Cystic fibrosis transmembrane conductance regulator: Actin(g) as a master regulator of cholangiocyte function
Authors: Andrew P. Feranchak
Journal: Hepatology (2017)

Potential conflict of interest: Nothing to report. See Article On Page 972 Cystic fibrosis (CF) is an autosomal‐recessive disease characterized by abnormal electrolyte transport in the epithelial cells of many organs. Affecting approximately 1 in every 2,400 live births, it is one of the most common genetic diseases in the white population. The disease is attributed to a mutation in the gene for the cystic fibrosis transmembrane conductance regulator (CFTR), a membrane protein that functions as an anion channel in the epithelial cells of the airway, pancreas, intestine, biliary tract, sweat ducts, and vas deferens. There are now over 2,000 recognized mutations of the CFTR gene (CF Mutation Database; http://www.genet.sickkids.on.ca). The various CFTR gene mutations may be classified according to their effect on CFTR protein function. Class I mutations cause impairment of CFTR mRNA production. Class II mutations result in defective processing or trafficking of CFTR protein to the apical membrane. The most common mutation, ΔF508, is of this class and results in an abnormally folded protein that is subsequently diverted from normal trafficking and subsequently degraded by the ubiquitin‐proteosome pathway. Class III mutations are associated with defective regulation of CFTR, which locates correctly to the apical membrane, but does not respond normally to cAMP. Class IV mutations demonstrate some residual Cl– conductance, but at a significantly decreased amplitude, whereas Class V mutations lead to abnormal splicing of CFTR with partial reduction in the number of functioning Cl– channels. Mutations of Classes I, II, and III are considered severe mutations because they result in an absence of functioning CFTR at the plasma membrane, whereas Classes IV and V are considered mild mutations attributed to the presence of some residual CFTR activity. Therapies for CF have been, or are being, developed directly targeting the effects of these mutations on the trafficking and regulation of the CFTR channel protein. Liver disease is a significant complication of CF and is now the third‐leading cause of death in patients with the disease.1 Although autopsy studies suggest that the majority of, if not all, patients with CF have some histological findings suggestive of liver disease, only 10%‐15% develop clinically significant liver disease with portal hypertension. Additionally, there is not an apparent genotype‐phenotype correlation, and patients with the same mutation in the gene for the CFTR may have significantly variable clinical manifestations of cystic fibrosis–associated liver disease (CFLD). Moreover, although pulmonary infection is a major contributor to the lung phenotype, there is no evidence that infection of the liver (e.g., cholangitis) modulates the development or progression of CFLD. In the liver, CFTR is only expressed in biliary epithelial cells (cholangiocytes), not in hepatocytes, where it functions as a cAMP‐activated Cl– channel in the apical membrane.2 The resulting efflux of Cl– from the apical membrane drives Cl–/ HCO3− exchange and resultant water efflux through aquaporin channels, contributing to the alkalinization and dilution of bile. Thus, Cl– efflux at the cholangiocyte apical membrane provides the driving force for ductular secretion. In addition to CFTR, cholangiocytes also express transmembrane member 16A (TMEM16A), also known as anoctamin or ANO‐1, a Ca2+‐activated Cl– channel.3 In contrast to CFTR, which is regulated by basolateral receptors, TMEM16A is regulated by extracellular adenosine triphosphate (ATP)‐binding apical purinergic (P2) receptors. Importantly, in single cells and biliary epithelial monolayers, the ATP‐stimulated Ca2+‐activated Cl– currents are 2‐3 times greater than the cAMP‐activated Cl– currents mediated by CFTR.3 Thus, it is unclear why an absent or abnormal CFTR protein leads to liver disease in some patients, given the existence of this alternative Cl– secretory pathway, suggesting additional "nonchannel" roles for CFTR in cholangiocytes. From studies in various epithelia, it is clear that CFTR regulates the activity of many diverse apical membrane proteins, including epithelial Na+ channel (ENaC), Cl–/ HCO3− exchangers, Ca2+‐activated Cl– channels, and ATP release mechanisms. Although the mechanism through which CFTR regulates these other transport pathways is far from understood, the formation of actin‐based macromolecular signaling complexes at the apical membrane appears to underlie many of these processes. CFTR forms these complexes, critical for membrane transport activity, through interactions with actin through intermediary actin‐binding proteins, thus forming a scaffolding network in which channels and regulatory kinases are held in close proximity. Loss of CFTR protein at the apical cholangiocyte membrane disrupts this network, resulting in abnormalities in several transport activities including ATP release5 and Na+ absorption through altered ENaC activity.6 This shift from a "secretory" to an "absorptive" phenotype may promote bile stasis and bile duct plugging, the histological hallmark of CFLD. In this issue, the studies by Fiorotto et al.7 build on their earlier work in mouse models and provide additional evidence that CFTR is indeed a master regulator at the cholangiocyte apical membrane in human‐derived biliary epithelial cells. For these elegant studies, the investigators have developed a new and novel model of human CF cholangiocytes using induced pluripotent stem cells (iPSCs) derived from healthy control and ΔF508 CFTR patients. Utilizing defined cell‐culture techniques, the investigators have demonstrated that they can differentiate human iPSCs into polarized cholangiocyte monolayers. Furthermore, these human cholangiocyte monolayers not only maintain polarized expression of biliary‐specific markers in culture, but also exhibit robust secretory responses as expected of primary cells. The significance of this model should not be underappreciated given that reliable human biliary epithelial models have been difficult to develop given the intrahepatic location and small number of cholangiocytes within the liver (representing < 5% of the nuclear mass of the liver). Utilizing this novel model, the investigators demonstrate that in human ΔF508‐expressing biliary epithelia, a number of abnormalities exist that directly affects biliary function (Fig. 1). First, the absence of a normal CFTR protein in the apical membrane leads to disruption of actin. Second, the investigators show that loss of CFTR leads to the phosphorylation of the tyrosine kinase, Src. The activation of Src appears to result from the disruption of the actin cytoskeleton, allowing Src, usually bound by actin in an inactive state, to self‐assemble. Activated Src leads to a series of downstream events, including activation of Toll‐like receptor 4 (TLR‐4) and nuclear factor kappa B (NF‐κB) signaling pathways, resulting in the elaboration of the proinflammatory cytokines, monocyte chemoattractant protein 1 (MCP‐1) and interleukin‐8 (IL‐8). Importantly, the investigators demonstrate that pharmacological inhibition of Src prevents the downstream effects, reverses the cytoskeletal changes, and improves CFTR‐mediated fluid secretion in response to small‐molecule drugs used to correct and potentiate ΔF508 CFTR.Figure 1: Model of CFTR as a master regulator of cholangiocyte transport. Proposed mechanism of CFTR‐regulated cholangiocyte transport during normal conditions (top panel) and during cystic fibrosis (bottom panel). CFTR is a cAMP‐ and PKA‐activated Cl– channel in the apical membrane of cholangiocytes. Through interactions with the actin cytoskeleton, CFTR also regulates other membrane transport activities, including ATP release, activation of the Ca2+‐activated channel, TMEM16A, and inhibition of the ENaC. In CF, absence of CFTR in the apical membrane leads to disruption of the actin cytoskeleton and loss of cAMP‐mediated Cl– secretion, ATP release, and ATP‐stimulated TMEM16A Cl– secretion. Loss of CFTR also leads to increased Na+ absorption through ENaC. Additionally, the disruption of the actin cytoskeleton results in phosphorylation of Src tyrosine kinase, leading to activation of TLR4 and NF‐κB signaling with elaboration of the proinflammatory cytokines, MCP‐1 and IL‐8 (see Fiorotto et al., this issue).If these studies, performed in human in vitro epithelial models, translate to in vivo conditions, several implications arise. First, it is still unclear why only a subset of patients with CF, even with the same mutation (e.g., ΔF508), develop clinically significant CFLD. Presumably, the loss of CFTR, leading to actin disruption and increased TLR4 and NF‐κB activity, would affect all patients with CFTR mutations that result in absent CFTR protein in the membrane. However, based on the investigators' work, one could speculate that perhaps individual differences in the immune response may account for some of the differences in the clinical manifestations of CFLD. Second, it is unknown in humans whether the absence of CFTR, with subsequent Src‐mediated TLR4 and NF‐κB signaling, is enough to develop CFLD or if a "second hit" is required. For example, the investigators have previously demonstrated that similar operative pathways are found in mice, though Cftr–/– mice or ΔF508‐expressing mice failed to develop histological evidence of liver injury unless a second inflammatory hit was applied (dextran sodium sulfate–induced colitis in their model).8 If a second hit is required, what might this be? Pulmonary infection? Altered intestinal microbiome? Other? Regardless, the investigators' work provides evidence that, within the liver, CFTR should be considered an important member of the innate immune response. Third, the cellular mechanism by which CFTR regulates ATP release, and thus alternate secretory pathways, requires further investigation. The investigators have previously shown that Cftr–/– mouse cholangiocytes demonstrate abnormal ATP release,5 though the mechanism is unknown. Given that the exocytosis of ATP‐containing vesicles is a pathway for cholangiocyte ATP release,9 disruption of the actin cytoskeleton would be expected to alter the exocytic machinery and thereby affect normal vesicle fusion and/or release. Elucidating these pathways will be an important area for future investigation. Last, an important implication of these studies is that restoring the Cl– secretory function of CFTR at the apical membrane may not be sufficient to prevent CFLD. Additionally, the finding that the compounds VX‐770 and VX‐809, correctors and potentiators, respectively, did not improve fluid secretion in ΔF508‐expressing human cholangiocytes raises questions about their potential usefulness in the treatment of CFLD, though clinical results are needed. The finding that addition of a Src inhibitor to the VX‐770 and VX‐809 regimen restored secretion in the ΔF508 cholangiocytes is exciting, and demonstrates that targeting the respective regulatory kinases is as important as targeting the trafficking and gating of the Cl– channel itself. Overall, the work of Fiorotto et al. advances the field and provides further evidence that in human biliary epithelium, the CFTR, through actin‐dependent interactions, is a master orchestrator conducting apical membrane transport, immunity, and protein networks with fidelity and efficiency. Moreover, the development of iPSCs to model human liver disease is a powerful tool that can be used to screen potential treatments for CFLD as well as study the cellular mechanisms of other human cholangiopathies. Author names in bold designate shared co‐first authorship.