Cholangiocyte Cell Research Articles

Abstract 361: Loss of cholangiocyte primary cilia induces LKB1 downregulation and defective AMPK signaling

Abstract Cholangiocarcinoma (CCA) is a malignancy arising from cholangiocytes, the epithelial cells lining the biliary tree. CCA is an uncommon, but devastating cancer that is increasing in incidence. Over the past 3 decades, 5-years survival rates have remained at 10%. Although surgical resection and liver transplantation are potentially curative therapies, most patients are diagnosed at late stages and are not eligible for these options. Therefore, it is imperative to identify novel targets leading to new therapeutic strategies for this devastating disease. Cholangiocytes express primary cilia that function as chemo, mechano, and osmosensors controlling several molecular pathways. We showed cilia are absent in CCA cells, and experimental deciliation of normal cholangiocytes induced a malignant like phenotype, with significant invasion and proliferation, suggesting the loss of cilia could be associated with CCA development. LKB1 is a tumor suppressor described to be expressed in primary cilia in MDCK cells, and is involved in AMPK activation through a ciliary dependent mechanism. AMPK functions as metabolic and stress check points. Interestingly, patients with intrahepatic CCA and low expression of LKB1 have poor prognosis. Therefore, we hypothesized that primary cilia function as tumor suppressor organelles through a LKB1-AMPK-p53 pathway. To test this hypothesis, first LKB1 subcellular localization was evaluated by confocal microscopy. We show for the first time that LKB1 is enriched in cholangiocyte primary cilia in a normal human cholangiocyte cell line (NHC). However, in experimental deciliated NHC and the CCA cell line KMCH, LKB1 is found dispersed in the cell cytoplasm. Next, we analyzed cellular expression levels of LKB1, AMPK (T172), p53 (s15), p53 and p21 by western blot. We found experimentally deciliated cholangiocytes and CCA cells exhibit defective AMPK signaling characterized by lower levels of LKB1 (-70% and -85%), AMPK (T172) (-72% and -71%), p53 (-37% and -83%), p53 (S15) (-38% and -100%) and p21 (-59% and -87%) relative to NHC. These molecular characteristics correlated with increased cell proliferation in deciliated cells (28%) and CCA cells (27%). Finally, we attempted to rescue the phenotype by using the AMPK activator, ampkinone. This treatment induced 20% inhibition of proliferation on experimental deciliated cholangiocytes and 18% in CCA cells, while not affecting NHC cells and the inhibition was associated with phosphorylation of AMPK and p21 up-regulation. In summary, our data suggest cholangiocyte primary cilia may normally function as tumor suppressors via a mechanism involving LKB1 and AMPK. The loss of cilia in CCA impairs LKB1-AMPK-p53-p21 signaling inducing a proliferative phenotype that may be rescued by specific activation of AMPK, warranting further studies to assess its use as a potential therapeutic approach. Citation Format: Adrian P. Mansini, Kristen M. Thelen, Sergio A. Gradilone. Loss of cholangiocyte primary cilia induces LKB1 downregulation and defective AMPK signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 361. doi:10.1158/1538-7445.AM2017-361

Development of a Potent Wound Healing Agent Based on the Liver Fluke Granulin Structural Fold.

Granulins are a family of protein growth factors that are involved in cell proliferation. An orthologue of granulin from the human parasitic liver fluke Opisthorchis viverrini, known as Ov-GRN-1, induces angiogenesis and accelerates wound repair. Recombinant Ov-GRN-1 production is complex and poses an obstacle for clinical development. To identify the bioactive region(s) of Ov-GRN-1, four truncated N-terminal analogues were synthesized and characterized structurally using NMR spectroscopy. Peptides that contained only two native disulfide bonds lack the characteristic granulin β-hairpin structure. Remarkably, the introduction of a non-native disulfide bond was critical for formation of β-hairpin structure. Despite this structural difference, both two and three disulfide-bonded peptides drove proliferation of a human cholangiocyte cell line and demonstrated potent wound healing in mice. Peptides derived from Ov-GRN-1 are leads for novel wound healing therapeutics, as they are likely less immunogenic than the full-length protein and more convenient to produce.

Regeneration and Cell Recruitment in an Improved Heterotopic Auxiliary Partial Liver Transplantation Model in the Rat

Auxiliary partial liver transplantation (APLT) in humans is a therapeutic modality used especially to treat liver failure in children or congenital metabolic disease. Animal models of APLT have helped to explore therapeutic options. Though many groups have suggested improvements, standardizing the surgical procedure has been challenging. Additionally, the question of whether graft livers are reconstituted by recipient-derived cells after transplantation has been controversial. The aim of this study was to improve experimental APLT in rats and to assess cell recruitment in the liver grafts. To inhibit recipient liver regeneration and to promote graft regeneration, we treated recipients with retrorsine and added arterial anastomosis. Using green fluorescence protein transgenic rats as recipients, we examined liver resident cell recruitment within graft livers by immunofluorescence costaining. In the improved APLT model, we achieved well-regenerated grafts that could maintain regeneration for at least 4 weeks. Regarding the cell recruitment, there was no evidence of recipient-derived hepatocyte, cholangiocyte, or hepatic stellate cell recruitment into the graft. Macrophages/monocytes, however, were consistently recruited into the graft and increased over time, which might be related to inflammatory responses. Very few endothelial cells showed colocalization of markers. We have successfully established an improved rat APLT model with arterial anastomosis as a standard technique. Using this model, we have characterized cell recruitment into the regenerating grafts.

Foetal hepatic progenitor cells assume a cholangiocytic cell phenotype during two-dimensional pre-culture.

Liver consists of parenchymal hepatocytes and other cells. Liver progenitor cell (LPC) is the origin of both hepatocytes and cholangiocytic cells. The analyses of mechanism regulating differentiation of LPCs into these functional cells are important for liver regenerative therapy using progenitor cells. LPCs in adult livers were found to form cysts with cholangiocytic characteristics in 3D culture. In contrast, foetal LPCs cannot form these cholangiocytic cysts in the same culture. Thus, the transition of foetal LPCs into cholangiocytic progenitor cells might occur during liver development. Primary CD45−Ter119−Dlk1+ LPCs derived from murine foetal livers formed ALBUMIN (ALB)+CYTOKERATIN (CK)19− non-cholangiocytic cysts within 3D culture. In contrast, when foetal LPCs were pre-cultured on gelatine-coated dishes, they formed ALB−CK19+ cholangiocytic cysts. When hepatocyte growth factor or oncostatin M, which are inducers of hepatocytic differentiation, was added to pre-culture, LPCs did not form cholangiocytic cysts. These results suggest that the pre-culture on gelatine-coated dishes changed the characteristics of foetal LPCs into cholangiocytic cells. Furthermore, neonatal liver progenitor cells were able to form cholangiocytic cysts in 3D culture without pre-culture. It is therefore possible that the pre-culture of mid-foetal LPCs in vitro functioned as a substitute for the late-foetal maturation step in vivo.

Immunohistochemical study of hepatocyte, cholangiocyte and stem cell markers of hepatocellular carcinoma: the second report: relationship with tumor size and cell differentiation

BackgroundThe purpose of this study is to investigate whether ordinary hepatocellular carcinomas (HCCs) show positivity of stem/progenitor cell markers and cholangiocyte markers during the process of tumor progression.MethodsNinety‐four HCC lesions no larger than 8 cm from 94 patients were immuno‐histochemically studied using two hepatocyte markers (Hep par 1 and α‐fetoprotein), five cholangiocyte markers (cytokeratin CK7, CK19, Muc1, epithelial membrane antigen and carcinoembryonic antigen) and three hepatic stem/progenitor cell markers (CD56, c‐Kit and EpCAM). The tumors were classified into three groups by tumor size: S1, < 2.0 cm; S2, 2.0–5.0 cm; S3, 5.0–8.0 cm. The tumors were also classified according to tumor differentiation: well, moderately and poorly differentiated. The relationship between the positive ratios of these markers, tumor size and tumor differentiation was examined.ResultsThe positive ratios of cholangiocyte markers tended to be higher in larger sized and more poorly differentiated tumors (except for CK7). The positive ratios of stem/progenitor cell markers tended to be higher in larger sized and more poorly differentiated tumors (except for c‐Kit).ConclusionOrdinary HCC can acquire the characteristic of positivity of cholangiocyte and stem/progenitor cell markers during the process of tumor progression.

Effects of 5-Aza-2'-Deoxycytidine, Bromodeoxyuridine, Interferons and Hydrogen Peroxide on Cellular Senescence in Cholangiocarcinoma Cells.

Cellular senescence, a barrier to tumorigenesis, controls aberrant proliferation of cells. We here aimed to investigate cellular senescence in immortalized cholangiocyte and cholangiocarcinoma cell lines using five different inducing agents: 5-aza-2'deoxycytidine, bromodeoxyuridine, interferons (IFNβ and IFNγ), and hydrogen peroxide. We analyzed senescence characteristics, colony formation ability, expression of genes involved in cell cycling and interferon signaling pathways, and protein levels. Treatment with all five agents decreased cell proliferation and induced cellular senescence in immortalized cholangiocyte and cholangiocarcinoma cell lines with different degrees of growth-inhibitory effects depending on cell type and origin. Bromodeoxyuridine gave the strongest stimulus to inhibit growth and induce senescence in most cell lines tested. Expression of p21 and interferon related genes was upregulated in most conditions. The fact that bromodeoxyuridine had the strongest effects on growth inhibition and senescence induction implies that senescence in cholangiocarcinoma cells is likely controlled by DNA damage response pathways relating to the p53/p21 signaling. In addition, interferon signaling pathways may partly regulate this mechanism in cholangiocarcinoma cells.

MEK-ERK Activity Regulates the Proliferative Activity of Fetal Hepatoblasts Through Accumulation of p16/19(cdkn2a).

Hepatoblasts are somatic progenitor cells in the fetal liver, which retain a high proliferative capacity and differentiate into both hepatocytes and cholangiocytes in vivo. Although efficient expansion of hepatoblasts in vitro has been difficult without genetic modification, we have previously demonstrated that the interaction with mesenchymal cells is important for expansion of hepatoblasts in vitro. In this study, we show cell signaling pathways regulating the long-term proliferative ability of hepatoblasts. Individual primary hepatoblasts derived from mouse fetal livers formed large colonies when cocultured with mesenchymal feeder cells; however, secondary colony formation was unsuccessful, indicating that in vitro culture could induce short-term, but not long-term, proliferation. When the MEK inhibitor, PD0325901, was added to these cultures, hepatoblasts formed large colonies containing many Ki-67-positive cells. Expression of p16/19(cdkn2a), a cyclin-dependent kinase inhibitor, was induced after 3-6 days culture of hepatoblasts, whereas PD0325901 significantly suppressed this expression. Consistent with these observations, fetal hepatoblasts derived from p16/19(cdkn2a) knockout mice showed long-term proliferation without PD0325901, suggesting that MEK activity induced cell cycle arrest through accumulation of p16/19(cdkn2a). In transplantation assays, we could demonstrate that in vitro expanded hepatoblasts could proliferate and differentiate into hepatocytic and cholangiocytic cells in injured livers. It should also be noted that ERK in primary hepatoblasts was not highly activated during fetal liver development. Collectively, all these findings suggest that the MEK/ERK-independent pathway in the fetal liver is involved in hepatoblast proliferation to avoid accumulation of cyclin-dependent kinase inhibitor.

Abstract 192: Exportin-5 downregulation induces miRNA dysregulation and HDAC6 overexpression in cholangiocarcinoma

Abstract Cholangiocarcinoma (CCA) is a malignancy arising from the epithelial cells lining the biliary tract in the liver, the cholangiocytes. While cholangiocytes normally express cilia, we showed that they are significantly reduced in CCA by a mechanism involving overexpression of histone deacetylase 6 (HDAC6), a molecule that deacetylates the ciliary axoneme and promotes ciliary disassembly. Even though HDAC6 protein is overexpressed in CCA, we found no differences in its messenger RNA (mRNA) level, suggesting a post-transcriptional regulation, possibly involving microRNA (miRNA). Therefore, we hypothesized that decreased levels of specific miRNAs induce overexpression of HDAC6 in CCA. We compared miRNA levels between normal and CCA cholangiocyte cell lines and found that three miRNAs potentially targeting HDAC6 are downregulated (40-55% decrease) in CCA: miR-433, miR-22, and miR-200a. Experimental overexpression of miR-433, miR-22, or miR-200a in normal cholangiocytes decreased HDAC6 expression by 82%, 33%, and 39%, respectively. We selected miR-433 and miR-22 for further analysis and the downregulation of both miRNAs were confirmed by qRT-PCR on normal and six different CCA cell lines, and by in situ hybridization on control and CCA human liver samples. To further investigate the dysregulation of these miRNAs, we measured its precursor forms by qRT-PCR and, in contrast to the mature miR-433 and miR-22, the levels of the precursors were increased in malignant cells. In situ hybridization on human samples showed that the miR-433 probe was accumulated in the nucleus of malignant cholangiocytes. Taken together, these data suggested a potential impairment of nuclear export; therefore, we assessed the expression of the protein responsible for transporting microRNA precursors out of the nucleus, exportin-5. By western blotting, we found that exportin-5 is decreased by 50% in five different CCA cell lines compared to normal cholangiocyte cell lines. We confirmed the downregulation of exportin-5 protein by immunofluorescence of normal and CCA human liver samples. Finally, the experimental overexpression of exportin-5 in CCA cell lines restores the precursor and mature forms of these miRNAs to normal levels, inducing a decrease in the expression of HDAC6, and decreased proliferation and anchorage independent growth of the malignant cells. In summary, these data suggest that downregulation of exportin-5, preventing the nuclear export of the precursor forms of miR-433 and miR-22, may be the underlying mechanism explaining the decrease of miR-433 and miR-22, and the overexpression of HDAC6 in CCA. Thus, our work further clarifies the molecular mechanisms accounting for the absence of cilia in CCA and also identifies multiple potential molecules for targeted therapies like HDAC6, miR-433, miR-22, or exportin-5. Citation Format: Maria J. Lorenzo Pisarello, Brynn N. Howard, Christy E. Trussoni, Sergio Gradilone. Exportin-5 downregulation induces miRNA dysregulation and HDAC6 overexpression in cholangiocarcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 192. doi:10.1158/1538-7445.AM2015-192

Proteomics profiling of cholangiocarcinoma exosomes: A potential role of oncogenic protein transferring in cancer progression

Cholangiocarcinoma (CCA), a common primary malignant tumor of bile duct epithelia, is highly prevalent in Asian countries and unresponsive to chemotherapeutic drugs. Thus, a newly recognized biological entity for early diagnosis and treatment is highly needed. Exosomes are small membrane bound vesicles found in body fluids and released by most cell types including cancer cells. The vesicles contain specific subset of proteins and nucleic acids corresponding to cell types and play essential roles in pathophysiological processes. The present study aimed to assess the protein profiles of CCA-derived exosomes and their potential roles. We have isolated exosomes from CCA cells namely KKU-M213 and KKU-100 derived from Thai patients and their roles were investigated by incubation with normal human cholangiocyte (H69) cells. Exosomes were internalized into H69 cells and had no effects on viability or proliferation of the host cells. Interestingly, the exosomes from KKU-M213 cells only induced migration and invasion of H69 cells. Proteomic analysis of the exosomes from KKU-M213 cells disclosed multiple cancer related proteins that are not present in H69 exosomes. Consistent with the protein profile, treatment with KKU-M213 exosomes induced β-catenin and reduced E-cadherin expressions in H69 cells. Collectively, our results suggest that a direct cell-to-cell transfer of oncogenic proteins via exosomal pathway may be a novel mechanism for CCA progression and metastasis.

Development and characterization of a hydrogen peroxide-resistant cholangiocyte cell line: A novel model of oxidative stress-related cholangiocarcinoma genesis

Oxidative stress is a cause of inflammation–related diseases, including cancers. Cholangiocarcinoma is a liver cancer with bile duct epithelial cell phenotypes. Our previous studies in animal and human models indicated that oxidative stress is a major cause of cholangiocarcinoma development. Hydrogen peroxide (H2O2) can generate hydroxyl radicals, which damage lipids, proteins, and nucleic acids, leading to cell death. However, some cells can survive by adapting to oxidative stress conditions, and selective clonal expansion of these resistant cells would be involved in oxidative stress-related carcinogenesis. The present study aimed to establish H2O2-resistant cell line from an immortal cholangiocyte cell line (MMNK1) by chronic treatment with low-concentration H2O2 (25 μM). After 72 days of induction, H2O2-resistant cell lines (ox-MMNK1-L) were obtained. The ox-MMNK1-L cell line showed H2O2-resistant properties, increasing the expression of the anti-oxidant genes catalase (CAT), superoxide dismutase-1 (SOD1), superoxide dismutase-2 (SOD2), and superoxide dismutase-3 (SOD3) and the enzyme activities of CAT and intracellular SODs. Furthermore, the resistant cells showed increased expression levels of an epigenetics-related gene, DNA methyltransferase-1 (DNMT1), when compared to the parental cells. Interestingly, the ox-MMNK1-L cell line had a significantly higher cell proliferation rate than the MMNK1 normal cell line. Moreover, ox-MMNK1-L cells showed pseudopodia formation and the loss of cell-to-cell adhesion (multi-layers) under additional oxidative stress (100 μM H2O2). These findings suggest that H2O2-resistant cells can be used as a model of oxidative stress-related cholangiocarcinoma genesis through molecular changes such as alteration of gene expression and epigenetic changes.

Plasma IgG autoantibody against actin-related protein 3 in liver fluke Opisthorchis viverrini infection.

Opisthorchiasis secondary to Opisthorchis viverrini infection leads to cholangiocellular carcinoma through chronic inflammation of the bile ducts and possibly inducing autoimmunity. It was hypothesized that plasma autoantibodies directed against self-proteins are biomarkers for opisthorchiasis. Plasma from patients with opisthorchiasis was tested using proteins derived from immortalized cholangiocyte cell lines, and spots reacting with plasma were excised and subjected to LC-MS/MS. Seven protein spots were recognized by IgG autoantibodies, and the highest matching scored protein was actin-related protein 3 (ARP3). The antibody against ARP3 was tested in plasma from 55 O. viverrini-infected patients, 24 patients with others endemic parasitic infections and 17 healthy controls using Western blot and ELISA. Immunoreactivity against recombinant ARP3 was significantly more prevalent in opisthorchiasis compared to healthy controls at Western blotting and ELISA (P < 0.05). Plasma ARP3 autoantibody titres were also higher in opisthorchiasis compared to healthy individuals (P < 0.01) and other parasitic infections including Strongyloides stercoralis (P < 0.001), echinostome (P < 0.05), hookworms (P < 0.001) and Taenia spp. (P < 0.05). It was further characterized in that the ARP3 autoantibody titre had a sensitivity of 78.18% and specificity of 100% for opisthorchiasis. In conclusion, it may be suggested that plasma anti-ARP3 might represent a new diagnostic antibody for opisthorchiasis.

The biliary epithelium presents antigens to and activates natural killer T cells.

Cholangiocytes express antigen-presenting molecules, but it has been unclear whether they can present antigens. Natural killer T (NKT) cells respond to lipid antigens presented by the major histocompatibility complex class I-like molecule CD1d and are abundant in the liver. We investigated whether cholangiocytes express CD1d and present lipid antigens to NKT cells and how CD1d expression varies in healthy and diseased bile ducts. Murine and human cholangiocyte cell lines as well as human primary cholangiocytes expressed CD1d as determined by flow cytometry and western blotting. Murine cholangiocyte cell lines were able to present both exogenous and endogenous lipid antigens to invariant and noninvariant NKT cell hybridomas and primary NKT cells in a CD1d-dependent manner. A human cholangiocyte cell line, cholangiocarcinoma cell lines, and human primary cholangiocytes also presented exogenous CD1d-restricted antigens to invariant NKT cell clones. CD1d expression was down-regulated in the biliary epithelium of patients with late primary sclerosing cholangitis, primary biliary cirrhosis, and alcoholic cirrhosis compared to healthy controls. Cholangiocytes express CD1d and present antigens to NKT cells and CD1d expression is down-regulated in diseased biliary epithelium, findings which show that the biliary epithelium can activate an important lymphocyte subset of the liver. This is a potentially important immune pathway in the biliary system, which may be capable of regulating inflammation in the context of biliary disease.

Human pluripotent stem cell-derived cholangiocytes: current status and future applications.

Pluripotent stem cells, such as embryonic stem cells and inducible pluripotent stem (iPS) cells, have high proliferative multipotency for differentiation into mature functional cells that are useful for treatment and basic research on several diseases. Cholangiocytes are differentiated from fetal hepatic progenitor cells (hepatoblasts) and are important for transport of bile acids that are synthesized by mature hepatocytes in the liver. However, the molecular mechanisms of development and function of human cholangiocytes remain unknown. This review mentions the potential of human cholangiocytic culture from pluripotent stem cells to contribute to the analyses of the human bile duct system and diseases. Recent studies found that human hepatic cholangiocytic cells can be differentiated from human embryonic stem and iPS cells in a suitable culture condition. Cholangiocytic cysts have epithelial cell polarity formed in a three-dimensional cell culture system using extracellular matrices. Disease pathogenesis was elucidated in vitro using differentiated cells from disease-related iPS cells. Using genome-editing enzymes, iPS cells with disease-specific gene mutations can be easily and rapidly established. These disease-related iPS cells and cholangiocytic culture system may be useful for analyses and drug screening of human bile duct diseases.

Wnt/Beta‐Catenin Cooperates With Yap Signaling To Promote Hepatobiliary Repair In A Model Of Chronic Liver Injury

Proliferation of atypical ductules and compensatory hepatocyte transdifferentiation to cholangiocytes is a hallmark of 3,5‐diethoxycarbonyl‐ 1, 4‐dihydro‐collidine (DDC) diet‐induced injury in mice. Because liver‐specific beta‐catenin conditional KO mice exhibit fewer proliferating ductules after DDC, we hypothesized that Wnt signaling plays a role in this process. To test this, WT animals on control and DDC diet were analyzed for Wnt expression. We found upregulation of 3 Wnts – 7a, 7b, and 10a – in the portal triad of DDC‐fed mice, as determined by tissue microdissection. Cell sorting showed expression of these three Wnts exclusively in the EpCAM+ cell compartment. Addition of Wnt7b and 10a to small cholangiocyte cell line induced proliferation in an autocrine manner, consistent with an increase in CK19+/Ki67+ cells after DDC. In contrast, direct co‐culture of Hek293 cells expressing recombinant Wnt7a significantly increased Sox‐9 gene expression in AML12 cells and primary hepatocytes. To elucidate the role of beta‐catenin in transdifferentiation, we analyzed liver‐specific transgenic (TG) ser45‐mutant b‐catenin‐expressing mice after DDC diet. Compared to WT, TG had significantly upregulated expression of EpCAM and CK19, and more Sox‐9+ hepatocytes. Further, Yap, which regulates liver cell fate, is increased in TG hepatocyte nuclei, concomitant with increased expression of Yap target genes. Thus, Wnt/beta‐catenin signaling cooperates with Yap pathway to regulate hepatobiliary repair after DDC injury.

Sa1687 Modeling Cholangiocarcinoma Desmoplasia for Rapidly Identifying Stromal Targeting Agents

A S L D A b st ra ct s sham or bile duct ligation (BDL) and were then treated with chloroquine (an inhibitor of autophagy; 60 mg/kg/day), rapamycin (an activator of autophagy; 30 mg/kg/day) or recombinant PGRN (5 nmol/kg/day) via intraperitoneal implanted minipump for 3 days. Cholangiocyte proliferation and intrahepatic biliary mass were assessed in vivo by immunohistochemistry using proliferating cellular nuclear antigen (PCNA) or cytokeratin 19 (CK19) antibodies respectively. In vitro, a parental mouse cholangiocyte cell line and cells overexpressing the pro-autophagic effector Sirt1 were treated with chloroquin and rapamycin and PGRN for up to 48 hr and proliferation assessed using MTS assays. Expression of the autophagy markers, Beclin, ATG5, ATG7 and LAMP1, and Sirt1 were assessed by qPCR, immunoblotting and immunohistochemistry in livers and cholangiocytes after the above-mentioned treatments. Autophagy was also assessed in vitro in mouse cholangiocyte cell lines using a commercially available lysotracker staining kit. Results: After BDL, markers of autophagy were increased, specifically in cholangiocytes. Strategies to inhibit autophagy (chloroquine) increased intrahepatic biliary mass and induced cholangiocyte proliferation in vivo and in vitro, whereas strategies to stimulate autophagy (rapamycin) reduced intrahepatic biliary mass after BDL and reduced cholangiocyte proliferation in vivo and in vitro. Treatment with recombinant PGRN reduced the expression of autophagy markers in cholangiocytes and increased cholangiocyte proliferation. Associated with these effects was a PGRN-induced suppression of the pro-autophagic molecule, Sirt1. Indeed, the inhibitory effect of PGRN on autophagy was alleviated in cholangiocytes overexpressing Sirt1. Conclusion: Our data suggest that autophagy is upregulated during hyperplastic cholangiocyte proliferation, presumably as a compensatory mechanism to prevent overt proliferation. Furthermore, PGRN may induce cholangiocyte proliferation by inhibiting autophagy via the suppression of Sirt1 expression.

Nuclear Factor, Erythroid 2-Like 2 Regulates Expression of Type 3 Inositol 1,4,5-Trisphosphate Receptor and Calcium Signaling in Cholangiocytes

Most cholestatic disorders are caused by defects in cholangiocytes. The type 3 isoform of the inositol 1,4,5-trisphosphate receptor (ITPR3) is the most abundant intracellular calcium release channel in cholangiocytes. ITPR3 is required for bicarbonate secretion by bile ducts, and its expression is reduced in intrahepatic bile ducts of patients with cholestatic disorders. We investigated whether the nuclear factor, erythroid 2-like 2 (NFE2L2 or NRF2), which is sensitive to oxidative stress, regulates expression of ITPR3. The activity of the ITPR3 promoter was measured in normal human cholangiocyte (NHC) cells and primary mouse cholangiocytes. Levels of ITPR3 protein and messenger RNA were examined by immunoblot and polymerase chain reaction analyses, respectively. ITPR3 activity was determined by measuring calcium signaling in normal human cholangiocyte cells and secretion in isolated bile duct units. Levels of NRF2 were measured in liver tissues from rats with cholestasis (induced by administration of α-napthylisothiocyanate) and from patients with biliary diseases. We identified a musculo-aponeurotic fibrosarcoma recognition element in the promoter of ITPR3 that bound NRF2 directly in NHC cells and mouse cholangiocytes. Increasing binding of NRF2 at this site resulted in chromatin remodeling that reduced promoter activity. Mutant forms of the musculo-aponeurotic fibrosarcoma recognition element did not bind NRF2. Activation of NRF2 with quercetin or by oxidative stress reduced expression of ITPR3 and calcium signaling in NHC cells; quercetin also reduced secretion by bile duct units isolated from rats. Knockdown of NRF2 with small interfering RNAs restored expression and function of ITPR3 in NHC cells incubated with quercetin. Bile ducts from rats with cholestasis and patients with cholangiopathic disorders expressed higher levels of NRF2 and lower levels of ITPR3 than ducts from control rats or patients with other liver disorders. The transcription factor NRF2 binds to the promoter of ITPR3 to inhibit its expression in cholangiocytes, leading to reduced calcium signaling and bile duct secretion. This could be a mechanism by which oxidative stress inhibits these processes and contributes to cholangiopathies.

Gonadotropin-Releasing Hormone Stimulates Biliary Proliferation by Paracrine/Autocrine Mechanisms

During cholestatic liver disease, there is dysregulation in the balance between biliary growth and loss in bile duct-ligated (BDL) rats modulated by neuroendocrine peptides via autocrine/paracrine pathways. Gonadotropin-releasing hormone (GnRH) is a trophic peptide hormone that modulates reproductive function and proliferation in many cell types. We evaluated the autocrine role of GnRH in the regulation of cholangiocyte proliferation. The expression of GnRH receptors was assessed in a normal mouse cholangiocyte cell line (NMC), sham, and BDL rats. The effect of GnRH administration was evaluated in normal rats and in NMC. GnRH-induced biliary proliferation was evaluated by changes in intrahepatic bile duct mass and the expression of proliferation and function markers. The expression and secretion of GnRH in NMC and isolated cholangiocytes was assessed. GnRH receptor subtypes GnRHR1 and GnRHR2 were expressed in cholangiocytes. Treatment with GnRH increased intrahepatic bile duct mass as well as proliferation and function markers in cholangiocytes. Transient knockdown and pharmacologic inhibition of GnRHR1 in NMC decreased proliferation. BDL cholangiocytes had increased expression of GnRH compared with normal rats, accompanied by increased GnRH secretion. In vivo and in vitro knockdown of GnRH decreased intrahepatic bile duct mass/cholangiocyte proliferation and fibrosis. GnRH secreted by cholangiocytes promotes biliary proliferation via an autocrine pathway. Disruption of GnRH/GnRHR signaling may be important for the management of cholestatic liver diseases.

Breast tumor kinase/protein tyrosine kinase 6 (Brk/PTK6) activity in normal and neoplastic biliary epithelia

Breast tumor kinase (BRK) augments proliferation and promotes cell survival in breast cancers via interactions with SH2 and SH3 ligand-containing proteins, such as receptor tyrosine kinases (RTK; e.g. EGFR, ErbB2/neu). Since RTK contribute to cholangiocarcinoma (CC) evolution we probed BRK protein expression and function in normal and CC livers. Immunohistochemical staining of normal livers and CC (n=93) in a tissue microarray and three CC and an immortalized human cholangiocyte cell lines (real-time PCR, Western blotting, siRNA) were used to study the functional relationships between BRK, EGFR, ErbB2, SAM68, and SPRR2a. BRK protein was expressed in normal human intrahepatic bile ducts; all CC cell lines and a majority of CC showed strong BRK protein expression. Multiplex immunostaining/tissue cytometry and immunoprecipitation studies showed: 1) BRK co-localized with EGFR and ErbB2/neu; 2) BRK(high)/EGFR(high)-co-expressing CC cells had significantly higher Ki67 labeling and; 3) stronger BRK protein expression was seen in perihilar and distal CC than intrahepatic CC and directly correlated with CC differentiation. In cell lines, BRK expression augmented proliferation in response to exogenous EGF, whereas BRK siRNA significantly reduced growth. The SH3 ligand-containing, SPRR2A activated pTyr342 BRK, which in turn, phosphorylated SAM68, causing nuclear localization and increased cell proliferation similar to observations in breast cancers. BRK expression in a majority of CC can interact with RTK, augmenting growth and interfering with proliferation inhibitors (SAM68). Therapeutically targeting BRK function (in addition to RTK) should be of benefit for CC treatment.

Activation of the renin-angiotensin system stimulates biliary hyperplasia during cholestasis induced by extrahepatic bile duct ligation.

Cholangiocyte proliferation is regulated in a coordinated fashion by many neuroendocrine factors through autocrine and paracrine mechanisms. The renin-angiotensin system (RAS) is known to play a role in the activation of hepatic stellate cells and blocking the RAS attenuates hepatic fibrosis. We investigated the role of the RAS during extrahepatic cholestasis induced by bile duct ligation (BDL). In this study, we used normal and BDL rats that were treated with control, angiotensin II (ANG II), or losartan for 2 wk. In vitro studies were performed in a primary rat cholangiocyte cell line (NRIC). The expression of renin, angiotensin-converting enzyme, angiotensinogen, and angiotensin receptor type 1 was evaluated by immunohistochemistry (IHC), real-time PCR, and FACs and found to be increased in BDL compared with normal rat. The levels of ANG II were evaluated by ELISA and found to be increased in serum and conditioned media of cholangiocytes from BDL compared with normal rats. Treatment with ANG II increased biliary mass and proliferation in both normal and BDL rats. Losartan attenuated BDL-induced biliary proliferation. In vitro, ANG II stimulated NRIC proliferation via increased intracellular cAMP levels and activation of the PKA/ERK/CREB intracellular signaling pathway. ANG II stimulated a significant increase in Sirius red staining and IHC for fibronectin that was blocked by angiotensin receptor blockade. In vitro, ANG II stimulated the gene expression of collagen 1A1, fibronectin 1, and IL-6. These results indicate that cholangiocytes express a local RAS and that ANG II plays an important role in regulating biliary proliferation and fibrosis during extraheptic cholestasis.

Post-translational Regulation of the Type III Inositol 1,4,5-Trisphosphate Receptor by miRNA-506

The type III isoform of the inositol 1,4,5-trisphosphate receptor (InsP3R3) is apically localized and triggers Ca(2+) waves and secretion in a number of polarized epithelia. However, nothing is known about epigenetic regulation of this InsP3R isoform. We investigated miRNA regulation of InsP3R3 in primary bile duct epithelia (cholangiocytes) and in the H69 cholangiocyte cell line, because the role of InsP3R3 in cholangiocyte Ca(2+) signaling and secretion is well established and because loss of InsP3R3 from cholangiocytes is responsible for the impairment in bile secretion that occurs in a number of liver diseases. Analysis of the 3'-UTR of human InsP3R3 mRNA revealed two highly conserved binding sites for miR-506. Transfection of miR-506 mimics into cell lines expressing InsP3R3-3'UTR-luciferase led to decreased reporter activity, whereas co-transfection with miR-506 inhibitors led to enhanced activity. Reporter activity was abrogated in isolated mutant proximal or distal miR-506 constructs in miR-506-transfected HEK293 cells. InsP3R3 protein levels were decreased by miR-506 mimics and increased by inhibitors, and InsP3R3 expression was markedly decreased in H69 cells stably transfected with miR-506 relative to control cells. miR-506-H69 cells exhibited a fibrotic signature. In situ hybridization revealed elevated miR-506 expression in vivo in human-diseased cholangiocytes. Histamine-induced, InsP3-mediated Ca(2+) signals were decreased by 50% in stable miR-506 cells compared with controls. Finally, InsP3R3-mediated fluid secretion was significantly decreased in isolated bile duct units transfected with miR-506, relative to control IBDU. Together, these data identify miR-506 as a regulator of InsP3R3 expression and InsP3R3-mediated Ca(2+) signaling and secretion.