γ-Catenin at adherens junctions: mechanism and biologic implications in hepatocellular cancer after ß-catenin knockdown.

ß-Catenin is important in liver homeostasis as a part of Wnt signaling and adherens junctions (AJs), while its aberrant activation is observed in hepatocellular carcinoma (HCC). We have reported hepatocyte-specific ß-catenin knockout (KO) mice to lack adhesive defects as γ-catenin compensated at AJ. Because γ-catenin is a desmosomal protein, we asked if its increase in KO might deregulate desmosomes. No changes in desmosomal proteins or ultrastructure other than increased plakophilin-3 were observed. To further elucidate the role and regulation of γ-catenin, we contemplate an in vitro model and show γ-catenin increase in HCC cells upon ß-catenin knockdown (KD). Here, γ-catenin is unable to rescue ß-catenin/T cell factor (TCF) reporter activity; however, it sufficiently compensates at AJs as assessed by scratch wound assay, centrifugal assay for cell adhesion (CAFCA), and hanging drop assays. γ-Catenin increase is observed only after ß-catenin protein decrease and not after blockade of its transactivation. γ-Catenin increase is associated with enhanced serine/threonine phosphorylation and abrogated by protein kinase A (PKA) inhibition. In fact, several PKA-binding sites were detected in γ-catenin by in silico analysis. Intriguingly γ-catenin KD led to increased ß-catenin levels and transactivation. Thus, γ-catenin compensates for ß-catenin loss at AJ without affecting desmosomes but is unable to fulfill functions in Wnt signaling. γ-Catenin stabilization after ß-catenin loss is brought about by PKA. Catenin-sensing mechanism may depend on absolute ß-catenin levels and not its activity. Anti-ß-catenin therapies for HCC affecting total ß-catenin may target aberrant Wnt signaling without negatively impacting intercellular adhesion, provided mechanisms leading to γ-catenin stabilization are spared.

Hepatocyte γ-catenin compensates for conditionally deleted ß-catenin at adherens junctions.

BACKGROUND & AIMS: Wnt/ß-catenin signaling is important in liver physiology. Moreover, ß-catenin is also pivotal in adherens junctions (AJ). Here, we investigate hepatocyte-specific ß-catenin conditional null mice (KO) for any alterations in AJ and related tight junctions (TJ). METHODS: Using gene array, PCR, Western blot, immunohistochemistry, immunofluorescence, and co-immunoprecipitation, we compare and contrast the composition of AJ and TJ in KO and littermate wild-type (WT) control livers. RESULTS: We show association of E-cadherin with ß-catenin in epithelial cells of WT livers, which is lost in the KOs. While total levels of α-catenin, E-cadherin, and F-actin were modestly decreased, KO livers show increased γ-catenin/plakoglobin. By co-immunoprecipitation, E-cadherin/ß-catenin/F-actin association was observed in WT livers, while the association of E-cadherin/γ-catenin/F-actin was evident in KO livers. γ-Catenin was localized at the hepatocyte membrane at baseline in the KO liver. While γ-catenin gene expression remained unaltered, an increase in serine- and threonine-phosphorylated, but not tyrosine-phosphorylated γ-catenin was observed in KO livers. A continued presence of γ-catenin at the hepatocyte membrane, without any nuclear localization, was observed in liver regeneration after partial hepatectomy at 40 and 72 h, in both KO and WT. Analysis of TJ revealed lack of claudin-2 and increased levels of JAM-A and claudin-1 in KO livers. CONCLUSIONS: ß-Catenin adequately maintains AJ in the absence of ß-catenin in hepatocytes; however, it lacks nuclear localization. Moreover, ß-catenin/claudin-2 may be an important mechanism of crosstalk between the AJ and TJ.