Ezrin finds its groove in cholangiocytes
Authors: Laura Fouassier, Romina Fiorotto
Journal: Hepatology (2014)

Ezrin was first detected in epidermal growth factor–treated skin epithelial cells as a protein tyrosine kinase substrate prior to its purification and characterization as a structural component of microvilli of chicken intestinal epithelial cell brush borders.1, 2 Subsequently, other closely related proteins, including radixin and moesin, were identified that together with ezrin constitute the ERM protein family. Family members have the ability to crosslink proteins of the plasma membrane with the subcortical cytoskeleton and are important for the stabilization of the cell cortex structure and regulation of several signal transduction pathways.3 The ERM proteins are localized at the interface between the plasma membrane and the cortical actin cytoskeleton and organized into three functional domains: an N-terminal domain (four point one ERM, or FERM), an extended coiled-coil region, and a short C-terminal domain. Through the C-terminal region, ERM proteins bind directly to F-actin, whereas binding to transmembrane proteins that occurs directly or indirectly (i.e., NHE3, cystic fibrosis conductance transmembrane regulator [CFTR]) is achieved through the FERM domain. Indirect binding of ERM proteins with transmembrane proteins occurs through adaptor proteins such as ERM-binding phosphoprotein 50 (EBP50, also termed NHERF1 for Na+/H+ exchanger regulatory factor 1) or NHERF2 (i.e., E3KARP). Interestingly, the expression and localization of ezrin and EBP50 are interdependent. Indeed, in intestinal epithelial cells of Ez–/– mice, EBP50 is no longer apically concentrated and instead is diffusely localized in the cytoplasm.4 Conversely, in EBP50–/– mice, expression of ezrin is reduced in the brush border membrane of both kidney proximal tubules and small intestine cells.5 In the liver, ERM proteins are differentially expressed between the epithelial cell populations, i.e., hepatocytes and biliary cells. Moesin is expressed in hepatocytes and not in biliary epithelial cells (BECs).6 However, both populations express radixin, whereas ezrin is exclusively expressed in the apical domain of BECs.6, 7 The specific expression pattern of ezrin already exists in the developing liver, where ezrin is detected only in cells of the ductal plate and early bile ducts, indicating that ezrin is specific to the biliary lineage. However, thus far, the biliary functions of ezrin have remained unexplored. In this issue of Hepatology, Hatano et al.8 shed some light on this matter by analyzing the consequences of ezrin deficiency on liver physiology. Ezrin-deficient mice (Vil2kd/kd) exhibit a marked liver phenotype characterized by a cholestatic liver injury. Cholestasis observed in ezrin-deficient mice is primarily caused by a deregulation of biliary secretory function since no morphological abnormalities were observed in BECs, suggesting that ezrin may not be essential for the integrity of the biliary epithelium. This observation should be more carefully addressed since it is slightly in contrast with what has been previously described in the intestine,4 where deletion of the mouse ezrin gene yields severe morphological consequences both in the developing intestinal epithelium and in intestinal homeostasis in the adult (i.e., incomplete villus morphogenesis, junctional remodeling, cell geometry). A few points could be raised to explain this incongruence. First, a very low residual level of ezrin expression is detectable in Vil2kd/kd mice, which could be responsible for a milder phenotype in the liver. Second, ezrin has different roles in different tissues, and other ERM members (i.e., radixin) may partially substitute for its function in biliary cells. Moreover, ezrin deletion in the intestine affects not only the apical villi structure but also the cell-junction organization. In fact, functional ERM proteins are also important for the organization of the F-actin cytoskeleton, which is tightly associated with proteins of the apical junction complex. One can speculate that evaluation of the morphology of the apical junction complex in the liver of ezrin-defective mice might have revealed a similar defect. Indeed, it is well known that the functional integrity of cell junctions is frequently impaired in cholestasis.9 The work from Hatano et al.8 clearly shows that the absence of ezrin interferes with the physiological function of BECs or cholangiocytes. The biliary epithelium is mainly involved in the regulation of the fluidity and alkalinity of the primary canalicular bile secreted by the hepatocytes. This function depends on a number of specific transport systems and ion channels, the most important of which are the anion exchanger-2 (AE-2) and the protein kinase A (PKA)–regulated chloride channel CFTR, both expressed in the apical membrane of cholangiocytes and whose activities are tightly coupled.10 Hatano et al. show that the apical expression of CFTR and EBP50 proteins is reduced in BECs of ezrin-deficient mice, suggesting that in the absence of ezrin, the CFTR–EBP50–ezrin complex is disrupted and PKA-dependent signaling is impaired, leading to mislocalization and deregulation of CFTR. Thus, ezrin appears to be an essential functional organizer of the subapical membrane. This is in line with evidence demonstrating that ezrin controls PKA-mediated phosphorylation of CFTR by completing the function of A-kinase anchoring protein, a protein binding the regulatory subunit of PKA, therefore localizing the kinase in proximity to CFTR.11, 12 But what happens if EBP50 or CFTR is removed from the complex? Does the liver phenotype reproduce the one described in ezrin-deficient mice? Although the phenotype and the secretory function of BECs have not been investigated in the liver of EBP50-deficient mice, in vitro studies in a human BEC cell line demonstrate that EBP50 regulates PKA-dependent chloride secretion.13 Moreover, since EBP50 is also expressed in hepatocytes, loss of EBP50 also leads to a reduced bile acid–independent bile flow linked to a lower expression of the MRP2 protein in hepatocytes,14 which would imply a more severe cholestatic phenotype. However, in contrast to ezrin-deficient mice, no significant elevation of plasma concentrations of liver enzymes is observed in EBP50-deficient mice (L. Fouassier, unpublished data), indicating an absence of cholestasis. One explanation that may be raised is the existence of compensatory mechanisms such as the up-regulation of EBP50-related proteins (NHERF2).5 Interestingly, EBP50 expression has been shown to be aberrantly distributed in the liver from patients with cystic fibrosis, primary biliary cholangitis, and primary sclerosing cholangitis and a similar redistribution was also confirmed in the proliferating biliary cells of bile duct–ligated rats, a model of intrahepatic cholestasis.15 Similar to ezrin, CFTR is selectively expressed by BECs in the liver; and although CFTR has a major role in bile secretion, CFTR-deficient mice do not spontaneously develop liver disease.16 Accordingly, only a small percentage of cystic fibrosis patients progress to severe liver disease, in spite of a defective biliary secretory function. In this regard, it has been recently reported17 that absence of CFTR at the membrane affects the innate immune properties of the biliary epithelium in response to bacterial products. In fact, lack of CFTR increases the TLR4-mediated response to endotoxins of the biliary epithelium, causing biliary damage and inflammation. Induction of chemical colitis in Cftr-knockout mice by treatment with dextran sodium sulfate causes a biliary injury with proliferation of bile ducts similar to the ezrin-defective mouse, suggesting that the secretory defect is a predisposing factor to further damage and that a second hit is necessary to develop the disease. However, differently from Hatano et al.,8 a strong periportal infiltration of neutrophils and macrophages was described in Cftr-knockout mice treated with dextran sodium sulfate. There is a possibility that ezrin-defective mice exposed, for example, to endotoxins would potentially develop an inflammatory phenotype as well. How is CFTR linked with TLR4 and innate immunity in biliary cells? Unpublished data suggest that CFTR, through its association with EBP50, might participate in the regulation of TLR4 signaling transduction.18 In ezrin-deficient mice, additional transporters other than CFTR are deregulated, which may explain the development of cholestasis. About a decade ago, it was suggested that CFTR, AE-2, and aquaporin 1 (AQP1) were coregulated since they colocalized in intracellular vesicles in the subapical domain of BECs. Furthermore, the cyclic adenosine monophosphate/PKA pathway regulated insertion of these vesicles to the apical plasma membrane of the cells.19 Interestingly, ezrin-deficient mice display an accumulation of subapical vesicles in BECs, which is correlated with decreased apical localization of CFTR, AE-2, AQP1, and EBP50. In vitro, loss of ezrin function in isolated BECs causes an impairment of translocation in the apical membrane of the transporters in basal and stimulated conditions after PKA activation. Ezrin may therefore be an underlying mechanism for trafficking and/or stabilization of the transporters to the apical membrane and for their regulation through discrete compartmentalization of PKA. Nonetheless, the molecular mechanisms by which ezrin anchors AQP1 and AE-2 to the cortical F-actin cytoskeleton deserve further investigations. In conclusion, the study by Hatano et al.8 adds another piece to the puzzle of the pathogenesis of ductal cholestasis by defining the importance of ezrin in the regulation of bile secretory mechanisms. Indeed, multiple studies suggest that transporters involved in bile secretion are organized in macromolecular complexes and that their interaction with the cytoskeleton mediated by accessory proteins (i.e., EBP50, ezrin) plays a critical role in coordinating their function (Fig. 1). Moreover, these macromolecular complexes also contain signaling molecules and kinases, and therefore, their regulation may provide a link with other key functions of biliary cells (i.e., cell polarity, innate immunity, proliferation, inflammation). In future studies, it will be important to dissect these interactions and how they account for the broad spectrum of cholangiopathies. Proposed model for the regulation of bile secretion by ezrin in cholangiocytes. Ezrin is localized at the apical actin-rich region of cholangiocytes, where it contributes to the organization of multiprotein complexes. Ezrin regulates the membrane localization and the activation of CFTR. The FERM domain of ezrin anchors CFTR indirectly through the PDZ protein EBP50, whereas the C-terminal domain interacts with F-actin to promote the stabilization of the channel at the membrane. Ezrin can bind additional proteins such as PKA, thereby contributing to the regulation of CFTR. By acting as a protein kinase A anchoring protein, ezrin positions the PKA near CFTR to be phosphorylated and activated. Furthermore, the activation of CFTR is spatially and functionally coordinated with the activation of AE-2 and AQP1. This secretory complex already colocalizes in intracellular vesicles whose trafficking and membrane insertion are regulated through the interaction with actin cytoskeleton and by PKA activation. The involvement of ezrin in the insertion/stabilization and regulation of AE-2 and AQP1 is currently undetermined. Thus, the architecture of the subcortical cytoskeleton and the distribution and retention of proteins at the apical membrane are important signals to maintain the apical polarity and the secretory functions of the epithelium. Abbreviations: AE-2, anion exchanger 2; AQP1, aquaporin 1; CFTR, cystic fibrosis conductance transmembrane regulator; EBP50, ERM-binding phosphoprotein 50; ERM, ezrin-radixin-moesin; FERM, four point one ERM; PKA, protein kinase A; PDZ, postsynaptic density protein (PSD95)-Drosophila disc large tumor suppressor (Dlg1)-zonula occludens-1 protein (ZO-1). We thank Yves Chrétien for the graphic support. Laura Fouassier, Ph.D.1,2Romina Fiorotto, Ph.D.3 1INSERM, UMR S 938 Centre de Recherche Saint-Antoine Paris, France 2Sorbonne Universités UPMC Université de Paris 6 UMR S 938 Centre de Recherche Saint-Antoine Paris, France 3Section of Digestive Diseases Liver Center Yale University School of Medicine New Haven, Connecticut Author names in bold designate shared co-first authorship.