Abstract
We previously described a cell surface reactive monoclonal antibody, MAb OC.10, which recognizes an epitope shared by rat fetal liver ductal cells, hepatic progenitor cells, mature cholangiocytes, and hepatocellular carcinomas (HCC). Here, intrasplenic injection of MAb OC.10 into newborn rats was shown by immunofluorescence microscopy to strongly label intrahepatic bile ducts. Furthermore, the in situ labeling of intrahepatic cholangiocytes by injecting MAb OC.10 increased the number of intraportal and intralobular bile ducts with well-defined lumens when compared to IgM-injected control animals. The antigen for MAb OC.10 was identified by mass spectrometry as Hsc70, a constitutively expressed heat shock protein belonging to the HSP70 family. Immunoblot analysis demonstrated that MAb OC.10 reacted with recombinant bovine Hsc70 protein, with protein immunoprecipitated from rat bile duct epithelial (BDE) cell lysates with monoclonal anti-Hsc70 antibody, and with Hsc70-FLAG protein over-expressed in human 293T cells. In addition, Hsc70-specific small interfering RNA reduced the amount of OC.10 antigen expressed in nucleofected BDE cells. Consistent with the specificity of MAb OC.10 for Hsc70, heat shock did not induce OC.10 expression in BDE cells, a characteristic of Hsp70. Immunofluorescence with BDE cells further suggested that MAb OC.10 binds a novel cell surface epitope of Hsc70. This was in contrast to a commercially available monoclonal anti-Hsc70 antibody that showed strong cytosolic reactivity. These findings demonstrate that presentation of the OC.10 epitope differs between cytosolic and surface forms of Hsc70 and may suggest distinct differences in protein conformation or epitope availability determined in part by protein–protein or protein–lipid interactions. Phage display and pepscan analysis mapped the epitope for MAb OC.10 to the N-terminal 340–384 amino acids of the ATPase domain of rat Hsc70. These findings suggest that MAb OC.10 recognizes an epitope on rat Hsc70 when presented on the cell surface that promotes morphogenic maturation of bile ducts in newborn rat liver. Furthermore, since we have shown previously that the OC.10 antigen is expressed on HCC subpopulations with oval cell characteristics, our current results indicate that Hsc70 has the potential to be expressed on the surface of certain tumor cells.
Similar content being viewed by others
References
Arispe N, De Maio A (2000) ATP and ADP modulate a cation channel formed by Hsc70 in acidic phospholipid membranes. J Biol Chem 275:30839–30843. doi:10.1074/jbc.M005226200
Arispe N, Doh M, De Maio A (2002) Lipid interaction differentiates the constitutive and stress-induced heat shock proteins Hsc70 and Hsp70. Cell Stress Chaperones 7:330–338. doi:10.1379/1466-1268(2002)007<0330:LIDTCA>2.0.CO;2
Arispe N, Doh M, Simakova O, Kurganov B, De Maio A (2004) Hsc70 and Hsp70 interact with phosphatidylserine on the surface of PC12 cells resulting in a decrease of viability. FASEB J 18:1636–1645. doi:10.1096/fj.04-2088com
Bisgaard HC, Parmelee DC, Dunsford HA, Sechi S, Thorgeirsson SS (1993) Keratin 14 protein in cultured nonparenchymal rat hepatic epithelial cells: characterization of keratin 14 and keratin 19 as antigens for the commonly used mouse monoclonal antibody OV-6. Mol Carcinog 7:60–66. doi:10.1002/mc.2940070110
Botzler C, Issels R, Multhoff G (1996) Heat-shock protein 72 cell-surface expression on human lung carcinoma cells in associated with an increased sensitivity to lysis mediated by adherent natural killer cells. Cancer Immunol Immunother 43:226–230. doi:10.1007/s002620050326
Botzler C, Li G, Issels RD, Multhoff G (1998) Definition of extracellular localized epitopes of Hsp70 involved in an NK immune response. Cell Stress Chaperones 3:6–11. doi:10.1379/1466-1268(1998)003<0006:DOELEO>2.3.CO;2
Broquet AH, Thomas G, Masliah J, Trugnan G, Bachelet M (2003) Expression of the molecular chaperone Hsp70 in detergent-resistant microdomains correlates with its membrane delivery and release. J Biol Chem 278:21601–21606. doi:10.1074/jbc.M302326200
Clifton JG, Brown MK, Huang F et al (2006) Identification of members of the annexin family in the detergent-insoluble fraction of rat Morris hepatoma plasma membranes. J Chromatogr A 1123:205–211. doi:10.1016/j.chroma.2006.02.020
Coleman W, McCullough K, Esch G, Faris R, Hixson D, Smith G, Grisham J (1997) Evaluation of the differentiation potential of WB-F344 rat liver epithelial stem-like cells in vivo: differentiation to hepatocytes following transplantation into dipeptidylpeptidase IV-deficient rat liver. Am J Pathol 151:353–359
Dabeva MD, Hwang SG, Vasa SR et al (1997) Differentiation of pancreatic epithelial progenitor cells into hepatocytes following transplantation into rat liver. Proc Natl Acad Sci U S A 94:7356–7361. doi:10.1073/pnas.94.14.7356
Dastoor Z, Dreyer J (2000) Nuclear translocation and aggregate formation of heat shock cognate protein 70 (Hsc70) in oxidative stress and apoptosis. J Cell Sci 113(Pt 16):2845–2854
Daugaard M, Rohde M, Jaattela M (2007) The heat shock protein 70 family: highly homologous proteins with overlapping and distinct functions. FEBS Lett 581:3702–3710. doi:10.1016/j.febslet.2007.05.039
de la Rosa EJ, Vega-Nunez E, Morales AV, Serna J, Rubio E, de Pablo F (1998) Modulation of the chaperone heat shock cognate 70 by embryonic (pro) insulin correlates with prevention of apoptosis. Proc Natl Acad Sci U S A 95:9950–9955. doi:10.1073/pnas.95.17.9950
De Maio A (1999) Heat shock proteins: facts, thoughts, and dreams. Shock 11:1–12. doi:10.1097/00024382-199901000-00001
Dunsford HA, Karnasuta C, Hunt JM, Sell S (1989) Different lineages of chemically induced hepatocellular carcinoma in rats defined by monoclonal antibodies. Cancer Res 49:4894–4900
Eisenberg E, Greene LE (2007) Multiple roles of auxilin and hsc70 in clathrin-mediated endocytosis. Traffic 8:640–646. doi:10.1111/j.1600-0854.2007.00568.x
Elefant F, Palter KB (1999) Tissue-specific expression of dominant negative mutant Drosophila HSC70 causes developmental defects and lethality. Mol Biol Cell 10:2101–2117
Fan GH, Yang W, Sai J, Richmond A (2002) Hsc/Hsp70 interacting protein (hip) associates with CXCR2 and regulates the receptor signaling and trafficking. J Biol Chem 277:6590–6597. doi:10.1074/jbc.M110588200
Ferrarini M, Heltai S, Zocchi MR, Rugarli C (1992) Unusual expression and localization of heat-shock proteins in human tumor cells. Int J Cancer 51:613–619. doi:10.1002/ijc.2910510418
Fishelson Z, Hochman I, Greene LE, Eisenberg E (2001) Contribution of heat shock proteins to cell protection from complement-mediated lysis. Int Immunol 13:983–991. doi:10.1093/intimm/13.8.983
Gehrmann M, Liebisch G, Schmitz G et al (2008) Tumor-specific Hsp70 plasma membrane localization is enabled by the glycosphingolipid Gb3. PLoS ONE 3:e1925
Gordon GJ, Coleman WB, Grisham JW (2000) Temporal analysis of hepatocyte differentiation by small hepatocyte-like progenitor cells during liver regeneration in retrorsine-exposed rats. Am J Pathol 157:771–786
Hanzal-Bayer MF, Hancock JF (2007) Lipid rafts and membrane traffic. FEBS Lett 581:2098–2104. doi:10.1016/j.febslet.2007.03.019
Hirai I, Sato N, Qi W, Ohtani S, Torigoe T, Kikuchi K (1998) Localization of pNT22 70 kDa heat shock cognate-like protein in the plasma membrane. Cell Struct Funct 23:153–158
Hixson DC, Fowler LC (1997) Development and phenotypic heterogeneity of intrahepatic biliary epithelial cells. In: Sirica AE, Longnecker DS (eds) Biliary and pancreatic ductal epithelium. Marcel Dekker, New York, pp 1–40
Hixson DC, Brown J, McBride AC, Affigne S (2000) Differentiation status of rat ductal cells and ethionine-induced hepatic carcinomas defined with surface-reactive monoclonal antibodies. Exp Mol Pathol 68:152–169. doi:10.1006/exmp. 2000.2302
Kamiguchi K, Torigoe T, Fujiwara O et al (2008) Disruption of the association of 73 kDa heat shock cognate protein with transporters associated with antigen processing (TAP) decreases TAP-dependent translocation of antigenic peptides into the endoplasmic reticulum. Microbiol Immunol 52:94–106. doi:10.1111/j.1348-0421.2008.00017.x
Kubota H, Reid LM (2000) Clonogenic hepatoblasts, common precursors for hepatocytic and biliary lineages, are lacking classical major histocompatibility complex class I antigen. Proc Natl Acad Sci U S A 97:12132–12137. doi:10.1073/pnas.97.22.12132
Lawson EL, Clifton JG, Huang F, Li X, Hixson DC, Josic D (2006) Use of magnetic beads with immobilized monoclonal antibodies for isolation of highly pure plasma membranes. Electrophoresis 27:2747–2758. doi:10.1002/elps.200600059
Mayer MP, Bukau B (2005) Hsp70 chaperones: cellular functions and molecular mechanism. Cell Mol Life Sci 62:670–684. doi:10.1007/s00018-004-4464-6
Multhoff G, Botzler C, Wiesnet M, Muller E, Meier T, Wilmanns W, Issels RD (1995) A stress-inducible 72-kDa heat-shock protein (HSP72) is expressed on the surface of human tumor cells, but not on normal cells. Int J Cancer 61:272–279. doi:10.1002/ijc.2910610222
Multhoff G, Botzler C, Jennen L, Schmidt J, Ellwart J, Issels R (1997) Heat shock protein 72 on tumor cells: a recognition structure for natural killer cells. J Immunol 158:4341–4350
Multhoff G, Mizzen L, Winchester CC et al (1999) Heat shock protein 70 (Hsp70) stimulates proliferation and cytolytic activity of natural killer cells. Exp Hematol 27:1627–1636. doi:10.1016/S0301-472X(99)00104-6
Multhoff G, Pfister K, Gehrmann M, Hantschel M, Gross C, Hafner M, Hiddemann W (2001) A 14-mer Hsp70 peptide stimulates natural killer (NK) cell activity. Cell Stress Chaperones 6:337–344. doi:10.1379/1466-1268(2001)006<0337:AMHPSN>2.0.CO;2
Newmyer SL, Christensen A, Sever S (2003) Auxilin–dynamin interactions link the uncoating ATPase chaperone machinery with vesicle formation. Dev Cell 4:929–940. doi:10.1016/S1534-5807(03)00157-6
Novikoff PM, Yam A, Oikawa I (1996) Blast-like cell compartment in carcinogen-induced proliferating bile ductules. Am J Pathol 148:1473–1492
Patra SK (2008) Dissecting lipid raft facilitated cell signaling pathways in cancer. Biochim Biophys Acta 1785:182–206
Rajapandi T, Greene LE, Eisenberg E (2000) The molecular chaperones Hsp90 and Hsc70 are both necessary and sufficient to activate hormone binding by glucocorticoid receptor. J Biol Chem 275:22597–22604. doi:10.1074/jbc.M002035200
Sarrio S, Casado V, Escriche M et al (2000) The heat shock cognate protein hsc73 assembles with A(1) adenosine receptors to form functional modules in the cell membrane. Mol Cell Biol 20:5164–5174. doi:10.1128/MCB.20.14.5164-5174.2000
Schlossman DM, Schmid SL, Braell WA, Rothman JE (1984) An enzyme that removes clathrin coats: purification of an uncoating ATPase. J Cell Biol 99:723–733. doi:10.1083/jcb.99.2.723
Sell S (1998) Comparison of liver progenitor cells in human atypical ductular reactions with those seen in experimental models of liver injury. Hepatology 27:317–331. doi:10.1002/hep.510270202
Sell S, Ilic Z (eds) (1997) Response to injury. Liver stem cells. Landes Company, Austin, 223–286
Sell S, Pierce GB (1994) Maturation arrest of stem cell differentiation is a common pathway for the cellular origin of teratocarcinomas and epithelial cancers. Lab Invest 70:6–22
Shin BK, Wang H, Yim AM et al (2003) Global profiling of the cell surface proteome of cancer cells uncovers an abundance of proteins with chaperone function. J Biol Chem 278:7607–7616. doi:10.1074/jbc.M210455200
Shrivastav A, Varma S, Lawman Z et al (2008) Requirement of N-myristoyltransferase 1 in the development of monocytic lineage. J Immunol 180:1019–1028
Simons K, Ikonen E (1997) Functional rafts in cell membranes. Nature 387:569–572. doi:10.1038/42408
Simper-Ronan R, Brilliant K, Flanagan D, Carreiro M, Callanan H, Sabo E, Hixson DC (2006) Cholangiocyte marker-positive and -negative fetal liver cells differ significantly in their ability to regenerate the livers of adult rats exposed to retrorsine. Development 133:4269–4279. doi:10.1242/dev.02589
Sorger PK, Pelham HR (1987) Cloning and expression of a gene encoding hsc73, the major hsp70-like protein in unstressed rat cells. Embo J 6:993–998
Suzuki A, Zheng YY, Kaneko S, Onodera M, Fukao K, Nakauchi H, Taniguchi H (2002) Clonal identification and characterization of self-renewing pluripotent stem cells in the developing liver. J Cell Biol 156:173–184. doi:10.1083/jcb.200108066
Tateno C, Yoshizato K (1996) Growth and differentiation in culture of clonogenic hepatocytes that express both phenotypes of hepatocytes and biliary epithelial cells. Am J Pathol 149:1593–1605
Tavaria M, Gabriele T, Kola I, Anderson RL (1996) A hitchhiker’s guide to the human Hsp70 family. Cell Stress Chaperones 1:23–28. doi:10.1379/1466-1268(1996)001<0023:AHSGTT>2.3.CO;2
Triantafilou K, Triantafilou M, Dedrick RL (2001) A CD14-independent LPS receptor cluster. Nat Immunol 2:338–345. doi:10.1038/86342
Tsuboi N, Ishikawa M, Tamura Y et al (1994) Monoclonal antibody specifically reacting against 73-kilodalton heat shock cognate protein: possible expression on mammalian cell surface. Hybridoma 13:373–381
Tsukahara F, Maru Y (2004) Identification of novel nuclear export and nuclear localization-related signals in human heat shock cognate protein 70. J Biol Chem 279:8867–8872. doi:10.1074/jbc.M308848200
Vega-Nunez E, Pena-Melian A, de la Rosa EJ, de Pablo F (1999) Dynamic restricted expression of the chaperone Hsc70 in early chick development. Mech Dev 82:199–203. doi:10.1016/S0925-4773(99)00015-5
Yang L, Faris RA, Hixson DC (1993a) Characterization of a mature bile duct antigen expressed on a subpopulation of biliary ductular cells but absent from oval cells. Hepatology 18:357–366
Yang L, Faris RA, Hixson DC (1993b) Long-term culture and characteristics of normal rat liver bile duct epithelial cells. Gastroenterology 104:840–852
Zhou D, Li P, Lin Y et al (2005) Lamp-2a facilitates MHC class II presentation of cytoplasmic antigens. Immunity 22:571–581. doi:10.1016/j.immuni.2005.03.009
Acknowledgments
We wish to thank Laura Bangs and Sandy DeAngelis for clerical assistance and Dr. James G. Clifton for technical assistance with the QSTAR XL mass spectrometry studies. This work was supported by grants CA93840, CA42715, and RRP20RR017695 (to D.C.H.) from the National Institutes of Health and RI-INBRE Grant P20RR016457 (to D.R.M.) from the National Center for Research Resources (NCRR), a component of the National Institutes of Health (NIH). Its contents are solely the responsibility of the authors and do not necessarily represent the official views of NCRR or NIH.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Mills, D.R., Haskell, M.D., Callanan, H.M. et al. Monoclonal antibody to novel cell surface epitope on Hsc70 promotes morphogenesis of bile ducts in newborn rat liver. Cell Stress and Chaperones 15, 39–53 (2010). https://doi.org/10.1007/s12192-009-0120-2
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12192-009-0120-2