The cystic fibrosis transmembrane conductance regulator controls biliary epithelial inflammation and permeability by regulating Src tyrosine kinase activity

Abstract

In the liver, CFTR regulates bile secretion and other functions at the apical membrane of biliary epithelial cells (i.e cholangiocytes). CF-related liver disease (CFLD) is a major cause of death in patients with CF. CFTR dysfunction affects innate immune pathways, generating a para-inflammatory status in the liver, and other epithelia. This study investigates the mechanisms linking CFTR to TLR4 activity. We found that CFTR is associated in a multi-protein complex at the apical membrane of normal mouse cholangiocytes, with proteins that negatively control Src activity. In CFTR-defective cholangiocytes, Src tyrosine kinase self-activates and phosphorylates TLR4, resulting in activation of NF-κB, and increased pro-inflammatory cytokines production in response to endotoxins. This Src/NF-κB-dependent inflammatory process attracts inflammatory cells, but also generates changes in the apical junctional complex and loss of epithelial barrier function. Inhibition of Src decreased the inflammatory response of CF-cholangiocytes to LPS, rescued the junctional defect in-vitro and significantly attenuated endotoxin-induced biliary damage and inflammation in vivo (Cftr-KO mice). Conclusion Our findings reveal a novel function of CFTR as regulator of TLR4 responses and cell polarity in biliary epithelial cells. This mechanism is pathogenetic, as shown by the protective effects of Src inhibition in vivo and maybe a novel therapeutic target in CFLD and other inflammatory cholangiopathies.

Animal models of biliary injury and altered bile acid metabolism

“…LPS [75,77]. In addition, aberrant activation of Src decreases the epithelia barrier function by destabilizing cell junctional complexes [77,78]. …”

Diet‐Induced Dysbiosis and Genetic Background Synergize With Cystic Fibrosis Transmembrane Conductance Regulator Deficiency to Promote Cholangiopathy in Mice

“…Likewise, it has been reported that intestinal permeability was abnormal in virtually all (i.e., 96%) CF subjects . Acting as a hub, CFTR also stabilizes the apical junction complex through actin‐dependent interactions in epithelial cells so that the loss of apical protein networks orchestrated by CFTR has emerged as a common mechanism of increased permeability in CF epithelia, including gut and bile ducts . A disruption of tight junctions that could explain increased permeability has thus been demonstrated in the small intestine of CF mice …”

“…In addition to its channel function, CFTR was also recently shown to operate as a hub for cytoskeletal elements, transmembrane proteins, and signaling molecules . In cholangiocytes, CFTR promotes the assembly of a protein complex that maintains the tyrosine kinase Rous sarcoma oncogene cellular homolog (Src) in an inactive state . Thus, in the absence of CFTR at the apical membrane, this complex does not assemble, resulting in the self‐activation of Src and subsequent increase in TLR4 and NF‐B signaling in response to endotoxins .…”

“…In cholangiocytes, CFTR promotes the assembly of a protein complex that maintains the tyrosine kinase Rous sarcoma oncogene cellular homolog (Src) in an inactive state . Thus, in the absence of CFTR at the apical membrane, this complex does not assemble, resulting in the self‐activation of Src and subsequent increase in TLR4 and NF‐B signaling in response to endotoxins . Nonetheless, CF mouse models in which these abnormalities were demonstrated did not develop bile duct damage or portal inflammation unless a second hit (i.e., dextran sodium sulfate–induced colitis) was applied .…”

Recent Advances in Practical Methods for Liver Cell Biology: A Short Overview

“…In contrast, BEC can be more purified but cannot proliferate in culture. Recently, it has been shown that primary cultures of mouse cholangiocytes could be cultured on rat tail collagen for several passages [81] and used for specific functional studies with transgenic mice [82].…”