Cultured Gallbladder Epithelial Cells Research Articles

Bile acid accelerates erbB2-induced pro-tumorigenic activities in biliary tract cancer.

Although very few studies have addressed the molecular and cellular mechanisms underlying the development of biliary tract cancer (BTC), several lines of evidence suggest a role for the erbB receptor family. Overexpression and activation of erbB2 has been reported in a significant percentage of human BTC. Further, we previously reported that overexpression of erbB2 basal epithelial cells of the biliary tract (BK5.erbB2 mouse) led to the development of BTC. However, the mechanisms by which erbB2 overexpression led to the spontaneous development of tumors specifically in the biliary tract are not completely understood. The goals of the current study were to (1) determine whether a cooperative relationship between bile acid exposure and erbB2 activation exists during biliary tract carcinogenesis and (2) to characterize the mechanism(s) underlying bile acid-mediated biliary tract carcinogenesis in cells with activated erbB2. In this study, we demonstrated that the secondary conjugated bile acid, taurochenodeoxycholic acid (TCDC), increased proliferation of primary cultured gallbladder epithelial cells from BK5.erbB2 mice and human BTC cells. TCDC treatment activated EGFR/erbB2 and downstream signaling molecules in both primary cultured cells and human BTC cells. TCDC also increased the expression of epidermal growth factor receptor (EGFR) ligands and TACE activity in human BTC cells. Inhibition of src activation led to attenuation of bile-induced upregulation of TACE activity as well as signaling through the EGFR/erbB2, suggesting that during the development of BTC erbB2 overexpression/activation accelerates the bile acid-induced signaling cascade: bile acid → src → TACE → EGFR/erbB2 → downstream signaling. We also provide direct evidence that bile acids possess tumor promoting capacity in epithelial cells overexpressing erbB2 using the two-stage skin carcinogenesis model. Collectively these findings suggest cooperative roles for bile acid and erbB2 activation in epithelial cell proliferation; bile acid appears to accelerate erbB2-induced pro-tumorigenic activities in the biliary tract and skin.

Pravastatin activates PPARα/PPARγ expression in the liver and gallbladder epithelium of hamsters

Our earlier study with cultured gallbladder epithelial cells demonstrated that statins (HMG-CoA reductase inhibitors) activate the expression of PPARalpha and PPARgamma, consequently blocking the production of pro-inflmmatory cytokines. The present study used hamsters to investigate the effects of pavastatin on PPARalpha/PPARgamma expression in the liver and gallbladder epithelium, and to determine whether pravastatin suppresses cholesterol crystal formation in the gallbladder. A total of 40 Golden Syrian male hamsters (4 weeks old) were randomly assigned to four groups (basal diet control; basal diet+pavastatin; high cholesterol diet; high cholesterol diet+pravastatin). All hamsters were 11 weeks old at the end of the experiment. The liver, gallbladder and bile were harvested. Immunohistochemical staining and Western blotting for PPARalpha and PPARgamma were performed in the liver and gallbladder. A drop of fresh bile was examined for cholesterol crystals under a microscope. In the gallbladder and liver of the hamsters, pravastatin activated the PPARalpha and PPARgamma expression of gallbladder epithelial cells and hepatocytes, and particularly the response of PPARgamma was much stronger than that of PPARalpha. Pravastatin suppressed the formation of cholesterol gallstones or crystals in the gallbladder. Pravastatin is an effective medication to activate PPARs (especially PPARgamma) in the liver and the gallbladder epithelium of hamsters, and contributes to the prevention of gallstone formation.

HMG-CoA Reductase Inhibitors (Statins) Activate Expression of PPARα/PPARγ and ABCA1 in Cultured Gallbladder Epithelial Cells

In gallbladder epithelial cells (GBEC), PPARalpha and PPARgamma ligands modulate inflammation by suppression of TNFalpha production and prevent excessive accumulation of cholesterol by ABCA1 activation. Recently, HMG-CoA reductase inhibitors (statins) were shown to activate PPARalpha and PPARgamma in various cells but no studies of their effects in GBEC have been conducted. The objective of this study was, therefore, to determine the effects of statins on PPAR and ABCA1 expression and the anti-inflammatory effect of statins in GBEC. Canine GBEC were cultured on Petri dishes. Expression of the proteins PPARalpha, PPARgamma, and ABCA1 was measured by western blotting analysis after treatment with simvastatin, pravastatin, NO-pravastatin, PPARalpha ligand, or PPARgamma ligand in the culture media. Expression of ABCA1 and LXRalpha mRNAs was estimated by RT-PCR. Expression of TNFalpha mRNA was measured by RT-PCR after 24 h pre-treatment with the statins, preceding 1 h of lipopolysaccharide (LPS) loading. Simvastatin, pravastatin, and NO-pravastatin increased expression of the proteins PPARalpha, PPARgamma, and ABCA1, and expression of the mRNA of ABCA1 and LXRalpha in GBEC. Pre-treatment with simvastatin, pravastatin, and NO-pravastatin suppressed the production of TNFalpha mRNA induced by LPS. In conclusion, statins probably contribute to the preservation of GBEC function by activation of PPARalpha and PPARgamma, which have anti-inflammatory effects by suppression of pro-inflammatory cytokines, and ABCA1 activation mediated by LXRalpha, which prevents the accumulation of cholesterol in GBEC.

The Effect of PPARα and PPARγ Ligands on Inflammation and ABCA1 Expression in Cultured Gallbladder Epithelial Cells

The preservation of gallbladder function by control of inflammation and elimination of cholesterol accumulation in gallbladder epithelial cells (GBEC) could contribute to the prevention of gallstone formation and cholecystitis. Peroxisome proliferator-activated receptors (PPARs) modulate inflammation and lipid metabolism in various cells and GBEC efflux of excessive amounts of absorbed cholesterol through the ATP-binding cassette transporter A1 (ABCA1)-mediated pathway. The aim of this study was to determine whether ligands of PPARalpha and PPARgamma modulate inflammation and have an effect on ABCA1 expression in GBEC. Canine GBEC were cultured on dishes coated with collagen matrix. We performed Western blot analysis for the expression of specific protein and/or RT-PCR for the expression of specific mRNA. PPARalpha and PPARgamma expression was observed and increased in GBEC treated with WY-14643 (PPARalpha ligand), troglitazone (PPARgamma ligand), and lipopolysaccharide (LPS) compared to the no-treatment control and PPARalpha( antagonist (GW-9662) treatment group. WY-14643, troglitazone, and LPS also induced an increase in the expression of ABCA1 protein and mRNA in cultured GBEC. LPS-induced TNFalpha mRNA expression was suppressed by pretreatment with WY-14643 and troglitazone preceding LPS treatment in GBEC. PPAR ligands, especially PPARgamma, may preserve gallbladder function by suppression of inflammatory reaction and prevention of cholesterol accumulation in GBEC, contributing to the prevention of gallstone formation and progression to cholecystitis.

Lipid peroxidation and mucin secretagogue activity in bile of gallstone patients

Chronic inflammation of the gallbladder wall and mucin hypersecretion are considered to be important factors in the pathogenesis of cholesterol gallstone disease. The aim of the study was to compare mucin concentration and mucin secretagogue activity with lipid peroxidation in gallbladder bile of patients with cholesterol or pigment stones. We studied mucin concentration and, as a marker of lipid peroxidation, malondialdehyde concentration in 11 rapid (1 to 3 days) and eight non-nucleating (> 21 days) gallbladder biles of patients with cholesterol or pigment stones. Furthermore, the mucin secretagogue activity of rapid and non-nucleating gallbladder biles, as well as 1-5 micromol L(-1) malondialdehyde on cultured gallbladder epithelial cells, was determined. Our data show an increased malondialdehyde (7.2 +/- 1.8 vs. 3.8 +/- 0.5 micromol L(-1), P = 0.01) and mucin concentration (0.9 +/- 0.09 vs. 0.41 +/- 0.03 mg mL(-1), P = 0.01) and an increased mucin secretagogue activity (2.0 +/- 0.5 vs. 1.1 +/- 0.3 mucin secretion/control, P = 0.04) and cholesterol saturation index (1.2 +/- 0.1 vs. 08 +/- 0.1, P = 0.04) in rapid as compared to non-nucleating gallbladder biles. Malondialdehyde stimulated mucin secretion of cultured gallbladder epithelial cells in a concentration dependent manner. Our results support a promoting effect of gallbladder mucin hypersecretion by lipid peroxidation leading to rapid formation of cholesterol crystals in gallbladder bile. These findings suggest that besides hypersecretion of cholesterol in bile, chronic inflammation of the gallbladder wall is implicated in the pathogenesis of cholesterol gallstone disease.

Agonists of protease-activated receptors 1 and 2 stimulate electrolyte secretion from mouse gallbladder

Cholecystitis is one of the most common gastrointestinal diseases. Inflammation induces the activation of proteases that can signal to cells by cleaving protease-activated receptors (PARs) to induce hemostasis, inflammation, pain, and repair. However, the distribution of PARs in the gallbladder is unknown, and their effects on gallbladder function have not been fully investigated. We localized immunoreactive PAR(1) and PAR(2) to the epithelium, muscle, and serosa of mouse gallbladder. mRNA transcripts corresponding to PAR(1) and PAR(2), but not PAR(4), were detected by RT-PCR and sequencing. Addition of thrombin and a PAR(1)-selective activating peptide (TFLLRN-NH(2)) to the serosal surface of mouse gallbladder mounted in an Ussing chamber stimulated an increase in short-circuit current in wild-type but not PAR(1) knockout mice. Similarly, serosally applied trypsin and PAR(2) activating peptide (SLIGRL-NH(2)) increased short-circuit current in wild-type but not PAR(2) knockout mice. Proteases and activating peptides strongly inhibited electrogenic responses to subsequent stimulation with the same agonist, indicating homologous desensitization. Removal of HCO(3)(-) ions from the serosal buffer reduced responses to thrombin and trypsin by >80%. Agonists of PAR(1) and PAR(2) increase intracellular Ca(2+) concentration in isolated and cultured gallbladder epithelial cells. The COX-2 inhibitor meloxicam and an inhibitor of CFTR prevented the stimulatory effect of PAR(1) but not PAR(2). Thus PAR(1) and PAR(2) are expressed in the epithelium of the mouse gallbladder, and serosally applied proteases cause a HCO(3)(-) secretion. The effects of PAR(1) but not PAR(2) depend on generation of prostaglandins and activation of CFTR. These mechanisms may markedly influence fluid and electrolyte secretion of the inflamed gallbladder when multiple proteases are generated.

Absence of CFTR is associated with pleiotropic effects on mucins in mouse gallbladder epithelial cells

Mucus of cystic fibrosis patients exhibits altered biochemical composition and biophysical behavior, but the causal relationships between altered cystic fibrosis transmembrane conductance regulator (CFTR) function and the abnormal mucus seen in various organ systems remain unclear. We used cultured gallbladder epithelial cells (GBEC) from wild-type and Cftr((-/-)) mice to investigate mucin gene and protein expression, kinetics of postexocytotic mucous granule content expansion, and biochemical and ionic compositions of secreted mucins. Muc1, Muc3, Muc4, Muc5ac, and Muc5b mRNA levels were significantly lower in Cftr((-/-)) GBEC compared with wild-type cells, whereas Muc2 mRNA levels were higher in Cftr((-/-)) cells. Quantitative immunoblotting demonstrated a trend toward lower MUC1, MUC2, MUC3, MUC5AC, and MUC5B mucin levels in Cftr((-/-)) cells compared with cells from wild-type mice. In contrast, the levels of secreted MUC1, MUC3, MUC5B, and MUC6 mucins were significantly higher from Cftr((-/-)) cells; a trend toward higher levels of secreted MUC2 and MUC5AC was also noted from Cftr((-/-)) cells. Cftr((-/-)) cells demonstrated slower postexocytotic mucous granule content expansion. Calcium concentration was significantly elevated in the mucous gel secreted by Cftr((-/-)) cells compared with wild-type cells. Secreted mucins from Cftr((-/-)) cells contained higher sulfate concentrations. Thus absence of CFTR is associated with pleiotropic effects on mucins in murine GBEC.

Cultured gallbladder epithelial cells synthesize apolipoproteins A-I and E.

Gallbladder epithelial cells (GBEC) are exposed to high and fluctuating concentrations of biliary cholesterol on their apical (AP) surface. GBEC absorb and efflux cholesterol, but the mechanisms of cholesterol uptake, intracellular trafficking, and efflux in these cells are not known. We previously reported that ATP binding cassette (ABC)A1 mediates basolateral (BL) cholesterol efflux in cultured polarized GBEC. In addition, the nuclear hormone receptors liver X receptor (LXR)alpha and retinoid X receptor (RXR) mediate both AP and BL cholesterol efflux. An interesting finding from our previous study was that apolipoprotein (apo)A-I applied to the AP surfaces of cells elicited BL ABCA1-mediated cholesterol efflux. Because ABCA1-mediated cholesterol efflux requires the presence of a cholesterol acceptor, we hypothesized that GBEC synthesize and secrete endogenous apo into the BL compartment. Here, we demonstrate that cholesterol loading of cells with model bile and AP apoA-I treatment is associated with an increase in the synthesis of apoE mRNA and protein. Furthermore, apoE is secreted into the BL compartment. LXRalpha/RXR ligands stimulate the synthesis of endogenous apoA-I mRNA and protein, as well as apoE mRNA. BL secretion of apoA-I is elicited by LXRalpha/RXR ligands. Therefore, GBEC synthesize apoA-I and -E and efflux cholesterol using ABCA1- and non-ABCA1- mediated pathways. These processes may alter gallbladder biliary cholesterol concentrations and thereby influence gallstone formation.

Cultured gallbladder epithelial cells synthesize apolipoproteins A-I and E

Cultured gallbladder epithelial cells synthesize apolipoproteins A-I and E

Regulation of electrogenic anion secretion in normal and cystic fibrosis gallbladder mucosa.

Fluid and ion transport across biliary epithelium contributes to bile flow. Alterations of this function may explain hepatobiliary complications in cystic fibrosis (CF). We investigated electrogenic anion transport across intact non-CF and CF human gallbladder mucosa in Ussing-type chambers. In non-CF tissues, baseline transmural potential difference (PD), short-circuit current (Isc), and resistance (R) were -2.2 +/- 0.3 mV (lumen negative), 40.7 +/- 7.8 microA/cm2, and 66.5 +/- 9.6 Omega. cm2, respectively (n = 14). The addition of forskolin (10(-5) mol/L) to the apical and basolateral baths and that of adenosine 5'-triphosphate (ATP) (10(-4) mol/L) to the apical bath induced significant increases in Isc by 8.0 +/- 1.4 and 10.3 +/- 1.8 microA/cm2, respectively. Depletion of bathing solutions in Cl- and HCO3- significantly reduced baseline Isc and the forskolin- and ATP-induced increases in Isc. Anion secretion was stimulated by extracellular ATP via P2Y2 purinoceptors, as indicated by the effects of different nucleotides on Isc and on 36Cl efflux in cultured gallbladder epithelial cells. This effect was mediated by cytosolic calcium increase and Ca2+/calmodulin-dependent protein kinase II, as ascertained by using inhibitors. In CF preparations, basal PD and Isc were lower than in non-CF, and the response to forskolin was abolished, whereas the response to ATP was enhanced (P <.05 for all). We conclude that electrogenic anion secretion occurs in human gallbladder mucosa under basal state and is stimulated by an adenosine 3',5'-cyclic monophosphate (cAMP)-dependent pathway mediated by cystic fibrosis transmembrane conductance regulator (CFTR), and by exogenous ATP via a CFTR-independent pathway that is up-regulated in CF and involves P2Y2 purinoceptors and a calcium-dependent pathway.

A delta F508 mutation in mouse cystic fibrosis transmembrane conductance regulator results in a temperature-sensitive processing defect in vivo.

The most prevalent mutation (delta F508) in cystic fibrosis patients inhibits maturation and transfer to the plasma membrane of the mutant cystic fibrosis transmembrane conductance regulator (CFTR). We have analyzed the properties of a delta F508 CFTR mouse model, which we described recently. We show that the mRNA levels of mutant CFTR are normal in all tissues examined. Therefore the reduced mRNA levels reported in two similar models may be related to their intronic transcription units. Maturation of mutant CFTR was greatly reduced in freshly excised oviduct, compared with normal. Accumulation of mutant CFTR antigen in the apical region of jejunum crypt enterocytes was not observed, in contrast to normal mice. In cultured gallbladder epithelial cells from delta F508 mice, CFTR chloride channel activity could be detected at only two percent of the normal frequency. However, in mutant cells that were grown at reduced temperature the channel frequency increased to over sixteen percent of the normal level at that temperature. The biophysical characteristics of the mutant channel were not significantly different from normal. In homozygous delta F508 mice we did not observe a significant effect of genetic background on the level of residual chloride channel activity, as determined by the size of the forskolin response in Ussing chamber experiments. Our data show that like its human homologue, mouse delta F508-CFTR is a temperature sensitive processing mutant. The delta F508 mouse is therefore a valid in vivo model of human delta F508-CFTR. It may help us to elucidate the processing pathways of complex membrane proteins. Moreover, it may facilitate the discovery of new approaches towards therapy of cystic fibrosis.

Mechanism of bile salt-induced mucin secretion by cultured dog gallbladder epithelial cells

1. Hypersecretion of gallbladder mucin has been proposed to be a pathogenic factor in cholesterol gallstone formation. Using cultured gallbladder epithelial cells, we demonstrated that bile salts regulate mucin secretion by the gallbladder epithelium. In the present study we have investigated whether established second messenger pathways are involved in bile salt-induced mucin secretion. 2. The effect of activators and inhibitors on mucin secretion was studied by measuring the secretion of [3H]N-acetyl-D-glucosamine-labelled glycoproteins. Intracellular cAMP content of the cells was measured using a radioimmunoassay. 3. Incubation of the cells with 10 mM taurocholate did not increase the intracellular cAMP content (25.7 versus control 22.8 pmol of cAMP/mg of protein). No stimulation of mucin secretion was observed after incubation with 1-100 microM concentrations of the calcium ionophores ionomycin and A23187. The stimulatory effect of 10 mM tauroursodeoxycholate (TUDC) on mucin secretion could not be inhibited by the addition of EDTA. Activation of protein kinase C (PKC) by 1 microgram/ml phorbol 12-myristate 13-acetate (PMA) caused an increase in mucin secretion (342% versus control 100%), comparable with the effect of 40 mM TUDC. The effect of 10 ng/ml PMA could partially be inhibited by a concentration of 2 microM of the PKC inhibitor staurosporin. Staurosporin had no inhibitory effect on mucin secretion induced by TUDC. 4. In gallbladder epithelial cells bile salts do not stimulate mucin secretion via one of the classical signal transduction pathways. We hypothesize that bile salts act on mucin secretion via a direct interaction with the apical membrane.