Gene expression patterns in the progression of canine copper-associated chronic hepatitis

Abstract

Copper is an essential trace element, but can become toxic when present in abundance. The severe effects of copper-metabolism imbalance are illustrated by the inherited disorders Wilson disease and Menkes disease. The Labrador retriever dog breed is a novel non-rodent model for copper-storage disorders carrying mutations in genes known to be involved in copper transport. Besides disease initiation and progression of copper accumulation, the molecular mechanisms and pathways involved in progression towards copper-associated chronic hepatitis still remain unclear. Using expression levels of targeted candidate genes as well as transcriptome micro-arrays in liver tissue of Labrador retrievers in different stages of copper-associated hepatitis, pathways involved in progression of the disease were studied. At the initial phase of increased hepatic copper levels, transcriptomic alterations in livers mainly revealed enrichment for cell adhesion, developmental, inflammatory, and cytoskeleton pathways. Upregulation of targeted MT1A and COMMD1 mRNA shows the liver's first response to rising intrahepatic copper concentrations. In livers with copper-associated hepatitis mainly an activation of inflammatory pathways is detected. Once the hepatitis is in the chronic stage, transcriptional differences are found in cell adhesion adaptations and cytoskeleton remodelling. In view of the high similarities in copper-associated hepatopathies between men and dog extrapolation of these dog data into human biomedicine seems feasible.

Fig 1. Cellular copper metabolism.

Copper enters the cell via CTR1 and is immediately bound by metallothioneins (MT) and/or GSH to prevent cellular damage. COX17, CCS, and ATOX1 transfer copper to its destination molecules CCO, SOD1, and ATP7A/ATP7B respectively. In the enterocyte ATP7A facilitates copper transport over the basolateral membrane into the portal circulation[92,93], while in the hepatocyte it mobilizes hepatic copper stores in the case of peripheral copper deficiency[94]. ATP7B functions in the export of copper to the blood bound to ceruloplasmin (CP) or to the bile when copper levels are high[11,12]. The biliary excretion of copper also depends on COMMD1, which interacts with the amino terminus of ATP7B. In addition COMMD1 may be involved in quality control of ATP7A and ATP7B[14,15,95]. COMMD1 interacts with also with other proteins, including SOD1 and CCS, in the regulation of intracellular copper levels. XIAP inhibits COMMD1 functioning by promoting its degradation, resulting in rising cellular copper levels[96]. In turn, XIAP is regulated by intracellular copper levels. Under basal copper conditions XIAP-mediated ubiquitination of CCS leads to enhanced copper acquisition and positively regulates SOD1 activation by CCS[90]. When copper levels are elevated, CCS delivers copper to XIAP, resulting in degradation of CCS and XIAP and decrease in caspase inhibition, which may result in enhanced apoptosis[90,91]. APP is proposed to have a role in the copper efflux pathway, and intracellular copper levels have shown to modulate cellular APP trafficking in neuronal cells[83,84,97]. APP, amyloid beta (A4) precursor protein; ATOX1, antioxidant 1 copper chaperone; ATP7A, ATPase, Cu++ transporting, alpha polypeptide; ATP7B, ATPase, Cu++ transporting, beta polypeptide; CCO, cytochrome C oxidase; CCS, copper chaperone for superoxide dismutase; COMMD1, copper metabolism (Murr1) domain containing 1; COX17, cytochrome C oxidase copper chaperone; CP, ceruloplasmin; CTR1, copper transporter 1; GSH, glutathione; MT1A, metallothionein 1A; MT2A, metallothionein 2A; SOD1, Cu,Zn superoxide dismutase 1; XIAP, X-linked inhibitor of apoptosis.

Fig 2. Quantitative PCR results.

Liver tissue of Labrador retrievers with normal copper (N; n = 7), high copper (HC; n = 8), high copper hepatitis (HCH, n = 8), and high copper chronic hepatitis (HCCH; n = 8) was used for mRNA quantification of genes involved in copper metabolism. Gene expression of APP (A), CCS (B), COMMD1 (C), MAT1A (D), MT1A (E), MT2A (F), and GSTP1 (G) was significantly changed between two successive stages of the disease. The thick black line represents the median (50th percentile), also the first and third quartile (25th and 75th percentile respectively) are displayed. Outliers are depicted with an open dot, representing values higher than 1.5 times the interquartile range. APP, amyloid beta (A4) precursor protein; CCS, copper chaperone for superoxide dismutase; COMMD1, copper metabolism (Murr1) domain containing 1; GSTP1, glutathione s-transferase pi 1; MAT1A, methionine adenosyltransferase I alpha; MT1A, metallothionein 1A; MT2A, metallothionein 2A. * P<0.05, ** P<0.01.

Fig 3. Copper metabolism adaptation in disease progression.

A schematic overview of up- or downregulated of genes, involved in copper metabolism and oxidative stress within the different stages of disease progression. APP, amyloid beta (A4) precursor protein; CCS, copper chaperone for superoxide dismutase; COMMD1, copper metabolism (Murr1) domain containing 1; GSTP1, glutathione s-transferase pi 1; MAT1A, methionine adenosyltransferase I alpha; MT1A, metallothionein 1A; MT2A, metallothionein 2A.

Fig 4. Heatmap of genes regulated through disease progression.

216 probes (listed in rows) were expressed significantly (P<0.001) different samples in the four different stages (listed in columns) compared with the common reference pool (Beagles). HC, high copper; HCH, high copper hepatitis; HCCH, high copper chronic hepatitis; N, normal liver.

Fig 5. Total number of genes involved in specific stages of disease progression.

The Venn-diagram depicts the number of genes that are differentially expressed compared to the normal liver group (N) within the specific phenotypes. Selected genes were filtered on log2 Fold Change under -0.5 or over 0.5, and a P value of <0.001. HC, high copper; HCH, high copper hepatitis; HCCH, high copper chronic hepatitis.

Fig 6. Process networks enriched in the different stages of disease.

The unique genes involved in the different stages of disease were used to determine functional enrichment using Metacore. The most involved and significant process networks are depicted for high copper (A), high copper hepatitis (B), and high copper chronic hepatitis (C).