The hypothesis to be tested in these studies is that all chondrocytes are derived from a common progenitor cell which undergoes variations in patterns of gene expression during cartilage development. The objective of these studies will be to define at a molecular level both the variations and the similarities between chondrocytes that ultimately have different developmental fates. Avian chondrocytes isolated from either caudal sterna which remains as hyaline cartilage in vivo, or ventral vertebra which undergoes bone replacement in vivo will be grown in vitro. Four parameters central to the development of these cells will be examined to explain the phenotypic variations between these two types of chondrocytes: a) Collagen gene expression (types I and II) and its regulation; b) Cartilage-specific collagen synthesis and mRNA levels (for collagen types II, IX, and X) in chondrocytes under different experimental condition; c) Ability of these cells to respond to specific vitamin D metabolites (1,25 (OH)2 versus 24,25 (OH)2 and express a calcified matrix in vitro; d) Cytoskeletal variations (actin and fibronectin expression) between these cell types. Previous data demonstrate that while both chondrocyte types express high levels of collagen pro alpha 1(II) mRNAs and protein only vertebral chondrocytes express pro alpha 1(I) and pro alpha 2(I) mRNAs. These mRNAs, however, are not translated into protein. An analysis of type I collagen transcription rates and mRNA stability will be undertaken to determine at what level mRNA quantities are regulated in these cells. Type II collagen and gamma-actin transcription rates and their stability will be similarly examined and compared as tissue- specific and nontissue-specific genes which are expressed in chondrocytes. The in vivo level of translational regulation will be defined (i.e., initiation versus elongation). Cell lysates will be made in other to identify whether specific factors exist that regulate type I collagen translation in chondrocytes. A quantitative comparison of the protein synthesis and steady state mRNA levels for collagen types I, II, IX and X will be undertaken in either sternal cell, vertebral cells or in sternal cells treated with 1,25(OH)2D3. In conjunction with these experiments, the effect of in vitro calcification of chondrocyte cultures on the collagen phenotype, collagen synthesis and processing, and the expression of bone specific proteins will be undertaken. The final aspect of these studies is directed at determining the functional significance of the observed modulation of chondrocyte morphology that has been shown to be related to the ability of chondrocytes to express their differentiated phenotype.
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