Gastroenterology

Gastroenterology

Volume 156, Issue 4, March 2019, Pages 1173-1189.e5
Gastroenterology

Original Research
Full Report: Basic and Translational—Liver
Activation of Autophagy, Observed in Liver Tissues From Patients With Wilson Disease and From ATP7B-Deficient Animals, Protects Hepatocytes From Copper-Induced Apoptosis

https://doi.org/10.1053/j.gastro.2018.11.032Get rights and content

Background & Aims

Wilson disease (WD) is an inherited disorder of copper metabolism that leads to copper accumulation and toxicity in the liver and brain. It is caused by mutations in the adenosine triphosphatase copper transporting β gene (ATP7B), which encodes a protein that transports copper from hepatocytes into the bile. We studied ATP7B-deficient cells and animals to identify strategies to decrease copper toxicity in patients with WD.

Methods

We used RNA-seq to compare gene expression patterns between wild-type and ATP7B-knockout HepG2 cells exposed to copper. We collected blood and liver tissues from Atp7b−/− and Atp7b+/− (control) rats (LPP) and mice; some mice were given 5 daily injections of an autophagy inhibitor (spautin-1) or vehicle. We obtained liver biopsies from 2 patients with WD in Italy and liver tissues from patients without WD (control). Liver tissues were analyzed by immunohistochemistry, immunofluorescence, cell viability, apoptosis assays, and electron and confocal microscopy. Proteins were knocked down in cell lines using small interfering RNAs. Levels of copper were measured in cell lysates, blood samples, liver homogenates, and subcellular fractions by spectroscopy.

Results

After exposure to copper, ATP7B-knockout cells had significant increases in the expression of 103 genes that regulate autophagy (including MAP1LC3A, known as LC3) compared with wild-type cells. Electron and confocal microscopy visualized more autophagic structures in the cytoplasm of ATP7B-knockout cells than wild-type cells after copper exposure. Hepatocytes in liver tissues from patients with WD and from Atp7b−/− mice and rats (but not controls) had multiple autophagosomes. In ATP7B-knockout cells, mammalian target of rapamycin (mTOR) had decreased activity and was dissociated from lysosomes; this resulted in translocation of the mTOR substrate transcription factor EB to the nucleus and activation of autophagy-related genes. In wild-type HepG2 cells (but not ATP7B-knockout cells), exposure to copper and amino acids induced recruitment of mTOR to lysosomes. Pharmacologic inhibitors of autophagy or knockdown of autophagy proteins ATG7 and ATG13 induced and accelerated the death of ATP7B-knockout HepG2 cells compared with wild-type cells. Autophagy protected ATP7B-knockout cells from copper-induced death.

Conclusion

ATP7B-deficient hepatocytes, such as in those in patients with WD, activate autophagy in response to copper overload to prevent copper-induced apoptosis. Agents designed to activate this autophagic pathway might decrease copper toxicity in patients with WD.

Section snippets

Antibodies and Other Reagents

The full list of antibodies, small interfering RNAs (siRNAs), and primers is provided in the Supplementary Methods.

Cell Culture and Construction of Recombinant Adenoviruses

Wild-type (WT) HepG2 cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum, L-glutamine 2 mmol/L, and 1% penicillin and streptomycin. ATP7B-knockout (ko) HepG2 cells13 were grown in RPMI medium supplemented with 10% fetal calf serum, L-glutamine 2 mmol/L, and 1% penicillin and streptomycin. To transduce WT or ATP7B-ko cells, adenoviruses that

Loss of ATP7B and Cu Overload Trigger Autophagy in Hepatic Cells in Livers of Atp7b−/− Animals and Patients With WD

To investigate which mechanisms might be activated in ATP7B-deficient hepatocytes to protect against Cu toxicity in WD, we used an ATP7B-ko HepG2 cell line.13 Control (WT) and ATP7B-ko HepG2 cells were incubated with Cu for 24 hours and their transcriptional responses were analyzed using QuantSeq 3′ mRNA sequencing, which allows precise measurements of quantitative differences in gene expression.19 We found that Cu increased the expression levels of 1244 genes in ATP7B-ko cells vs 480 in

Discussion

The protective function of autophagy has been documented in a wide range of disorders, including conditions in which oxidative damage precipitates cell death and organismal disease.26, 27 In contrast, the role of autophagy in WD has remained obscure, although recent reports have documented activation of autophagy by Cu salts or Cu-containing anticancer compounds.10, 11, 12 In this article, we report on several novel findings demonstrating that autophagy operates as a prosurvival mechanism in WD.

Acknowledgments

The authors thank Telethon Institute of Genetics and Medicine members Cathal Wilson for critical reading of the manuscript; Andrea Ballabio, Chiara di Malta, and Gennaro Napolitano for helpful suggestions; and Angela Zampelli, Sergio Attanasio, Leandro Soria, and Edoardo Nusco for technical assistance. The authors are grateful to ITMO University/IEM members Yurii Orlov and Ekaterina Skomorokhova for help with specimen analysis. They also acknowledge support from the American and Italian Wilson

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    Conflicts of interest The authors disclose no conflicts.

    Funding This work was funded by Telethon, Italy (grant TIGEM-CBDM9 to Roman S. Polishchuk); AIRC, Italy (grant IG 17118 to Roman S. Polishchuk); CNR/RFBR Collaboration Program, Italy and Russia (grant 18-515-7811 to Elia Di Schiavi and Ludmila V. Puchkova); RFBR (grants 16-34-60219 and МК 2718.2018.4 to Ekaterina Y. Ilyechova); ACRI Travel Fellowship, Italy (to Ekaterina Y. Ilyechova), and in part by the Deutsche Forschungsgemeinschaft (DFG grant ZI1386/2-1 to Hans Zischka).

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    Authors share co-senior authorship.

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