ciliary subcellular localization of tgr5 determines the ... · in biliary epithelia. in the liver,...

Ciliary subcellular localization of TGR5 determines the cholangiocytefunctional response to bile acid signaling

Anatoliy I. Masyuk, Bing Q. Huang, Brynn N. Radtke, Gabriella B. Gajdos, Patrick L. Splinter,Tatyana V. Masyuk, Sergio A. Gradilone, and Nicholas F. LaRussoMayo Clinic College of Medicine, Department of Internal Medicine, Rochester, Minnesota

Submitted 28 September 2012; accepted in final form 22 March 2013

Masyuk AI, Huang BQ, Radtke BN, Gajdos GB, Splinter PL,Masyuk TV, Gradilone SA, LaRusso NF. Ciliary subcellular localizationof TGR5 determines the cholangiocyte functional response to bile acidsignaling. Am J Physiol Gastrointest Liver Physiol 304: G1013–G1024,2013. First published April 11, 2013; doi:10.1152/ajpgi.00383.2012.—TGR5, the G protein-coupled bile acid receptor that transmits bile acidsignaling into a cell functional response via the intracellular cAMPsignaling pathway, is expressed in human and rodent cholangiocytes.However, detailed information on the localization and function ofcholangiocyte TGR5 is limited. We demonstrated that in human(H69 cells) and rat cholangiocytes, TGR5 is localized to multiple,diverse subcellular compartments, with its strongest expression onthe apical plasma, ciliary, and nuclear membranes. To evaluate therelationship between ciliary TGR5 and the cholangiocyte functionalresponse to bile acid signaling, we used a model of ciliated andnonciliated H69 cells and demonstrated that TGR5 agonists induceopposite changes in cAMP and ERK levels in cells with and withoutprimary cilia. The cAMP level was increased in nonciliated cholan-giocytes but decreased in ciliated cells. In contrast, ERK signaling wasinduced in ciliated cholangiocytes but suppressed in cells without cilia.TGR5 agonists inhibited proliferation of ciliated cholangiocytes butactivated proliferation of nonciliated cells. The observed differentialeffects of TGR5 agonists were associated with the coupling of TGR5 toG�i protein in ciliated cells and G�s protein in nonciliated cholangio-cytes. The functional responses of nonciliated and ciliated cholangiocytesto TGR5-mediated bile acid signaling may have important pathophysio-logical significance in cilia-related liver disorders (i.e., cholangiociliopa-thies), such as polycystic liver disease. In summary, TGR5 is expressedon diverse cholangiocyte compartments, including a primary cilium, andits ciliary localization determines the cholangiocyte functional responseto bile acid signaling.

cholangiocytes; primary cilia; TGR5; G protein-coupled bile acidreceptor; bile acid signaling

BILE ACIDS act as signaling molecules primarily via activation ofnuclear farnesoid X receptor (FXR) and pregnane X receptor(PXRs) and a plasma membrane-bound G protein-coupledreceptor, TGR5 (13, 32). In the liver, FXR and PXR are highlyexpressed in hepatocytes and function as ligand-modulatedtranscriptional factors that regulate target genes implicatedmainly in hepatic bile acid transport and metabolism (13, 32).Cholangiocytes show weak or no expression of FXR and PXR(7, 41). In contrast, TGR5, which mediates the rapid, transcrip-tion-independent actions of bile acids, is not expressed inhepatocytes (22) but is expressed in cholangiocytes (16–18,20) and is, presumably, a major receptor for bile acid signalingin biliary epithelia. In the liver, TGR5 is also expressed inKupffer cells (16) and sinusoidal endothelial cells (19). Func-

tionally, TGR5 is linked to the cAMP signaling pathway, andits activation in different cell types by free, taurine-conjugated,and glycine-conjugated bile acids induces an increase in intra-cellular cAMP levels followed by a cell-specific response (14,17, 18, 22).

In cholangiocytes, TGR5 has been reported to be localizedto the apical plasma membrane, subapical compartment, andprimary cilia (15, 17, 18, 20). However, the functional signif-icance of this diverse localization of TGR5 remains unknown.The expression of TGR5 in cholangiocyte cilia, which we havedemonstrated are mechano-, chemo-, and osmosensory organ-elles (9, 25, 27, 28), suggests the potential existence of a novelciliary-associated TGR5-mediated bile acid signaling pathwayin biliary epithelia. This pathway may have physiologicalimportance given the fact that cholangiocyte cilia are con-stantly exposed to varying amount and types of bile acidsmoving through the intrahepatic bile ducts (IBDs). The local-ization of TGR5 to the cholangiocyte apical plasma membrane,primary cilia, and subapical compartment also suggested to usthat the cell functional response to bile acid signaling mightdepend on the subcellular localization of TGR5.

Based on these observations, the aims of the present studywere to further characterize the localization of TGR5 in humanand rodent cholangiocytes and to evaluate the relationshipbetween its subcellular expression and the cholangiocyte func-tional response to TGR5-mediated bile acid signaling. Wedemonstrated that TGR5 is localized to multiple, diversecholangiocyte subcellular compartments, with its strongestexpression on the apical plasma, ciliary and nuclear mem-branes. To assess the functional role of ciliary-associatedTGR5, we used experimental models of ciliated and noncili-ated cholangiocytes, which allowed us to observe the differ-ential effects of TGR5 agonists on cAMP and ERK levels incells with and without cilia as well as their proliferation. Thedifferential effects of TGR5 agonists were associated with thecoupling of TGR5 to G�i protein in ciliated cells and G�s

protein in nonciliated cells. Our results indicate that the sub-cellular localization of TGR5 determines the cholangiocytefunctional response to bile acid signaling.

MATERIALS AND METHODS

Chemicals. All chemicals were of the highest purity commerciallyavailable and were purchased from Sigma Chemical (St. Louis, MO)unless otherwise indicated.

Models. IBDs dissected from male Sprague-Dawley rat livers(Harlan Sprague Dawley, Indianapolis, IN) (24) and H69 cells, whichare simian virus 40-transformed cholangiocytes originally derivedfrom the normal human liver (10), were used in this study. H69 cellswere grown in 96-well plates (Corning, Corning, NY) for measuringcAMP levels and the rate of cell proliferation and in 24-well plates(Becton Dickinson, Franklin Lakes, NJ) for ERK-related experiments.

Address for reprint requests and other correspondence: N. F. LaRusso, MayoClinic College of Medicine, 200 First St. SW, Rochester, MN 55905 (e-mail:[email protected]).

Am J Physiol Gastrointest Liver Physiol 304: G1013–G1024, 2013.First published April 11, 2013; doi:10.1152/ajpgi.00383.2012.

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TGR5 agonists were added to a monolayer of H69 cells, i.e., to theapical side of the cells. Dissected IBDs were placed in a microperfu-sion apparatus, as we have previously described in detail (24), and theTGR5 agonist 6�-ethyl-23(S)-methyl-cholic acid (INT-777) was de-livered to the lumen of IBDs using a system of micropipettes; thistechnique allows stimulation of the apical side of naturally ciliatedcholangiocytes lining the IBDs. All experimental procedures usinganimals were approved by the Institutional Animal Care and UseCommittee of Mayo Clinic.

TGR5 ligands. Two strongly potent natural TGR5 agonists, tauro-lithocholic acid (TLCA) and lithocholic acid (LCA) (14, 22, 32), andtwo well-characterized selective agonists, INT-777 (Intercept Phar-maceuticals, New York, NY) (38), which is a semisynthetic derivativeof cholic acid, and oleanolic acid (OA), which is a triterpene naturallypresent in leaves of the olive tree (33), were used in this study.Ursodeoxycholic acid (UDCA), which does not have ligand capacityfor TGR5, was used as a negative control. Forskolin (FSK; 10 �M),which increases cAMP levels directly through adenylyl cyclases, wasused as a positive control. EGF (20 ng/ml) was used as a positivecontrol for ERK signaling and cholangiocyte proliferation. H69 cellsand isolated rat IBDs were treated for 30 min with 25 �M TGR5agonists and UDCA, a concentration that is commonly used in TGR5functional studies (15, 16, 19).

TGR5-green fluorescent protein construct. Total RNA was pre-pared from H69 cells using TRIzol (Invitrogen) and reverse tran-scribed to cDNA using a Superscript III First Strand Synthesis kit(Invitrogen). TGR5 was amplified using the forward primer 5=-CCGGAATTCCTCCAGGACTCCCCTGTCCC-3= and the reverseprimer: 5=-CGCGGATCCGCGTTCAAGTCCAGGTCGACACTG-3=. The forward primer was designed to contain an EcoRI restrictionsite and a BamH1 site that was included in the reverse primer(underlined). The TGR5 stop codon was eliminated, and two nucle-otides (GC, bold) were inserted to put the TGR5 coding sequence inframe with the green fluorescent protein (GFP) coding sequence. ThePCR product was cloned to the pEGFP-N1 vector using the EcoRI andBamH1 restriction sites. The TGR5-GFP recombinant clone wasconfirmed by sequencing.

Apical sodium-dependent bile acid transporter small interferingRNA transfection. H69 cells at 70–80% confluence were transfectedwith apical sodium-dependent bile acid transporter (ASBT) smallinterfering (si)RNA (sc-141294, Santa Cruz Biotechnology, SantaCruz, CA) or the corresponding scrambled siRNA control (sc-36869,Santa Cruz Biotechnology) using siPORT Lipid Transfection Agent(Ambion, Austin, TX) according to the manufacturer’s instruction.The transfection required the use of Opti-MEM1 medium, whichstimulates rapid (i.e., in 48 h) growth of primary cilia in cultured cells(31). In our experiments, H69 cells were treated with 5 nmol ASBTsiRNA or the corresponding scrambled siRNA control for 72 h. Thus,using this protocol, we generated ciliated ASBT siRNA-transfectedH69 cells. The transfection efficacy was determined by fluorescentmicroscopy using control siRNA (FITC conjugate)-A (sc-36869,Santa Cruz Bioltechnology) and approached 80% at 72 h. The ex-pression of ASBT protein in ASBT siRNA-transfected H69 cells wasdetected by Western blot analysis with actin as a loading control.

Immunofluorescence confocal microscopy. Livers from normal rats(Harlan Sprague Dawley) and mice (C57BL/6, our own colony) werefixed and embedded in paraffin. Normal human liver tissues wereobtained from the Mayo Clinical Core and National Disease ResearchInterchange. H69 cells, H69 cells transfected with the TGR5-GFPconstruct, and paraffin-embedded sections of rat, mouse, and humanlivers were incubated with the following antibodies: acetylated �-tu-bulin, a ciliary marker (T6793, Sigma Chemical); lamin B2, a nuclearmembrane marker (ab84366, Abcam, Cambridge, MA); TGR5 (sc-48687, Santa Cruz Biotechnology); G�s (ab83735, Abcam); and G�i

(5290S, Cell Signaling Technology, Danvers, MA) and then incubatedfor 1 h at room temperature with fluorescent secondary antibodies(Molecular Probes, Eugene, OR). Nuclei were stained blue with

Prolong Gold Antifade Reagent with 4=,6-diamidino-2-phenylindole(P36935, Invitrogen). The corresponding negative controls (i.e., noprimary antibodies) were performed for all experiments. A ZeissLSM-510 microscope (Carl Zeiss, Thornwood, NY) was used in thisstudy.

Immunogold transmission electron microscopy. Isolated rat IBDsand H69 cells grown to 7 days postconfluence on collagen-coatedcoverslips (BD Biosciences, Sparks, MD) were fixed in 4% parafor-maldehyde and 0.2% glutaraldehyde for 1 h at room temperature.After fixation, samples were washed in 0.1 M phosphate buffer (PB;pH 7.2–7.4) and in PB containing 0.1% sodium borohydride (10 min,4 times) to inactivate residual aldehyde groups. Samples were thenwashed four times in PB and treated for 5 min with PB containing0.1% Triton X-100. After being washed four times in PB, sampleswere incubated for 60 min at 4°C in blocking solution, which was PBScontaining 1% FCS. Samples were then incubated overnight at 4°Cwith TGR5 (ab72608, Abcam) and G�i (5290S, Cell Signaling Tech-nology) primary antibodies diluted 1:20 with PBS containing 2%FCS. After six washes with PBS containing 2% FCS, samples wereincubated for 1 h at room temperature with a secondary goat anti-mouse antibody conjugated with ultrasmall gold (Electron Micros-copy Sciences, diluted 1:100 with blocking solution). Samples werewashed in PBS, postfixed with 2.5% glutaraldehyde in PB for 2 h,enhanced with silver enhancement mixture (R-Gent SE-EM) for 30min, and the osmicated with 1% osmium tetroxide for 30 min. In thecorresponding controls, the incubation of samples with primary anti-bodies was omitted. For transmission electron microscopy (TEM),samples were dehydrated, embedded in Spurrs resin, sectioned at 90nm, and observed using a Joel 12 electron microscope (Joel USA,Peabody, MA).

Negative staining immunogold TEM of biliary exosomes. Exo-somes were isolated from rat bile and processed for negative stainingimmunogold TEM, as we have previously described in detail (26).The expression of the exosomal marker CD63 in biliary exosomeswas detected with a corresponding primary antibody (sc-51662, SantaCruz Biotechnology).

Immunogold scanning electron microscopy. Isolated rat IBDs wereimmersed in 4% phosphate-buffered glutaraldehyde (pH 7.4) for 1 h,treated with 0.1% Triton X-100 for 5 min, and rinsed with PB threetimes. Samples were incubated overnight at 4°C with antibodies toTGR5 (sc-48687, Santa Cruz Biotechnology, dilution: 1:100) and thenincubated for 2 h at room temperature with anti-rabbit IgG conjugatedwith 1.4-nm nanogold. Samples were fixed in 1% glutaraldehyde for10 min, and gold enhancement was performed with a gold enhance-ment kit (Nanoprobes). Samples were dehydrated, critical point dried,and carbon coated. Images were generated by a Hitachi S-4700microscope (Hitachi, Pleasanton, CA).

Western blot analysis. Proteins were separated by 4–15% SDS-PAGE, transferred to nitrocellulose membranes (Bio-Rad, Hercules,CA), and the probed with antibodies against phosphorylated (p-)ERK(612359, BD Biosciences, San Jose, CA), total ERK (ab36991,Abcam), TGR5 (sc-48687, Santa Cruz Biotechnology), CD63 (sc-51662, Santa Cruz Biotechnology), ASBT (sc-27493, Santa CruzBiotechnology), G�s (ab83735, Abcam), and G�i (5290S, Cell Sig-naling Technology) overnight at 4°C. Actin staining was used for thenormalization of protein loading. Membranes were washed and incu-bated for 1 h at room temperature with the corresponding horseradishperoxidase-conjugated (Invitrogen) or IRdye 680 or 800 (Odyssey)secondary antibodies. Bands were visualized with the ECL PlusWestern Blotting Detection kit (BD Biosciences) or Odyssey Li-CorScanner (Li-Cor, Lincoln, NE).

Measurement of cAMP. cAMP levels in cholangiocytes were mea-sured by the Bridge-It cAMP designer cAMP assay (Mediomics, St.Louis, MO), as we have previously described (25), and expressed inpicomoles of cAMP per microgram of protein (H69 cells) or inpicomoles of cAMP per microgram of DNA (microdissected ratIBDs).

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Cell proliferation assay. H69 cells were grown at 37°C, 5% CO2,and 100% humidity. Twenty-four hours before the assay, cells weretreated with TGR5 agonists (25 �M). The rate of cell proliferationwas determined using a CellTiter 96 Aqueous One Solution CellProliferation Assay (Promega). The relative rates of cholangiocyteproliferation are expressed as percentages of the proliferation ofnontreated cholangiocytes (100%).

Statistical analysis. GraphPadPrism software (version 4.0, SanDiego, CA) was used for statistical analysis. The statistical signifi-cance between two groups was tested by a two-tailed Student’s t-test.All values are expressed as means � SE, and differences of P � 0.05were considered significant.

RESULTS

TGR5 is localized to cholangiocyte cilia. The localization ofTGR5 to cholangiocyte cilia was demonstrated by severalcomplementary experimental approaches. By immunofluores-

cence confocal microscopy of paraffin-embedded sections ofrat, mouse, and human livers (Fig. 1A), cilia and ciliary-associated TGR5 were identified with antibodies to a ciliarymarker, acetylated �-tubulin, and TGR5, respectively. Theimages shown in Fig. 1A show acetylated �-tubulin- andTGR5-positive cilia. The overlay images show colocalizationof these two proteins, i.e., the localization of TGR5 to cholan-giocyte cilia in rat, mouse, and human livers.

To generate additional evidence on the localization of TGR5to cholangiocyte cilia, H69 cells were transiently transfectedwith a TGR5-GFP construct, and its presence in cilia wasanalyzed by confocal immunofluorescence microscopy (Fig.1B). The ciliary expression of TGR5-GFP was determined byits colocalization with a ciliary marker, acetylated �-tubulin. Astrong accumulation of TGR5-GFP was found on the cholan-giocyte plasma membrane and cilia. In contrast, no specific

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Fig. 1. TGR5 is localized to cholangiocyte cilia. A: paraffin-embedded sections of rat, mouse, and human livers were stained with antibodies to the ciliary markeracetylated �-tubulin (red) and TGR5 (green). Cholangiocyte nuclei were visualized with 4=,6-diamidino-2-phenylindole (DAPI; blue). The overlay images showcolocalization of acetylated �-tubulin and TGR5 in yellow. L, lumen of the intrahepatic bile duct (IBD). B: H69 cells transiently transfected with a recombinanthuman TGR5-green fluorescent protein (GFP) construct showed localization of TGR5-GFP to the plasma membrane (PM) and primary cilium. In contrast, theGFP-empty vector (control) was distributed homogeneously in the cytoplasm of transfected H69 cells. Primary cilia were visualized with antibodies to acetylated�-tubulin (red) and colocalization with TGR5-GFP shown in yellow. Insets show higher-magnification images. C: immunogold tranmission electron microscopyshowed localization of TGR5 on the ciliary membrane in rat IBD and H69 cells. A TGR5-positive exosome (inset) was attached to the ciliary membrane.Exosomes isolated from rat bile were positive for the exosomal marker CD63 and TGR5. D: conventional scanning electron microscopy image of an isolatedsplit-open rat IBD showing primary cilia and microvilli extending from the cholangiocyte apical PM. The image generated by backscattered electrons showednumerous white dots, revealing the presence of TGR5 on cholangiocyte cilia and microvilli.

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subcellular distribution of GFP was found in H69 cells trans-fected with the empty vector.

The localization of TGR5 to cholangiocyte cilia was furtherconfirmed by immunogold TEM (Fig. 1C) and scanning elec-tron microscopy (SEM; Fig. 1D). By immunogold TEM,TGR5 was found on the ciliary membrane of freshly micro-dissected rat IBDs and cultured human cholangiocytes (H69cells). The TGR5-positive vesicle shown in Fig. 1C (and alsoin the inset) attaching to the cholangiocyte cilium is likely anexosome. This interpretation is further supported by immuno-gold TEM and Western blot analysis results (Fig. 1C) demon-strating that exosomes isolated from rat bile contain both theexosomal marker CD63 and TGR5 and by our previous obser-vation that exosomes interact with cholangiocyte cilia (26).

Finally, cilia extending from the cholangiocyte apical plasmamembrane could be clearly seen by conventional SEM of thelumen of a microdissected split-open rat IBD (Fig. 1D). Theimage of the same field generated by backscattered electronsshows the gold-labeled secondary antibodies, which are shownon the ciliary membrane as bright white dots. The bright whitedots can also be seen on the cholangiocyte microvilli, support-ing our and others (18) observations that TGR5 is distributed

on cilia and on the nonciliary portion of the cholangiocyteapical plasma membrane.

TGR5 is localized to diverse cholangiocyte compartments.The localization of TGR5 was not limited to the apical surfaceof cholangiocytes. By immunogold TEM of both microdissectedrat IBDs and ciliated H69 cells, TGR5 immunoreactivity wasobserved in the endoplasmic reticulum (ER), nucleus, multive-sicular bodies (MVBs), and intracellular vesicles (Fig. 2A). Thepattern of TGR5 labeling in rat and human (H69 cells) cholan-giocytes was similar. Notably, TGR5 was present at highdensities on the cholangiocyte nuclear membrane. As shown inthe high-power photomicrographs of cholangiocyte nuclei(Fig. 2A), TGR5 immunoreactivity was associated with bothouter and inner nuclear membranes (ONM and INM, respec-tively). Because the ONM is continuous with the ER, theobserved TGR5 immunoreactivity may reflect both the ER andnuclear localization of TGR5. However, the TGR5 immuno-reactivity on the cholangiocyte INM strongly implies the nu-clear localization of this protein.

The localization of TGR5 to the cholangiocyte INM wasfurther confirmed by confocal immunofluorescence micros-copy of H69 cells and H69 cells transiently transfected with a

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Fig. 2. TGR5 is localized to diverse cholangio-cyte compartments. A: TGR5 was localized onthe apical PM, microvilli, multivesicular body(MVB), endoplasmic reticulum (ER), vesicles,and outer and inner nuclear membranes (ONMand INM, respectively) of H69 cells and micro-dissected rat IBDs. B: strong expression of thenuclear membrane marker lamin B2 (red) andTGR5 (green) was observed on the nuclearmembrane of H69 cells, and their colocalizationis shown in yellow. C: lamin B2 (red) wascolocalized with TGR5-GFP in H69 cells tran-siently transfected with a TGR5-GFP construct(shown in yellow). The nuclei of H69 cellswere stained with DAPI (blue).



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Fig. 3. TGR5 agonists differentially affect cAMP levels in nonciliated and ciliated cholangiocytes. A: immunofluorescence confocal microscopy images showingnonciliated and ciliated H69 cells. Cells were stained with the ciliary marker acetylated �-tubulin (red). DAPI (blue) indicates nuclei. Multiple cilia were foundin H69 cells cultured for 7 days after confluence (ciliated) but not in confluent H69 cells (day 0, nonciliated). B: the cAMP level was higher in nonciliated H69cells compared with ciliated cells under basal and forskolin (FSK; 10 �mol)-induced conditions. C: in response to taurolithocholic acid (TLCA), lithocholic acid(LCA), INT-777, and oleanolic acid (OA) (all 25 �mol), cAMP levels were increased in nonciliated cells but decreased in ciliated H69 cells. Ursodeoxycholicacid (UDCA; 25 �mol), which does not have affinity for TGR5 (negative control), did not affect cAMP levels in both nonciliated and ciliated cholangiocytes.Also, INT-777 (25 �mol) inhibited cAMP levels in ciliated cholangiocytes of rat IBDs microperfused through their lumen. n � 12 wells from 3 separateexperiments; n � 7 bile ducts isolated from 3 rats. *P � 0.05. D: representative Western blots (selected portions of the original blots are shown) and quantitativedata demonstrating suppression of apical sodium-dependent bile acid transporter (ASBT) expression in H69 cells transfected with ASBT small interfering(si)RNAs. Suppression of ASBT expression in H69 cells did not abolish the inhibitory effects of the TGR5 agonists TLCA and INT-777 (both 25 �mol) on cAMPlevels. n � 6 wells from 3 separate experiments. *P � 0.05.



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TGR5-GFP construct. To identify the expression of TGR5 onthe nuclear membrane of H69 cells, cells were stained withantibodies to a marker of the INM, lamin B2, and TGR5.Figure 2B shows the strong expression of lamin B2 (red) andTGR5 (green) on the cholangiocyte nuclear membrane. Thecolocalization of these two proteins is shown in yellow. In H69cells transiently transfected with a TGR5-GFP construct (Fig.2C), TGR5-GFP was colocalized with lamin B2 on the cholan-giocyte INM.

Taken together, these data demonstrate that TGR5 is local-ized to diverse cholangiocyte compartments, with its strongestexpression on the apical, ciliary, and nuclear membranes.

TGR5 agonists decrease cAMP levels in ciliated cholangiocytes.To address the role of ciliary TGR5 in bile acid signaling incholangiocytes, we used H69 cells and compared the effects ofTGR5 agonists on cAMP levels in cells with and without cilia(Fig. 3). H69 cells, when grown on the appropriate matrixes,form polarized monolayers and retain the phenotypic andfunctional characteristics of cholangiocytes in vivo (10). Theformation of primary cilia in cholangiocytes depends on cellconfluence and the duration of cell growth (12). H69 cells atday 0 after confluence are naturally occurring nonciliatedcholangiocytes (Fig. 3A). H69 cells on day 7 after confluencepossess well-developed primary cilia, i.e., are naturally occur-ring ciliated cholangiocytes (Fig. 3A).

Nonciliated and ciliated H69 cells are different with regardto their cAMP levels. In particular, basal and FSK-inducedcAMP levels in ciliated H69 cells were 3.4 and 4.2 times lowercompared with nonciliated cells (i.e., 1.64 � 0.08 vs. 5.65 �0.25 pmol/�g protein and 12.76 � 0.44 vs. 54.08 � 2.54pmol/�g protein, respectively; Fig. 3B). In response to the twomost potent natural TGR5 agonists, TLCA and LCA, and twoselective TGR5 agonists, INT-777 and OA, cAMP levelsdecreased in ciliated H69 cells by 29.9 � 6.0%, 40.8 � 8.7%,43.6 � 10.2%, and 33.0 � 8.2%, respectively (Fig. 3C),whereas treatment of nonciliated H69 cells with TGR5 agonistsunder similar experimental conditions resulted in an increase of

cAMP levels by 63.8 � 10.7%, 49.2 � 7.9%, 39.3 � 5.9%,and 72.2 � 8.7%, respectively (Fig. 3C). UDCA (a negativecontrol), as expected, did not affect cAMP levels in nonciliatedand ciliated cholangiocytes (Fig. 3C). In all experiments, theeffects of FSK, which activates selective adenylyl cyclases viaa TGR5-independent mechanism (a positive control), did notdepend on the ciliary status of H69 cells and resulted in similar(i.e., by 9.6- and 7.8-fold) increases in cAMP levels in non-ciliated and ciliated cholangiocytes, respectively (Fig. 3B).Taken together, these data demonstrate that in ciliated cholan-giocytes, TGR5 agonists inhibit cAMP signaling.

Consistent with our results in cultured human cholangio-cytes, an inhibitory effect of the TGR5 selective agonistINT-777 was also observed in isolated rat IBDs, which arelined by ciliated cholangiocytes. The cAMP level in ratIBDs in response to INT-777 was decreased by 69.7 �31.1% (Fig. 3C).

Taking into account the multiple intracellular locations ofTGR5 that could be activated by TGR5 agonists delivered intothe cell via the bile acid transporter ASBT, rather than at theciliary membrane, we tested the effects of two TGR5 agonists,TLCA and INT-777, on cAMP levels in ciliated H69 cells inwhich ASBT expression was suppressed by specific siRNAs.ASBT siRNAs inhibited the expression of ASBT protein inciliated H69 cells by 54.5 � 7.9% (Fig. 3D), but this decreasein ASBT expression did not affect the inhibitory action ofTGR5 agonists on cAMP levels. Similar to previously ob-served effects, TLCA and INT-777 inhibited cAMP levels inciliated H69 cells transfected with ASBT siRNAs by 41.4 �4.2% and 41.9 � 2.5%, respectively, which were not signifi-cantly different (i.e., inhibition by 49.1 � 2.8% and 38.9 �1.7%, respectively) from H69 cells transfected with ASBTscrambled control siRNAs.

In conclusion, the effects of TGR5 agonists on cAMP levelsin cholangiocytes occur at the apical membrane and depend onthe presence of cilia. In ciliated cholangiocytes, TGR5 agonistsinduce a decrease in cAMP levels, whereas they provoke an

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Fig. 4. TGR5 agonists differentially affectERK signaling in nonciliated and ciliatedcholangiocytes. The TGR5 agonists TLCAand INT-777 (both 25 �mol) decreased theratio of phosphorylated ERK to total ERK(p-ERK/t-ERK ratio) in nonciliated H69 cellsbut increased this ratio in ciliated cholangio-cytes. UDCA (25 �mol, negative control) didnot affect ERK signaling in both nonciliatedand ciliated cholangiocytes, whereas EGF(20 ng/ml, positive control) increased thep-ERK/t-ERK ratio in both cell types. Rep-resentative Western blots and quantitativedata from 3 separate experiments are shown.*P � 0.05.



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opposite effect, i.e., stimulate cAMP production, in nonciliatedcholangiocytes.

TGR5 agonists differentially affect ERK1/2 in nonciliatedand ciliated cholangiocytes. Because signal transduction by Gprotein-coupled receptors involves the activation of ERK, westudied whether TGR5 agonists affect ERK signaling in non-ciliated and ciliated cholangiocytes and how. We found that theeffects of TGR5 agonists on ERK1/2 in nonciliated and ciliatedcholangiocytes were different and opposite to their effects oncAMP levels. A natural TGR5 agonist, TLCA, and a selectiveTGR5 agonist, INT-777, decreased the p-ERK-to-total ERKratio in nonciliated H69 cells by 48.2 � 8.4% and 36.6 �6.1%, respectively, but increased this ratio in ciliated H69 cellsby 39.2 � 7.1% and 35.7 � 6.3% (Fig. 4). The effects ofUDCA and EGF, which were used as negative and positive

controls, respectively, did not depend on the ciliation status ofcholangiocytes. UDCA did not affect ERK in both nonciliatedand ciliated cholangiocytes, whereas EGF increased the p-ERK-to-total ERK ratio in both nonciliated and ciliated cellsby 39.5 � 9.0% and 37.7 � 8.1%, respectively (Fig. 4). Thus,these data demonstrate that the cholangiocyte ERK signalingresponse to TGR5 agonists also depends on cilia.

The effects of TGR5 agonists on cholangiocyte proliferationdepend on the presence of cilia. To determine whether ciliary-associated TGR5 contributes to a bile acid-regulated functionalcellular response, we tested the effects of TGR5 agonists on theproliferation of nonciliated and ciliated H69 cells. UDCA andEGF were used as negative and positive controls, respectively.

The data shown in Fig. 5 demonstrate that all three TGR5agonists, TLCA, INT-777, and OA, increased the proliferation

Relative rate of cell proliferation, %


Non-ciliated H69 cells Ciliated H69 cells


Ciliated H69 cells transfected with siRNAs

* * *

* * *

* * Control siRNAs ASBT siRNAs

* * * *

Fig. 5. TGR5 agonists differentially affect the proliferation of nonciliated and ciliated H69 cells. The TGR5 agonists TLCA, INT-777, and OA (all 25 �mol)increased the proliferation of nonciliated H69 cells but decreased the proliferation of ciliated cells. UDCA (25 �mol, negative control) did not affect theproliferation of nonciliated or ciliated cholangiocytes. In contrast, EGF (20 ng/ml, positive control) increased the proliferation of both cell types. Suppressionof ASBT expression in H69 cells did not abolish the inhibitory effects of the TGR5 agonists TLCA and INT-777 (both 25 �mol) on cell proliferation. n � 16wells from 3 separate experiments. *P � 0.05.

A

Gαiα-tubulin overlay

Gαsα-tubulin overlay

BH69 cells Rat IBD

Gαi

Basal body

Ciliary shaft

Fig. 6. G�i protein is expressed on cholangiocyte cilia. A: expression of G�i protein and its localization at the base of cilia in H69 cells detected byimmunofluorescence confocal microscopy. B: expression and localization of G�i protein at the base of cholangiocyte cilia detected by immunogold tranmissionelectron microscopy in isolated rat IBDs. Numerous black dots reveal the expression of G�i in the basal body.



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of nonciliated H69 cells by 38.0 � 7.3%, 31.4 � 4.5%, and25.8 � 5.2%, respectively. The proliferative effects of TGR5agonists were comparable with the effect of EGF (positivecontrol), which increased the proliferation of nonciliated H69cells by 45.3 � 4.7%. UDCA did not affect cholangiocyteproliferation.

The proliferative response of ciliated H69 cells to TGR5agonists was opposite: all three TGR5 agonists inhibitedcholangiocyte proliferation by 24.6.0 � 6.5%, 35.0 � 7.7%,and 27.0 � 6.7%, respectively. UDCA did not affect cellproliferation, whereas EGF (positive control) increased theproliferation of ciliated H69 cells by 31.3.3 � 6.8% (Fig. 5).

Given the importance of the bile acid transporter ASBT incholangiocyte proliferation (40), we tested the effects of twoTGR5 agonists, TLCA and INT-777, on the proliferation ofciliated H69 cells in which ASBT expression was suppressedby specific siRNAs. Our data (Fig. 5) show that ASBT sup-pression did not affect the inhibitory effects of TGR5 agonistson cholangiocyte proliferation. TLCA and INT-777 inhibitedthe proliferation of ciliated H69 cells transfected with ASBTsiRNAs by 48.5 � 5.7% and 49.2 � 4.8%, respectively.Similar effects of TGR5 agonists (i.e., inhibition by 40.9 �1.1% and 41.6 � 3.6%, respectively) were observed in H69cells transfected with ASBT scrambled control siRNAs.

These data show that the proliferative response of H69 cellsto TGR5 agonists depends on the presence of cilia.

Differential effects of TGR5 agonists in ciliated and noncili-ated cholangiocytes are associated with the coupling of TGR5to different G� proteins. One of the potential mechanismsthrough which TGR5 agonists may induce differential func-tional responses of ciliated and nonciliated cholangiocytes isassociated with the coupling of TGR5 to different G� proteins.To test if the inhibitory effects of TGR5 agonists on cAMPlevels in ciliated cholangiocytes are associated with G�i,whereas their stimulatory effects in nonciliated cells are asso-ciated with G�s, we studied the expression of G�i and G�s

proteins on cholangiocyte cilia and their colocalization withTGR5 in nonciliated and ciliated H69 cells under basal condi-tions and in response to a specific TGR5 agonist, INT-777.

The images shown in Fig. 6A demonstrate that G�i but notG�s protein is localized to primary cilia in H69 cells. In theseimages, G�i localization appeared to be restricted to the base ofcilia but not over the entire length of the ciliary shaft. Acharacteristic localization of G�i protein to the base of cholan-giocyte cilia was further confirmed on freshly isolated rat IBDsby immunogold TEM (Fig. 6B).

As demonstrated by Western blot analysis (Fig. 7) andconfocal immunofluorescence microscopy (Fig. 8), both G�s

Gαi

TGR5

Gαs

Actin

Non-ciliatedH69 cells

Control INT-777 Control INT-777

CiliatedH69 cells

Long (45 kDa)Short (43 kDa)

A B

C

Non-ciliated H69 cells

CiliatedH69 cells

Gα proteins/actin

INT-777Control

Control INT-777 Control INT-777 0.00

0.05

0.10

0.15

0.20

TGR5/actin


CiliatedH69 cells

**

*

* *

*

**


CiliatedH69 cells

1.00

0.75

0.50

0.25

0.00Gαs Gαi Gαs Gαi Gαs Gαi Gαs Gαi

Fig. 7. G�s and G�i proteins are differentiallyexpressed in nonciliated and ciliated H69 cells.Representative Western blots of G� proteins andTGR5 (A) and quantitative data (B and C) areshown. Actin was used as a loading control. G�s

was present in H69 cells in two isoforms: long (45kDa) and short (43 kDa) (A). The amount of TGR5(B) and G� proteins (C) increased in ciliated cellscompared with nonciliated cells. The amount ofG�s in nonciliated cholangiocytes was larger thanthe amount of G�i (C). The TGR5-specific agonistINT-777 (25 �mol) did not affect the amount ofTGR5 (B) and G� proteins (C) in nonciliated andciliated cholangiocytes.



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and G�i proteins were expressed in nonciliated and ciliatedcholangiocytes; however, in nonciliated cells, the amount ofG�s protein normalized to actin (i.e., 0.16 � 0.02) was twotimes higher than the amount of G�i protein (i.e., 0.08 � 0.01).Compared with nonciliated H69 cells, ciliated cholangiocytesexpressed larger amount of both G�s and G�i proteins (theamount of G�s and G�i proteins was increased by 4.8- and8.5-fold, respectively; Fig. 7, A and C). The amount of TGR5was also increased in ciliated cholangiocytes compared withnonciliated cells by twofold, i.e., less dramatically than theamount of G�s or G�i proteins (Fig. 7B). Stimulation ofnonciliated and ciliated H69 cells with INT-777 did not affectthe amount of both G�s and G�i proteins as well as TGR5 (Fig.7) but induced changes in their colocalization (Fig. 8). Innonciliated cholangiocytes, INT-777 increased colocalizationof TGR5 with G�s protein but not with G�i protein, whereas inciliated cells the effect of INT-777 was opposite: TGR5 wasprimarily colocalized with G�i protein but not with G�s pro-tein. These data support our hypothesis that differential effectsof TGR5 agonists in ciliated and nonciliated cholangiocytes areassociated with the coupling of TGR5 to G�i and G�s proteins,respectively.

DISCUSSION

The key findings of this study relate to the localizationand function of the G protein-coupled bile acid receptorTGR5 in cholangiocytes. We demonstrated that 1) TGR5 islocalized to diverse cholangiocyte compartments, with its

strongest expression on the cholangiocyte apical plasma,ciliary, and nuclear membranes; 2) activation of TGR5 inciliated and nonciliated cholangiocytes resulted in oppositechanges in cAMP levels (i.e., in a decrease and an increase,respectively), demonstrating that ciliary TGR5 is associatedwith inhibition of cAMP signaling; 3) TGR5 is linked toERK signaling, which is activated in ciliated cholangiocytesbut inhibited in nonciliated cholangiocytes; 4) activation ofTGR5 promotes the proliferation of nonciliated cholangio-cytes but inhibits the proliferation of ciliated cholangio-cytes; and 5) the differential effects of TGR5 agonists inciliated and nonciliated cholangiocytes are associated withthe differential coupling of TGR5 to G�i and G�s proteins,respectively.

TGR5 is recognized as a novel bile acid receptor that isexpressed in different cells exposed to bile acids. The highestlevels of TGR5 expression have been detected in the gallblad-der (39) and in the intestine, primarily in the ileum and colon(22, 23, 39). TGR5 is also expressed in human and rodentcholangiocytes (16, 18, 20). The initial observations on TGR5suggested that in different cell types, this bile acid receptor ispredominantly localized in a subapical compartment and, to asmaller extent, on the plasma membrane (14, 16). The intra-cellular localization of TGR5 was proposed to be a result of itsinternalization into the cytoplasm in response to bile acids,whereas its expression on the plasma membrane links thesignaling properties of bile acids to the intracellular cAMPsignaling pathway (14).

TGR5 Gαs overlay

TGR5 Gαi overlay TGR5 Gαi overlay

TGR5 Gαs overlay

TGR5 Gαi overlay

TGR5 Gαs overlay

TGR5 Gαi overlay

TGR5 Gαs overlay

Ciliated H69 cellsNon-ciliated H69 cells

Control

Control

INT-777

INT-777

A B

Fig. 8. G�s and G�i proteins are differentially involved in the effects of the TGR5 agonist INT-777 in nonciliated and ciliated H69 cells. A: in nonciliated H69cells, INT-777 increased the colocalization of TGR5 with G�s protein but not G�i protein. B: in ciliated cholangiocytes, the effect of INT-777 was opposite:colocalization of TGR5 increased with G�i protein but not with G�s protein.



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Our study demonstrates that the cellular localization ofTGR5 in human and rat cholangiocytes is complex. We foundthat in addition to its localization to the plasma and ciliarymembranes, TGR5 is also expressed on the cholangiocyte ER,in MVBs and exosomes, and on the nuclear membrane. Thepresence of TGR5 on the cholangiocyte ER may simply reflectits synthesis in this organelle. However, based on the observa-tions that bile acids can affect the ER via organelle-associatedsignaling mechanisms (3, 8), we cannot exclude that TGR5 inthe cholangiocyte ER is functional. The expression of TGR5 inMVBs and intracellular vesicles suggests that TGR5 mayundergo internalization, recycling, and release from the cell asa component of exosomes, which are small extracellular ves-icles containing numerous biologically active molecules (26).However, the functional significance of exosomal TGR5 re-mains unclear. The expression of TGR5 on the cholangiocytenuclear membrane may provide a novel mechanism for in-tranuclear bile acid signaling. The expression and function of anumber of the G protein-coupled receptors on the nuclearmembrane in different cell types are known physiologic phe-nomena (21, 37), and these phenomena could be extended toTGR5 localized to the cholangiocyte nuclear membrane.

We hypothesized in this study that the localization of TGR5in different cellular compartments may provide mechanismsfor subcellular, site-specific bile acid signaling. As an initialattempt to test this hypothesis, we addressed the functionalsignificance of TGR5 localized to the cholangiocyte ciliarymembrane. We found that cAMP levels in ciliated cholangio-cytes differ from cAMP levels in nonciliated cholangiocytes,being higher in nonciliated H69 cells compared with ciliatedcells. These data are in agreement with recently publishedobservations showing that nonciliated and ciliated renal epi-thelial cells differ with regard to cAMP levels and that primarycilia in renal epithelial cells negatively control intracellularcAMP signaling (5). Our present and previous work on sensoryfunctions of cholangiocyte cilia (25, 27, 28) also suggest thatthese organelles are negatively linked to cAMP signaling.Activation of ciliary TGR5 by bile acids and selective agonistsinhibits cAMP signaling in cholangiocytes similarly to otherextracellular stimuli, such as bile flow and biliary nucleotides,which act via ciliary polycystin-1/polycystin-2 and P2Y12/adenylate cyclase 6 complexes (25, 28). Thus, there appears tobe a consistent inhibitory ciliary-associated signaling pathway,at least as it relates to the cAMP response.

Our observations showing that in nonciliated H69 cellsTGR5 agonists increase cAMP levels are in agreement withpreviously observed TGR5-mediated cAMP increases in dif-ferent cell types in culture since in all of these reports, cellswere cultured no longer than 24 h (i.e., not grown long enoughto develop cilia). Thus, an increase in cAMP levels in responseto TGR5 agonists in such cells should be considered as afunctional response of naturally occurring nonciliated cells toTGR5 agonists.

TGR5 agonists also affect the ERK signaling pathway incholangiocytes. In general, the linkage of TGR5 to ERKsignaling is not yet well established. It has been mentioned thatactivation of TGR5 in TGR5-transfected Chinese hamsterovary (CHO) cells resulted in changes in ERK1/2 (14) and thatin gastric carcinoma cells TGR5 is involved in the activation ofERK1/2 by deoxycholic acid (42). The precise mechanisms ofTGR5-mediated regulation of ERK signaling remain unknown.

It has been shown that cAMP is involved in both activation andinhibition of ERK signaling depending on the functional stateof the cell (6, 36). For example, cAMP-induced inactivation ofERK was observed in NIH-3T3-A14 cells, whereas in CHOand OVCAR3 cells, ERK was activated by cAMP (6). Also,inhibition of cAMP production and activation of ERK inresponse to progestin hormones were observed in MDA-MB-231 cells (43). In our experiments, an increase in cAMP levelsin response to TGR5 agonists resulted in inhibition of ERKsignaling and vice versa, suggesting a potential TGR5-medi-ated cross talk between cAMP and ERK signaling pathways.The changes in the p-ERK-to-total ERK ratio in response toTGR5 agonists are presumably secondary to changes in cAMPlevels.

Cholangiocytes are epithelial cells with a great capacity toproliferate under different experimental conditions and in thecourse of chronic liver diseases, including cholangiociliopa-thies, a group of diseases that are caused by abnormalities inthe structure and function of cholangiocyte cilia (29). We (29)have previously reported that cholangiocyte proliferation in-creases when cilia are absent or structurally or functionallyabnormal. This observation is in agreement with the existingconcept that primary cilia are involved in the inhibition of cellproliferation in a healthy adult organism (2). Our data demon-strating that activation of ciliary-associated TGR5 triggersinhibition of cholangiocyte proliferation further support thisconcept. In contrast, nonciliated cholangiocytes respond toTGR5 agonists by an increase in proliferation. The involve-ment of TGR5 in bile acid-induced proliferation of nonciliatedcells has been recently observed in human Barrett’s adenocar-cinoma cell line FLO (11) and cholangiocytes isolated fromwild-type and TGR5 knockout mice (18). It is likely thatnonciliary TGR5 transduces bile acid signaling via mecha-nisms that are distinct from the ciliary-associated TGR5-me-diated mechanisms controlling cholangiocyte proliferation.

TGR5 TGR5

Non-ciliatedcholangiocytes

Ciliatedcholangiocytes

cAMP ERK1/2

proliferation

cAMP ERK1/2

proliferation

BAs BAs

TGR5

Fig. 9. Working model of TGR-mediated bile acid signaling in nonciliated andciliated cholangiocytes. The localization of TGR5 on the PM or ciliarymembrane determines the cholangiocyte functional response to bile acid (BA)signaling. In nonciliated cholangiocytes, TGR5 agonists increase cAMP levelsand cell proliferation but inhibit ERK signaling. In ciliated cholangiocytes, theeffects of TGR5 agonists are opposite, i.e., cAMP levels and cell proliferationdecreased, but ERK signaling is activated.



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As for any G protein-coupled receptor, coupling to G�proteins is a major mechanism in TGR5-mediated bile acidsignaling. TGR5 is primarily coupled to G�s protein; however,its coupling to G�i protein has also been reported (4, 11). Bystudying the relevance of G�s and G�i proteins in TGR5-mediated bile acid signaling in nonciliated and ciliated cholan-giocytes, we found that G�s protein is likely responsible for theTGR5-mediated effects of bile acids in nonciliated cholangio-cytes, whereas G�i protein is involved in mechanisms ofTGR5-mediated bile acid signaling in ciliated cells. The ex-pression of G�s and G�i proteins was not changed in bothnonciliated and ciliated cholangiocytes in response to a TGR5agonist, but the colocalization and redistribution of G� pro-teins and TGR5 were specifically and differentially affected incells with and without cilia. However, the cellular and molec-ular mechanisms that determine the coupling of TGR5 todifferent G� proteins in nonciliated and ciliated cells remain tobe elucidated.

In the present study, we showed that in cholangiocytes, G�i

is localized at the base of cilia. Several published observationssupport our findings. For example, G�i2, a member of the G�i

family, is localized to motile cilia in the rat ependyma, oviduct,and trachea (35) and in the human endometrium and fallopiantubes (30). G�i1, G�i2, and G�i3 are distributed between thebase and shaft of the primary cilium when expressed incultured cerebellar granular neuronal precursors (1).

The primary cilium is currently recognized as a dynamicorganelle consisting of several morphological and functionalcompartments. The proximal segment of the primary ciliumand the ciliary base are considered as a continuous functionalcompartment where different signaling molecules cycle andinteract with each other in a dynamic manner (1, 34). Thus, thelocalization of TGR5 and G�i protein to cholangiocyte ciliasuggests that these organelles function as a main integrationcenter that determines cholangiocyte functional responses tobile acid signaling.

Taking into account the increasing evidence for a role ofTGR5 in liver diseases (17), the observed differential func-tional responses of nonciliated and ciliated cholangiocytes toTGR5-mediated bile acid signaling may have important patho-physiological significance. Our preliminary data (unpublishedobservations) suggest that in cilia-related liver disorders suchas autosomal dominant and autosomal recessive polycystickidney diseases, TGR5 is overexpressed in cholangiocytes butnot localized on cilia. On the other hand, autosomal dominantand autosomal recessive polycystic kidney disease-affectedcholangiocytes are characterized by malformed cilia and in-creased levels of cAMP (27), suggesting that they have lostciliary-associated mechanisms negatively controlling cAMPlevels. This may contribute to a cAMP increase in cysticcholangiocytes and, thus, reflect the potential involvement ofTGR5 in cholangiociliopathies.

In a working model (Fig. 9), we summarized the results ofthis study, indicating that the subcellular localization of TGR5determines cholangiocyte functional responses to bile acidsignaling.

ACKNOWLEDGMENTS

The authors thank Dr. Mark Pruzanski and Dr. Luciano Adorini (InterceptPharmaceuticals, New York, NY, and Perugia, Italy) for providing the selec-tive TGR5 agonist INT-777.

GRANTS

This work was supported by National Institute of Diabetes and Digestiveand Kidney Diseases (NIDDK) Grant DK-24031 (to N. F. LaRusso), by theOptical Microscopy Core of the Mayo Clinic Center for Cell Signaling inGastroenterology (through NIDDK Grant P30-DK-084567), and by the MayoFoundation.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

Author contributions: A.I.M. and N.F.L. conception and design of research;A.I.M., B.Q.H., B.N.R., G.B.G., P.L.S., T.V.M., and S.A.G. performed exper-iments; A.I.M., B.N.R., T.V.M., and S.A.G. analyzed data; A.I.M., B.Q.H.,and T.V.M. interpreted results of experiments; A.I.M. and B.Q.H. preparedfigures; A.I.M. drafted manuscript; A.I.M. and N.F.L. edited and revisedmanuscript; A.I.M. and N.F.L. approved final version of manuscript.

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