Cholangiocyte Apoptosis Research Articles

Activation of the PI3K/mTOR Pathway Is Involved in Cystic Proliferation of Cholangiocytes of the PCK Rat

The polycystic kidney (PCK) rat is an animal model of Caroli’s disease as well as autosomal recessive polycystic kidney disease (ARPKD). The signaling pathways involving the mammalian target of rapamycin (mTOR) are aberrantly activated in ARPKD. This study investigated the effects of inhibitors for the cell signaling pathways including mTOR on cholangiocyte proliferation of the PCK rat. Cultured PCK cholangiocytes were treated with rapamycin and everolimus [inhibitors of mTOR complex 1 (mTOC1)], LY294002 [an inhibitor of phosphatidylinositol 3-kinase (PI3K)] and NVP-BEZ235 (an inhibitor of PI3K and mTORC1/2), and the cell proliferative activity was determined in relation to autophagy and apoptosis. The expression of phosphorylated (p)-mTOR, p-Akt, and PI3K was increased in PCK cholangiocytes compared to normal cholangiocytes. All inhibitors significantly inhibited the cell proliferative activity of PCK cholangiocytes, where NVP-BEZ235 had the most prominent effect. NVP-BEZ235, but not rapamycin and everolimus, further inhibited biliary cyst formation in the three-dimensional cell culture system. Rapamycin and everolimus induced apoptosis in PCK cholangiocytes, whereas NVP-BEZ235 inhibited cholangiocyte apoptosis. Notably, the autophagic response was significantly induced following the treatment with NVP-BEZ235, but not rapamycin and everolimus. Inhibition of autophagy using siRNA against protein-light chain3 and 3-methyladenine significantly increased the cell proliferative activity of PCK cholangiocytes treated with NVP-BEZ235. In vivo, treatment of the PCK rat with NVP-BEZ235 attenuated cystic dilatation of the intrahepatic bile ducts, whereas renal cyst development was unaffected. These results suggest that the aberrant activation of the PI3K/mTOR pathway is involved in cystic proliferation of cholangiocytes of the PCK rat, and inhibition of the pathway can reduce cholangiocyte proliferation via the mechanism involving apoptosis and/or autophagy.

GABA induces the differentiation of small into large cholangiocytes by activation of Ca2+/CaMK I-dependent adenylyl cyclase 8

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/Ca(2+) /calmodulin-dependent protein kinase (CaMK) I. gamma-Aminobutyric acid (GABA) affects cell functions by modulation of Ca(2+) -dependent signaling and AC. We hypothesized that GABA induces the differentiation of small into large cholangiocytes by the activation of Ca(2+) /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. 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.

Histidine decarboxylase regulates biliary homeostasis via autocrine histamine signaling

Cholangiocyte homeostasis is regulated by both autocrine/paracrine signaling. In bile duct ligated (BDL) rats large cholangiocyte proliferate, whereas CCl4 induces large cholangiocyte apoptosis. Histidine decarboxylase (HDC) regulates histamine synthesis. We have shown that: (i) cholangiocytes express HDC and secrete histamine; (ii) histamine stimulates biliary growth; and (ii) HDC inhibition decreases biliary growth. No information exists that HDC/histamine regulates cholangiocyte homeostasis. In vivo, normal and 1 wk BDL rats were treated with saline or histamine (0.5 mg/kg) and given CCl4 by gavage (0.5 ml/100 gm). Liver blocks and large cholangiocytes were obtained. HDC, PCNA, TUNEL and CK‐19 biliary expression was measured by immunohistochemistry in liver sections and by qPCR and immunoblots in large cholangiocytes. In vitro, control (MSE‐neg) or stable transfected HDC knockdown large cholangiocytes (MSE‐HDC) were treated with CCl4 (5 mM) in the absence/presence of histamine (10 μM) before evaluating apoptosis and proliferation. After BDL, there was enhanced cholangiocyte proliferation, ductal mass and biliary HDC expression. CCl4 treatment (i) enhanced biliary apoptosis; and (ii) decreased HDC and serum/biliary histamine levels and biliary proliferation and ductal mass, changes that were prevented by histamine. In MSE‐HDC cells there was reduced biliary growth and enhanced apoptosis compared to Mz‐neg. In MSE‐HDC cells treated with CCl4, apoptosis increased compared to MSE‐neg cells treated with CCl4. In Mz‐HDC, CCl4‐induced apoptosis and loss of proliferation was prevented by histamine. HDC is a key regulator of cholangiocyte homeostasis and histamine may be a protective factor after toxic injury. Treatment of cholangiopathies characterized by proliferation or loss may be regulated by the modulation of histamine levels.

Clues to the Etiology of Bile Duct Injury in Biliary Atresia

Biliary atresia (BA) is an infantile obstructive cholangiopathy of unknown etiology with suboptimal therapy, which is responsible for 40 to 50% of all pediatric liver transplants. Although the etiology of bile duct injury in BA in unknown, it is postulated that a pre- or perinatal viral infection initiates cholangiocyte apoptosis and release of antigens that trigger a Th1 immune response that leads to further bile duct injury, inflammation, and obstructive fibrosis. Humoral immunity and activation of the innate immune system may also play key roles in this process. Moreover, recent investigations from the murine BA model and human data suggest that regulatory T cells and genetic susceptibility factors may orchestrate autoimmune mechanisms. What controls the coordination of these events, why the disease only occurs in the first few months of life, and why a minority of infants with perinatal viral infections develop BA are remaining questions to be answered.

Fn14 is expressed on neoplastic cholangiocytes in intrahepatic cholangiocarcinoma and promotes necrosis after interaction with TWEAK

BackgroundCholangiocarcinoma is the second most common primary hepatic malignancy worldwide. The incidence of intrahepatic cholangiocarcinoma in the UK has steadily increased over the past 40 years. The main treatment is chemotherapy, and 5-year survival after radical surgery is only 25%. The carcinogenic mechanisms involved in cholangiocarcinoma remain elusive. The fibroblast growth factor-inducible 14 (Fn14)/tumour necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) receptor-ligand system has been shown to be of importance in cellular proliferation and tumour angiogenesis in hepatocellular carcinoma. The aim of this study was to demonstrate the expression of Fn14 and TWEAK in cholangiocarcinoma and determine the functional significance of Fn14/TWEAK interaction on neoplastic cholangiocytes. MethodsHuman liver samples were obtained with consent from the Queen Elizabeth Hospital liver transplant programme. Sections were stained for Fn14 with immunohistochemical techniques. Expression of Fn14 on a cholangiocarcinoma cell line (CC-LP-1) stimulated with TNFα, interferon γ and fibroblast growth factor (FGF) basic was established quantitatively with flow cytometry. Stimulated CC-LP-1 were exposed to different concentrations of TWEAK for 24h. Apoptosis, necrosis, autophagy, and reactive oxygen species production at 24 h were determined by flow cytometry using annexin, 7AAD, dansylcadaverine, and dichlorofluorescein assays, respectively. Proliferation was determined with Ki69 nuclear staining. FindingsImmunohistochemistry revealed Fn14 on the intrahepatic malignant small bile ducts in cholangiocarcinoma. Exposure of neoplastic cholangiocytes to TWEAK for 24h induced necrosis and reduced apoptosis in FGF-activated neoplastic cholangiocytes in a dose-dependent manner. InterpretationFn14 is expressed on neoplastic cholangiocytes in intrahepatic cholangiocarcinoma. Activation of the Fn14/TWEAK receptor-ligand system induces necrosis. The role of Fn14/TWEAK in cholangiocarcinoma needs further investigation to ascertain the mechanisms involved and outcome on overall tumour viability. FundingUK Medical Research Council.

Role of secretin in the growth of the biliary epithelium in normal and cholestatic mice

Background: Secretin stimulates bicarbonate secretion in large ducts by interaction with receptors by a cAMP-dependent pathway [1]. In BDL rats, the growth of large (but not small) cholangiocytes is activated by the cAMP/ERK1/2/Elk-1 pathway; and is associated with increased secretin receptor (SR) expression [2]. The aim of our study was to define the role of secretin in experimental models of cholestatic mice. Methods: We used (SEC+/+) (wild-type, WT) and SEC-/- male mice that underwent sham surgery or BDL for 7 days treated with saline or secretin. Then, we used: (i) liver sections to evaluate secretin protein expression, cholangiocyte growth, apoptosis, the presence of VEGF-A/C and NGF; (ii) purified and immortalized small and large BDL cholangiocytes to measure PCNA protein expression and the same previous growth factors. Results: In vivo, we demonstrated that: (i) secretin is expressed in normal liver by large bile ducts; and (ii) secretin expression increases in large bile ducts following BDL. Isolated large cholangiocytes from BDL WT mice express higher levels of the mRNA for secretin and secrete greater amounts of this hormone compared to normal large cholangiocytes. Knockout of the secretin gene reduced BDL-induced increase in large IBDM compared to WT BDL mice, an event that was associated with enhanced apoptosis of large cholangiocytes and decreased expression of VEGF-A/C and NGF. Concomitant with reduced large IBDM there was increased small IBDM in SEC-/- KO BDL mice compared to WT BDL mice. After prolonged treatment, secretin increased the biliary expression of PCNA, VEGF-A/C and NGF. Conclusions: The study introduces the novel concept that cholangiocytes express and secrete secretin and that manipulation of this gene may be an important target for the management of biliary disorders.

Effects of Autologous Bone Marrow Mononuclear Cells Implantation on Cholangiocytes Apoptosis in Model of Intrahepatic Ischemic Type Biliary Lesion in Rabbits

AimTo investigate the effects of autologous bone marrow mononuclear cells (BM-MNCs) implantation on regulation of cholangiocyte apoptosis in a model of intrahepatic ischemic type biliary lesion (ITBL) in rabbits. Materials and MethodsThirty Japanese white rabbits were divided randomly into 3 groups (10 per group) including controls (group A), ITBL model (group B), and BM-MNCs implantation groups (group C). All rabbits underwent the same surgical procedure to prepare the liver for graft removal during transplantation. Subsequently, no additional vascular intervention was performed in group A. In group B, the hepatic artery and common bile duct were clamped with microvascular clips for 2 hours, where after the clips were removed to recover the blood supply. Group C received, BM-MNCs (108 cells per rabbit) injected through the hepatic artery after removing the clips. The animals were killed 4 weeks after operation. The survival rate, histopathologic examination, cholangiocyte apoptosis with terminal uridine nick-end labeling (TUNEL) staining and expressions of Bcl-2 and Bax proteins were examined using immunohistochemical staining. ResultsGroup A animals showed a survival of 100%; the rates in groups B and C were both 90%. Histopathologic examination revealed normal intrahepatic cholangiocytes in group A, obviously damaged ones in group B, and alleviated damage in group C. TUNEL staining indicated apoptosis of cholangiocytes in group B was more serious than that in group A or group C. Immunohistochemical staining demonstrated significantly decreased Bcl-2 expression in group B compared with that in group A; Bcl-2 expression in group C returned to the level of group A. Simultaneously, the Bax expression presented adverse results; the ratios of Bcl-2/Bax were ranked as group A > group C > group B. ConclusionImplantation of autologous BM-MNCs significantly reduced apoptosis of intrahepatic cholangiocytes and prevented or abated intrahepatic ITBL.

The biliary HCO3− umbrella

To critically review most recent experimental evidence for the protective action of biliary HCO(3)(-) secretion against bile acid-induced bile duct damage and development of fibrosing cholangiopathy in humans and experimental animals. Studies in human cholangiocytes in vitro indicate that a biliary HCO(3)(-) umbrella protects against bile acid-induced cholangiocyte damage and apoptosis in humans. The Cl(-)/HCO(3)(-) exchanger, AE2, and an intact biliary glycocalyx appear crucial for its stability. Related studies with experimental animal models in vivo have to be interpreted with caution as humans and mice differ not only with regard to bile salt pool, but also their expression patterns of transport proteins and signalling molecules. Adequate biliary HCO(3)(-) secretion may protect against bile salt-induced cholangiopathies. Future therapeutic strategies in biliary diseases will aim at stabilizing the biliary HCO(3)(-) umbrella.

Etiopathogenesis of primary biliary cirrhosis: an overview of recent developments.

Substantial advancements in the field of primary biliary cirrhosis (PBC) research have broadened our understanding of this enigmatic disease. Genome-wide studies have identified several new candidate genes involved in the immunoregulatory process, particularly those responsible for antigen presentation and lymphocyte signaling. Examples include the HLA class-II region and genes implicated in IL12-JAK/STAT signaling, and the NF-κB and TNF signaling pathways. Environmental triggers appear to disrupt the pre-existing, unstable immune tolerance in genetically susceptible individuals, and molecular mimics of the PBC-specific autoantigen (PDC) may be derived from microbes or xenobiotic compounds, which modify native proteins, making them immunogenic. Although the vast majority of patients with PBC are AMA-positive, a variety of disease-specific antinuclear antibodies have been recognized in conferring a worse clinical outcome. There has also been a revived interest in the role of antibody-secreting B cells in murine models suggesting that depletion of these cells paradoxically exacerbates cholangiopathy. Biliary specificity in PBC is most likely driven by the uniqueness of cholangiocyte apoptosis in which the PDC-E2 autoantigen undergoes differential glutathiolation. Cholangiocytes also possess the ability to phagocytose neighboring apoptotic cells, present intact immunoreactive antigen, and undergo attack from autoantibodies, the innate immune system, and autoreactive lymphocytes. Cellular senescence and a lack of functioning T-regulatory cells are proposed mechanisms by which this multi-lineage process is thought to be enhanced. This review summarizes these key advances as the true complexities of the disease process begin to be unraveled.

Liver fluke-induced hepatic oxysterols stimulate DNA damage and apoptosis in cultured human cholangiocytes

Oxysterols are cholesterol oxidation products that are generated by enzymatic reactions through cytochrome P450 family enzymes or by non-enzymatic reactions involving reactive oxygen and nitrogen species. Oxysterols have been identified in bile in the setting of chronic inflammation, suggesting that biliary epithelial cells are chronically exposed to these compounds in certain clinical settings. We hypothesized that biliary oxysterols resulting from liver fluke infection participate in cholangiocarcinogenesis. Using gas chromatography/mass spectrometry, we identified oxysterols in livers from hamsters infected with Opisthorchis viverrini that develop cholangiocarcinoma. Five oxysterols were found: 7-keto-cholesta-3,5-diene (7KD), 3-keto-cholest-4-ene (3K4), 3-keto-cholest-7-ene (3K7), 3-keto-cholesta-4,6-diene (3KD), and cholestan-3β,5α,6β-triol (Triol). Triol and 3K4 were found at significantly higher levels in the livers of hamsters with O. viverrini-induced cholangiocarcinoma. We therefore investigated the effects of Triol and 3K4 on induction of cholangiocarcinogenesis using an in vitro human cholangiocyte culture model. Triol- and 3K4-treated cells underwent apoptosis. Western blot analysis showed significantly increased levels of Bax and decreased levels of Bcl-2 in these cells. Increased cytochrome c release from mitochondria was found following treatment with Triol and 3K4. Triol and 3K4 also induced formation of the DNA adducts 1,N6-etheno-2′-deoxyadenosine, 3,N4-etheno-2′-deoxycytidine and 8-oxo-7,8-dihydro-2′-deoxyguanosine in cholangiocytes. The data suggest that Triol and 3K4 cause DNA damage via oxidative stress. Chronic liver fluke infection increases production of the oxysterols Triol and 3K4 in the setting of chronic inflammation in the biliary system. These oxysterols induce apoptosis and DNA damage in cholangiocytes. Insufficient and impaired DNA repair of such mutated cells may enhance clonal expansion and further drive the change in cellular phenotype from normal to malignant.

Sorafenib before liver transplantation for hepatocellular carcinoma: risk or give up

Dear Sirs, We read with interest the article by Truesdale et al. [1] regarding their experience in the use of sorafenib before liver transplantation (LT) in patients with hepatocellular carcinoma (HCC). The authors compare ten patients who were administered sorafenib during waiting time for LT with twenty-three patients to whom the drug was not given. No difference in terms of overall and HCC recurrence-free survival was seen between the two groups. Indeed a higher incidence of biliary complications (67% vs. 17%) and acute cellular rejections (67% vs. 22%) was observed in the first group. The authors assume that the higher amount of complications is secondary to the use of sorafenib because of its main effect in inhibiting the vascular endothelial growth factor (VEGFR), reducing the resistance to apoptosis of cholangiocytes and thus both hindering the integrity of biliary vascularization and altering the immune pathway leading to cellular rejection. However, some major issues arise reading this paper: the authors do not specify the inclusion criteria for the use of sorafenib, the extension of the tumour burden before LT, the number of locoregional treatments before the drug was given and in general the efficacy of down-sizing procedures in this group of patients. An interesting matter of debate is the time to stop the administration of sorafenib. Infact the authors assert that the drug was stopped on the day of LT. In our experience [2], it seems not to be safe to continue with the administration of the drug until the day of LT: although there is no international agreement about this point [2–5], we usually interrupt the drug at least 3 weeks before a surgical procedure such as LT, because of the increased risk of bleeding of impaired wound healing and of liver dysfunction in the perioperative period in this setting of patients. Another intriguing issue is that doubtlessly sorafenib cannot still be considered nowadays in the group of the standard down-staging procedures of HCC in terms of safety and cost–effectiveness, but it should be reserved to the patients with advanced HCC traditionally treated with locoregional therapies/hepatic resection in whom sorafenib plays as important role in bringing them within the accepted criteria for LT with HCC. So as to exploit as much as possible the potential of the drug, we believe that sorafenib should not be reserved to patients with advanced HCC to whom any other curative treatment is not possible, but it should be offered also to a different setting of patients such as young patients outside standard criteria to LT. In the future, the challenge will be to break the Damocles sword of medical therapies for HCC, until now no effective therapy exists. The integration of sorafenib in the medical practice and its fully comprehension may permit the best management of these patients as possible.

TAK1: Another mesh in the NF-κB – JNK controlled network causing hepatocellular carcinoma

The MAP3-kinase TGF-beta-activated kinase 1 (TAK1) critically modulates innate and adaptive immune responses and connects cytokine stimulation with activation of inflammatory signaling pathways. Here, we report that conditional ablation of TAK1 in liver parenchymal cells (hepatocytes and cholangiocytes) causes hepatocyte dysplasia and early-onset of hepatocarcinogenesis, coinciding with biliary ductopenia and cholestasis. TAK1-mediated cancer suppression is exerted through activating NF-kappaB in response to tumor necrosis factor (TNF) and through preventing Caspase-3-dependent hepatocyte and cholangiocyte apoptosis. Moreover, TAK1 suppresses a procarcinogenic and pronecrotic pathway, which depends on NF-kappaB-independent functions of the I kappaB-kinase (IKK)-subunit NF-kappaB essential modulator (NEMO). Therefore, TAK1 serves as a gatekeeper for a protumorigenic, NF-kappaB-independent function of NEMO in parenchymal liver cells.

Cholangiocytes of Cirrhotic Rats are More Tolerant to Ischemia than Normal Rats: A Role for Abnormal Hepatic Arteriovenous Communications?

To assess whether cholangiocytes of rats with liver cirrhosis are more tolerant to ischemic changes than normal rats, and whether this is due to arteriovenous fistulas. Ninety-eight Sprague-Dawley rats were divided into the normal group (n = 30) and the cirrhosis group (n = 68), and then each group was divided into controls and those with bile duct ischemia. At 0 h, 6 h, 3 d, and 14 d after the induction of bile duct ischemia, the liver of each rat was removed and stained with toluidine blue to compare cholangiocyte morphology. Cholangiocyteapoptosis was evaluated by a deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) assay. Expression of VEGF, HIF1, NF-kB(p65) was assessed by quantitative analysis of the products of reverse-transcriptase polymerase chain reaction. Resin casts were used to reproduce the intrahepatic vasculature of cirrhotic rats, and the presence of communications between the portal vein and hepatic artery was assessed with stereomicroscopy. Rats with liver cirrhosis were more tolerant than normal rats 6 h after bile duct ischemia (P < 0.05); at 3 and 14 d after the ischemic insult, there was no significant difference between the cirrhotic rats and normal rats. Levels of expression of VEGF, HIF1, and NF-kB(p65) genes, either in normal rats or cirrhotic rats, were significantly elevated compared with those in the control group (∗, ∗∗P < 0.05), but a lower extent changes appeared in the cirrhotic rats (∗∗∗P < 0.05). Several communications could be observed between the portal vein and hepatic artery. Cholangiocytes of cirrhotic rats appear to be more tolerant to ischemia of bile duct than non-cirrhotic rats. This may be due to the protective role of arterioportal fistulas.

Primary and malignant cholangiocytes undergo CD40 mediated Fas dependent apoptosis, but are insensitive to direct activation with exogenous Fas ligand.

IntroductionCholangiocarcinoma is a rare malignancy of the biliary tract, the incidence of which is rising, but the pathogenesis of which remains uncertain. No common genetic defects have been described but it is accepted that chronic inflammation is an important contributing factor. We have shown that primary human cholangiocyte and hepatocyte survival is tightly regulated via co-operative interactions between two tumour necrosis family (TNF) receptor family members; CD40 and Fas (CD95). Functional deficiency of CD154, the ligand for CD40, leads to a failure of clearance of biliary tract infections and a predisposition to cholangiocarcinoma implying a direct link between TNF receptor-mediated apoptosis and the development of cholangiocarcinoma.AimsTo determine whether malignant cholangiocytes display defects in CD40 mediated apoptosis. By comparing CD40 and Fas-mediated apoptosis and intracellular signalling in primary human cholangiocytes and three cholangiocyte cell lines.ResultsPrimary cholangiocytes and cholangiocyte cell lines were relatively insensitive to direct Fas-mediated killing with exogenous FasL when compared with Jurkat cells, which readily underwent Fas-mediated apoptosis, but were extremely sensitive to CD154 stimulation. The sensitivity of cells to CD40 activation was similar in magnitude in both primary and malignant cells and was STAT-3 and AP-1 dependent in both.Conclusions1) Both primary and malignant cholangiocytes are relatively resistant to Fas–mediated killing but show exquisite sensitivity to CD154, suggesting that the CD40 pathway is intact and fully functional in both primary and malignant cholangiocytes 2) The relative insensitivity of cholangiocytes to Fas activation demonstrates the importance of CD40 augmentation of Fas dependent death in these cells. Agonistic therapies which target CD40 and associated intracellular signalling pathways may be effective in promoting apoptosis of malignant cholangiocytes.

Prostate Apoptosis Response-4 Is Expressed in Normal Cholangiocytes, Is Down-Regulated in Human Cholangiocarcinoma, and Promotes Apoptosis of Neoplastic Cholangiocytes When Induced Pharmacologically

Prostate apoptosis response-4 (Par-4) is a tumor suppressor protein that sensitizes cells to apoptosis; therefore, Par-4 modulation has therapeutic potential. No data currently exist on Par-4 expression in cholangiocarcinoma (CCA). We evaluated the expression of Par-4 in normal and neoplastic cholangiocytes and the effects of its pharmacological or genetic modulation. The study was performed in human and rat liver, CCA patient biopsies, and two CCA cell lines. PAR-4 was expressed in normal rat and human cholangiocytes, but its expression levels decreased in both human CCA and CCA cell lines. In both intrahepatic and extrahepatic CCA, Par-4 expression (as shown by immunohistochemistry) was inversely correlated with markers of proliferation (eg, proliferating cellular nuclear antigen) and directly correlated with apoptotic markers (eg, Bax and Bax/BCL2 ratio). Par-4 expression was decreased during CCA cell proliferation but was enhanced after apoptosis induction. Pharmacological induction of Par-4 expression in CCA cell lines by diindolymethane or withaferin A promoted activation of apoptosis and inhibition of proliferation. In contrast, specific Par-4 silencing by small-interfering RNA determined activation of CCA cell line proliferation. Par-4 is expressed in rat and human cholangiocytes and is down-regulated in both human CCA and CCA cell lines. Par-4 protein levels decrease during cell proliferation but increase during apoptosis. Pharmacological or genetic induction of Par-4 determines apoptosis of CCA cells, suggesting Par-4 targeting as a CCA treatment strategy.

The MAP3K TAK1: A Chock Block to Liver Cancer Formation

Abstract TGF-β-activated kinase 1 (TAK1) is a MAP3K family member that activates NF-κB and JNK via Toll-like receptors and the receptors for IL-1, TNF-α, and TGF-β. Because the TAK1 downstream molecules NF-κB and JNK have opposite effects on cell death and carcinogenesis, the role of TAK1 in the liver is unpredictable. To address this issue, we generated hepatocyte-specific Tak1 -deficient ( Tak1ΔHEP ) mice. The Tak1ΔHEP mice displayed spontaneous hepatocyte death, compensatory proliferation, inflammatory cell infiltration, and perisinusoidal fibrosis at age 1 month. Older Tak1ΔHEP mice developed multiple cancer nodules characterized by increased expression of fetal liver genes including α-fetoprotein. Cultures of primary hepatocytes deficient in Tak1 exhibited spontaneous cell death that was further increased in response to TNF-α. TNF-α increased caspase-3 activity but activated neither NF-κB nor JNK in Tak1 -deficient hepatocytes. Genetic abrogation of TNF receptor type I (TNFRI) in Tak1ΔHEP mice reduced liver damage, inflammation, and fibrosis compared with unmodified Tak1ΔHEP mice. In conclusion, hepatocyte-specific deletion of TAK1 in mice resulted in spontaneous hepatocyte death, inflammation, fibrosis, and carcinogenesis that was partially mediated by TNFR signaling, indicating that TAK1 is an essential component for cellular homeostasis in the liver. Abstract The MAP3-kinase TGF-β-activated kinase 1 (TAK1) critically modulates innate and adaptive immune responses and connects cytokine stimulation with activation of inflammatory signaling pathways. Here, we report that conditional ablation of TAK1 in liver parenchymal cells (hepatocytes and cholangiocytes) causes hepatocyte dysplasia and early-onset hepatocarcinogenesis, coinciding with biliary ductopenia and cholestasis. TAK1-mediated cancer suppression is exerted through activating NF-κB in response to tumor necrosis factor (TNF) and through preventing Caspase-3-dependent hepatocyte and cholangiocyte apoptosis. Moreover, TAK1 suppresses a procarcinogenic and pronecrotic pathway, which depends on NF-κB-independent functions of the IκB-kinase (IKK)-subunit NF-κB essential modulator (NEMO). Therefore, TAK1 serves as a gatekeeper for a protumorigenic, NF-κB-independent function of NEMO in parenchymal liver cells.

Bombesin and neurotensin exert antiproliferative effects on oval cells and augment the regenerative response of the cholestatic rat liver

The regenerative capacity of the cholestatic liver is significantly attenuated. Oval cells are hepatic stem cells involved in liver's regeneration following diverse types of injury. The present study investigated the effect of the neuropeptides bombesin (BBS) and neurotensin (NT) on oval cell proliferation as well as on hepatocyte and cholangiocyte proliferation and apoptosis in the cholestatic rat liver. Seventy male Wistar rats were randomly divided into five groups: controls, sham operated, bile duct ligated (BDL), BDL + BBS (30 μg/kg/d), BDL + NT (300 μg/kg/d). Ten days later, alpha-fetoprotein (AFP) mRNA ( in situ hybridization), cytokeratin-19 and Ki67 antigen expression (immunohistochemistry) and apoptosis (TUNEL) were evaluated on liver tissue samples. Cells with morphologic features of oval cells that were cytokeratin-19(+) and AFP mRNA(+) were scored in morphometric analysis and their proliferation was recorded. In addition, the proliferation and apoptotic rates of hepatocytes and cholangiocytes were determined. Alanine aminotransferase (ALT) levels and hepatic oxidative stress (lipid peroxidation and glutathione redox state) were also estimated. The neuropeptides BBS and NT significantly reduced ALT levels and hepatic oxidative stress. Both agents exerted similar and cell type-specific effects on oval cells, hepatocytes and cholangiocytes: (a) oval cell proliferation and accumulation in the cholestatic liver was attenuated, (b) hepatocyte proliferation was increased along with a decreased rate of their apoptosis and (c) cholangiocyte proliferation was attenuated and their apoptosis was increased. These observations might be of potential value in patients with extrahepatic cholestasis.

A concentration-dependent effect of ursodeoxycholate on apoptosis and caspases activities of HepG2 hepatocellular carcinoma cells

Clinical observations suggest that ursodeoxycholate (UDCA) may protect from hepatocellular carcinoma in cirrhotic patients. Increased apoptosis of malignant cells is a candidate mechanism. Decreased apoptosis of cholangiocytes is proposed as a mechanism for the favourable effect of UDCA in primary biliary cirrhosis. We therefore studied the effects of different concentrations of UDCA on HepG2 cell proliferation, apoptosis and caspases activities. Apoptotic features and activities of the effector or initiator caspases-8, -9, -3 and -2 after treatment of HepG2 cells with different concentrations of UDCA alone or in combination with TNF-alpha were examined. Apoptosis was detected by DNA fragmentation and flow cytometric determination of apoptotic cells with Annexin-V/PI. UDCA significantly inhibits cell proliferation only at high concentrations, but increases apoptosis at low concentrations and protects from apoptosis at higher concentrations. TNF-alpha induced DNA fragmentation is potentiated by UDCA, but flow cytometry indicates protection from early apoptosis and increase in cell survival by low and intermediate UDCA concentrations. UDCA differentially activates initiator and effector caspases in different concentrations. These data demonstrate that the effect of UDCA on caspase activation and apoptosis of HepG2 cells is concentration-dependent and activation of the caspase cascade is not always translated into increased apoptosis. Serum levels of UDCA should be possibly monitored and dosage of the drug adjusted according to the required effect.

TAK1 Suppresses a NEMO-Dependent but NF-κB-Independent Pathway to Liver Cancer

The MAP3-kinase TGF-beta-activated kinase 1 (TAK1) critically modulates innate and adaptive immune responses and connects cytokine stimulation with activation of inflammatory signaling pathways. Here, we report that conditional ablation of TAK1 in liver parenchymal cells (hepatocytes and cholangiocytes) causes hepatocyte dysplasia and early-onset hepatocarcinogenesis, coinciding with biliary ductopenia and cholestasis. TAK1-mediated cancer suppression is exerted through activating NF-kappaB in response to tumor necrosis factor (TNF) and through preventing Caspase-3-dependent hepatocyte and cholangiocyte apoptosis. Moreover, TAK1 suppresses a procarcinogenic and pronecrotic pathway, which depends on NF-kappaB-independent functions of the I kappaB-kinase (IKK)-subunit NF-kappaB essential modulator (NEMO). Therefore, TAK1 serves as a gatekeeper for a protumorigenic, NF-kappaB-independent function of NEMO in parenchymal liver cells.

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

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.