Regulation of biliary homeostasis by the scaffold protein EBP50 – EBPatobile

Scaffold proteins assemble and orchestrate cellular events such as the regulation of integral membrane protein activity, e.g. transporters, receptors, and signal transduction pathways. EBP50 (Ezrin-radixin-moesin (ERM)-Binding Phosphoprotein 50)/NHERF1 (NHE3 exchanger Regulatory Factor 1) is a PDZ-scaffold protein highly expressed in liver. At cellular level, EBP50 is localized in the sub-plasma membranous region of epithelial cells along with the cortical actin cytoskeleton. In cell, EBP50 plays a role in epithelial integrity by regulating apical structure and transporters essential for epithelial secretory functions and, in regulation of cell signaling pathways including EGFR. Biliary function is essential for the intestinal absorption of fat and cholesterol homeostasis. Bile primarily formed by the hepatocyte is modified downstream by absorptive and secretory properties of biliary epithelial cells (BEC) in liver and gallbladder (GB). In physiological conditions, both hepatocytes and BEC are virtually quiescent. However, in case of injury, they proliferate in response to diverse regulatory factors which contribute to liver cell mass restoration. Alterations of the absorptive and secretory functions of the biliary system caused by the dysregulation of apical transporters contribute to cholestatic diseases. Therefore, establishment and maintenance of epithelial integrity is crucial to properly ensure the biliary secretory function, which depends on the anchorage and regulation of transporters at the apical domain of liver epithelial cells. EBP50 is expressed by hepatocytes and by BEC along the biliary tree including in the GB in which EBP50 is expressed at higher levels. In hepatocytes and BEC, EBP50 regulates cell surface expression, stability and functions of transporters essential for bile secretory function comprising MRP2, MDR3 and CFTR. Another potentially important regulator of bile composition and volume through an impact on GB epithelium is the membrane G protein-coupled bile acid (BA) receptor TGR5. Interestingly, like EBP50, TGR5 is highly enriched in the GB epithelium and is reported to control CFTR function and expression. However, the precise mechanisms by which TGR5-mediated BA impacts secretory processes in GB have not been explored yet. In particular, potential interactions between EBP50 and TGR5 may couple BA signaling on the one hand, to channels and transporters function and expression on the other hand.
Preliminary results strongly suggest a regulatory role of EBP50 in biliary homoeostasis, and therefore will provide the scientific basis of the current project. Mice with targeted EBP50 deletion are viable, develop normally and do not show any gross nor histological signs of liver disease. However, EBP50-KO mice have a significantly larger GB than their WT littermate controls with decreased bile flow, increased plasma total BA concentration and hydrophobicity and, obvious signs of GB inflammation. In line with epithelial hyperplasia observed on GB tissue sections, BEC proliferation is in addition significantly increased in EBP50-KO mice GB.
All together, these preliminary data let us to hypothesize that EBP50 regulates biliary homeostasis by acting on hepatobiliary secretion and/or BEC proliferation. Our objective is to study the mechanisms by which EBP50 operates in this context. In addition, cross-talk between EBP50 and TGFR5 will be analyzed since the two proteins are highly expressed in the GB and are both coupled to CFTR and EGFR regulation. Biliary homeostasis will be studied in EBP50-KO mice in basal conditions and in pathophysiological settings during which a biliary adaptive response to BA overload is shown to occur, along with BEC proliferation (e.g. liver regeneration after partial hepatectomy and bile duct-ligation). In a translational perspective, studies on human tumor and non-tumor cholangiopathies will be performed.