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Am J Physiol Gastrointest Liver Physiol 294: G506-G519, 2008. First published December 20, 2007; doi:10.1152/ajpgi.00304.2007
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LIVER AND BILIARY TRACT

Anandamide inhibits cholangiocyte hyperplastic proliferation via activation of thioredoxin 1/redox factor 1 and AP-1 activation

Sharon DeMorrow,1,2 Heather Francis,1 Eugenio Gaudio,3 Yoshiyuki Ueno,4 Julie Venter,5 Paolo Onori,5 Antonio Franchitto,3 Bradley Vaculin,6 Shelley Vaculin,7 and Gianfranco Alpini2,6,7

1Division of Research and Education, Scott & White Hospital, Temple; 2Department of Medicine, Texas A&M Health Science Center, College of Medicine, Temple, Texas; 3Division of Anatomy, University "La Sapienza", Rome, Italy; 4Division of Gastroenterology, Tohoku University Hospital, Aobaku, Sendai, Japan; 5Department of Experimental Medicine, University of L'Aquila, L'Aquila, Italy; 6Systems Biology and Translational Medicine, The Texas A&M University System Health Science Center, College of Medicine, Temple; and 7Division of Research, Central Texas Veterans Health Care System, Temple, Texas

Submitted 6 July 2007 ; accepted in final form 16 December 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The endocannabinoid system regulates various aspects of hepatic fibrosis; however, nothing is known about its role in regulating cholangiocyte proliferation and function. We evaluated the effects of anandamide (AEA) on cholangiocyte proliferation and explored the effects of AEA on the thioredoxin 1 (TRX1)/redox factor 1 (Ref1)/activator protein-1 (AP-1) pathway. Mice underwent bile duct ligation (BDL) and were infused with AEA for 3 days postsurgery. Proliferation and apoptosis were evaluated in liver sections. Effects of in vitro AEA treatment on cholangiocyte proliferation and apoptosis were studied in purified cholangiocytes. The relative expression of cannabinoid receptors was also assessed in liver sections and cholangiocytes. mRNA expression of the cannabinoid receptors Cb1 and VR1 was decreased after BDL, whereas there was an upregulation of Cb2 mRNA. AEA decreased cholangiocyte growth and induced accumulation of reactive oxygen species, upregulation of TRX1, Ref1, c-Fos, and c-Jun expression, increased nuclear localization of TRX1, and increased AP-1 transcriptional activity. Specific knockdown of TRX1 or Ref1 expression ablated the AP-1 transcriptional activity and AEA-induced cell death but not expression of c-Fos and c-Jun. Knockdown of c-Fos and c-Jun expression also ablated AEA-induced apoptosis. We conclude that AEA suppresses cholangiocyte proliferation during cholestasis via a Cb2-dependent mechanism. Modulation of the endocannabinoid system may be important in the treatment of cholangiopathies.

endocannabinoids; biliary epithelia; cell proliferation; apoptosis; Fos; Jun

CHOLANGIOCYTES THAT ARE CONSTITUTIVELY mitotically dormant possess marked proliferative capacity (4), which is apparent during experimental conditions such as cholestasis induced by bile duct ligation (BDL) (3) as well as in human cholangiopathies (4). Cholangiocytes can also be damaged in several experimental models such as acute administration of carbon tetrachloride (CCl4) (44, 45) or interruption of the parasympathetic innervation by vagotomy (43). In humans, cholangiocyte proliferation occurs in extrahepatic biliary obstruction, in the course of chronic cholestatic liver diseases (e.g., primary sclerosing cholangitis, primary biliary cirrhosis, liver allograft rejection, and graft-vs.-host disease) (4), and in many forms of liver injury (e.g., in response to alcohol, toxin, or drugs) (4, 61).

The finding of specific G protein-coupled receptors for the psychoactive component of Cannabis sativa (–)-{Delta}9-tetrahydrocannabinol (28) led to the discovery of a whole endogenous signaling system now known as the endocannabinoid system (11). This system consists of the cannabinoid receptors (Cb1 and Cb2, as well as a putative involvement of the vanilloid receptor, VR1), their endogenous ligands (endocannabinoids), and the proteins for their synthesis and inactivation (11). The cannabinoid receptors are seven-transmembrane domain proteins coupled to Gi/o types of G proteins (11). Cb1 receptors are found predominantly in the central nervous system but also in most peripheral tissues including immune cells, the reproductive system, the gastrointestinal tract, and the lung (15, 51, 54). Cb2 receptors are found predominantly in the immune system, i.e., in tonsils, spleen, macrophages, and lymphocytes (15, 51, 54). To date, many endocannabinoids have been identified with varying affinities for the receptors and all of which are lipid molecules. Anandamide (AEA) was the first endogenous ligand to be identified (15), which acts as a partial Cb1 agonist and weak Cb2 agonist. Although the physiological roles of many of the other ligands have not yet been fully clarified, AEA has been implicated in a wide variety of physiological and pathological processes.

The function of the endocannabinoid system within the liver is a subject of ongoing research. Evidence indicates that the endogenous cannabinoid system could be of relevance in the pathogenesis of liver fibrosis and portal hypertension (39). For example, studies have demonstrated that AEA is a selective killer of hepatic activated stellate cells in vitro (64). Furthermore, Cb2 receptors are strongly induced during human liver cirrhosis and are expressed in nonparenchymal cells and cholangiocytes located within and at the edges of fibrotic septa (40). Cb2 receptor activation has resulted in growth inhibition and apoptosis (40). Moreover, CCl4-treated mice lacking Cb2 receptors develop more fibrosis than CCl4-treated wild-type mice (40). Although many factors have been shown to regulate cholangiocyte hyperplastic growth (8), the role of the endocannabinoid system is unknown. However, we (14) recently demonstrated an antiproliferative effect of AEA on cholangiocarcinoma cell growth.

Immediate-early genes encode transcription factors that play a role in converting short-term extracellular signals into long-term alterations in the pattern of gene expression (13). There are three families of immediate-early genes commonly studied: the Fos, Jun, and Egr families. Transcription of these genes is rapid and transient, depends on posttranslational modifications of existing transcription factors, and therefore does not require protein synthesis (13). Members of the Fos and Jun families of leucine zipper proteins form hetero- or homodimeric complexes that constitute the activator protein-1 (AP-1) transcription factor and interact with the AP-1 consensus DNA binding sequence (13). Besides the expression and protein composition of the AP-1 dimer, many other factors influence the DNA-binding activity and transactivation potential. One such factor is the oxidation state of the AP-1 proteins (63). It is believed that with an increase in reactive oxygen species (ROS) comes an activation of AP-1 transcription activity (63), possibly via an upregulation and translocation of thioredoxin 1 (TRX1) and the subsequent activation of redox factor 1 (Ref1) (70).

The ability of cannabinoids (both endogenous and plant derived) to activate AP-1 transcriptional activity has been shown previously (36, 58, 62). In the liver, AEA has been shown to upregulate the expression of c-Jun and Jun B, as well as an upregulation of AP-1 DNA-binding activity containing c-Jun, Jun B, and c-Fos (30) in Chang liver cells. Furthermore, this AP-1 activation was associated with AEA-induced apoptosis in these cells (30). The activation of AP-1 in cholangiocytes by AEA has not been demonstrated, and little is known about the functional consequences of such activation on cholangiocytes. Thus our aims were 1) to determine the effect of AEA treatment on hyperplastic cholangiocyte growth [in vivo, using the classical BDL model of cholangiocyte hyperplasia (2, 32), and in vitro] and 2) to determine the role of the TRX1/Ref1/AP-1 pathway on the AEA-mediated effects on cholangiocyte proliferation.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials

All reagents were purchased from Sigma Chemical (St. Louis, MO) unless otherwise stated. AEA and AMG9810 were purchased from Tocris Bioscience (Ellisville, MO). The specific Cb1 and Cb2 inhibitors SR141716 and SR144528 were generously supplied by the National Institute on Drug Abuse drug supply program (Bethesda, MD). All primers for real-time PCR and all short hairpin (sh)RNA plasmids were purchased from SuperArray (Frederick, MD). Antibodies directed against TRX1, TRX2, Ref1, all members of the Fos and Jun families, H3 histone, and proliferating cellular nuclear antigen (PCNA) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Specific antibodies against the Cb1 and Cb2 receptors were purchased from Cayman Chemicals (Ann Arbor, MI). The cytokeratin 7 (CK-7) antibody was obtained from Caltag Laboratories (Burlingame, CA).

Animal Treatment

Male C57/Bl6N mice (20–25 g) were purchased from Charles River (Wilmington, MA). The animals were kept in a temperature-controlled environment (22°C) with a 12:12-h light-dark cycle and free access to standard rodent chow and water. The studies were performed in 1) sham-operated mice that were treated via intraperitoneally implanted Alzet osmotic minipumps with 10 mg·kg–1·day–1 AEA (dissolved in 1:4 Tocrisolve-NaCl) (66) or vehicle for 3 and 7 days and 2) mice that underwent BDL surgery (3) and were immediately treated via intraperitoneally implanted Alzet osmotic minipumps with 10 mg·kg–1·day–1 AEA (dissolved in 1:4 Tocrisolve-NaCl) or vehicle for 3 and 7 days. Study protocols were performed with strict adherence to institution guidelines.

Cholangiocyte Isolation

Freshly isolated cholangiocytes (97–100% pure as shown by CK-7 immunohistochemistry) from the selected group of animals were obtained by immunoaffinity bead separation (5, 6) using a mouse monoclonal antibody (IgM; kindly provided by Dr. R. Faris, Brown University, Providence, RI) against an unidentified membrane antigen expressed by all intrahepatic cholangiocytes. Cell number and viability (>97%) were assessed using standard trypan blue exclusion.

In Vitro Studies

To dissect the effects of AEA treatment on intracellular signaling pathways in vitro, we resuspended the affinity-purified, freshly isolated cholangiocytes (from 3-day BDL mice) in RPMI buffer containing DNase to prevent cell clumping. Cholangiocytes were then stimulated with AEA (10–5 M) for various time points up to 2 h in the presence or absence of specific cannabinoid receptor inhibitors: SR141716, a Cb1 antagonist (10 nM) (60); SR144528, a Cb2 antagonist (10 nM) (35); or AMG9810, a VR1 antagonist (10 nM) (18). However, because freshly isolated cholangiocytes have a very limited survival time in culture (Alpini G, unpublished observations) and because they cannot be transfected and thus are not conducive to genetic manipulation studies, we also performed our studies in an SV40-transformed mouse cholangiocyte cell line that retains cholangiocyte phenotypes (49).

Cannabinoid Receptor Expression

Cannabinoid receptor expression (Cb1, Cb2, and VR1) was evaluated by 1) real-time PCR and 2) immunoblotting in freshly isolated cholangiocytes from BDL and sham-operated mice, as well as in situ by immunofluorescent staining of frozen liver sections. Specific details of the methodology can be found in the supplemental information. Supplemental data for this article is available online at the American Journal of Physiology-Gastrointestinal and Liver Physiology website.

Analysis of Cholangiocyte Proliferation

In vivo. Cholangiocyte proliferation was determined from 1) the number of CK-7- and PCNA-positive cholangiocytes in liver sections (5 µm thick, 6 slides evaluated for each group) (7) and 2) PCNA protein expression (an index of cell replication) (21) in purified cholangiocytes from the selected group of animals. Immunohistochemical staining for PCNA and CK-7 were performed as previously described (7). For all immunoreactions, negative controls were also included. After staining, sections were counterstained with hematoxylin and examined with a microscope (Olympus BX 40; Olympus Optical). Over 100 cholangiocytes were counted in a random, blinded fashion in three different slides for each group of animals by analyzing six nonoverlapping fields from each slide. Data are expressed as the number of CK-7- and PCNA-positive cholangiocytes per portal tract. For PCNA immunoblots, the comparability of the protein loaded was normalized to immunoblots for β-actin. The intensity of the bands was determined by scanning video densitometry using the phosphorimager Storm 860 (Amersham Biosciences) with ImageQuant TLV 2003.02.

In vitro. Cholangiocyte proliferation was determined in vitro by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4- sulfophenyl)-2H-tetrazolium (MTS) inner salt cell proliferation assay (14, 24) in the SV40 cholangiocyte cell line. After trypsinization, cells were seeded into 96-well plates (10,000 cells/well) in a final volume of 200 µl of medium. Cells were stimulated for 24, 48, and 72 h with AEA (1 pM to 10 µM). Cell proliferation was assessed using a colorimetric cell proliferation assay (CellTiter 96Aqueous; Promega, Madison, WI), and absorbance was measured at 490 nm by a microplate spectrophotometer (Versamax; Molecular Devices, Sunnyvale, CA).

To determine possible mechanisms of action, SV40 cholangiocytes were first preincubated for 1 h with various inhibitors before the addition of AEA for 48 h. The inhibitors utilized were SR141716 [a Cb1 antagonist, 10 nM (60)], SR144528 [a Cb2 antagonist, 10 nM (35)], and AMG9810 [a VR1 antagonist, 10 nM (18)].

In all cases, data are expressed as the degree of change of treated cells compared with vehicle-treated controls. Statistical significance was determined using a t-test, and P < 0.05 was considered significant.

Analysis of Apoptosis

In vivo. We evaluated cholangiocyte apoptosis using terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) analysis (46) in paraffin-embedded liver sections (5 µm, 3 slides evaluated for each group) from the selected group of animals using a commercially available kit (Wako Chemicals, Tokyo, Japan). After counterstaining with hematoxylin solution, liver sections were examined by light microscopy with an Olympus BX-40 microscope (Tokyo, Japan) equipped with a camera. Approximately 100 cells per slide were counted in a coded fashion in 10 nonoverlapping fields.

In vitro. Annexin V staining was used as an indicator of apoptosis after in vitro treatment of freshly isolated BDL cholangiocytes as well as in the SV40-transformed cholangiocytes with AEA (10 µM) in the absence or presence of specific cannabinoid receptor inhibitors as described previously (14). Approximately 100 cells per slide were counted in a coded fashion in 10 nonoverlapping fields. Data are expressed as the percentage of Annexin V-positive cells; statistical significance was determined using a t-test, and P < 0.05 was considered significant.

Intracellular Signaling Mechanisms

ROS detection. We evaluated the intracellular ROS accumulation in freshly isolated cholangiocytes from sham-operated and BDL mice as well as in our SV40-transformed cell line. These cells were incubated in the presence or absence of AEA (10 µM) for 2 h at 37°C. ROS accumulation was detected using the indicator dye 5-(and-6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate (DCF; Invitrogen, Carlsbad, CA) as described previously (46). Details of the methodology can be found in the supplemental information. The relative fluorescence was detected using the Olympus IX71 inverted confocal microscope and quantitated using Adobe Photoshop, where the green pixel intensity per cell was assessed. Data are expressed as means ± SE in at least five random fields per treatment from three independent experiments.

Thioredoxin activation. The effects of AEA on the expression of TRX1 was evaluated in 1) freshly isolated cholangiocytes from vehicle- and AEA-treated BDL mice in vivo, 2) in vitro stimulated freshly isolated cholangiocytes from BDL mice, and 3) an in vitro stimulated SV40 mouse cholangiocyte cell line, using real-time PCR and immunoblotting as described above. In addition, the nuclear translocation of TRX1 was evaluated by immunofluorescence microscopy in vitro in SV40-transformed cholangiocytes. Cells were seeded into six-well dishes containing a sterile coverslip on the bottom of each well. Cells were allowed to adhere overnight and incubated with AEA (10 µM) for various times up to 2 h. Cells were then washed once in cold PBS and fixed to the coverslip with 4% paraformaldehyde (in PBS) at room temperature for 5 min, and stained for immunofluorescence as described above using an anti-TRX1 antibody. Nuclear translocation of TRX1 was also assessed using cellular fractionation followed by immunoblotting of the nuclear and cytoplasmic fractions. Briefly, SV40-transformed cells were stimulated with AEA (10 µM) for 2 h, and the cells were scraped and pelleted in ice-cold PBS. Cells were then lysed in a lysis buffer containing 10 mM HEPES, pH 7.4, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, and 0.1% Igepal and incubated on ice for 15 min. The intact nuclei were centrifuged, and the supernatant was retained as the cytoplasmic fraction. The nuclear pellet was washed once with lysis buffer and centrifuged again. Nuclear proteins were extracted in 2 volumes of a high-salt buffer containing 20 mM HEPES, pH 7.9, 20% glycerol, 0.42 M NaCl, 1 mM EDTA, 0.5 mM PMSF, and 0.5 mM DTT on ice for 30 min, sonicated, and centrifuged at maximum speed for 10 min. Equal amounts of nuclear and cytoplasmic proteins were loaded onto an SDS-PAGE gel and processed for Western blotting using the TRX1 antibody as well as β-actin as a cytoplasmic protein marker and H3 histone as a nuclear marker to assess the purity of the samples.

Thioredoxin reductase activity. Changes in the activity of thioredoxin reductase were assessed using a commercially available kit (Sigma). Briefly, freshly isolated cholangiocytes and SV40-transformed cells were incubated for various times up to 1 h with AEA (10 µM), after which time cells were scraped in lysis buffer containing 20 mM Tris·HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% NP-40, 1 mM aprotinin, 1 mM PMSF, and 1 mM leupeptin. Thioredoxin reductase activity was assessed according to the manufacturer's instructions and is expressed as the relative increase in units per minute per milligram of protein.

Ref1 activation. Ref1 expression was also determined in the three experimental models described for TRX1 by performing real-time PCR and immunoblotting. In addition, relative binding activity of Ref1 and TRX1 after AEA treatment was assessed using coimmunoprecipitation studies. Briefly, SV40 cholangiocytes were plated onto 10-cm dishes and allowed to adhere overnight. Cells were treated with AEA for 2 h and lysed in lysis buffer as described for Western blotting. Protein G-Sepharose (100 µl per sample) was washed in PBS. The pellet was resuspended in 500 µl of PBST (PBS containing 0.1% Tween 20). Either TRX1- or Ref-1-specific antibody (1 µg) and total protein (30 µg) were used in the immunoprecipitation reaction that was allowed to proceed overnight at 4°C. The immunoprecipitation was washed in PBST, and the resulting pellet was resuspended in 100 µl of SDS-PAGE denaturing buffer and denatured for 5 min at 95°C. Each sample (20 µl) was loaded onto an SDS-PAGE gel and processed for immunoblotting as described above.

AP-1 activation. The relative expression of the members of the AP-1 transcription factor family (c-Fos, Fos B, Fra-1, Fra-2, c-Jun, Jun B, and Jun D) was determined in the three AEA treatment models described by performing real-time PCR and immunoblotting. In addition, relative AP-1 DNA-binding activity was assessed using EMSA as described previously (34). Double-stranded oligonucleotides containing either the consensus binding motif for AP-1 (5'-CGC TTG ATG AGT CAG CCG GAA-3') or Octamer consensus binding motif (5'-TGT CGA ATG CAA ATC ACT AGA A-3'; Promega) was end labeled with [32P]dATP using T4 polynucleotide kinase for 10 min at room temperature.

To identify the AP-1 protein(s) involved in the AP-1 DNA-binding activity, supershift analysis was performed (34). Equal aliquots (2 µg) of nuclear protein were incubated with 1 µg of specific antibody directed against each of the members of the Fos and Jun families. The binding activities were resolved through a 1.6% glycerol-6% polyacrylamide gel for 4 h at 130 V. A positive supershift can be identified as either a retardation of the band in the gel or as a decrease in the intensity of the binding activity compared with the control (no antibody) due to the interference of the antibody with the DNA-binding site on the protein.

In addition, the AP-1 transcriptional activity was assessed in SV40 cholangiocytes using a luciferase reporter construct coupled to a promoter region containing the AP-1 consensus sequence (Panomics, Fremont, CA). SV40-transformed mouse cholangiocytes were plated onto 96-well plates at a density of 10,000 cells/well and allowed to adhere overnight. Cells were then transfected with the AP-1-luciferase reporter construct (0.1 µg DNA/well) with 0.28 µl of TransIT-LT1 transfection reagent (Mirus, Madison, WI) overnight at 37°C. After this time, the cells were stimulated with various concentrations of AEA (10 µM) in the absence or presence of the specific cannabinoid receptor antagonists mentioned previously. After 24 h, cells were assayed for luciferase activity using the luciferase assay kit (Promega) and a Fluoroskan Ascent FL luminometer. Treatments were done at least in quadruplicate, and results are expressed as the degree of change of luciferase activity per microgram of protein.

Stable Transfected Knockdown Cell Lines

The roles of TRX1, Ref1, c-Fos, and c-Jun expression in the antiproliferative actions of AEA were demonstrated using cells that have the expression of each of these genes stably knocked down. These cell lines were established using SureSilencing shRNA (SuperArray, Frederick, MD) plasmids for mouse TRX1, Ref1, c-Fos, and c-Jun, containing a marker for neomycin resistance for the selection of stably transfected cells, according to the instructions provided by the vendor. Specific details as to the methodology used to establish these cell lines can be found in the supplemental information. The three cell lines, mock-transfected clone ("neo neg"), the TRX1 knockdown clone, and the Ref1 knockdown clone were treated with AEA (10 µM) for 0, 30, and 60 min, and c-fos and c-jun mRNA expression was determined using real-time PCR. In addition, AP-1 transcriptional activity was determined by luciferase assay as described previously after the following treatments: 1) basal, 2) AEA treatment, 3) simultaneous stimulation with AEA and 10 mM DTT; and 4) AEA treatment for 2 h before the addition of DTT. The roles of TRX1, Ref1, c-Fos, and c-Jun in AEA-induced cell death were determined in these cell lines by MTS assay following AEA (10 µM) treatment as described above.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Cannabinoid Receptor Expression

Cb1 and VR1 mRNA and protein expression were decreased in proliferating BDL cholangiocytes at 3 and 7 days compared with normal cholangiocytes, whereas Cb2 expression was upregulated at 3 and 7 days ~50-fold after BDL (Fig. 1, A and B). With immunofluorescence microscopy, all of the cannabinoid receptors were predominantly found in cholangiocytes (colocalized with CK-7 staining), with weak expression in the surrounding hepatocytes (Fig. 1C). There was no staining observed after substitution of preimmune serum for the primary antibodies as a negative control (not shown).


Figure 1
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  		Fig. 1. Cannabinoid receptor expression in cholangiocytes from sham-operated (normal) or bile duct ligation (BDL) mice. As shown by real-time PCR (A), immunoblotting (B), and immunofluorescence (C), cannabinoid receptor 1 (Cb1) and vanilloid receptor (VR1) expression are significantly decreased in cholangiocytes isolated from BDL mice, whereas Cb2 expression is upregulated. Data are expressed as relative cannabinoid receptor expression (means ± SE; n = 4) after correction for the housekeeping genes GAPDH (A) or β-actin (B). *P < 0.05 vs. normal cholangiocytes. As shown by immunofluorescence in liver sections (C) cannabinoid receptor expression (red) was colocalized with cytokeratin 7 (CK-7; green). Liver sections were counterstained with 4,6-diamidino-2-phenylindole (DAPI; blue). Scale bar, 20 µm. No immunofluorescent staining was observed when normal and BDL liver sections were incubated with preimmune serum (not shown).

 
AEA Effects on Cholangiocyte Proliferation

Systemic administration of AEA for 3 or 7 days to normal mice had no effect on biliary mass (Fig. 2A). In contrast, treatment of BDL mice with AEA for 3 and 7 days resulted in a significant decreased in biliary mass (Fig. 2A). To support this, we also evaluated PCNA protein immunoreactivity and protein expression as a marker of proliferative activity and observed no changes in PCNA expression in normal cholangiocytes after AEA treatment (Fig. 2, B and C). However, consistent with previous studies (25, 46), there was an increase in PCNA expression 3 and 7 days after BDL, indicating enhanced cholangiocyte proliferation, which was significantly reduced after in vivo AEA treatment for 3 and 7 days (Fig. 2, B and C).


Figure 2
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  		Fig. 2. Anandamide (AEA) treatment suppresses cholangiocyte growth in BDL mice. Mice were treated with 10 mg·kg–1·day–1 AEA for 3 and 7 days immediately after either sham or BDL surgery. Measurement of the number of CK-7 cholangiocytes (A) and PCNA-expressing cholangiocytes (B) in liver sections (indicated by arrows) revealed a decrease in cholangiocyte growth in BDL but not normal livers after AEA treatment. Data are means ± SE. *P < 0.05 vs. vehicle-treated BDL mice. C: proliferating cellular nuclear antigen (PCNA) protein expression was assessed by immunoblotting in cholangiocytes isolated from mice that underwent BDL or sham surgery and were then immediately treated with either vehicle or AEA for 3 and 7 days. Data are means ± SE of n = 4 experiments. AEA (10–5 M) also had antiproliferative effects on SV40-transformed cholangiocytes in vitro, as shown by 3-(4,5dimethylthiazol-2-yl)-5-(3-carboxy- methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assays (D), which was blocked by pretreatment with the Cb2 antagonist SR144528 but not by either the Cb1 (SR141716) or VR1 antagonist (AMG9810) (E). Data are expressed as fold change (means ± SE, n = 7). *P < 0.05 vs. basal treatment or cholangiocytes treated with receptor antagonist alone. #P < 0.05 vs. AEA-treated cholangiocytes.

 
To confirm that the antiproliferative effects of AEA in our in vivo model are a result of the direct effects of AEA on cholangiocytes, we treated our two in vitro cell culture models with AEA. Using MTS proliferation assays in our SV40 mouse cell line, we showed that AEA had a marked antiproliferative effect on cholangiocyte growth at a concentration of 10 µM but not at a lower dose (Fig. 2D), possibly due to the very short half-life of AEA (17). Furthermore, this growth-suppressing effect of AEA could be partially blocked by pretreatment with a specific Cb2 receptor antagonist but not with Cb1 and VR1 antagonists, suggesting a Cb2-dependent mechanism (Fig. 2E). A Cb2-dependent, growth-suppressing effect of AEA was confirmed by in vitro treatment of the freshly isolated mouse cholangiocytes, followed by the evaluation of PCNA protein expression (data not shown).

AEA Induces Cholangiocyte Apoptosis

We evaluated apoptosis in vivo by TUNEL staining and in vitro by Annexin V staining. No TUNEL-positive cholangiocytes were observed in normal mouse liver treated with either vehicle or AEA for 3 and 7 days (Fig. 3A). However, TUNEL-positive cholangiocytes were evident after BDL, the incidence of which was increased with subsequent in vivo AEA treatment (Fig. 3A). The incidence of apoptosis was also increased after in vitro treatment of SV40 cholangiocytes as demonstrated by Annexin V staining (Fig. 3B). This effect could be blocked with pretreatment of the cholangiocytes with the specific Cb2 antagonist SR144528 but not with specific antagonists of Cb1 or VR1 (Fig. 3B), further supporting a Cb2-dependent mechanism.


Figure 3
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  		Fig. 3. AEA treatment increases cholangiocyte apoptosis. A: mice were treated with 10 mg·kg–1·day–1 AEA for 3 and 7 days immediately after either sham or BDL surgery. Apoptosis was detected by terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) staining (represented by the arrow) in liver sections. Data are expressed as the percentage of TUNEL-positive cholangiocytes per portal tract (means ± SE). *P < 0.05 vs. vehicle-treated BDL mice. B: measurement of apoptosis (by Annexin V staining) in SV40-transformed cholangiocytes, which were treated with AEA (10–5 M) in the absence or presence of specific cannabinoid receptor antagonists for 24 h before apoptosis was evaluated by Annexin V staining. The number of Annexin-positive cells was expressed as a percentage of total cells. Data are means ± SE from 5 random fields from 3 independent experiments. *P < 0.05 vs. basal treatment.

 
AEA Increases ROS

Because AEA-induced apoptosis is associated with the generation of ROS (30), we evaluated the effects of AEA on ROS accumulation in both in vitro models. Cholangiocytes isolated from BDL (but not normal) mice showed a marked increase in the intracellular accumulation of ROS after AEA treatment in vitro (Fig. 4). This increase was also observed after AEA treatment of our SV40 cell line (not shown).


Figure 4
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  		Fig. 4. AEA treatment increases intracellular accumulation of ROS in cholangiocytes isolated from BDL mice. Cholangiocytes were isolated from BDL and control mice and stimulated in vitro with AEA (10–5 M) for 2 h before the addition of the indicator dye 5-(and-6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate (DCF). Data are expressed as fold change in pixel intensity of the DCF staining per nuclei (means ± SE) in 5 different fields from 3 independent experiments. *P < 0.05 vs. vehicle-treated BDL cholangiocytes.

 
AEA-Induced Cell Death is via a TRX1/Ref1/AP-1-Dependent Pathway

We next evaluated the downstream signaling events associated with the AEA-induced cell death. Chronic administration of AEA to BDL mice in vivo resulted in an upregulation of TRX1 mRNA and protein expression (Fig. 5, A and B) compared with vehicle treatment. Acute AEA treatment in vitro to cholangiocytes isolated from BDL mice as well as to the SV40 cell line also resulted in an upregulation of TRX1 expression (Fig. 5, A and B, and Supplemental Fig. S1). No difference was observed in the expression of the related TRX2 (data not shown). One of the actions of TRX1 is to translocate from the cytoplasm to the nucleus, where it interacts and facilitates the actions of several proteins (22, 38, 41, 48, 52). With the use of immunofluorescence microscopy, it is evident that after AEA treatment, TRX1 translocates from the cytoplasm to the nucleus in the SV40 cell line (Fig. 5C). Furthermore, by subcellular fractionation of SV40 cholangiocytes after AEA treatment, followed by immunoblotting for TRX1, H3 histone [as a nuclear marker (53)] and β-actin [as a cytoplasmic marker (56)], it can be seen that there is a decrease in TRX1 protein in the cytoplasmic fraction and an increase in the nuclear fraction after AEA treatment (Fig. 5C), further indicating that TRX1 translocates into the nucleus of cholangiocytes after AEA treatment in vitro. The activity of TRX1 requires this protein to remain in the reduced form (10), which is the function of TRX reductase. Activity of this enzyme was upregulated 1 and 2 h after AEA treatment in the SV40 cholangiocyte cell line (Fig. 5D). Together, these data suggest that AEA increases not only the expression of TRX1 in cholangiocytes but also the nuclear activity of this protein.


Figure 5
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  		Fig. 5. AEA treatment increases thioredoxin 1 (TRX1) expression and activity. AEA was administered to BDL mice in vivo and to the SV40-transformed cholangiocyte cell line in vitro. Relative TRX1 expression was determined by real-time PCR (A) and immunoblotting (B). Data are expressed as relative TRX1 expression (means ± SE) after correction for the housekeeping genes GAPDH (A) or β-actin (B). *P < 0.05 vs. basal treatment. TRX1 nuclear translocation was demonstrated in SV40-transformed cholangiocytes after AEA treatment by immunofluorescence and by subcellular fractionation followed by immunoblotting (C). TRX1 immunoreactivity (red) was predominantly in the cytoplasm under basal conditions but was found in the nucleus after AEA treatment. Nuclei were counterstained with DAPI (blue). The relative activity of TRX reductase was determined in SV40-transformed cholangiocytes after AEA treatment (D). Data are expressed as fold change in the rate of TRX activity (means ± SE, n = 4). *P < 0.05 vs. basal treatment.

 
One of the nuclear targets of TRX1 activation is Ref1 (67). Ref1 mRNA and protein expression are increased after chronic in vivo AEA treatment of BDL mice as well as in both in vitro models of acute AEA stimulation (Fig. 6, A and B, and Supplemental Fig. S2). In addition, because the modulation of Ref1 activity by TRX1 normally requires a physical interaction between the two molecules (67), we performed coimmunoprecipitation studies to evaluate this interaction. As shown in Fig. 6C, there is an increased association of Ref1 and TRX1 after AEA treatment in the SV40-transformed cholangiocytes.


Figure 6
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  		Fig. 6. AEA treatment increases redox factor 1 (Ref1) expression. AEA was administered to BDL mice in vivo and to the SV40-transformed cholangiocyte cell line in vitro. Relative Ref1 expression was determined by real-time PCR (A) and immunoblotting (B). Data are expressed as relative Ref1 expression (means ± SE, n = 4) after correction for the housekeeping genes GAPDH (A) or β-actin (B). *P < 0.05 vs. basal treatment. Relative association of Ref1 and TRX1 was determined by coimmunoprecipitation (C). Extracts from SV40-transformed cholangiocytes treated with AEA were immunoprecipitated with either TRX1- or Ref1-specific antibodies. Precipitates were then immunoblotted for Ref1.

 
We next evaluated the effects of AEA treatment on the expression of the Fos and Jun members of the AP-1 transcription factor family. Chronic treatment of BDL mice with AEA resulted in an upregulation of c-Fos and c-Jun mRNA and protein (Fig. 7, A and B, and Supplemental Figs. S3 and S4) with no observable effect on any other family member (Fos B, Fra-1, Fra-2, Jun B, or Jun D; data not shown). Furthermore, acute AEA treatment in both freshly isolated cholangiocytes from BDL mice and the SV40-transformed cell line also resulted in an upregulation of both c-Fos and c-Jun expression (Fig. 7, A and B). To confirm that this increase in protein expression translated into an increase in AP-1 DNA-binding activity, we performed EMSA and demonstrated that there was an increase in AP-1 DNA-binding activity after acute AEA treatment of freshly isolated cholangiocytes and SV40-transformed cholangiocytes (data not shown) as well as after chronic AEA treatment of BDL mice (Fig. 7C). Octamer DNA-binding activity was assessed as a control to ensure that the increase in AP-1 DNA-binding activity was specific. In addition, using a supershift analysis and specific antibodies to the AP-1 family members, we determined that c-Fos and c-Jun are the major contributors to the AP-1 DNA-binding activity observed (Fig. 7C) as shown by an apparent decrease in AP-1 DNA binding due to the interference of the added antibody in the interaction between AP-1 and its consensus oligonucleotide. We then assessed the AP-1 transactivation potential in our SV40-transformed cell line after AEA treatment, using an AP-1 reporter construct. As is evident in Fig. 7D, AEA treatment increases the AP-1-driven luciferase activity, which can be blocked by pretreatment with SR144528, the specific Cb2 receptor antagonist, but not by pretreatment with specific antagonists of Cb1 or VR1, offering further support of a Cb2-dependent mechanism.


Figure 7
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  		Fig. 7. AEA treatment increases c-Fos and c-Jun expression. AEA was administered to BDL mice in vivo and to the SV40-transformed cholangiocyte cell line in vitro. Relative c-Fos and c-Jun expression was determined by real-time PCR (A) and immunoblotting (B). Data are expressed as relative c-Fos or c-Jun expression (means ± SE, n = 4) after correction for the housekeeping genes GAPDH (A) or β-actin (B). *P < 0.05 vs. basal treatment. The effects of AEA treatment on AP-1 DNA-binding activity in vivo were determined by EMSA (C) using labeled oligonucleotides containing the AP-1 consensus sequence. Oct DNA-binding activity was assessed as a loading control. The identity of the proteins contributing to the AP-1 DNA-binding activity after in vivo AEA treatment of BDL mice was determined by supershift analysis using specific antibodies against each of the AP-1 proteins. A positive supershift (reduction in band intensity) was evident with the c-Fos and c-Jun antibodies. AP-1 transcriptional activity was assessed using an AP-1 luciferase construct transfected in the SV40-transformed cholangiocyte cell lines (D). Transfected cells were treated with AEA (10–5 M) or vehicle (Veh) in the presence or absence of specific cannabinoid receptor antagonists. Data are expressed as average luciferase activity per µg of protein (means ± SE, n = 7). *P < 0.05 vs. vehicle-treated cholangiocytes.

 
To define the roles of TRX1 and Ref1 expression in the AEA mediated events, we established subclones of the SV40 mouse cholangiocyte cell line that were stably transfected with shRNA expression plasmids for TRX1 and Ref1. The degree and specificity of this knockdown is shown in Supplemental Fig. S5. The TRX1 shRNA clone knocked expression down by 85% compared with cells transfected with an empty vector containing neomycin resistance (designated neo neg), without effecting the expression of TRX2, whereas the Ref1 shRNA clone knocked down Ref1 mRNA expression by 96% compared with neo neg cells. We then determined what effect silencing of TRX1 or Ref1 expression has on the induction of c-fos and c-jun mRNA expression and AP-1 activity. Interestingly, although the absence of Ref1 or TRX1 expression had no effect on the induction of c-fos or c-jun mRNA expression (Fig. 8A), there was no concomitant upregulation of AP-1 luciferase activity (Fig. 8A), suggesting a posttranslational effect of TRX1 and Ref1 on the AP-1 protein. Because the main action of Ref1 on AP-1 activity is to maintain the redox state of the proteins, thus facilitating the DNA-binding activity (69, 70), and because AEA treatment causes an accumulation of ROS (Fig. 4A), we performed the AP-1 luciferase assay in the presence of the reducing agent DTT. When we stimulated the cells with AEA and DTT simultaneously, we observed no increase in AP-1 luciferase activity in any cell line studied (data not shown); however, when we first treated the cell lines with AEA for 2 h, to allow the induction of c-Fos and c-Jun proteins, and then added the reducing agent DTT, we effectively restored the AEA-induced AP-1 luciferase activity in the TRX1 shRNA and Ref1 shRNA clones (Fig. 8A), indicating a regulatory role for Ref1 and TRX1 in the maintenance of AP-1 proteins in the reduced state. We then determined whether, indeed, Ref1 and TRX1 expression was essential for the AEA-induced cell death by using MTS assays. By knocking down the expression of either Ref1 or TRX1, we effectively prevented the AEA-mediated cell death (Fig. 8B).


Figure 8
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  		Fig. 8. Stable knockdown of TRX1 and Ref1 expression inhibits AEA-induced AP-1 transcriptional activity but not c-Fos and c-Jun expression. Knockdown of TRX1 and Ref1 expression was achieved by the stable transfection of TRX1 or Ref1 short hairpin (sh)RNA plasmid constructs into SV40-transformed cholangiocytes. Stable transfected cell lines were then treated with AEA (10–5 M), and the relative expression of c-fos and c-jun mRNA was assessed using real-time PCR (A). Data are expressed as relative mRNA expression (means ± SE, n = 4) after correction for GAPDH expression. *P < 0.05 vs. basal treatment. AP-1 transcriptional activity was assessed using an AP-1 luciferase construct transfected into the stable knockdown cell lines (A). Transfected cells were treated with AEA (10–5 M) or AEA for 2 h, followed by the addition of DTT. Data are expressed as average luciferase activity per µg of protein (means ± SE, n = 7). *P < 0.05 vs. basal treatment within each cell line. The effect of AEA on cell proliferation was assessed in these cell lines by performing MTS assays (B). Specific knockdown of TRX1 and Ref1 prevented the antiproliferative effects of AEA. Data are expressed as fold change (means ± SE, n = 7). *P < 0.05 vs. basal treatment within each cell line.

 
To further characterize the signaling pathways associated with AEA-mediated cholangiocyte cell death, we established shRNA clones that have reduced c-Fos or c-Jun expression. By stably transfecting our SV40 mouse cholangiocyte cell line with either c-Fos or c-Jun shRNA plasmids, we specifically knocked down the expression of these two proteins (91 and 98%, respectively) but not any other member of the Fos or Jun families (Supplemental Fig. S6). By eliminating the expression of either of these two proteins, we were able to block the AEA-mediated cell death pathway (Fig. 9), again suggesting an integral role for the TRX1/Ref1/AP-1 pathway in the AEA-mediated suppression of biliary growth.


Figure 9
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  		Fig. 9. Specific knockdown of c-Fos and c-Jun expression inhibits AEA-induced cell death. Knockdown of c-Fos and c-Jun expression was achieved by the stable transfection of c-Fos or c-Jun shRNA plasmid constructs into SV40-transformed cholangiocytes. The effect of AEA on cell proliferation was assessed in these cell lines by performing MTS assays. Specific knockdown of c-Fos and c-Jun prevented the antiproliferative effects of AEA. Data are expressed as fold change (means ± SE, n = 7). *P < 0.05 vs. basal treatment within each cell line.

 

   DISCUSSION
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 ABSTRACT
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The major findings of this study relate to the involvement of the endocannabinoid system in the regulation of cholangiocyte growth. Using a mouse model of cholestatic liver disease, we showed that 1) chronic AEA treatment in vivo inhibited biliary growth after BDL; 2) specific inhibitors of the cannabinoid receptor Cb2 prevented the growth-suppressing effects of AEA; 3) the actions of AEA on proliferating cholangiocytes involve an increased accumulation of ROS, upregulation of TRX1 expression, activation, and nuclear translocation, and subsequent increased association with Ref1; and 4) transcriptional activity of AP-1 increased, comprising c-Fos and c-Jun. A schematic diagram summarizing the data presented in this report is shown in Fig. 10. Altogether, we interpreted our data to suggest that activation of Cb2 by AEA results in increased intracellular ROS accumulation. This, in turn, results in an increased expression and nuclear translocation of TRX1 (but not TRX2), where it interacts with Ref1. In addition, increased ROS results in an upregulation of c-Fos and c-Jun expression, which together constitute the AP-1 DNA-binding activity. The AEA-induced AP-1 transcriptional activity is inhibited in the absence of the TRX1/Ref1 complex, thus suggesting posttranslational control of TRX1/Ref1 in AP-1 transcriptional activity.


Figure 10
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  		Fig. 10. Schematic diagram of the potential cell signaling mechanisms responsible for the AEA-induced cell death of proliferating cholangiocytes after BDL. Activation of Cb2 by AEA results in increased intracellular ROS accumulation (red asterisks). This, in turn, results in an increased expression and nuclear translocation of TRX1 (but not TRX2), where it interacts with Ref1. In addition, increased ROS results in an upregulation of c-Fos and c-Jun expression, which together constitute the AP-1 DNA-binding activity. However, this transcription factor, under oxidized conditions such as that shown (i.e., in the presence of increased ROS) fails to retain AP-1 transcriptional activity. This apparent dichotomy is resolved by the reducing properties of the TRX1/Ref1 complex, which restores the AP-1 complex to its reduced form, thereby allowing DNA-binding activity and the subsequent transcription of AP-1 target genes that are responsible for the AEA-induced cell death.

 
To date, our knowledge concerning the roles of the endocannabinoid system in liver function and disease is scant; however, an emerging role of the endocannabinoid system in the regulation of liver fibrosis is evident. Experimental evidence suggests that there are opposing functions of the cannabinoid receptors Cb1 and Cb2 in the fibrogenic process (65); however, the direct role of cannabinoids on the hyperplastic growth of cholangiocytes is unknown.

In the present studies, the antiproliferative effects of AEA were associated with increased accumulation of ROS. This is supported by previous studies in which AEA induced apoptosis in Chang liver cells, which could be prevented by the addition of a thiol-reducing agent that maintains the intracellular glutathione level in the reduced state, thus counteracting the effects of ROS accumulation (30). It is believed that the generation of ROS by AEA may occur as a result of increased ceramide production from AEA-mediated induction of sphingomyelin hydrolysis (12, 14, 27, 37). Indeed, AEA treatment of cholangiocarcinoma cells results in an increased ceramide production (14). In addition, increased ROS accumulation is associated with cholangiocyte apoptosis induced by the cessation of {alpha}-naphthylisothiocyanate feeding (46). Furthermore, ROS generation is associated with fibrogenesis seen as a result of CCl4 administration (55). Together, these data support a role of ROS accumulation in cholangiocyte apoptosis induced by AEA.

The activation of AP-1 by cannabinoids has been demonstrated previously and regulates a wide variety of intracellular responses (30, 36, 58, 62). Members of the Fos and Jun families are upregulated in response to a large number of stimulants, including oxidative stressors (59). Indeed, the AEA-induced AP-1 transcriptional activity could be abolished by the simultaneous addition of the reducing agent DTT, which supports a role of AEA-induced ROS accumulation in the activation of AP-1 activity in cholangiocytes. Similar to our studies, Giuliano et al. (30) recently demonstrated that AEA treatment of Chang liver cells induced apoptosis via the activation of AP-1. However, in contrast to the present data, the AP-1 complex comprised mainly c-Jun and Jun B, with c-Fos representing only a minor component (30). In our cellular models of cholangiocyte apoptosis, c-Fos and c-Jun were the main components of the AP-1 complex, both of which were required for AEA-induced cell death. Because the AP-1 complex is a heterodimer made up of members of the Fos and Jun families, the cellular outcome of AP-1 activity is varied (9). A role for c-Jun in AP-1-mediated apoptosis is well established and supported by a vast array of data in many different organs and cellular models (19, 20, 33, 42), whereas the importance of c-Fos in the apoptotic process is less evident (23, 26, 29). However, the data generated from our c-Fos and c-Jun shRNA knockdown clones clearly indicate that both of these molecules are essential for AEA-induced apoptosis in cholangiocytes. In addition, when interpreting the present data, it must be acknowledged that the AP-1 pathway does not function in isolation but has been shown previously to interact with such signaling pathways as STAT (47), NF-{kappa}B (16), and MAPK (68), all of which have been shown previously to regulate cholangiocyte growth (1, 50, 57).

The main function of TRX1 is to maintain the intracellular environment in a reduced form (10); therefore, it is not surprising that TRX1 expression and activation are upregulated in response to AEA treatment in our in vivo and in vitro models. Furthermore, TRX1 has been shown to modulate the activity of Ref1 to enhance AP-1 activity after ionizing radiation (41) in a manner similar to that observed presently. In our knockdown experiments, we showed that both TRX1 and Ref1 are essential for the transcriptional activity of AP-1. The main site of action of these two molecules on the AP-1 complex is at the redox-sensitive cysteine motifs that regulate activity in response to oxidative stress (41). As stated earlier, the expression of Fos and Jun are rapidly induced in response to oxidative stimuli (59). However, the DNA-binding activity of the resulting AP-1 complex is inhibited by the same oxidative stimuli (31). This apparent dichotomy is resolved by the reducing properties of the TRX1/Ref1 complex (41, 67, 69, 70), which is strongly supported by the present data, showing that simultaneous stimulation of both the mock-transfected and TRX1 and Ref1 shRNA cholangiocyte clones with AEA and DTT completely abolished AP-1 luciferase activity, whereas AEA administration 2 h before the addition of DTT restored the AP-1 luciferase activity in the TRX1 and Ref1 shRNA clones.

In conclusion, the present data describe a novel pathway by which AEA treatment prohibits cholangiocyte proliferation observed after extrahepatic biliary obstruction. The pathway involves the accumulation of ROS and activation and nuclear translocation of TRX1, where it interacts with Ref1 to modulate the DNA-binding activity of the AP-1 complex. These data suggest that modulation of the endocannabinoid system and/or the ROS/TRX1/Ref1/AP-1 pathway may have important therapeutic implications in the treatment of cholangiocyte proliferation seen after biliary obstruction and in the early stages of cholestatic liver diseases.


   GRANTS
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 ABSTRACT
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This study was supported by an award from Scott & White Hospital (to S. DeMorrow), Health and Labour Sciences Research Grants for the Research on Measures for Intractable Diseases (from the Ministry of Health, Labour and Welfare of Japan), a Grant-in-Aid for Scientific Research C (19590744) from the Japan Society for the Promotion of Science (to Y. Ueno), the Dr. Nicholas C. Hightower Centennial Chair of Gastroenterology, a Veterans Affairs Research Scholar Award, and a Veterans Affairs Merit Award (to G. Alpini).


   ACKNOWLEDGMENTS
 
We acknowledge Glen Cryer of the Scott & White Hospital, Grants Administration Office, for assistance with proofreading and the Scott & White Hospital animal facility staff for assistance with animal surgical models. We also acknowledge the Texas A&M Health Science Center Microscopy Imaging Center for assistance with the confocal microscopy imaging.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. DeMorrow, Division of Research and Education, Scott & White Hospital, Dept. of Internal Medicine, Texas A&M Health Science Center, Medical Research Bldg., 702 SW H. K. Dodgen Loop, Temple, TX 76504 (e-mail: demorrow{at}medicine.tamhsc.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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