Hepatocyte GP73 expression in Wilson disease

Abstract

Background/aims: Wilson disease (WD) is a disorder of copper transport caused by mutations within the ATP7B gene. WD is phenotypically variable and can present with predominantly hepatic or neurologic manifestations. The mechanisms responsible for this variability are unknown. GP73, a Golgi membrane protein, is expressed in hepatocytes in response to acute and chronic liver disease.

Methods: Hepatocyte GP73 expression was examined in the livers of WD patients by semiquantitative immunohistochemistry. GP73 mRNA levels were measured in mice with a deletion of the WD gene (Atp7b(-/-)) by real-time PCR, and these values were compared to the concomitant histological abnormalities and previously reported copper levels.

Results: Hepatocyte GP73 expression was more frequently observed in patients with hepatic versus neurologic presentation (79% vs. 30%, p<0.05). Furthermore, GP73 expression was significantly higher (44.7+/-14.0 vs. 2.0+/-0.81, p<0.05) in patients with hepatic phenotype. In Atp7b(-/-) mice, GP73 mRNA was significantly elevated at 20-46 weeks of age, coincident with extensive hepatic inflammation and fibrosis, but not at 6 weeks, when hepatic histology was normal despite significant copper overload. GP73 mRNA levels normalized concomitantly with the resolution of hepatic injury at 60-weeks. However, in tumor-like nodules GP73 was strikingly elevated.

Conclusion: Increased hepatocyte GP73 expression is more commonly a feature of hepatic than neurologic WD, and is triggered in response to inflammation, fibrosis, and dysplasia, rather than copper overload.

Fig. 1

Distribution of histological liver disease in patients with hepatic vs. neurologic presentations of WD. Percent of WD patients with predominantly hepatic (open bars) or neurologic (hatched bars) involvement with the diagnosis shown.

Fig. 2

Hepatocyte expression of GP73 in WD with predominant hepatologic or neurologic manifestation. GP73 IH staining in livers from patients with primary hepatologic (A) or neurologic (B) presentations of WD. In (A), robust immunoreactivity is present in hepatocytes, with a pericanalicular pattern. Little or no immunoreactivity is present in fibrous septa and in non-hepatocyte cell types within the lobules. In (B) minimal or no immunoreactivity is present in hepatocytes. Individual perisinusoidal cells show GP73 immunoreactivity. Original Magnification 20×. (C) GP73 IH staining intensity scores (mean ± standard error) of hepatocyte GP73 expression in WD patients with primary liver and neurological involvement. Significance levels; *, p<0.05.

Fig. 3

Upregulation of GP73 mRNA in Atp7b^−/− mice. Whole-liver GP73 mRNA levels (mean ± SD) were quantified in wild-type and Atp7b^−/− mice by RT-PCR relative to the internal standard (GAPDH). GP73 mRNA values from Atp7b^−/− mice are expressed relative to normalized values from wild-type mice at each time point. GP73 mRNA levels were measured separately in micro-dissected tumor tissues from 60-weeks old Atp7b^−/− mice (open bar). Statistical significances were calculated using REST gene software with a Taylor algorithm. Significance levels; *, p < 0.05.

Fig. 4

Histology of Atp7b^−/− mouse livers. H&E stained liver sections from a 6 week-old wild-type female (A), a 32 week old wild-type female (B), a 6 week-old Atp7b^−/− female showing normal histology (C), a 20 week-old Atp7b^−/− female showing hepatitis and a bile duct lesion (D), a 32 week-old Atp7b^−/− female showing severe hepatitis and a microabcess (E), a 46 week-old Atp7b^−/− female showing severe necrosis and hepatitis (F), a tumor from a 60 week-old Atp7b^−/− female showing neoplastic bile duct proliferation (G), and regenerating liver tissue with normal histology (H) from the same animal as (G). Original magnification 100× (A, B), 200× (C–G).