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Regulation of ceruloplasmin in human hepatic cells by redox active copper: identification of a novel AP-1 site in the ceruloplasmin gene.

ABSTRACT: 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.

SUBMITTER: Das D

PROVIDER: S-EPMC1783982 | biostudies-other | 2007 Feb

REPOSITORIES: biostudies-other

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Project description:Background & aimsWilson disease (WD) is caused by accumulation of copper primarily in the liver and brain. During maintenance therapy of WD with D-penicillamine, current guidelines recommend on-treatment ranges of urinary copper excretion (UCE) of 200-500 μg/24 h and serum non-ceruloplasmin-bound copper (NCC) of 50-150 μg/L. We compared NCC (measured by two novel assays) and UCE from patients with clinically stable WD on D-penicillamine therapy with these recommendations.MethodsThis is a secondary analysis of data from the Chelate trial (NCT03539952) that enrolled physician-selected patients with clinically stable WD on D-penicillamine maintenance therapy (at an unaltered dose for at least 4 months). We analyzed laboratory samples from the first screening visit, prior to interventions. NCC was measured by either protein speciation (NCC-Sp) using anion exchange high-performance liquid chromatography protein speciation followed by copper determination with inductively coupled plasma mass spectroscopy or as exchangeable copper (NCC-Ex). NCC-Sp was also analyzed in healthy controls (n = 75).ResultsIn 76 patients with WD with 21.3±14.3 average treatment-years, NCC-Sp (mean±SD: 56.6±26.2 μg/L) and NCC-Ex (mean±SD: 57.9±24.7 μg/L) were within the 50-150 μg/L target in 61% and 54% of patients, respectively. In addition, 36% and 31%, respectively, were even below the normal ranges (NCC-Sp: 46-213 μg/L, NCC-Ex: 41-71 μg/L). NCC-Ex positively correlated with NCC-Sp (r2 = 0.66, p <0.001) but with systematic deviation. UCE was outside the 200-500 μg/24 h target range in 58%. Only 14/69 (20%) fulfilled both the NCC-Sp and UCE targets. Clinical or biochemical signs of copper deficiency were not detected.ConclusionClinically stable patients with WD on maintenance D-penicillamine therapy frequently have lower NCC-Sp or higher UCE than current recommendations without signs of overtreatment. Further studies are warranted to identify appropriate target ranges of NCC-Sp, NCC-Ex and UCE in treated WD.Impact and implicationsChelator treatment of patients with Wilson disease (WD) is currently guided by measurements of non-ceruloplasmin-bound copper (NCC) and 24 h urinary copper excretion (UCE) but validation is limited. In 76 adults with ≈21 years history of treated WD and clinically stable disease on D-penicillamine therapy, NCC was commonly found to be below normal values and recommended target ranges whether measured by protein speciation (NCC-Sp) or as exchangeable copper (NCC-Ex), while UCE values were above the recommended target range in 49%. Common wisdom would suggest overtreatment in these cases, but no clinical or biochemical signs of copper deficiency were observed. Exploratory analysis of liver enzymes suggested that NCC below levels seen in controls may be beneficial, while the relation to UCE was less clear. The data calls for critical re-evaluation of target ranges for treatment of WD, specific for drug and laboratory methodology.Clinical trial number(NCT03539952).

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Regulation of ceruloplasmin in human hepatic cells by redox active copper: identification of a novel AP-1 site in the ceruloplasmin gene.

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