Alam T, An M R, Mifflin R C, Hsieh C C, Ge X, Papaconstantinou J
Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston 77555-0643.
J Biol Chem. 1993 Jul 25;268(21):15681-8.
alpha 1-Acid glycoprotein (AGP) is a major acute phase protein synthesized primarily by the liver. The AGP gene is transcriptionally activated in hepatocytes during the acute phase response to bacterial lipopolysaccharide. In this study, we analyzed an acute phase responsive element (APRE) located between nucleotide residues -127 to -104 relative to the transcription initiation site of the mouse AGP gene. Binding studies show that several trans-acting factors interact with the APRE. Using monospecific antibodies we demonstrate that three isoforms of the CCAAT/enhancer-binding protein (C/EBP) family, namely C/EBP alpha, C/EBP beta, and C/EBP delta, bind to the APRE. Furthermore, with liver nuclear protein from control animals, C/EPB alpha is the predominant form that binds to the APRE, whereas with nuclear proteins from acute phase-induced animals, C/EBP alpha is replaced by C/EBP beta. The mechanism of activation of the AGP gene during the acute phase response appears to involve an exchange of C/EBP alpha by C/EBP beta. C/EBP delta does not play a role in this reaction. Interestingly, the C/EBP binding site of the APRE partially overlaps a functional glucocorticoid responsive element. We present evidence that both purified C/EBP alpha and glucocorticoid receptor bind strongly to the APRE. By site-specific mutation, we have identified the C/EBP and glucocorticoid receptor binding sites in the APRE. These mutants were used in expression vectors to demonstrate that both C/EBP and glucocorticoid receptor are essential for maximal response to interleukin-6 and dexamethasone. These results demonstrate that the APRE is a composite binding site for multiple factors that are responsible for the transcriptional control of the mouse AGP. Finally, functional analyses indicate that C/EBP alpha, C/EBP beta, and C/EBP delta are strong transcriptional trans-activators of the AGP APRE in hepatoma cells. These data suggest that the regulatory activity of the C/EBP with the APRE in the liver may require interactions with adjacent proteins.
α1-酸性糖蛋白（AGP）是一种主要由肝脏合成的急性期蛋白。在对细菌脂多糖的急性期反应过程中，AGP基因在肝细胞中被转录激活。在本研究中，我们分析了相对于小鼠AGP基因转录起始位点核苷酸残基-127至-104之间的急性期反应元件（APRE）。结合研究表明，几种反式作用因子与APRE相互作用。使用单特异性抗体，我们证明CCAAT/增强子结合蛋白（C/EBP）家族的三种异构体，即C/EBPα、C/EBPβ和C/EBPδ，与APRE结合。此外，对于来自对照动物的肝核蛋白，C/EPBα是与APRE结合的主要形式，而对于来自急性期诱导动物的核蛋白，C/EBPα被C/EBPβ取代。急性期反应期间AGP基因的激活机制似乎涉及C/EBPα被C/EBPβ替换。C/EBPδ在该反应中不起作用。有趣的是，APRE的C/EBP结合位点部分与功能性糖皮质激素反应元件重叠。我们提供的证据表明，纯化的C/EBPα和糖皮质激素受体都与APRE强烈结合。通过位点特异性突变，我们确定了APRE中的C/EBP和糖皮质激素受体结合位点。这些突变体被用于表达载体，以证明C/EBP和糖皮质激素受体对于对白介素-6和地塞米松的最大反应都是必不可少的。这些结果表明，APRE是多个因子的复合结合位点，这些因子负责小鼠AGP的转录控制。最后，功能分析表明，C/EBPα、C/EBPβ和C/EBPδ是肝癌细胞中AGP APRE的强转录反式激活因子。这些数据表明，C/EBP在肝脏中与APRE的调节活性可能需要与相邻蛋白相互作用。
