Abstract
Copper is essential for human physiology, but in excess it causes the severe metabolic disorder Wilson disease. Elevated copper is thought to induce pathological changes in tissues by stimulating the production of reactive oxygen species that damage multiple cell targets. To better understand the molecular basis of this disease, we performed genome-wide mRNA profiling as well as protein and metabolite analysis for Atp7b-/- mice, an animal model of Wilson disease. We found that at the presymptomatic stages of the disease, copper-induced changes are inconsistent with widespread radical-mediated damage, which is likely due to the sequestration of cytosolic copper by metallothioneins that are markedly up-regulated in Atp7b-/- livers. Instead, copper selectively up-regulates molecular machinery associated with the cell cycle and chromatin structure and down-regulates lipid metabolism, particularly cholesterol biosynthesis. Specific changes in the transcriptome are accompanied by distinct metabolic changes. Biochemical and mass spectroscopy measurements revealed a 3.6-fold decrease of very low density lipoprotein cholesterol in serum and a 33% decrease of liver cholesterol, indicative of a marked decrease in cholesterol biosynthesis. Consistent with low cholesterol levels, the amount of activated sterol regulatory-binding protein 2 (SREBP-2) is increased in Atp7b-/- nuclei. However, the SREBP-2 target genes are dysregulated suggesting that elevated copper alters SREBP-2 function rather than its processing or re-localization. Thus, in Atp7b-/- mice elevated copper affects specific cellular targets at the transcription and/or translation levels and has distinct effects on liver metabolic function, prior to appearance of histopathological changes. The identification of the network of specific copper-responsive targets facilitates further mechanistic analysis of human disorders of copper misbalance.
Highlights
Copper plays an essential role in human physiology
Evaluation of the Quality of the Array Data—To characterize molecular consequences of hepatic copper accumulation in the Atp7bϪ/Ϫ (KO) mice, we initially performed a comparative genome-wide analysis of transcripts isolated from livers of 6-week-old animals
The mRNA was isolated from three of each control (WT) and Atp7bϪ/Ϫ livers, and for each of these samples two technical replicates were run. The quality of generated data was initially verified by scatter plots, in which the overall distribution of normalized signal intensities was plotted for each pair of the biological replicates
Summary
Animals—The generation of the Atp7bϪ/Ϫ mouse has been described previously [16]. Mice were maintained on strain C57BL ϫ 129S6/SvEv, and female animals at 6 weeks of age were used for microarray and RT-PCR studies. This analysis identified those GO terms that were most strongly represented among the differentially expressed genes (probe sets). Analysis of Soluble Proteins by Two-dimensional Gel Electrophoresis—The liver tissue (50 –100 mg wet weight) from control and Atp7bϪ/Ϫ mice was homogenized in 800 l of buffer containing 10 mM HEPES, 10 mM NaCl, 1 mM KH2PO4, 5 mM NaHCO3, 5 mM EDTA, 1 mM CaCl2, and 0.5 mM MgCl2 using a glass Dounce homogenizer with tight pestle. Soluble proteins were obtained by centrifuging samples at 100,000 ϫ g for 30 min and collecting the supernatant. Immunodetection was visualized with an Odyssey infrared scanner (LI-COR, Lincoln, NE)
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