Molecular pathophysiology of cholangiopathies: lessons from polycystic and fibropolycystic liver disease

Cholangiocytes, the epithelial cells lining the intra- and extrahepatic biliary tree, play an essential role in regulating the production of bile and digestive functions. Cholangiocytes are also involved in the repair from liver damage and are also the target of cholangiopathies a group of acquired, congenital, or genetic complex chronic liver diseases , that represents a substantial cause of morbidity and mortality and a frequent indication for liver transplant. Treatment of cholangiopathies is unsatisfactory and these diseases are among the main unmet needs in modern hepatology. Cholangiocytes in addition of being the target of the disease, also contribute to disease development. After biliary damage cholangiocytes, become “reactive”, acquire the ability to produce a number of cytokines and inflammatory mediators and growth factors and to promote further inflammation and portal fibrosis that ultimately may lead to cirrhosis and chronic liver failure. A hallmark of this process is the activation of intracellular morphogenetic signaling, such as the Notch, Wnt/β-catenin and VEGF pathways that are transiently involved in the development of the biliary epithelium during embryogenesis. The understanding of the mechanism underlying the reactivation of these intracellular signaling pathways has been boosted thanks to the availability of animal models that phenocopy the congenital and genetic cholangiopathies. These translational tools improved the knowledge of the pathophysiology of cholangiocytes and provided experimental therapeutic strategies. Acquired cholangiopathies are complex diseases for which only unsatisfactory animal and cellular models are available. Therefore, the approach taken by our laboratory has been to study the pathogenesis of genetic cholangiopathies with an identified causative gene and derive “lessons” that can be applied to the more general field of biliary and liver diseases. During the Ph.D. program, I mainly focused on investigating the molecular mechanism that drives the progression of two inherited cholangiopathies: 1) the polycystic liver disease associated with autosomal dominant polycystic kidney disease (PLD-ADPKD), in which the abnormal proliferation of cystic cholangiocytes is associated with changes in intracellular Ca2+ homeostasis and with the reactivation of biliary angiogenetic signals and, 2) the fibro-polycystic disease (Caroli Disease and Congenital Hepatic Fibrosis), where the aberrant lack of fibrocystin leads to activation of β-Catenin, chemokine secretion and recruitment of macrophages and fibrogenic mesenchymal cells , leading to liver fibrosis and portal hypertension. The thesis is divided in two parts; for both parts, the experimental methodology used is reported in the method section. In part I, we show that polycystin 2 (PC2), the ciliary protein affected in PLD-ADPKD, is a regulator of Ca2+ homeostasis and cAMP signaling in cholangiocytes. Expression of PC2 is essential for cytoplasmic and endoplasmic reticulum (ER) calcium handling. As PC2-deficient cells are also characterized by increased cAMP/PKA signaling and by Ras/Raf/ ERK/mTOR/HIF1α-dependent VEGF secretion, we aimed at understanding the relationships between altered Ca2+ homeostasis and increased cAMP production. We found that increased cAMP production is sustained by Adenylyl Cyclase 5 (AC5), an AC that is inactive at the normal intracellular Ca2+ concentrations,but it is activated at the decreased intracellular Ca2+ concentrations measured in PC2-deficient cystic cholangiocytes. As activation of AC5 was also dependent on STIM1 (a ER calcium sensor protein responsible for activation of the store-operated Ca2+ entry) we also concluded that the absence of PC2, intracellular Ca2+ store depletion promotes the interaction of STIM1 with AC5, resulting in aberrant cAMP production, stimulation of cystic cholangiocyte proliferation and cyst growth via Ras/Raf/ERK/mTOR/HIF1α-dependent VEGF secretion. These findings indicate that AC5 is an attractive target for therapy, as also demonstrated by inhibition of cyst growth in mice treated with the AC5 inhibitor SQ22,536. (Part of this work has been published. Spirli C., et al., Journal of Hepatology, 2017) In part II, we studied how fibrosis develops in Caroli Disease and Congenital Hepatic Fibrosis, two variants of the fibropolycystic diseases resulting from congenital deficiency of fibrocystin and characterized by biliary cysts and severe liver fibrosis. In prior studies we showed that in this condition, fibrosis was not a consequence of necroinflammatory damage to the biliary tree, but rather of a low grade chronic inflammation originating from the fibrocystin-deficient cholangiocytes and the consequent recruitment of macrophages. As macrophages recruitment depended on increased secretion of CXCL10 by fibrocystin-defective cholangiocytes we aimed to better understand the link between fibrocystin deficiency and increased CXCL10 secretion. We found that in the absence of fibrocystin, CXCL10 secretion is mediated by β-catenin nuclearization through phosphorylation at the Serine675 and by the abnormal secretion of IL-1β through the activation of NLRP3 inflammasome complex. These findings suggest that periportal fibrosis can be generated by a chronic low level inflammation originating from the fibrocystin deficient cholangiocytes and open new therapeutic avenues aiming at targeting β-catenin signaling and the inflammasome. (This work has been published. Kaffe E. et al., Hepatology, 2018) The identification of these pathobiological mechanisms has clarified some general aspects of the sequence of events leading from cholangiocyte dysfunction to hyperplasia and biliary fibrosis. This process is the result of increased cAMP levels, secretion of pro-inflammatory chemokines and angiogenetic factors, and macrophage recruitment; all these steps which may be targeted also for the treatment of acquired cholangiopathies that share common pathobiological traits