During development and fracture repair, endochondral bone formation is preceded by an orderly process of cartilage cell (chondrocyte) enlargement and matrix calcification. Since some cartilages do not, comparing mineralizing and non-mineralizing chondrocytes is an excellent way to learn about the requirements for matrix mineralization and factors controlling this process. Analysis of calcifying vs. non-calcifying cartilage has identified several differences in matrix proteins; among these are appearance of a novel type X collagen and decreased synthesis of the major component of cartilage matrix: Type II collagen. In addition, there is a marked increase in alkaline phosphatase, an enzyme expressed at high levels in all mineralizing tissues. Cultured chondrocytes can be induced to undergo changes in gene expression and to produce calcified matrix by exposure to ascorbic acid (vitamin C). The primary goal of the proposed research is to identify those changes required for mineralization and to define the mechanism by which ascorbate accomplishes these changes. The application proposes four specific aims to address these issues: l) Transcriptional regulatory mechanisms responsive to ascorbate are to be identified by studying ascorbate effects on transcript ion of reporter gene constructs containing regulatory regions from type II collagen, type X collagen and alkaline phosphatase genes. 2) The hypothesis that ascorbate induces expression of genes coding for proteolytic enzymes required for matrix degradation, and genes for matrix proteins involved in altered cell-matrix interaction, are to be tested by analysis of mRNAs from control and ascorbate-treated chondrocytes. 3) The hypothesis that matrix components present in non-calcifying cartilage can inhibit mineralization is to be investigated. 4) The role of alkaline phosphatase in chondrocyte maturation and mineralization will be examined by infecting non-mineralizing chondrocytes with viral vectors engineered to express high levels of alkaline phosphatase (AP).
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