Purification and Characterization of Growth Factor Extracted From Bone Tetsuma Uchida Department of Maxillofacial Surgery, Faculty of Dentistry, Tokyo

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1 Purification and Characterization of Growth Factor Extracted From Bone Tetsuma Uchida Department of Maxillofacial Surgery, Faculty of Dentistry, Tokyo Medical and Dental University (Director : Prof. Teruo Amagasa) Department of Biochemistry, Faculty of Dentistry, Tokyo Medical and Dental University (Director : Prof. Satoshi Sasaki) Local metabolic balance between the bone formation and resorption is modulated by many growth factors present in the bone. The mechanism of this regulation has been investigated by many researchers but is not completely clarified yet. In this study, the author found the presence of several growth factors in the 1 M NaCl extract from the bovine bone and one of them was confirmed to be different from the factors previously reported in the bone tissue. This factor was purified by means of gel filtration and heparin-affinity chromatography and characterized in detail. The purified factor stimulated the proliferation of the fibroblast cell line, Balb/c3T3, but neither stimulated nor inhibited that of the osteoblast cell line, MC3T3-E1. The factor was stable against heat (65t), acid (2N HCl), 6M urea or 4M guanidine-hcl treatments. However, it was inactivated by trypsin. The molecular size of this factor was about 15,000 and had a high affinity for heparin. In contrast to afgf or bfgf, the fibroblast-proliferating activity of this factor was suppressed dose-dependently by heparin addition. According to those properties, this factor was indicated to be different from TGF-ƒÀ, afgf, bfgf, PDGF, IGF- I, IGF-II or BMP which were reported to be present in the bone tissue. It suggested the presence of a new factor in the bone which may regulate the proliferation of fibroblasts in vivo.

2) Mohan, S. and Baylink, D.J.: Bone Growth Factors. Clinical Orthopaedics and Related Research. 263: 30-48, 1991. 4) Seyedin, S.M., Thomas, T.C., Thompson, A.

10 2) Mohan, S. and Baylink, D.J.: Bone Growth Factors. Clinical Orthopaedics and Related Research. 263: 30-48, ) Seyedin, S.M., Thomas, T.C., Thompson, A.Y., Rosen, D.M. and Piez, K.A.: Purification and characterization of two cartilage-inducing factors from bovine demineralized bone. Proc. Natl. Acad. Sci. USA 82: , ) Seyedin, S.M., Thompson, A.Y., Bentz, H., Rosen, D.M., McPherson, J.M., Conti, A., Siegel, N.R., Galluppi, G.R. and Piez, K.A.: Cartilage-inducing factor-a; apparent identity to transforming growth factor-ƒà. J. Biol. Chem. 261: , ) Seyedin, S. M., Segarini, P.R., Rosen, D.M., Thompson, A.Y., Bentz, H. and Grycar, J.: Cartilage-inducing factor-b is a unique protein structurally and functionally related to transforming growth factor-ƒà. J.Biol. Chem. 262 : , 1987.

7) Hauschka, P.V., Mavrakos, A.E., Iafrati, M.D., Doleman, S.E, and Klagsbrun, M.: Growth factors in bone matrix. J.Biol. Chem. 261: 12665-12674, 1986. 8) Farley, JR. and Baylink, D.J.: Purification of a skeletal growth factor from human bone.

11 7) Hauschka, P.V., Mavrakos, A.E., Iafrati, M.D., Doleman, S.E, and Klagsbrun, M.: Growth factors in bone matrix. J.Biol. Chem. 261: , ) Farley, JR. and Baylink, D.J.: Purification of a skeletal growth factor from human bone. Biochemistry 21: , ) Farley, J.R., Masuda, T., Wergedal, J.E. and Baylink, D.J.: Human skeletal growth factor: Characterization of the mitogenic effect on bone cells in vitro. Biochemistry 21: , ) Mohan, S., Jennings, J.C., Linkhart, TA. and Baylink, D.J.: Primary structure of human skeletal growth factor; homology with human insulin-like growth factor-ii, Biochim. Biophys. Acta. 966: 44-55, ) Urist, M.R. and Strates, B.S.: Bone morphogenetic protein. J.Dent. Res. 50: , ) Urist, MR., Hou, Y.K., Brownell, A.G., Hohl, W.M., Buyske, J., Lietze, A., Tempst, P., Hunkapiller, M. and DeLange, R.J.: Purification of bovine bone morphogenetic protein by hydroxyapatite chromatography. Proc. Natl. Acad. Sci. USA 81: , ) Jingushi, S., Heydemann, A., Kana, S.K., Macey, L.R. and Bolander, ME.: Acidic fibroblast growth factor injection stimulates cartilage enlargement and inhibits cartilage gene expression in rat fracture healing. J.Orthop. Res. 8: , proliferation and cytotoxicity assays. J.Immunol. Methods. 65: 55-63, ) Carmichael, J., DeGraff, W.G., Gazdar, A.F., Minna, J.D. and Mitchell, J.B.: Evaluation of a tetrazolium-based semiautomated colorimetric assay; Assessment of chemosensitivity testing. Cancer Res. 47: , ) Laemmli, U.K.: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: , ) Termine, J.D., Belcourt, A.B., Conn, K.M. and Kleinman, H.K.: Mineral and collagen-binding proteins of fetal calf bone. J. Biol: Chem. 256 : , ) Sampath, T.K. and Reddi, A.H.: Dissociative extraction and reconstitution of extracellular matrix components involved in local bone differentiation. Proc. Natl. Acad. Sci. USA. 78 : , ) Sampath, T.K., Muthukumaran, N. and Reddi, A.H.: Isolation of osteogenin. An extracellular matrix-associated, bone-inductive protein, by heparin affinity chromatography. Proc. Natl. Acad. Sci. USA. 84: , ) Ooi, G.T. and Herington, A.C.: The biological and structural characterization of specific serum binding proteins for the insulin-like growth factors. J.Endocrinol. 118: 7-18, ) Rubin, J.S., Osada, H., Finch, P.W., Taylor, W. 15) Noda, M, and Camilliere, J.J.: In vivo stimulation G., Rudikoff, S. and Aaronson, S.A.: Purifica- of bone formation by transforming tion and characterization of a newly growth growth factor-ƒà. Endocrinology 124: factor specific for epithelial cells. Proc. Natl , Acad. Sci. USA. 86: , ) Gospodarowicz, D., Bialecki, H. and Greenburg, 29) Thornton, S.C., Mueller, S.N. and Levine, E. G.: Purification of the fibroblast growth M.: Human endothelial cells; Use of heparin factor activity from bovine brain. J.Biol. Chem. in cloning and long-term serial cultivation. 253: , Science 222: , ) Gospodarowicz, D. and Cheng, J.: Heparin protects basic and acidic FGF from inactivation. J.Cell. Physiol. 128: , ) Ichijo, H., Momose, F. and Miyazono, K.: 31) Uhlrich, S., Lagente, O., Lenfant, M. and Courtois, Biological effects and binding properities of Y.: Effect of heparin on the stimulation of transforming growth factor-ƒà on human oral non-vascular cells by human acidic and basic squamous cell carcinoma cells. Exp. Cell Res. FGF. Biochem. Biophys. Res. Commun. 137: 187: , , ) Mosmann, T.: Rapid colorimetric assay for 32) Klagsbrun, M.: The affinity of fibroblast cellular growth and survival; application to growth factors (FGFs) for heparin; FGFheparan sulfate interactions in cells and

12 extracellular matrix. Curr. Opinion Cell Biol. 2: , ) Yayon, A., Klagsbrun, M., Esko, J.D., Leder, P. and Ornitz, D.M.: Cell surface, heparin-like molecules are required for binding of basic fibroblast growth factor to its high affinity receptor. Cell 64: , ) Sakaguchi, K., Yanagishita, M., Takeuchi, Y. and Aurbach, G.D.: Identification of heparan sulfate proteoglycan as a high affinity receptor for acidic fibroblast growth factor (afgf) in a parathyroid cell line. J.Biol. Chem. 266: , 1991.

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Database Center for Life Science Online Service 2) Shin, S. I., Fredman, V. H., Risser, P., Pol lack,r.: Proc. Natl. Acad. Sci. USA, 72, 4435-4439 (1975) 3) Marquardt, H., Todaro, G. J.: J. Biol. Chem., 256, 6859-6865 (1981) 4) Dicker, P., Pohjanpelto,