During endochondral ossification, cartilage undergoes a complex process of maturation. As this process unfolds, resting and proliferating chondrocytes develop into hypertrophic chondrocytes, synthesis of a unique collagen (type X) is activated, and the matrix surrounding hypertrophic chondrocytes is mineralized. Eventually, the mature mineralized cartilage undergoes involution and is replaced by bone. Very little is currently known of the mechanisms that regulate this complex developmental process. A growing body of evidence indicates that retinoic acid (RA) is involved in cartilage development but the specific roles this microenvironmental cue could play are unknown. This project aims to test the hypothesis that RA has a role in cartilage maturation and, in particular, is an inducer of type X collagen gene expression during chondrocyte maturation. In preliminary experiments, we have found that treatment of maturing chick sternal chondrocytes with physiological doses of RA rapidly induces initiation of type X collagen gene expression. Maturing sternal chondrocytes harvested from 1 week-old primary cultures were treated with 15-50 nM RA for 1, 2 or 3 days. Whereas control untreated cultures as well as cultures treated for 1 day only contained undetectable levels of type X collagen mRNA and legs than 0.5% type X collagen-producing cells, the cultures treated for 2-3 days contained large amounts of type X collagen mRNA and 30-35% type X collagen-producing cells. In this project we will test the hypothesis that RA treatment increases production of the cartilage-characteristic gamma isoform of the RA receptor (RAR) and that increased gamma RAR production induces transcriptional activation of type X collagen gene expression. We propose to isolate cDNA clones for chick gamma RAR (a) to map the gene expression of this RAR during chondrocyte maturation and (b) to establish a functional relationship between gamma RAR and type X collagen gene expression. The information resulting from this project will shed light on previously unsuspected roles of RA in cartilage maturation. The findings will have important implications for the understanding of the normal progression of the endochondral process as well as its abnormalities.
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