Ovariectomy impairs the proliferative and secretory capacity of cholangiocytes from bile duct ligated rats. New evidence for the role of estrogens in modulating the function of the intrahepatic biliary epithelium

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AASLD ABSTRACTS

HEPATOLOGY October 2001

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TRANSLATIONAL REGULATION OF RAT HEPATIC MRP2. Mary Vore, Tim Hoffman, Jingsong Cao, Graduate Center for Toxicology, University of Kentucky, Lexington, ICY

TRANSCRIPTION OF HUMAN A B C B l l IS REGULATED BY THE FARNESOID X RECEPTOR AND BILE SALTS. Jacqueline R Plass, Olaf Mol, Janette Heegsma, Mariska Geuken, Peter L Jansen, University Hospital Groningen, Groningen Netherlands; Michael Muller, University Wageningen, Wageningen Netherlands

Mrp2 mediates the biliary excretion of glutathione, glucuronide and sulfate conjugates. Hepatic expression of Mrp2 is decreased in pregnancy, while Mrp2 mRNA is unchanged, indicative of post-transcriptional regulation of expression. To determine if Mrp2 undergoes translational regulation, we examined expression of Mrp2 rotein and mRNA and the polysomal distribution of Mrp2 mRNA in female control C) and pregnant (P) rats, and in rats treated with corn oil (CO) or pregnenolone16c~-carbonitrile (PCN). Methods: Livers from C and P (19-20 d pregnant) rats, and rats treated with CO or PCN (75 mg/kg/d, 2d; ip) were used for Western analysis of Mrp2 protein in total homogenate. Mrp2 mRNA in total liver and in sucrose gradient fractions was quantitated by Northern analysis. Separation of polyribosomes (polysomes) from single ribosomes (monosomes)by differential sedimentation through a sucrose gradient, followed by Northern analysis of RNA purified from individual fractions of the sucrose gradient, identifies RNA that is being actively translated based on the number of ribosomes with which it is associated. Posmuclear supernatants were fractionated on a continuous sucrose gradient (20-47%) and the 24 fractions (0.5 ml) analyzed for Mrp2 mRNA. Results are expressed as % of total Mrp2 mRNA in the bottom (1; 1-7), middle (II; 8-14) and top (III; 15-21) of the gradient and the monosome (M; 22-24) fractions. Data are shown as Mean + SE where n = 3-5; *, p < 0.05 in C vs P or CO vs PCN, t-test. Results: Total Mrp2 mRNA was the same in C, P, CO and PCN groups; the relative proportion of the three transcripts, 8.3, 6.5 and 5.5 kb, in C was 41.9 + 9.0, 8.3 - 1.5 and 49.8 - 10.5%, respectively, and was not different among the groups. Mrp2 protein was decreased by 50% (*) in P vs C and increased two-fold (*) in PCN vs CO. Polysomal distribution analysis (Table) demonstrated that in C, Mrp2 mRNA is almost uniformly distributed throughout the gradient. In P, there was a marked shift of Mrp2 mRNA from fraction I, containing the heavier polysomes, to fractions II and Ill, relative to C. In PCN, the association of Mrp2 mRNA with fraction I was increased two-fold relative to CO, reflecting a shift of Mrp2 mRNA from fractions 1I and III. Conclusions: The decreased enrichment of Mrp2 mRNA in the denser polysomal fractions in liver from pregnant rats is consistent with a decrease in protein synthesis and decreased Mrp2 protein expression in pregnancy. Conversely, the increased association of Mrp2 mRNA with the fraction most actively engaged in protein Synthesis is consistent with increased Mrp2 protein expression in PCN. Taken together, these data strongly support the hypothesis that in pregnancy and following PCN treatment, hepatic Mrp2 expression is translationally regulated.

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Mrp2 mRNA (% of total in 9radient) Fraction C l 25,5_+3.9 1~ 34,0+3.0 Ill 23.4_+3.3 M t6,8±3.9

P 8.0±1,5" 46,1±2.6' 40.5+..+.2,3" 5.4_+4.1

CO 23.8_+4.8 25.0+3.2 45,2_+5.0 6.0_+3.2

PCN ' 47.3_+6,2* tL4±4.4 29.1±4.7 6.2+2.0

Introduction: ABCB11, formerly known as the Bile Salt Export Pump (BSEP) or Sister of P-glycoprotein (SPGP) mediates the ATP-dependent transport of bile salts across the canalicular membrane of the hepatocyte. A number of bile salts, such as chenodeoxycholic acid (CDCA), have been shown to bind and activate the nuclear hormone receptor, the farnesoid X receptor (FXR). In mice, disruption of the Fxr gene results in a decreased Abcbl I mRNA level A cholate rich diet substantially increased A b c b l l mRNA levels in wild-type mice but not in the Fxr-nun mice. These data indicate that FXR and bile salts are involved in the regulation of routine Abcb 11 expression. The aim of this study was to investigate the regulation of human ABCB11 gene transcription by FXR and bile salts. Methods: W e have cloned a h7-kb promoter region of the human ABCBI1 gene. Analysis for possible regulatory elements revealed a candidate farnesoid X receptor responsive element (FXRE) at position -180 bp relative to the startcodon. The promoter sequence was subcloned into a luciferase reporter gene vector. Site-directed mutations were introduced in the proposed FXRE to determine its involvement in the activation of the ABCB11 promoter. HepG2 and Hek293 cells were cotransfected with these constructs together with an FXR expression vector or a control vector and treated with or without 100/~M CDCA for 24 hours. Subsequently, the luciferase activity was assayed using a Luciferase Reporter Assay System. Next, we investigated whether the endogenous ABCB11 mRNA levels reflected the luciferase activities. HepG2 cells were transfeeted with an FXR expression vector or a control vector and treated with or without 100/xM CDCA for 48 hours. Using realtime quantitative (Taqman) RT-PCR the mRNA level of ABCBll was determined. Results: Cotransfection of the ABCB11 reporter constructs and stimulation with 100/zM CDCA resulted in a 64-fold (HepG2 cells) and 44-fold (Hek293 cells) induction of the basal promoter activity. Mutation of the putative FXRE resulted in a clear reduction of this activity in HepG2 cells (22-fold induction) and in an almost basal activity in Hek293 ceils. Concurrently, significantly increased ABCB11 mRNA levels were detected in FXR transfected and CDCA stimulated HepG2 cells, using real-time quantitative (Taqman) RT-PCR analysis. Conclusion: These results show that the human A B C B l l promoter contains an FXRE and that FXR and bile salts are involved in the transcriptional regulation of the human ABCB 11 gene.

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DOUBLE-STRANDED RNA ACTIVATES A P38 MITOGEN ACTIVATED PROTEIN KINASE (MAPK) DEPENDENT CELL SURVIVAL PROGRAM IN BILIARY EPITHELIA. Laura Tadlock, Yoko Yamagiwa, Tushar Pate1, Scott and White Clinic, Texas A&M University System HSC College of Medicine, Temple, TX

OVARIECTOMY IMPAIRS THE PROLIFERATIVE AND SECRETORY CAPACITY OF CItOLANGIOCYTES FROM BILE DUCT LIGATED RATS. N E W EVIDENCE FOR THE ROLE OF ESTROGENS IN MODULATING THE FUNCTION OF THE INTRAHEPATIC BILIARY EPITHELIUM: Domenico Alvaro, University of Rome, Rome Italy; Gianfranco Alpini, The Texas A&M University System and VA Hospital, Temple, TX; Paolo Onori, University of Rome, Rome Italy; Shannon G]aser, Gene Lesage, Scott & White Hospital, Temple, TX; Antonio Franchitto, Adolfo Attili, Eugenio Gaudio, University of Rome, Rome Italy

The epithelial lining of the biliary tract is exposed to and susceptible to infection with hepatotrophic viruses such as the Hepatitis C virus. However, bile duct damage is infrequently observed during chronic viral hepatitis. Double stranded RNA (dsRNA) produced during replicative viral infection activates the dsRNA-activated protein kinase PKR and triggers host cell antiviral responses that lead to the elimination of infected cells by apoptosis. We postulated that the biliary tract epithelium avoids injury during replicative viral infection by abrogation of apoptotic signaling in response to dsRNA. To test our hypothesis, we assessed the cellular responses of biliary epithelia to dsRNA in vitro. METHODS: MzChA-1 human biliary epithelial cells were incubated with dsRNA (polyIepolyC). Cell proliferation or toxicity was assessed using the MTS viable cell assay. Caspase activity was assessed fluorometrically in cytoplasmic extracts. Activation of p38 MAPK, p44/p42 MAPK, JNK and PKR was assessed by immunoblot analysis using phospho-specific antibodies. SB203580 (25/zM), PD098059 (25 ~M) and 2-aminopurine (1 mM) were used as inhibitors of p38 MAPK, p44/p42 MAPK and PKR respectively. NFt~B activation was assessed using the TRANS-AM kit, and Lactacystin (25/xM) used to inhibit NFJcB activation. RESULTS: Incubation with dsRNA (0-100/xg/ml) for 24 hours did not induce cytotoxicity. Furthermore, incubation with dsRNA for up to 7 days did not affect cell proliferation. However, incubation with 5/~g/ml dsRNA for 24 hours decreased basal caspase 3, 8 and 9- like activities by 55 +4%, 52 +- 13% and 4 8 - 1 % of controls respectively, and decreased cytotoxicity due to 100 nM camptothecin by 58+1%. DsRNA (1 and 5/xg/ml for 24 hours) increased the activation of p38 andJNK but not p44/p42 MAPK. In ceils treated with 5 p~g/ml dsRNA, basal caspase-3 like activity increased from 3 9 +- 5 oB of untreated controls to 8 2 --~-3 o~ in the presence of SB203580, but only to 45+3% in the presence of PD098059. Actinomycin D (10/zg/ml) did not aher cell viability or the decrease in caspase-3 like activity due to dsRNA. Although PKR was constitutively expressed, dsRNA did not increase PKR phosphorylation Furthermore, dsRNA did not activate NFfcB. Neither 2-aminopurine (PKR inhibitor) nor ]actacystin (inhibitor of NFKB activation) altered basal caspase-3 like activity or cen viability in dsRNA treated cells. SUMMARY: These novel observations indicate that dsRNA stimulates a p38 MAPK dependent cell-survival pathway in biliary epithelia that suppresses the activation of the caspase effector machinery independent of de novo protein synthesis or NF~cB activation. CONCLUSIONS: Biliary epithelia exhibit a dominant protective effect over apoptotic signaling in response to dsRNA. We speculate that avoidance of cell death during viral infection may contribute to persistent infection and the establishment of a viral reservoir in the biliary tract.

Cholangiocyteproliferation is a repair mechanism, which counterbalances the progression of chronic cholestaticliver diseasestoward the terminal ductopenicstage.In animalmodels,cholangiocytesproliferate in response to a number of pathologicalstimuli including bile duct ligation (BDL). FollowingBDL, cholangiocyteproliferationis associatedwith increasedductal secretion evidencedby enhanced secretin stimulated cAMPlevelsand secretin-stimulatedbicarbonate-rich eholeresis. Chronic cholestaticliver diseases (e.g., primary bilialy cirrhosis) predominate in femaleswhere estrogensor their metabofitesmay play a potentialpathogenicrole. We haverecently shown that intrahepaticrat cholangiocytesexpressalpha and beta estrogenreceptors(ER)and that estrogensstimulatein vitro cholangiocyteproliferation(Gastroenterology2000; 119:1681-1691). To obtain more insights on the involvement of estrogens on the modulation of cholangiocyte functions,we evaluatedthe effectsof ovariectomyand estrogenreplacementtreatmenton cholangiocyteproliferationand secretion in rats with BDL.BDL (2 weeks) was performedin ovariectomized rats or in sham-ovariectomizedcontrols, and the proliferative,apoptotic and secretory activity of bile ducts were then compared. Cholangiocyteproliferation was evaluated in fiver sectionsby quantitativemeasurementof ino'ahepaticductal mass and m pure cbolangiocytesby PCNA immunoblots. Apoptosis was determined by morphology and TUNEL analysis in liver sectionsand immunoblotsfor Fas in purified eholangiocytes.Basaland secretin-stimulatedbicarbonate-rich choleresis (in bile fistula rats) and basal and secretin-stimulatedintracefiularcAMP levels (in purified cholangiocytes)were also evaluated.In separatesets of experiments, 17/8-estradio] was administered for 2 weeks (28/~g/daily,subcutaneously)in BDL-ovariectomizedrats and the effecton cholangiocyteproliferationand apoptosiswas compared with sham-ovariectomizedBDLrats. Results:Ovariectomyinduced a significantreduction of bile duct mass in 2 week BDL rats in comparisonwith sham operated rats (p< 0.05, n = 3-6). The reduction of bile duct mass was due to a significantimpairment of cholangiocyteproliferation (as demonstrated by significantdecreasedPCNAexpression,p<0.05 vs. controls) and a significantincreasein apoptosis, as indicatedby Fas and TUNE]-positivity (p<0.05 vs. controls). Secretinstimulationof bile flow, bicarbonate secretion and cAMPlevels of BDL rats was significantly(p<0.01) ablated by ovariectomy. Chronic administration of 17,8-estradiolto BDL-ovariectomizedrats completely restored bile duct mass, PCNAprotein expressionand the markers of apoptosis to valuessimilar to the appropriate controls (n= 3-5). Summary/conclusion:We demonstrated that ovariectomy impairscholangiocyteprofiferadonand increasesapoptosisin BDLrats. The effectsof ovariectomy on cholangiocyteproliferation and apoptosis are prevented by chronic administration of 17/8estradiol. At the functional lever, ovariectomycompletelyablated secretin-stimulatedductal secretion. These findings support the role of endogenous estrogens in sustaining the enhanced proliferativeand secretoryactivitiesof cbolangiocytein cholestasis.On the basisof these data, the hypothesis of an estrogenicfunctional deficiencyin chronic cholestaticliver diseasesin females should be carefullystudied. This work was supported by MM06215421/2grant to D. A., by a VA Merit Award (to G. A.), and by grants DK 54208 (G.L.) and DK 58411 (to G. A.).