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Nicholas F. Larusso - One of the best experts on this subject based on the ideXlab platform.
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genetic or pharmacological reduction of Cholangiocyte senescence improves inflammation and fibrosis in the mdr2 mouse
JHEP reports : innovation in hepatology, 2021Co-Authors: Mohammed Alsuraih, Steven P Ohara, Julie Woodrum, Nicholas E Pirius, Nicholas F. LarussoAbstract:Summary Background & Aims Cholangiocyte senescence is important in the pathogenesis of primary sclerosing cholangitis (PSC). We found that CDKN2A (p16), a cyclin-dependent kinase inhibitor and mediator of senescence, was increased in Cholangiocytes of patients with PSC and from a PSC mouse model (multidrug resistance 2; Mdr2-/-). Given that recent data suggest that a reduction of senescent cells is beneficial in different diseases, we hypothesised that inhibition of Cholangiocyte senescence would ameliorate disease in Mdr2-/- mice. Methods We used 2 novel genetic murine models to reduce Cholangiocyte senescence: (i) p16Ink4a apoptosis through targeted activation of caspase (INK-ATTAC)xMdr2-/-, in which the dimerizing molecule AP20187 promotes selective apoptotic removal of p16-expressing cells; and (ii) mice deficient in both p16 and Mdr2. Mdr2-/- mice were also treated with fisetin, a flavonoid molecule that selectively kills senescent cells. p16, p21, and inflammatory markers (tumour necrosis factor [TNF]-α, IL-1β, and monocyte chemoattractant protein-1 [MCP-1]) were measured by PCR, and hepatic fibrosis via a hydroxyproline assay and Sirius red staining. Results AP20187 treatment reduced p16 and p21 expression by ~35% and ~70% (p >0.05), respectively. Expression of inflammatory markers (TNF-α, IL-1β, and MCP-1) decreased (by 60%, 40%, and 60%, respectively), and fibrosis was reduced by ~60% (p >0.05). Similarly, p16-/-xMdr2-/- mice exhibited reduced p21 expression (70%), decreased expression of TNF-α, IL-1β (60%), and MCP-1 (65%) and reduced fibrosis (~50%) (p >0.05) compared with Mdr2-/- mice. Fisetin treatment reduced expression of p16 and p21 (80% and 90%, respectively), TNF-α (50%), IL-1β (50%), MCP-1 (70%), and fibrosis (60%) (p >0.05). Conclusions Our data support a pathophysiological role of Cholangiocyte senescence in the progression of PSC, and that targeted removal of senescent Cholangiocytes is a plausible therapeutic approach. Lay summary Primary sclerosing cholangitis is a fibroinflammatory, incurable biliary disease. We previously reported that biliary epithelial cell senescence (cell-cycle arrest and hypersecretion of profibrotic molecules) is an important phenotype in primary sclerosing cholangitis. Herein, we demonstrate that reducing the number of senescent Cholangiocytes leads to a reduction in the expression of inflammatory, fibrotic, and senescence markers associated with the disease.
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the transcription factor ets1 promotes apoptosis resistance of senescent Cholangiocytes by epigenetically up regulating the apoptosis suppressor bcl2l1
Journal of Biological Chemistry, 2019Co-Authors: Steven P Ohara, Patrick L. Splinter, Christy E Trussoni, Maria Eugenia Guicciardi, Noah P Splinter, Mohammed Al S Suraih, Navine Nasserghodsi, Deborah Stollenwerk, Gregory J Gores, Nicholas F. LarussoAbstract: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.
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ets proto oncogene 1 transcriptionally up regulates the Cholangiocyte senescence associated protein cyclin dependent kinase inhibitor 2a
Journal of Biological Chemistry, 2017Co-Authors: Steven P Ohara, Patrick L. Splinter, Christy E Trussoni, Noah P Splinter, Maria Lorenzo J Pisarello, Lorena Loarca, Bryce F Schutte, Nicholas F. LarussoAbstract: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.
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Cholangiocyte senescence by way of n ras activation is a characteristic of primary sclerosing cholangitis
Hepatology, 2014Co-Authors: James H Tabibian, Patrick L. Splinter, Steven P Ohara, Christy E Trussoni, Nicholas F. LarussoAbstract: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 p16INK4a 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. (Hepatology 2014;59:2263–2275)
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ciliary subcellular localization of tgr5 determines the Cholangiocyte functional response to bile acid signaling
American Journal of Physiology-gastrointestinal and Liver Physiology, 2013Co-Authors: Anatoliy I Masyuk, Sergio A Gradilone, Bing Q Huang, Tatyana V Masyuk, Patrick L. Splinter, Brynn N Radtke, Garbriella B Gajdos, Nicholas F. LarussoAbstract: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.
Heather Francis - One of the best experts on this subject based on the ideXlab platform.
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knockout of secretin receptor reduces biliary damage and liver fibrosis in mdr2 mice by diminishing senescence of Cholangiocytes
Laboratory Investigation, 2018Co-Authors: Tianhao Zhou, Heather Francis, Fanyin Meng, Julie Venter, Konstantina Kyritsi, Thao Giang, Antonio FranchittoAbstract:Secretin receptor (SR), only expressed by Cholangiocytes, plays a key role in the regulation of biliary damage and liver fibrosis. The aim of this study was to determine the effects of genetic depletion of SR in Mdr2−/− mice on intrahepatic biliary mass, liver fibrosis, senescence, and angiogenesis. 12 wk SR−/−, Mdr2−/−, and SR−/−/Mdr2−/− mice with corresponding wild-type mice were used for the in vivo studies. Immunohistochemistry or immunofluorescence was performed in liver sections for (i) biliary expression of SR; (ii) hematoxylin and eosin; (iii) intrahepatic biliary mass by CK-19; (iv) fibrosis by Col1a1 and α-SMA; (v) senescence by SA-β-gal and p16; and (vi) angiogenesis by VEGF-A and CD31. Secretin (Sct) and TGF-β1 levels were measured in serum and Cholangiocyte supernatant by ELISA. In total liver, isolated Cholangiocytes or HSCs, we evaluated the expression of fibrosis markers (FN-1 and Col1a1); senescence markers (p16 and CCL2); microRNA 125b and angiogenesis markers (VEGF-A, VEGFR-2, CD31, and vWF) by immunoblots and/or qPCR. In vitro, we measured the paracrine effect of Cholangiocyte supernatant on the expression of senescent and fibrosis markers in human hepatic stellate cells (HHSteCs). The increased level of ductular reaction, fibrosis, and angiogenesis in Mdr2−/− mice was reduced in SR−/−/Mdr2−/− mice. Enhanced senescence levels in Cholangiocytes from Mdr2−/− mice were reversed to normal in SR−/−/Mdr2−/− mice. However, senescence was decreased in HSCs from Mdr2−/− mice but returned to normal values in SR−/−/Mdr2−/− mice. In vitro treatment of HHSteCs with supernatant from Cholangiocyte lacking SR (containing lower biliary levels of Sct-dependent TGF-β1) have decreased fibrotic reaction and increased cellular senescence. Sct-induced TGF-β1 secretion was mediated by microRNA 125b. Our data suggest that differential modulation of angiogenesis-dependent senescence of Cholangiocytes and HSCs may be important for the treatment of liver fibrosis in cholangiopathies.
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selective activation of the serotonin receptor 2b isoform increases Cholangiocyte growth and induces the expression of the fibroblast growth factor 1 fgf1
Gastroenterology, 2011Co-Authors: S. Demorrow, Yoshiyuki Ueno, Heather Francis, Antonio Franchitto, Paolo Onori, Romina Mancinelli, Julie Venter, Mellanie White, Wendy Butler, Eugenio GaudioAbstract:Serotonin (5HT) exerts its effects through 16 different receptor isoforms. The activation of 5-HT receptors (5HTR) 1A and 1B inhibits biliary hyperplasia in bile duct ligated (BDL) rats, however, the effects of other 5HTR subtypes on biliary growth are unknown. Cholangiocytes secrete several angiogenic factors including VEGF that regulates biliary mass in an autocrine fashion. FGF1 modulates the function of hepatocytes, although the effects on Cholangiocyte growth are unknown. We aim to: (i) assess the effects of activation of other 5HTR subtypes on biliary growth; and (ii) identify a role for FGF1 in 5HTR2B-induced biliary growth. Methods: The expression of 5HTRs and FGF receptors were assessed in liver sections and Cholangiocytes. Normal and BDL rats were treated with specific agonists for the 5HTR subtypes, 2A, 2B, 2C, 3, 4, 5, 6 and 7, for 1 wk and intrahepatic bile duct mass (IBDM) was assessed. Normal rat Cholangiocyte (NRC) and large mouse Cholangiocyte (LMC) lines were treated In Vitro with 5HTR 2A (TCB2), 2B (BW 723C86) and 2C (WAY 161503) agonists in the absence/presence of BAPTA/AM (Ca2+ chelator), H89 (PKA inhibitor) or Go6976 (PKC inhibitor) and proliferation was assessed by MTS assays and PCNA immunoblots. The expression of FGF1 was assessed by qPCR and immunoblots in: (i) Cholangiocytes from normal and BDL rats; and (ii) NRC and LMC cells following treatments with 5HTR 2A, 2B and 2C agonists. The effects of FGF1 on biliary growth was studied by: i) measuring IBDM in normal and BDL rats treated In Vivo with FGF1 (2.5 nmoles/hr/Kg BW by osmotic minipumps) for 1 wk; and ii) PCNA immunoblots in NRC and LMC treated In Vitro with FGF1 for 48 hr. Results: Bile ducts (in liver sections), NRC and LMC express all serotonin receptors. Activation of 5HTR 2B increased Cholangiocyte growth in normal and BDL rats In Vivo and biliary lines In Vitro. The In Vivo administration of SB 204741 (antagonist for 5HTR 2B) to BDL rats for 1 wk decreased biliary hyperplasia. There were no changes in Cholangiocyte proliferation after the In Vivo administration of agonists for 5HTR 2A, 2C, 3, 4, 5, or 6. 5HTR7 agonist inhibited biliary growth and will be evaluated further in future experiments. The effects of 5HTR 2B agonists were blocked by H89 suggesting a cAMPdependent pathway. There was a concomitant increase in the expression of FGF1 in Cholangiocytes treated with 5HTR 2B In Vivo and In Vitro. Cholangiocytes express all FGFR isoforms (I to IV) and treatment of Cholangiocytes with FGF1 In Vivo and In Vitro increased biliary proliferation. Conclusions: Activation of 5HTR 2B increased Cholangiocyte growth via a cAMP-dependent pathway. There was a parallel increase in FGF1 expression, which may in turn stimulate biliary growth in an autocrine manner. The activation/deactivation of 5HTR 2B may be important for the treatment of cholangiopathies.
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after damage of large bile ducts by gamma aminobutyric acid small ducts replenish the biliary tree by amplification of calcium dependent signaling and de novo acquisition of large Cholangiocyte phenotypes
American Journal of Pathology, 2010Co-Authors: Shannon Glaser, Heather Francis, Antonio Franchitto, Paolo Onori, Romina Mancinelli, Eugenio GaudioAbstract:Large Cholangiocytes secrete bicarbonate in response to secretin and proliferate after bile duct ligation by activation of cyclic adenosine 3′, 5′-monophosphate signaling. The Ca2+-dependent adenylyl cyclase 8 (AC8, expressed by large Cholangiocytes) regulates secretin-induced choleresis. Ca2+-dependent protein kinase C (PKC) regulates small Cholangiocyte function. Because γ-aminobutyric acid (GABA) affects cell functions by activation of both Ca2+ signaling and inhibition of AC, we sought to develop an in vivo model characterized by large Cholangiocyte damage and proliferation of small ducts. Bile duct ligation rats were treated with GABA for one week, and we evaluated: GABAA, GABAB, and GABAC receptor expression; intrahepatic bile duct mass (IBDM) and the percentage of apoptotic Cholangiocytes; secretin-stimulated choleresis; and extracellular signal-regulated kinase1/2 (ERK1/2) phosphorylation and activation of Ca2+-dependent PKC isoforms and AC8 expression. We found that both small and large Cholangiocytes expressed GABA receptors. GABA: (i) induced apoptosis of large Cholangiocytes and reduced large IBDM; (ii) decreased secretin-stimulated choleresis; and (iii) reduced ERK1/2 phosphorylation and AC8 expression in large Cholangiocytes. Small Cholangiocytes: (i) proliferated leading to increased IBDM; (ii) displayed activation of PKCβII; and (iii) de novo expressed secretin receptor, cystic fibrosis transmembrane regulator, Cl−/HCO3− anion exchanger 2 and AC8, and responded to secretin. Therefore, in pathologies of large ducts, small ducts replenish the biliary epithelium by amplification of Ca2+-dependent signaling and acquisition of large Cholangiocyte phenotypes.
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repair related activation of hedgehog signaling promotes Cholangiocyte chemokine production
Hepatology, 2009Co-Authors: Alessia Omenetti, Heather Francis, Wingkin Syn, Youngmi Jung, Alessandro Porrello, Rafal P Witek, Steve S Choi, Liu Yang, Marlyn J MayoAbstract:The mechanisms mediating hepatic accumulation of inflammatory cells in cholestatic liver disease remain enigmatic. Our thesis is that Hedgehog (Hh) pathway activation promotes hepatic accumulation of immune cells that interact with Cholangiocytes. We believe that myofibroblastic hepatic stellate cells (MF-HSC) release soluble Hh ligands that stimulate Cholangiocytes to express chemokines that recruit mononuclear cell types with cognate receptors for these chemokines, thereby orchestrating a repair-related mechanism for liver inflammation. To address this thesis, we used three experimental systems that allow definition of Hh-dependent mechanisms that induce phenotypic changes in Cholangiocytes. First, Cholangiocytes were cultured alone or in the presence of Hh-producing MF-HSC in a transwell co-culture system and/or treated with MF-HSC-conditioned medium with or without Hh-neutralizing antibodies. Changes in Cholangiocyte phenotype were then evaluated by microarray analysis, QRT-PCR, and/or ELISA for Cxcl16. Bile duct ligation was chosen to model biliary fibrosis in mice with an overly-active Hh pathway, control littermates, and healthy rats, and the gene profile was evaluated by QRT-PCR in whole liver tissue. Second, a transwell chemotaxis assay was used to examine NKT cell migration in response to Cholangiocytes, particularly Cholangiocyte-derived Cxcl16. Finally, we studied liver samples from PBC patients and controls by QRT PCR to compare differences in the Hh pathway and Cxcl16. Co-immunostaining of CK-7 and Cxcl16 was then performed to localize the phenotypic source of the Cxcl16. We found that MF-HSC release soluble Hh ligands that stimulate Cholangiocytes to produce Cxcl16 and recruit NKT cells. Hh pathway activation during cholestatic liver injury also induces Cholangiocyte expression of Cxcl16. Conclusion During biliary injury, Hh pathway activation induces Cholangiocyte production of chemokines that recruit NKT cells to portal tracts.
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diferentially expressed adenylyl cyclase isoforms mediate secretory functions in Cholangiocyte subpopulation
Hepatology, 2009Co-Authors: Gianfranco Alpini, Shannon Glaser, Heather Francis, Mario Strazzabosco, Romina Fiorotto, Saida Melero, Carlo SpirliAbstract:Cyclic adenosine monophosphate (cAMP) is generated by adenylyl cyclases (ACs), a group of enzymes with different tissue specificity and regulation. We hypothesized that AC isoforms are heterogeneously expressed along the biliary tree, are associated with specific secretory stimuli, and are differentially modulated in cholestasis. Small duct and large duct Cholangiocytes were isolated from controls and from lipopolysaccharide-treated or α-naphthylisothiocyanate–treated rats. AC isoform expression was assessed via real-time polymerase chain reaction. Secretion and cAMP levels were measured in intrahepatic bile duct units after stimulation with secretin, forskolin, HCO3−/CO2, cholinergic agonists, and β-adrenergic agonists, with or without selected inhibitors or after silencing of AC8 or soluble adenylyl cyclase (sAC) with small interfering RNA. Gene expression of the Ca2+-insensitive isoforms (AC4, AC7) was higher in small duct Cholangiocytes, whereas that of the Ca2+-inhibitable (AC5, AC6, AC9), the Ca2+/calmodulin-stimulated AC8, and the soluble sAC was higher in large duct Cholangiocytes. Ca2+/calmodulin inhibitors and AC8 gene silencing inhibited choleresis and cAMP production stimulated by secretin and acetylcholine, but not by forskolin. Secretion stimulated by isoproterenol and calcineurin inibitors was cAMP-dependent and γ-aminobutyric acid–inhibitable, consistent with activation of AC9. Cholangiocyte secretion stimulated by isohydric changes in [HCO3−]i was cAMP-dependent and inhibited by sAC inhibitor and sAC gene silencing. Treatment with lipopolysaccharide or α-naphthylisothiocyanate increased expression of AC7 and sAC but decreased expression of the other ACs. Conclusion: These studies demonstrate a previously unrecognized role of ACs in biliary pathophysiology. In fact: (1) AC isoforms are differentially expressed in Cholangiocyte subpopulations; (2) AC8, AC9, and sAC mediate Cholangiocyte secretion in response to secretin, β-adrenergic agonists, or changes in [HCO3−]i, respectively; and (3) AC gene expression is modulated in experimental cholestasis. (HEPATOLOGY 2009)
Gianfranco Alpini - One of the best experts on this subject based on the ideXlab platform.
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Role of lactoferrin and its receptors on biliary epithelium.
Biometals : an international journal on the role of metal ions in biology biochemistry and medicine, 2018Co-Authors: Romina Mancinelli, Gianfranco Alpini, Antonio Franchitto, Guido Carpino, Francesca Olivero, Diletta Overi, Luigi Rosa, Maria Stefania Lepanto, Antimo Cutone, Paolo OnoriAbstract:Human lactoferrin is an iron-binding glycoprotein present at high concentrations in breast milk and colostrum. It is produced by many exocrine glands and widely distributed in a variety of body fluids. This protein has antimicrobial, immunomodulatory, antioxidant, and anticancer properties. Two important hLf receptors have been identified: LDL receptor related protein (LRP1), a low specificity receptor, and intelectin-1 (ITLN1), a high specificity receptor. No data are present on the role of hLf on the biliary epithelium. Our aims have been to evaluate the expression of Lf and its receptors in human and murine Cholangiocytes and its effect on proliferation. Immunohistochemistry and immunofluorescence (IF) were conducted on human healthy and primary biliary cholangitis (PBC) liver samples as well as on liver samples obtained from normal and bile duct ligated (BDL) mice to evaluate the expression of Lf, LRP1 and ITLN1. Cell proliferation in vitro studies were performed on human Cholangiocyte cell lines via 3-(4,5-dimetiltiazol-2-il)-2,5-diphenyltetrazolium assay as well as IF to evaluate proliferating cell nuclear antigen (PCNA) expression. Our results show that mouse and human Cholangiocytes express Lf, LRP1 and ITLN1, at higher extent in Cholangiocytes from BDL and PBC samples. Furthermore, the in vitro addition of bovine Lf (bLf) has a proliferative effect on human Cholangiocyte cell line. The results support a proliferative role of hLf on the biliary epithelium; this pro-proliferative effect of hLf and bLf on Cholangiocytes could be particularly relevant in human cholangiopathies such as PBC, characterized by Cholangiocyte death and ductopenia.
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the role of the secretin secretin receptor axis in inflammatory Cholangiocyte communication via extracellular vesicles
Scientific Reports, 2017Co-Authors: Keisaku Sato, Gianfranco Alpini, Shannon Glaser, Fanyin Meng, Julie Venter, Thao GiangAbstract:Small and large intrahepatic bile ducts consist of small and large Cholangiocytes, respectively, and these Cholangiocytes have different morphology and functions. The gastrointestinal peptide hormone, secretin (SCT) that binds to secretin receptor (SR), is a key mediator in Cholangiocyte pathophysiology. Extracellular vesicles (EVs) are membrane-bound vesicles and cell-cell EV communication is recognized as an important factor in liver pathology, although EV communication between Cholangiocytes is not identified to date. Cholangiocytes secrete proinflammatory cytokines during bacterial infection leading to biliary inflammation and hyperplasia. We demonstrate that Cholangiocytes stimulated with lipopolysaccharide (LPS), which is a membrane component of gram-negative bacteria, secrete more EVs than Cholangiocytes incubated with vehicle. These LPS-derived EVs induce inflammatory responses in other Cholangiocytes including elevated cytokine production and cell proliferation. Large but not small Cholangiocytes show inflammatory responses against large but not small Cholangiocyte-derived EVs. Large Cholangiocytes with knocked down either SCT or SR by short hairpin RNAs show reduced EV secretion during LPS stimulation, and EVs isolated from SCT or SR knocked down Cholangiocytes fail to induce inflammatory reactions in control large Cholangiocytes. This study identifies Cholangiocyte EV communication during LPS stimulation, and demonstrates that the SCT/SR axis may be important for this event.
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mechanisms of Cholangiocyte responses to injury
Biochimica et Biophysica Acta, 2017Co-Authors: Keisaku Sato, Gianfranco Alpini, Shannon Glaser, Fanyin Meng, Thao GiangAbstract:Cholangiocytes, epithelial cells that line the biliary epithelium, are the primary target cells for cholangiopathies including primary sclerosing cholangitis and primary biliary cholangitis. Quiescent Cholangiocytes respond to biliary damage and acquire an activated neuroendocrine phenotype to maintain the homeostasis of the liver. The typical response of Cholangiocytes is proliferation leading to bile duct hyperplasia, which is a characteristic of cholestatic liver diseases. Current studies have identified various signaling pathways that are associated with Cholangiocyte proliferation/loss and liver fibrosis in cholangiopathies using human samples and rodent models. Although recent studies have demonstrated that extracellular vesicles and microRNAs could be mediators that regulate these messenger/receptor axes, further studies are required to confirm their roles. This review summarizes current studies of biliary response and Cholangiocyte proliferation during cholestatic liver injury with particular emphasis on the secretin/secretin receptor axis. This article is part of a Special Issue entitled: Cholangiocytes in Health and Diseaseedited by Jesus Banales, Marco Marzioni, Nicholas LaRusso and Peter Jansen.
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regulators of Cholangiocyte proliferation
Gene Expression, 2017Co-Authors: Chad Hall, Shannon Glaser, Fanyin Meng, Konstantina Kyritsi, Tianhao Zhou, Keisaku Sato, Gianfranco AlpiniAbstract:Cholangiocytes, a small population of cells within the normal liver, have been the focus of a significant amount of research over the past two decades because of their involvement in cholangiopathies such as primary sclerosing cholangitis and primary biliary cholangitis. This article summarizes landmark studies in the field of Cholangiocyte physiology and aims to provide an updated review of biliary pathogenesis. The historical approach of rodent extrahepatic bile duct ligation and the relatively recent utilization of transgenic mice have led to significant discoveries in Cholangiocyte pathophysiology. Cholangiocyte physiology is a complex system based on heterogeneity within the biliary tree and a number of signaling pathways that serve to regulate bile composition. Studies have expanded the list of neuropeptides, neurotransmitters, and hormones that have been shown to be key regulators of proliferation and biliary damage. The peptide histamine and hormones, such as melatonin and angiotensin, angiotensin, as well as numerous sex hormones, have been implicated in Cholangiocyte proliferation during cholestasis. Numerous pathways promote Cholangiocyte proliferation during cholestasis, and there is growing evidence to suggest that Cholangiocyte proliferation may promote hepatic fibrosis. These pathways may represent significant therapeutic potential for a subset of cholestatic liver diseases that currently lack effective therapies.
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inhibition of the liver expression of arylalkylamine n acetyltransferase increases the expression of angiogenic factors in Cholangiocytes
Hepatobiliary surgery and nutrition, 2014Co-Authors: Anastasia Renzi, Gianfranco Alpini, Shannon Glaser, Antonio Franchitto, Paolo Onori, Romina Mancinelli, Eugenio GaudioAbstract:Background and aims: Reduction of biliary serotonin N-acetyltransferase (AANAT) expression and melatonin administration/secretion in Cholangiocytes increases biliary proliferation and the expression of SR, CFTR and Cl – /HCO 3 – AE2. The balance between biliary proliferation/damage is regulated by several autocrine neuroendocrine factors including vascular endothelial growth factor-A/C (VEGF-A/C). VEGFs are secreted by several epithelia, where they modulate cell growth by autocrine and paracrine mechanisms. No data exists regarding the effect of AANAT modulation on the expressions of VEGFs by Cholangiocytes. Methods: In this study, we evaluated the effect of local modulation of biliary AANAT expression on the Cholangiocytes synthesis of VEGF-A/C. Results: The decrease in AANAT expression and subsequent lower melatonin secretion by Cholangiocytes was associated with increased expression of VEGF-A/C. Overexpression of AANAT in Cholangiocyte lines decreased the expression of VEGF-A/C. Conclusions: Modulation of melatonin synthesis may affect the expression of VEGF-A/C by Cholangiocytes and may modulate the hepatic microvascularization through the regulation of VEGF-A/C expression regulating biliary functions.
Shannon Glaser - One of the best experts on this subject based on the ideXlab platform.
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the role of the secretin secretin receptor axis in inflammatory Cholangiocyte communication via extracellular vesicles
Scientific Reports, 2017Co-Authors: Keisaku Sato, Gianfranco Alpini, Shannon Glaser, Fanyin Meng, Julie Venter, Thao GiangAbstract:Small and large intrahepatic bile ducts consist of small and large Cholangiocytes, respectively, and these Cholangiocytes have different morphology and functions. The gastrointestinal peptide hormone, secretin (SCT) that binds to secretin receptor (SR), is a key mediator in Cholangiocyte pathophysiology. Extracellular vesicles (EVs) are membrane-bound vesicles and cell-cell EV communication is recognized as an important factor in liver pathology, although EV communication between Cholangiocytes is not identified to date. Cholangiocytes secrete proinflammatory cytokines during bacterial infection leading to biliary inflammation and hyperplasia. We demonstrate that Cholangiocytes stimulated with lipopolysaccharide (LPS), which is a membrane component of gram-negative bacteria, secrete more EVs than Cholangiocytes incubated with vehicle. These LPS-derived EVs induce inflammatory responses in other Cholangiocytes including elevated cytokine production and cell proliferation. Large but not small Cholangiocytes show inflammatory responses against large but not small Cholangiocyte-derived EVs. Large Cholangiocytes with knocked down either SCT or SR by short hairpin RNAs show reduced EV secretion during LPS stimulation, and EVs isolated from SCT or SR knocked down Cholangiocytes fail to induce inflammatory reactions in control large Cholangiocytes. This study identifies Cholangiocyte EV communication during LPS stimulation, and demonstrates that the SCT/SR axis may be important for this event.
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mechanisms of Cholangiocyte responses to injury
Biochimica et Biophysica Acta, 2017Co-Authors: Keisaku Sato, Gianfranco Alpini, Shannon Glaser, Fanyin Meng, Thao GiangAbstract:Cholangiocytes, epithelial cells that line the biliary epithelium, are the primary target cells for cholangiopathies including primary sclerosing cholangitis and primary biliary cholangitis. Quiescent Cholangiocytes respond to biliary damage and acquire an activated neuroendocrine phenotype to maintain the homeostasis of the liver. The typical response of Cholangiocytes is proliferation leading to bile duct hyperplasia, which is a characteristic of cholestatic liver diseases. Current studies have identified various signaling pathways that are associated with Cholangiocyte proliferation/loss and liver fibrosis in cholangiopathies using human samples and rodent models. Although recent studies have demonstrated that extracellular vesicles and microRNAs could be mediators that regulate these messenger/receptor axes, further studies are required to confirm their roles. This review summarizes current studies of biliary response and Cholangiocyte proliferation during cholestatic liver injury with particular emphasis on the secretin/secretin receptor axis. This article is part of a Special Issue entitled: Cholangiocytes in Health and Diseaseedited by Jesus Banales, Marco Marzioni, Nicholas LaRusso and Peter Jansen.
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regulators of Cholangiocyte proliferation
Gene Expression, 2017Co-Authors: Chad Hall, Shannon Glaser, Fanyin Meng, Konstantina Kyritsi, Tianhao Zhou, Keisaku Sato, Gianfranco AlpiniAbstract:Cholangiocytes, a small population of cells within the normal liver, have been the focus of a significant amount of research over the past two decades because of their involvement in cholangiopathies such as primary sclerosing cholangitis and primary biliary cholangitis. This article summarizes landmark studies in the field of Cholangiocyte physiology and aims to provide an updated review of biliary pathogenesis. The historical approach of rodent extrahepatic bile duct ligation and the relatively recent utilization of transgenic mice have led to significant discoveries in Cholangiocyte pathophysiology. Cholangiocyte physiology is a complex system based on heterogeneity within the biliary tree and a number of signaling pathways that serve to regulate bile composition. Studies have expanded the list of neuropeptides, neurotransmitters, and hormones that have been shown to be key regulators of proliferation and biliary damage. The peptide histamine and hormones, such as melatonin and angiotensin, angiotensin, as well as numerous sex hormones, have been implicated in Cholangiocyte proliferation during cholestasis. Numerous pathways promote Cholangiocyte proliferation during cholestasis, and there is growing evidence to suggest that Cholangiocyte proliferation may promote hepatic fibrosis. These pathways may represent significant therapeutic potential for a subset of cholestatic liver diseases that currently lack effective therapies.
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inhibition of the liver expression of arylalkylamine n acetyltransferase increases the expression of angiogenic factors in Cholangiocytes
Hepatobiliary surgery and nutrition, 2014Co-Authors: Anastasia Renzi, Gianfranco Alpini, Shannon Glaser, Antonio Franchitto, Paolo Onori, Romina Mancinelli, Eugenio GaudioAbstract:Background and aims: Reduction of biliary serotonin N-acetyltransferase (AANAT) expression and melatonin administration/secretion in Cholangiocytes increases biliary proliferation and the expression of SR, CFTR and Cl – /HCO 3 – AE2. The balance between biliary proliferation/damage is regulated by several autocrine neuroendocrine factors including vascular endothelial growth factor-A/C (VEGF-A/C). VEGFs are secreted by several epithelia, where they modulate cell growth by autocrine and paracrine mechanisms. No data exists regarding the effect of AANAT modulation on the expressions of VEGFs by Cholangiocytes. Methods: In this study, we evaluated the effect of local modulation of biliary AANAT expression on the Cholangiocytes synthesis of VEGF-A/C. Results: The decrease in AANAT expression and subsequent lower melatonin secretion by Cholangiocytes was associated with increased expression of VEGF-A/C. Overexpression of AANAT in Cholangiocyte lines decreased the expression of VEGF-A/C. Conclusions: Modulation of melatonin synthesis may affect the expression of VEGF-A/C by Cholangiocytes and may modulate the hepatic microvascularization through the regulation of VEGF-A/C expression regulating biliary functions.
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involvement of Cholangiocyte proliferation in biliary fibrosis
World Journal of Gastrointestinal Pathophysiology, 2010Co-Authors: Sally Priester, Candace Wise, Shannon GlaserAbstract:Cholangiocytes are the epithelial cells that line the biliary tree. In the adult liver, they are a mitotically dormant cell population, unless ductular reaction is triggered by injury. The ability of Cholangiocytes to proliferate is important in many different human pathological liver conditions that target this cell type, which are termed cholangiopathies (i.e. primary biliary cirrhosis, primary sclerosing cholangitis and biliary atresia). In our article, we provide background information on the morphological and functional heterogeneity of Cholangiocytes, summarize what is currently known about their proliferative processes, and briefly describe the diseases that target these cells. In addition, we address recent findings that suggest Cholangiocyte involvement in epithelial-to-mesenchymal transformation and liver fibrosis, and propose directions for future studies.
Tatyana V Masyuk - One of the best experts on this subject based on the ideXlab platform.
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tgr5 contributes to hepatic cystogenesis in rodents with polycystic liver diseases through cyclic adenosine monophosphate gαs signaling
Hepatology, 2017Co-Authors: Tatyana V Masyuk, Anatoliy I Masyuk, Bing Q Huang, Maria Lorenzo J Pisarello, Brynn N Howard, Pui Yuen Lee, Xavier Fung, Eduard Sergienko, Robert J Ardecky, Thomas D Y ChungAbstract:Hepatic cystogenesis in polycystic liver disease is associated with increased levels of cyclic adenosine monophosphate (cAMP) in Cholangiocytes lining liver cysts. Takeda G protein receptor 5 (TGR5), a G protein-coupled bile acid receptor, is linked to cAMP and expressed in Cholangiocytes. Therefore, we hypothesized that TGR5 might contribute to disease progression. We examined expression of TGR5 and Gα proteins in cultured Cholangiocytes and in livers of animal models and humans with polycystic liver disease. In vitro, we assessed Cholangiocyte proliferation, cAMP levels, and cyst growth in response to (1) TGR5 agonists (taurolithocholic acid, oleanolic acid [OA], and two synthetic compounds), (2) a novel TGR5 antagonist (m-tolyl 5-chloro-2-[ethylsulfonyl] pyrimidine-4-carboxylate [SBI-115]), and (3) a combination of SBI-115 and pasireotide, a somatostatin receptor analogue. In vivo, we examined hepatic cystogenesis in OA-treated polycystic kidney rats and after genetic elimination of TGR5 in double mutant TGR5-/- ;Pkhd1del2/del2 mice. Compared to control, expression of TGR5 and Gαs (but not Gαi and Gαq ) proteins was increased 2-fold to 3-fold in cystic Cholangiocytes in vitro and in vivo. In vitro, TGR5 stimulation enhanced cAMP production, cell proliferation, and cyst growth by ∼40%; these effects were abolished after TGR5 reduction by short hairpin RNA. OA increased cystogenesis in polycystic kidney rats by 35%; in contrast, hepatic cystic areas were decreased by 45% in TGR5-deficient TGR5-/- ;Pkhd1del2/del2 mice. TGR5 expression and its colocalization with Gαs were increased ∼2-fold upon OA treatment. Levels of cAMP, cell proliferation, and cyst growth in vitro were decreased by ∼30% in cystic Cholangiocytes after treatment with SBI-115 alone and by ∼50% when SBI-115 was combined with pasireotide. Conclusion TGR5 contributes to hepatic cystogenesis by increasing cAMP and enhancing Cholangiocyte proliferation; our data suggest that a TGR5 antagonist alone or concurrently with somatostatin receptor agonists represents a potential therapeutic approach in polycystic liver disease. (Hepatology 2017;66:1197-1218).
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centrosomal abnormalities characterize human and rodent cystic Cholangiocytes and are associated with cdc25a overexpression
American Journal of Pathology, 2014Co-Authors: Tatyana V Masyuk, Anatoliy I Masyuk, Jesus M Banales, Angela J Stroope, Seung Ok Lee, Brynn N Radtke, Bing Huang, Patrick L. SplinterAbstract: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 (Pkd2WS25/− 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.
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ciliary subcellular localization of tgr5 determines the Cholangiocyte functional response to bile acid signaling
American Journal of Physiology-gastrointestinal and Liver Physiology, 2013Co-Authors: Anatoliy I Masyuk, Sergio A Gradilone, Bing Q Huang, Tatyana V Masyuk, Patrick L. Splinter, Brynn N Radtke, Garbriella B Gajdos, Nicholas F. LarussoAbstract: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.
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the role of cilia in the regulation of bile flow
Digestive Diseases, 2011Co-Authors: Nicholas F. Larusso, Tatyana V MasyukAbstract:Cholangiocytes, the epithelial cells lining intrahepatic bile ducts, are ciliated cells. Each Cholangiocyte has a primary cilium consisting of (i) a microtubule-based axoneme and (ii) the basal body, centriole-derived, microtubule-organizing center from which the axoneme emerges. Primary cilia in Cholangiocytes were described decades ago, but their physiological and pathophysiological significance remained unclear until recently. We now recognize that Cholangiocyte cilia extend from the apical plasma membrane into the bile duct lumen and, as such, are ideally positioned to detect changes in bile flow, bile composition and bile osmolality. These sensory organelles act as cellular antennae that can detect and transmit signals that influence Cholangiocyte function. Indeed, recent data show that Cholangiocyte primary cilia can activate intracellular signaling pathways when they sense modifications in the flow, molecular constituents and osmolarity of bile. Their ability to sense and transmit signals depends on the participation of a growing number of specific ciliary-associated proteins that act as receptors, channels and transporters. Cholangiocyte cilia, in addition to being important in normal biliary physiology, likely contribute to the cholangiopathies when their normal structure or function is disturbed. Indeed, the polycystic liver diseases that occur in combination with autosomal dominant and recessive polycystic kidney disease (i.e. ADPKD and ARPKD) are two important examples of such conditions. Recent insights into the role of Cholangiocyte cilia in cystic liver disease using in vitro and animal models have already resulted in clinical trials that have influenced the management of cystic liver disease.
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the camp effectors epac and protein kinase a pka are involved in the hepatic cystogenesis of an animal model of autosomal recessive polycystic kidney disease arpkd
Hepatology, 2009Co-Authors: Juan F. Medina, Sergio A Gradilone, Anatoliy I Masyuk, Tatyana V Masyuk, Jesus M Banales, Nicholas F. LarussoAbstract:Autosomal recessive polycystic kidney disease (ARPKD) is a genetic disorder in which affected infants often die at birth or shortly thereafter, primarily as the result of markedly enlarged kidney cysts and impaired lung function. In surviving patients, hepatic fibrosis, bile duct dilatation (Caroli’s disease) and/or cyst development becomes progressively more severe and may be the major cause of morbidity and mortality.1 ARPKD is linked to mutations in the PKHD1 gene which encodes fibrocystin, a large trans-membrane protein with unknown function,2, 3 located on Cholangiocyte primary cilia;4 these non-motile long tubular organelles extend from the Cholangiocyte apical membrane and function as mechano-,5 chemo-6 and osmo-sensors,7 detecting and transmitting luminal stimuli into intracellular signals. Although the pathophysiology of hepatic cystogenesis in ARPKD is unclear, abnormalities in Cholangiocyte fluid secretion and proliferation likely contribute.1, 8 Indeed, in the PCK rat, a well-characterized animal model of ARPKD,9 liver cysts originate from intrahepatic bile ducts and their growth is associated with increased proliferative activity of cystic Cholangiocytes.10 However, the mechanism underlying this benign hyperproliferative process is not fully understood. Accumulated evidence suggests that two intracellular signaling mediators, cAMP and Ca2+, regulate proliferation in different cell types,11, 12 including Cholangiocytes.13 We recently demonstrated that cAMP levels are increased in Cholangiocytes of the PCK rat and that octreotide, a somatostatin analog known to inhibit cAMP, decreases hepatic cyst volume, hepatic fibrotic scores and mitotic indices.10 cAMP has two downstream targets, cAMP-GEF/Epac (Epac) and protein kinase A (PKA).14 Epac proteins, a family of Rap guanine nucleotide exchange factors, regulate many cellular processes via PKA-independent mechanisms.15 There are two different Epac isoforms (Epac1 and Epac2, also known as RapGEF3 and RapGEF4, respectively) which are expressed from different genes in a variety of tissues.15–17 PKA is a heterotetrameric holoenzyme with two regulatory and two catalytic subunits, that responds to intracellular changes of cAMP regulating a wide range of intracellular processes.14 There exist several members of PKA regulatory (RIα, RIβ, RIIα and RIIβ) and catalytic (Cα, Cβ, Cγ and PrKX) subunits. The biochemical and functional features of PKA are largely determined by the structure and properties of the regulatory subunits, which are differentially expressed depending on the tissue and the cellular state.14 cAMP binds to the PKA regulatory subunits, leading to dissociation and activation of the catalytic subunits that may regulate the phosphorylation of a number of proteins and the expression of different genes.14 Both cAMP downstream effectors, Epac and PKA, may be involved in regulation of proliferation in different cell types. In polycystic diseases, however, only PKA has been shown to be responsible for hyperproliferation of epithelial cells lining renal cysts from patients with autosomal dominant polycystic kidney disease (ADPKD).18 But no data exist regarding the expression, intracellular localization and function of PKA regulatory subunits and Epac isoforms in Cholangiocytes, and the involvement of these effectors in cystic Cholangiocyte proliferation remains unknown. Intracellular Ca2+ [Ca2+]i may also participate in the control of cell growth.11, 12 In fact, renal cystic cells of patients with ARPKD have low calcium levels,11 and silencing of Pkhd1 in cultured normal kidney cells results in decreased intracellular calcium and hyperproliferation.19 The interconnection between cAMP and [Ca2+]i signaling pathways under normal and pathological conditions is currently attracting considerable attention. In particular, it has been shown that in renal cells from ADPKD patients, intracellular calcium modulates cAMP-dependent proliferation. Moreover, these cells were characterized by decreased [Ca2+]i and exhibited higher rates of cell proliferation in response to cAMP, while experimental restoration of [Ca2+]i inhibited their cAMP-stimulated growth.11 We recently demonstrated in normal rat Cholangiocytes that elevated [Ca2+]i is able to terminate cAMP signaling (i.e., turn off regulation) initially activated by choleretic stimuli.5 However, it is unclear whether intracellular Ca2+ levels are altered in cystic Cholangiocytes and what role, if any, intracellular Ca2+ plays in hepatic cystogenesis. Thus, we examined the: i) expression and intracellular localization of two cAMP downstream effectors, PKA and Epac, in cultured Cholangiocytes from normal and PCK rats; ii) role of PKA and Epac activation in proliferation of normal and PCK Cholangiocytes; iii) intracellular calcium levels in PCK Cholangiocytes; and iv) relationships between cAMP-stimulated proliferation and calcium levels in PCK Cholangiocytes.