Hamm J K, Park B H, Farmer S R
Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 2118, USA.
J Biol Chem. 2001 May 25;276(21):18464-71. doi: 10.1074/jbc.M100797200. Epub 2001 Feb 27.
The differentiation of 3T3-L1 preadipocytes is regulated in part by a cascade of transcriptional events involving activation of the CCAAT/enhancer-binding proteins (C/EBPs) and peroxisome proliferator-activated receptor gamma (PPARgamma) by dexamethasone (DEX), 3-isobutyl-1-methylxanthine (MIX), and insulin. In this study, we demonstrate that exposure of 3T3-L1 preadipocytes to DEX and insulin fails to induce adipogenesis as indicated by a lack of C/EBPalpha, PPARgamma2, and adipose protein 2/fatty acid-binding protein expression; however, PPARgamma1 is expressed. Treatment of these MIX-deficient cells with a PPARgamma ligand, troglitazone, induces C/EBPalpha expression and rescues the block in adipogenesis. In this regard, we also show that induction of C/EBPalpha gene expression by troglitazone in C3H10T1/2 cells ectopically expressing PPARgamma occurs in the absence of ongoing protein synthesis, suggesting a direct transactivation of the C/EBPalpha gene by PPARgamma. Furthermore, ectopic expression of a dominant negative isoform of C/EBPbeta (liver-enriched transcriptional inhibitory protein (LIP)) inhibits the induction of C/EBPalpha, PPARgamma2, and adipose protein 2/fatty acid-binding protein by DEX, MIX, and insulin in 3T3-L1 cells without affecting the induction of PPARgamma1 by DEX. Exposure of LIP-expressing preadipocytes to troglitazone along with DEX, MIX, and insulin induces differentiation into adipocytes. Additionally, we show that sustained expression of C/EBPalpha in these LIP-expressing adipocytes requires constant exposure to troglitazone. Taken together, these observations suggest that inhibition of C/EBPbeta activity not only blocks C/EBPalpha and PPARgamma2 expression, but it also renders the preadipocytes dependent on an exogenous PPARgamma ligand for their differentiation into adipocytes. We propose, therefore, an additional role for C/EBPbeta in regulating PPARgamma activity during adipogenesis, and we suggest an alternative means of inducing preadipocyte differentiation that relies on the dexamethasone-associated induction of PPARgamma1 expression.
3T3-L1前脂肪细胞的分化部分受一系列转录事件调控，这些事件包括地塞米松（DEX）、3-异丁基-1-甲基黄嘌呤（MIX）和胰岛素激活CCAAT/增强子结合蛋白（C/EBP）和过氧化物酶体增殖物激活受体γ（PPARγ）。在本研究中，我们证明，将3T3-L1前脂肪细胞暴露于DEX和胰岛素下无法诱导脂肪生成，这表现为缺乏C/EBPα、PPARγ2和脂肪蛋白2/脂肪酸结合蛋白的表达；然而，PPARγ1却有表达。用PPARγ配体曲格列酮处理这些缺乏MIX的细胞，可诱导C/EBPα表达并挽救脂肪生成的阻滞。在这方面，我们还表明，在异位表达PPARγ的C3H10T1/2细胞中，曲格列酮诱导C/EBPα基因表达是在没有持续蛋白质合成的情况下发生的，这表明PPARγ可直接反式激活C/EBPα基因。此外，C/EBPβ的显性负性异构体（肝脏富集转录抑制蛋白（LIP））的异位表达可抑制DEX、MIX和胰岛素在3T3-L1细胞中诱导C/EBPα、PPARγ2和脂肪蛋白2/脂肪酸结合蛋白的表达，但不影响DEX诱导PPARγ1的表达。将表达LIP的前脂肪细胞暴露于曲格列酮以及DEX、MIX和胰岛素下可诱导其分化为脂肪细胞。此外，我们表明，在这些表达LIP的脂肪细胞中，C/EBPα的持续表达需要持续暴露于曲格列酮。综上所述，这些观察结果表明，抑制C/EBPβ活性不仅会阻断C/EBPα和PPARγ2的表达，还会使前脂肪细胞依赖外源性PPARγ配体来分化为脂肪细胞。因此，我们提出C/EBPβ在脂肪生成过程中调节PPARγ活性的另一个作用，并且我们提出了一种诱导前脂肪细胞分化的替代方法，该方法依赖于地塞米松相关的PPARγ1表达诱导。
