The present research proposal concerns the investigation of the mechanisms of action through which a bone matrix derived protein factor stimulates chondrogenesis. This purified 31,000 dalton protein component has been shown to possess Chondrogenic Stimulating Activity (CSA) when tested in stage 24 chick limb bud cell cultures. Protein fractions containing this agent also initiate chondrogenesis in minced skeletal muscle explant cultures. Our primary objective is to evaluate the CSA mediated cellular response mechanisms which initiate the mesenchyme-to-chondrocyte transition. The availability of a monoclonal antibody to CSA makes possible the construction of an immunoaffinity column which can be utilized to prepare relatively large quantities of purified CSA that will be required to perform these studies. Purified CSA will then be further investigated in two ways. First, a detailed study of its physical-chemical properties will be conducted including the determination of its amino acid composition, N-terminal amino acid sequence and carbohydrate composition. The second series of experiments with purified CSA will involve the identification of limb bud mesenchymal cell receptor complexes using 125I-labeled CSA. These studies will include the determination of CSA binding affinities, the number and types of receptors and what physiological or developmental parameters may influence the CSA-receptor interaction. The data generated by such investigations should provide the basis for a more thorough biochemical and molecular understanding of the chondro-inductive process for which there is currently little or no information.
