Differential modulation of cellular death and survival pathways by conjugated bile acids
© Torchia et al; licensee BioMed Central Ltd. 2001
Received: 10 September 2001
Accepted: 15 October 2001
Published: 15 October 2001
The liver-derived McNtcp.24 cells transport bile acids and show distinctive responses to the two classes of conjugated bile acids. Whereas taurine-conjugated bile acids are non-toxic, glycine-conjugated bile acids efficiently induce apoptosis. The aim of this study was to determine if the differential sensitivity is limited to cells that normally transport bile acids and if bile acid binding proteins could reduce bile acid-mediated apoptosis. The apical sodium/bile acid co-transporter (asbt) was expressed in Chinese hamster ovary (CHO) cells to establish active bile acid transport in a non-liver-derived cell model (CHO.asbt). A high-affinity bile acid binder was expressed in McNtcp.24 cells.
The tolerance of McNtcp.24 cells to taurine-conjugated bile acids was associated with the stimulation of phosphatidylinositol 3-kinase (PI3K) activity. Treatment of CHO.asbt cells with taurine- and glycine-conjugated bile acids resulted in apoptosis. Unlike in McNtcp.24 cells, PI3K activity was not increased in CHO.asbt cells treated with taurine-conjugated bile acids. High level expression of a bile acid binder did not attenuate bile acid-induced cytotoxicity in McNtcp.24 cells.
The data suggest that McNtcp.24 cells possess a mechanism that can elaborate distinctive responses to the different classes of bile acids. Additionally, activation of a signaling pathway involving PI3K appears to be the dominant mechanism responsible for the tolerance of McNtcp.24 cells to taurine-conjugated bile acids.
The liver exclusively synthesizes bile acids from cholesterol via two biosynthetic pathways. The "classical" pathway begins with the 7α-hydroxylation of cholesterol and operates entirely in the liver . The "alternative" pathway begins with the hydroxylation of the cholesterol side chain. Although this reaction occurs in a wide variety of cells, the completion of bile acid synthesis takes place in liver cells. The classical pathway is responsible for the bulk of the bile acids synthesized by the liver [2, 3].
The major bile acid species produced by the liver are cholic and chenodeoxycholic acids. These bile acids are conjugated to either taurine or glycine before secretion into bile , which is subsequently released into the small intestine. Bile acids are reclaimed from the lumen of the intestine and returned to the liver via portal blood for reuse . The recovery of bile acids from the intestine is an efficient process that involves both passive transport along the entire axis of the intestine, and active transport in the terminal ileum . The apical sodium/bile acid co-transporter (asbt) mediates the active uptake of conjugated bile acids in the ileum [5, 6]. In hepatocytes, both sodium-dependent and sodium-independent transport systems on the sinusoidal membranes extract bile acids from portal blood. Of these, the sodium/bile acid co-transporting polypeptide (ntcp) is considered to be quantitatively the most important transporter of conjugated bile acids in these cells [7, 8]. The transport of bile acids from the basolateral to the canalicular membrane of liver cells is a poorly understood process. Intracellular bile acid binding proteins have been postulated to participate in the transcellular transport as well as to buffer against the cytotoxic effects of bile acids. ATP-dependent transporters on the canalicular membranes of hepatocytes mediate the secretion of bile acids into bile .
Recently, it was discovered that bile acids are the natural ligands for a nuclear receptor termed farnesoid x receptor (FXR; NR1H4) [10–12]. Therefore, bile acids may be important regulators of gene expression in the liver and intestines. To date, the genes that have been shown to be responsive to regulation by FXR encode proteins involved in the biosynthesis and transport of bile acids . Bile acids have been shown to modulate a variety of other cellular functions, such as secretion of lipoproteins from hepatocytes [14, 15] and translocation of bile acid transporters to the hepatocyte canalicular membrane . In McNtcp cells, which are liver-derived cells engineered to transport bile acids actively , taurine-conjugated bile acids induce the formation of intracellular vesicles that resembles structures observed in cholestatic liver cells without apparent cellular cytotoxicity [1, 18]. In contrast, glycine-conjugated bile acids promote apoptosis in these cells . Thus, different classes of conjugated bile acids stimulate distinctive responses in McNtcp cells. Whereas taurine-conjugated bile acids are well tolerated, glycine-conjugated bile acids are extremely cytotoxic. Here we examined if this differential response is limited to cells that normally metabolize bile acids, and whether high level expression of an intracellular bile acid binding protein can attenuate the cytotoxic response of McNtcp.24 cells to glycine-conjugated bile acids.
Differential cytotoxicity of glycine-conjugated bile acids in McNtcp.24 cells
Expression of bile acid uptake activity in CHO cells
Cytotoxic potential of bile acids in CHO.asbt.35 cells
Mechanism of cell death
Cytotoxic potential of bile acids in HBAB-expressing McNtcp.24 cells
In this study, we determined if the differential sensitivity elaborated by the liver-derived McNtcp.24 cell line to different classes of conjugated bile acids is limited to cells that normally transport bile acids. We previously demonstrated that taurine-conjugated bile acids are not toxic to McNtcp.24 cells . In contrast, glycine-conjugated bile acids induce apoptosis in McNtcp.24 cells and primary mouse hepatocytes, through activation of the Fas receptor in a FasL-independent manner [19, 25]. Moreover, GCDCA was demonstrated to activate both initiator and effector caspases, and that the apoptotic response can be blocked by Crm A, a caspase 8 inhibitor . The mechanism by which bile acids stimulate the activation of Fas receptor remains unclear. In Fas deficient hepatocytes, toxic bile acids were still able to induce apoptosis, suggesting that bile acids are capable of stimulating multiple pathways to initiate cell death .
To gain further insight into the specificity of bile acid cytotoxicity, we conferred active bile acid uptake capacity in CHO cells, a cell type that does not normally metabolize bile acids, by expressing a bile acid transporter. CHO.asbt cells produce recombinant asbt with a molecular mass and kinetic parameters that are similar to previously reported values . It is not known if CHO cells have pathways capable of facilitating the efflux of bile acids out of the cell. The apparent efflux observed in CHO.asbt.35 could be attributed to the fact that asbt is capable a bidirectional transport of bile acids . However, it is conceivable that an efflux mechanism that recognizes bile acids exists in CHO cells and other mammalian cells. Indeed, yeast and plants express proteins capable of active bile acid transport . As these organisms do not normally synthesize bile acids, their ability to transport bile acids may illustrate a generalized mechanism for cellular transport of hydrophobic organic anions.
CHO.asbt.35 cells were used as a model to assess the cytotoxic potential of taurine- and glycine-conjugated bile acids in non-hepatic derived and non-bile acid metabolizing cells. Unlike McNtcp.24 cells, treatment of CHO.asbt clones with taurine-conjugated bile acids resulted in cell death. TCA was the most potent of taurine-conjugated bile acid tested. It is unlikely that the observed cytotoxicity of the conjugated bile acids could be attributed to the generalized intracellular accumulation of bile acids because unconjugated bile acids were transported by CHO.asbt cells but did not cause significant release of LDH into the culture medium (Torchia and Agellon, unpublished observations). In contrast, glycine-conjugated bile acids (GCDCA in particular) were highly effective at causing the increase in LDH activity in the culture medium of both McNtcp.24 and CHO.asbt.35 cells. Primary hepatocytes exhibit differential sensitivity to taurine- and glycine-conjugated bile acids in culture . The bile acid-transporting WIF-B cells, which are rat hepatoma-human fibroblast hybrids , remain viable even when grown in TCA-containing culture medium for up to 14 days . In contrast, our study shows that taurine-conjugated bile acids is toxic to a cell type that is not normally exposed to bile acids. Liver cells, which normally metabolize bile acids, apparently possess a mechanism that can distinguish and differentially elaborate distinct biological activities of taurine- and glycine-conjugated bile acids.
Both taurine- and glycine-conjugated bile acids induce apoptotic cell death in CHO.asbt.35 cells. The involvement of the caspase cascade in CHO.asbt.35 cells was illustrated by the fact that conjugated bile acids activated caspase 8 in a time dependent manner. However, the different profile of caspase 8 activation suggests that taurine- and glycine-conjugated bile acids may trigger apoptosis by different pathways. At present, it is not known how conjugated bile acids activate caspases in CHO.asbt.35 cells. A protein that is immunoreactive to a Fas receptor antiserum and has a molecular mass similar to the rat Fas receptor is detectable in CHO cell extracts (Torchia and Agellon, unpublished results). This leaves the possibility that conjugated bile acids may activate the apoptotic cascade through Fas receptor in this cell line, as glycine-conjugated bile acids do in hepatocytes . However, the activation of caspases does not require a specific kind of bile acid transporter since TCA is not toxic to McArdle RH-7777 cells irrespective of whether these cells internalize bile acids via ntcp or asbt.
The differential response of McNtcp.24 cells to different classes of conjugated bile acids is apparently due to the selective activation of either a PI3K-dependent survival pathway [22, 23] or caspase-dependent death pathway . Indeed, neither taurine- nor glycine-conjugated bile acids stimulated PI3K activity in CHO.asbt.35 cells and both classes of bile acids were toxic. In addition, pre-treatment of McNtcp.24 cells with a PI3K inhibitor rendered these cells susceptible to TCA- and TCDCA- mediated cell death. The latter finding indicates that taurine-conjugated bile acids are capable of inducing apoptosis when PI3K is inhibited, probably by utilizing the same mechanism activated by glycine-conjugated bile acids. That TCA is able to activate PI3K in plasma membrane fractions from McNtpc.24 agrees with a previous study showing that TCA increases PI3K activity in canalicular and sinusoidal membrane vesicles . The activation of PI3K in that report was also associated with recruitment of sister of P-glycoprotein (the bile acid export pump) and other multidrug resistance proteins to the canalicular membrane . Extended treatment of McNtcp.24 cells does not appear to have detrimental and permanent consequences on cellular function. The marked morphological changes that occur during long term incubation with TCA  dissipate after the withdrawal of the bile acid from the culture medium, and the cells remain viable.
The significance of an intracellular and high affinity bile acid binder in bile acid mediated cytotoxicity was also evaluated. Several proteins capable of binding bile acids in vitro have been identified (reviewed in ref. ). It is thought that the major bile acid binding protein in the cytosol of rat hepatocytes is 3α-hydroxysteroid dehydrogenase, a member of the aldo keto reductase supergene family . However, the exact role of this protein in intracellular transport of bile acids and cytoprotection was undefined. HBAB, a member of the human aldo keto reductase gene family, has been shown to bind bile acids with high affinity (<1 μM) and is postulated to play a similar role as 3α-hydroxysteroid dehydrogenase in human hepatocytes . Expression of HBAB in McNtcp.24 cells was expected to increase the capacity of these cells to sequester bile acids in the cytoplasm, and thereby reduce their toxicity. High level expression of HBAB had a negligible effect on the ability of the BN clones to take up or secrete bile acids, suggesting that HBAB has a minor role in the net transport of bile acids in these cells. More importantly however, expression of HBAB in McNtcp.24 cells had no effect on GCDCA-induced cell death, even at low bile acid concentrations, indicating that this class of protein does not provide cytoprotection against toxic bile acids as previously suspected. This finding is consistent with the idea that bile acids directly modulate specific signaling cascades that control cellular death and survival.
In summary, the failure of CHO.asbt cells to tolerate taurine-conjugated bile acids has revealed the existence of a mechanism that may be specific for cells that normally metabolize bile acids. Since bile acids undergo enterohepatic circulation, it would be reasonable to expect the existence of the same mechanism in other cell types that naturally transport bile acids, such as ileocytes and cholangiocytes [34, 35]. The last few years have brought significant advances in our understanding of bile acid transport in liver and intestinal cells. However, the regulatory potential of bile acids is only beginning to be realized. With the recent discovery that bile acids represent the natural ligands for the nuclear receptor FXR [10–12], there is heightened interest in identifying the genes that are regulated by bile acids. Insight into how bile acids directly modulate a variety of other cellular functions will be important in understanding the significance of cellular responses to bile acids.
Materials and Methods
Tissue culture reagents were purchased from Canadian Life Technologies (Burlington, ON, Canada). [3H]-Taurocholic acid (2.6 Ci/mmol), [32P]-dCTP (3000 Ci/mmol), and [32P]-ATP (3000 Ci/mmol) were purchased from Amersham Pharmacia Biotech (Baie d'Urfe, QC, Canada). Bile acids were obtained from Sigma-Aldrich Canada Ltd. (Oakville, ON, Canada) and Calbiochem-NovaBiochem Corp. (San Diego, CA, USA). Other reagents purchased from various commercial suppliers were of analytical grade.
McNtcp.24 cells were cultured as described previously . Chinese hamster ovary K1 cells were cultured in Ham's F12 medium supplemented with 10% fetal bovine serum. To establish cell lines stably expressing asbt, a cDNA encoding the hamster asbt  was cloned into pBK-CMV expression vector (Stratagene, La Jolla, CA). The prokaryotic promoter embedded in the 5'-untranslated region of the encoded recombinant eukaryotic mRNA was removed from the resulting plasmid. The human bile acid binder (HBAB) cDNA  was cloned into a derivative of pCep4 (Invitrogen Corp., Carlsbad, CA), that had been previously modified by deleting the EBNA 1 gene . The lipofectamine reagent (Canadian Life Technologies) was used to transfect the expression plasmid into CHO, McArdle RH7777, and McNtcp.24 cells. The cell medium was replaced 24 h post-transfection with medium containing G418 (400 μg/ml) or hygromycin (500 μg/ml) to select for stable transfectants. Surviving colonies were expanded and assayed for the ability to take up [3H]-TCA from the culture medium as described previously  or assayed for the expression of HBAB by immunoblotting.
RNA and immunoblot analyses
Total RNA from CHO.asbt and BN cells was prepared and analyzed by RNA blotting as previously described . Cell lysates were prepared by lysing cells in 50 mM Tris-HCl (pH 7.4), 1% NP-40%, 0.25% sodium deoxycholate; 150 mM NaCl, 1 mM EGTA, 1 mM Na3VO4, 1 mM sodium fluoride, containing a protease inhibitor cocktail (Sigma-Aldrich Canada Ltd.). The lysates were fractionated by polyacrylamide gel electrophoresis and then transferred to Immobilon-P membrane (Millipore Ltd., Nepean, ON). Asbt was detected using a rabbit antiserum directed against the last 14 amino acids of the carboxyl terminal of the hamster asbt protein. HBAB protein was detected as previously described .
Bile acid uptake activity assay
Lactate dehydrogenase (LDH) assay
Cells (104 per well) were grown on 24 well multiwell plates for 16 h. The cells were then washed with phosphate buffered saline (PBS) and then incubated in medium (Ham's F12 or Dulbecco's modified Eagle's medium (DMEM)) containing 100 μM bile acids for 1 to 5 h as detailed in the Figure legends. The media was collected and adjusted to 0.1% Triton X-100. Cells remaining on the dish were dissolved in medium (Ham's F12 or DMEM) containing 0.1% Triton X-100. LDH activities in both the cell lysates and media were measured as described previously .
Visualization of genomic DNA fragmentation
Cells (106 per 60 mm dish) were grown for 16 h. After washing with PBS, the cells were incubated with 50 μM bile acids for 3 h at 37°C. Adherent and non-adherent cells were dissolved in Tris-buffered saline (pH 7.6) containing 10 mM EDTA and 0.5% sarkosyl, and 1 mg/ml proteinase K. After digestion (~16 h) of cellular proteins at 55°C, DNA was sequentially extracted with phenol/chloroform and chloroform. The DNA was precipitated, washed in 70% ethanol, and dissolved in buffer containing 10 mM Tris-HCL (pH 7.5) and 1 mM EDTA. The isolated DNA was treated with RNase A and then analyzed by conventional agarose gel electrophoresis . DNA was visualized with ultraviolet light after ethidium bromide staining.
Caspase activity assays
Cells (106 per 60 mm dish) were grown for 16 h. After washing with PBS, the cells were incubated with 50 to 100 μM bile acids for 1 to 2 h. After the incubation period, the medium was discarded and adherent cells were harvested in PBS and sedimented by centrifugation (500 × g). The cells were resuspended in buffer containing 50 mM Tris-HCl (pH 7.5), 1% Nonidet P-40, and 150 mM NaCl. Following 10 min incubation on ice, lysed cells were centrifuged for 10 min, 10,000 × g at 4°C. The supernatant was assayed for caspase activity in 20 mM Piperazine-N,N'-bis(2-ethanesulfonic acid) (pH 6.8), 100 mM NaCl, 1 mM EDTA, 1% CHAPS, and 10% sucrose with Z-IETD-AFC for caspase 8 [42, 43]. The release of the fluorogenic-leaving group was detected by excitation at 400 nm and emission at 505 nm. Free AFC was used to generate standard curve.
PI3K activity assay
Crude membrane fractions were isolated from CHO.asbt and McNtcp.24 cells as described previously . To prepare lipid suspension, phosphatidylserine and phosphatidylinositol (1:1) were sonicated in 25 mM 3-(N-morpholino)propanesulfonic acid (MOPS; pH 7.0), 1 mM EGTA, 1 mM Na3VO4. Cell extracts were assayed in 25 mM MOPS (pH 7.5), 5 mM MgCl2, 1 mM EGTA, 1 mM Na3VO4, 130 μM ATP, 20 μCi [32P]-ATP (3000 Ci/mmol) and 0.2 mg/ml sonicated lipids. The reaction was allowed to proceed for 10 min at 37°C and stopped by addition of 300 μl of 1N HCl:methanol (1:1). Lipids were extracted twice with 350 μl chloroform and the organic phases from both extractions were combined then washed once with 700 μl 1 N HCl/methanol (1:1). Lipids were dried, and then taken up in chloroform:methanol (1:1). The samples were spotted along with pure phosphoinositol phosphate standards (Avanti Polar Lipids, Alabaster, AL, USA) onto 1.2% potassium oxalate impregnated TLC plates . The chromatogram was developed in chloroform: methanol: water: aqueous ammonia (90:70:17:3) . The incorporation of [32P] into phosphatidylinositol was quantitated by phosphorimaging using a Fuji BAS1000 phosphorimager. Visualization of standards was done by dippingTLC plates in 10% CuSO4,10% H3PO4 and heating at 200°C for 10 min.
Each set of experiments was repeated at least twice. The data is represented by means ± SD. Differences between control and test cells were evaluated by a Student's t test. Differences in the slope of regression lines were evaluated by analysis of variance. P values of <0.05 were considered statistically significant.
List of abbreviations used
apical sodium/bile acid cotransporter
human bile acid binder
taurocholic acid, TCDCA, taurochenodeoxycholic acid
This research was supported by grants from the Canadian Institutes of Health Research, the Heart and Stroke Foundation of Alberta & NWT, and in part by Novartis Pharmaceuticals Canada Ltd. ECT was supported by a Studentship from the Alberta Heritage Foundation for Medical Research. LBA is a Senior Medical Scholar of the Alberta Heritage Foundation for Medical Research.
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