Transforming growth factor beta (TGF beta) is a polypeptide growth regulator with bifunctional effects on cell proliferation. The TGF beta homodimer (25,000 daltons) has been purified from platelets and from bone. We have found that TGF beta may act as a coupling factor which links osteoblast activity with prior bone resorption, and thereby plays an important role in bone remodeling. TGF beta is released from organ cultures of murine calvaria in an inactive large molecular weight complex and is activated by decreasing pH, suggesting the presence of a binding protein attached through hydrogen bonding. Our crosslinking studies using I125 TGF beta and the crosslinking agent disuccinimidyl suberate have revealed by autoradiography on SDS-PAGE a binding protein (BP) greater than 200,000 daltons. We have coupled purified BSA-free TGF beta to 6% cross-linked agarose beads followed by incubation with the BP-TGF beta complex fraction from a Sephadex G-200 column. The material eluted from the TGF beta coupled beads was applied to SDS-PAGE and the major molecular species was approximately 300,000 MW. We plan to examine the mechanism whereby TGF beta is released from its binding protein and thereby activated. We have found that bone-- derived. TGF beta is activated by low pH. Mineral removal is dependent on acid production, optimal activity of many enzymes is at acid pH and acid conditions exist under the ruffled border of the actively resorbing osteoclast. Enzymes such as plasmin may also play a role in TGF beta activation as osteoblasts have been shown to secrete plasminogen activator. In this proposal we plan to investigate potential mechanisms of regulation of TGF beta activity via dissociation of the TGF beta monomer from it binding protein. We also plan to use the techniques we have developed in our preliminary data to purify the binding protein for bone-derived TGF beta to homogeneity.

National Institute of Health (NIH)
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
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Pathobiochemistry Study Section (PBC)
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University of Texas Health Science Center San Antonio
Schools of Medicine
San Antonio
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Bonewald, L F; Schwartz, Z; Swain, L D et al. (1992) Stimulation of matrix vesicle enzyme activity in osteoblast-like cells by 1,25(OH)2D3 and transforming growth factor beta (TGF beta). Bone Miner 17:139-44
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Bonewald, L F; Wakefield, L; Oreffo, R O et al. (1991) Latent forms of transforming growth factor-beta (TGF beta) derived from bone cultures: identification of a naturally occurring 100-kDa complex with similarity to recombinant latent TGF beta. Mol Endocrinol 5:741-51
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Bonewald, L F; Schwartz, Z; Swain, L D et al. (1990) Stimulation of plasma membrane and matrix vesicle enzyme activity by transforming growth factor-beta in osteosarcoma cell cultures. J Cell Physiol 145:200-6
Mundy, G R; Bonewald, L F (1990) Role of TGF beta in bone remodeling. Ann N Y Acad Sci 593:91-7
Bonewald, L F; Mundy, G R (1990) Role of transforming growth factor-beta in bone remodeling. Clin Orthop Relat Res :261-76