential mesenchymal stem cells (NIH 3T3 fibroblasts) is dependent on elevated levels of C/EBPbeta throughout the differentiation process, as well as an initial exposure to glucocorticoids. C/EBPdelta may function by synergizing with C/EBPbeta to enhance the level of PPARgamma expression.
3T3前脂肪细胞向脂肪细胞的分化伴随着C/EBPβ和C/EBPδ表达的短暂诱导,这分别是细胞用甲基异丁基黄嘌呤(MIX)和地塞米松(DEX)处理后的反应。在本报告中,我们证明3T3-L1前脂肪细胞中过氧化物酶体增殖物激活受体γ(PPARγ)的表达由MIX和DEX诱导,这表明C/EBPβ和C/EBPδ可能参与了这一过程。使用四环素反应性表达系统,我们最近发现,在DEX存在的情况下,NIH 3T3成纤维细胞(β2细胞)中C/EBPβ的条件性异位表达激活了过氧化物酶体PPARγ mRNA的合成。随后将这些细胞暴露于PPAR激活剂中会刺激它们转化为脂肪细胞;然而,单独的C/EBPβ表达或DEX暴露都不能在β2细胞系中诱导PPARγ表达。我们发现,与3T3前脂肪细胞不同,在这些3T3成纤维细胞中,C/EBPδ不会被DEX诱导,因此不会将这种糖皮质激素的作用传递给PPARγ基因。为了确定糖皮质激素在调节PPARγ表达中的作用以及C/EBPδ可能的参与情况,我们使用四环素反应系统建立了另一组NIH 3T3细胞系,这些细胞系分别单独表达C/EBPδ(δ23细胞)或同时表达C/EBPδ和C/EBPβ(β/δ39细胞)。在含有DEX、MIX、胰岛素和胎牛血清的四环素缺乏培养基中培养这些细胞,结果显示β/δ39细胞中PPARγ和aP2 mRNA的表达水平几乎与在完全分化的3T3-L1脂肪细胞中观察到的水平相当。这些水平比它们在β2细胞中的表达水平高约三倍。尽管这些β/δ39细胞(在没有四环素的情况下)产生大量的C/EBPβ和C/EBPδ,但它们仍然需要糖皮质激素来实现PPARγ mRNA的最大表达。此外,将这些细胞暴露于DEX诱导PPARγ mRNA的过程中并不需要持续的蛋白质合成。另一方面,δ23细胞在暴露于相同的成脂诱导剂时不能激活PPARγ基因表达。最后,在分化过程的各个阶段异位C/EBPβ产生的减弱会导致PPARγ和脂肪生成程序的相应抑制。这些数据强烈表明,多能间充质干细胞(NIH 3T3成纤维细胞)中PPARγ基因表达的诱导在整个分化过程中依赖于C/EBPβ水平的升高,以及最初对糖皮质激素的暴露。C/EBPδ可能通过与C/EBPβ协同作用来增强PPARγ表达水平而发挥作用。