Human nuclear pregnane X receptor cross-talk with CREB to repress cAMP activation of the glucose-6-phosphatase gene
Human nuclear pregnane X receptor cross-talk with CREB to repress cAMP activation of the glucose-6-phosphatase gene
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DOI：
10.1042/bj20070481
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发表时间：
2007-11-01
影响因子：
4.1
通讯作者：
Negishi, Masahiko
中科院分区：
文献类型：
--
作者：
Kodama, Susumu;Moore, Rick;Negishi, Masahiko
核PXR(孕烷X受体)最初被认为是一种关键的转录因子，激活编码药物代谢酶的肝脏基因。我们现在已经证明，PXR还通过直接与CREB[cAMP反应元件结合蛋白]结合来抑制高血糖素激活的G6Pase(葡萄糖-6-磷酸酶)基因的转录。腺病毒介导的人PXR(HPXR)表达及利福平激活人PXR在Huh7细胞中强烈抑制cAMP依赖的内源性G6Pase基因的诱导。将G6Pase启动子构建的-259用于细胞转录检测，将hPXR对PKA(cAMP依赖蛋白激酶)介导的启动子激活的抑制作用定位于Cre位点。GST(谷胱甘肽转移酶)下拉和免疫沉淀实验表明PXR与CREB直接结合，而凝胶位移实验证明这种结合阻止了CREB与CRE的相互作用。这些结果与PXR通过抑制CREB的DNA结合能力而抑制G6Pase基因转录的假设一致。为了支持这一假设，用小鼠PXR激活剂PCN(孕烯醇酮16α-碳腈)治疗抑制了野生型(但不是PXR基因敲除)小鼠制备的原代肝细胞中cAMP依赖的G6Pase基因的诱导，也抑制了禁食野生型小鼠的肝脏中G6Pase基因的诱导，但不抑制PXR基因敲除小鼠的肝脏中的G6Pase基因。此外，染色质免疫沉淀(ChIP)检测显示，PCN处理后禁食野生型小鼠的CREB与G6Pase启动子的结合减少。因此，药物激活PXR可以抑制CREB的转录活性，下调糖异生。
The nuclear PXR (pregnane X receptor) was originally characterized as a key transcription factor that activated hepatic genes encoding drug-metabolizing enzymes. We have now demonstrated that PXR also represses glucagon-activated transcription of the G6Pase (glucose-6-phosphatase) gene by directly binding to CREB [CRE (CAMP-response element)-binding protein]. Adenoviral-mediated expression of human PXR (hPXR) and its activation by rifampicin strongly repressed cAMP-dependent induction of the endogenous G6Pase gene in Huh7 cells. Using the - 259 by G6Pase promoter construct in cell-based transcription assays, repression by hPXR of PKA (CAMP-dependent protein kinase)-mediated promoter activation was delineated to CRE sites. GST (glutathione transferase) pull-down and immunoprecipitation assays were employed to show that PXR binds directly to CREB, while gel-shift assays were used to demonstrate that this binding prevents CREB interaction with the CRE. These results are consistent with the hypothesis that PXR represses the transcription of the G6Pase gene by inhibiting the DNA-binding ability of CREB. In support of this hypothesis, treatment with the mouse PXR activator PCN (pregnenolone 16 alpha-carbonitrile) repressed CAMP-dependent induction of the G6Pase gene in primary hepatocytes prepared from wild-type, but not from PXR-knockout, mice, and also in the liver of fasting wildtype, but not PXR-knockout, mice. Moreover, ChIP (chromatin immunoprecipitation) assays were performed to show a decreased CREB binding to the G6Pase promoter in fasting wild-type mice after PCN treatment. Thus drug activation of PXR can repress the transcriptional activity of CREB, down-regulating gluconeogenesis.