Elevated copper impairs hepatic nuclear receptor function in Wilson’s disease
Clavia Ruth Wooton-Kee, … , Svetlana Lutsenko, David D. Moore
Clavia Ruth Wooton-Kee, … , Svetlana Lutsenko, David D. Moore
Published August 4, 2015
Citation Information: J Clin Invest. 2015;125(9):3449-3460. https://doi.org/10.1172/JCI78991.
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Research Article Hepatology

Elevated copper impairs hepatic nuclear receptor function in Wilson’s disease

Abstract

Wilson’s disease (WD) is an autosomal recessive disorder that results in accumulation of copper in the liver as a consequence of mutations in the gene encoding the copper-transporting P-type ATPase (ATP7B). WD is a chronic liver disorder, and individuals with the disease present with a variety of complications, including steatosis, cholestasis, cirrhosis, and liver failure. Similar to patients with WD, Atp7b–/– mice have markedly elevated levels of hepatic copper and liver pathology. Previous studies have demonstrated that replacement of zinc in the DNA-binding domain of the estrogen receptor (ER) with copper disrupts specific binding to DNA response elements. Here, we found decreased binding of the nuclear receptors FXR, RXR, HNF4α, and LRH-1 to promoter response elements and decreased mRNA expression of nuclear receptor target genes in Atp7b–/– mice, as well as in adult and pediatric WD patients. Excessive hepatic copper has been described in progressive familial cholestasis (PFIC), and we found that similar to individuals with WD, patients with PFIC2 or PFIC3 who have clinically elevated hepatic copper levels exhibit impaired nuclear receptor activity. Together, these data demonstrate that copper-mediated nuclear receptor dysfunction disrupts liver function in WD and potentially in other disorders associated with increased hepatic copper levels.

Authors

Clavia Ruth Wooton-Kee, Ajay K. Jain, Martin Wagner, Michael A. Grusak, Milton J. Finegold, Svetlana Lutsenko, David D. Moore

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Figure 1

Copper disruption of DNA binding is reversed by zinc.

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Copper disruption of DNA binding is reversed by zinc. (A) EMSA of in vit...
(A) EMSA of in vitro–translated FXR and RXR with (+) or without 4 μM copper sulfate (Cu+2), or 1–10 μM nickel (Ni+2) or cobalt (Co+2) chloride in the RXR reaction. One hundred–fold excess cold competition with WT (CC) but not mutant cold oligonucleotide (mCC). (B) Nuclear extracts from HepG2 cells treated with 1–4 μM Cu+2 or 4 μM Cu+2 plus 5–40 μM ZnSO4 (Zn+2) were incubated with a radiolabeled probe containing the FXRE on the BSEP promoter. (C) HepG2 cells were treated overnight with DMSO, 75 μM CDCA, 10 μM Cu+2, and 10–40 μM Zn+2, as indicated. Error bars represent the mean ± SEM. n = 3; *P < 0.05 and #P < 0.01 by 1-way ANOVA, followed by Bonferroni’s post-hoc test. (D) EMSA analysis of binding to the human FXRE on the BSEP promoter with nuclear extracts harvested from HepG2 cells treated with 10 μM Cu+2, 10 μM Cu+2 plus 40 μM Zn+2, and 40 μM Zn+2. Binding of nuclear extracts from HepG2 cells treated with 10 μM Cu+2 or 10 μM Cu+2 plus 40 μM Zn+2 to radiolabeled oligonucleotides containing an (E) HNF4α, (F) TR, (G) SP1 (complex I), (H) GATA, or (I) CREB response element. NE, nuclear extract.