Das Dola, Tapryal Nisha, Goswami Shyamal K, Fox Paul L, Mukhopadhyay Chinmay K
Special Centre for Molecular Medicine, Jawaharlal Nehru University, New Delhi 110 067, India.
Biochem J. 2007 Feb 15;402(1):135-41. doi: 10.1042/BJ20060963.
Cp (ceruloplasmin), a copper containing plasma protein, mainly synthesized in the liver, is known to be functional between the interface of iron and copper metabolism. We have reported previously that Cp is regulated by cellular iron status, but the process of the regulation of Cp by copper still remains a subject for investigation. In the present paper, we show that PDTC (pyrrolidine dithiocarbamate), a thiol compound widely known to increase intracellular redox copper, regulates Cp expression in hepatic cells by a copper-dependent transcriptional mechanism. To find out the mechanism of induction, chimeric constructs of the Cp 5'-flanking region driving luciferase were transfected into human hepatic cells. Deletion and mutational analyses showed the requirement of a novel APRE [AP-1 (activator protein-1) responsive element] present about 3.7 kb upstream of the translation initiation site. The role of AP-1 was confirmed by electrophoretic mobility-shift analysis. Western blot and overexpression studies detected the AP-1 as a heterodimer of c-jun and c-fos proteins. The activation of AP-1 was found to be copper-dependent as a specific extracellular chelator bathocuproine disulfonic acid blocked PDTC-mediated induction of AP-1-DNA binding and increased reporter gene activity. Whereas, in a copper-free medium, PDTC failed to activate either AP-1 or Cp synthesis, supplementation of copper could reverse AP-1 activation and Cp synthesis. Our finding is not only the first demonstration of regulation of Cp by redox copper but may also explain previous findings of increased Cp expression in cancers like hepatocarcinoma, where the intracellular copper level is higher in a redox compromised environment.
铜蓝蛋白（Cp）是一种含铜血浆蛋白，主要在肝脏中合成，已知其在铁和铜代谢的界面发挥作用。我们之前报道过Cp受细胞铁状态的调节，但铜对Cp的调节过程仍是一个有待研究的课题。在本文中，我们表明吡咯烷二硫代氨基甲酸盐（PDTC），一种广为人知的可增加细胞内氧化还原铜的硫醇化合物，通过一种铜依赖的转录机制调节肝细胞中的Cp表达。为了找出诱导机制，将驱动荧光素酶的Cp 5'侧翼区的嵌合构建体转染到人肝细胞中。缺失和突变分析表明，在翻译起始位点上游约3.7 kb处存在一个新的APRE [AP-1（激活蛋白-1）反应元件]是必需的。通过电泳迁移率变动分析证实了AP-1的作用。蛋白质印迹和过表达研究检测到AP-1是c-jun和c-fos蛋白的异二聚体。发现AP-1的激活是铜依赖的，因为一种特异性细胞外螯合剂bathocuproine disulfonic acid可阻断PDTC介导的AP-1与DNA结合的诱导并增加报告基因活性。然而，在无铜培养基中，PDTC未能激活AP-1或Cp合成，补充铜可逆转AP-1激活和Cp合成。我们的发现不仅首次证明了氧化还原铜对Cp的调节作用，还可能解释了之前在肝癌等癌症中Cp表达增加的发现，在这些癌症中，细胞内铜水平在氧化还原受损的环境中较高。
