TYPE III COLLAGEN GENE EXPRESSION IN CARTILAGE

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Type III collagen mRNA and protein are present in limb mesenchyme, but are not found in normal growth cartilages; thus synthesis of type III collagen must cease during chondrogenesis. However, a small amount of type III collagen is found in adult articular cartilage, and the amount appears to increase in osteoarthritic articular cartilage. The chick type III collagen gene contains two functional promoters which display different patterns of expression during embryonic development and initiate production of transcripts with very different structures and functions. The upstream promoter, which is responsible for production of the type III collagen mRNA, is active in skin fibroblasts, but inactive in chondrocytes. An internal, chondrocyte-specific promoter has been identified which is responsible for production of an alternative transcript in which exons 1-23 are replaced by a previously undescribed exon. The alternative transcript displays a complex structure in terms of protein coding potential, containing at least three possible open reading frames, one of which is a truncated collagen; thus the alternative transcript cannot encode type III collagen. Three specific aims are proposed to investigate the complex regulation of type III collagen gene expression during chondrogenesis. 1) In situ hybridization and immunohistochemistry will be used to define the spatial and temporal expression of type III collagen mRNA and protein, as well as the alternative transcript, in the developing limb. 2) The molecular mechanisms that regulate transcription of the type III collagen gene from dual promoters during chondrogenesis will be characterized. 3) Several complementary strategies will be utilized to define potential functions of the alternative transcript. The effects of overexpression and reduced expression of the alternative transcript on in vivo chondrogenesis will be determined. In addition, if the alternative transcript seems likely to serve a protein coding function in limb mesenchyme, the protein will be identified and characterized. These experiments will define the mechanisms that prevent production of type III collagen in cartilage and provide insight into the mechanisms resulting in reactivation of type III collagen synthesis in chondrocytes in endochondral fracture repair and osteoarthritis. These strategies will also increase our understanding of potential additional functions of the type III collagen gene mediated by the alternative transcript.