Search

Background: α-Calcitonin gene-related peptide (α-CGRP) is a 37-amino acid neuropeptide involved in several pathophysiological processes. α-CGRP is involved in the regulation of cholangiocyte proliferation during cholestasis. In this study, we aimed to evaluate if α-CGRP regulates bile duct ligation (BDL)-induced liver fibrosis by using a α-CGRP knockout (α-CGRP−/−) mouse model. Methods: α-CGRP−/− and wild-type (WT) mice were subjected to sham surgery or BDL for 7 days. Then, liver fibrosis and cellular senescence as well as the expression of kinase such as p38 and C-Jun N-terminal protein kinase (JNK) in mitogen-activated protein kinases (MAPK) signaling pathway were evaluated in total liver, together with measurement of cellular senescence in cholangiocytes or hepatic stellate cells (HSCs). Results: There was enhanced hepatic expression of Calca (coding α-CGRP) and the CGRP-receptor components (CRLR, RAMP-1 and RCP) in BDL and in both WT α-CGRP−/− and BDL α-CGRP−/− mice, respectively. Moreover, there was increased CGRP serum levels and hepatic mRNA expression of CALCA and CGRP receptor components in late-stage PSC samples compared to healthy control samples. Depletion of α-CGRP reduced liver injury and fibrosis in BDL mice that was associated with enhanced cellular senescence of hepatic stellate cells and reduced senescence of cholangiocytes as well as decreased activation of p38 and JNK MAPK signaling pathway. Cholangiocyte supernatant from BDL α-CGRP−/− mice inhibited the activation and increased cellular senescence of cultured human HSCs (HHSCs) compared to HHSCs stimulated with BDL cholangiocyte supernatant. Taken together, endogenous α-CGRP promoted BDL-induced cholestatic liver fibrosis through differential changes in senescence of HSCs and cholangiocytes and activation of p38 and JNK signaling. Modulation of α-CGRP/CGRP receptor signaling may be key for the management of biliary senescence and liver fibrosis in cholangiopathies.

Cholangiopathies are diseases that affect cholangiocytes, the cells lining the biliary tract. Liver stem cells (LSCs) are able to differentiate into all cells of the liver and possibly influence the surrounding liver tissue by secretion of signaling molecules. One way in which cells can interact is through secretion of extracellular vesicles (EVs), which are small membrane-bound vesicles that contain proteins, microRNAs (miRNAs), and cytokines. We evaluated the contents of liver stem cell–derived EVs (LSCEVs), compared their miRNA contents to those of EVs isolated from hepatocytes, and evaluated the downstream targets of these miRNAs. We finally evaluated the crosstalk among LSCs, cholangiocytes, and human hepatic stellate cells (HSCs). We showed that LSCEVs were able to reduce ductular reaction and biliary fibrosis in multidrug resistance protein 2 (MDR2)(−/−) mice. Additionally, we showed that cholangiocyte growth was reduced and HSCs were deactivated in LSCEV-treated mice. Evaluation of LSCEV contents compared with EVs derived from hepatocytes showed a large increase in the miRNA, lethal-7 (let-7). Further evaluation of let-7 in MDR2(−/−) mice and human primary sclerosing cholangitis samples showed reduced levels of let-7 compared with controls. In liver tissues and isolated cholangiocytes, downstream targets of let-7 (identified by ingenuity pathway analysis), Lin28a (Lin28 homolog A), Lin28b (Lin28 homolog B), IL-13 (interleukin 13), NR1H4 (nuclear receptor subfamily 1 group H member 4) and NF-κB (nuclear factor kappa B), are elevated in MDR2(−/−) mice, but treatment with LSCEVs reduced levels of these mediators of ductular reaction and biliary fibrosis through the inhibition of NF-κB and IL-13 signaling pathways. Evaluation of crosstalk using cholangiocyte supernatants from LSCEV-treated cells on cultured HSCs showed that HSCs had reduced levels of fibrosis and increased senescence. CONCLUSION: Our studies indicate that LSCEVs could be a possible treatment for cholangiopathies or could be used for target validation for future therapies.

BACKGROUND AND AIMS: Serotonin (5HT) is a neuroendocrine hormone synthetized in the central nervous system (CNS) as well as enterochromaffin cells of the gastrointestinal tract. Tryptophan hydroxylase (TPH1) and monoamine oxidase (MAO-A) are the key enzymes for the synthesis and catabolism of 5HT, respectively. Previous studies demonstrated that 5-hydroxytryptamine receptor (5HTR)1A/1B receptor agonists inhibit biliary hyperplasia in bile-duct ligated (BDL) rats, whereas 5HTR2B receptor antagonists attenuate liver fibrosis (LF) in mice. Our aim was to evaluate the role of 5HTR2A/2B/2C agonists/antagonists in cholestatic models. APPROACH AND RESULTS: While in vivo studies were performed in BDL rats and the multidrug resistance gene 2 knockout (Mdr2(−/−)) mouse model of PSC, in vitro studies were performed in cell lines of cholangiocytes and hepatic stellate cells (HSCs). 5HTR2A/2B/2C and MAO-A/TPH1 are expressed in cholangiocytes and HSCs from BDL rats and Mdr2(−/−) mice. Ductular reaction, LF, as well as the mRNA expression of proinflammatory genes increased in normal, BDL rats, and Mdr2(−/−) mice following treatment 5HTR2A/2B/2C agonists, but decreased when BDL rats and Mdr2(−/−) mice were treated with 5HTR2A/2B/2C antagonists compared to BDL rats and Mdr2(−/−) mice, respectively. 5HT levels increase in Mdr2(−/−) mice and in PSC human patients compared to their controls and decrease in serum of Mdr2(−/−) mice treated with 5HTR2A/2B/2C antagonists compared to untreated Mdr2(−/−) mice. In vitro, cell lines of murine cholangiocytes and human HSCs express 5HTR2A/2B/2C and MAO-A/TPH1; treatment of these cell lines with 5HTR2A/2B/2C antagonists or TPH1 inhibitor decreased 5HT levels as well as expression of fibrosis and inflammation genes compared to controls. CONCLUSIONS: Modulation of the TPH1/MAO-A/5HT/5HTR2A/2B/2C axis may represent a therapeutic approach for management of cholangiopathies, including PSC.

α7-nAChR is a nicotinic acetylcholine receptor [specifically expressed on hepatic stellate cells (HSCs), Kupffer cells, and cholangiocytes] that regulates inflammation and apoptosis in the liver. Thus, targeting α7-nAChR may be therapeutic in biliary diseases. Bile duct ligation (BDL) was performed on wild-type (WT) and α7-nAChR−/− mice. We first evaluated the expression of α7-nAChR by immunohistochemistry (IHC) in liver sections. IHC was also performed to assess intrahepatic bile duct mass (IBDM), and Sirius Red staining was performed to quantify the amount of collagen deposition. Immunofluorescence was performed to assess colocalization of α7-nAChR with bile ducts (costained with CK-19) and HSCs (costained with desmin). The mRNA expression of α7-nAChR, Ki-67/PCNA (proliferation), fibrosis genes (TGF-β1, fibronectin-1, Col1α1, and α-SMA), and inflammatory markers (IL-6, IL-1β, and TNF-α) was measured by real-time PCR. Biliary TGF-β1 and hepatic CD68 (Kupffer cell marker) expression was assessed using IHC. α7-nAChR immunoreactivity was observed in both bile ducts and HSCs and increased following BDL. α7-nAChR−/− BDL mice exhibited decreased (i) bile duct mass, liver fibrosis, and inflammation, and (ii) immunoreactivity of TGF-β1 as well as expression of fibrosis genes compared to WT BDL mice. α7-nAChR activation triggers biliary proliferation and liver fibrosis and may be a therapeutic target in managing extrahepatic biliary obstruction.

Substance P (SP) is involved in the proliferation of cholangiocytes in bile duct–ligated (BDL) mice and human cholangiocarcinoma growth by interacting with the neurokinin-1 receptor (NK-1R). To identify whether SP regulates liver fibrosis during cholestasis, wild-type or NK-1R knockout (NK-1R–/–) mice that received BDL or sham surgery and multidrug resistance protein 2 knockout (Mdr2–/–) mice treated with either an NK-1R antagonist (L-733,060) or saline were used. Additionally, wild-type mice were treated with SP or saline intraperitoneally. In vivo, there was increased expression of tachykinin precursor 1 (coding SP) and NK-1R in both BDL and Mdr2–/–mice compared to wild-type mice. Expression of tachykinin precursor 1 and NK-1R was significantly higher in liver samples from primary sclerosing cholangitis patients compared to healthy controls. Knockout of NK-1R decreased BDL-induced liver fibrosis, and treatment with L-733,060 resulted in decreased liver fibrosis in Mdr2–/–mice, which was shown by decreased sirius red staining, fibrosis gene and protein expression, and reduced transforming growth factor-β1 levels in serum and cholangiocyte supernatants. Furthermore, we observed that reduced liver fibrosis in NK-1R–/–mice with BDL surgery or Mdr2–/–mice treated with L-733,060 was associated with enhanced cellular senescence of hepatic stellate cells and decreased senescence of cholangiocytes. In vitro, L-733,060 inhibited SP-induced expression of fibrotic genes in hepatic stellate cells and cholangiocytes; treatment with L-733,060 partially reversed the SP-induced decrease of senescence gene expression in cultured hepatic stellate cells and the SP-induced increase of senescence-related gene expression in cultured cholangiocytes. Conclusion: Collectively, our results demonstrate the regulatory effects of the SP/NK-1R axis on liver fibrosis through changes in cellular senescence during cholestatic liver injury. (Hepatology 2017;66:528–541)

Melatonin, a neuroendocrine hormone synthesized by the pineal gland and cholangiocytes, decreases biliary hyperplasia and liver fibrosis during cholestasis-induced biliary injury via melatonin-dependent autocrine signaling through increased biliary arylalkylamine N-acetyltransferase (AANAT) expression and melatonin secretion, downregulation of miR-200b and specific circadian clock genes. Melatonin synthesis is decreased by pinealectomy (PINX) or chronic exposure to light. We evaluated the effect of PINX or prolonged light exposure on melatonin-dependent modulation of biliary damage/ductular reaction/liver fibrosis. Studies were performed in male rats with/without BDL for 1 week with 12:12 h dark/light cycles, continuous light or after 1 week of PINX. The expression of AANAT and melatonin levels in serum and cholangiocyte supernatant were increased in BDL rats, while decreased in BDL rats following PINX or continuous light exposure. BDL-induced increase in serum chemistry, ductular reaction, liver fibrosis, inflammation, angiogenesis and ROS generation were significantly enhanced by PINX or light exposure. Concomitant with enhanced liver fibrosis, we observed increased biliary senescence and enhanced clock genes and miR-200b expression in total liver and cholangiocytes. In vitro, the expression of AANAT, clock genes and miR-200b was increased in PSC human cholangiocyte cell lines (hPSCL). The proliferation and activation of HHStecs (human hepatic stellate cell lines) were increased after stimulating with BDL cholangiocyte supernatant and further enhanced when stimulated with BDL rats following PINX or continuous light exposure cholangiocyte supernatant via intracellular ROS generation. Conclusion: Melatonin plays an important role in the protection of liver against cholestasis-induced damage and ductular reaction.

Activation of the secretin (Sct)/secretin receptor (SR) axis stimulates ductular reaction and liver fibrosis, which are hallmarks of cholangiopathies. Our aim was to define the role of Sct-regulated cellular senescence, and we demonstrated that both ductular reaction and liver fibrosis are significantly reduced in Sct(−/−), SR(−/−), and Sct(−/−)/SR(−/−) bile duct ligated (BDL) mice compared with BDL wild-type mice. The reduction in hepatic fibrosis in Sct(−/−), SR(−/−), and Sct(−/−)/SR(−/−) BDL mice was accompanied by reduced transforming growth factor-β1 levels in serum and cholangiocyte supernatant, as well as decreased expression of markers of cellular senescence in cholangiocytes in contrast to enhanced cellular senescence in hepatic stellate cells compared with BDL wild-type mice. Secretin directly stimulated the senescence of cholangiocytes and regulated, by a paracrine mechanism, the senescence of hepatic stellate cells and liver fibrosis via modulation of transforming growth factor-β1 biliary secretion. Targeting senescent cholangiocytes may represent a novel therapeutic approach for ameliorating hepatic fibrosis during cholestatic liver injury.

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.

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.

Background Cholangiocarcinoma is a deadly biliary tumour with limited treatment strategies. Vitamin (1,25(OH)2D) has anti-proliferative effects on several cancers. Vitamin D3 is synthesized by the enzyme, CYP27B1, and signals via the nuclear vitamin D3 receptor. The enzyme, CYP24A1, degrades vitamin D3. Aims (i) Measure the expression of CYP27B1, CYP24A1, and vitamin D3 receptor in human nonmalignant and cholangiocarcinoma lines and biopsy control or tumour samples; and (ii) evaluate the effects of vitamin D3 on vitamin D3 synthesis and cholangiocarcinoma growth. Methods In vitro studies were performed in malignant and nonmalignant cholangiocytes. Vitamin D3 receptor, CYP24 and CYP27 expression was measured in cell lines and biopsy samples. Cell lines were stimulated with vehicle or vitamin D3 from 30 min to 48 h. Cell viability was assessed by MTS assays and BrdU incorporation. Vitamin D3 receptor, CYP24A1 and CYP27B1 expression was measured in cholangiocarcinoma cells stimulated with vehicle or vitamin D3. Results In cholangiocarcinoma lines and biopsy samples, vitamin D3 receptor and CYP24A1 expression increased compared to controls, whereas CYP27B1 expression was decreased or unchanged. Vitamin D3 induced nuclear translocation of vitamin D3 receptor in cholangiocarcinoma and decreased cholangiocarcinoma growth. Conclusion Treatment with vitamin D3 decreased CYP24A1, whereas CYP27B1 expression increased. Modulation of vitamin D3 synthesis may be important in the management of cholangiocarcinoma.

Background In several tumours the endogenous activity of histidine decarboxylase (HDC), the enzyme stimulating histamine synthesis, sustains the autocrine trophic effect of histamine on cancer progression. Cholangiocarcinoma is a biliary cancer with limited treatment options. Histamine interacts with four G-protein coupled receptors, H1–H4 histamine receptors (HRs). Objective To determine the effects of histamine stimulation and inhibition of histamine synthesis (by modulation of HDC) on cholangiocarcinoma growth. Methods In vitro studies were performed using multiple human cholangiocarcinoma lines. The expression levels of the histamine synthetic machinery and HRs were evaluated along with the effects of histamine stimulation and inhibition on cholangiocarcinoma proliferation. A xenograft tumour model was used to measure tumour volume after treatment with histamine or inhibition of histamine synthesis by manipulation of HDC. Vascular endothelial growth factor (VEGF) expression was measured in cholangiocarcinoma cells concomitant with the evaluation of the expression of CD31 in endothelial cells in the tumour microenvironment. Results Cholangiocarcinoma cells display (1) enhanced HDC and decreased monoamine oxidase B expression resulting in increased histamine secretion; and (2) increased expression of H1–H4 HRs. Inhibition of HDC and antagonising H1HR decreased histamine secretion in Mz-ChA-1 cells. Long-term treatment with histamine increased proliferation and VEGF expression in cholangiocarcinoma that was blocked by HDC inhibitor and the H1HR antagonist. In nude mice, histamine increased tumour growth (up to 25%) and VEGF expression whereas inhibition of histamine synthesis (by reduction of HDC) ablated the autocrine stimulation of histamine on tumour growth (∼80%) and VEGF expression. No changes in angiogenesis (evaluated by changes in CD31 immunoreactivity) were detected in the in vivo treatment groups. Conclusion The novel concept that an autocrine loop (consisting of enhanced histamine synthesis by HDC) sustains cholangiocarcinoma growth is proposed. Drug targeting of HDC may be important for treatment of patients with cholangiocarcinoma.

Ischemic injury by hepatic artery ligation (HAL) during obstructive cholestasis induced by bile duct ligation (BDL) results in bile duct damage, which can be prevented by administration of VEGF-A. The potential regulation of VEGF and VEGF receptor expression and secretion by bile acids in BDL with HAL is unknown.We evaluated whether taurocholic acid (TC) can prevent HAL-induced cholangiocyte damage via the alteration of VEGFR-2 and/or VEGF-A expression.Utilizing BDL, BDL+TC, BDL+HAL, BDL+HAL+TC, and BDL+HAL+wortmannin+TC treated rats, we evaluated cholangiocyte apoptosis, proliferation, and secretion as well VEGF-A and VEGFR-2 expression by immunohistochemistry. In vitro, we evaluated the effects of TC on cholangiocyte secretion of VEGF-A and the dependence of TC-induced proliferation on the activity of VEGFR-2.In BDL rats with HAL, chronic feeding of TC prevented HAL-induced loss of bile ducts and HAL-induced decreased cholangiocyte secretion. TC also prevented HAL-inhibited VEGF-A and VEGFR-2 expression in liver sections and HAL-induced circulating VEGF-A levels, which were blocked by wortmannin administration. In vitro, TC stimulated increased VEGF-A secretion by cholangiocytes, which was blocked by wortmannin and stimulated cholangiocyte proliferation that was blocked by VEGFR-2 kinase inhibitor.TC prevented HAL-induced biliary damage by upregulation of VEGF-A expression.

Adenosine triphosphate (ATP) is released from cholangiocytes into bile and is a potent secretogogue by increasing intracellular Ca2+ and stimulating fluid and electrolyte secretion via binding purinergic (P2) receptors on the apical membrane. Although morphological differences exist between small and large cholangiocytes (lining small and large bile ducts, respectively), the role of P2 signaling has not been previously evaluated along the intrahepatic biliary epithelium. The aim of these studies therefore was to characterize ATP release and P2-signaling pathways in small (MSC) and large (MLC) mouse cholangiocytes. The findings reveal that both MSCs and MLCs express P2 receptors, including P2X4 and P2Y2. Exposure to extracellular nucleotides (ATP, uridine triphosphate, or 2′,3′-O-[4-benzoyl-benzoyl]-ATP) caused a rapid increase in intracellular Ca2+ concentration and in transepithelial secretion (Isc) in both cell types, which was inhibited by the Cl− channel blockers 5-nitro-2-(-3-phenylpropylamino)-benzoic acid (NPPB) or niflumic acid. In response to mechanical stimulation (flow/shear or cell swelling secondary to hypotonic exposure), both MSCs and MLCs exhibited a significant increase in the rate of exocytosis, which was paralleled by an increase in ATP release. Mechanosensitive ATP release was two-fold greater in MSCs compared to MLCs. ATP release was significantly inhibited by disruption of vesicular trafficking by monensin in both cell types. Conclusion: These findings suggest the existence of a P2 signaling axis along intrahepatic biliary ducts with the “upstream” MSCs releasing ATP, which can serve as a paracrine signaling molecule to “downstream” MLCs stimulating Ca2+-dependent secretion. Additionally, in MSCs, which do not express the cystic fibrosis transmembrane conductance regulator, Ca2+-activated Cl− efflux in response to extracellular nucleotides represents the first secretory pathway clearly identified in these cholangiocytes derived from the small intrahepatic ducts. (HEPATOLOGY 2010)

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.

Large cholangiocytes secrete bicarbonate in response to secretin and proliferate after bile duct ligation by activation of cyclic adenosine 3', 5'-monophosphate signaling. The Ca(2+)-dependent adenylyl cyclase 8 (AC8, expressed by large cholangiocytes) regulates secretin-induced choleresis. Ca(2+)-dependent protein kinase C (PKC) regulates small cholangiocyte function. Because gamma-aminobutyric acid (GABA) affects cell functions by activation of both Ca(2+) 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: GABA(A), GABA(B), and GABA(C) 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 Ca(2+-)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 PKCbetaII; and (iii) de novo expressed secretin receptor, cystic fibrosis transmembrane regulator, Cl(-)/HCO(3)(-) anion exchanger 2 and AC8, and responded to secretin. Therefore, in pathologies of large ducts, small ducts replenish the biliary epithelium by amplification of Ca(2+)-dependent signaling and acquisition of large cholangiocyte phenotypes.

Cholangiocarcinomas are cancers that have poor prognosis and limited treatment options. The noncanonical Wnt pathway is mediated predominantly by Wnt 5a, which activates a Ca2+-dependent pathway involving protein kinase C, or a Ca2+-independent pathway involving the orphan receptor Ror2 and subsequent activation of Jun NH2-terminal kinase (JNK). This pathway is associated with growth-suppressing effects in numerous cell types. We have shown that anandamide decreases cholangiocarcinoma growth in vitro. Therefore, we determined the effects of anandamide on cholangiocarcinoma tumor growth in vivo using a xenograft model and evaluated the effects of anandamide on the noncanonical Wnt signaling pathways. Chronic administration of anandamide decreased tumor growth and was associated with increased Wnt 5a expression in vitro and in vivo. Treatment of cholangiocarcinoma cells with recombinant Wnt 5a decreased cell proliferation in vitro. Neither anandamide nor Wnt 5a affected intracellular calcium release, but both increased the JNK phosphorylation. Stable knockdown of Wnt 5a or Ror2 expression in cholangiocarcinoma cells abolished the effects of anandamide on cell proliferation and JNK activation. Modulation of the endocannabinoid system may be important in cholangiocarcinoma treatment. The antiproliferative actions of the noncanonical Wnt signaling pathway warrants further investigation to dissect the mechanism by which this may occur.

Histamine regulates many functions by binding to four histamine G-coupled receptor proteins (H1R, H2R, H3R and H4R). As H3R exerts their effects by coupling to Galpha(i/o) proteins reducing adenosine 3', 5'-monophosphate (cAMP) levels (a key player in the modulation of cholangiocyte hyperplasia/damage), we evaluated the role of H3R in the regulation of biliary growth. We posed the following questions: (1) Do cholangiocytes express H3R? (2) Does in vivo administration of (R)-(alpha)-(-)-methylhistamine dihydrobromide (RAMH) (H3R agonist), thioperamide maleate (H3R antagonist) or histamine, in the absence/presence of thioperamide maleate, to bile duct ligated (BDL) rats regulate cholangiocyte proliferation? and (3) Does RAMH inhibit cholangiocyte proliferation by downregulation of cAMP-dependent phosphorylation of protein kinase A (PKA)/extracellular signal-regulated kinase 1/2 (ERK1/2)/ets-like gene-1 (Elk-1)? The expression of H3R was evaluated in liver sections by immunohistochemistry and immunofluorescence, and by real-time PCR in cholangiocyte RNA from normal and BDL rats. BDL rats (immediately after BDL) were treated daily with RAMH, thioperamide maleate or histamine in the absence/presence of thioperamide maleate for 1 week. Following in vivo treatment of BDL rats with RAMH for 1 week, and in vitro stimulation of BDL cholangiocytes with RAMH, we evaluated cholangiocyte proliferation, cAMP levels and PKA, ERK1/2 and Elk-1 phosphorylation. Cholangiocytes from normal and BDL rats express H3R. The expression of H3R mRNA increased in BDL compared to normal cholangiocytes. Histamine decreased cholangiocyte growth of BDL rats to a lower extent than that observed in BDL RAMH-treated rats; histamine-induced inhibition of cholangiocyte growth was partly blocked by thioperamide maleate. In BDL rats treated with thioperamide maleate, cholangiocyte hyperplasia was slightly higher than that of BDL rats. In vitro, RAMH inhibited the proliferation of BDL cholangiocytes. RAMH inhibition of cholangiocyte growth was associated with decreased cAMP levels and PKA/ERK1/2/Elk-1 phosphorylation. Downregulation of cAMP-dependent PKA/ERK1/2/Elk-1 phosphorylation (by activation of H3R) is important in the inhibition of cholangiocyte growth in liver diseases.

Cholangiocarcinomas are devastating cancers of biliary origin with limited treatment options. Modulation of the endocannabinoid system is being targeted to develop possible therapeutic strategies for a number of cancers; therefore, we evaluated the effects of the two major endocannabinoids, anandamide and 2-arachidonylglycerol, on numerous cholangiocarcinoma cell lines. Although anandamide was antiproliferative and proapoptotic, 2-arachidonylglycerol stimulated cholangiocarcinoma cell growth. Specific inhibitors for each of the cannabinoid receptors did not prevent either of these effects nor did pretreatment with pertussis toxin, a Gi/o protein inhibitor, suggesting that anandamide and 2-arachidonylglycerol did not exert their diametric effects through any known cannabinoid receptor or through any other Gi/o protein-coupled receptor. Using the lipid raft disruptors methyl-β-cyclodextrin and filipin, we demonstrated that anandamide, but not 2-arachidonylglycerol, requires lipid raft-mediated events to inhibit cellular proliferation. Closer inspection of the lipid raft structures within the cell membrane revealed that although anandamide treatment had no observable effect 2-arachidonylglycerol treatment effectively dissipated the lipid raft structures and caused the lipid raft-associated proteins lyn and flotillin-1 to disperse into the surrounding membrane. In addition, anandamide, but not 2-arachidonylglycerol, induced an accumulation of ceramide, which was required for anandamide-induced suppression of cell growth. Finally we demonstrated that anandamide and ceramide treatment of cholangiocarcinoma cells recruited Fas and Fas ligand into the lipid rafts, subsequently activating death receptor pathways. These findings suggest that modulation of the endocannabinoid system may be a target for the development of possible therapeutic strategies for the treatment of this devastating cancer.

The role of the thyroid hormone agonist 3,3′,5 l-tri-iodothyronine (T3) on cholangiocytes is unknown. We evaluated the in vivo and in vitro effects of T3 on cholangiocyte proliferation of bile duct-ligated (BDL) rats. We assessed the expression of α1-, α2-, β1-, and β2-thyroid hormone receptors (THRs) by immunohistochemistry in liver sections from normal and BDL rats. BDL rats were treated with T3 (38.4 μg/day) or vehicle for 1 wk. We evaluated 1) biliary mass and apoptosis in liver sections and 2) proliferation in cholangiocytes. Serum-free T3 levels were measured by chemiluminescence. Purified BDL cholangiocytes were treated with 0.2% BSA or T3 (1 μM) in the absence/presence of U-73122 (PLC inhibitor) or BAPTA/AM (intracellular Ca2+chelator) before measurement of PCNA protein expression by immunoblots. The in vitro effects of T3 (1 μM) on 1) cAMP, IP3, and Ca2+levels and 2) the phosphorylation of Src Tyr139 and Tyr530 (that, together, regulate Src activity) and ERK1/2 of BDL cholangiocytes were also evaluated. α1-, α2-, β1-, and β2-THRs were expressed by bile ducts of normal and BDL rats. In vivo, T3 decreased cholangiocyte proliferation of BDL rats. In vitro, T3 inhibition of PCNA protein expression was blocked by U-73122 and BAPTA/AM. Furthermore, T3 1) increased IP3and Ca2+levels and 2) decreased Src and ERK1/2 phosphorylation of BDL cholangiocytes. T3 inhibits cholangiocyte proliferation of BDL rats by PLC/IP3/Ca2+-dependent decreased phosphorylation of Src/ERK1/2. Activation of the intracellular signals triggered by T3 may modulate the excess of cholangiocyte proliferation in liver diseases.

Chronic cholestatic diseases are characterized by morphological changes involving cholangiocyte proliferation and functional alterations of secretory capacity. The plant polyphenol tannic acid inhibits the growth of malignant human cholangiocytes. However, the mechanisms by which tannic acid limits excessive cholangiocyte proliferation are unknown. In this study we assessed the effect of tannic acid on cholangiocyte proliferation after bile duct ligation in rats. Tannic acid feeding decreased cholangiocyte proliferation and ductal mass in vivo after bile duct ligation. These changes were associated with functional changes in bile secretion and with decreases of intracellular cyclic adenosine 5',3'-monophosphate. The anti-proliferative effect of tannic acid was associated with a reduction of ERK1,2 phosphorylation. Additionally, tannic acid feeding decreased protein kinase A phosphorylation and activity. Similar changes were observed in isolated cholangiocytes during in vitro incubation with tannic acid. Furthermore, forskolin abolished the anti-proliferative effect of tannic acid on cholangiocyte proliferation after bile duct ligation. In conclusion, the anti-proliferative effects of tannic acid in cholangiocytes involve modulation of ERK1,2 by a cyclic adenosine 5',3'-monophosphate-protein kinase A-dependent pathway. These data suggest that tannic acid may be useful in limiting excessive cholangiocyte proliferation and modulating secretion during cholestasis.

Background/Aims To evaluate if increased cholangiocyte cAMP levels alone are sufficient to enhance cholangiocyte proliferation and secretion. Methods Normal rats were treated in vivo with forskolin for two weeks. Cholangiocyte apoptosis, proliferation and secretion were evaluated. Purified cholangiocytes from normal rats were treated in vitro with forskolin in the absence or presence of Rp-cAMPs (a PKA inhibitor), PP2 (an Src inhibitor) or PD98059 (a MEK inhibitor). Subsequently, we evaluated cholangiocyte proliferation by determination of proliferating cellular nuclear antigen (PCNA) protein expression by immunoblots. We evaluated if the effects of forskolin on cholangiocyte functions are associated with changes in the cAMP/PKA/Src/MEK/ERK1/2 pathway. Results Chronic administration of forskolin to normal rats increased the number of ducts, cAMP levels, and secretin-induced choleresis compared to controls. Forskolin-induced increases in cholangiocyte proliferation and secretion were devoid of cholangiocyte necrosis, inflammation and apoptosis. In vitro, in pure isolated cholangiocytes, forskolin increased cholangiocyte proliferation, which was ablated by Rp-cAMPs, PP2 and PD98059. The effects of forskolin on cholangiocyte proliferation were associated with increased activity of PKA, Src Tyrosine 139 (Tyr 139) and ERK1/2. Conclusions Modulation of the PKA/Src/MEK/ERK1/2 pathway may be important in the regulation of cholangiocyte growth and secretion observed in cholestatic liver diseases.

Large, but not small, cholangiocytes (1) secrete bicarbonate by interaction with secretin receptors (SRs) through activation of cystic fibrosis transmembrane regulator (CFTR), Cl−/HCO3− (apex) anion exchanger 2 (Cl−/HCO3− AE2), and adenylyl cyclase (AC)8 (proteins regulating large biliary functions) and (2) proliferate in response to bile duct ligation (BDL) by activation of cyclic adenosine monophosphate (cAMP) signaling. Small, mitotically dormant cholangiocytes are activated during damage of large cholangiocytes by activation of D-myo-inositol 1,4,5-trisphosphate/Ca2+/calmodulin-dependent protein kinase (CaMK) I. gamma-Aminobutyric acid (GABA) affects cell functions by modulation of Ca2+-dependent signaling and AC. We hypothesized that GABA induces the differentiation of small into large cholangiocytes by the activation of Ca2+/CaMK I-dependent AC8. In vivo, BDL mice were treated with GABA in the absence or presence of 1,2-bis-(o-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid, tetraacetoxymethyl ester (BAPTA/AM) or N-(6-aminohexyl)-5-chloro-1-naphtalenesulfonamide (W7) before evaluating apoptosis and intrahepatic bile ductal mass (IBDM) of small and large cholangiocytes. In vitro, control- or CaMK I-silenced small cholangiocytes were treated with GABA for 3 days before evaluating apoptosis, proliferation, ultrastructural features, and the expression of CFTR, Cl−/HCO3− AE2, AC8, and secretin-stimulated cAMP levels. In vivo administration of GABA induces the apoptosis of large, but not small, cholangiocytes and decreases large IBDM, but increased de novo small IBDM. GABA stimulation of small IBDM was blocked by BAPTA/AM and W7. Subsequent to GABA in vitro treatment, small cholangiocytes de novo proliferate and acquire ultrastructural and functional phenotypes of large cholangiocytes and respond to secretin. GABA-induced changes were prevented by BAPTA/AM, W7, and stable knockdown of the CaMK I gene. Conclusion: GABA damages large, but not small, cholangiocytes that differentiate into large cholangiocytes. The differentiation of small into large cholangiocytes may be important in the replenishment of the biliary epithelium during damage of large, senescent cholangiocytes. (HEPATOLOGY 2013;)

Although large cholangiocytes exert their functions by activation of cyclic adenosine 3',5'-monophosphate (cAMP), Ca(2+)-dependent signaling regulates the function of small cholangiocytes. Histamine interacts with four receptors, H1-H4HRs. H1HR acts by Gαq activating IP(3)/Ca(2+), whereas H2HR activates Gα(s) stimulating cAMP. We hypothesize that histamine increases biliary growth by activating H1HR on small and H2HR on large cholangiocytes. The expression of H1-H4HRs was evaluated in liver sections, isolated and cultured (normal rat intrahepatic cholangiocyte culture (NRIC)) cholangiocytes. In vivo, normal rats were treated with histamine or H1-H4HR agonists for 1 week. We evaluated: (1) intrahepatic bile duct mass (IBDM); (2) the effects of histamine, H1HR or H2HR agonists on NRIC proliferation, IP(3) and cAMP levels and PKCα and protein kinase A (PKA) phosphorylation; and (3) PKCα silencing on H1HR-stimulated NRIC proliferation. Small and large cholangiocytes express H1-H4HRs. Histamine and the H1HR agonist increased small IBDM, whereas histamine and the H2HR agonist increased large IBDM. H1HR agonists stimulated IP(3) levels, as well as PKCα phosphorylation and NRIC proliferation, whereas H2HR agonists increased cAMP levels, as well as PKA phosphorylation and NRIC proliferation. The H1HR agonist did not increase proliferation in PKCα siRNA-transfected NRICs. The activation of differential signaling mechanisms targeting small and large cholangiocytes is important for repopulation of the biliary epithelium during pathologies affecting different-sized bile ducts.

In bile duct-ligated (BDL) rats, cholangiocyte proliferation is regulated by neuroendocrine factors such as α-calcitonin gene-related peptide (α-CGRP). There is no evidence that the sensory neuropeptide substance P (SP) regulates cholangiocyte hyperplasia. Wild-type (WT,+/+) and NK-1 receptor (NK-1R) knockout (NK-1R−/−) mice underwent sham or BDL for 1 wk. Then we evaluated 1) NK-1R expression, transaminases, and bilirubin serum levels; 2) necrosis, hepatocyte apoptosis and steatosis, and the number of cholangiocytes positive by CK-19 and terminal deoxynucleotidyl transferase biotin-dUTP nick-end labeling in liver sections; 3) mRNA expression for collagen 1α and α-smooth muscle (α-SMA) actin in total liver samples; and 4) PCNA expression and PKA phosphorylation in cholangiocytes. In cholangiocyte lines, we determined the effects of SP on cAMP and d-myo-inositol 1,4,5-trisphosphate levels, proliferation, and PKA phosphorylation. Cholangiocytes express NK-1R with expression being upregulated following BDL. In normal NK-1R−/−mice, there was higher hepatocyte apoptosis and scattered hepatocyte steatosis compared with controls. In NK-1R−/−BDL mice, there was a decrease in serum transaminases and bilirubin levels and the number of CK-19-positive cholangiocytes and enhanced biliary apoptosis compared with controls. In total liver samples, the expression of collagen 1α and α-SMA increased in BDL compared with normal mice and decreased in BDL NK-1R−/−compared with BDL mice. In cholangiocytes from BDL NK-1R−/−mice there was decreased PCNA expression and PKA phosphorylation. In vitro, SP increased cAMP levels, proliferation, and PKA phosphorylation of cholangiocytes. Targeting of NK-1R may be important in the inhibition of biliary hyperplasia in cholangiopathies.

Caffeic acid phenethyl ester (CAPE) inhibits the growth of tumor cells and is a known inhibitor of nuclear factor kappa beta (NF-kappaB), which is constitutively active in cholangiocarcinoma (CCH) cells. We evaluated the effects of CAPE on CCH growth both in vitro and in vivo. Inhibition of NF-kappaB DNA-binding activity was confirmed in nuclear extracts treated with CAPE at 50, 40 and 20 microM. CAPE decreases the expression of NF-kappaB1 (p50) and RelA (p65). CAPE decreased the growth of a number of CCH cells but not normal cholangiocytes. Cell cycle decrease was seen by a decrease in PCNA protein expression and the number of BrdU-positive cells treated with CAPE at 20 microM compared to vehicle. Inhibition of growth and increased cell cycle arrest of Mz-ChA-1 cells by CAPE were coupled with increased apoptosis. Bax expression was increased, whereas Bcl-2 was decreased in cells treated with CAPE compared to vehicle. In vivo studies were performed in BALB/c nude (nu/nu) mice implanted subcutaneously with Mz-ChA-1 cells and treated with daily IP injections of DMSO or CAPE (10 mg/kg body weight in DMSO) for 77 days. Tumor growth was decreased and tumor latency was increased 2-fold in CAPE compared to vehicle-treated nude mice. In tumor samples, decreased CCH growth by CAPE was coupled with increased apoptosis. CAPE both in vivo and in vitro decreases the growth of CCH cells by increasing apoptosis. These results demonstrate that CAPE might be an important therapeutic tool in the treatment of CCH.

Cholangiopathies are characterized by the heterogeneous proliferation of different-sized cholangiocytes. Large cholangiocytes proliferate by a cAMP-dependent mechanism. The function of small cholangiocytes may depend on the activation of inositol trisphosphate (IP3)/Ca2+-dependent signaling pathways; however, data supporting this speculation are lacking. Four histamine receptors exist (HRH1, HRH2, HRH3, and HRH4). In several cells: 1) activation of HRH1 increases intracellular Ca2+concentration levels; and 2) increased [Ca2+]ilevels are coupled with calmodulin-dependent stimulation of calmodulin-dependent protein kinase (CaMK) and activation of cAMP-response element binding protein (CREB). HRH1 agonists modulate small cholangiocyte proliferation by activation of IP3/Ca2+-dependent CaMK/CREB. We evaluated HRH1 expression in cholangiocytes. Small and large cholangiocytes were stimulated with histamine trifluoromethyl toluidide (HTMT dimaleate; HRH1 agonist) for 24–48 h with/without terfenadine, BAPTA/AM, or W7 before measuring proliferation. Expression of CaMK I, II, and IV was evaluated in small and large cholangiocytes. We measured IP3, Ca2+and cAMP levels, phosphorylation of CaMK I, and activation of CREB (in the absence/presence of W7) in small cholangiocytes treated with HTMT dimaleate. CaMK I knockdown was performed in small cholangiocytes stimulated with HTMT dimaleate before measurement of proliferation and CREB activity. Small and large cholangiocytes express HRH1, CaMK I, and CaMK II. Small (but not large) cholangiocytes proliferate in response to HTMT dimaleate and are blocked by terfenadine (HRH1 antagonist), BAPTA/AM, and W7. In small cholangiocytes, HTMT dimaleate increased IP3/Ca2+levels, CaMK I phosphorylation, and CREB activity. Gene knockdown of CaMK I ablated the effects of HTMT dimaleate on small cholangiocyte proliferation and CREB activation. The IP3/Ca2+/CaMK I/CREB pathway is important in the regulation of small cholangiocyte function.

The endocannabinoid system regulates various aspects of hepatic fibrosis; however, nothing is known about its role in regulating cholangiocyte proliferation and function. We evaluated the effects of anandamide (AEA) on cholangiocyte proliferation and explored the effects of AEA on the thioredoxin 1 (TRX1)/redox factor 1 (Ref1)/activator protein-1 (AP-1) pathway. Mice underwent bile duct ligation (BDL) and were infused with AEA for 3 days postsurgery. Proliferation and apoptosis were evaluated in liver sections. Effects of in vitro AEA treatment on cholangiocyte proliferation and apoptosis were studied in purified cholangiocytes. The relative expression of cannabinoid receptors was also assessed in liver sections and cholangiocytes. mRNA expression of the cannabinoid receptors Cb1 and VR1 was decreased after BDL, whereas there was an upregulation of Cb2 mRNA. AEA decreased cholangiocyte growth and induced accumulation of reactive oxygen species, upregulation of TRX1, Ref1, c-Fos, and c-Jun expression, increased nuclear localization of TRX1, and increased AP-1 transcriptional activity. Specific knockdown of TRX1 or Ref1 expression ablated the AP-1 transcriptional activity and AEA-induced cell death but not expression of c-Fos and c-Jun. Knockdown of c-Fos and c-Jun expression also ablated AEA-induced apoptosis. We conclude that AEA suppresses cholangiocyte proliferation during cholestasis via a Cb2-dependent mechanism. Modulation of the endocannabinoid system may be important in the treatment of cholangiopathies.

A S L D A b st ra ct s GDNF suppresses expression of genes associated with hepatic steatosis Real-time PCR analyses of gene expression in liver from WT and GDNF Tg mice maintained on RD or HFD for 12 weeks. Plotted are means + SEM. *, P 5 mice per group.

Background & Aims: Vascular endothelial growth factor (VEGF) is secreted by several epithelia and modulates cellular functions by autocrine and paracrine mechanisms. The role of VEGF in cholangiocyte pathophysiology is unknown. We evaluated the role of VEGF in the regulation of cholangiocyte proliferation in rats that underwent bile duct ligation. Methods: The expression of VEGF-A and VEGF-C and their receptors in cholangiocytes from normal and BDL rats was evaluated. Normal or BDL rats were treated with recombinant-VEGF-A or recombinant-VEGF-C or anti-VEGF antibodies, and proliferation of cholangiocytes was evaluated in situ by morphometry and in vitro by proliferating cell nuclear antigen immunoblots and MTS assay. In vitro, normal rat cholangiocyte cultures were stimulated with r-VEGF-A or r-VEGF-C and proliferation and signal transduction were evaluated. Results: We found that (1) cholangiocytes express messenger RNA and protein for VEGF-A, VEGF-C, VEGF receptor 2 (VEGFR-2), and VEGF receptor 3 (VEGFR-3) and secrete VEGF; (2) secretion of VEGF and expression of VEGFR-2 and VEGFR-3 increases in BDL cholangiocytes; (3) blocking VEGF in vivo by anti–VEGF-A or anti–VEGF-C antibodies decreases cholangiocyte proliferation; (4) the in vivo administration of r-VEGF-A or r-VEGF-C induces cholangiocyte proliferation in normal rats; and (5) in vitro, VEGF-A increases normal rat cholangiocyte culture proliferation by activation of inositol 1,4,5-triphosphate/Ca 2+ /protein kinase C α and phosphorylation of Src/ERK1/2. Conclusions: Cholangiocytes secrete VEGF and express VEGFR-2 and VEGFR-3, all of which are amplified in BDL cholangiocytes. VEGF induces cholangiocyte proliferation by activation of inositol 1,4,5-triphosphate/[Ca 2+ ] i /protein kinase C α and phosphorylation of Src/ERK1/2. VEGF mediates the adaptive proliferative response of cholangiocytes to cholestasis.

A S L D A b st ra ct s increase in circulating Galanin in the serum. Galanin immunoreactivity was observed in both cholangiocytes and hepatocytes, whereas GalR1 was found predominantly in cholangiocytes. Systemic treatment of rats with M617 increased both CK-19 expression and IBDM in both sham and BDL-treated animals. In vitro, treatment of the mouse cholangiocyte cell line with M617 increased ERK1/2 and RSK-1 activity. There was a concomitant increase in cholangiocyte proliferation after M617 treatment that could be blocked by pretreatment with inhibitors for ERK1/2 and RSK-1. Conclusions: Data presented here demonstrate a direct stimulatory role for Galanin on cholangiocyte proliferation under physiological (sham) and pathological (BDL) conditions via a mechanism involving the activation of ERK1/2 and subsequent phosphorylation of RSK-1. Targeting Galanin or GalR1 may prove a useful strategy to regulate biliary mass during cholestatic liver injury.

A S L D A b st ra ct s increase in circulating Galanin in the serum. Galanin immunoreactivity was observed in both cholangiocytes and hepatocytes, whereas GalR1 was found predominantly in cholangiocytes. Systemic treatment of rats with M617 increased both CK-19 expression and IBDM in both sham and BDL-treated animals. In vitro, treatment of the mouse cholangiocyte cell line with M617 increased ERK1/2 and RSK-1 activity. There was a concomitant increase in cholangiocyte proliferation after M617 treatment that could be blocked by pretreatment with inhibitors for ERK1/2 and RSK-1. Conclusions: Data presented here demonstrate a direct stimulatory role for Galanin on cholangiocyte proliferation under physiological (sham) and pathological (BDL) conditions via a mechanism involving the activation of ERK1/2 and subsequent phosphorylation of RSK-1. Targeting Galanin or GalR1 may prove a useful strategy to regulate biliary mass during cholestatic liver injury.