Dual ablation of β- and γ-catenin: Critical regulators of junctions and their functions

Abstract

The duality of β- and γ–catenin during liver injury has not been defined. It’s well known that loss of β-catenin plays a critical role in overall liver health and is a major component of adherens junctions (AJ). Further, γ-catenin has been shown to regulate β-catenin and vice-versa. In this excellent manuscript, the authors investigated the effects of knocking out both β- and γ–catenin creating a β-;γ-catenin-double knockout (DKO). The result of this interbreeding revealed a model that is reminiscent of early childhood cholestatic liver diseases (CLD) like Progressive Familial Intrahepatic Cholestasis (PFIC). The authors provide both in vivo and in vitro data to demonstrate the important role of these catenin genes in the regulation of hepatocyte-junctions. The experiments show partially redundant function of catenin’s at hepatocyte AJ in regulating tight junctions (TJ) and contributing to a disrupted blood-bile barrier. Further, concomitant hepatic loss of β- and γ-catenin disrupts structural and functional integrity of AJ and TJ via transcriptional and posttranslational mechanisms. Overall, these studies shed important light on junctional protein dysregulation during CLD.

The Role of β- and γ-catenin in Disease

Numerous groups, including the authors of the current manuscript, have shown the importance of β-catenin in liver development and disease progression. β-catenin is a dual function protein that is expressed in numerous tissues and organs including the liver and is part of the Wnt signaling pathway. With respect to chronic liver injury, the Wnt/β-catenin signaling pathway is regarded as a key regulator of disease progression, especially when β-catenin is overexpressed. Recently, β-catenin has been highlighted as a regulator of bile acid metabolism during cholestatic liver injury. In this study, Thompson et al. found that loss of β-catenin attenuated cholestatic liver injury by inhibition of the β-catenin/FXR complex (1). Further, overexpression and activation of the Wnt/β-catenin pathway leads to the progression of tumor growth and cancer, including hepatocellular cancer as shown by Puliga, et al. (2). Since β-catenin activation is found in a number of hepatocellular carcinoma (HCC) tumors, agents that are able to target HCC without inducing alteration in β-catenin are attractive candidates as demonstrated in this study which found that the thyromimetic agent, GC-1, reduced tumor growth without activating β-catenin (2).

In contrast to β-catenin, the role of γ-catenin in liver disease is less known; however, there are recent studies that demonstrate that this protein may also be a key regulator of chronic liver injury (3, 4). Unlike β-catenin, γ-catenin appears to have both positive and negative impacts on Wnt signaling depending upon the injury (3). However, the role of γ-catenin in the Wnt signaling pathway is controversial and unknown. In normal liver loss of γ-catenin does not induce alterations in hepatic damage; however, when γ-catenin knockout mice are subjected to bile duct ligation (BDL), there is an exacerbation of liver injury and hepatic fibrosis (4). In addition, chemical-induced carcinogenesis is further enhanced in γ-catenin knockout mice compared to wild-type mice (4) demonstrating that the role of γ-catenin may be protective in certain liver pathologies.

Studies have also evaluated the potential compensatory action between β- and γ-catenin during injurious models and development. Sun et al. reported that there is a redundant mechanism between β-catenin and γ-catenin during human embryonic stem cell adhesion and body formation; however, both catenin genes are required for the maintenance of these cells (5). Further, in chronic myeloid leukemic cells that have homozygous deletion of β-catenin, nuclear γ-catenin was found to promote survivin transcription thereby increasing tumorigenic pathways (6) suggesting that there is a compensatory action between these genes that might allow for the persistence of diseased phenotypes.

Catenin-Regulation of Junction Function

Adherens junctions (AJs) and tight junctions (TJs) make up cell-cell adhesion components, but with very different functions, and the formation of AJs leads to TJ assembly. With regards to catenin function, β-catenin localizes to AJs and allows for the creation of barriers between blood and other components (7, 8) and γ-catenin is a major component of desmosomes and AJs (7). How β- and γ-catenin regulate junctional integrity in chronic liver disease has not been fully examined; however, numerous studies demonstrate that there is a critical requirement for these genes in other diseased models.

The formation of AJ is largely dependent on β-catenin interaction as has been demonstrated in chronic inflammatory disease and disruption of β-catenin signaling induces a penetration of the AJ barrier, which results in pathological implications (7). In a separate study examining hepatocellular carcinoma, loss of β-catenin within AJ resulted in γ-catenin stabilization, but γ-catenin had no impact on functional Wnt/β-catenin signaling (9). These studies (and others) demonstrate the important role of catenin genes in maintaining the integrity and function of junctions along with tight regulation of junctional components including E-cadherin, claudin-2 and occludin (3, 7, 9).

Summary of Current Findings and Future Perspectives

In the current study, the authors expand on their previous work, which demonstrates the multifaceted role for both β- and γ-catenin in liver disease. Here they demonstrate that loss of both catenin genes induced a phenotype that clinically resembles Progressive Familial Intrahepatic Cholestasis (PFIC), but that also might be relevant to a number of liver pathologies. The resulting DKO mice were associated with failure to thrive, impaired hepatocyte differentiation, cholestasis/cholemia, ductular reaction, fibrosis, and increased serum enzymes including ALP and bilirubin (8). These findings represent a potential new model of injury to allow for the study of junctional function during chronic liver disease as well as opening the window for new exploration of hepatocyte malfunction. For instance, no studies have been performed to understand if there is dysregulation of junctional barriers and proteins (regulated by catenin signaling) in pathologies such as non-alcoholic fatty liver disease, which targets hepatocytes.

To take this study further, the authors delved into the mechanistic forces driving this phenotype. By performing temporal and hepatocyte-specific elimination of the two catenin genes, Pradhan-Sundd et al. discovered a loss of TJ integrity that was also associated with AJ assembly dysfunction (8). These findings were further supported by alterations in transcriptional regulators of TJ proteins including E-cadherin and occludin. With regards to claudin-2, a primary target of β-catenin, the authors found that depletion of β-catenin and γ-catenin was associated with a loss of claudin-2 leading to TJ disruption and promotion of chronic liver injury. This complicated interaction demonstrates the critical role that β-catenin and γ-catenin play in maintaining junctional integrity, most importantly when there is depletion of β-catenin,.

In adding another twist to their story, the authors reveal that loss of both β- and γ-catenin resulted in a large increase in total bile acids coupled with leaky hepatocyte junctions and disruption of blood-bile barriers. Disruption of hepatocyte junctions leads to a mixing of bile and blood (i.e. cholemia), which is regulated by both β- and γ-catenin as demonstrated in the DKO mouse model. These studies further demonstrated a loss of expression in numerous bile acid transporters and related genes, such as NTCP, Cyp7A and PPARγ in DKO mice, further highlighting the complexity of both β- and γ-catenin in liver disease pathology and specifically in the role of hepatocytes (8).

Conclusions

The study by Pradhan-Sundd et al. provides substantial evidence of a dual regulation provided by both β- and γ-catenin in chronic liver disease. These downstream mediators of Wnt signaling have been implicated numerous times in a variety of liver injury (10), but this is the first evidence that, together, they regulate junctional integrity and function. The DKO model that resulted in profound chronic liver injury also provides a novel tool to further study mechanisms of Wnt signaling with regards to both AJs and TJs. The authors demonstrate that simultaneous loss of β- and γ-catenin induces hepatocyte leakage, disruption of the blood-bile barrier, and decreased activation of numerous structural proteins that may be targets of future therapies. Through manipulation of β- and γ-catenin, junctional integrity might be restored, which may result in amelioration of chronic liver injury. While this study highlighted the role of β- and γ-catenin in chronic liver injury, many questions still remain to be answered. Would manipulation by overexpression of just β-catenin in the DKO provide protection from the loss of γ-catenin or vice-versa? Additionally, what might be the role of β- and γ-catenin loss in a model where total bile acids are already upregulated, such as the multi-drug resistance 2-knockout model (Mdr2^−/−)? It’s clear that β- and γ-catenin are critical to maintaining junctional integrity and dual loss of these genes results in a devastating phenotype; however, their full impact on junction function in chronic liver disease remains elusive.

Abbreviations

AJ

adheren junctions

β-catenin

beta catenin

BDL

bile duct ligation

CLD

cholestatic liver disease

DKO

double knockout

γ-catenin

gamma catenin

HCC

hepatocellular carcinoma

PFIC

Progressive Familial Intrahepatic Cholestasis

TJ

tight junctions