Elucidating the cellular and molecular processes involved in growth and remodeling of skeletal elements is of paramount importance for our understanding of congenital limb deformities, such as chondrodystrophies. These processes can be advantageously studied in the epiphyseal growth zone, the region in which all of the increase in length of a developing long bone is achieved. Here, young growth cartilage matures, becomes hypertrophic, and ultimately is removed. During cartilage hypertrophy, a number of changes occur including the acquisition of synthesis of a number of components, such as type X collagen, and cessation of synthesis of others. In an in vitro system, exogenously added type X collagen rapidly moves through the matrix of non-hypertrophic cartilage, and mimics certain changes observed in hypertrophic cartilage in vivo. These include altering the cartilage collagen fibrils and effecting both quantitative and qualitative changes in proteoglycan (PG) accumulation. These results suggest that type X collagen, in addition to being a structural molecule, also performs regulatory functions during the process of hypertrophy. Much of the proposed work will be to investigate these functions. The changes in PGs in response to type X will be examined to determine whether they are due to posttranslational modifications, translation of core proteins, or accumulation or synthesis of mRNAs for the core proteins. Certain of the responses in PGs depend on the collagenous triple-helical domain of the type X molecule, whereas others appear to depend on cellular responses to its COOH-terminal globular domain. These cellular responses will be further examined, as well as ones involving direct type X collagen-PG interactions. It will also be determined whether the responses to the isolated triple-helical domain (an artificial form of the molecule) can be produced by the major vertebrate collagenase cleavage product, a natural form of the molecule likely to be generated in vivo during cartilage degradation. In addition, we will determine, during hypertrophy, how many and which genes are up-regulated, how many are down-regulated, and what are the magnitudes of these changes? These studies will employ a recently devised subtractive hybridization procedure based on the polymerase chain reaction, which in addition will provide cDNA probes for each of the regulated genes. Lastly, more long-term goals will be to use these cDNA probes to further determine, during hypertrophy, which genes are coordinately regulated and which appear sequentially? Which are regulated inherently within the chondocytes themselves, and which require exogenous development factors? These parameters will be determined by examining and comparing the changes in gene expression elicited by chondrocytes cultured under various, selected conditions.
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