Studies conducted over the last 14 years relate to the regulation of the cartilage phenotype. Over the current grant period emphasis has been placed on the regulation of gene expression of the chondrocyte phenotype and the function of the type hA procollagen splice form. The two most significant discoveries of this grant period are: (1) type hA procollagen is synthesized as a pN procollagen and binds to Bone Morphogenetic Proteins (BMP) and TGFbI; and (2) discovery of a new cartilage phenotype-characteristic molecule, CD-RAP. The binding of BMPs to type IIA procollagen may be critical to the regulation of BMPs during differentiation. We propose that type IIA procollagen is secreted into the extracellular matrix and binds BMPs. The binding of BMPs serves to localize the BMP in regions of potential chondrogenesis or in storage tissues such as periosteum and perichondrium. Subsequent cleavage of the N-propeptide by matrix metalloproteinases or astacin proteinases could release active BMP into the extracellular environment. A similar paradigm exists in the regulation of BMP presentation in Xenopus and Drosophila. The studies proposed in this competitive renewal seek to test this hypothesis.
The specific aims are: (1) investigate the binding of BMPs and TGFbs by type hA procollagen N-propeptide; (2) investigate the enzymic processing of type IIA and type IIB procollagens; and (3) investigate the function of type IIA procollagen in vitro and in vivo. Traditional biochemical techniques along with expression of recombinant native and mutated proteins will be used for the binding studies; classic enzyme digestion and protein sequencing, pulse-chase in cell culture and histology will be used to investigate enzymic processing; and transgenic mice and injection into Xenopus embryos will be used to study function
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