Valeria Mariotti

Adenylyl cyclase 5 links changes in calcium homeostasis to cAMP-dependent cyst growth in polycystic liver disease

BACKGROUND & AIMS Genetic defects in polycystin-1 or -2 (PC1 or PC2) cause polycystic liver disease associated with autosomal dominant polycystic kidney disease (PLD-ADPKD). Progressive cyst growth is sustained by a cAMP-dependent Ras/ERK/HIFα pathway, leading to increased vascular endothelial growth factor A (VEGF-A) signaling. In PC2-defective cholangiocytes, cAMP production in response to [Ca2+]ER depletion is increased, while store-operated Ca2+ entry (SOCE), intracellular and endoplasmic reticulum [Ca2+]ER levels are reduced. We investigated whether the adenylyl cyclases, AC5 and AC6, which can be inhibited by Ca2+, are activated by the ER chaperone STIM1. This would result in cAMP/PKA-dependent Ras/ERK/HIFα pathway activation in PC2-defective cells, in response to [Ca2+]ER depletion. METHODS PC2/AC6 double conditional knockout (KO) mice were generated (Pkd2/AC6 KO) and compared to Pkd2 KO mice. The AC5 inhibitor SQ22,536 or AC5 siRNA were used in isolated cholangiocytes while the inhibitor was used in biliary organoid and animals; liver tissues were harvested for histochemical analysis. RESULTS When comparing Pkd2/AC6 KO to Pkd2 KO mice, no decrease in liver cyst size was found, and cellular cAMP after [Ca2+]ER depletion only decreased by 12%. Conversely, in PC2-defective cells, inhibition of AC5 significantly reduced cAMP production, pERK1/2 expression and VEGF-A secretion. AC5 inhibitors significantly reduced growth of biliary organoids derived from Pkd2 KO and Pkd2/AC6 KO mice. In vivo treatment with SQ22,536 significantly reduced liver cystic area and cell proliferation in PC2-defective mice. After [Ca2+]ER depletion in PC2-defective cells, STIM1 interacts with AC5 but not with Orai1, the Ca2+ channel that mediates SOCE. CONCLUSION [Ca2+]ER depletion in PC2-defective cells activates AC5 and results in stimulation of cAMP/ERK1-2 signaling, VEGF production and cyst growth. This mechanism may represent a novel therapeutic target. LAY SUMMARY Polycystic liver diseases are characterized by progressive cyst growth until their complications mandate surgery or liver transplantation. In this manuscript, we demonstrate that inhibiting cell proliferation, which is induced by increased levels of cAMP, may represent a novel therapeutic target to slow the progression of the disease.

Posttranslational regulation of polycystin-2 protein expression as a novel mechanism of cholangiocyte reaction and repair from biliary damage.

Polycystin‐2 (PC2 or TRPPC2), a member of the transient receptor potential channel family, is a nonselective calcium channel. Mutations in PC2 are associated with polycystic liver diseases. PC2‐defective cholangiocytes show increased production of cyclic adenosine monophosphate, protein kinase A–dependent activation of the extracellular signal–regulated kinase 1/2 (ERK1/2) pathway, hypoxia‐inducible factor 1α (HIF‐1α)–mediated vascular endothelial growth factor (VEGF) production, and stimulation of cyst growth and progression. Activation of the ERK/HIF‐1α/VEGF pathway in cholangiocytes plays a key role during repair from biliary damage. We hypothesized that PC2 levels are modulated during biliary damage/repair, resulting in activation of the ERK/HIF‐1α/VEGF pathway. PC2 protein expression, but not its gene expression, was significantly reduced in mouse livers with biliary damage (Mdr2–/– knockout, bile duct ligation, 3,5‐diethoxycarbonyl‐1,4‐dihydrocollidine treatment). Treatment of cholangiocytes with proinflammatory cytokines, nitric oxide donors, and endoplasmic reticulum stressors increased ERK1/2 phosphorylation, HIF‐1α transcriptional activity, secretion of VEGF, and VEGF receptor type 2 phosphorylation and down‐regulated PC2 protein expression without affecting PC2 gene expression. Expression of homocysteine‐responsive endoplasmic reticulum–resident ubiquitin‐like domain member 1 protein and NEK, ubiquitin‐like proteins that promote proteosomal PC2 degradation, was increased. Pretreatment with the proteasome inhibitor MG‐132 restored the expression of PC2 in cells treated with cytokines but not in cells treated with nitric oxide donors or with endoplasmic reticulum stressors. In these conditions, PC2 degradation was instead inhibited by interfering with the autophagy pathway. Treatment of 3,5‐diethoxycarbonyl‐1,4‐dihydrocollidine mice and of Mdr2–/– mice with the proteasome inhibitor bortezomib restored PC2 expression and significantly reduced the ductular reaction, fibrosis, and phosphorylated ERK1/2. Conclusion: In response to biliary damage, PC2 expression is modulated posttranslationally by the proteasome or the autophagy pathway, and PC2 down‐regulation is associated with activation of ERK1/2 and an increase of HIF‐1α‐mediated VEGF secretion; treatments able to restore PC2 expression and to reduce ductular reaction and fibrosis may represent a new therapeutic approach in biliary diseases.(Hepatology 2015;62:1828–1839)

Src kinase inhibition reduces inflammatory and cytoskeletal changes in ΔF508 human cholangiocytes and improves cystic fibrosis transmembrane conductance regulator correctors efficacy

Cystic fibrosis transmembrane conductance regulator (CFTR), the channel mutated in cystic fibrosis (CF), is expressed by the biliary epithelium (i.e., cholangiocytes) of the liver. Progressive clinical liver disease (CF‐associated liver disease; CFLD) occurs in around 10% of CF patients and represents the third leading cause of death. Impaired secretion and inflammation contribute to CFLD; however, the lack of human‐derived experimental models has hampered the understanding of CFLD pathophysiology and the search for a cure. We have investigated the cellular mechanisms altered in human CF cholangiocytes using induced pluripotent stem cells (iPSCs) derived from healthy controls and a ΔF508 CFTR patient. We have devised a novel protocol for the differentiation of human iPSC into polarized monolayers of cholangiocytes. Our results show that iPSC‐cholangiocytes reproduced the polarity and the secretory function of the biliary epithelium. Protein kinase A/cAMP‐mediated fluid secretion was impaired in ΔF508 cholangiocytes and negligibly improved by VX‐770 and VX‐809, two small molecule drugs used to correct and potentiate ΔF508 CFTR. Moreover, ΔF508 cholangiocytes showed increased phosphorylation of Src kinase and Toll‐like receptor 4 and proinflammatory changes, including increased nuclear factor kappa‐light‐chain‐enhancer of activated B cells activation, secretion of proinflammatory chemokines (i.e., monocyte chemotactic protein 1 and interleukin‐8), as well as alterations of the F‐actin cytoskeleton. Treatment with Src inhibitor (4‐amino‐5‐(4‐chlorophenyl)‐7‐(t‐butyl)pyrazolo[3,4‐d]pyramidine) decreased the inflammatory changes and improved cytoskeletal defects. Inhibition of Src, along with administration of VX‐770 and VX‐809, successfully restored fluid secretion to normal levels. Conclusion: Our findings have strong translational potential and indicate that targeting Src kinase and decreasing inflammation may increase the efficacy of pharmacological therapies aimed at correcting the basic ΔF508 defect in CF liver patients. These studies also demonstrate the promise of applying iPSC technology in modeling human cholangiopathies. (Hepatology 2018;67:972–988)

Pathophysiologic implications of innate immunity and autoinflammation in the biliary epithelium

The most studied physiological function of biliary epithelial cells (cholangiocytes) is to regulate bile flow and composition, in particular the hydration and alkalinity of the primary bile secreted by hepatocytes. After almost three decades of studies it is now become clear that cholangiocytes are also involved in epithelial innate immunity, in inflammation, and in the reparative processes in response to liver damage. An increasing number of evidence highlights the ability of cholangiocyte to undergo changes in phenotype and function in response to liver damage. By participating actively to the immune and inflammatory responses, cholangiocytes represent a first defense line against liver injury from different causes. Indeed, cholangiocytes express a number of receptors able to recognize pathogen- or damage-associated molecular patterns (PAMPs/DAMPs), such as Toll-like receptors (TLR), which modulate their pro-inflammatory behavior. Cholangiocytes can be both the targets and the initiators of the inflammatory process. Derangements of the signals controlling these mechanisms are at the basis of the pathogenesis of different cholangiopathies, both hereditary and acquired, such as cystic fibrosis-related liver disease and sclerosing cholangitis. This article is part of a Special Issue entitled: Cholangiocytes in Health and Diseaseedited by Jesus Banales, Marco Marzioni, Nicholas LaRusso and Peter Jansen.

Src kinase inhibition reduces inflammatory and cytoskeletal changes in ΔF508 human cholangiocytes and improves CFTR correctors efficacy

Cystic fibrosis transmembrane conductance regulator (CFTR), the channel mutated in cystic fibrosis (CF), is expressed by the biliary epithelium (i.e., cholangiocytes) of the liver. Progressive clinical liver disease (CF‐associated liver disease; CFLD) occurs in around 10% of CF patients and represents the third leading cause of death. Impaired secretion and inflammation contribute to CFLD; however, the lack of human‐derived experimental models has hampered the understanding of CFLD pathophysiology and the search for a cure. We have investigated the cellular mechanisms altered in human CF cholangiocytes using induced pluripotent stem cells (iPSCs) derived from healthy controls and a ΔF508 CFTR patient. We have devised a novel protocol for the differentiation of human iPSC into polarized monolayers of cholangiocytes. Our results show that iPSC‐cholangiocytes reproduced the polarity and the secretory function of the biliary epithelium. Protein kinase A/cAMP‐mediated fluid secretion was impaired in ΔF508 cholangiocytes and negligibly improved by VX‐770 and VX‐809, two small molecule drugs used to correct and potentiate ΔF508 CFTR. Moreover, ΔF508 cholangiocytes showed increased phosphorylation of Src kinase and Toll‐like receptor 4 and proinflammatory changes, including increased nuclear factor kappa‐light‐chain‐enhancer of activated B cells activation, secretion of proinflammatory chemokines (i.e., monocyte chemotactic protein 1 and interleukin‐8), as well as alterations of the F‐actin cytoskeleton. Treatment with Src inhibitor (4‐amino‐5‐(4‐chlorophenyl)‐7‐(t‐butyl)pyrazolo[3,4‐d]pyramidine) decreased the inflammatory changes and improved cytoskeletal defects. Inhibition of Src, along with administration of VX‐770 and VX‐809, successfully restored fluid secretion to normal levels. Conclusion: Our findings have strong translational potential and indicate that targeting Src kinase and decreasing inflammation may increase the efficacy of pharmacological therapies aimed at correcting the basic ΔF508 defect in CF liver patients. These studies also demonstrate the promise of applying iPSC technology in modeling human cholangiopathies. (Hepatology 2018;67:972–988)

β-catenin and IL-1β dependent CXCL10 production drives progression of disease in a mouse model of Congenital Hepatic Fibrosis

Congenital hepatic fibrosis (CHF), a genetic disease caused by mutations in the polycystic kidney and hepatic disease 1 (PKHD1) gene, encoding for the protein fibrocystin/polyductin complex, is characterized by biliary dysgenesis, progressive portal fibrosis, and a protein kinase A–mediated activating phosphorylation of β‐catenin at Ser675. Biliary structures of Pkhd1del4/del4 mice, a mouse model of CHF, secrete chemokine (C‐X‐C motif) ligand 10 (CXCL10), a chemokine able to recruit macrophages. The aim of this study was to clarify whether CXCL10 plays a pathogenetic role in disease progression in CHF/Caroli disease and to understand the mechanisms leading to increased CXCL10 secretion. We demonstrate that treatment of Pkhd1del4/del4 mice for 3 months with AMG‐487, an inhibitor of CXC chemokine receptor family 3, the cognate receptor of CXCL10, reduces the peribiliary recruitment of alternative activated macrophages (cluster of differentiation 45+F4/80+ cells), spleen size, liver fibrosis (sirius red), and cyst growth (cytokeratin 19–positive area), consistent with a pathogenetic role of CXCL10. Furthermore, we show that in fibrocystin/polyductin complex–defective cholangiocytes, isolated from Pkhd1del4/del4 mice, CXCL10 production is mediated by Janus kinase/signal transducer and activator of transcription 3 in response to interleukin 1beta (IL‐1β) and β‐catenin. Specifically, IL‐1β promotes signal transducer and activator of transcription 3 phosphorylation, whereas β‐catenin promotes its nuclear translocation. Increased pro‐IL‐1β was regulated by nuclear factor kappa‐light‐chain‐enhancer of activated B cells, and increased secretion of active IL‐1β was mediated by the activation of Nod‐like receptors, pyrin domain containing 3 inflammasome (increased expression of caspase 1 and Nod‐like receptors, pyrin domain containing 3). Conclusion: In fibrocystin/polyductin complex–defective cholangiocytes, β‐catenin and IL‐1β are responsible for signal transducer and activator of transcription 3–dependent secretion of CXCL10; in vivo experiments show that the CXCL10/CXC chemokine receptor family 3 axis prevents the recruitment of macrophages, reduces inflammation, and halts the progression of the disease; the increased production of IL‐1β highlights the autoinflammatory nature of CHF and may open novel therapeutic avenues. (Hepatology 2018;67:1903‐1919).

Animal models of cholangiocarcinoma: What they teach us about the human disease.

Despite recent advances, pathogenesis of cholangiocarcinoma, a highly lethal cancer, remains enigmatic. Furthermore, treatment options are still limited and often disappointing. For this reason, in the last few years there has been a mounting interest towards the generation of experimental models able to reproduce the main features associated with this aggressive behavior. Toxic and infestation-induced, genetically engineered and cell implantation rodent models have been generated, contributing to a deeper understanding of the complex cell biology of the tumor, sustained by multiple cell interactions and driven by a huge variety of molecular perturbations. Herein, we will overview the most relevant animal models of biliary carcinogenesis, highlighting the methodological strategy, the molecular, histological and clinical phenotypes consistent with the human condition, their particular strengths and weaknesses and the novel therapeutic approaches that have been developed.

Liver diseases in the dish: iPSC and organoids as a new approach to modeling liver diseases

Liver diseases negatively impact the quality of life and survival of patients, and often require liver transplantation in cases that progress to organ failure. Understanding the cellular and molecular mechanisms of liver development and pathogenesis has been a challenging task, in part for the lack of adequate cellular models directly relevant to the human diseases. Recent technological advances in the stem cell field have shown the potentiality of induced pluripotent stem cells (iPSC) and liver organoids as the next generation tool to model in vitro liver diseases. Hepatocyte-like cells and cholangiocyte are currently being generated from skin fibroblasts and mononuclear blood cells reprogrammed into iPSC and have been successfully used for disease modeling, drug testing and gene editing, with the hope to be able to find application also in regenerative medicine. Protocols to generate other liver cell types are still under development, but the field is advancing rapidly. On the other end, liver cells can now be isolated from liver specimens (liver explants or liver biopsies) and cultured in specific conditions to form polarized 3D organoids. The purpose of this review is to summarize all these recent technological advances and their potential applications but also to analyze the current issues to be addressed before the technology can reach its full potential.

Animal models for cystic fibrosis liver disease (CFLD)

Liver disease is a severe complication in patients with Cystic Fibrosis (CF), a genetic disease caused by mutations in the gene encoding for cystic fibrosis transmembrane conductance regulator (CFTR) channel. The sequence of events leading to CFLD is still unclear and has limited the development of more specific treatments other than the bile acid UDCA. However, in the last twenty years, several gaps have been filled, which have mainly been possible due to the availability of different animal models that mimic CF. CF mice, although they lack a spontaneous liver manifestation, have been essential to better understand the multiple functions of CFTR expression on the apical membrane of cholangiocytes, from chloride channel to regulator of epithelial innate immunity. Additionally, we have learned that the gut microbiota might be a pathogenetic factor for the development of liver disease. The recent creation of novel CF animal models (i.e. pig and ferret) that better reproduce the human disease, will allow for comparative studies with species that spontaneously develop the liver disease and will hopefully lead to novel therapeutic treatments. In this review, we have compared and summarized the main features of the current available CF animal models and their applicability for the study of the liver phenotype.

Animal models of cholestasis: An update on inflammatory cholangiopathies

Cholestasis is a frequent clinical condition initiating or complicating chronic liver diseases, particularly cholangiopathies, where the biliary epithelium is the primary target of the pathogenetic sequence. Until a few decades ago, understanding of cholestasis relied mostly on the experimental model of bile duct ligation in rodents. However, a simple model of biliary obstruction cannot reproduce the complex mechanisms and networks leading to cholestasis in cholangiopathies. These networks are underpinned by an intricate dysregulation of pro-inflammatory and pro-fibrotic signals involving besides cholangiocytes, multiple cell elements of both innate and adaptive immunity. Therefore, in the last years, a wide range of animal models of biliary injury have been developed, mostly in mice, following three main approaches, chemical induction, immunization and genetic manipulation. In this review, we will give an update of the animal models of the two main cholangiopathies, primary sclerosing cholangitis and primary biliary cholangitis, which have provided us with the most relevant insights into the pathogenesis of these still controversial diseases.