Abstract

Background: Wilson’s disease (WD) is an autosomal recessive disorder caused by inactivating mutations in the copper (Cu⁺⁺) transporting P-type ATPase. Loss of Atp7b function prevents translocation of Cu⁺⁺ into the trans-Golgi network and excretion into bile. Cu⁺⁺ exposure results in oxidative stress, inflammation, and mitochondrial dysfunction and fibrosis, cirrhosis, and liver failure. WD patients and Atp7b^-/- mice have a progressive course of hepatocellular pathology, which includes regenerative hepatic nodule growth. An abundance of pregnane X receptor (PXR) response elements were recently identified in down-regulated gene sets identified by microarray analysis in pre-symptomatic Atp7b^-/- mice, suggesting a link between xenobiotic and metabolic pathways in WD. Further, PXR mRNA expression was decreased in Atp7b^-/- mice. We tested the hypothesis that decreased PXR activity exacerbates the progressive hepatic pathology in Atp7b^-/- mice. Methods: H&E, Ki-67, and CK-19 staining was performed on liver sections of Atp7b^-/-, DKO^Atp7b/PXR, and wild-type mice. RNA-Sequencing analysis was performed with 3-, 6-, and 12-month-old liver tissues from Atp7b^-/-, DKO^Atp7b/PXR, and wild-type mice. Results: Hepatocellular pathology and liver nodule development was more advanced in DKO^7b:PXR mice vs. Atp7b^-/- mice. At 12 months of age DKO^Atp7b:PXR and Atp7b^-/- livers displayed prominent histologic abnormalities including mild microsteatosis, hepatocellular ballooning, apoptosis, and mild lobular inflammation. H&E staining revealed clear demarcation between nodules and normal tissue. Nodules of Atp7b^-/- mice displayed more Ki-67 staining than the adjacent normal tissue (Ki-67 index 5.5% normal tissue vs. 28% nodule). Ki-67 staining in nodules of DKO^Atp7b:PXR livers was less than Atp7b^-/- mice, which is suggestive of dynamic molecular shifts to limit further cell proliferation. Normal tissue areas in the Atp7b^-/- and DKO^Atp7b:PXR livers displayed increased and diffuse glutamine synthetase (GS) staining, which is characteristic of hepatocellular stress; however, normalized GS staining around the central vein was displayed in adjacent nodular sections. Atp7b^-/- and DKO^Atp7b:PXR livers displayed increased CK-19 staining and ductular reaction with bile duct proliferation and inflammation. Chemical Carcinogenesis, Metabolic, PPAR Signaling, and Cell Signaling pathways were enriched in 3-, 6-, and 12-month-old Atp7b^-/- mice. We found unique and abundant enrichment of pathways related to protein metabolism and cytoskeleton regulation in DKO^Atp7b:PXR relative with Atp7b^-/- mice. Pathway analysis of nodules revealed an abundance of immune function and cell adhesion pathways in nodules of DKO^Atp7b:PXR relative to nodules of Atp7b^-/- mice. Conclusion: Impaired PXR activity increases copper-induced hepatocellular pathology. These findings reveal a new target in the development of hepatic pathology in WD.