Effects of Therapeutically Approved Individual Bile Acids on The Development of Metabolic Dysfunction-Associated Steatohepatitis a Low Bile Acid Mouse Model.

Bile acid (BA) signaling dysregulation is an important etiology for the development of Metabolic Dysfunction-associated Steatotic Liver Disease (MASLD). As diverse signaling molecules synthesized in the liver by pathways initiated with CYP7A1 and CYP27A1, BAs are endogenous modulators of farnesoid x receptor (FXR). FXR activation is crucial in maintaining BA homeostasis, regulating lipid metabolism, and suppressing inflammation. Additionally, BAs interact with membrane receptors and gut microbiota to regulate energy expenditure and intestinal health. Complex modulation of BAs in vivo and the lack of suitable animal models impede our understanding of the functions of individual BAs, especially during MASLD development. Previously, we determined that acute feeding of individual BAs differentially affects lipid, inflammation, and oxidative stress pathways in a low-BA mouse model, Cyp7a1/Cyp27a1 double knockout (DKO) mice. Currently, we investigated to what degree that cholic acid (CA), deoxycholic acid (DCA) or ursodeoxycholic acid (UDCA) at physiological concentrations impact MASLD development in DKO mice. The results showed that these three BAs varied in ability to activate hepatic and intestinal FXR, disrupt lipid homeostasis, and modulate inflammation and fibrosis. Additionally, UDCA activated intestinal FXR in these low-BA mice. Significant alterations in lipid uptake and metabolism in DKO mice following CA and DCA feeding indicate differences in cholesterol and lipid handling across genotypes. Overall, the DKO were less susceptible to weight gain, but more susceptible to MASH diet induced inflammation and fibrosis on CA and DCA supplement, while WT mice were more vulnerable to CA-induced fibrosis on control diet.

The arginine and nitric oxide metabolic pathway regulate the gut colonization and expansion of Ruminococcous gnavus.

Ruminococcus gnavus is a mucolytic commensal bacterium whose increased gut colonization has been associated with chronic inflammatory and metabolic diseases in humans. Whether R. gnavus metabolites can modulate host intestinal physiology remains largely understudied. We performed untargeted metabolomic and bulk RNA-seq analyses using R. gnavus monocolonization in germ-free mice. Based on transcriptome-metabolome correlations, we tested the impact of specific arginine metabolites on intestinal epithelial production of nitric oxide (NO) and examined the effect of NO on the growth of various strains of R. gnavus in vitro and in nitric oxide synthase 2 (Nos2)-deficient mice. R. gnavus produces specific arginine, tryptophan, and tyrosine metabolites, some of which are regulated by the environmental richness of sialic acid and mucin. R. gnavus colonization promotes expression of amino acid transporters and enzymes involved in metabolic flux of arginine and associated metabolites into NO. R. gnavus induced elevated levels of NOS2, while Nos2 ablation resulted in R. gnavus expansion in vivo. The growth of various R. gnavus strains can be inhibited by NO. Specific R. gnavus metabolites modulate intestinal epithelial cell NOS2 abundance and reduce epithelial barrier function at higher concentrations. Intestinal colonization and interaction with R. gnavus are partially regulated by an arginine-NO metabolic pathway, whereby a balanced control by the gut epithelium may restrain R. gnavus growth in healthy individuals. Disruption in this arginine metabolic regulation will contribute to the expansion and blooming of R. gnavus.

Characterization of individual bile acids in vivo utilizing a novel low bile acid mouse model.

Bile acids (BAs) are signaling molecules synthesized in the liver initially by CYP7A1 and CYP27A1 in the classical and alternative pathways, respectively. BAs are essential for cholesterol clearance, intestinal absorption of lipids, and endogenous modulators of farnesoid x receptor (FXR). FXR is critical in maintaining BA homeostasis and gut-liver crosstalk. Complex reactions in vivo and the lack of suitable animal models impede our understanding of the functions of individual BAs. In this study, we characterized the in vivo effects of three-day feeding of cholic acid (CA), deoxycholic acid (DCA), or ursodeoxycholic acid (UDCA) at physiological/non-hepatotoxic concentrations in a novel low-BA mouse model (Cyp7a1-/-/Cyp27a1-/-, DKO). Liver injury, BA levels and composition and BA signaling by the FXR-fibroblast growth factor 15 (FGF15) axis were determined. Overall, higher basal inflammation and altered lipid metabolism in DKO mice might be associated with low BAs. CA, DCA, and UDCA feeding activated FXR signals with tissue specificity. Dietary CA and DCA similarly altered tissue BA profiles to be less hydrophobic, while UDCA promoted a more hydrophobic tissue BA pool with the profiles shifted toward non-12α-OH BAs and secondary BAs. However, UDCA did not offer any overt protective effects as expected. These findings allow us to determine the precise effects of individual BAs in vivo on BA-FXR signaling and overall BA homeostasis in liver physiology and pathologies.

FXR Friend-ChIPs in the Enterohepatic System.

Chronic liver diseases encompass a wide spectrum of hepatic maladies that often result in cholestasis or altered bile acid secretion and regulation. Incidence and cost of care for many chronic liver diseases are rising in the United States with few Food and Drug Administration-approved drugs available for patient treatment. Farnesoid X receptor (FXR) is the master regulator of bile acid homeostasis with an important role in lipid and glucose metabolism and inflammation. FXR has served as an attractive target for management of cholestasis and fibrosis; however, global FXR agonism results in adverse effects in liver disease patients, severely affecting quality of life. In this review, we highlight seminal studies and recent updates on the FXR proteome and identify gaps in knowledge that are essential for tissue-specific FXR modulation. In conclusion, one of the greatest unmet needs in the field is understanding the underlying mechanism of intestinal versus hepatic FXR function.

FXR and NASH: an avenue for tissue-specific regulation.

NASH is within the spectrum of NAFLD, a liver condition encompassing liver steatosis, inflammation, hepatocyte injury, and fibrosis. The prevalence of NASH-induced cirrhosis is rapidly rising and has become the leading indicator for liver transplantation in the US. There is no Food and Drug Administration (FDA)-approved pharmacological intervention for NASH. The farnesoid X receptor (FXR) is essential in regulating bile acid homeostasis, and dysregulation of bile acids has been implicated in the pathogenesis of NASH. As a result, modulators of FXR that show desirable effects in mitigating key characteristics of NASH have been developed as promising therapeutic approaches. However, global FXR activation causes adverse effects such as cholesterol homeostasis imbalance and pruritus. The development of targeted FXR modulation is necessary for ideal NASH therapeutics, but information regarding tissue-specific and cell-specific FXR functionality is limited. In this review, we highlight FXR activation in the regulation of bile acid homeostasis and NASH development, examine the current literature on tissue-specific regulation of nuclear receptors, and speculate on how FXR regulation will be beneficial in the treatment of NASH.

p16 INK4A drives nonalcoholic fatty liver disease phenotypes in high fat diet fed mice through biliary E2F1/FOXO1/IGF-1 signaling.

BACKGROUND AND AIMS: NAFLD is characterized by steatosis, hepatic inflammation, and fibrosis, which can develop into NASH. Patients with NAFLD/NASH have increased ductular reaction (DR) and biliary senescence. High fat/high cholesterol diet feeding increases biliary senescence, DR, and biliary insulin-like growth factor-1 (IGF-1) expression in mice. p16/IGF-1 converges with fork-head box transcription factor O1 (FOXO1) through E2F1. We evaluated p16 inhibition on NAFLD phenotypes and biliary E2F1/FOXO1/IGF-1 signaling. APPROACH AND RESULTS: 4-week wild-type (C57BL/6J) male mice were fed a control diet (CD) or high fat/high cholesterol diet and received either p16 or control Vivo Morpholino (VM) by tail vein injection 2× during the 16th week of feeding. We confirmed p16 knockdown and examined: (i) NAFLD phenotypes; (ii) DR and biliary senescence; (iii) serum metabolites; and (iv) biliary E2F1/FOXO1/IGF-1 signaling. Human normal, NAFLD, and NASH liver samples and isolated cholangiocytes treated with control or p16 VM were evaluated for p16/E2F1/FOXO1/IGF-1 signaling. p16 VM treatment reduced cholangiocyte and hepatocyte p16. In wild-type high fat/high cholesterol diet mice with control VM, there were increased (i) NAFLD phenotypes; (ii) DR and biliary senescence; (iii) serum metabolites; and (iv) biliary E2F1/FOXO1/IGF-1 signaling; however, p16 VM treatment reduced these parameters. Biliary E2F1/FOX-O1/IGF-1 signaling increased in human NAFLD/NASH but was blocked by p16 VM. In vitro , p16 VM reduced biliary E2f1 and Foxo1 transcription by inhibiting RNA pol II binding and E2F1 binding at the Foxo1 locus, respectively. Inhibition of E2F1 reduced biliary FOXO1 in vitro. CONCLUSION: Attenuating hepatic p16 expression may be a therapeutic approach for improving NAFLD/NASH phenotypes.

Loss of apical sodium bile acid transporter alters bile acid circulation and reduces biliary damage in cholangitis.

Primary sclerosing cholangitis (PSC) is characterized by increased ductular reaction (DR), liver fibrosis, hepatic total bile acid (TBA) levels, and mast cell (MC) infiltration. Apical sodium BA transporter (ASBT) expression increases in cholestasis, and ileal inhibition reduces PSC phenotypes. FVB/NJ and multidrug-resistant 2 knockout (Mdr2-/-) mice were treated with control or ASBT Vivo-Morpholino (VM). We measured 1) ASBT expression and MC presence in liver/ileum; 2) liver damage/DR; 3) hepatic fibrosis/inflammation; 4) biliary inflammation/histamine serum content; and 5) gut barrier integrity/hepatic bacterial translocation. TBA/BA composition was measured in cholangiocyte/hepatocyte supernatants, intestine, liver, serum, and feces. Shotgun analysis was performed to ascertain microbiome changes. In vitro, cholangiocytes were treated with BAs ± ASBT VM, and histamine content and farnesoid X receptor (FXR) signaling were determined. Treated cholangiocytes were cocultured with MCs, and FXR signaling, inflammation, and MC activation were measured. Human patients were evaluated for ASBT/MC expression and histamine/TBA content in bile. Control patient- and PSC patient-derived three-dimensional (3-D) organoids were generated; ASBT, chymase, histamine, and fibroblast growth factor-19 (FGF19) were evaluated. ASBT VM in Mdr2-/- mice decreased 1) biliary ASBT expression, 2) PSC phenotypes, 3) hepatic TBA, and 4) gut barrier integrity compared with control. We found alterations between wild-type (WT) and Mdr2-/- mouse microbiome, and ASBT/MC and bile histamine content increased in cholestatic patients. BA-stimulated cholangiocytes increased MC activation/FXR signaling via ASBT, and human PSC-derived 3-D organoids secrete histamine/FGF19. Inhibition of hepatic ASBT ameliorates cholestatic phenotypes by reducing cholehepatic BA signaling, biliary inflammation, and histamine levels. ASBT regulation of hepatic BA signaling offers a therapeutic avenue for PSC.NEW & NOTEWORTHY We evaluated knockdown of the apical sodium bile acid transporter (ASBT) using Vivo-Morpholino in Mdr2KO mice. ASBT inhibition decreases primary sclerosing cholangitis (PSC) pathogenesis by reducing hepatic mast cell infiltration, altering bile acid species/cholehepatic shunt, and regulating gut inflammation/dysbiosis. Since a large cohort of PSC patients present with IBD, this study is clinically important. We validated findings in human PSC and PSC-IBD along with studies in novel human 3-D organoids formed from human PSC livers.

Protective and aggressive bacterial subsets and metabolites modify hepatobiliary inflammation and fibrosis in a murine model of PSC.

OBJECTIVE: Conflicting microbiota data exist for primary sclerosing cholangitis (PSC) and experimental models. GOAL: define the function of complex resident microbes and their association relevant to PSC patients by studying germ-free (GF) and antibiotic-treated specific pathogen-free (SPF) multidrug-resistant 2 deficient (mdr2-/- ) mice and microbial profiles in PSC patient cohorts. DESIGN: We measured weights, liver enzymes, RNA expression, histological, immunohistochemical and fibrotic biochemical parameters, faecal 16S rRNA gene profiling and metabolomic endpoints in gnotobiotic and antibiotic-treated SPF mdr2-/- mice and targeted metagenomic analysis in PSC patients. RESULTS: GF mdr2-/- mice had 100% mortality by 8 weeks with increasing hepatic bile acid (BA) accumulation and cholestasis. Early SPF autologous stool transplantation rescued liver-related mortality. Inhibition of ileal BA transport attenuated antibiotic-accelerated liver disease and decreased total serum and hepatic BAs. Depletion of vancomycin-sensitive microbiota exaggerated hepatobiliary disease. Vancomycin selectively decreased Lachnospiraceae and short-chain fatty acids (SCFAs) but expanded Enterococcus and Enterobacteriaceae. Antibiotics increased Enterococcus faecalis and Escherichia coli liver translocation. Colonisation of GF mdr2-/- mice with translocated E. faecalis and E. coli strains accelerated hepatobiliary inflammation and mortality. Lachnospiraceae colonisation of antibiotic pretreated mdr2-/- mice reduced liver fibrosis, inflammation and translocation of pathobionts, and SCFA-producing Lachnospiraceae and purified SCFA decreased fibrosis. Faecal Lachnospiraceae negatively associated, and E. faecalis/ Enterobacteriaceae positively associated, with PSC patients' clinical severity by Mayo risk scores. CONCLUSIONS: We identified novel functionally protective and detrimental resident bacterial species in mdr2-/- mice and PSC patients with associated clinical risk score. These insights may guide personalised targeted therapeutic interventions in PSC patients.

Secretin alleviates biliary and liver injury during late-stage primary biliary cholangitis via restoration of secretory processes.

BACKGROUND & AIMS: Primary biliary cholangitis (PBC) is characterised by ductopenia, ductular reaction, impairment of anion exchanger 2 (AE2) and the 'bicarbonate umbrella'. Ductulo-canalicular junction (DCJ) derangement is hypothesised to promote PBC progression. The secretin (Sct)/secretin receptor (SR) axis regulates cystic fibrosis transmembrane receptor (CFTR) and AE2, thus promoting choleresis. We evaluated the role of Sct/SR signalling on biliary secretory processes and subsequent injury in a late-stage PBC mouse model and human samples. METHODS: At 32 weeks of age, female and male wild-type and dominant-negative transforming growth factor beta receptor II (late-stage PBC model) mice were treated with Sct for 1 or 8 weeks. Bulk RNA-sequencing was performed in isolated cholangiocytes from mouse models. RESULTS: Biliary Sct/SR/CFTR/AE2 expression and bile bicarbonate levels were reduced in late-stage PBC mouse models and human samples. Sct treatment decreased bile duct loss, ductular reaction, inflammation, and fibrosis in late-stage PBC models. Sct reduced hepatic bile acid levels, modified bile acid composition, and restored the DCJ and 'bicarbonate umbrella'. RNA-sequencing identified that Sct promoted mature epithelial marker expression, specifically anterior grade protein 2 (Agr2). Late-stage PBC models and human samples exhibited reduced biliary mucin 1 levels, which were enhanced by Sct treatment. CONCLUSION: Loss of Sct/SR signalling in late-stage PBC results in a faulty 'bicarbonate umbrella' and reduced Agr2-mediated mucin production. Sct restores cholangiocyte secretory processes and DCJ formation through enhanced mature cholangiocyte phenotypes and bile duct growth. Sct treatment may be beneficial for individuals with late-stage PBC. IMPACT AND IMPLICATIONS: Secretin (Sct) regulates biliary proliferation and bicarbonate secretion in cholangiocytes via its receptor, SR, and in mouse models and human samples of late-stage primary biliary cholangitis (PBC), the Sct/SR axis is blunted along with loss of the protective 'bicarbonate umbrella'. We found that both short- and long-term Sct treatment ameliorated ductular reaction, immune cell influx, and liver fibrosis in late-stage PBC mouse models. Importantly, Sct treatment promoted bicarbonate and mucin secretion and hepatic bile acid efflux, thus reducing cholestatic and toxic bile acid-associated injury in late-stage PBC mouse models. Our work perpetuates the hypothesis that PBC pathogenesis hinges on secretory defects, and restoration of secretory processes that promote the 'bicarbonate umbrella' may be important for amelioration of PBC-associated damage.

Mast cells selectively target large cholangiocytes during biliary injury via H2HR-mediated cAMP/pERK1/2 signaling.

Bile ducts are heterogenous in structure and function, and primary sclerosing cholangitis (PSC) damages specific bile ducts leading to ductular reaction (DR), mast cell (MC) infiltration, increased histamine release, inflammation, and fibrosis. Bile duct ligation (BDL) induces large duct damage via cyclic adenosine monophosphate (cAMP)/extracellular signal-related protein kinase (ERK) signaling, and large cholangiocytes express H2 histamine receptor (H2HR). We evaluated how MCs interact with large cholangiocytes during cholestasis. Male wild-type (WT) and MC-deficient (KitW-sh ) mice 10-12 weeks of age were subjected to BDL for 7 days. Select KitW-sh mice were injected with MCs pretreated with control or H2HR antagonist (ranitidine, 25 µm, 48 h) via tail vein injection. In vitro, MC migration toward small mouse cholangiocytes (SMCCs) and large mouse cholangiocytes (LMCCs) treated with lipopolysaccharide or histamine (±ranitidine) was measured. LMCCs were stimulated with MC supernatants pretreated with control, α-methyl-dl-histidine (to block histamine release), or ranitidine. Liver damage, large duct DR/senescence, inflammation, fibrosis, and cAMP/ERK immunoreactivity increased in BDL WT and KitW-sh +MC mice but decreased in BDL KitW-sh and KitW-sh +MC-H2HR mice. In vitro, MCs migrate toward damaged LMCCs (but not SMCCs) blocked by inhibition of H2HR. Loss of MC histamine or MC-H2HR decreases LMCC proliferation, senescence, H2HR, and cAMP/ERK levels. Human PSC livers have increased MC number found near DR, senescent ducts, and H2HR-positive ducts. Conclusion: Infiltrating MCs preferentially interact with large ducts via H2HR signaling promoting biliary and liver damage. Mediation of MCs may be a therapeutic strategy for PSC.

The Effects of Taurocholic Acid on Biliary Damage and Liver Fibrosis Are Mediated by Calcitonin-Gene-Related Peptide Signaling.

BACKGROUND & AIMS: Cholangiocytes are the target cells of liver diseases that are characterized by biliary senescence (evidenced by enhanced levels of senescence-associated secretory phenotype, SASP, e.g., TGF-ß1), and liver inflammation and fibrosis accompanied by altered bile acid (BA) homeostasis. Taurocholic acid (TC) stimulates biliary hyperplasia by activation of 3',5'-cyclic cyclic adenosine monophosphate (cAMP) signaling, thereby preventing biliary damage (caused by cholinergic/adrenergic denervation) through enhanced liver angiogenesis. Also: (i) α-calcitonin gene-related peptide (α-CGRP, which activates the calcitonin receptor-like receptor, CRLR), stimulates biliary proliferation/senescence and liver fibrosis by enhanced biliary secretion of SASPs; and (ii) knock-out of α-CGRP reduces these phenotypes by decreased cAMP levels in cholestatic models. We aimed to demonstrate that TC effects on liver phenotypes are dependent on changes in the α-CGRP/CALCRL/cAMP/PKA/ERK1/2/TGF-ß1/VEGF axis. METHODS: Wild-type and α-CGRP-/- mice were fed with a control (BAC) or TC diet for 1 or 2 wk. We measured: (i) CGRP levels by both ELISA kits in serum and by qPCR in isolated cholangiocytes (CALCA gene for α-CGRP); (ii) CALCRL immunoreactivity by immunohistochemistry (IHC) in liver sections; (iii) liver histology, intrahepatic biliary mass, biliary senescence (by ß-GAL staining and double immunofluorescence (IF) for p16/CK19), and liver fibrosis (by Red Sirius staining and double IF for collagen/CK19 in liver sections), as well as by qPCR for senescence markers in isolated cholangiocytes; and (iv) phosphorylation of PKA/ERK1/2, immunoreactivity of TGF-ß1/TGF- ßRI and angiogenic factors by IHC/immunofluorescence in liver sections and qPCR in isolated cholangiocytes. We measured changes in BA composition in total liver by liquid chromatography/mass spectrometry. RESULTS: TC feeding increased CALCA expression, biliary damage, and liver inflammation and fibrosis, as well as phenotypes that were associated with enhanced immunoreactivity of the PKA/ERK1/2/TGF-ß1/TGF-ßRI/VEGF axis compared to BAC-fed mice and phenotypes that were reversed in α-CGRP-/- mice fed TC coupled with changes in hepatic BA composition. CONCLUSION: Modulation of the TC/ α-CGRP/CALCRL/PKA/ERK1/2/TGF-ß1/VEGF axis may be important in the management of cholangiopathies characterized by BA accumulation.

Molecular Mechanisms Linking Risk Factors to Cholangiocarcinoma Development.

The poor prognosis of cholangiocarcinoma in humans is related to several factors, such as (i) the heterogeneity of the disease, (ii) the late onset of symptoms and (iii) the limited comprehension of the carcinogenic pathways determining neoplastic changes, which all limit the pursuit of appropriate treatment. Several risk factors have been recognized, including different infective, immune-mediated, and dysmorphogenic disorders of the biliary tree. In this review, we report the details of possible mechanisms that lead a specific premalignant pathological condition to become cholangiocarcinoma. For instance, during liver fluke infection, factors secreted from the worms may play a major role in pathogenesis. In primary sclerosing cholangitis, deregulation of histamine and bile-acid signaling may determine important changes in cellular pathways. The study of these molecular events may also shed some light on the pathogenesis of sporadic (unrelated to risk factors) forms of cholangiocarcinoma, which represent the majority (nearly 75%) of cases.

Melatonin receptor 1A, but not 1B, knockout decreases biliary damage and liver fibrosis during cholestatic liver injury.

BACKGROUND AND AIMS: Melatonin reduces biliary damage and liver fibrosis in cholestatic models by interaction with melatonin receptors 1A (MT1) and 1B (MT2). MT1 and MT2 can form heterodimers and homodimers, but MT1 and MT2 can heterodimerize with the orphan receptor G protein-coupled receptor 50 (GPR50). MT1/GPR50 dimerization blocks melatonin binding, but MT2/GPR50 dimerization does not affect melatonin binding. GPR50 can dimerize with TGFß receptor type I (TGFßRI) to activate this receptor. We aimed to determine the differential roles of MT1 and MT2 during cholestasis. APPROACH AND RESULTS: Wild-type (WT), MT1 knockout (KO), MT2KO, and MT1/MT2 double KO (DKO) mice underwent sham or bile duct ligation (BDL); these mice were also treated with melatonin. BDL WT and multidrug resistance 2 KO (Mdr2-/- ) mice received mismatch, MT1, or MT2 Vivo-Morpholino. Biliary expression of MT1 and GPR50 increases in cholestatic rodents and human primary sclerosing cholangitis (PSC) samples. Loss of MT1 in BDL and Mdr2-/- mice ameliorated biliary and liver damage, whereas these parameters were enhanced following loss of MT2 and in DKO mice. Interestingly, melatonin treatment alleviated BDL-induced biliary and liver injury in BDL WT and BDL MT2KO mice but not in BDL MT1KO or BDL DKO mice, demonstrating melatonin's interaction with MT1. Loss of MT2 or DKO mice exhibited enhanced GPR50/TGFßR1 signaling, which was reduced by loss of MT1. CONCLUSIONS: Melatonin ameliorates liver phenotypes through MT1, whereas down-regulation of MT2 promotes liver damage through GPR50/TGFßR1 activation. Blocking GPR50/TGFßR1 binding through modulation of melatonin signaling may be a therapeutic approach for PSC.

Feedback Signaling between Cholangiopathies, Ductular Reaction, and Non-Alcoholic Fatty Liver Disease.

Fatty liver diseases, such as non-alcoholic fatty liver disease (NAFLD), are global health disparities, particularly in the United States, as a result of cultural eating habits and lifestyle. Pathological studies on NAFLD have been mostly focused on hepatocytes and other inflammatory cell types; however, the impact of other biliary epithelial cells (i.e., cholangiocytes) in the promotion of NAFLD is growing. This review article will discuss how cholestatic injury and cholangiocyte activity/ductular reaction influence NAFLD progression. Furthermore, this review will provide informative details regarding the fundamental properties of cholangiocytes and bile acid signaling that can influence NAFLD. Lastly, studies relating to the pathogenesis of NAFLD, cholangiopathies, and ductular reaction will be analyzed to help gain insight for potential therapies.

Cyclic AMP Signaling in Biliary Proliferation: A Possible Target for Cholangiocarcinoma Treatment?

Cholangiocarcinoma is a lethal disease with scarce response to current systemic therapy. The rare occurrence and large heterogeneity of this cancer, together with poor knowledge of its molecular mechanisms, are elements contributing to the difficulties in finding an appropriate cure. Cholangiocytes (and their cellular precursors) are considered the liver component giving rise to cholangiocarcinoma. These cells respond to several hormones, neuropeptides and molecular stimuli employing the cAMP/PKA system for the translation of messages in the intracellular space. For instance, in physiological conditions, stimulation of the secretin receptor determines an increase of intracellular levels of cAMP, thus activating a series of molecular events, finally determining in bicarbonate-enriched choleresis. However, activation of the same receptor during cholangiocytes' injury promotes cellular growth again, using cAMP as the second messenger. Since several scientific pieces of evidence link cAMP signaling system to cholangiocytes' proliferation, the possible changes of this pathway during cancer growth also seem relevant. In this review, we summarize the current findings regarding the cAMP pathway and its role in biliary normal and neoplastic cell proliferation. Perspectives for targeting the cAMP machinery in cholangiocarcinoma therapy are also discussed.

Mast Cells Regulate Ductular Reaction and Intestinal Inflammation in Cholestasis Through Farnesoid X Receptor Signaling.

BACKGROUND AND AIMS: Cholestasis is characterized by increased total bile acid (TBA) levels, which are regulated by farnesoid X receptor (FXR)/FGF15. Patients with primary sclerosing cholangitis (PSC) typically present with inflammatory bowel disease (IBD). Mast cells (MCs) (i) express FXR and (ii) infiltrate the liver during cholestasis promoting liver fibrosis. In bile-duct-ligated (BDL) MC-deficient mice (B6.Cg-KitW-sh /HNihrJaeBsmJ [KitW-sh ]), ductular reaction (DR) and liver fibrosis decrease compared with BDL wild type, and MC injection exacerbates liver damage in normal mice. APPROACH AND RESULTS: In this study, we demonstrated that MC-FXR regulates biliary FXR/FGF15, DR, and hepatic fibrosis and alters intestinal FXR/FGF15. We found increased MC number and biliary FXR expression in patients with liver injury compared with control. Histamine and FGF19 serum levels and small heterodimer partner expression increase in patients PSC and PSC-IBD compared with healthy controls. MC injection increased liver damage, DR, inflammation, biliary senescence/senescence-associated secretory phenotype (SASP), fibrosis, and histamine in KitW-sh mice. Inhibition of MC-FXR before injection reduced these parameters. BDL and KitW-sh mice injected with MCs displayed increased TBA content, biliary FXR/FGF15, and intestinal inflammation, which decreased in BDL KitW-sh and KitW-sh mice injected with MC-FXR. MCs increased ileal FXR/FGF15 expression in KitW-sh mice that was reduced following FXR inhibition. BDL and multidrug resistance 2/ATP-binding cassette family 2 member 4 knockout (Mdr2-/- ) mice, models of PSC, displayed increased intestinal MC infiltration and FXR/FGF15 expression. These were reduced following MC stabilization with cromolyn sodium in Mdr2-/- mice. In vitro, MC-FXR inhibition decreased biliary proliferation/SASP/FGF and hepatic stellate cell activation. CONCLUSIONS: Our studies demonstrate that MC-FXR plays a key role in liver damage and DR, including TBA regulation through alteration of intestinal and biliary FXR/FGF15 signaling.

Inhibition of Secretin/Secretin Receptor Axis Ameliorates NAFLD Phenotypes.

BACKGROUND AND AIMS: Human NAFLD is characterized at early stages by hepatic steatosis, which may progress to NASH when the liver displays microvesicular steatosis, lobular inflammation, and pericellular fibrosis. The secretin (SCT)/secretin receptor (SCTR) axis promotes biliary senescence and liver fibrosis in cholestatic models through down-regulation of miR-125b signaling. We aim to evaluate the effect of disrupting biliary SCT/SCTR/miR-125b signaling on hepatic steatosis, biliary senescence, and liver fibrosis in NAFLD/NASH. APPROACH AND RESULTS: In vivo, 4-week-old male wild-type, Sct-/- and Sctr-/- mice were fed a control diet or high-fat diet (HFD) for 16 weeks. The expression of SCT/SCTR/miR-125b axis was measured in human NAFLD/NASH liver samples and HFD mouse livers by immunohistochemistry and quantitative PCR. Biliary/hepatocyte senescence, ductular reaction, and liver angiogenesis were evaluated in mouse liver and human NAFLD/NASH liver samples. miR-125b target lipogenesis genes in hepatocytes were screened and validated by custom RT2 Profiler PCR array and luciferase assay. Biliary SCT/SCTR expression was increased in human NAFLD/NASH samples and in livers of HFD mice, whereas the expression of miR-125b was decreased. Biliary/hepatocyte senescence, ductular reaction, and liver angiogenesis were observed in human NAFLD/NASH samples as well as HFD mice, which were decreased in Sct-/- and Sctr-/- HFD mice. Elovl1 is a lipogenesis gene targeted by miR-125b, and its expression was also decreased in HFD mouse hepatocytes following Sct or Sctr knockout. Bile acid profile in fecal samples have the greatest changes between wild-type mice and Sct-/- /Sctr-/- mice. CONCLUSION: The biliary SCT/SCTR/miR-125b axis promotes liver steatosis by up-regulating lipid biosynthesis gene Elovl1. Targeting the biliary SCT/SCTR/miR-125b axis may be key for ameliorating phenotypes of human NAFLD/NASH.

Mast Cells Promote Nonalcoholic Fatty Liver Disease Phenotypes and Microvesicular Steatosis in Mice Fed a Western Diet.

BACKGROUND AND AIMS: Nonalcoholic fatty liver disease (NAFLD) is simple steatosis but can develop into nonalcoholic steatohepatitis (NASH), characterized by liver inflammation, fibrosis, and microvesicular steatosis. Mast cells (MCs) infiltrate the liver during cholestasis and promote ductular reaction (DR), biliary senescence, and liver fibrosis. We aimed to determine the effects of MC depletion during NAFLD/NASH. APPROACH AND RESULTS: Wild-type (WT) and KitW-sh (MC-deficient) mice were fed a control diet (CD) or a Western diet (WD) for 16 weeks; select WT and KitW-sh WD mice received tail vein injections of MCs 2 times per week for 2 weeks prior to sacrifice. Human samples were collected from normal, NAFLD, or NASH mice. Cholangiocytes from WT WD mice and human NASH have increased insulin-like growth factor 1 expression that promotes MC migration/activation. Enhanced MC presence was noted in WT WD mice and human NASH, along with increased DR. WT WD mice had significantly increased steatosis, DR/biliary senescence, inflammation, liver fibrosis, and angiogenesis compared to WT CD mice, which was significantly reduced in KitW-sh WD mice. Loss of MCs prominently reduced microvesicular steatosis in zone 1 hepatocytes. MC injection promoted WD-induced biliary and liver damage and specifically up-regulated microvesicular steatosis in zone 1 hepatocytes. Aldehyde dehydrogenase 1 family, member A3 (ALDH1A3) expression is reduced in WT WD mice and human NASH but increased in KitW-sh WD mice. MicroRNA 144-3 prime (miR-144-3p) expression was increased in WT WD mice and human NASH but reduced in KitW-sh WD mice and was found to target ALDH1A3. CONCLUSIONS: MCs promote WD-induced biliary and liver damage and may promote microvesicular steatosis development during NAFLD progression to NASH through miR-144-3p/ALDH1A3 signaling. Inhibition of MC activation may be a therapeutic option for NAFLD/NASH treatment.