Wu Z, Xie Y, Bucher N L, Farmer S R
Department of Biochemistry, Boston University School of Medicine, Massachusetts 02118, USA.
Genes Dev. 1995 Oct 1;9(19):2350-63. doi: 10.1101/gad.9.19.2350.
Activation of adipogenesis in 3T3 preadipocytes by exposure to the adipogenic inducers dexamethasone, methylisobutylxanthine, insulin, and fetal bovine serum is accompanied by a transient burst of C/EBP beta protein expression that precedes the induction of the fat gene program. In this study we have investigated the role of C/EBP beta in initiating the adipogenic program by overexpressing C/EBP beta in multipotential NIH-3T3 fibroblasts. Conditional ectopic expression of C/EBP beta was accomplished by using an artificial transcriptional regulatory system based on the Escherichia coli tetracycline repressor to generate a stable 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 dexamethasone in the presence of insulin and fetal bovine serum activates the expression of an adipocyte-specific nuclear hormone receptor, PPAR gamma, that stimulates the conversion of these fibroblasts into committed preadipocytes. Either ectopic expression of C/EBP beta or treatment with dexamethasone alone is incapable of inducing PPAR gamma expression, but when present together, they have a synergistic effect on the adipogenic program. Exposure of these stimulated cells to a PPAR activator 5,8,11,14-eicosatetraynoic acid (ETYA) results in the accumulation of fat droplets and expression of the adipocyte-enriched genes aP2 and glycerol phosphate dehydrogenase (GPD). The number of beta 2 cells that can differentiate into adipocytes is related to the concentration of tetracycline and, therefore, the amount of the exogenous C/EBP beta protein expressed. C/EBP beta can induce PPAR gamma mRNA in the absence of ETYA; however, expression of aP2 mRNA and maximum fat deposition is dependent on the PPAR activator. Our results suggest that enhanced expression of C/EBP beta converts multipotential mesenchymal precursor cells into preadipocytes that respond to adipogenic inducers, including dexamethasone and PPAR activators to differentiate into adipocytes.
通过暴露于成脂诱导剂地塞米松、甲基异丁基黄嘌呤、胰岛素和胎牛血清，3T3前脂肪细胞中的脂肪生成被激活，同时伴随着C/EBPβ蛋白表达的短暂爆发，这一爆发先于脂肪基因程序的诱导。在本研究中，我们通过在多能NIH-3T3成纤维细胞中过表达C/EBPβ，研究了C/EBPβ在启动脂肪生成程序中的作用。通过使用基于大肠杆菌四环素阻遏物的人工转录调控系统来实现C/EBPβ的条件性异位表达，从而产生一个稳定的细胞系β2，该细胞系以严格受控的四环素剂量依赖性方式表达C/EBPβ mRNA和蛋白。在胰岛素和胎牛血清存在的情况下，将NIH-3T3 β2细胞暴露于地塞米松中诱导C/EBPβ DNA结合活性，可激活脂肪细胞特异性核激素受体PPARγ的表达，该受体刺激这些成纤维细胞转化为定型前脂肪细胞。单独异位表达C/EBPβ或用地塞米松处理均不能诱导PPARγ表达，但当两者同时存在时，它们对脂肪生成程序具有协同作用。将这些受刺激的细胞暴露于PPAR激活剂5,8,11,14-二十碳四烯酸（ETYA）中，会导致脂滴积累以及富含脂肪细胞的基因aP2和甘油磷酸脱氢酶（GPD）的表达。能够分化为脂肪细胞的β2细胞数量与四环素浓度相关，因此也与外源表达的C/EBPβ蛋白量相关。在没有ETYA的情况下，C/EBPβ可诱导PPARγ mRNA；然而，aP2 mRNA的表达和最大脂肪沉积依赖于PPAR激活剂。我们的结果表明，C/EBPβ表达增强可将多能间充质前体细胞转化为对包括地塞米松和PPAR激活剂在内的成脂诱导剂有反应的前脂肪细胞，进而分化为脂肪细胞。
