C/EBPbeta contributes to cAMP-activated transcription of phosphoenolpyruvate carboxykinase in LLC-PK(1)-F+ cells.
C/EBPbeta contributes to cAMP-activated transcription of phosphoenolpyruvate carboxykinase in LLC-PK(1)-F+ cells.
复制标题
C/EBPbeta 有助于 LLC-PK(1)-F 细胞中磷酸烯醇丙酮酸羧激酶的 cAMP 激活转录。
DOI：
10.1152/ajprenal.2001.281.4.f649
复制
发表时间：
2001
期刊：
影响因子：
--
通讯作者：
Curthoys,NP
中科院分区：
文献类型：
--
作者：
Liu,X;Wall,QT;Taylor,L;Curthoys,NP
磷酸烯醇丙酮酸羧激酶（PEPCK）是肾脏糖异生的关键调控酶。在猪肾近端小管样细胞的糖异生系LLC-PK1-F+细胞中，蛋白激酶a （PKA）通过PEPCK启动子的camp反应元件(CRE)-1介导多种PEPCK−2300Luc报告基因的激活。含有寡核苷酸的cre1与lc - pk1 - f +细胞的核提取物孵育产生多个条带，所有这些条带都被C/EBPβ特异性抗体阻断，而不是C/EBPα或C/EBPδ。用cAMP处理细胞不影响C/EBPβ的表达，但观察到其结合活性增加了近三倍。CRE-1在Gal-4结合位点的突变使pka依赖性的PEPCK−2300Luc的激活降低到野生型构建体的40%。共表达含有Gal-4结合域和C/EBPβ反激活域的嵌合蛋白，但不含C/EBPα或CRE结合蛋白（CREB），可以恢复PKA的完全激活。缺乏C/EBPβ激活域的缺失结构体作为显性阴性抑制剂发挥作用。因此，C/EBPβ与CRE-1的结合可能有助于肾细胞中camp依赖性的PEPCK启动子的激活。
Phosphoenolpyruvate carboxykinase (PEPCK) is a key regulatory enzyme in renal gluconeogenesis. Activation of various PEPCK−2300Luc reporter constructs in LLC-PK1-F+cells, a gluconeogenic line of porcine renal proximal tubule-like cells, by protein kinase A (PKA) is mediated, in part, through the cAMP-response element (CRE)-1 of the PEPCK promoter. Incubation of a CRE-1 containing oligonucleotide with nuclear extracts from LLC-PK1-F+cells produced multiple bands, all of which were blocked by antibodies that are specific for C/EBPβ but not for C/EBPα or C/EBPδ. Treatment of cells with cAMP did not affect the expression of C/EBPβ, but the observed binding activity was increased nearly threefold. Mutation of CRE-1 to a Gal-4 binding site reduced the PKA-dependent activation of PEPCK−2300Luc to 40% of that observed with the wild-type construct. Coexpression of a chimeric protein containing a Gal-4 binding domain and the transactivation domain of C/EBPβ, but not of C/EBPα or CRE binding protein (CREB), restored full activation by PKA. A deletion construct that lacks the activation domain of C/EBPβ functions as a dominant negative inhibitor. Thus the binding of C/EBPβ to the CRE-1 may contribute to the cAMP-dependent activation of the PEPCK promoter in kidney cells.