Augmenter of liver regeneration (ALR) activates cytoplasmic β-Catenin by GSK-3β independent mechanisms

Abstract

Liver regeneration is orchestrated by a variety of factors amongst them ß-Catenin and Augmenter of Liver Regeneration (ALR), the latter is known for its anti-oxidative, anti-apoptotic and anti-inflammatory properties. ß-Catenin acts as a transduction molecule within the Wnt signaling pathway and is part of a membrane bound E-cadherin-protein complex, released upon EGF receptor activation. In the absence of a Wnt/Wingless signal, cytoplasmic ß-catenin interacts with GSK-3ß in the degradation complex, which competes with E-cadherin for binding to ß-catenin. Previously we have shown that ALR activates the EGF-receptor-PI3/Akt signaling pathway and thereby inhibiting GSK-3ß. Therefore we aimed to analyze the impact of ALR (exogenous and endogenous) on cellular ß-catenin signaling. We performed in vitro studies treating cell lines (Hep3B, Huh7), w/o stably expressing short form ALR (sfALR), with specific inhibitors and/or recombinant ALR (rALR) followed by western blotting. Treatment with rALR phosphorylates PI3/Akt and GSK-3ß, but did not change ß-catenin degradation or activation, detected by specific antibodies against ß-catenin phosphorylation at S33/S37/T41 or S552. Interestingly, cells expressing sfALR revealed increased total and reduced phosphorylated ß-catenin. On the other hand, rALR treatment enhanced phosphorylation of ß-catenin (Y654) even more than EGF compared to control cells. As previously shown for EGF treatment, phospho-(Y654)-ß-catenin (Y654) dissociates from E-cadherin, translocation to the nucleus, and increase transactivation by GSK-3ß independent mechanisms. Contrary to this potential mitogenic mechanism we found increased ß-catenin in sfALR expressing cells in agreement with our previous report of increased E-cadherin and ZO-1 expression in these cells and their anti-EMT properties.