Abstract
Bone cells produce a variety of glycoproteins that contribute to bone health, and function in cell adhesion, stabilizing the extracellular matrix, promoting growth and differentiation, and the induction of apoptosis. Some of these processes appear to be disturbed in arthritis. In this chapter, in vitro studies aimed at an understanding of the biological effects of inflammatory stimuli in the bone of arthritis patients are described. The glycodynamics of cells can be studied using primary cultures of osteoblasts or bone cancer cell cultures, to examine the relationship between the biosynthesis of cell-surface glycoproteins and inflammatory stimuli affecting cell growth and cell death. Cell-surface carbohydrates are assessed by lectin staining of cells, and the potential of cells to synthesize glycoproteins is determined by glycosyltransferase assays. These parameters are then related to [3H]thymidine incorporation as a measure of cell proliferation, and to flow cytometry of terminal deoxynucleotidyl transferase dUTP-mediated nick end labeling (TUNEL) and annexin V-stained cells as a measure of apoptosis. These in vitro studies are aimed at an understanding of the role of glycosylation in the bone of arthritis patients, but they can also be applied to other diseases.
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References
Gehron Robey, P., Fedarko, N. S., Hefferan, T. E., et al. (1993) Structure and molecular regulation of bone matrix proteins. J. Bone Miner. Res. 8, S483–S487.
Midura, R. J. and Hascall, V. C. (1996) Bone sialoprotein—a mucin in disguise? Glycobiology 6, 677–681.
de Pollak, C., Arnaud, E., Renier, D., and Marie, P. J. (1997) Age-related changes in bone formation, osteoblastic cell proliferation and differentiation during postnatal osteogenesis in human calvaria. J. Cell. Biochem. 128, 128–139.
Malaval, L., Modrowski, D., Gupta, A. K., and Aubin, J. E. (1994) Cellular expression of bone-related proteins during in vitro osteogenesis in rat bone marrow stromal cell cultures. J. Cell. Physiol. 158, 555–572.
Mundy, G. R. (1996) Regulation of bone formation by bone morphogenic proteins and other growth factors. Clin. Orthop. Relat. Res. 323, 24–28.
Brockhausen, I., Schutzbach, J., and Kuhns, W. (1998) Glycoproteins and their relationship to human disease. Acta Anat. 161, 36–78.
Brockhausen, I. and Kuhns, W. (1997) Glycoproteins and human disease. Medical Intelligence Unit, CRC Press and Mosby-Year Book, Chapman & Hall NY, Springer-Verlag, Heidelberg, Germany.
Varki, A. (1993) Biological roles of oligosaccharides: all of the theories are correct. Glycobiology 3, 97–130.
Hanasaki, K., Varki, A., Stamenkovic, I., and Bevilacqua, M. P. (1994) Cytokine-induced beta-galactoside alpha-2,6-sialyltransferase in human endothelial cells mediates alpha 2,6-sialylation of adhesion molecules and CD22 ligands. J. Biol. Chem. 269, 10,637–10,643.
Keppler, O. T., Peter, M. E., Hinderlich, S., et al. (1999) Differential sialylation of cell surface glycoconjugates in a human B lymphoma cell line regulates susceptibility for CD95 (APO-1/Fas)-mediated apoptosis and for infection by a lymphotropic virus. Glycobiology 9, 557–569.
Koya, D., Dennis, J. W., Warren, C. E., et al. (1999) Overexpression of core 2 N-acetylglycosaminyltransferase enhances cytokine actions and induces hypertrophic myocardium in transgenic mice. FASEB J. 13, 2329–2337.
Hiraishi, K., Suzuki, K., Hakomori, S., and Adachi, M. (1993) Le y antigen expression is correlated with apoptosis (programmed cell death). Glycobiology 3, 381–390.
Leist, M. and Wendel, A. (1995) Tunicamycin potently inhibits tumor necrosis factor-induced hepatocyte apoptosis. Eur. J. Pharmacol. 292, 201–204.
Brockhausen, I., Lehotay, M., Yang, J., et al. (2002) Glycoprotein biosynthesis in porcine aortic endothelial cells and changes in the apoptotic cell population. Glycobiology 12, 33–45.
Yang, X., Lehotay, M., Anastassiades, T., Harrison, M., and Brockhausen, I. (2004) The effect of TNFalpha on glycosylation pathways in bovine synoviocytes. Biochem. Cell Biology 82, 559–568.
Delmotte, P., Degroote, S., Lafitte, J. J., Lamblin, G., Perini, J. P., and Roussel, P. (2002) Tumor Necrosis Factor alpha increases the expression of glycosyltransferases and sulfotransferases responsible for the biosynthesis of sialylated and sulfated Lewis x epitopes in the human bronchial mucosa. J. Biol. Chem. 277, 424–431.
Brockhausen, I., Yang, X., and Harrison, M. (2005) Glycodynamics of human osteoblastic cells. Glycobiology (Abstract 223) 15, 1242.
Kassem, M., Risteli, L., Mosekilde, L., Melsen, F., and Eriksen, E. F. (1991) Formation of osteoblast-like cells from human mononuclear bone marrow cultures. APMIS 99, 269–274.
Brockhausen, I. (2000) O-linked chain glycosyltransferases. Mucin methods and protocols. Methods Mol. Biol. 125, 273–293.
Brockhausen, I., Yang, J., Lehotay, M., Ogata, S., and Itzkowitz, S. (2001) Pathways of mucin O-glycosylation in normal and malignant rat colonic epithelial cells reveal a mechanism for cancer-associated Sialyl-Tn antigen expression. Biol. Chemistry 382, 219–232.
National Academy of Sciences—National Research Council, Washington, DC (1972) Specifications and Criteria for Biochemical Compounds, 3rd ed.
Möller, G., Reck, F., Paulsen, H., et al. (1992) Control of glycoprotein synthesis: Properties and substrate specificity of UDP-GlcNAc: Man α3R β2-N-acetylglucosaminyl-transferase I using synthetic substrates. Glycoconj. J. 9, 180–190.
Paulsen, H., Meinjohanns, E., Reck, F., and Brockhausen, I. (1993) Building units of oligosaccharides CVIII. Synthesis of modified oligosaccharides of N-glycoproteins for substrate specificity studies of N-acetylglucosaminyltransferase III to V. Liebigs Ann. Chemie 737–750.
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Brockhausen, I., Yang, X., Harrison, M. (2006). Analysis of the Glycodynamics of Primary Osteoblasts and Bone Cancer Cells. In: Brockhausen, I. (eds) Glycobiology Protocols. Methods in Molecular Biology, vol 347. Humana Press. https://doi.org/10.1385/1-59745-167-3:211
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DOI: https://doi.org/10.1385/1-59745-167-3:211
Publisher Name: Humana Press
Print ISBN: 978-1-58829-553-8
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