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Characterization of endocytic compartments after holo-high density lipoprotein particle uptake in HepG2 cells

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Abstract

Holo-high density lipoprotein (HDL) particle uptake, besides selective lipid uptake, constitutes an alternative pathway to regulate cellular cholesterol homeostasis. In the current study, the cellular path of holo-HDL particles was investigated in human liver carcinoma cells (HepG2) using combined light and electron microscopical methods. The apolipoprotein moiety of HDL was visualized with different markers: horseradish peroxidase, colloidal gold and the fluorochrome Alexa568, used in fluorescence microscopy and after photooxidation correlatively at the ultrastructural level. Time course experiments showed a rapid uptake of holo-HDL particles, an accumulation in endosomal compartments, with a plateau after 1–2 h of continuous uptake, and a clearance 1–2 h upon replacement by unlabeled HDL. Correlative microscopy, using HDL-Alexa568-driven diaminobenzidine (DAB) photooxidation, identified the fluorescent organelles as DAB-positive multivesicular bodies (MVBs) in the electron microscope; their luminal contents but not the internal vesicles were stained. Labeled MVBs increased in numbers and changed shapes along with the duration of uptake, from polymorphic organelles with multiple surface domains and differently shaped protrusions dominating at early times of uptake to compact bodies with mainly tubular appendices and densely packed vesicles after later times. Differently shaped and labeled surface domains and appendices, as revealed by three dimensional reconstructions, as well as images of homotypic fusions indicate the dynamics of the HDL-positive MVBs. Double staining visualized by confocal microscopy, along with the electron microscopic data, shows that holo-HDL particles after temporal storage in MVBs are only to a minor degree transported to lysosomes, which suggests that different mechanisms are involved in cellular HDL clearance, including resecretion.

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References

  • Acton S, Rigotti A, Landschulz KT, Xu S, Hobbs HH, Krieger M (1996) Identification of scavenger receptor SR-BI as a high density lipoprotein receptor. Science 271:518–520

    Article  CAS  PubMed  Google Scholar 

  • Assemat E, Vinot S, Gofflot F, Linsel-Nitschke P, Illien F, Chatelet F, Verroust P, Louvet-Vallee S, Rinninger F, Kozyraki R (2005) Expression and role of cubilin in the internalization of nutrients during the peri-implantation development of the rodent embryo. Biol Reprod 72:1079–1086

    Article  CAS  PubMed  Google Scholar 

  • Bierman EL, Stein O, Stein Y (1974) Lipoprotein uptake and metabolism by rat aortic smooth muscle cells in tissue culture. Circ Res 35:136–150

    CAS  PubMed  Google Scholar 

  • Brown MS, Goldstein JL (1986) A receptor-mediated pathway for cholesterol homeostasis. Science 232:34–47

    Article  CAS  PubMed  Google Scholar 

  • Conde-Vancells J, Rodriguez-Suarez E, Embade N, Gil D, Matthiesen R, Valle M, Elortza F, Lu SC, Mato JM, Falcon-Perez JM (2008) Characterization and comprehensive proteome profiling of exosomes secreted by hepatocytes. J Proteome Res 7:5157–5166

    Article  CAS  PubMed  Google Scholar 

  • Dantuma NP, Pijnenburg MA, Diederen JH, Van der Horst DJ (1998) Electron microscopic visualization of receptor-mediated endocytosis of Dil-labeled lipoproteins by diaminobenzidine photoconversion. J Histochem Cytochem 46:1085–1089

    CAS  PubMed  Google Scholar 

  • De Gassart A, Geminard C, Hoekstra D, Vidal M (2004) Exosome secretion: the art of reutilizing nonrecycled proteins? Traffic 5:896–903

    Article  PubMed  Google Scholar 

  • DeLamatre JG, Sarphie TG, Archibold RC, Hornick CA (1990) Metabolism of apoE-free high density lipoproteins in rat hepatoma cells: evidence for a retroendocytic pathway. J Lipid Res 31:191–202

    CAS  PubMed  Google Scholar 

  • Glass C, Pittman RC, Weinstein DB, Steinberg D (1983) Dissociation of tissue uptake of cholesterol ester from that of apoprotein A-I of rat plasma high density lipoprotein: selective delivery of cholesterol ester to liver, adrenal, and gonad. Proc Natl Acad Sci USA 80:5435–5439

    Article  CAS  PubMed  Google Scholar 

  • Glass C, Pittman RC, Civen M, Steinberg D (1985) Uptake of high-density lipoprotein-associated apoprotein A-I and cholesterol esters by 16 tissues of the rat in vivo and by adrenal cells and hepatocytes in vitro. J Biol Chem 260:744–750

    CAS  PubMed  Google Scholar 

  • Glomset JA (1973) The metabolic role of lecithin: cholesterol acyltransferase: perspectives from pathology. Adv Lipid Res 11:1–65

    CAS  Google Scholar 

  • Goldstein JL, Brown MS, Anderson RG, Russell DW, Schneider WJ (1985) Receptor-mediated endocytosis: concepts emerging from the LDL receptor system. Annu Rev Cell Biol 1:1–39

    Article  CAS  PubMed  Google Scholar 

  • Graham RC, Karnovsky MJ (1966) The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultrastructural cytochemistry by a new technique. J Histochem Cytochem 14:291–302

    CAS  PubMed  Google Scholar 

  • Hammad SM, Stefansson S, Twal WO, Drake CJ, Fleming P, Remaley A, Brewer HB Jr, Argraves WS (1999) Cubilin, the endocytic receptor for intrinsic factor-vitamin B(12) complex, mediates high-density lipoprotein holoparticle endocytosis. Proc Natl Acad Sci USA 96:10158–10163

    Article  CAS  PubMed  Google Scholar 

  • Handley DA, Arbeeny CM, Witte LD, Chien S (1981) Colloidal gold-low density lipoprotein conjugates as membrane receptor probes. Proc Natl Acad Sci USA 78:368–371

    Article  CAS  PubMed  Google Scholar 

  • Harding C, Heuser J, Stahl P (1983) Receptor-mediated endocytosis of transferrin and recycling of the transferrin receptor in rat reticulocytes. The Journal of cell biology 97:329–339

    Article  CAS  PubMed  Google Scholar 

  • Hasty AH, Plummer MR, Weisgraber KH, Linton MF, Fazio S, Swift LL (2005) The recycling of apolipoprotein E in macrophages: influence of HDL and apolipoprotein A-I. J Lipid Res 46:1433–1439

    Article  CAS  PubMed  Google Scholar 

  • Heeren J, Grewal T, Laatsch A, Rottke D, Rinninger F, Enrich C, Beisiegel U (2003) Recycling of apoprotein E is associated with cholesterol efflux and high density lipoprotein internalization. J Biol Chem 278:14370–14378

    Article  CAS  PubMed  Google Scholar 

  • Jacquet S, Malaval C, Martinez LO, Sak K, Rolland C, Perez C, Nauze M, Champagne E, Terce F, Gachet C, Perret B, Collet X, Boeynaems JM, Barbaras R (2005) The nucleotide receptor P2Y13 is a key regulator of hepatic high-density lipoprotein (HDL) endocytosis. Cell Mol Life Sci 62:2508–2515

    Article  CAS  PubMed  Google Scholar 

  • Klinger A, Reimann FM, Klinger MH, Stange EF (1997) Clathrin-mediated endocytosis of high density lipoprotein3 in human intestinal Caco-2 cells. A post-embedding immunocytochemical study. Biochim Biophys Acta 1345:65–70

    CAS  PubMed  Google Scholar 

  • Kozyraki R, Fyfe J, Kristiansen M, Gerdes C, Jacobsen C, Cui S, Christensen EI, Aminoff M, de la Chapelle A, Krahe R, Verroust PJ, Moestrup SK (1999) The intrinsic factor-vitamin B12 receptor, cubilin, is a high-affinity apolipoprotein A-I receptor facilitating endocytosis of high-density lipoprotein. Nat Med 5:656–661

    Article  CAS  PubMed  Google Scholar 

  • Krieger M (1999) Charting the fate of the “good cholesterol”: identification and characterization of the high-density lipoprotein receptor SR-BI. Annu Rev Biochem 68:523–558

    Article  CAS  PubMed  Google Scholar 

  • Malaval C, Laffargue M, Barbaras R, Rolland C, Peres C, Champagne E, Perret B, Terce F, Collet X, Martinez LO (2009) RhoA/ROCK I signalling downstream of the P2Y13 ADP-receptor controls HDL endocytosis in human hepatocytes. Cell Signal 21:120–127

    Article  CAS  PubMed  Google Scholar 

  • Maranto AR (1982) Neuronal mapping: a photooxidation reaction makes Lucifer yellow useful for electron microscopy. Science 217:953–955

    Article  CAS  PubMed  Google Scholar 

  • Martinez LO, Jacquet S, Esteve JP, Rolland C, Cabezon E, Champagne E, Pineau T, Georgeaud V, Walker JE, Terce F, Collet X, Perret B, Barbaras R (2003) Ectopic beta-chain of ATP synthase is an apolipoprotein A-I receptor in hepatic HDL endocytosis. Nature 421:75–79

    Article  CAS  PubMed  Google Scholar 

  • Meisslitzer-Ruppitsch C, Vetterlein M, Stangl H, Maier S, Neumüller J, Freissmuth M, Pavelka M, Ellinger A (2008) Electron microscopic visualization of fluorescent signals in cellular compartments and organelles by means of DAB-photoconversion. Histochem Cell Biol 130:407–419

    Article  CAS  Google Scholar 

  • Meisslitzer-Ruppitsch C, Röhrl C, Neumüller J, Pavelka M, Ellinger A (2009) Photooxidation technology for correlated light and electron microscopy. J Microscopy 235:322–335

    Article  CAS  Google Scholar 

  • Mobius W, Ohno-Iwashita Y, van Donselaar EG, Oorschot VM, Shimada Y, Fujimoto T, Heijnen HF, Geuze HJ, Slot JW (2002) Immunoelectron microscopic localization of cholesterol using biotinylated and non-cytolytic perfringolysin O. J Histochem Cytochem 50:43–55

    CAS  PubMed  Google Scholar 

  • Mobius W, van Donselaar E, Ohno-Iwashita Y, Shimada Y, Heijnen HF, Slot JW, Geuze HJ (2003) Recycling compartments and the internal vesicles of multivesicular bodies harbor most of the cholesterol found in the endocytic pathway. Traffic 4:222–231

    Article  CAS  PubMed  Google Scholar 

  • Moestrup SK, Nielsen LB (2005) The role of the kidney in lipid metabolism. Curr Opin Lipidol 16:301–306

    Article  CAS  PubMed  Google Scholar 

  • Pagler TA, Rhode S, Neuhofer A, Laggner H, Strobl W, Hinterndorfer C, Volf I, Pavelka M, Eckhardt ER, van der Westhuyzen DR, Schutz GJ, Stangl H (2006) SR-BI-mediated high density lipoprotein (HDL) endocytosis leads to HDL resecretion facilitating cholesterol efflux. J Biol Chem 281:11193–11204

    Article  CAS  PubMed  Google Scholar 

  • Pavelka M, Neumüller J, Ellinger A (2008) Retrograde traffic in the biosynthetic-secretory route. Histochem Cell Biol 129:277–288

    Article  CAS  PubMed  Google Scholar 

  • Rhode S, Breuer A, Hesse J, Sonnleitner M, Pagler TA, Doringer M, Schutz GJ, Stangl H (2004) Visualization of the uptake of individual HDL particles in living cells via the scavenger receptor class B type I. Cell Biochem Biophys 41:343–356

    Article  CAS  PubMed  Google Scholar 

  • Röhrl C, Fruhwürth S, Schreier SM, Lohninger A, Dolischka A, Hüttinger M, Zemann N, Hermann M, Strobl W, Stangl H (2009) Scavenger receptor, Class B, Type I provides an alternative means for beta-VLDL uptake independent of the LDL receptor in tissue culture. Biochim Biophy Acta. doi:10.1016/j.bbalip.2009.11.005

  • Schmitz G, Robenek H, Lohmann U, Assmann G (1985) Interaction of high density lipoproteins with cholesteryl ester-laden macrophages: biochemical and morphological characterization of cell surface receptor binding, endocytosis and resecretion of high density lipoproteins by macrophages. EMBO J 4:613–622

    CAS  PubMed  Google Scholar 

  • Schumaker VN, Puppione DL (1986) Sequential flotation ultracentrifugation. Methods Enzymol 128:155–170

    Article  CAS  PubMed  Google Scholar 

  • Silver DL, Jiang XC, Tall AR (1999) Increased high density lipoprotein (HDL), defective hepatic catabolism of ApoA-I and ApoA-II, and decreased ApoA-I mRNA in ob/ob mice. Possible role of leptin in stimulation of HDL turnover. J Biol Chem 274:4140–4146

    Article  CAS  PubMed  Google Scholar 

  • Silver DL, Wang N, Tall AR (2000) Defective HDL particle uptake in ob/ob hepatocytes causes decreased recycling, degradation, and selective lipid uptake. J Clin Invest 105:151–159

    Article  CAS  PubMed  Google Scholar 

  • Silver DL, Wang N, Xiao X, Tall AR (2001) High density lipoprotein (HDL) particle uptake mediated by scavenger receptor class B type 1 results in selective sorting of HDL cholesterol from protein and polarized cholesterol secretion. J Biol Chem 276:25287–25293

    Article  CAS  PubMed  Google Scholar 

  • Sosinsky GE, Giepmans BN, Deerinck TJ, Gaietta GM, Ellisman MH (2007) Markers for correlated light and electron microscopy. Methods Cell Biol 79:575–591

    Article  CAS  PubMed  Google Scholar 

  • Stangl H, Cao G, Wyne KL, Hobbs HH (1998) Scavenger receptor, class B, type I-dependent stimulation of cholesterol esterification by high density lipoproteins, low density lipoproteins, and nonlipoprotein cholesterol. J Biol Chem 273:31002–31008

    Article  CAS  PubMed  Google Scholar 

  • Tabas I, Lim S, Xu XX, Maxfield FR (1990) Endocytosed beta-VLDL and LDL are delivered to different intracellular vesicles in mouse peritoneal macrophages. J Cell Biol 111:929–940

    Article  CAS  PubMed  Google Scholar 

  • Woodman PG, Futter CE (2008) Multivesicular bodies: co-ordinated progression to maturity. Curr Opin Cell Biol 20:408–414

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

The authors would like to thank Mrs. Loredana Ionce, Mrs. Barbara Kornprat, Mrs. Beatrix Mallinger, and Mrs. Elfriede Scherzer for the excellent technical assistance, and thank Mr. Ulrich Kaindl for his valuable help with the artwork and the 3D model. This project is supported by the Austrian Science Fund (FWF) grant P20116.

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  1. Tamara A. Pagler

    Present address: Division of Molecular Medicine, Columbia University, New York, NY, USA

Authors and Affiliations

  1. Center for Physiology and Pathophysiology, Institute of Medical Chemistry, Medical University of Vienna, Vienna, Austria

    Clemens Röhrl, Tamara A. Pagler, Witta Strobl & Herbert Stangl

  2. Center for Anatomy and Cell Biology, Department of Cell Biology and Ultrastructure Research, Medical University of Vienna, Schwarzspanierstrasse 17, 1090, Vienna, Austria

    Adolf Ellinger, Josef Neumüller, Margit Pavelka & Claudia Meisslitzer-Ruppitsch

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  1. Clemens Röhrl
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Correspondence to Claudia Meisslitzer-Ruppitsch.

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Röhrl, C., Pagler, T.A., Strobl, W. et al. Characterization of endocytic compartments after holo-high density lipoprotein particle uptake in HepG2 cells. Histochem Cell Biol 133, 261–272 (2010). https://doi.org/10.1007/s00418-009-0672-3

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