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- Titel:
Activation of Autophagy, Observed in Liver Tissues From Patients With Wilson Disease and From ATP7B-Deficient Animals, Protects Hepatocytes From Copper-Induced Apoptosis.
- Dokumenttyp:
- Journal Article; Research Support, Non-U.S. Gov't
- Autor(en):
- Polishchuk, Elena V; Merolla, Assunta; Lichtmannegger, Josef; Romano, Alessia; Indrieri, Alessia; Ilyechova, Ekaterina Y; Concilli, Mafalda; De Cegli, Rossella; Crispino, Roberta; Mariniello, Marta; Petruzzelli, Raffaella; Ranucci, Giusy; Iorio, Raffaele; Pietrocola, Federico; Einer, Claudia; Borchard, Sabine; Zibert, Andree; Schmidt, Hartmut H; Di Schiavi, Elia; Puchkova, Ludmila V; Franco, Brunella; Kroemer, Guido; Zischka, Hans; Polishchuk, Roman S
- Abstract:
- 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.
- Zeitschriftentitel:
- Gastroenterology
- Jahr:
- 2019
- Band / Volume:
- 156
- Heft / Issue:
- 4
- Seitenangaben Beitrag:
- 1173-1189.e5
- Sprache:
- eng
- Volltext / DOI:
- doi:10.1053/j.gastro.2018.11.032
- PubMed:
- http://view.ncbi.nlm.nih.gov/pubmed/30452922
- Print-ISSN:
- 0016-5085
- TUM Einrichtung:
- Institut für Toxikologie und Umwelthygiene
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