Our long-term goal has been the rational development of therapeutic tools for the control of endochondral bone formation through characterization of critical control pathways in skeletogenesis. We previously postulated that apoptosis of hypertrophic chondrocytes at the interface between growth cartilage and developing bone provides a key regulatory element controlling the release of angiogenic factors. In the previous grant period we demonstrated that a multipotent growth factor with angiogenic activity, transforming growth factor beta 2 (TGFbeta2), was released from hypertrophic chondrocytes at about the time of their apoptosis. Others have shown that the TGFbeta1 isotype is released as a multimolecular inactive precursor, which is subsequently activated by proteolytic cleavage. Nothing was known about the TGFbeta2 complex or its activation. We have shown that TGFbeta2 is released from hypertrophic chondrocytes in large latent complexes that are activated in association with chondrocyte apoptosis. We propose to determine the molecular composition of this TGFbeta2 complex using antibody affinity purification and tandem affinity purification of dual-tagged TGFbeta2 inserted in a high efficiency recombinant competent avian retroviral (RCAS) vector. Isolated complex components will then be identified by mass spectrometry. Characterization and overexpression of proteins associated with the TGFbeta2 complex in cells in culture and in developing chick embryos, using RCAS expression, will provide an understanding of their function in skeletal development. The role of proteinases and apoptosis in processing of the components of the TGFbeta2 complex will be analyzed using specific inhibitors and overexpression of Bcl-2. Cleavage sites generated during processing of some of the components will be analyzed by mass spectrometry and enable preparation of constructs for the expression of proteins with mutations in the processing sites or peptide containing processing sites. Their expression in cell culture and chick embryos will determine the importance of specific processing steps in the activation of TGFbeta2 and assess the efficacy of developing inhibitors of endochondral bone formation targeted at inhibition of extracellular processing. The ability to modulate endochondral bone formation will find application in not only potential therapies to correct or control misshapen bone formation in children, but also (the applicant did not complete this sentence).
