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Expression, localization, and inducibility by bile acids of hepatobiliary transporters in the new polarized rat hepatic cell lines, Can 3−1 and Can 10

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Abstract

Sinusoidal and apical transporters are responsible for the uptake and biliary elimination of many compounds by hepatocytes. Few in vitro models are however available for analyzing such functions. The expression and bile-acid inducibility of 13 transporters and two nuclear receptors were investigated in the new rat polarized lines, Can 3−1 and Can 10, and in their unpolarized parent, Fao. The relative abundance of mRNA, the protein level, and their localization were examined by real-time quantitative PCR, Western blotting, immunofluorescence, and confocal microscopy. Compared with rat liver, mRNA levels of Fao cells were: negligible for Bsep/Abcb11; lower for the uptake transporters Ntcp and Oatps; similar for SHP, FXR, and Bcrp/Abcg2; and higher (four–fold to 160-fold) for the efflux pumps Mdr1b/Abcb1b, Mdr2/Abcb4, Mrp1/Abcc1, Mrp2/Abcc2, Mrp3/Abcc3, Abcg5, and Abcg8. This profile was mostly maintained (and improved for Bsep) in Can 10. Some transporters were less well expressed in Can 3−1. In both lines, sinusoidal (Ntcp, Mrp3) and canalicular transporters (Mdr-P-glycoproteins detected with C219 antibody, Mrp2) were localized at their correct poles. Bile-acid effects on polarity and mRNA levels of transporters were analyzed after a 6-day treatment with 50 μM taurocholic, chenodeoxycholic (CDCA), or ursodeoxycholic acid (UDCA). No polarization of Fao cells was induced; Can 10 and Can 3−1 polarity was maintained. CDCA and UDCA induced marked enhancement of the volume of Can 10 bile canaliculi. CDCA upregulated Bsep, Mdr2, SHP, Mdr1b, and Oatp2/1a4 in Can 10 (two- to seven-fold) and in Fao cells. Thus, Can 10 constitutes an attractive polarized model for studying vectorial hepatobiliary transport of endogenous and xenobiotic cholephilic compounds.

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Abbreviations

Abc/ABC:

ATP-binding cassette

BC:

bile canaliculi

Bcrp:

breast cancer resistance protein

Bsep/BSEP:

bile salt export pump

CDCA:

chenodeoxycholic acid

DPPIV:

dipeptidylpeptidase IV

FITC:

fluorescein isothiocyanate

FXR:

farnesoid X-activated receptor

Mdr/MDR:

multidrug resistance protein

Mrp/MRP:

multidrug resistance-associated protein

Ntcp:

Na+-taurocholate co-transporting polypeptide

PBS:

phosphate-buffered saline

Oatp/OATP:

organic anion transporting polypeptide

PM:

plasma membrane

SHP:

small heterodimer partner

TCA:

taurocholic acid

UDCA:

ursodeoxycholic acid

References

  • Ananthanarayanan M, Balasubramanian N, Makishima M, Mangelsdorf DJ, Suchy FJ (2001) Human bile salt export pump promoter is transactivated by the farnesoid X receptor/bile acid receptor. J Biol Chem 276:28857–28865

    Article  PubMed  CAS  Google Scholar 

  • Bender V, Buschlen S, Cassio D (1998) Expression and localization of hepatocyte domain-specific plasma membrane proteins in hepatoma-fibroblast hybrids and in hepatoma dedifferentiated variants. J Cell Sci 111:3437–3450

    PubMed  CAS  Google Scholar 

  • Bravo P, Bender V, Cassio D (1998) Efficient in vitro vectorial transport of a fluorescent conjugated bile acid analogue by polarized hepatic hybrid WIF-B and WIF-B9 cells. Hepatology 27:576–583

    Article  PubMed  CAS  Google Scholar 

  • Briz O, Macias RI, Vallejo M, Silva A, Serrano MA, Marin JJ (2003) Usefulness of liposomes loaded with cytostatic bile acid derivatives to circumvent chemotherapy resistance of enterohepatic tumors. Mol Pharmacol 63:742–750

    Article  PubMed  CAS  Google Scholar 

  • Briz O, Cassio D, Blazquez AG, Grosse B, Serrano MA, Marin JJ (2007) Characterization of WIF-B9/R cells as an in vitro model with hepatocyte-like polarity and enhanced expression of canalicular ABC transporters involved in phase III of hepatic detoxification. Toxicology 232:24-36

    Article  PubMed  CAS  Google Scholar 

  • Buchler M, Konig J, Brom M, Kartenbeck J, Spring H, Horie T, Keppler D (1996) cDNA cloning of the hepatocyte canalicular isoform of the multidrug resistance protein, cMrp, reveals a novel conjugate export pump deficient in hyperbilirubinemic mutant rats. J Biol Chem 271:15091–15098

    Article  PubMed  CAS  Google Scholar 

  • Cantz T, Nies AT, Brom M, Hofmann AF, Keppler D (2000) MRP2, a human conjugate export pump, is present and transports fluo 3 into apical vacuoles of HepG2 cells. Am J Physiol Gastrointest Liver Physiol 279:G522–G531

    Google Scholar 

  • Cassio D, Hamon-Benais C, Guerin M, Lecoq O (1991) Hybrid cell lines constitute a potential reservoir of polarized cells: isolation and study of highly differentiated hepatoma-derived hybrid cells able to form functional bile canaliculi in vitro. J Cell Biol 115:1397–1408

    Article  PubMed  CAS  Google Scholar 

  • Chan W, Calderon G, Swift AL, Moseley J, Li S, Hosoya H, Arias IM, Ortiz DF (2005) Myosin II regulatory light chain is required for trafficking of bile salt export protein to the apical membrane in Madin-Darby canine kidney cells. J Biol Chem 280:23741–23747

    Article  PubMed  CAS  Google Scholar 

  • Decaens C, Rodriguez P, Bouchaud C, Cassio D (1996) Establishment of hepatic cell polarity in the rat hepatoma-human fibroblast hybrid WIF-B9. A biphasic phenomenon going from a simple epithelial polarized phenotype to an hepatic polarized one. J Cell Sci 109:1623–1635

    PubMed  CAS  Google Scholar 

  • Deschatrette J, Weiss MC (1974) Characterization of differentiated and dedifferentiated clones from a rat hepatoma. Biochimie 56:1603–1611

    Article  PubMed  CAS  Google Scholar 

  • Donner MG, Keppler D (2001) Up-regulation of basolateral multidrug resistance protein 3 (Mrp3) in cholestatic rat liver. Hepatology 34:351–359

    Article  PubMed  CAS  Google Scholar 

  • Eloranta JJ, Kullak-Ublick GA (2005) Coordinate transcriptional regulation of bile acid homeostasis and drug metabolism. Arch Biochem Biophys 433:397–412

    Article  PubMed  CAS  Google Scholar 

  • Goodwin B, Jones SA, Price RR, Watson MA, McKee DD, Moore LB, Galardi C, Wilson JG, Lewis MC, Roth ME, Maloney PR, Willson TM, Kliewer SA (2000) A regulatory cascade of the nuclear receptors FXR, SHP-1, and LRH-1 represses bile acid biosynthesis. Mol Cell 6:517–526

    Article  PubMed  CAS  Google Scholar 

  • Huang L, Zhao A, Lew JL, Zhang T, Hrywna Y, Thompson JR, Pedro N de, Royo I, Blevins RA, Pelaez F, Wright SD, Cui J (2003) Farnesoid X receptor activates transcription of the phospholipid pump MDR3. J Biol Chem 278:51085–51090

    Article  PubMed  CAS  Google Scholar 

  • Ihrke G, Neufeld EB, Meads T, Shanks MR, Cassio D, Laurent M, Schroer T, Pagano RE, Hubbard AL (1993) WIF-B cells: an in vitro model for studies of hepatocyte polarity. J Cell Biol 123:1761–1775

    Article  PubMed  CAS  Google Scholar 

  • Ito K, Suzuki H, Horie T, Sugiyama Y (2005) Apical/basolateral surface expression of drug transporters and its role in vectorial drug transport. Pharm Res 22:1559–1577

    Article  PubMed  CAS  Google Scholar 

  • Knowles BB, Howe CC, Aden DP (1980) Human hepatocellular carcinoma cell lines secrete the major plasma proteins and hepatitis B surface antigen. Science 209:497–499

    Article  PubMed  CAS  Google Scholar 

  • Konieczko EM, Ralston AK, Crawford AR, Karpen SJ, Crawford JM (1998) Enhanced Na+-dependent bile salt uptake by WIF-B cells, a rat hepatoma hybrid cell line, following growth in the presence of a physiological bile salt. Hepatology 27:191–198

    Article  PubMed  CAS  Google Scholar 

  • Kubitz R, Sutfels G, Kuhlkamp T, Kolling R, Haussinger D (2004) Trafficking of the bile salt export pump from the Golgi to the canalicular membrane is regulated by the p38 MAP. Gastroenterology 126:541–553

    Article  PubMed  CAS  Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227:680–685

    Article  PubMed  CAS  Google Scholar 

  • Le Vee M, Jigorel E, Glaise D, Grippon P, Guguen-Guillouzo C, Fardel O (2006) Functional expression of sinusoidal and canalicular hepatic drug transporters in the differentiated human hepatoma HepaRG cell line. Eur J Pharm Sci 28:109–117

    Article  PubMed  Google Scholar 

  • Lee FY, Lee H, Hubbert ML, Edwards PA, Zhang Y (2006) FXR, a multipurpose nuclear receptor. Trends Biochem Sci 31:572–580

    Article  PubMed  CAS  Google Scholar 

  • Lew JL, Zhao A, Yu J, Huang L, Pedro N de, Pelaez F, Wright SD, Cui J (2004) The farnesoid X receptor controls gene expression in a ligand- and promoter-selective fashion. J Biol Chem 279:8856–8861

    Article  PubMed  CAS  Google Scholar 

  • Lu TT, Makishima M, Repa JJ, Schoonjans K, Kerr TA, Auwerx J, Mangelsdorf DJ (2000) Molecular basis for feedback regulation of bile acid synthesis by nuclear receptors. Mol Cell 6:507–515

    Article  PubMed  CAS  Google Scholar 

  • Makishima M, Okamoto AY, Repa JJ, Tu H, Learned RM, Luk A, Hull MV, Lustig KD, Mangelsdorf DJ, Shan B (1999) Identification of a nuclear receptor for bile acids. Science 284:1362–1365

    Article  PubMed  CAS  Google Scholar 

  • Monte MJ, El-Mir MY, Sainz GR, Bravo P, Marin JJG (1997) Bile acid secretion during synchronized rat liver regeneration. Biochim Biophys Acta 1362:56–66

    PubMed  CAS  Google Scholar 

  • Nakai D, Nakagomi R, Furuta Y, Tokui T, Abe T, Ikeda T, Nishimura K (2001) Human liver-specific organic anion transporter, LST-1, mediates uptake of pravastatin by human hepatocytes. J Pharmacol Exp Ther 297:861–867

    PubMed  CAS  Google Scholar 

  • Ng KH, Le Goascogne C, Amborade E, Stieger B, Deschatrette J (2000) Reversible induction of rat hepatoma cell polarity with bile acids. J Cell Sci 113:4241–4251

    PubMed  CAS  Google Scholar 

  • Nies AT, Cantz T, Brom M, Leier I, Keppler D (1998) Expression of the apical conjugate export pump, Mrp2, in the polarized hepatoma cell line, WIF-B. Hepatology 28:1332–1340

    Article  PubMed  CAS  Google Scholar 

  • Parent R, Marion MJ, Furio L, Trepo, Petit MA (2004) Origin and characterization of a human bipotent liver progenitor cell line. Gastroenterology 126:1147–1156

    Article  PubMed  Google Scholar 

  • Peng X, Grosse B, Le Tiec B, Nicolas V, Delagebeaudeuf C, Bedda T, Decaens C, Cassio D (2006) How to induce non-polarized cells of hepatic origin to express typical hepatocyte polarity: generation of new highly polarized cell models with developed and functional bile canaliculi. Cell Tissue Res 323:233–243

    Article  PubMed  Google Scholar 

  • Sai Y, Nies AT, Arias IM (1999) Bile acid secretion and direct targeting of mdr1-green fluorescent protein from Golgi to the canalicular membrane in polarized WIF-B cells. J Cell Sci 112:4535–4545

    PubMed  CAS  Google Scholar 

  • Scharschmidt BF, Lake JR (1989) Hepatocellular bile acid transport and ursodeoxycholic acid hypercholeresis. Dig Dis Sci 34:5S–15S

    Article  PubMed  CAS  Google Scholar 

  • Shanks MR, Cassio D, Lecoq O, Hubbard AL (1994) An improved polarized rat hepatoma hybrid cell line. J Cell Sci 107:813–825

    PubMed  Google Scholar 

  • St-Pierre MV, Kullak-Ublick GA, Hagenbuch B, Meier PJ (2001) Transport of bile acids in hepatic and non-hepatic tissues. J Exp Biol 204:1673–1686

    PubMed  CAS  Google Scholar 

  • Stevenson BR, Siliciano JD, Mooseker MS, Goodenough DA (1986) Identification of ZO-1: a high molecular weight polypeptide associated with the tight junction (zonula occludens) in a variety of epithelia. J Cell Biol 10:755–766

    Article  Google Scholar 

  • Stieger B, Hagenbuch B, Landmann L, Hochli M, Schroeder A, Meier PJ (1994) In situ localization of the hepatocytic Na+/taurocholate cotransporting polypeptide in rat liver. Gastroenterology 107:1781–1787

    PubMed  CAS  Google Scholar 

  • Trauner M, Boyer JL (2003) Bile salt transporters: molecular characterization, function, and regulation. Physiol Rev 83:633–671

    PubMed  CAS  Google Scholar 

  • Van Ijzendoorn SC, Van Der Wouden JM, Liebisch G, Schmitz G, Hoekstra D (2004) Polarized membrane traffic and cell polarity development is dependent on dihydroceramide synthase-regulated sphinganine turnover. Mol Biol Cell 15:4115–4124

    Article  PubMed  Google Scholar 

  • Yu J, Lo JL, Huang L, Zhao A, Metzger E, Adams A, Meinke PT, Wright SD, Cui J (2002) Lithocholic acid decreases expression of bile salt export pump through farnesoid X receptor antagonist activity. J Biol Chem 30:31441–31447

    Article  Google Scholar 

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Acknowledgments

We are grateful to A. Hubbard, D. Keppler, B.R. Stevenson, and B. Stieger for kindly providing antibodies. We also thank V. Nicolas (IFR141-ITFM, Chatenay Malabry, France) and Mickael Bourge (INSERM U757, Orsay, France) for help with confocal analysis and artwork, respectively.

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Authors and Affiliations

  1. INSERM, U757, University of Orsay, 91400, Orsay, France

    Doris Cassio & Brigitte Grosse

  2. University of Orsay, 91400, Orsay, France

    Doris Cassio & Brigitte Grosse

  3. Laboratory of Experimental Hepatology and Drug Targeting (HEVEFARM), CIBERehd, University of Salamanca, 37007, Salamanca, Spain

    Rocio I. R. Macias, Jose J. G. Marin & Maria J. Monte

  4. Unité INSERM 757, Bâtiment 443, Université Paris-Sud, 91405, Orsay Cedex, France

    Doris Cassio

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  1. Doris Cassio
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  2. Rocio I. R. Macias
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  3. Brigitte Grosse
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  4. Jose J. G. Marin
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  5. Maria J. Monte
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Correspondence to Doris Cassio.

Additional information

This work was supported by a grant from Egide (PAI Picasso) and the Acción Integrada Hispano-Francesa (HF2003-0089). This research group is part of the Network for Cooperative Research on Membrane Transport Proteins (REIT), co-funded by the Ministerio de Educación y Ciencia, Spain and the European Regional Development Fund (ERDF; grant BFU2005-24983-E/BFI) and belongs to the “Centro de Investigación Biomédica en Red” for Hepatology and Gastroenterology Research (CIBERehd), Instituto de Salud Carlos III, Spain.

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Cassio, D., Macias, R.I.R., Grosse, B. et al. Expression, localization, and inducibility by bile acids of hepatobiliary transporters in the new polarized rat hepatic cell lines, Can 3−1 and Can 10. Cell Tissue Res 330, 447–460 (2007). https://doi.org/10.1007/s00441-007-0494-3

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  • DOI: https://doi.org/10.1007/s00441-007-0494-3

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