Cholestasis induces expression of the oncofetal marker Nope in adult murine liver independent of Fxr

Background: Neighbor of Punc E11 (Nope) is an axonal guidance receptor that is strongly expressed in fetal and adult hepatic stem/progenitor cells and in hepatocellular carcinoma but not in terminally differentiated hepatocytes. We here investigated the expression pattern of the oncofetal marker Nope in adult mice in regenerating liver after biliary liver injury.

Methods: Liver tissue was extracted from adult C57Bl/6 mice and Fxr-/- mice 24 hours up to 4 weeks after bile duct ligation (BDL) or after a 1 week diet containing cholic (0.5%) or ursodeoxycholic acid (0.5%). Liver tissue was tested for expression levels of Nope via quantitative RT-PCR. Costainings were performed for Nope in combination with CK19 (biliary), E-cadherin (epithelial) or the canalicular marker dipeptidylpeptidase (DPP) IV. For in vitro experiments, primary isolated murine hepatocytes were incubated with the bile acids TC, TLC and TDC (25µM) for up to 5 days and expression of Nope was quantitatively analysed by RT-PCR.

Results: Bile duct ligation leads to a significantly increasing expression level of Nope (after 1 week 87-fold vs. adult liver, p < 0.0001, after 4 weeks 676-fold vs. adult liver, p < 0.001).

Costainings with E-cadherin and DPPIV demonstrate a sinusoidal expression pattern of Nope on hepatocytes, but no expression on CK19-positive cholangiocytes. At later stages after BDL, almost all of the hepatocytes stain positive for Nope. In Fxr-/- mice, Nope is expressed without additional biliary injury (28-fold vs. adult liver, p < 0.0001) and shows a further and significant increase after BDL (440-fold vs. no BDL, p = 0.03). A diet with cholic (26-fold vs. adult liver, p = 0.002) or ursodeoxycholic acid (8-fold vs. adult liver, p = 0.004) also leads to a significant expression of Nope with a membranous staining pattern on hepatocytes and a diet with hydrophobic acids results in a significantly higher expression of Nope (p = 0.04). In vitro, however, expression of Nope is increasing until day 5 independent of the presence of bile acids in the medium.

Conclusion: We here report the bile acid-induced expression of the oncofetal marker Nope on (in vivo) adult hepatocytes probably indicating their dedifferentiation. The induction of Nope is not mediated through Fxr but lack of the receptor leads to a higher expression level probably due to limited compensatory mechanisms of bile acid homeostasis in hepatocytes and cholangiocytes.

Corresponding author: Bowe, Andrea

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