Ca2+-dependent protein kinase C isoforms are critical to estradiol 17β-D-glucuronide–induced...

Ca2�-Dependent Protein Kinase C Isoforms Are Criticalto Estradiol 17�-D-Glucuronide–Induced

Cholestasis in the RatFernando A. Crocenzi,1,2 Enrique J. Sanchez Pozzi,1 Marıa Laura Ruiz,1 Andres E. Zucchetti,1 Marcelo G. Roma,1

Aldo D. Mottino,1,2 and Mary Vore2

The endogenous estradiol metabolite estradiol 17�-D-glucuronide (E217G) induces an acutecholestasis in rat liver coincident with retrieval of the canalicular transporters bile salt exportpump (Bsep, Abcc11) and multidrug resistance-associated protein 2 (Mrp2, Abcc2) and theirassociated loss of function. We assessed the participation of Ca2�-dependent protein kinase Cisoforms (cPKC) in the cholestatic manifestations of E217G in perfused rat liver (PRL) and inisolated rat hepatocyte couplets (IRHCs). In PRL, E217G (2 �mol/liver; intraportal, singleinjection) maximally decreased bile flow, total glutathione, and [3H] taurocholate excretion by61%, 62%, and 79%, respectively; incorporation of the specific cPKC inhibitor Go6976 (500nM) in the perfusate almost totally prevented these decreases. In dose-response studies usingIRHC, E217G (3.75-800 �M) decreased the canalicular vacuolar accumulation of the Bsepsubstrate cholyl-lysylfluorescein with an IC50 of 54.9 � 7.9 �M. Go6976 (1 �M) increased theIC50 to 178.4 � 23.1 �M, and similarly prevented the decrease in the canalicular vacuolaraccumulation of the Mrp2 substrate, glutathione methylfluorescein. Prevention of these changesby Go6976 coincided with complete protection against E217G-induced retrieval of Bsep andMrp2 from the canalicular membrane, as detected both in the PRL and IRHC. E217G alsoincreased paracellular permeability in IRHC, which was only partially prevented by Go6976.The cPKC isoform PKC�, but not the Ca2�-independent PKC isoform, PKC�, translocated tothe plasma membrane after E217G administration in primary cultured rat hepatocytes; Go6976completely prevented this translocation, thus indicating specific activation of cPKC. This isconsistent with increased autophosphorylation of cPKC by E217G, as detected via westernblotting. Conclusion: Our findings support a central role for cPKC isoforms in E217G-inducedcholestasis, by inducing both transporter retrieval from the canalicular membrane and openingof the paracellular route. (HEPATOLOGY 2008;48:1885-1895.)

Bile formation represents a key liver function bywhich xenobiotics and endogenous metabolitessuch as cholesterol, bilirubin, and hormones are

eliminated from the body.1,2 Efflux of solutes by adeno-

sine triphosphate–dependent transporters at the canalic-ular membrane of hepatocytes provide the driving forcefor osmotic bile formation; among these transporters, thebile salt export pump (Bsep, Abcc11) and multidrug re-

Abbreviations: BSA, bovine serum albumin; Bsep, bile salt export pump; CLF, cholyl-lysylfluorescein; CMFDA, 5-chloromethylfluorescein diacetate; cPKC, Ca2�-dependent protein kinase C isoforms; DMSO, dimethyl sulfoxide; E217G, estradiol 17�-D-glucuronide; GSH, glutathione; GS-MF, glutathione methylfluorescein; IRHC,isolated rat hepatocyte couplet; Mrp2, multidrug resistance-associated protein 2; PKC, protein kinase C; PMA, phorbol-12-myristate-13-acetate; PMSF, phenylmethyl-sulfonyl fluoride; PRL, perfused rat liver.

From the 1Institute of Experimental Physiology, National University of Rosario, Rosario, Argentina; and the 2Graduate Center for Toxicology, University of Kentucky,Lexington, KY.

Received January 17, 2008; accepted July 8, 2008.Supported by Public Health Service grant HD58299 (to Mary Vore) and by grants from Agencia Nacional de Promocion Cientıfica y Tecnologica (PICT N° 05-26115

and 05-26306), Consejo Nacional de Investigaciones Cientıficas y Tecnicas (PIP N° 6442), and Universidad Nacional de Rosario, Argentina (to Aldo Mottino andMarcelo Roma).

Address reprint requests to: Dr. Mary Vore, Graduate Center for Toxicology, 306 Health Sciences Research Building, University of Kentucky, Lexington, KY40536-0305. E-mail: [email protected]; fax: 859-323-1059.

Copyright © 2008 by the American Association for the Study of Liver Diseases.Published online in Wiley InterScience (www.interscience.wiley.com).DOI 10.1002/hep.22532Potential conflict of interest: Nothing to report.

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sistance-associated protein 2 (Mrp2, Abcc2), which areresponsible for transporting bile salts and glutathione(GSH) and glucuronide conjugates, respectively, play acentral role in this process.1,3

Alterations of canalicular transporter expression, localiza-tion, or activity can lead to cholestasis.3 Short-term endo-cytic retrieval of Bsep and Mrp2 occurs in severalexperimental models of cholestasis.3,4 This mechanism likelyimpairs secretory function by reducing the total number ofefflux transporters in the canalicular domain and thereforetheir transport capacity (Vmax) within minutes. Stimulationof exocytic insertion of transporter-containing vesicles by ad-ministration of tauroursodeoxycholate or cyclic adenosinemonophosphate prevents cholestasis, thus supporting thishypothesis.5

The endogenous estradiol metabolite, estradiol 17�-D-glucuronide (E217G), is a useful model to study the mani-festations associated with estrogen-induced cholestasis.Indeed, E217G induces a dose-dependent, acute and revers-ible cholestasis by impairing both Bsep-mediated efflux ofbile acids and the Mrp2-mediated efflux of GSH.6 As a likelymechanism, E217G causes a microtubule-independent, en-docytic retrieval of both Bsep and Mrp27,8; spontaneous exo-cytic reinsertion of these canalicular transporters in amicrotubule-dependent fashion is linked to restoration ofbile flow and canalicular transport activity.9

Recent evidence indicates that activation of proteinkinase C (PKC) is associated with impairment of the se-cretory function of hepatocytes, and ultimately leads tocholestasis.10-12 Thymeleatoxin-mediated selective activa-tion of the classical Ca2�-dependent PKC isoforms(cPKC), of which PKC� and PKC�II are expressed inhepatocytes,13 is cholestatic in perfused rat liver (PRL).14

Similarly, oxidative stress-induced impairment of bile saltsecretory function in isolated rat hepatocyte couplets(IRHCs) is dependent on activation of PKC�.15 Interest-ingly, this PKC� activation is also associated with re-trieval of Bsep and the associated loss of its secretoryfunction.14,15

In the present study, we determined whether cPKCisoforms mediate the cholestasis induced by E217G in thein situ PRL and in IRHCs. Our results unambiguouslydemonstrate that cPKC isoforms are critical to the im-pairment of localization and function of Bsep and Mrp2induced by E217G.

Materials and Methods

Materials. L-15 culture medium, leupeptin, aproti-nin, phenylmethylsulfonyl fluoride (PMSF), pepstatin A,E217G, collagenase, methylbutane (isopentane), bovineserum albumin (BSA), phorbol-12-myristate-13-acetate

(PMA) and dimethyl sulfoxide (DMSO) were obtainedfrom Sigma-Aldrich (St. Louis, MO). Go6976 was ob-tained from Calbiochem (San Diego, CA) and 5-chlo-romethylfluorescein diacetate (CMFDA) was obtainedfrom Molecular Probes (Carlsbad, CA). Cholyl-lysylfluo-rescein (CLF) was a generous gift of Dr. Charles O. Mills(Queen Elizabeth Hospital, Birmingham, UK). [3H]tau-rocholate (3.0 Ci/mmol) was obtained from Perkin ElmerLife and Analytical Sciences (Boston, MA). All otherchemicals were of analytical grade purity and were used assupplied.

Rat Liver Perfusion. Female Sprague-Dawley rats(180-210 g) (Harlan Industries, Indianapolis, IN) wereanesthetized with urethane (1,000 mg/kg intraperitone-ally) and the bile duct was cannulated with PE-10 tubing(Intramedic, Clay Adams). Livers were perfused in situ viathe portal vein in a non-recirculating single-pass designwith Krebs-Ringer bicarbonate at 37°C, equilibrated with5% CO2/95% O2, at a constant flow rate of 30 mL/min(�4 mL/min/g liver). [3H]taurocholate (2 �Ci/L; 0.7�mol/L) was added to the perfusion medium for bile saltsecretion studies. After a 20-minute equilibration, the se-lective cPKC inhibitor Go697616 (500 nM final concen-tration) or its solvent (DMSO, 370 �L/L) was added tothe reservoir. Fifteen minutes later, a 5-minute basal bilesample was collected, followed by administration ofE217G (2 �mol/liver, intraportal single injection over a1-minute period) or its solvent (DMSO/10% BSA in sa-line [4:96]), and bile collected at 5-minute intervals for anadditional 60 minutes. Experiments were consideredvalid only if initial bile flow (after equilibration) wasgreater than 30 �L/min/kg (�0.9 �L/min/g liver). Via-bility of the liver was monitored via lactate dehydrogenaseactivity in the perfusate outflow; experiments exhibitingactivities over 20 U/L were considered invalid.

Transport activity of Mrp2 and Bsep was evaluated bymeasuring biliary GSH and [3H]taurocholate excretion,respectively. Total GSH content was measured by therecycling method of Tietze.17 [3H]Taurocholate was de-termined by liquid scintillation spectroscopy.

Confocal Laser Scanning Microscopy and ImageAnalysis. A separate group of PRL was used for confocalimmunofluorescence microscopy of Mrp2, Bsep, ZO-1,and occludin localization. In these experiments, perfusionwas stopped 20 minutes after E217G, and a liver lobe wasexcised and frozen immediately in isopentane precooledin liquid nitrogen and stored at �80°C. Liver sampleswere sectioned and fixed as described.8,9 Mrp2 was labeledusing a monoclonal antibody to human MRP2 (M2 III-6;Alexis Biochemicals, Carlsbad, CA). Bsep was labeledwith a polyclonal antibody to mouse Bsep (Kamiya Bio-medical, Seattle, WA). ZO-1 and occludin were labeled

1886 CROCENZI ET AL. HEPATOLOGY, December 2008

with polyclonal and monoclonal antibodies directed tohuman ZO-1 and occludin, respectively (Zymed Labora-tories Inc., South San Francisco, CA). Immunostainingwas completed by treatment of the preparations with ap-propriate Cy2- or Cy3-conjugated donkey anti–immuno-globulin G (Jackson ImmunoResearch Laboratory, Inc.,West Grove, PA). All confocal studies were performed ina True Confocal Scanner Leica TCS SP II microscope(Heidelberg, Germany). To ensure comparable stainingand image capture performance for the different groupsbelonging to the same experimental protocol, liver sliceswere prepared on the same day, mounted on the sameglass slide in a single well, and subjected to the stainingprocedure and confocal microscopy analysis simulta-neously.

Assessment of Bsep and Mrp2 endocytic internaliza-tion in confocal images was performed using the Image J1.34m software (National Institutes of Health) as de-scribed.9 Immunodetection of the tight junctional-asso-ciated proteins ZO-1 and occludin was performed tovisualize the border of bile canalicular structures in sam-ples stained for Mrp2 or Bsep, respectively.

Isolation and Culture of Rat Hepatocytes. Isolatedhepatocytes were obtained by collagenase perfusion andmechanical disruption of rat liver, as described.18 To ob-tain a preparation enriched in IRHCs, livers were per-fused according to the two-step collagenase perfusionprocedure and further enriched via centrifugal elutria-tion.19 Viability of final preparations (assessed via trypanblue exclusion) was greater than 90%.

Localization and Function of Bsep and Mrp2 inIRHCs. IRHCs were plated in 24-well plastic plates at adensity of 0.5 � 105 U/mL in L-15 culture medium, andcultured for 5 hours to allow restoration of couplet polar-ity. They were then exposed to DMSO (control group) orE217G (3.75-800 �M) for 20 minutes. To evaluate therole of cPKC isoforms, Go6976 (1 �M) was added to theculture medium 15 minutes before the addition ofE217G, and maintained throughout the period of expo-sure to E217G (20 minutes).

The canalicular vacuolar accumulation of the fluores-cent CLF was used to quantify Bsep transport; similarly,canalicular accumulation of glutathione methylfluores-cein (GS-MF), derived from CMFDA, was used to quan-titate Mrp2 transport.7 CLF is a bile salt analoguetransported selectively by Bsep,20 whereas CMFDA is alipophilic compound taken up by passive diffusion acrossthe basolateral membrane and converted into GS-MF bythe action of both intracellular esterases and glutathioneS-transferases. For transport studies, cells were washedtwice with L-15 and exposed to 2 �M CLF21,22 or 2.5 �MCMFDA21,23 for 15 minutes. Finally, cells were washed

twice with L-15, and canalicular transport activity forboth substrates assessed by fluorescence microscopy21 un-der an inverted microscope (Zeiss Axiovert 25) and im-ages captured with a digital camera (Zeiss AxioCamMRc5). Quantitation was deternined as the percentage ofIRHC in the images displaying visible green fluorescencein their canalicular vacuoles from a total analysis of at least200 couplets per preparation.

For intracellular localization of Bsep and Mrp2,IRHCs were cultured on glass coverslips (12 mm) placedin 24-well plates, treated with E217G and/or Go6976 asdescribed above, and then fixed and stained as reported.21

Briefly, cells were incubated with the specific antibodiesto Bsep or Mrp2 (1:200) for 2 hours, followed by incu-bation with Cy2-conjugated donkey anti–immunoglobu-lin G (1:100) for 40 minutes. Densitometric analysis ofimages (taken with a Zeiss Axiovert 25 fluorescence mi-croscope) was made along a line perpendicular to the can-alicular vacuole using the Image J 1.34m software, asdescribed.9

Evaluation of Paracellular Permeability in IRHCs.We assessed the permeability of tight junctions by quan-titating the retention of CLF in the canalicular vacuoles.24

CLF was added for 15 minutes to the culture media (L-15) to allow it to accumulate in canalicular vacuoles, andthen removed from the incubation media by washingtwice with L-15 at 37°C. IRHCs were then incubatedwith Go6976 (1 �M) for 15 minutes before the additionof E217G (50 �M); exposure to E217G and/or Go6976was maintained for an additional 40 minutes. Cells werethen washed with cold (4°C) culture medium, and theproportion of IRHCs displaying CLF-associated greenfluorescence in their canalicular vacuoles was assessed asdescribed above.

Immunoblotting of PKC� and PKC�. Isolated rathepatocytes were cultured on 9-cm glass Petri dishes at adensity of 1 � 106 cells/mL. After culture for 5 hours,cells were exposed to E217G (50 �M) for 5, 10, 15, or 20minutes, washed with cold 0.3 M sucrose, resuspended in0.3 M sucrose plus protease inhibitors (25 �g/mL leupep-tin, 5 �g/mL pepstatin A, and 0.1 mM PMSF), and dis-rupted via sonication. In separate experiments, we testedthe effect of Go6976 (1 �M) by exposing the cells for 15minutes to the inhibitor, previous to the incubation withE217G (50 or 200 �M, 10 minutes) or its solvent;Go6976 was maintained throughout the period of expo-sure to E217G. Cytosolic (supernatant) and total mem-brane (pellet)-enriched fractions were obtained viaultracentrifugation for 60 minutes at 100,000g25 afterelimination of nuclei and cell fragments via centrifugationfor 10 minutes at 500g, and total protein determined,26

using BSA as a standard. Western blotting in total mem-

HEPATOLOGY, Vol. 48, No. 6, 2008 CROCENZI ET AL. 1887

brane and cytosolic fractions used an amount of proteinthat gave a densitometric signal in the linear range for theantibodies used. Proteins were separated via 10% sodiumdodecyl sulfate–polyacrylamide gel electrophoresis; mem-brane and cytosolic fractions from the same experimentwere loaded in the same gel. After electrotransfer, nitro-cellulose membranes (Sigma Chemical Co.) were incu-bated overnight with monoclonal antibodies to humanPKC� or PKC� (BD Biosciences Pharmingen; 1:1,000)followed by incubation with an alkaline phosphatase–linked secondary antibody (Bio-Rad, 1:5,000) for 1 hour,and further detection with a color development solution(Bio-Rad). Densitometry was performed with Gel-ProAnalyzer (Media Cybernetics) software. To estimate theamount of PKC associated with both cytosolic and mem-brane fractions, the relative intensity of each band wasdivided by micrograms of protein loaded in that lane,then multiplied by the total amount of protein recoveredin the corresponding fraction. The proportion of mem-brane-bound PKC isoforms was expressed as the amountin membranes (Amembrane) relative to the total cellularamount, according to: Amembrane/(Amembrane � Acytosol). Inseparate experiments, we compared the effect of treat-ment of hepatocytes for 10 minutes with E217G (50 �M)or PMA (1 �M), as a positive control, on PKC� translo-cation to membranes.

Phosphorylation of PKC at several aminoacidic resi-dues is necessary for acquisition of their full catalytic ac-tivity.27 PKC� undergoes phosphorylation at Thr497,Thr638, and Ser660, with Thr638 being an autophos-phorylation site. The degree of phosphorylation of PKCprovides an estimate of the extent of its activation.28 Iso-lated rat hepatocytes were exposed to E217G (50 �M) orPMA (1 �M) as a positive control, for 10 minutes,washed with cold 0.3 M sucrose, resuspended in sodiumvanadate buffer (Cell Lysis Buffer, Cell Signaling Tech-nology, Inc.) plus protease inhibitors (25 �g/mL leupep-tin, 5 �g/mL pepstatin A, and 0.1 mM PMSF), anddisrupted by sonication followed by centrifugation for 10minutes at 500g. The phosphorylated cPKC was quanti-tated by immunoblotting of the supernatants, using aspecific phospho-PKC�/�II (Thr638/641) antibody(Cell Signaling Technology, Inc.).

Statistical Analysis. Results are expressed as themean � standard error of the mean. Statistical analysiswas performed using one-way analysis of variance, fol-lowed by the Newman-Keuls test. Densitometric profilesof Bsep and Mrp2 were compared using the Mann-Whit-ney test. The four-parameter dose-response curves werecompared using GraphPad Prism software (F test). Valuesof P � 0.05 were considered statistically significant.

Results

Inhibition of cPKC Isoforms Prevents E217G-In-duced Impairment of Biliary Secretory Function. Asingle dose of E217G induced an acute 61% decrease inbile flow within 10 minutes that did not recover duringthe 60-minute perfusion period (Fig. 1A), consistent withthe 62% and 79% decreases induced by E217G in thebiliary excretion of total GSH and [3H]taurocholate, re-spectively (Fig. 1B,C). The selective cPKC isoform inhib-itor, Go6976, prevented the impairment of both bile flowand total GSH excretion, but provided only partial pro-tection against the initial decrease in [3H]taurocholatesecretion, followed by a total recovery of taurocholatesecretion 20 minutes after E217G administration.

The functional status of Bsep and Mrp2 was evaluatedin IRHC by monitoring efflux of Mrp2 (GS-MF) andBsep (CLF) substrates into the canalicular vacuole (Fig.2). In dose-response studies, Go6976 significantly in-creased the IC50 of E217G from 54.9 � 7.9 �M (E217Galone) to 178.4 � 23.1 �M (E217G � Go6976). Theminimal and maximal percent inhibition of CLF accumu-lation and the slope of the dose-response curve as derivedfrom nonlinear fit of the dose-response curves were notmodified by cPKC inhibition (Fig. 2). Similarly, canalic-ular accumulation of GS-MF in the presence of 50 �ME217G was inhibited 40 � 3%; this inhibition was re-duced to only 20 � 2% in the presence of Go6976.

Inhibition of cPKC Isoforms Prevents E217G-In-duced Retrieval of Bsep and Mrp2. E217G-inducedendocytic retrieval of both Bsep and Mrp2 from the can-alicular membrane to intracellular, vesicular compart-ments decreases their transport activity, and thereby bileflow.7-9 Figure 3 shows confocal images of Bsep (green)and occludin (red), or Mrp2 (red) and ZO-1 (green) 20minutes after E217G administration. In E217G-treatedlivers, both Bsep and Mrp2 were detected in intracellularstructures (white arrows), consistent with their endocyticretrieval from the canalicular membrane. Consistent withour earlier studies,9 this pattern of internalization wasevident only in some canalicular structures and coexistedwith preserved canalicular localization of the transportersat other sites. We analyzed profiles of distribution oftransporter-associated fluorescence along a line perpen-dicular to the canaliculus (Fig. 4). E217G-treated liversshowed a wider profile, consistent with increased fluores-cence at a greater distance from the canalicular mem-brane, indicative of retrieval of these transporters into theintracellular compartment. In livers perfused withE217G � Go6976, the distribution of both Bsep andMrp2 was almost identical to that in control livers. Thesedata demonstrate that cPKC inhibition protected against


E217G-induced retrieval of Mrp2 and Bsep. Analysis ofthe distribution profiles of the tight junctional proteinsZO-1 and occludin (Fig. 4) demonstrates a conservedwidth of the canaliculi in all the experimental groups, thus

eliminating the possible influence of changes in this pa-rameter on the distribution profiles of Bsep and Mrp2.

Similar results were obtained in IRHC exposed toE217G in the presence or absence of Go6976 (Fig. 5).Both in control cells and in those incubated withE217G � Go6976, Bsep and Mrp2 appeared to be local-ized within the canalicular space, whereas in cells treatedonly with E217G, Bsep and Mrp2 exhibited a fuzzy stain-ing pattern, compatible with their internalization. Thesepatterns of distribution were confirmed by densitometricanalysis of the images, which demonstrated an E217G-induced redistribution of both Bsep and Mrp2 over agreater distance from the canalicular vacuoles; Go6976prevented this redistribution. When given alone, Go6976

Fig. 1. Go6976 protects against E217G-induced inhibition of bile flowand biliary secretion of GSH and taurocholate in the perfused rat liver.Rat livers were perfused as described in Experimental Procedures and theeffects of E217G on bile secretory function determined in the presenceand absence of the PKC� inhibitor, Go6976 (500 nM), or its solvent(DMSO). The effect of the treatments on (A) bile flow, (B) total GSHexcretion, (C) and [3H]taurocholate excretion are shown. Go6976 alonedid not induce any changes in these parameters (data not shown).Results are expressed as the mean � standard error of the mean (n �4). aSignificantly different from E217G. bSignificantly different from con-trol.

Fig. 2. Go6976 partially prevents E217G-induced impairment of thecanalicular accumulation of CLF and E217G-induced increase in para-cellular permeability in IRHCs. (A) IRHCs were preincubated with Go6976(1 �M) for 15 minutes and then exposed to E217G (3.75-800 �M) foran additional 20 minutes. Canalicular accumulation of CLF was deter-mined as the percentage of couplets displaying visible fluorescence intheir canalicular vacuoles from a total of at least 200 couplets perpreparation and expressed as a percentage inhibition of canalicularaccumulation, where 0 correspond to controls. Data are expressed as themean � standard error of the mean (n � 3). (B) Effects of E217G onparacellular permeability and the protection provided by Go6976 weredetermined as described in Experimental Procedures. Results are ex-pressed as the mean � standard error of the mean (n � 4). aSignifi-cantly different from control. bSignificantly different from E217G.


did not cause any difference in these patterns of distribu-tion relative to DMSO (data not shown).

Inhibition of cPKC Isoforms Partially PreventsE217G-Induced Increased Paracellular Permeabilityin IRHCs. We assessed the importance of cPKC activa-tion in increasing the permeability of the paracellularpathway by quantitating the ability of IRHC to retainCLF already accumulated in the canalicular vacuoles. Ad-dition of E217G alone significantly diminished retentionof CLF in canalicular vacuoles (Fig. 2B); in the presenceof Go6976, the effect of E217G was only partially, but yetsignificantly blocked, so that retention of CLF was stillsignificantly less than in control IRHCs. Incubation withGo6976 alone did not affect this measure (data notshown). These data imply that E217G increases paracel-lular permeability, but that this effect is only partiallydependent on cPKC.

E217G Selectively Activates cPKC Isoforms. PKCactivation is associated with translocation of the proteinfrom the cytosol to cellular membranes. Figure 6A shows

representative immunoblots of the PKC� and PKC� inboth cytosolic and membrane fractions of primary cul-tured rat hepatocytes at various times after addition ofE217G (50 �M). Calculation of the percentage of eachPKC isoform in the membrane fraction revealed thatE217G increased membrane-bound PKC� by 60%within 5 minutes; this increase persisted for 15 minutesand returned to control values by 20 minutes after E217Gadministration (Fig. 6A). The proportion of PKC� asso-ciated with the membrane fraction was not modified byE217G (Fig. 6A). The selective translocation to mem-branes of PKC� induced by 50 and 200 �M E217G wasprevented by the specific cPKC inhibitor Go6976 (Fig.6B). Treatment with Go6976 alone did not affect thenormal pattern of distribution of PKC� (data notshown). To further assess the selective effect of E217G onPKC translocation to membranes, we compared the ef-fects of a 10-minute treatment with E217G and the diac-ylglycerol analog PMA on the proportion of PKC�associated with membranes (Fig. 6C); only PMA induced

Fig. 3. Confocal images of E217G-inducedretrieval of Mrp2 and Bsep and protection byGo6976. Representative confocal images of im-munostained liver samples displaying a costain-ing of Bsep (green) and occludin (red) (leftpanels), and Mrp2 (red) and ZO-1 (green) (rightpanels). In control livers, both Bsep and Mrp2were mainly confined to the canalicular spacedelineated by the tight junction-associated pro-teins occludin or ZO-1, respectively. FollowingE217G (2 �mol/liver), some canaliculi showintracellular fluorescence associated with Bsepor Mrp2 (arrows) at a greater distance from thecanalicular membrane, consistent with theirendocytic retrieval. Go6976 prevented the in-ternalization of canalicular transporters, as illus-trated by a control-like pattern of Bsep andMrp2 distribution. Go6976 itself did not induceany changes in transporters localization (imagesnot shown). Bar � 10 �m.


a significant increase (30%; P � 0.05) in PKC� associatedwith membranes.

Activation of cPKC isoforms by E217G was furtherassessed by detecting autophosphorylation of PKC�and PKC�II at positions Thr638 and Thr641, respec-

tively (Fig. 6D). There was a significant increase in thetotal cellular content of phosphorylated PKC� andPKC�II in response to treatment with both E217G andPMA (86% and 112% over control, respectively; P �0.05).

Fig. 4. Densitometric analysis of localization of Bsep, occludin, Mrp2, and ZO-1 in perfused livers. Profiles of Bsep (left, top) and Mrp2 (right, top)associated fluorescence in confocal images shown in Fig. 3. Graphs represent the intensity of fluorescence associated with the transporters alongan 8-�m line (from �4 �m to �4 �m of the canalicular center) perpendicular to the canaliculus. In control livers, transporter-associatedfluorescence was concentrated in the canalicular space (from �1 �m to �1 �m). E217G-induced retrieval of transporters from the canalicularmembrane (P � 0.0001 versus control) was detected as a decrease in the fluorescence intensity in the canalicular area together with an increasedfluorescence at a greater distance from the canaliculus (from � 1 �m to � 2 �m). Distribution profiles of livers treated with E217G � Go6976showed a significantly decreased Mrp2 internalization (P � 0.01 versus E217G; P � 0.0001 versus control). Bsep distribution did not differ fromcontrols in E217G � Go6976 group (P � 0.0001 versus E217G) (n � 20-50 canaliculi per preparation, three independent preparations). Statisticalanalysis of the distribution profiles of tight junction-associated proteins, used to demarcate limits of the canaliculi, occludin (left, bottom), and ZO-1(right, bottom) (for Bsep and Mrp2, respectively), showed no changes in the normal distribution by any of the treatments.


DiscussionThis study provides important insights regarding the

signaling pathways involved in cholestasis mediated byE217G that contribute to estrogen-induced cholestasis.The present data in both the PRL and IRHCs clearlyimplicate cPKC isoforms in the inhibition of bile forma-tion and Mrp2- and Bsep-mediated canalicular transport.

In the perfused liver, the selective cPKC inhibitorGo6976 completely prevented the E217G-induced de-crease in bile flow and GSH excretion and partially pre-vented the impairment in [3H]taurocholate excretion.Further support for the role of cPKC came from dose-response studies of canalicular accumulation of CLF inhepatocyte couplets, where Go6976 induced a parallelshift to the right of the dose-response curve, leading to athree-fold increase in the IC50 of E217G. Furthermore,both in perfused liver and hepatocyte couplets, E217Gactivated a cPKC-dependent signaling that was responsi-ble for retrieval of Bsep and Mrp2. Analysis of the fluo-rescence associated with Bsep and Mrp2 supports the

conclusion that cPKC inhibition almost completely pre-vented their E217G-induced retrieval.

The data also demonstrated that E217G selectively ac-tivated PKC�, which is the main cPKC present in rathepatocytes.13 PKC� activation was transient, with amaximum occurring between 5 and 10 minutes afterE217G administration, and returning to basal values after20 minutes. The novel, Ca2�-independent PKC�, whichis activated by oxidative stress to induce Mrp2 retrieval,29

was not activated by E217G at these time periods, furtherindicating that E217G activates specific isoforms of PKC.Interestingly, activation of cPKC at 5 minutes, as detectedin cultured hepatocytes, preceded maximal impairment ofbile flow in vivo and in the perfused liver,7,8,30 as well asretrieval of both Mrp2 and Bsep in vivo,7,8 which occurbetween 10 and 20 minutes after E217G administration.Although exposure of hepatocytes to E217G in culture couldlead to intracellular effects more rapidly than could exposureof the liver via the blood supply, the data are consistent witha role for cPKC activation in initiating transporter retrieval

Fig. 5. Go6976 prevents E217G-induced retrieval of Bsep and Mrp2 in isolated rat hepatocyte couplets. Isolated rat hepatocyte couplets werepreincubated with Go6976 (1 �M), exposed to E217G (50 �M), and fixed and immunostained as described in Experimental Procedures. Graphsdepict quantitative analysis of transporter-associated fluorescence along a line (8 �m) perpendicular to the canalicular vacuole. Statistical analysisof the profiles revealed a significant internalization of Bsep and Mrp2 under E217G treatment (P � 0.0001 versus control), which was completelyabolished by Go6976 (P � 0.0001 versus E217G). Results are expressed as the mean � standard error of the mean (n � 15-25 canalicularvacuoles per preparation, three independent preparations).


and inhibition of bile flow. Activation of PKC by phorbolesters, the Ca2�-elevating hormone vasopressin, or the cho-lestatic bile salt taurolithocholate is correlated with their abil-ity to induce cholestasis.10,12,31,32 Moreover, activation ofPKC� by the pro-oxidant compound t-butylhydroperox-ide15 or by the selective PKC� activator thymeleatoxin14

induces cholestasis associated with retrieval of Bsep. Our

data on E217G-induced cholestasis are consistent with thesefindings.

The intracellular targets for cPKC following its activa-tion by E217G are unknown. However, multiple lines ofevidence have long indicated that activation of PKC canregulate the permeability of epithelial tight junctions.33,34

PKC� participates in the disassembly of the tight junc-

Fig. 6. Specific PKC� activation by E217G in primary cultured hepatocytes. (A) Immunoblots of PKC� and PKC� in membrane (60 �g protein)and cytosolic (40 �g protein) fractions. Primary cultured hepatocytes were treated with E217G (50 �M) for 5, 10, 15, or 20 minutes, and PKC isoformdistribution was evaluated. The bar graph shows membrane bound PKC� and PKC�, expressed as percentage of total cellular content. (B)Immunoblots of PKC� in membrane and cytosolic fractions. Primary cultured hepatocytes were treated with E217G (50 or 200 �M) for 10 minutesin the presence or absence of Go6976 (1 �M). The bar graph shows membrane-bound PKC� expressed as a percentage of total cellular content.(C) Representative immunoblot of PKC� in membrane and cytosolic fractions of primary cultured hepatocytes treated for 10 minutes with E217G (50�M) or the PKC activator PMA (1 �M). The bar graph shows membrane-bound PKC� expressed as a percentage of total cellular content. (D)Representative immunoblots and quantitation of the phosphorylated forms of cPKC in total cellular extracts from E217G-treated (50 �M, 10 minutes)and PMA-treated (1 �M, 10 minutes) primary cultured rat hepatocytes. Results are expressed as the mean � standard error of the mean (n � 3).aSignificantly different from control. bSignificantly different from E217G 50 �M. cSignificantly different from E217G 200 �M.


tions, while nPKC� plays a role in tight junction forma-tion in some epithelial tissues.35 Tight junctions representthe apical portion of the junctional complex and serve as agatekeeper of the paracellular pathway, regulating the pas-sage of water and ions that, in liver, is a critical determi-nant of bile flow.36 PKC activation increases tightjunction permeability in IRHC,24,32 an event that con-tributes in part to cholestasis by leakage of osmoticallyactive solutes from the canalicular compartment. We re-cently demonstrated that E217G causes disruption oftight junctions, as indicated by increased paracellular per-meability to horseradish peroxidase, and by loss of the“fence” function between canalicular and sinusoidalmembranes, as indicated by redistribution of the canalic-ular transporter Mrp2 to the basolateral domain.37 In-creased paracellular permeability occurred simultaneouslywith endocytic retrieval of Mrp2 in vivo, suggesting acausal relationship. Kubitz et al.12 have similarly noted aPKC-dependent distribution of Mrp2 from the canalicu-lar to the basolateral membrane in human HepG2 cells.In the current studies, Go6976 only partially preventedthe E217G-induced increase in paracellular permeabilityin hepatocyte couplets, implying that cPKC-independentfactors also contribute to the disruption of the tight junc-tions observed in E217G-induced cholestasis. Furtherwork is needed to determine the extent or nature of thelink between endocytosis of Mrp2 or Bsep and increasedtight-junctional permeability.

Direct phosphorylation of the retrieved transportersBsep and Mrp2 may also be associated with PKC activa-tion. The adenosine triphosphate–binding cassette trans-porter, Mdr1, the closest Bsep homologue, isphosphorylated by PKC at three serine residues in theC-terminal, “linker” region that binds to the actin-associ-ated protein, HAX-1.38 There is also evidence thatHAX-1 participates in clathrin-mediated endocytosis ofBsep from the apical membrane in MDCK II cells.39

Whether this phosphorylation occurs also for Bsep, andwhether these changes improve Bsep binding to HAX-1so as to become more readily endocytosed, remains to beascertained.

In conclusion, the results indicate a key role for cPKCin the endocytic internalization of the canalicular trans-porters Bsep and Mrp2 in E217G-induced cholestasis.Further studies are needed to ascertain the specific targetsmodulated directly or indirectly by cPKC that lead totheir internalization.

References1. Trauner M, Boyer JL. Bile salt transporters: molecular characterization,

function, and regulation. Physiol Rev 2003;83:633-671.2. Bohan A, Boyer JL. Mechanisms of hepatic transport of drugs: implica-

tions for cholestatic drug reactions. Semin Liver Dis 2002;22:123-136.

3. Crocenzi F, Mottino A, Roma M. Regulation of synthesis and traffickingof canalicular transporters and its alteration in acquired hepatocellularcholestasis: experimental therapeutic strategies for its prevention. CurrMed Chem 2004;11:501-524.

4. Haussinger D, Kubitz R, Reinehr R, Bode JG, Schliess F. Molecular as-pects of medicine: from experimental to clinical hepatology. Mol AspectsMed 2004;25:221-360.

5. Haussinger D, Schmitt M, Weiergraber O, Kubitz R. Short-term regula-tion of canalicular transport. Semin Liver Dis 2000;20:307-321.

6. Meyers M, Slikker W, Pascoe G, Vore M. Characterization of cholestasisinduced by estradiol-17 beta-D-glucuronide in the rat. J Pharmacol ExpTher 1980;214:87-93.

7. Crocenzi FA, Mottino AD, Cao J, Veggi LM, Sanchez Pozzi EJ, Vore M,et al. Estradiol-17beta-D-glucuronide induces endocytic internalization ofBsep in rats. Am J Physiol Gastrointest Liver Physiol 2003;285:G449-G459.

8. Mottino AD, Cao J, Veggi LM, Crocenzi FA, Roma MG, Vore M. Alteredlocalization and activity of canalicular Mrp2 in estradiol-17beta-D-gluc-uronide-induced cholestasis. HEPATOLOGY 2002;35:1409-1419.

9. Mottino AD, Crocenzi FA, Pozzi EJ, Veggi LM, Roma MG, Vore M. Roleof microtubules in estradiol-17beta-D-glucuronide-induced alteration ofcanalicular Mrp2 localization and activity. Am J Physiol Gastrointest LiverPhysiol 2005;288:G327-G336.

10. Corasanti JG, Smith ND, Gordon ER, Boyer JL. Protein kinase C agonistsinhibit bile secretion independently of effects on the microcirculation inthe isolated perfused rat liver. HEPATOLOGY 1989;10:8-13.

11. Nathanson MH, Gautam A, Bruck R, Isales CM, Boyer JL. Effects ofCa2� agonists on cytosolic Ca2� in isolated hepatocytes and on bilesecretion in the isolated perfused rat liver. HEPATOLOGY 1992;15:107-116.

12. Kubitz R, Huth C, Schmitt M, Horbach A, Kullak-Ublick G, HaussingerD. Protein kinase C-dependent distribution of the multidrug resistanceprotein 2 from the canalicular to the basolateral membrane in humanHepG2 cells. HEPATOLOGY 2001;34:340-350.

13. Croquet F, Brehier A, Gil S, Davy J, Feger J. Five isoenzymes of proteinkinase C are expressed in normal and STZ-diabetic rat hepatocytes: effectof phorbol 12-myristate 13-acetate. Biochim Biophys Acta 1996;1315:163-168.

14. Kubitz R, Saha N, Kuhlkamp T, Dutta S, vom Dahl S, Wettstein M, et al.Ca2�-dependent protein kinase C-isoforms induce cholestasis in rat liver.J Biol Chem 2004;279:10323-10330.

15. Perez LM, Milkiewicz P, Elias E, Coleman R, Sanchez Pozzi EJ, RomaMG. Oxidative stress induces internalization of the bile salt export pump,Bsep, and bile salt secretory failure in isolated rat hepatocyte couplets: a rolefor protein kinase C and prevention by protein kinase A. Toxicol Sci2006;91:150-158.

16. Martiny-Baron G, Kazanietz MG, Mischak H, Blumberg PM, Kochs G,Hug H, et al. Selective inhibition of protein kinase C isozymes by theindolocarbazole Go 6976. J Biol Chem 1993 5;268(13):9194-9197.

17. Tietze F. Enzymic method for quantitative determination of nanogramamounts of total and oxidized glutathione: applications to mammalianblood and other tissues. Anal Biochem 1969;27:502-522.

18. Garcia F, Kierbel A, Larocca MC, Gradilone SA, Splinter P, LaRusso NF,et al. The water channel aquaporin-8 is mainly intracellular in rat hepato-cytes, and its plasma membrane insertion is stimulated by cyclic AMP.J Biol Chem 2001;276:12147-12152.

19. Wilton JC, Williams DE, Strain AJ, Parslow RA, Chipman JK, ColemanR. Purification of hepatocyte couplets by centrifugal elutriation. HEPATOL-OGY 1991;14:180-183.

20. Mills CO, Milkiewicz P, Muller M, Roma MG, Havinga R, Coleman R, etal. Different pathways of canalicular secretion of sulfated and non-sulfatedfluorescent bile acids: a study in isolated hepatocyte couplets and TR- rats.J Hepatol 1999;31:678-684.

21. Roma MG, Milkiewicz P, Elias E, Coleman R. Control by signaling mod-ulators of the sorting of canalicular transporters in rat hepatocyte couplets:role of the cytoskeleton. HEPATOLOGY 2000;32:1342-1356.

22. Crocenzi FA, Mottino AD, Sanchez Pozzi EJ, Pellegrino JM, RodrıguezGaray EA, Milkiewicz P, et al. Impaired localisation and transport function


of canalicular Bsep in taurolithocholate-induced cholestasis in the rat. Gut2003;52:1170-1177.

23. Roelofsen H, Soroka CJ, Keppler D, Boyer JL. Cyclic AMP stimulatessorting of the canalicular organic anion transporter (Mrp2/cMoat) to theapical domain in hepatocyte couplets. J Cell Sci 1998;111:1137-1145.

24. Roma MG, Orsler DJ, Coleman R. Canalicular retention as a marker oftight junctional permeability in isolated hepatocyte couplets: effects ofprotein kinase modulation and cholestatic agents. Fund Appl Toxicol1997;37:71-81.

25. Carreras FI, Gradilone SA, Mazzone A, Garcia F, Huang BQ, Ochoa JE, etal. Rat hepatocyte aquaporin-8 water channels are down-regulated in ex-trahepatic cholestasis. HEPATOLOGY 2003;37:1026-1033.

26. Lowry OH, Rosembrough NJ, Farr AL, Randall RJ. Protein measurementwith the Folin phenol reagent. J Biol Chem 1951;193:265-275.

27. Parekh DB, Ziegler W, Parker PJ. Multiple pathways control protein ki-nase C phosphorylation. EMBO J 2000;19:496-503.

28. Mitchell FE, Marais RM, Parker PJ. The phosphorylation of protein kinaseC as a potential measure of activation. Biochem J 1989;261:131-136.

29. Sekine S, Ito K, Horie T. Oxidative stress and Mrp2 internalization. FreeRadic Biol Med 2006;40:2166-2174.

30. Mottino AD, Veggi LM, Wood M, Roman JM, Vore M. Biliary secretionof glutathione in estradiol 17beta-D-glucuronide-induced cholestasis.J Pharmacol Exp Ther 2003;307:306-313.

31. Beuers U, Probst I, Soroka C, Boyer JL, Kullak-Ublick GA, PaumgartnerG. Modulation of protein kinase C by taurolithocholic acid in isolated rathepatocytes. HEPATOLOGY 1999;29:477-482.

32. Roma MG, Stone V, Shaw R, Coleman R. Vasopressin-induced disruptionof actin cytoskeletal organization and canalicular function in isolated rathepatocyte couplets: possible involvement of protein kinase C. HEPATOL-OGY 1998;28:1031-1041.

33. Gonzalez-Mariscal L, Tapia R, Chamorro D. Crosstalk of tight junctioncomponents with signaling pathways. Biochim Biophys Acta 2007;1778:729-756.

34. Andreeva AY, Krause E, Muller EC, Blasig IE, Utepbergenov DI. Proteinkinase C regulates the phosphorylation and cellular localization of occlu-din. J Biol Chem 2001;276:38480-38486.

35. Andreeva AY, Piontek J, Blasig IE, Utepbergenov DI. Assembly of tightjunction is regulated by the antagonism of conventional and novel proteinkinase C isoforms. Int J Biochem Cell Biol 2006;38:222-233.

36. Boyer JL. Tight junctions in normal and cholestatic liver: does the paracellularpathway have functional significance? HEPATOLOGY 1983;3:614-617.

37. Mottino AD, Hoffman T, Crocenzi FA, Sanchez Pozzi EJ, Roma MG,Vore M. Disruption of function and localization of tight junctional struc-tures and Mrp2 in sustained estradiol-17beta-D-glucuronide-inducedcholestasis. Am J Physiol Gastrointest Liver Physiol 2007;293:G391-G402.

38. Ortiz DF, Moseley J, Calderon G, Swift AL, Li S, Arias IM. Identificationof HAX-1 as a protein that binds bile salt export protein and regulates itsabundance in the apical membrane of Madin-Darby canine kidney cells.J Biol Chem 2004;279:32761-32770.

39. Chambers TC, Pohl J, Raynor RL, Kuo JF. Identification of specific sites inhuman P-glycoprotein phosphorylated by protein kinase C. J Biol Chem1993;268:4592-4595.


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