The Epigenetic Reader, Bromodomain Containing 2, Mediates Cholangiocyte Senescence via Interaction With ETS Proto-Oncogene 1.

BACKGROUND & AIMS: We reported that cholangiocyte senescence, regulated by the transcription factor ETS proto-oncogene 1 (ETS1), is a pathogenic feature of primary sclerosing cholangitis (PSC). Furthermore, histone 3 lysine 27 is acetylated at senescence-associated loci. The epigenetic readers, bromodomain and extra-terminal domain (BET) proteins, bind acetylated histones, recruit transcription factors, and drive gene expression. Thus, we tested the hypothesis that BET proteins interact with ETS1 to drive gene expression and cholangiocyte senescence. METHODS: We performed immunofluorescence for BET proteins (BRD2 and 4) in liver tissue from liver tissue from PSC patients and a mouse PSC model. Using normal human cholangiocytes (NHCs), NHCs experimentally induced to senescence (NHCsen), and PSC patient-derived cholangiocytes (PSCDCs), we assessed senescence, fibroinflammatory secretome, and apoptosis after BET inhibition or RNA interference depletion. We assessed BET interaction with ETS1 in NHCsen and tissues from PSC patient, and the effects of BET inhibitors on liver fibrosis, senescence, and inflammatory gene expression in mouse models. RESULTS: Tissue from patients with PSC and a mouse PSC model exhibited increased cholangiocyte BRD2 and 4 protein (∼5×) compared with controls without disease. NHCsen exhibited increased BRD2 and 4 (∼2×), whereas PSCDCs exhibited increased BRD2 protein (∼2×) relative to NHC. BET inhibition in NHCsen and PSCDCs reduced senescence markers and inhibited the fibroinflammatory secretome. ETS1 interacted with BRD2 in NHCsen, and BRD2 depletion diminished NHCsen p21 expression. BET inhibitors reduced senescence, fibroinflammatory gene expression, and fibrosis in the 3,5-diethoxycarbonyl-1,4-dihydrocollidine-fed and Mdr2-/- mouse models. CONCLUSION: Our data suggest that BRD2 is an essential mediator of the senescent cholangiocyte phenotype and is a potential therapeutic target for patients with PSC.

The transcription factor ETS1 promotes apoptosis resistance of senescent cholangiocytes by epigenetically up-regulating the apoptosis suppressor BCL2L1.

Primary sclerosing cholangitis (PSC) is an idiopathic, progressive cholangiopathy. Cholangiocyte senescence is important in PSC pathogenesis, and we have previously reported that senescence is regulated by the transcription factor ETS proto-oncogene 1 (ETS1) and associated with overexpression of BCL2 like 1 (BCL2L1 or BCL-xL), an anti-apoptotic BCL2-family member. Here, we further explored the mechanisms regulating BCL-xL-mediated, apoptosis resistance in senescent cholangiocytes and uncovered that ETS1 and the histone acetyltransferase E1A-binding protein P300 (EP300 or p300) both promote BCL-xL transcription. Using immunofluorescence, we found that BCL-xL protein expression is increased both in cholangiocytes of livers from individuals with PSC and a mouse model of PSC. Using an in vitro model of lipopolysaccharide-induced senescence in normal human cholangiocytes (NHCs), we found increased BCL-xL mRNA and protein levels, and ChIP-PCRs indicated increased occupancy of ETS1, p300, and histone 3 Lys-27 acetylation (H3K27Ac) at the BCL-xL promoter. Using co-immunoprecipitation and proximity ligation assays, we further demonstrate that ETS1 and p300 physically interact in senescent but not control NHCs. Additionally, mutagenesis of predicted ETS1-binding sites within the BCL-xL promoter blocked luciferase reporter activity, and CRISPR/Cas9-mediated genetic deletion of ETS1 reduced senescence-associated BCL-xL expression. In senescent NHCs, TRAIL-mediated apoptosis was reduced ∼70%, and ETS1 deletion or RNAi-mediated BCL-xL suppression increased apoptosis. Overall, our results suggest that ETS1 and p300 promote senescent cholangiocyte resistance to apoptosis by modifying chromatin and inducing BCL-xL expression. These findings reveal ETS1 as a central regulator of both cholangiocyte senescence and the associated apoptosis-resistant phenotype.

Senescent cholangiocytes release extracellular vesicles that alter target cell phenotype via the epidermal growth factor receptor.

BACKGROUND & AIMS: Primary sclerosing cholangitis (PSC) is a chronic liver disease characterized by peribiliary inflammation and fibrosis. Cholangiocyte senescence is a prominent feature of PSC. Here, we hypothesize that extracellular vesicles (EVs) from senescent cholangiocytes influence the phenotype of target cells. METHODS: EVs were isolated from normal human cholangiocytes (NHCs), cholangiocytes from PSC patients and NHCs experimentally induced to senescence. NHCs, malignant human cholangiocytes (MHCs) and monocytes were exposed to 108 EVs from each donor cell population and assessed for proliferation, MAPK activation and migration. Additionally, we isolated EVs from plasma of wild-type and Mdr2-/- mice (a murine model of PSC), and assessed mouse monocyte activation. RESULTS: EVs exhibited the size and protein markers of exosomes. The number of EVs released from senescent human cholangiocytes was increased; similarly, the EVs in plasma from Mdr2-/- mice were increased. Additionally, EVs from senescent cholangiocytes were enriched in multiple growth factors, including EGF. NHCs exposed to EVs from senescent cholangiocytes showed increased NRAS and ERK1/2 activation. Moreover, EVs from senescent cholangiocytes promoted proliferation of NHCs and MHCs, findings that were blocked by erlotinib, an EGF receptor inhibitor. Furthermore, EVs from senescent cholangiocytes induced EGF-dependent Interleukin 1-beta and Tumour necrosis factor expression and migration of human monocytes; similarly, Mdr2-/- mouse plasma EVs induced activation of mouse monocytes. CONCLUSIONS: The data continue to support the importance of cholangiocyte senescence in PSC pathogenesis, directly implicate EVs in cholangiocyte proliferation, malignant progression and immune cell activation and migration, and identify novel therapeutic approaches for PSC.

Targeting senescent cholangiocytes and activated fibroblasts with B-cell lymphoma-extra large inhibitors ameliorates fibrosis in multidrug resistance 2 gene knockout (Mdr2-/- ) mice.

Cholangiocyte senescence has been linked to primary sclerosing cholangitis (PSC). Persistent secretion of growth factors by senescent cholangiocytes leads to the activation of stromal fibroblasts (ASFs), which are drivers of fibrosis. The activated phenotype of ASFs is characterized by an increased sensitivity to apoptotic stimuli. Here, we examined the mechanisms of apoptotic priming in ASFs and explored a combined targeting strategy to deplete senescent cholangiocytes and ASFs from fibrotic tissue to ameliorate liver fibrosis. Using a coculture system, we determined that senescent cholangiocytes promoted quiescent mesenchymal cell activation in a platelet-derived growth factor (PDGF)-dependent manner. We also identified B-cell lymphoma-extra large (Bcl-xL) as a key survival factor in PDGF-activated human and mouse fibroblasts. Bcl-xL was also up-regulated in senescent cholangiocytes. In vitro, inhibition of Bcl-xL by the small molecule Bcl-2 homology domain 3 mimetic, A-1331852, or Bcl-xL-specific small interfering RNA induced apoptosis in PDGF-activated fibroblasts, but not in quiescent fibroblasts. Likewise, inhibition of Bcl-xL reduced the survival and increased apoptosis of senescent cholangiocytes, compared to nonsenescent cells. Treatment of multidrug resistance 2 gene knockout (Mdr2-/- ) mice with A-1331852 resulted in an 80% decrease in senescent cholangiocytes, a reduction of fibrosis-inducing growth factors and cytokines, decrease of α-smooth muscle actin-positive ASFs, and finally in a significant reduction of liver fibrosis. CONCLUSION: Bcl-xL is a key survival factor in ASFs as well as in senescent cholangiocytes. Treatment with the Bcl-xL-specific inhibitor, A-1331852, reduces liver fibrosis, possibly by a dual effect on activated fibroblasts and senescent cholangiocytes. This mechanism represents an attractive therapeutic strategy in biliary fibrosis. (Hepatology 2018;67:247-259).

ETS Proto-oncogene 1 Transcriptionally Up-regulates the Cholangiocyte Senescence-associated Protein Cyclin-dependent Kinase Inhibitor 2A.

Primary sclerosing cholangitis (PSC) is a chronic, fibroinflammatory cholangiopathy (disease of the bile ducts) of unknown pathogenesis. We reported that cholangiocyte senescence features prominently in PSC and that neuroblastoma RAS viral oncogene homolog (NRAS) is activated in PSC cholangiocytes. Additionally, persistent microbial insult (e.g. LPSs) induces cyclin-dependent kinase inhibitor 2A (CDKN2A/p16INK4a) expression and senescence in cultured cholangiocytes in an NRAS-dependent manner. However, the molecular mechanisms involved in LPS-induced cholangiocyte senescence and NRAS-dependent regulation of CDKN2A remain unclear. Using our in vitro senescence model, we found that LPS-induced CDKN2A expression coincided with a 4.5-fold increase in ETS1 (ETS proto-oncogene 1) mRNA, suggesting that ETS1 is involved in regulating CDKN2A This idea was confirmed by RNAi-mediated suppression or genetic deletion of ETS1, which blocked CDKN2A expression and reduced cholangiocyte senescence. Furthermore, site-directed mutagenesis of a predicted ETS-binding site within the CDKN2A promoter abolished luciferase reporter activity. Pharmacological inhibition of RAS/MAPK reduced ETS1 and CDKN2A protein expression and CDKN2A promoter-driven luciferase activity by ∼50%. In contrast, constitutively active NRAS expression induced ETS1 and CDKN2A protein expression, whereas ETS1 RNAi blocked this increase. Chromatin immunoprecipitation-PCR detected increased ETS1 and histone 3 lysine 4 trimethylation (H3K4Me3) at the CDKN2A promoter following LPS-induced senescence. Additionally, phospho-ETS1 expression was increased in cholangiocytes of human PSC livers and in the Abcb4 (Mdr2)-/- mouse model of PSC. These data pinpoint ETS1 and H3K4Me3 as key transcriptional regulators in NRAS-induced expression of CDKN2A, and this regulatory axis may therefore represent a potential therapeutic target for PSC treatment.

Macrophages contribute to the pathogenesis of sclerosing cholangitis in mice.

BACKGROUND & AIMS: Macrophages contribute to liver disease, but their role in cholestatic liver injury, including primary sclerosing cholangitis (PSC), is unclear. We tested the hypothesis that macrophages contribute to the pathogenesis of, and are therapeutic targets for, PSC. METHODS: Immune cell profile, hepatic macrophage number, localization and polarization, fibrosis, and serum markers of liver injury and cholestasis were measured in an acute (intrabiliary injection of the inhibitor of apoptosis antagonist BV6) and chronic (Mdr2-/- mice) mouse model of sclerosing cholangitis (SC). Selected observations were confirmed in liver specimens from patients with PSC. Because of the known role of the CCR2/CCL2 axis in monocyte/macrophage chemotaxis, therapeutic effects of the CCR2/5 antagonist cenicriviroc (CVC), or genetic deletion of CCR2 (Ccr2-/- mice) were determined in BV6-injected mice. RESULTS: We found increased peribiliary pro-inflammatory (M1-like) and alternatively-activated (M2-like) monocyte-derived macrophages in PSC compared to normal livers. In both SC models, genetic profiling of liver immune cells identified a predominance of monocytes/macrophages; immunohistochemistry confirmed peribiliary monocyte-derived macrophage recruitment (M1>M2-polarized), which paralleled injury onset and was reversed upon resolution in acute SC mice. PSC, senescent and BV6-treated human cholangiocytes released monocyte chemoattractants (CCL2, IL-8) and macrophage-activating factors in vitro. Pharmacological inhibition of monocyte recruitment by CVC treatment or CCR2 genetic deletion attenuated macrophage accumulation, liver injury and fibrosis in acute SC. CONCLUSIONS: Peribiliary recruited macrophages are a feature of both PSC and acute and chronic murine SC models. Pharmacologic and genetic inhibition of peribiliary macrophage recruitment decreases liver injury and fibrosis in mouse SC. These observations suggest monocyte-derived macrophages contribute to the development of SC in mice and in PSC pathogenesis, and support their potential as a therapeutic target. LAY SUMMARY: Primary sclerosing cholangitis (PSC) is an inflammatory liver disease which often progresses to liver failure. The cause of the disease is unclear and therapeutic options are limited. Therefore, we explored the role of white blood cells termed macrophages in PSC given their frequent contribution to other human inflammatory diseases. Our results implicate macrophages in PSC and PSC-like diseases in mice. More importantly, we found that pharmacologic inhibition of macrophage recruitment to the liver reduces PSC-like liver injury in the mouse. These exciting observations highlight potential new strategies to treat PSC.

Development and characterization of cholangioids from normal and diseased human cholangiocytes as an in vitro model to study primary sclerosing cholangitis.

Primary sclerosing cholangitis (PSC) is an incurable, fibroinflammatory biliary disease for which there is no effective pharmacotherapy. We recently reported cholangiocyte senescence as an important phenotype in PSC while others showed that portal macrophages accumulate in PSC. Unfortunately, our ability to explore cholangiocyte senescence and macrophage accumulation has been hampered by limited in vitro models. Thus, our aim was to develop and characterize a three-dimensional (3D) model of normal and diseased bile ducts (cholangioids) starting with normal human cholangiocytes (NHC), senescent NHC (NHC-sen), and cholangiocytes from PSC patients. In 3D culture, NHCs formed spheroids of ~5000 cells with a central lumen of ~150 µm. By confocal microscopy and western blot, cholangioids retained expression of cholangiocyte proteins (cytokeratin 7/19) and markers of epithelial polarity (secretin receptor and GM130). Cholangioids are functionally active, and upon secretin stimulation, luminal size increased by ~80%. Cholangioids exposed to hydrogen peroxide exhibited cellular senescence and the senescence-associated secretory phenotype (SASP; increased IL-6, p21, SA-ß-Gal, yH2A.x and p16 expression). Furthermore, cholangioids derived from NHC-sen or PSC patients were smaller and had slower growth than the controls. When co-cultured with THP-1 macrophages, the number of macrophages associated with NHC-sen or PSC cholangioids was five- to seven-fold greater compared to co-culture with non-senescent NHC. We observed that NHC-sen and PSC cholangioids release greater number of extracellular vesicles (EVs) compared to controls. Moreover, conditioned media from NHC-sen cholangioids resulted in an ~2-fold increase in macrophage migration. In summary, we developed a method to generate normal and diseased cholangioids, characterized them morphologically and functionally, showed that they can be induced to senescence and SASP, and demonstrated both EV release and macrophage attraction. This novel model mimics several features of PSC, and thus will be useful for studying the pathogenesis of PSC and potentially identifying new therapeutic targets.

Absence of the intestinal microbiota exacerbates hepatobiliary disease in a murine model of primary sclerosing cholangitis.

UNLABELLED: Primary sclerosing cholangitis (PSC) is a chronic, idiopathic, fibroinflammatory cholangiopathy. The role of the microbiota in PSC etiopathogenesis may be fundamentally important, yet remains obscure. We tested the hypothesis that germ-free (GF) mutltidrug resistance 2 knockout (mdr2(-/-) ) mice develop a distinct PSC phenotype, compared to conventionally housed (CV) mdr2(-/-) mice. Mdr2(-/-) mice (n = 12) were rederived as GF by embryo transfer, maintained in isolators, and sacrificed at 60 days in parallel with age-matched CV mdr2(-/-) mice. Serum biochemistries, gallbladder bile acids, and liver sections were examined. Histological findings were validated morphometrically, biochemically, and by immunofluorescence microscopy (IFM). Cholangiocyte senescence was assessed by p16(INK4a) in situ hybridization in liver tissue and by senescence-associated ß-galactosidase staining in a culture-based model of insult-induced senescence. Serum biochemistries, including alkaline phosphatase, aspartate aminotransferase, and bilirubin, were significantly higher in GF mdr2(-/-) (P < 0.01). Primary bile acids were similar, whereas secondary bile acids were absent, in GF mdr2(-/-) mice. Fibrosis, ductular reaction, and ductopenia were significantly more severe histopathologically in GF mdr2(-/-) mice (P < 0.01) and were confirmed by hepatic morphometry, hydroxyproline assay, and IFM. Cholangiocyte senescence was significantly increased in GF mdr2(-/-) mice and abrogated in vitro by ursodeoxycholic acid (UDCA) treatment. CONCLUSIONS: GF mdr2(-/-) mice exhibit exacerbated biochemical and histological features of PSC and increased cholangiocyte senescence, a characteristic and potential mediator of progressive biliary disease. UDCA, a commensal microbial metabolite, abrogates senescence in vitro. These findings demonstrate the importance of the commensal microbiota and its metabolites in protecting against biliary injury and suggest avenues for future studies of biomarkers and therapeutic interventions in PSC.

Centrosomal abnormalities characterize human and rodent cystic cholangiocytes and are associated with Cdc25A overexpression.

Hepatic cystogenesis in polycystic liver diseases is associated with abnormalities of cholangiocyte cilia. Given the crucial association between cilia and centrosomes, we tested the hypothesis that centrosomal defects occur in cystic cholangiocytes of rodents (Pkd2(WS25/-) mice and PCK rats) and of patients with polycystic liver diseases, contributing to disturbed ciliogenesis and cyst formation. We examined centrosomal cytoarchitecture in control and cystic cholangiocytes, the effects of centrosomal abnormalities on ciliogenesis, and the role of the cell-cycle regulator Cdc25A in centrosomal defects by depleting cholangiocytes of Cdc25A in vitro and in vivo and evaluating centrosome morphology, cell-cycle progression, proliferation, ciliogenesis, and cystogenesis. The cystic cholangiocytes had atypical centrosome positioning, supernumerary centrosomes, multipolar spindles, and extra cilia. Structurally aberrant cilia were present in cystic cholangiocytes during ciliogenesis. Depletion of Cdc25A resulted in i) a decreased number of centrosomes and multiciliated cholangiocytes, ii) an increased fraction of ciliated cholangiocytes with longer cilia, iii) a decreased proportion of cholangiocytes in G1/G0 and S phases of the cell cycle, iv) decreased cell proliferation, and v) reduced cyst growth in vitro and in vivo. Our data support the hypothesis that centrosomal abnormalities in cholangiocytes are associated with aberrant ciliogenesis and that accelerated cystogenesis is likely due to overexpression of Cdc25A, providing additional evidence that pharmacological targeting of Cdc25A has therapeutic potential in polycystic liver diseases.

Cholangiocyte senescence by way of N-ras activation is a characteristic of primary sclerosing cholangitis.

UNLABELLED: Primary sclerosing cholangitis (PSC) is an incurable cholangiopathy of unknown etiopathogenesis. Here we tested the hypothesis that cholangiocyte senescence is a pathophysiologically important phenotype in PSC. We assessed markers of cellular senescence and senescence-associated secretory phenotype (SASP) in livers of patients with PSC, primary biliary cirrhosis, hepatitis C, and in normals by fluorescent in situ hybridization (FISH) and immunofluorescence microscopy (IFM). We tested whether endogenous and exogenous biliary constituents affect senescence and SASP in cultured human cholangiocytes. We determined in coculture whether senescent cholangiocytes induce senescence in bystander cholangiocytes. Finally, we explored signaling mechanisms involved in cholangiocyte senescence and SASP. In vivo, PSC cholangiocytes expressed significantly more senescence-associated p16(INK4a) and γH2A.x compared to the other three conditions; expression of profibroinflammatory SASP components (i.e., IL-6, IL-8, CCL2, PAI-1) was also highest in PSC cholangiocytes. In vitro, several biologically relevant endogenous (e.g., cholestane 3,5,6 oxysterol) and exogenous (e.g., lipopolysaccharide) molecules normally present in bile induced cholangiocyte senescence and SASP. Furthermore, experimentally induced senescent human cholangiocytes caused senescence in bystander cholangiocytes. N-Ras, a known inducer of senescence, was increased in PSC cholangiocytes and in experimentally induced senescent cultured cholangiocytes; inhibition of Ras abrogated experimentally induced senescence and SASP. CONCLUSION: Cholangiocyte senescence induced by biliary constituents by way of N-Ras activation is an important pathogenic mechanism in PSC. Pharmacologic inhibition of N-Ras with a resultant reduction in cholangiocyte senescence and SASP is a new therapeutic approach for PSC.

Characterization of cultured cholangiocytes isolated from livers of patients with primary sclerosing cholangitis.

Primary sclerosing cholangitis (PSC) is a chronic, idiopathic cholangiopathy. The role of cholangiocytes (biliary epithelial cells) in PSC pathogenesis is unknown and remains an active area of research. Here, through cellular, molecular and next-generation sequencing (NGS) methods, we characterize and identify phenotypic and signaling features of isolated PSC patient-derived cholangiocytes. We isolated cholangiocytes from stage 4 PSC patient liver explants by dissection, differential filtration and immune-magnetic bead separation. We maintained cholangiocytes in culture and assessed for: (i) cholangiocyte, cell adhesion and inflammatory markers; (ii) proliferation rate; (iii) transepithelial electrical resistance (TEER); (iv) cellular senescence; and (v) transcriptomic profiles by NGS. We used two well-established normal human cholangiocyte cell lines (H69 and NHC) as controls. Isolated PSC cells expressed cholangiocyte (eg, cytokeratin 7 and 19) and epithelial cell adhesion markers (EPCAM, ICAM) and were negative for hepatocyte and myofibroblast markers (albumin, α-actin). Proliferation rate was lower for PSC compared with normal cholangiocytes (4 vs 2 days, respectively, P<0.01). Maximum TEER was also lower in PSC compared with normal cholangiocytes (100 vs 145 Ωcm(2), P<0.05). Interleukin-6 (IL-6) and IL-8 (protein and mRNA) were both increased compared with NHCs and H69s (all P<0.01). The proportion of cholangiocytes staining positive for senescence-associated ß-galactosidase was higher in PSC cholangiocytes compared with NHCs (48% vs 5%, P<0.01). Finally, NGS confirmed cholangiocyte marker expression in isolated PSC cholangiocytes and extended our findings regarding pro-inflammatory and senescence-associated signaling. In conclusion, we have demonstrated that high-purity cholangiocytes can be isolated from human PSC liver and grown in primary culture. Isolated PSC cholangiocytes exhibit a phenotype that may reflect their in vivo contribution to disease and serve as a vital tool for in vitro investigation of biliary pathobiology and identification of new therapeutic targets in PSC.

Micro-computed tomography and nuclear magnetic resonance imaging for noninvasive, live-mouse cholangiography.

The cholangiopathies are a diverse group of biliary tract disorders, many of which lack effective treatment. Murine models are an important tool for studying their pathogenesis, but existing noninvasive methods for assessing biliary disease in vivo are not optimal. Here we report our experience with using micro-computed tomography (microCT) and nuclear magnetic resonance (MR) imaging to develop a technique for live-mouse cholangiography. Using mdr2 knockout (mdr2KO, a model for primary sclerosing cholangitis (PSC)), bile duct-ligated (BDL), and normal mice, we performed in vivo: (1) microCT on a Siemens Inveon PET/CT scanner and (2) MR on a Bruker Avance 16.4 T spectrometer, using Turbo Rapid Acquisition with Relaxation Enhancement, IntraGate Fast Low Angle Shot, and Half-Fourier Acquisition Single-shot Turbo Spin Echo methods. Anesthesia was with 1.5-2.5% isoflurane. Scans were performed with and without contrast agents (iodipamide meglumine (microCT), gadoxetate disodium (MR)). Dissection and liver histology were performed for validation. With microCT, only the gallbladder and extrahepatic bile ducts were visualized despite attempts to optimize timing, route, and dose of contrast. With MR, the gallbladder, extra-, and intrahepatic bile ducts were well-visualized in mdr2KO mice; the cholangiographic appearance was similar to that of PSC (eg, multifocal strictures) and could be improved with contrast administration. In BDL mice, MR revealed cholangiographically distinct progressive dilation of the biliary tree without ductal irregularity. In normal mice, MR allowed visualization of the gallbladder and extrahepatic ducts, but only marginal visualization of the diminutive intrahepatic ducts. One mouse died during microCT and MR imaging, respectively. Both microCT and MR scans could be obtained in ≤20 min. We, therefore, demonstrate that MR cholangiography can be a useful tool for longitudinal studies of the biliary tree in live mice, whereas microCT yields suboptimal duct visualization despite requiring contrast administration. These findings support further development and application of MR cholangiography to the study of mouse models of PSC and other cholangiopathies.

The dynamic biliary epithelia: molecules, pathways, and disease.

Cholangiocytes, the cells lining bile ducts, are a heterogenous, highly dynamic population of epithelial cells. While these cells comprise a small fraction of the total cellular component of the liver, they perform the essential role of bile modification and transport of biliary and blood constituents. From a pathophysiological standpoint, cholangiocytes are the target of a diverse group of biliary disorders, collectively referred to as the cholangiopathies. To date, the cause of most cholangiopathies remains obscure. It is known, however, that cholangiocytes exist in an environment rich in potential mediators of cellular injury, express receptors that recognize potential injurious insults, and participate in portal tract repair processes following hepatic injury. As such, cholangiocytes may not be only a passive target, but are likely directly and actively involved in the pathogenesis of cholangiopathies. Here, we briefly summarize the characteristics of the reactive cholangiocyte and cholangiocyte responses to potentially injurious endogenous and exogenous molecules, and in addition, present emerging concepts in our understanding of the etiopathogenesis of several cholangiopathies.

Ciliary subcellular localization of TGR5 determines the cholangiocyte functional response to bile acid signaling.

TGR5, the G protein-coupled bile acid receptor that transmits bile acid signaling into a cell functional response via the intracellular cAMP signaling pathway, is expressed in human and rodent cholangiocytes. However, detailed information on the localization and function of cholangiocyte TGR5 is limited. We demonstrated that in human (H69 cells) and rat cholangiocytes, TGR5 is localized to multiple, diverse subcellular compartments, with its strongest expression on the apical plasma, ciliary, and nuclear membranes. To evaluate the relationship between ciliary TGR5 and the cholangiocyte functional response to bile acid signaling, we used a model of ciliated and nonciliated H69 cells and demonstrated that TGR5 agonists induce opposite changes in cAMP and ERK levels in cells with and without primary cilia. The cAMP level was increased in nonciliated cholangiocytes but decreased in ciliated cells. In contrast, ERK signaling was induced in ciliated cholangiocytes but suppressed in cells without cilia. TGR5 agonists inhibited proliferation of ciliated cholangiocytes but activated proliferation of nonciliated cells. The observed differential effects of TGR5 agonists were associated with the coupling of TGR5 to Gαi protein in ciliated cells and Gαs protein in nonciliated cholangiocytes. The functional responses of nonciliated and ciliated cholangiocytes to TGR5-mediated bile acid signaling may have important pathophysiological significance in cilia-related liver disorders (i.e., cholangiociliopathies), such as polycystic liver disease. In summary, TGR5 is expressed on diverse cholangiocyte compartments, including a primary cilium, and its ciliary localization determines the cholangiocyte functional response to bile acid signaling.

Up-regulation of microRNA 506 leads to decreased Cl-/HCO3- anion exchanger 2 expression in biliary epithelium of patients with primary biliary cirrhosis.

UNLABELLED: Cl(-) /HCO3- anion exchanger 2 (AE2) participates in intracellular pH homeostasis and secretin-stimulated biliary bicarbonate secretion. AE2/SLC4A2 gene expression is reduced in liver and blood mononuclear cells from patients with primary biliary cirrhosis (PBC). Our previous findings of hepatic and immunological features mimicking PBC in Ae2-deficient mice strongly suggest that decreased AE2 expression might be involved in the pathogenesis of PBC. Here, we tested the potential role of microRNA 506 (miR-506) - predicted as candidate to target AE2 mRNA - for the decreased expression of AE2 in PBC. Real-time quantitative polymerase chain reaction showed that miR-506 expression is increased in PBC livers versus normal liver specimens. In situ hybridization in liver sections confirmed that miR-506 is up-regulated in the intrahepatic bile ducts of PBC livers, compared with normal and primary sclerosing cholangitis livers. Precursor-mediated overexpression of miR-506 in SV40-immortalized normal human cholangiocytes (H69 cells) led to decreased AE2 protein expression and activity, as indicated by immunoblotting and microfluorimetry, respectively. Moreover, miR-506 overexpression in three-dimensional (3D)-cultured H69 cholangiocytes blocked the secretin-stimulated expansion of cystic structures developed under the 3D conditions. Luciferase assays and site-directed mutagenesis demonstrated that miR-506 specifically may bind the 3'untranslated region (3'UTR) of AE2 messenger RNA (mRNA) and prevent protein translation. Finally, cultured PBC cholangiocytes showed decreased AE2 activity, together with miR-506 overexpression, compared to normal human cholangiocytes, and transfection of PBC cholangiocytes with anti-miR-506 was able to improve their AE2 activity. CONCLUSION: miR-506 is up-regulated in cholangiocytes from PBC patients, binds the 3'UTR region of AE2 mRNA, and prevents protein translation, leading to diminished AE2 activity and impaired biliary secretory functions. In view of the putative pathogenic role of decreased AE2 in PBC, miR-506 may constitute a potential therapeutic target for this disease.

Cholangiocyte N-Ras protein mediates lipopolysaccharide-induced interleukin 6 secretion and proliferation.

Cholangiocytes, the epithelial cells lining the bile ducts in the liver, are periodically exposed to potentially injurious microbes and/or microbial products. As a result, cholangiocytes actively participate in microbe-associated, hepatic proinflammatory responses. We previously showed that infection of cultured human cholangiocytes with the protozoan parasite, Cryptosporidium parvum, or treatment with gram-negative bacteria-derived LPS, activates NFκB in a myeloid differentiation 88 (MyD88)-dependent manner. Here, we describe a novel signaling pathway initiated by Toll-like receptors (TLRs) involving the small GTPase, Ras, that mediates cholangiocyte proinflammatory cytokine production and induction of cholangiocyte proliferation. Using cultured human cholangiocytes and a Ras activation assay, we found that agonists of plasma membrane TLRs (TLR 1, 2, 4, 5, and 6) rapidly (<10 min) activated N-Ras, but not other p21 Ras isoforms, resulting in the rapid (<15 min) phosphorylation of the downstream Ras effector, ERK1/2. RNA interference-induced depletion of TRAF6, a downstream effector of MyD88 and known activator of MAPK signaling, had no effect on N-Ras activation. Following N-Ras activation the proinflammatory cytokine, IL6, is rapidly secreted. Using a luciferase reporter, we demonstrated that LPS treatment induced IL6 promoter-driven luciferase which was suppressed using MEK/ERK pharmacologic inhibitors (PD98059 or U0126) and RNAi-induced depletion of N-Ras. Finally, we showed that LPS increased cholangiocyte proliferation (1.5-fold), which was inhibited by depletion of N-Ras; TLR agonist-induced proliferation was also inhibited following pretreatment with an IL6 receptor-blocking antibody. Together, our results support a novel signaling axis involving microbial activation of N-Ras likely involved in the cholangiocyte pathogen-induced proinflammatory response.

Aquaporin-1 promotes angiogenesis, fibrosis, and portal hypertension through mechanisms dependent on osmotically sensitive microRNAs.

Changes in hepatic vasculature accompany fibrogenesis, and targeting angiogenic molecules often attenuates fibrosis in animals. Aquaporin-1 (AQP1) is a water channel, overexpressed in cirrhosis, that promotes angiogenesis by enhancing endothelial invasion. The effect of AQP1 on fibrogenesis in vivo and the mechanisms driving AQP1 expression during cirrhosis remain unclear. The purpose of this study was to test the effect of AQP1 deletion in cirrhosis and explore mechanisms regulating AQP1. After bile duct ligation, wild-type mice overexpress AQP1 that colocalizes with vascular markers and sites of robust angiogenesis. AQP1 knockout mice demonstrated reduced angiogenesis compared with wild-type mice, as evidenced by immunostaining and endothelial invasion/proliferation in vitro. Fibrosis and portal hypertension were attenuated based on immunostaining, portal pressure, and spleen/body weight ratio. AQP1 protein, but not mRNA, was induced by hyperosmolality in vitro, suggesting post-transcriptional regulation. Endothelial cells from normal or cirrhotic mice were screened for microRNA (miR) expression using an array and a quantitative PCR. miR-666 and miR-708 targeted AQP1 mRNA and were decreased in cirrhosis and in cells exposed to hyperosmolality, suggesting that these miRs mediate osmolar changes via AQP1. Binding of the miRs to the untranslated region of AQP1 was assessed using luciferase assays. In conclusion, AQP1 promotes angiogenesis, fibrosis, and portal hypertension after bile duct ligation and is regulated by osmotically sensitive miRs.

NFkappaB p50-CCAAT/enhancer-binding protein beta (C/EBPbeta)-mediated transcriptional repression of microRNA let-7i following microbial infection.

MicroRNAs, central players of numerous cellular processes, regulate mRNA stability or translational efficiency. Although these molecular events are established, the mechanisms regulating microRNA function and expression remain largely unknown. The microRNA let-7i regulates Toll-like receptor 4 expression. Here, we identify a novel transcriptional mechanism induced by the protozoan parasite Cryptosporidium parvum and Gram(-) bacteria-derived lipopolysaccharide (LPS) mediating let-7i promoter silencing in human biliary epithelial cells (cholangiocytes). Using cultured cholangiocytes, we show that microbial stimulus decreased let-7i expression, and promoter activity. Analysis of the mechanism revealed that microbial infection promotes the formation of a NFkappaB p50-C/EBPbeta silencer complex in the regulatory sequence. Chromatin immunoprecipitation assays (ChIP) demonstrated that the repressor complex binds to the let-7i promoter following microbial stimulus and promotes histone-H3 deacetylation. Our results provide a novel mechanism of transcriptional regulation of cholangiocyte let-7i expression following microbial insult, a process with potential implications for epithelial innate immune responses in general.

MicroRNA15a modulates expression of the cell-cycle regulator Cdc25A and affects hepatic cystogenesis in a rat model of polycystic kidney disease.

Hyperproliferation of bile duct epithelial cells due to cell-cycle dysregulation is a key feature of cystogenesis in polycystic liver diseases (PCLDs). Recent evidence suggests a regulatory role for microRNAs (miRNAs) in a variety of biological processes, including cell proliferation. We therefore hypothesized that miRNAs may be involved in the regulation of selected components of the cell cycle and might contribute to hepatic cystogenesis. We found that the cholangiocyte cell line PCK-CCL, which is derived from the PCK rat, a model of autosomal recessive polycystic kidney disease (ARPKD), displayed global changes in miRNA expression compared with normal rat cholangiocytes (NRCs). More specific analysis revealed decreased levels of 1 miRNA, miR15a, both in PCK-CCL cells and in liver tissue from PCK rats and patients with a PCLD. The decrease in miR15a expression was associated with upregulation of its target, the cell-cycle regulator cell division cycle 25A (Cdc25A). Overexpression of miR15a in PCK-CCL cells decreased Cdc25A levels, inhibited cell proliferation, and reduced cyst growth. In contrast, suppression of miR15a in NRCs accelerated cell proliferation, increased Cdc25A expression, and promoted cyst growth. Taken together, these results suggest that suppression of miR15a contributes to hepatic cystogenesis through dysregulation of Cdc25A.

Activation of Trpv4 reduces the hyperproliferative phenotype of cystic cholangiocytes from an animal model of ARPKD.

BACKGROUND & AIMS: In polycystic liver diseases, cyst formation involves cholangiocyte hyperproliferation. In polycystic kidney (PCK) rats, an animal model of autosomal-recessive polycystic kidney disease (ARPKD), decreased intracellular calcium [Ca(2+)](i) in cholangiocytes is associated with hyperproliferation. We recently showed transient receptor potential vanilloid 4 (Trpv4), a calcium-entry channel, is expressed in normal cholangiocytes and its activation leads to [Ca(2+)](i) increase. Thus, we hypothesized that pharmacologic activation of Trpv4 might reverse the hyperproliferative phenotype of PCK cholangiocytes. METHODS: Trpv4 expression was examined in liver of normal and PCK rats, normal human beings, and patients with autosomal-dominant polycystic kidney disease or ARPKD. Trpv4 activation effect on cell proliferation and cyst formation was assessed in cholangiocytes derived from normal and PCK rats. The in vivo effects of Trpv4 activation on kidney and liver cysts was analyzed in PCK rats. RESULTS: Trpv4 was overexpressed both at messenger RNA (8-fold) and protein (3-fold) levels in PCK cholangiocytes. Confocal and immunogold electron microscopy supported Trpv4 overexpression in the livers of PCK rats and ARPKD or autosomal-dominant polycystic kidney disease patients. Trpv4 activation in PCK cholangiocytes increased [Ca(2+)](i) by 30%, inhibiting cell proliferation by approximately 25%-50% and cyst growth in 3-dimensional culture (3-fold). Trpv4-small interfering RNA silencing blocked effects of Trpv4 activators by 70%. Trpv4 activation was associated with Akt phosphorylation and beta-Raf and Erk1/2 inhibition. In vivo, Trpv4 activation induced a significant decrease in renal cystic area and a nonsignificant decrease in liver cysts. CONCLUSIONS: Taken together, our in vitro and in vivo data suggest that increasing intracellular calcium by Trpv4 activation may represent a potential therapeutic approach in PKD.