Bile-acid-induced calcium signaling in mouse esophageal epithelial cells

In patients with gastroesophageal reflux disease (GERD), esophageal exposure to both acid and duodenogastroesophageal reflux are more common than to acid reflux alone, suggesting that acidic bile acid in duodenal juice may contribute to the pathophysiology and severity of GERD. However, the mechanism whereby esophageal mucosal epithelial cells react to bile acid remains unclear. We visually examined the real-time response of mouse esophageal epithelial cells to bile acids using calcium (Ca²⁺)-imaging methods. We investigated the effects of seven different bile acids. After stimulation for a few minutes, only Deoxycholate (DCA) under acidic conditions caused a elevation of intracellular Ca²⁺ concentration ([Ca²⁺]_i)in the cells in dose- and pH-dependent manners. Conjugated bile acids had no effect on the cells. Viability assay of the cells in the presence of DCA was in good agreement with the calcium imaging data. Besides, DCA-induced [Ca²⁺]_i increase in acidic conditions was observed not only in isolated primary cultured cells, but also in cells in the stratified squamous epithelium. This study suggests that DCA can pass through the anatomical barrier of the esophageal epithelium and induce calcium signaling in epithelial cells in a pH-dependent manner. This supports the hypothesis that bile acid reflux together with gastric acid can affect the esophageal mucosa, even under reflux times of a few minutes.     

Chemical synthesis of 24-beta-D-galactopyranosides of bile acids: a new type of bile acid conjugates in human urine

A method is reported for the preparation of the C-24 carboxyl-linked beta-D-galactopyranosides of lithocholic, deoxycholic, chenodeoxycholic, ursodeoxycholic, and cholic acids, two of which were recently identified as a novel type of the metabolites of bile acids excreted in human urine. Direct esterification (galactosidation) of the unprotected bile acids with 2,3,4,6-tetra-O-benzyl-D-galactopyranose in the presence of 2-chloro-1,3,5-trinitrobenzene as a coupling agent and subsequent hydrogenolysis of the resulting benzyloxy-protected bile acid 24-beta-D-galactopyranosides over 10% palladium on charcoal under atmospheric pressure afforded the title compounds. The structures of the bile acid acyl galactosides were confirmed by measuring several (1)H-(1)H and (1)H-(13)C shift correlated 2D NMR.     

Atorvastatin reduces lipopolysaccharide-induced expression of cyclooxygenase-2 in human pulmonary epithelial cells

Objective

To explore the effects of atorvastatin on expression of cyclooxygenase-2 (COX-2) in human pulmonary epithelial cells (A549).

Methods

A549 cells were incubated in DMEM medium containing lipopolysaccharide (LPS) in the presence or absence of atorvastatin. After incubation, the medium was collected and the amount of prostaglandin E₂ (PGE₂) was measured by enzyme-linked immunosorbent assay (ELISA). The cells were harvested, and COX-2 mRNA and protein were analyzed by RT-PCR and western-blot respectively.

Results

LPS increased the expression of COX-2 mRNA and production of PGE₂ in a dose- and time-dependent manner in A549. Induction of COX-2 mRNA and protein by LPS were inhibited by atorvastatin in a dose-dependent manner. Atorvastatin also significantly decreased LPS-induced production of PGE₂. There was a positive correlation between reduced of COX-2 mRNA and decreased of PGE₂ (r = 0.947, P < 0.05).

Conclusion

Atorvastatin down-regulates LPS-induced expression of the COX-2 and consequently inhibits production of PGE₂ in cultured A549 cells.     

The enhancement of the human immunoglobulin G Fc fragment-binding activity of Mycoplasma salivarium cells by trypsin treatment is ascribed to the binding of trypsin to the Fc fragment

Abstract Human immunoglobulin G Fc fragment-binding activity of Mycoplasma salivarium cells was remarkably enhanced by trypsin treatment of the cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis profile of proteins of the cells treated wtrypsin was the same as that of the cells treated with pronase, although pronase treatment had been shown to reduce the activity in our previous study (FEMS Microbiol. Lett. 123, 305–310, 1994). This contradiction was clarified by the finding that trypsin bound the Fc fragment more strongly than the cells, and a small amount of trypsin remained in the cells treated with trypsin and washed well. On the basis of these results, it was concluded that the enhancement of cell activity by trypsin treatment was ascribed to binding of the Fc fragment to trypsin remaining in the trypsin-treated cells.     

Synthesis of tenascin and laminin beta2 chain in human bronchial epithelial cells is enhanced by cysteinyl leukotrienes via CysLT1 receptor

Background

Cysteinyl leukotrienes (CysLTs) are key mediators of asthma, but their role in the genesis of airway remodeling is insufficiently understood. Recent evidence suggests that increased expression of tenascin (Tn) and laminin (Ln) β2 chain is indicative of the remodeling activity in asthma, but represents also an example of deposition of extracellular matrix, which affects the airway wall compliance. We tested the hypothesis that CysLTs affect production of Tn and Ln β2 chain by human bronchial epithelial cells and elucidated, which of the CysLT receptors, CysLT₁ or CysLT₂, mediate this effect.

Methods

Cultured BEAS-2B human bronchial epithelial cells were stimulated with leukotriene D₄ (LTD₄) and E₄ (LTE₄) and evaluated by immunocytochemistry, Western blotting, flow cytometry, and RT-PCR. CysLT receptors were differentially blocked with use of montelukast or BAY u9773.

Results

LTD₄ and LTE₄ significantly augmented the expression of Tn, whereas LTD₄, distinctly from LTE₄, was able to increase also the Ln β2 chain. Although the expression of CysLT₂ prevailed over that of CysLT₁, the up-regulation of Tn and Ln β2 chain by CysLTs was completely blocked by the CysLT₁-selective antagonist montelukast with no difference between montelukast and the dual antagonist BAY u9773 for the inhibitory capacity.

Conclusion

These findings suggest that the CysLT-induced up-regulation of Tn and Ln β2 chain, an important epithelium-linked aspect of airway remodeling, is mediated predominantly by the CysLT₁ receptor. The results provide a novel aspect to support the use of CysLT₁ receptor antagonists in the anti-remodeling treatment of asthma.     

Antiproliferation and apoptosis induced by tamoxifen in human bile duct carcinoma QBC939 cells via upregulated p53 expression

Tamoxifen (TAM) is a nonsteroidal antiestrogen that has been used in the treatment of breast cancer for over 30 years. Recently, it was shown that TAM also has efficacy on gastrointestinal neoplasms such as hepatocarcinoma and pancreatic carcinoma, and that the chemopreventive activities of TAM might be due to its abilities to inhibit cell growth and induce apoptosis. In the present study, we investigated the effects of tamoxifen on growth and apoptosis in the human bile duct carcinoma (BDC) cell line QBC939 using MTT assay, inverted microscopy, fluorescence microscopy, transmission electron microscopy, classic DNA fragmentation agarose gel electrophoresis assay, PI single- and FITC/PI double-staining flow cytometry, and Western blotting. Our data revealed that TAM could significantly inhibit growth and induce apoptosis in QBC939 cells. Increased expression of p53 was observed in TAM-treated cells, indicating that p53 might play an important role in TAM-induced apoptosis in QBC939 cells. These results provide significant insight into the anticarcinogenic action of TAM on BDC.     

Measurement of bile acid in serum and bile with arylamine-glass-bound 3alpha-hydroxysteroid dehydrogenase and diaphorase

3Alpha-hydroxysteroid dehydrogenase (3-HSD) from Pseudomonas testosteroni and diaphorase (lipoyl dehydrogenase) from Clostridium spp. have been immobilized individually onto arylamine glass beads through diazotization. A cost-effective enzymic colorimetric method for determination of bile acid in serum and bile employing a mixture of these immobilized enzymes was developed. The method is based on measurement of reduced nicotinamide adenine dinucleotide generated from bile acid in serum/bile by immobilized 3alpha-HSD with a color reagent consisting of nitro blue tetrazolium chloride salt, oxidized nicotinamide adenine dinucleotide, and immobilized lipoyl dehydrogenase in 0.065 M sodium phosphate buffer, pH 7.0. Analytical recovery of added bile acid (50 and 200 micromol/L) was 95.57 and 85.46% in serum and 97.6 and 91.6% in bile, respectively. Within- and between-batch coefficients of variation (CV) for bile acid determination were <1.2 and <0.2% in serum and >0.1 and <0.1% in bile, respectively. Good correlations for bile acid in serum (r1=0.92) and in bile (r2=0.97) were obtained by use of a standard chemical method and the present method. The mixture of immobilized 3alpha-HSD dehydrogenase and lipoyl dehydrogenase lost 50% of its initial activity after 6 months of regular use. The cost of bile acid determination in 100 serum and bile samples by the present method has been compared with that of the Sigma kit method.     

Morphogenesis of human endothelial cells is inhibited by DAB2 via Src

Disabled-2 (DAB2) is an adaptor protein implicated in signal transduction pathways and in protein traffic regulation. Here, we show that DAB2 is highly expressed in human endothelial cells. DAB2 silencing in endothelial cells by lentiviral-mediated small hairpin RNA expression affects cell migration and differentiation into capillary-like structures while increasing cell proliferation and viability. DAB2 knockdown causes activation of the Src-FAK signal pathway, extracellular-signal regulated kinase and c-Jun NH2-terminal kinase activation, and inhibition of p38 phosphorylation. In DAB2 silenced endothelial cells, pharmacological inhibition of Src with its specific inhibitor PP2 abolishes focal adhesion kinase activation and restores differentiation of endothelial cells. These results suggest that DAB2, via Src and focal adhesion signaling, plays a role in human endothelial cell function.     

Bile acid N-acetylglucosaminides. Formation by microsomal N-acetylglucosaminyltransferases in human liver and kidney

Bile acid N-acetylglucosaminyltransferase activity has been identified in microsomes from human liver and kidney. In both organs the transferases required UDP-N-aeetylglucosamine as sugar donor and were mainly active towards ursodeoxycholic acid. Minor activities were observed towards amidated ursodeoxycholic, hyodeoxycholic and β-muricholic acids. No N-acetylglucosaminidation was detectable with the major primary and secondary bile acids suggesting a specific requirement of the enzymes for bile acids containing 7β- or 6-hydroxyl groups. Kinetic parameters and other catalytic properties of liver and kidney microsomal N-acetylglucosaminyltransferase activities towards ursodeoxycholic acid are described.     

Increased cyclooxygenase-2 and thromboxane synthase expression is implicated in diosgenin-induced megakaryocytic differentiation in human erythroleukemia cells

Differentiation induction as a therapeutic strategy has, so far, the greatest impact in hematopoietic malignancies, most notably leukemia. Diosgenin is a very interesting natural product because, depending on the specific dose used, its biological effect is very different in HEL (human erythroleukemia) cells. For example, at 10 μM, diosgenin induced megakaryocytic differentiation, in contrast to 40 μM diosgenin, which induced apoptosis in HEL cells previously demonstrated using sedimentation field-flow fractionation (SdFFF). The goal of this work focused on the correlation between cyclooxygenase-2 (COX-2) and thromboxane synthase (TxS) and megakaryocytic differentiation induced by diosgenin in HEL cells. Furthermore, the technique of SdFFF, having been validated in our models, was used in this new study as an analytical tool that provided us with more or less enriched differentiated cell fractions that could then be used for further analyses of enzyme protein expression and activity for the first time. In our study, we showed the implication of COX-2 and TxS in diosgenin-induced megakaryocytic differentiation in HEL cells. Furthermore, we showed that the analytical technique of SdFFF may be used as a tool to confirm our results as a function of the degree of cell differentiation.     

Inhibition of butyrate uptake by the primary bile salt chenodeoxycholic acid in intestinal epithelial cells

Colorectal cancer (CRC) is one of the most common cancers worldwide. Epidemiological and experimental studies suggest that bile acids may play a role in CRC etiology. Our aim was to characterize the effect of the primary bile acid chenodeoxycholic acid (CDCA) upon(14) C-BT uptake in tumoral (Caco-2) and non-tumoral (IEC-6) intestinal epithelial cell lines. A 2-day exposure to CDCA markedly and concentration-dependently inhibited (14) C-BT uptake by IEC-6 cells (IC(50) = 120 μM), and, less potently, by Caco-2 cells (IC(50) = 402 μM). The inhibitory effect of CDCA upon (14) C-BT uptake did not result from a decrease in cell proliferation or viability. In IEC-6 cells: (1) uptake of (14) C-BT involves both a high-affinity and a low-affinity transporter, and CDCA acted as a competitive inhibitor of the high-affinity transporter; (2) CDCA inhibited both Na(+)-coupled monocarboxylate cotransporter 1 (SMCT1)- and H(+)-coupled monocarboxylate transporter 1 (MCT1)-mediated uptake of (14) C-BT; (3) CDCA significantly increased the mRNA expression level of SMCT1; (4) inhibition of (14) C-BT uptake by CDCA was dependent on CaM, MAP kinase (ERK1/2 and p38 pathways), and PKC activation, and reduced by a reactive oxygen species scavenger. Finally, BT (5 mM) decreased IEC-6 cell viability and increased IEC-6 cell differentiation, and CDCA (100 μM) reduced this effect. In conclusion, CDCA is an effective inhibitor of (14) C-BT uptake in tumoral and non-tumoral intestinal epithelial cells, through inhibition of both H(+) -coupled MCT1- and SMCT1-mediated transport. Given the role played by BT in the intestine, this mechanism may contribute to the procarcinogenic effect of CDCA at this level.     

The effect of ascorbic acid on Helicobacter pylori induced cyclooxygenase 2 expression and prostaglandin E2 production by gastric epithelial cells in vitro

BACKGROUND: Cyclooxygenase 2 (COX-2) is induced by the presence of Helicobacter pylori (H. pylori) on the gastric mucosa as part of the inflammatory response; this results in the synthesis of prostaglandins that amplify the local inflammatory response. The presence of H. pylori inhibits the secretion of ascorbate into the gastric lumen. Interestingly, ascorbate inhibits the growth of H. pylori and low dietary levels are associated with an increased risk of gastric adenocarcinoma. We therefore investigated the effect of ascorbate on H. pylori mediated COX-2 induction and prostaglandin production in vitro. METHODS: H. pylori was cocultured with gastric epithelial cells in the presence of ascorbate at physiological concentrations. The expression of COX-2 was assessed by Western blotting and prostaglandin E(2) (PGE(2)) was assessed by ELISA. RESULTS: Ascorbate inhibited gastric cell PGE(2) synthesis but not in COX-2 expression in response to H. pylori. In the absence of the organism, ascorbate also reduced PGE(2) expression in cells that constitutively express COX-2, again with no reduction of COX-2 protein expression. CONCLUSIONS: Physiological concentrations of ascorbate inhibit PGE(2) but not COX-2 expression in response to H. pylori in gastric epithelial cells.     

Mitochondrial genome depletion in human liver cells abolishes bile acid-induced apoptosis: Role of the Akt/mTOR survival pathway and Bcl-2 family proteins

Acute accumulation of bile acids in hepatocytes may cause cell death. However, during long-term exposure due to prolonged cholestasis, hepatocytes may develop a certain degree of chemoresistance to these compounds. Because mitochondrial adaptation to persistent oxidative stress may be involved in this process, here we have investigated the effects of complete mitochondrial genome depletion on the response to bile acid-induced hepatocellular injury. A subline (Rho) of human hepatoma SK-Hep-1 cells totally depleted of mitochondrial DNA (mtDNA) was obtained, and bile acid-induced concentration-dependent activation of apoptosis/necrosis and survival signaling pathways was studied. In the absence of changes in intracellular ATP content, Rho cells were highly resistant to bile acid-induced apoptosis and partially resistant to bile acid-induced necrosis. In Rho cells, both basal and bile acid-induced generation of reactive oxygen species (ROS), such as hydrogen peroxide and superoxide anion, was decreased. Bile acid-induced proapoptotic signals were also decreased, as evidenced by a reduction in the expression ratios Bax-α/Bcl-2, Bcl-xS/Bcl-2, and Bcl-xS/Bcl-xL. This was mainly due to a downregulation of Bax-α and Bcl-xS. Moreover, in these cells the Akt/mTOR pathway was constitutively activated in a ROS-independent manner and remained similarly activated in the presence of bile acid treatment. In contrast, ERK1/2 activation was constitutively reduced and was not activated by incubation with bile acids. In conclusion, these results suggest that impaired mitochondrial function associated with mtDNA alterations, which may occur in liver cells during prolonged cholestasis, may activate mechanisms of cell survival accounting for an enhanced resistance of hepatocytes to bile acid-induced apoptosis.     

Nicotinic acid suppresses sebaceous lipogenesis of human sebocytes via activating hydroxycarboxylic acid receptor 2 (HCA2)

Nicotinic acid (NA) activates hydroxycarboxylic acid receptor 2 (HCA₂), and it is widely used in treating dyslipidaemias. Since its side effects include skin dryness, whereas its deficiency can be accompanied by dyssebacia, characterized by sebaceous gland enlargement, we asked if HCA₂ is expressed on human sebocytes, and if NA influences sebocyte functions. By using human immortalized SZ95 sebocytes, we found that non‐cytotoxic (≤100 μmol/L; MTT‐assay) concentrations of NA had no effect on the homeostatic sebaceous lipogenesis (SLG; Nile Red), but normalized excessive, acne‐mimicking SLG induced by several lipogenic agents (arachidonic acid, anandamide, linoleic acid + testosterone; Nile Red; 48‐hr treatments). Moreover, it exerted significant anti‐proliferative actions (CyQUANT‐assay), and increased [Ca²⁺]_IC (Fluo‐4 AM‐based Ca²⁺‐measurement). Although NA did not prevent the lipopolysaccharide‐induced pro‐inflammatory response (up‐regulation [Q‐PCR] and release [ELISA] of several pro‐inflammatory cytokines) of the sebocytes, collectively, these data support the concept that NA may be effective in suppressing sebum production in vivo. While exploring the mechanism of the sebostatic actions, we found that sebocytes express HCA₂ (Q‐PCR, immunofluorescent labelling), siRNA‐mediated silencing of which prevented the NA‐induced Ca²⁺‐signal and the lipostatic action. Collectively, our data introduce NA, and HCA₂ activators in general, as novel, potent and most likely safe sebostatic agents, with possible anti‐acne potential.     

Bile acid N-acetylglucosaminides: Formation by microsomal N-acetylglucosaminyltransferases in human liver and kidney

Bile acid N-acetylglucosaminyltransferase activity has been identified in microsomes from human liver and kidney. In both organs the transferases required UDP-N-aeetylglucosamine as sugar donor and were mainly active towards ursodeoxycholic acid. Minor activities were observed towards amidated ursodeoxycholic, hyodeoxycholic and β-muricholic acids. No N-acetylglucosaminidation was detectable with the major primary and secondary bile acids suggesting a specific requirement of the enzymes for bile acids containing 7β- or 6α-hydroxyl groups. Kinetic parameters and other catalytic properties of liver and kidney microsomal N-acetylglucosaminyltransferase activities towards ursodeoxycholic acid are described.