Liver, Pancreas, and Biliary TractRole of ATP7B in biliary copper excretion in a human hepatoma cell line and normal rat hepatocytes☆,☆☆
Section snippets
Cells
Monolayer Huh7 cells were cultured in RPMI 1640 (Life Technologies, New York, NY) supplemented with 10% fetal calf serum (Wako Pure Chemical Industries, Ltd., Osaka, Japan), penicillin (100 U/mL, crystalline penicillin G Meiji; Meiji Seika Kaisya, Tokyo, Japan), and streptomycin (0.1 mg/mL; Meiji Seika Kaisya).
IRHs were obtained from male Sprague–Dawley rats weighing 200–250 g (8–10 weeks of age). Rats received humane care in accordance with the guidelines of Kurume University (Kurume, Japan,
Results
The immunofluorescent signals of endogenous and transfected ATP7B showed juxtanuclear patterns in Huh 7 cells (Figure 1A and B).
Discussion
This study shows that ATP7B, a copper transporter, localizes in the endocytic compartments, especially in the late endosomes in hepatocytes.
We constructed a chimeric GFP-ATP7B protein, yielding bright green fluorescence without any cofactors, for examining the intracellular localization. We did this for the following reasons. First, the intensity of fluorescent signals obtained from endogenous ATP7B in Huh7 cells by indirect immunofluorescent study is not sufficient in comparison with those of
Acknowledgements
The authors thank Dr. B. Hoflack for providing the anti-MPR polyclonal antibody, Dr. J. T. August for providing the anti-lamp1 and anti-lamp2 antibodies, and M. Inayoshi and K. Maeda for expert technical assistance.
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Cited by (57)
Copper induces hepatocyte injury due to the endoplasmic reticulum stress in cultured cells and patients with Wilson disease
2016, Experimental Cell ResearchCopper mediated neurological disorder: Visions into amyotrophic lateral sclerosis, Alzheimer and Menkes disease
2015, Journal of Trace Elements in Medicine and BiologyCitation Excerpt :This localization is consistent with their function in cuproenzyme biosynthesis, as several cuproenzymes are synthesized within the secretory pathway. Some controversy exists about the localization of ATP7B, as it has also been suggested that this protein resides in an endosomal compartment [145,146]. In addition, a smaller isoform of ATP7B exists, which has been localized to mitochondria [155].
Niemann-Pick C1 protein transports copper to the secretory compartment from late endosomes where ATP7B resides
2009, Experimental Cell ResearchCould ATP7B Export Cu(I) at the Tight Junctions and the Apical Membrane?
2008, Gastroenterology
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Address requests for reprints to: Masaru Harada, M.D., Second Department of Medicine, Kurume University School of Medicine, 67 Asahi-Machi, Kurume 830-0011, Japan.
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Supported in part by a grant from the alumni association of Kurume University School of Medicine (to M.H.).