Cram E J, Ramos R A, Wang E C, Cha H H, Nishio Y, Firestone G L
Department of Molecular and Cell Biology, University of California, Berkeley 94720, USA.
J Biol Chem. 1998 Jan 23;273(4):2008-14. doi: 10.1074/jbc.273.4.2008.
The preceding paper (Cha, H. H., Cram, E. J., Wang, E. C., Huang, A. J., Kasler, H. G., and Firestone, G. L. (1998) J. Biol. Chem. 273, 0000-0000(478563) defined a glucocorticoid responsive region within teh promoter of the p21 CDK inhibitor gene that contains a putative DNA-binding site for the transcription factor CCAAT/ enhancer binding protein-alpha (C/EBP alpha). Wild type rat BDS1 hepatoma cells as well as as4 hepatoma cells, which express antisense sequences to C/EBP alpha and ablate its protein production, were utilized to investigate the role of this transcription factor in the glucocorticoid regulation of p21 gene expression. The stimulation of p21 protein levels and promoter activity, as well as inhibition of CDK2-mediated retinoblastoma protein phosphorylation, by the synthetic glucocorticoid, dexamethasone, required the expression of C/EBP alpha. Overexpression of C/EBP alpha in as4 cells rescued the dexamethasone responsiveness of the p21 promoter. Site-directed mutagenesis of the p21 promoter revealed that dexamethasone stimulation of p21 promoter activity required the C/EBP consensus DNA-binding site. Furthermore, in glucocorticoid receptor-defective EDR1 hepatoma cells, dexamethasone failed to stimulate C/EBP alpha and p21 protein expression and promoter activities. Our results have established a functional link between the glucocorticoid receptor signaling pathway that mediates a G1 cell cycle arrest of rat hepatoma cells and the transcriptional control of p21 by a cascade that requires the steroid induction of C/EBP alpha gene expression.
前文（Cha, H. H., Cram, E. J., Wang, E. C., Huang, A. J., Kasler, H. G., and Firestone, G. L. (1998) J. Biol. Chem. 273, 0000 - 0000(478563)）确定了p21细胞周期蛋白依赖性激酶抑制剂基因启动子内的一个糖皮质激素反应区域，该区域含有转录因子CCAAT/增强子结合蛋白α（C/EBPα）的一个假定DNA结合位点。利用野生型大鼠BDS1肝癌细胞以及as4肝癌细胞（其表达C/EBPα的反义序列并消除其蛋白质产生）来研究该转录因子在糖皮质激素调节p21基因表达中的作用。合成糖皮质激素地塞米松对p21蛋白水平和启动子活性的刺激以及对CDK2介导的视网膜母细胞瘤蛋白磷酸化的抑制需要C/EBPα的表达。在as4细胞中过表达C/EBPα可挽救p21启动子对地塞米松的反应性。p21启动子的定点诱变表明，地塞米松对p21启动子活性的刺激需要C/EBP共有DNA结合位点。此外，在糖皮质激素受体缺陷的EDR1肝癌细胞中，地塞米松未能刺激C/EBPα和p21蛋白表达及启动子活性。我们的结果在介导大鼠肝癌细胞G1细胞周期停滞的糖皮质激素受体信号通路与p21的转录控制之间建立了功能联系，该转录控制由一个需要类固醇诱导C/EBPα基因表达的级联反应介导。
