Protein kinase C activity is required for aryl hydrocarbon receptor pathway-mediated signal transduction
Protein kinase C activity is required for aryl hydrocarbon receptor pathway-mediated signal transduction
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DOI：
10.1124/mol.53.4.691
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发表时间：
1998-04-01
影响因子：
3.6
通讯作者：
Perdew, GH
中科院分区：
文献类型：
--
作者：
Long, WP;Pray-Grant, M;Perdew, GH
在稳定转染pGUDLUC6.1的细胞系中，研究了蛋白激酶C(PKC)在人芳香烃受体(HAHR)信号转导通路中的作用。这些细胞系P5A11和HG40/6分别来自HeLa和HepG2细胞。用2，3，7，8，-四氯二苯并-对二恶英(TCDD)和佛波醇-12-肉豆蔻酸酯-13-醋酸酯(PMA)同时处理这些细胞，与单独使用TCDD处理的细胞相比，报告结构的反式激活增加了几倍。PKC抑制剂阻断PMA效应和HAHR介导的信号转导，表明这些过程需要PKC活性。对其他稳定转染pGUDLUC6.1的独立生成的HeLa来源的细胞系的检测表明，P5A11细胞中的PMA效应不是克隆人工制品。瞬时转染表明，PMA效应不是由于荧光素酶信息/基因产物稳定机制或基础转录机制的刺激。胞浆制剂检测显示，PKC刺激或抑制不会改变HAHR和HAHR核转位蛋白水平，也不会改变TCDD诱导的HAHR水平下调。同样，对核提取物的检测表明，PKC的刺激或抑制不会改变核AhR水平或HAHR/HAHR核转位蛋白DRE结合活性，如通过凝胶迁移率改变分析评估的那样。这些结果表明，HAHR需要一个PKC介导的事件来形成一个功能转录复合体，从而导致反式激活，而DRE是PMA增强AhR介导的反式激活所需的最低DNA元件。
The role of protein kinase C (PKC) in the human aryl hydrocarbon receptor (hAhR) signal transduction pathway was examined in cell lines stably transfected with pGUDLUC6.1, in which luc(+) is solely controlled by four dioxin-responsive elements (DREs). These cell lines, P5A11 and HG40/6, were derived from HeLa and HepG2 cells respectively. Simultaneous treatment of these cells with 2,3,7,8,-tetrachlorodibenzo-p-dioxin (TCDD) and phorbol-12-myristate-13-acetate (PMA) enhanced transactivation of the reporter construct several-fold relative to cells treated with TCDD alone. PKC inhibitors block the PMA effect and hAhR-mediated signal transduction, demonstrating these processes require PKC activity. Examination of other independently generated, HeLa-derived cell lines stably transfected with pGUDLUC6.1 demonstrates the PMA effect in P5A11 cells is not a clonal artifact. Transient transfections indicate the PMA effect is not due to a luciferase message/gene product stabilization mechanism or stimulation of the basal transcription machinery. Examination of cytosolic preparations demonstrates PKC stimulation or inhibition does not alter hAhR and hAhR nuclear translocator protein levels or TCDD-induced down-regulation of hAhR levels. Similarly, examination of nuclear extracts indicated PKC stimulation or inhibition does not alter nuclear AhR levels or hAhR/hAhR nuclear translocator protein heterodimer DRE-binding activity as assessed by electrophoretic mobility shift assay. These results demonstrate a PKC-mediated event is required for the hAhR to form a functional transcriptional complex that leads to trans-activation and that the DRE is the minimal DNA element required for PMA to enhance AhR-mediated trans-activation.