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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Several studies have argued that G-protein–coupled receptors (GPCR) have the capacity to promote activation of receptor tyrosine kinases. The current studies were performed to examine the regulation of the extracellular regulated kinase (ERK)1/2 and AKT pathways by conjugated and unconjugated bile acids in primary hepatocytes. Deoxycholic acid (DCA), chenodeoxycholic acid (CDCA), taurodeoxycholic acid (TDCA), glycodeoxycholic acid (GDCA), taurochenodeoxycholic acid (TCDCA), glycochenodeoxycholic acid (GCDCA), taurocholic acid (TCA), glycocholic acid (GCA), and tauroursodeoxycholic acid (TUDCA) all activated ERK1/2 in primary rat hepatocytes that was abolished by inhibition of ERBB1, and significantly reduced by ROS quenching agents. Bile acid–induced AKT activation was blunted by preventing ERBB1 activation and ROS generation. Treatment of rat hepatocytes with pertussis toxin (PTX) did not alter ERK1/2 and AKT activation induced by DCA or CDCA but abolished pathway activations by conjugated bile acids. Similar data to those with PTX were obtained when a dominant negative form of Gi was overexpressed. Treatment of rat hepatocytes with TDCA and TCA promoted guanosine triphosphate (GTP) loading of Gi, Gi, and Giin vitro. Treatment of rat hepatocytes with PTX abolished TDCA-induced tyrosine phosphorylation of ERBB1. Similar findings to those in rat hepatocytes were also obtained in primary mouse and human hepatocytes, but not in established rodent or human hepatoma cell lines. In conclusion, collectively our findings demonstrate that unconjugated bile acids activate hepatocyte receptor tyrosine kinases and intracellular signaling pathways in a ROS-dependent manner. In contrast, conjugated bile acids primarily activate receptor tyrosine kinases and intracellular signaling pathways in a GPCR (Giα) –dependent and ROS-dependent manner. (HEPATOLOGY 2005;42:1291–1299.)

Bile acids are detergent molecules, synthesized from cholesterol in the liver, actively secreted into bile, stored in the gallbladder, and released into the gut on feeding. After feeding, bile acids re-enter the liver via the portal vein together with digested nutrients and are re-circulated back into the gall bladder for use during the next feeding cycle.1 The levels of individual bile acids and their conjugation status relative to glycine or taurine changes between individuals based on diet and age2, 3: individuals eating a meat diet tend to have more taurine-conjugated bile acids in their bile acid pool than persons eating a vegetarian diet. Individually, when retained within the liver because of impaired secretion into the bile canaliculi, bile acids are also known to have hepatocellular-toxicity both in vivo and in vitro.4

Recently, we reported that treatment of primary rodent or human hepatocytes with deoxycholic acid (DCA) caused ligand-independent activation of ERBB1 (EGF receptor), which was responsible for activation of the ERK1/2 and AKT pathways.5–7 In addition to ERBB1, bile acids also have been shown to induce ligand-independent activation of the insulin receptor, but not the insulin-like growth factor 1 receptor, in primary rodent hepatocytes.8 Activation of the phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) pathway by DCA was more strongly linked to activation of the insulin receptor than to ERBB1. DCA-induced insulin receptor/PI3K/AKT signaling was linked to activation of glycogen synthase and a strong cytoprotective signal against activation of death receptors and mitochondrial dysfunction. Reactive oxygen species (ROS) scavengers and inhibitors of mitochondrial ROS generation blocked DCA-induced inhibition of protein tyrosine phosphatase (SHP-1) activity as well as activation of ERBB1 and the insulin receptor; several studies by other laboratories have argued that protein tyrosine phosphatase activity is sensitive to cellular redox status.9, 10 Blockade of DCA-induced ERK1/2 or PI3K activation, with inhibitors of Ras, PI3K, or MEK1/2, increased hepatocyte apoptosis 10-fold within 6 hours of exposure.6, 7 Apoptosis was dependent on bile acid–induced, ligand-independent activation of the FAS death receptor. Recent studies by our group have demonstrated that FAS receptor and JNK1/2 pathway activation are largely independent of bile acid–induced ROS/RNS generation.11 Other laboratories have reported that bile acids can activate ERBB1, the membrane-associated tyrosine kinase Src, and the FAS receptor.12–14

Several studies over the last 10 years have shown that ligand-independent activation of ERBB1 can occur via G-protein–coupled receptor-induced transactivation.15–20 In these studies, activation of membrane-associated non-receptor tyrosine kinases, such as src family members, has been linked to G-protein–coupled receptors (GPCR)-induced ERBB1 tyrosine phosphorylation as well as activation of the receptors by paracrine mechanisms. Because bile acids have been shown to activate some G-protein–coupled receptors in non-hepatocyte cell types, which has been linked to altered proliferation states, some of our prior data showing activation of the ERK1/2 and AKT pathways could have been influenced by bile acid–induced activation of hepatic GPCRs, which in turn promote activation of receptor tyrosine kinases.21–24

The studies in this manuscript demonstrate that conjugated forms of bile acids, but not unconjugated forms, interact with hepatic Gi-coupled GPCRs to promote activation of intracellular signaling pathways in cultures of primary rodent and human hepatocytes. Hence, transactivation of receptor tyrosine kinases by conjugated bile acid–stimulated GPCRs potentially represents a novel paradigm for bile acid signaling in hepatocytes.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Materials

All bile acids and 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS) were obtained from Sigma Chemical (St. Louis, MO). Phospho-/total-ERK1/2, Phospho-/total-JNK1/2, Anti-S473 AKT, and total AKT antibodies were purchased from Cell Signaling Technologies (Worcester, MA). Phospho-ERBB1 (Y1173) was purchased from Cell Signaling Technologies. All the secondary antibodies (anti-rabbit horseradish peroxidase [HRP], anti-mouse HRP, and anti-goat HRP) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Src family kinase inhibitor (PP2) and ERBB1 inhibitor (AG1478) were supplied by Calbiochem (San Diego, CA) as powder, dissolved in sterile dimethylsulfoxide (DMSO), and stored frozen under light-protected conditions at −80°C. Enhanced chemiluminescence (ECL) kits were purchased from Amersham Enhanced Chemi-Luminescence (ECL) system (Bucks, England) and NEN Life Science Products (NEN Life Science Products, Boston, MA). Trypsin-EDTA, Williams Medium E, and penicillin-streptomycin were purchased from GIBCOBRL (GIBCOBRL Life Technologies, Grand Island, NY). Other reagents were purchased or generated as described.5–8

Methods

Primary Culture of Rodent Hepatocytes.

Hepatocytes were isolated from adult male Sprague-Dawley rats or C57/BL6 mice by the 2-step collagenase perfusion technique. Human primary hepatocytes were obtained through the Liver Tissue Procurement and Distribution System (Pittsburgh, PA and Richmond, VA), which was funded by National Institutes of Health contract N01-DK-92310. The freshly isolated hepatocytes (approximately 90% viability) were plated on rat-tail collagen (Vitrogen)-coated plates at a density of 2 × 105 cells/well, and cultured in Williams E medium supplemented with 0.1 μmol/L dexamethasone, 1 μmol/L thyroxine, and 100 μg/mL penicillin/streptomycin, at 37°C in a humidified atmosphere containing 5% CO2. The initial medium change was performed 3 hours after cell seeding to minimize the contamination of dead or mechanically damaged cells. Unless otherwise indicated, cells were treated with 100 μmol/L bile acid approximately 24 hours after isolation.

Cell Treatments, SDS-PAGE, and Western Blot Analysis.

Cells were treated with either pertussis toxin (PTX, 300 ng/mL) or vehicle phosphate-buffered saline (PBS) diluent 16 hours or 6 hours as indicated before bile acid addition, and Vehicle (DMSO), AG1478 (1.0 μmol/L) or PP2 (10 μmol/L) 30 minutes before bile acid addition. The concentration of DMSO vehicle in the growth media was always less than 0.02% (vol/vol). Cells were then exposed to DCA/taurodeoxycholic acid (TDCA)/taurocholic acid (TCA) (100 μmol/L) or water diluent as indicated. Water diluent or treatment of hepatocytes with CHAPS did not alter the activation of signaling pathways, in agreement with Rao et al.5 (data not shown). One hundred μmol/L was chosen for most of these studies to observe large statistically significant alterations in hepatocyte biological behavior. For SDS-PAGE and immunoblotting, at various times after indicated treatment, hepatocytes were lysed in either a non-denaturing lysis buffer, and prepared for immunoprecipitation as previously described9–11 or in whole-cell lysis buffer (0.5 mol/L Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 1% β-mercaptoethanol, 0.02% bromophenol blue), and the samples were boiled for 30 minutes. After immunoprecipitation, samples were boiled in whole-cell lysis buffer. The boiled samples were loaded onto 7% to 10% SDS-PAGE, and electrophoresis was run overnight. Proteins were electrophoretically transferred onto 0.22-μm nitrocellulose, and immunoblotted with various primary antibodies against different proteins. All immunoblots were visualized by ECL. For presentation, immunoblots were digitally scanned at 600 dpi using Adobe PhotoShop 7.0, and their color removed and figures generated in MicroSoft PowerPoint.

Poly-L-Lysine Adenoviral Vectors: Generation and Infection In Vitro.

A psoralen-treated replication defective adenovirus was conjugated to poly-L-lysine and a cDNA plasmid construct to express a dominant negative Gαi1, as described.7, 8, 10, 11 Hepatocytes were transfected/infected with adenovirus at an approximate multiplicity of infection of 250. Cells were further incubated for 24 hours to ensure adequate expression of the transduced gene product.

Assays for ERK1/2 and AKT Activities.

ERK1/2 and AKT were immunoprecipitated from bile acid–treated hepatocytes using established procedures.5–8 Immunoprecipitates were suspended in a final volume of 50 μL of 25 mmol/L β-glycerophosphate pH 7.4, 1 mmol/L sodium orthovanadate, containing 0.2 mmol/L [γ-32P]ATP (2,000 cpm/pmol), 1 μmol/L microcystin-LR, containing either 0.5 mg/mL myelin basic protein (MBP) for ERK1/2 assays or 10 mg/mL RRGRPRTSSFAEG for AKT assays, which initiated reactions and incubated at 37°C. After 20 minutes, 40 μL of the reaction mixtures were spotted onto 2-cm circles of P81 phosphocellulose paper (Whatman, Maidstone, England) and immediately placed into 180 mmol/L phosphoric acid. Papers were washed 4 times (10 minutes each) with phosphoric acid, and once with acetone, and 32P-incorporation into MBP or peptide substrate was quantified by liquid scintillation spectroscopy. Pre-immune controls were performed to ensure phosphorylation was dependent on specific immunoprecipitation of ERK1/2 or AKT.

Identification of Receptor-Activated G-Proteins by [35S]GTPγS Binding Assay.

G-protein activation by bile acids was measured by an adaptation of the method of Okamoto et al.25 Cells were homogenized in 20 mmol/L HEPES (pH 7.4) containing 2 mmol/L MgCl2, 1 mmol/L EDTA, and 2 mmol/L 1,4-dithiothreitol. The homogenate was centrifuged at 30,000g for 30 minutes at 4°C, and the membranes were solubilized at 4°C in 20 mmol/L HEPES (pH 7.4) buffer containing 0.5% CHAPS. The cell membranes were incubated with 100 nmol/L [35S]guanosine triphosphate (GTP)γS in a solution containing 10 mmol/L HEPES (pH 7.4), 0.1 mmol/L EDTA, and 10 mmol/L MgCl2 for 20 minutes at 37°C in the presence or absence of bile acid agonist. The reaction was stopped with 10 volumes of 100 mmol/L Tris · HCl (pH 8.0) containing 10 mmol/L MgCl2, 100 mmol/L NaCl, and 20 μmol/L GTP. The membranes were incubated for 2 hours on ice in wells pre-coated with specific antibodies to Gi1α, Gi2α, and Gi3α. The wells were washed with phosphate buffer containing 0.05% Tween 20, and the radioactivity from each well was counted by liquid scintillation spectrometry.

Data Analysis.

Comparison of the effects of various treatments was performed using one-way analysis of variance and a two-tailed t test. Differences with a P value of < .05 were considered statistically significant. Experiments shown are the means of multiple individual points (±SEM).

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Initial studies examined the ability of DCA and TDCA to alter ERBB1 receptor autophosphorylation. TDCA and DCA activated the ERBB1 receptor in primary hepatocytes, which was blocked by the tyrphostin inhibitor of the ERBB1 tyrosine kinase, AG1478 (Fig. 1). TDCA, glycodeoxycholic acid (GDCA), chenodeoxycholic acid (CDCA), taurochenodeoxycholic acid (TCDCA), glycochenodeoxycholic acid (GCDCA), tauroursodeoxycholic acid (TUDCA), TCA, and DCA activated ERK1/2 and AKT in primary hepatocytes, which was blocked by AG1478 (Fig. 2).

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Figure 1. Regulation of receptor activities by deoxycholic acid (DCA) and taurodeoxycholic acid (TDCA). Primary rat hepatocytes were treated 6 hours after plating with Vehicle (VEH) (DMSO) or AG1478 (1.0 μmol/L), as indicated in the figure, 30 minutes before treatment with either DCA (100 μmol/L) or TDCA (100 μmol/L). Cells were isolated at the indicated time points after bile acid treatment and prepared for SDS-PAGE and immunoblotting to determine the phosphorylation of ERBB1 and the total expression of ERBB1. Data are from a representative experiment (n = 3).

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Figure 2. Conjugated bile acids activate ERK1/2 and AKT by a PTX- sensitive mechanism that is also dependent on signaling by ERBB1 and src family enzymes. (A) Hepatocytes were treated 6 hours after plating with phosphate-buffered saline (PBS) vehicle (VEH) or with PTX (300 ng/mL) 16 hours before bile acid exposure. Cells were then treated with vehicle control (dimethylsulfoxide, DMSO), PP2 (10 μmol/L), or AG1478 (1.0 μmol/L) 30 minutes before treatment with the bile acid as indicated; DCA, TDCA, CDCA, TCDCA, GCDCA, TCA, and TUDCA (100 μmol/L, each). Cells were isolated 20 minutes after treatment, and prepared for SDS-PAGE and immunoblotting to determine the phosphorylation of ERK1/2 and AKT (S473). Data are from a representative experiment (n = 4). (B) Hepatocytes were treated 6 hours after plating with PBS vehicle (VEH) or with PTX (300 ng/mL) 16 hours before bile acid exposure. Cells were then treated with CDCA, TCDCA, GCDCA, CA, TCA and GCA (100 μmol/L, each). Cells were isolated 20 minutes after treatment and prepared for SDS-PAGE and immunoblotting to determine the phosphorylation of ERK1/2 and AKT (S473). Data are from a representative experiment (n = 3). (C) Hepatocytes were treated 6 hours after plating with PBS vehicle (VEH) or with PTX (300 ng/mL) 16 hours before bile acid exposure. Cells were then treated with the indicated bile acids: DCA, TDCA, TCA, GDCA, and TUDCA (100 μmol/L, each). Cells were isolated 20 minutes after treatment, and ERK1/2 and AKT isolated by immunoprecipitation. Immune complex kinase assays to determine ERK1/2 activity (using MBP) and AKT activity (using a synthetic peptide) were performed as described in Materials and Methods. Data are mean ± SEM (n = 4). DCA, deoxycholic acid; CDCA, chenodeoxycholic acid; TDCA, taurodeoxycholic acid; GDCA, glycodeoxycholic acid; TCDCA, taurochenodeoxycholic acid; GCDCA, glycochenodeoxycholic acid; TCA, taurocholic acid; GCA, glycocholic acid; TUDCA, tauroursodeoxycholic acid; PTX, pertussis toxin.

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Further studies also determined in parallel whether PTX pretreatment of hepatocytes inhibited the activation of ERK1/2 and AKT by bile acids. PTX treatment did not alter DCA- or CDCA-induced phosphorylation of AKT or ERK1/2, whereas inhibition of Gi function after PTX treatment almost abolished the induction of AKT or ERK1/2 phosphorylation by glycine and taurine conjugated forms of these bile acids as well as TUDCA (Fig. 2A–B). ERK1/2 and AKT were activated in hepatocytes treated with cholic acid (CA), as previously noted,5 as well as with conjugated forms of CA to taurine or glycine (Fig. 2B). PTX treatment abolished ERK1/2 and AKT activation by TCA and glycocholic acid (GCA), but not by CA (Fig. 2A–B). Comparable data to those obtained using phospho-immunoblotting were noted when ERK1/2 and AKT protein kinase activity was assayed in immune complex kinase assays (Fig. 2C). Because PTX pretreatment of hepatocytes inhibited the activation of ERK1/2 and AKT by conjugated bile acids, we determined whether PTX treatment also altered ERBB1 phosphorylation; PTX pretreatment of hepatocytes inhibited the activation of ERBB1 by TDCA (Fig. 3).

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Figure 3. Pertussis toxin (PTX) treatment blocks TDCA-induced activation of ERBB1. Primary rat hepatocytes were treated 6 hours after plating with PBS vehicle (VEH) or with PTX (300 ng/mL) as indicated, 16 hours before treatment with water or with water containing TDCA (100 μmol/L). Cells were isolated at the indicated times after bile acid treatment and prepared for SDS-PAGE and immunoblotting to determine the phosphorylation of ERBB1 and the total expression of ERBB1. Data are from a representative experiment (n = 4).

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In general agreement with our findings using PTX, expression of a dominant negative Gαi1 protein in hepatocytes had an almost identical inhibitory impact on TDCA-, TUDCA-, GDCA-, and TCA-induced activation of the signaling pathways (Fig. 4). However, in general agreement with previous findings that demonstrated bile acid–induced JNK1/2 activation was dependent on the acidic sphingomyelinase and the FAS receptor, and not dependent on receptor tyrosine kinases, PTX treatment did not alter bile acid–induced activation of the JNK1/2 pathway (Fig. 5). Collectively, these findings demonstrate that conjugated bile acids, but not unconjugated bile acids such as DCA or CDCA, activate a Gi-coupled G-protein–coupled receptor (GPCR), which can promote activation of ERBB1, and the ERK1/2 and AKT pathways in primary rat hepatocytes.

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Figure 4. Expression of dominant negative Gαi1 inhibits conjugated, but not unconjugated, bile acid–induced activation of ERK1/2 and AKT. Hepatocytes were infected 4 hours after plating with poly-L-lysine conjugated adenoviruses to express either a control plasmid (CMV) or a plasmid to express dominant negative Gαi1 (dn Gαi). Twenty-four hours after infection, infected hepatocytes were treated with water (control) or with the bile acids DCA, GDCA, GCA, TDCA, TCA, and TUDCA (100 μmol/L, each). Cells were isolated 20 minutes after treatment, and prepared for SDS-PAGE and immunoblotting to determine the phosphorylation of ERK1/2 and AKT (S473). Data are from a representative experiment (n = 4). DCA, deoxycholic acid; TDCA, taurodeoxycholic acid; TCA, taurocholic acid; TUDCA, tauroursodeoxycholic acid; GDCA, glycodeoxycholic acid; GCA, glycocholic acid.

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Figure 5. Pertussis toxin (PTX) treatment does not alter bile acid–induced activation of JNK1/2 in primary hepatocytes. Hepatocytes were treated 6 hours after plating with PBS vehicle (VEH) or with PTX (300 ng/mL) 16 hours before treatment with water (control) or with the bile acids DCA, TCA, and GDCA (100 μmol/L each). Cells were isolated 20 minutes after treatment and prepared for SDS-PAGE and immunoblotting to determine the phosphorylation of JNK1/2. Data are from a representative experiment (n = 4). DCA, deoxycholic acid; TCA, taurocholic acid; GDCA, glycodeoxycholic acid.

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As both PTX and dominant negative Gαi1 blocked TDCA- and TCA-induced activation of intracellular signaling pathways, we next investigated whether bile acids promote GTP binding of Gαi proteins in primary hepatocytes. Treatment of hepatocyte membranes with TDCA and TCA, but not DCA, promoted GTP loading of Gαi1 and to a similar extent Gαi2 and Gαi3 (Fig. 6A–C). Thus, together with data presented in Figs. 2 through 4, these findings strongly argue that conjugated bile acids, but not unconjugated bile acids, promote GTP loading/activation of PTX sensitive G-proteins.

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Figure 6. Conjugated, but not unconjugated, bile acids promote G alpha i activation, which is blocked by pertussis toxin (PTX) pretreatment. Hepatocytes were treated 6 hours after plating with PBS vehicle (VEH) or with PTX (300 ng/mL) 16 hours. Hepatocyte membranes were isolated and treated with water control (CO) or with DCA, TCA, or GDCA (100 μmol/L each). (A) GTP binding for Gαi-1. (B) GTP binding for Gαi-2. (C) GTP binding for Gαi-3. Data are the means of 3 values per experiment from 3 independent experiments ± SEM. PBS, phosphate-buffered saline; DCA, deoxycholic acid; TCA, taurocholic acid; GDCA, glycodeoxycholic acid.

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In a previous study, we noted that DCA-induced activation of ERK1/2 in primary hepatocytes was dependent on the generation of ROS by mitochondria (Supplementary Fig. 1A and Fang et al.10). In agreement with these findings, activation of ERK1/2 and AKT by TDCA and TCA was also noted to be ROS dependent and ROS generation dependent on mitochondrial function (Supplementary Fig. 1A–B). Also in agreement with our prior findings, activation of ERBB1 by both DCA and TDCA was also noted to be dependent on mitochondria-dependent ROS generation (Supplementary Fig. 1C).

Because other laboratories have tended to perform studies in established hepatocyte cell lines, additional experiments then examined whether DCA, TDCA, and TCA activated ERK1/2 in an established rat hepatoma cell line, McArdle cells (McARH-7777), transfected with a control vector or with the Ntcp bile acid transporter. DCA, but not TCA or TDCA, activated ERK1/2 in control transfected cells (Fig. 7A–C). DCA, TCA, and TDCA were all competent to activate ERK1/2 in Ntcp transfected cells: however, in contrast to data in primary rat hepatocytes, TCA- and TDCA-induced ERK1/2 activation was PTX insensitive while still being ROS-dependent in the established rat hepatoma cell line (Fig. 7C). Similar data to those generated in McArdle cells also were obtained in studies using human hepatoma cells (HuH7.Ntcp) (data not shown and Fang et al.10). These findings argue that established rodent and human hepatoma cell lines are unlikely to express the Gi-coupled GPCR, which is expressed in primary rodent hepatocytes.

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Figure 7. Conjugated and unconjugated bile acids promote activation of intracellular signaling pathways in an ROS-dependent fashion in hepatoma cells: a lack of a PTX-sensitive GPCR signaling effect. (A) The established rat hepatoma cell line McArdle (McARH) were stably transfected with either a control plasmid or a plasmid to express the bile acid transporter Ntcp. As indicated, cells were pretreated for 6 hours with PBS vehicle (VEH) or PTX (300 ng/mL) or pretreated for 30 minutes with N-acetyl cysteine (NAC, 20 mmol/L) followed by exposure to DCA (100 μmol/L). After bile acid exposure (45 minutes), cells were lysed, and the cell lysates subjected to SDS-PAGE. Immunoblotting was performed to determine the tyrosine/threonine phosphorylation of ERK1/2. (B) The established rat hepatoma cell line McARH was stably transfected with either a control plasmid or a plasmid to express the bile acid transporter Ntcp. As indicated, cells were pretreated for 6 hours with PBS vehicle (VEH) or PTX (300 ng/mL) followed by exposure to TCA (100 μmol/L). After bile acid exposure (45 minutes), cells were lysed, and the cell lysates subjected to SDS-PAGE. Immunoblotting was performed to determine the tyrosine/threonine phosphorylation of ERK1/2 and JNK1/2. (C) The established rat hepatoma cell line McARH was stably transfected with either a control plasmid or a plasmid to express the bile acid transporter Ntcp. As indicated, cells were pretreated for 30 minutes with NAC (20 mmol/L) followed by exposure to TCA or TDCA (100 μmol/L each). After bile acid exposure (45 minutes), cells were lysed, and the cell lysates subjected to SDS-PAGE. Immunoblotting was performed to determine the tyrosine/threonine phosphorylation of ERK1/2 and JNK1/2. Data are from a representative experiment (n = 2). ROS, reactive oxygen species; TDCA, taurodeoxycholic acid; TCA, taurocholic acid; PTX, pertussis toxin.

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Based on our data in the McArdle established hepatoma cell line derived from rats, and from human HuH7 cells, it is possible that the GPCR-dependent signaling effects we have been observing for conjugated bile acids are only found in primary rat hepatocytes. To test this possibility, we examined the regulation of ERK1/2 activity by DCA, TDCA, and TCA in primary mouse and primary human hepatocytes. In both primary mouse and human hepatocytes, TCA induced ERK1/2 activation in a PTX-sensitive manner (Fig. 8). These findings argue that conjugated bile acids promote activation of a Gi-coupled GPCR in primary hepatocytes from multiple species.

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Figure 8. Conjugated bile acids activate ERK1/2 and AKT by a PTX- sensitive mechanism in primary mouse and primary human hepatocytes. Primary mouse and primary human hepatocytes were cultured for 6 hours followed by PBS vehicle (VEH) or PTX treatment (300 ng/mL) for 16 hours and then exposed to water vehicle (VEH) or the bile acids DCA, TCA, TDCA, or TUDCA (100 μmol/L each). Cells were isolated 20 minutes after bile acid addition, lysed, and subjected to SDS-PAGE. Immunoblotting was performed to determine the tyrosine/threonine phosphorylation of ERK1/2 and serine phosphorylation of AKT. Data are from a representative experiment (n = 2). PBS, phosphate-buffered saline; DCA, deoxycholic acid; TCA, taurocholic acid; TDCA, taurodeoxycholic acid; TUDCA, tauroursodeoxycholic acid; PTX, pertussis toxin.

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Two GPCRs have been reported in the literature as receptors for conjugated and to a lesser extent unconjugated bile acids: muscarinic family receptors and the orphan receptor TGR5, with neither report using primary hepatocytes in their studies.21–24, 26, 27 However, the liver is known to only express high levels of the muscarinic M3 receptor, and this was stated to be in hepatocyte progenitor cells, not adult hepatocytes.28 In addition, the M3 receptor is reported to be coupled to Gq, and the TGR5 receptor was noted to be Gs coupled, which would tend to negate both of these GPCRs as part of our Gi-dependent response. To exclude muscarinic receptors in our effects, incubation of hepatocytes with atropine (10 μmol/L) did not alter the activation of either ERK1/2 or of AKT by conjugated or unconjugated bile acids (data not shown). Furthermore, use of M3 receptor−/− primary mouse hepatocytes failed to modify the activation of ERK1/2 and AKT compared with wild-type cells (data not shown). Thus, the identity of the novel bile acid–responsive GPCR is unknown.

Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Previous studies by this group have linked bile acid–induced activation of ERBB1 and the insulin receptor to elevated signaling by the ERK1/2 and AKT pathways, and bile acid–induced activation of the FAS receptor to elevated signaling by the JNK1/2 pathway. Other laboratories have reported that bile acid–induced ERK1/2, JNK2, and AKT signaling are cytoprotective responses versus bile acid–induced FAS receptor/caspase activation and activation of JNK1.29

DCA activated ERBB1 and the ERK1/2 and AKT pathways, which was blocked by the tyrphostin AG1478. Similarly, AG1478 inhibited ERBB1, ERK1/2, and AKT activation induced by treatment of hepatocytes with TCA, TDCA, and GDCA. Studies by several laboratories have argued that activation of ERBB1, in addition to exogenous ligand- or ROS-induced activation of the receptor, can be due to the actions of GPCRs, which result in either a paracrine/autocrine form of receptor tyrosine kinase activation or a direct activation of ERBB1 via non-receptor tyrosine kinases.15–24 In our studies, incubation of hepatocytes with either marimastat or GM6001, to inhibit cell surface proteases that release ERBB1 receptor ligands, did not alter conjugated bile acid–induced ERK1/2 and AKT activation (unpublished studies and Zhang et al.30), whereas inhibition of Src family tyrosine kinase function blunted activation of ERK1/2 and AKT. These data would suggest a direct intra-membrane interaction between GPCR and ERBB1 activation, and enhanced activity in downstream signaling pathways (Supplementary Fig. 2).

In addition, we have recently shown in primary hepatocytes that DCA and TDCA could generate ROS that was responsible for the inhibition of the protein tyrosine phosphatase SHP-1, increased ERBB1 tyrosine phosphorylation, and activation of ERK1/2 and AKT.10 In the current studies, and in contrast to our findings with DCA, conjugated bile acids activated ERBB1, ERK1/2, and AKT in both a PTX-sensitive and an ROS-dependent manner. PTX effects are generally viewed by the heterotrimeric G-protein field as being specific to inhibition of Gαi proteins, and to confirm our findings we expressed a dominant negative Gαi in hepatocytes. Using this molecular tool, we corroborated that conjugated bile acids were primarily activating ERK1/2 and AKT via a Gαi -dependent mechanism (Fig. 4).

Our initial findings demonstrated that conjugated bile acids, in a PTX-dependent manner, activated ERK1/2 and AKT in primary rat hepatocytes, and subsequent studies confirmed that primary mouse and human hepatocytes also activated ERK1/2 and AKT via a similar mechanism. Thus, the novel GPCR being stimulated by conjugated bile acids is expressed in primary hepatocytes derived from multiple species. However, an established rat hepatoma cell line did not promote ERK1/2 and AKT activation in a PTX-dependent manner, arguing that expression of the novel/putative bile acid–binding GPCR is lost on establishment of rat hepatocytes in culture. Studies of adrenergic receptor expression in the liver show that, based on the proliferative state of the hepatocyte, alpha- and beta-adrenergic GPCRs are differentially expressed.31, 32 Similar changes in adrenergic receptor expression are observed when hepatocytes are placed into primary culture. The expression of multiple other proteins, such as the Ntcp bile acid transporter, cytochrome P450 enzymes, also changes during the primary culture of hepatocytes and in established cell lines.33–35 Thus, prior and future studies using established hepatocyte cell lines lacking bile acid–binding GPCR proteins may not permit an accurate description of bile acid signaling processes to be developed as they pertain to non-transformed primary hepatocytes.

GPCRs are the largest family of cell surface molecules involved in signal transduction, and they can be activated by a wide variety of ligands. Within the last several years, 16 different orphan GPCRs have been shown to be activated by lipid mediators, including bile salts, lysophospholipids, arachidonic acid, and short-, medium-, and long-chain fatty acids.36–38 Approximately 35 GPCRs are in the phylogenetic tree of lipid-activated GPCRs, and only 2 GPCRs have been definitively reported to be activated by bile salts, TGR5 and the muscarinic receptor family. However, TGR5 is not expressed in hepatocytes. TGR5 appears to be expressed mostly in immune cells and is thought to be Gs coupled, leading to the generation of cyclic AMP, and thus is not PTX sensitive. Phylogenetically, TGR5 is most closely related to GPCRs activated by sphingosine-1-phosphate, lysophosphatidic acid, and cannabinoid receptors.22, 26, 37 Inhibition of sphingosine-1-phosphate kinase function did not alter the activation of ERK1/2 and AKT by conjugated bile acids (unpublished findings). The M3 muscarinic receptor is the only muscarinic receptor expressed in the liver but is not detected by immunohistochemistry in mature adult hepatocytes. In one study, muscarinic receptors were most highly activated by taurine and glycine conjugates of lithocholic acid but also by unconjugated bile acids. However, the M3 receptor has been shown in many studies to be predominantly coupled to Gq, suggesting that this receptor does not signal through a PTX-sensitive pathway.23 In our hands, a muscarinic receptor antagonist, atropine, and use of primary hepatocytes from M3 receptor−/− mice have strongly argued that conjugated bile acid–induced activation of ERK1/2 and AKT remained PTX sensitive regardless of the expression of the M3 or any muscarinic receptor. Further analyses will be needed to determine which, if any, of the approximately 35 GPCRs in the phylogenic tree for GPCR lipid molecule binding are involved in the PTX-sensitive activation of the ERK1/2 and AKT pathways in primary hepatocytes.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

The authors thank Dr. Karnam S. Murthy and Dr. Huiping Zhou (Departments of Physiology and Medicine, VCU) for assistance during the initial GTP loading experiments. The authors thank Dr. J. Grandis (University of Pittsburgh) for supplying Marimastat. PD is the holder of the Universal Inc. Professorship in Signal Transduction Research.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information
  • 1
    Roberts MS, Magnusson BM, Burczynski FJ, Weiss M. Enterohepatic circulation: physiological, pharmacokinetic and clinical implications. Clin Pharmacokinet 2002; 41: 751790.
  • 2
    Hardison NGM. Hepatic taurine concentration and dietary taurine as regulators of bile acid conjugation with taurine. Gastroenterology 1970; 75: 7175.
  • 3
    Trautwein EA, Siddiqui A, Hayes KC. Modeling plasma lipoprotein-bile lipid relationships: differential impact of psyllium and cholestyramine in hamsters fed a lithogenic diet. Metabolism 1993; 42: 15311540.
  • 4
    Poupon R, Chazouilleres O, Poupon RE. Chronic cholestatic diseases. J Hepatol 2000; 32: 129140.
  • 5
    Rao YP, Studer EJ, Stravitz RT, Gupta S, Qiao L, Dent P, et al. Activation of the Raf-1/MEK/ERK cascade by bile acids occurs via the epidermal growth factor receptor in primary rat hepatocytes. HEPATOLOGY 2002; 35: 307314.
  • 6
    Qiao L, Yacoub A, Studer E, Gupta S, Pei XY, Grant S, et al. Inhibition of the MAPK and PI3K pathways enhances UDCA-induced apoptosis in primary rodent hepatocytes. HEPATOLOGY 2002; 35: 779789.
  • 7
    Qiao L, Studer E, Leach K, McKinstry R, Gupta S, Decker R, et al. Deoxycholic acid (DCA) causes ligand-independent activation of epidermal growth factor receptor (EGFR) and FAS receptor in primary hepatocytes: inhibition of EGFR/mitogen-activated protein kinase-signaling module enhances DCA-induced apoptosis. Mol Biol Cell 2001; 12: 26292645.
  • 8
    Han SI, Studer E, Gupta S, Fang Y, Qiao L, Li W, et al. Bile acids enhance the activity of the insulin receptor and glycogen synthase in primary rodent hepatocytes. HEPATOLOGY 2004; 39: 456463.
  • 9
    van Montfort RL, Congreve M, Tisi D, Carr R, Jhoti H. Oxidation state of the active-site cysteine in protein tyrosine phosphatase 1B. Nature 2003; 423: 773777.
  • 10
    Fang Y, Han SI, Mitchell C, Gupta S, Studer E, Grant S, et al. Bile acids induce mitochondrial ROS, which promote activation of receptor tyrosine kinases and signaling pathways in rat hepatocytes. HEPATOLOGY 2004; 40: 961971.
  • 11
    Gupta S, Natarajan R, Payne SG, Studer EJ, Spiegel S, Dent P, et al. Deoxycholic acid activates the c-Jun N-terminal kinase pathway via FAS receptor activation in primary hepatocytes: role of acidic sphingomyelinase-mediated ceramide generation in FAS receptor activation. J Biol Chem 2004; 279: 58215828.
  • 12
    Werneburg NW, Yoon JH, Higuchi H, Gores GJ. Bile acids activate EGF receptor via a TGF-alpha-dependent mechanism in human cholangiocyte cell lines. Am J Physiol Gastrointest Liver Physiol 2003; 285: G31G36.
  • 13
    Qiao D, Stratagouleas ED, Martinez JD. Activation and role of mitogen-activated protein kinases in deoxycholic acid-induced apoptosis. Carcinogenesis 2001; 22: 3541.
  • 14
    Carter S, Auer KL, Reardon DB, Birrer M, Fisher PB, Valerie K, et al. Inhibition of the mitogen activated protein (MAP) kinase cascade potentiates cell killing by low dose ionizing radiation in A431 human squamous carcinoma cells. Oncogene 1998; 16: 27872796.
  • 15
    Melien O, Sandnes D, Johansen EJ, Christoffersen T. Effects of pertussis toxin on extracellular signal-regulated kinase activation in hepatocytes by hormones and receptor-independent agents: evidence suggesting a stimulatory role of G(i) proteins at a level distal to receptor coupling. J Cell Physiol 2000; 184: 2736.
  • 16
    Shida D, Kitayama J, Yamaguchi H, Yamashita H, Mori K, Watanabe T, et al. Sphingosine 1-phosphate transactivates c-Met as well as epidermal growth factor receptor (EGFR) in human gastric cancer cells. FEBS Lett 2004; 577: 333338.
  • 17
    Slomiany BL, Slomiany A. Src-kinase-dependent epidermal growth factor receptor transactivation in salivary mucin secretion in response to beta-adrenergic G-protein-coupled receptor activation. Inflammopharmacology 2004; 12: 233245.
  • 18
    Melien O, Thoresen GH, Sandenes D, Ostby E, Christoffersen T. Activation of p42/44 mitogen-activated protein kinase by angiotensin II, vasopressin, norepinephrine and prostaglandin F2 alpha in hepatocytes is sustained and like the effect of epidermal growth factor, mediated through pertussis toxin sensitive mechanisms. J Cell Physiol 1998; 175: 348358.
  • 19
    Luttrell LM, Della Rocca GJ, van Biesen T, Luttrell DK, Lefkowitz RJ. Gbetagamma subunits mediate Src-dependent phosphorylation of the epidermal growth factor receptor: a scaffold for G protein-coupled receptor-mediated Ras activation. J Biol Chem 1997; 272: 46374644.
  • 20
    Luttrell LM, Daaka Y, Della Rocca GJ, Lefkowitz RJ. G protein-coupled receptors mediate two functionally distinct pathways of tyrosine phosphorylation in rat 1a fibroblasts. Shc phosphorylation and receptor endocytosis correlate with activation of Erk kinases. J Biol Chem 1997; 272: 3164831656.
  • 21
    Raufman JP, Cheng K, Zimniak P. Activation of muscarinic receptor signaling by bile acids: physiological and medical implications. Dig Dis Sci 2003; 48: 14311444.
  • 22
    Kawamata Y, Fujii R, Hosoya M, Harada M, Yoshida H, Miwa M, et al. A G protein-coupled receptor responsive to bile acids. J Biol Chem 2003; 278: 94359440.
  • 23
    Cheng K, Chen Y, Zimniak P, Raufman JP, Xiao Y, Frucht H. Functional interaction of lithocholic acid conjugates with M3 muscarinic receptors on a human colon cancer cell line. Biochim Biophys Acta 2002; 1588: 4855.
  • 24
    Cheng K, Khurana S, Chen Y, Kennedy RH, Zimniak P, Raufman JP. Lithocholylcholine, a bile acid/acetylcholine hybrid, is a muscarinic receptor antagonist. J Pharmacol Exp Ther 2002; 303: 2935.
  • 25
    Okamoto H, Takuwa N, Yokomizo T, Sugimoto N, Sakurada S, Shigematsu H, et al. Inhibitory regulation of Rac activation, membrane ruffling, and cell migration by the G protein-coupled sphingosine-1-phosphate receptor EDG5 but not EDG1 or EDG3. Mol Cell Biol 2000; 20: 92479261.
  • 26
    Katsuma S, Hirasawa A, Tsujimoto G. Bile acids promote glucagon-like peptide-1 secretion through TGR5 in a murine enteroendocrine cell line STC-1. Biochem Biophys Res Commun 2005; 329: 386390.
  • 27
    Raufman JP, Chen Y, Zimniak P, Cheng K. Deoxycholic acid conjugates are muscarinic cholinergic receptor antagonists. Pharmacology 2002; 65: 215221.
  • 28
    Cassiman D, Libbrecht L, Sinelli N, Desmet V, Denef C, Roskams T. The vagal nerve stimulates activation of the hepatic progenitor cell compartment via muscarinic acetylcholine receptor type 3. Am J Pathol 2002; 161: 521530.
  • 29
    Qiao L, Han SI, Fang Y, Park JS, Gupta S, Gilfor D, et al. Bile acid regulation of C/EBPbeta, CREB, and c-Jun function, via the extracellular signal-regulated kinase and c-Jun NH2-terminal kinase pathways, modulates the apoptotic response of hepatocytes. Mol Cell Biol 2003; 23: 30523066.
  • 30
    Zhang Q, Thomas SM, Xi S, Smithgall TE, Siegfried JM, Kamens J, et al. SRC family kinases mediate epidermal growth factor receptor ligand cleavage, proliferation, and invasion of head and neck cancer cells. Cancer Res 2004; 64: 61666173.
  • 31
    Spector MS, Auer KL, Jarvis WD, Ishac EJ, Gao B, Kunos G, et al. Differential regulation of the mitogen-activated protein and stress-activated protein kinase cascades by adrenergic agonists in quiescent and regenerating adult rat hepatocytes. Mol Cell Biol 1997; 17: 35563565.
  • 32
    Gao B, Jiang L, Kunos G. Transcriptional regulation of alpha(1b) adrenergic receptors (alpha(1b)AR) by nuclear factor 1 (NF1): a decline in the concentration of NF1 correlates with the downregulation of alpha(1b)AR gene expression in regenerating liver. Mol Cell Biol 1996; 16: 59976008.
  • 33
    Rippin SJ, Hagenbuch B, Meier PJ, Stieger B. Cholestatic expression pattern of sinusoidal and canalicular organic anion transport systems in primary cultured rat hepatocytes. HEPATOLOGY 2001; 33: 776782.
  • 34
    Clayton RF, Rinaldi A, Kandyba EE, Edward M, Willberg C, Klenerman P, et al. Liver cell lines for the study of hepatocyte functions and immunological response. Liver Int 2005; 25: 389402.
  • 35
    Trautwein M, Boyer JM. Bile salt transporters; molecular characterization, function, and regulation. Physiol Rev 2003; 83: 633671.
  • 36
    Im DS. Discovery of new G protein coupled receptors for lipid mediators. J Lipid Res 2004; 45: 410418.
  • 37
    Brown AJ, Jupe S, Briscoe CP. A family of fatty acid binding receptors. DNA Cell Biol 2005; 24: 5461.
  • 38
    Spiegel S, Milstien S. Sphingosine-1-phosphate: an enigmatic signalling lipid. Nat Rev Mol Cell Biol 2003; 4: 397407.

Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Supplementary material for this article can be found on the H EPATOLOGY website ( http://www.interscience.wiley.com/jpages/0270-9139/suppmat/index.html ).

FilenameFormatSizeDescription
jws-hep.20942.fig1AB.tif65K Conjugated and unconjugated bile acids promote activation of intracellular signaling pathways in an ROS-dependent fashion. (A) Twenty-four hours after plating, primary rat hepatocytes were pretreated with vehicle (VEH) (DMSO), Trolox (TX) (10 &&num;x03bc;mol/L) or bongkrekic acid (BA) (50 &&num;x03bc;mol/L) for 30 minutes prior to addition of DCA (100 &&num;x03bc;mol/L). Cells were isolated 20 minutes after bile acid addition, lysed, and subjected to SDS-PAGE. Immunoblotting was performed to determine the tyrosine/threonine phosphorylation of ERK1/2. (B) Twenty-four hours after plating primary rat hepatocytes were pretreated with vehicle (VEH) (DMSO), cyclosporin A (CsA) (1 &&num;x03bc;mol/L), Trolox (TX) (10 &&num;x03bc;mol/L),N -acetyl cysteine (NAC) (20 mmol/L) or bongkrekic acid (BA) (50 &&num;x03bc;mol/L) for 30 minutes prior to addition of TDCA or TCA (100 &&num;x03bc;mol/L, each, as indicated). Cells were isolated 20 minutes after bile acid addition, lysed, and subjected to SDS-PAGE. Immunoblotting was performed to determine the tyrosine/threonine phosphorylation of ERK1/2.
jws-hep.20942.fig1C.tif60K Conjugated and unconjugated bile acids promote activation of intracellular signaling pathways in an ROS-dependent fashion. (C) Twenty-four hours after plating primary rat hepatocytes were pretreated with vehicle (VEH) (DMSO), Trolox (TX) (10 &&num;x03bc;mol/L) or bongkrekic acid (BA) (50 &&num;x03bc;mol/L) for 30 minutes prior to addition of DCA or TDCA (100 &&num;x03bc;mol/L each). Cells were isolated at the indicated times after bile acid addition, lysed, ERBB1 immunoprecipitated, and subjected to SDS-PAGE. Immunoblotting was performed to determine the tyrosine phosphorylation of ERBB1.
jws-hep.20942.fig2.TIF79KRegulation of cell signaling by bile acids in primary hepatocytes and established hepatocyte cell lines. Unconjugated bile acids ( e.g. , DCA) can enter hepatocytes either through membrane transporters ( e.g. , Ntcp) or diffuse through the lipid bilayer directly into the cell. In contrast, conjugated bile acids ( e.g. , TDCA) require expression of Ntcp or other transporters to rapidly enter hepatocytes, and thus in cells lacking transporter expression ( e.g. , McARH cells), conjugated bile acids do not significantly activate intracellular pathways. In the presence of a transporter, McARH cells become permissive for conjugated bile acids to enter these cells and to activate intracellular pathways in an ROS-dependent and PTX-independent manner. In primary hepatocytes that express endogenous bile acid transporters, conjugated bile acids activate intracellular pathways via two concerted mechanisms: by binding to a G i- coupled GPCR and by generating ROS.

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