Regulation of aggrecan gene promoter during endochondral bone formation and fracture repair

During bone fracture healing the skeletal defect is initially stabilized via the formation of a cartilage callus. Consequently, there is significant interest in unraveling the complex regulation of cartilage formation and chondrocyte lineage progression. The focus of this thesis is on the molecular regulators that control chondrocyte differentiation. The first set of studies used an in vivo approach and assessed cartilage tissue development during normal fracture healing in comparison to a hypophosphatemic state generated by dietary phosphate restriction. While hypophosphatemia is known to impair normal chondrocyte development, the specific influence of phosphate restriction on mesenchymal stem cell recruitment to the fracture site, and its influence on the molecular mechanisms that regulate subsequent chondrocyte lineage progression are not well understood.;Histomorphometric and microcomputed tomography analysis showed that hypophosphatemia resulted in a significant reduction in fracture callus size, cartilage composition, and mineralization. Examination of the mRNA expression profiles that define chondrogenic differentiation by real-time PCR showed that hypophosphatemia resulted in a stage-specific delay in chondrogenic lineage progression. Taken together, these studies suggest that hypophosphatemia impairs fracture repair primarily through a delay in chondrocyte maturation, which translated into decreased callus mineralization and bone formation. Our assessment of known transcriptional regulators of cartilage development identified that the Runt-Related factor X (Runx) family of transcription factors were uniquely regulated during fracture healing. Interestingly, hypophosphatemia resulted in a significant reduction in both Runx2 and Runx3 expression in a stage-specific manner.;Subsequent in vitro experiments were directed at identifying the mechanism by which Runx proteins might mediate their effects on chondrocyte differentiation. Using the large chondroitin sulfate proteoglycan aggrecan, as a prototypical cartilage gene, these in vitro experiments examined the role that Runx(s) play in regulating chondrocyte specific gene expression differentiation. Both gain (Runx1, 2, and 3 over-expression) and loss (selective Runx shRNA knock-down) of function studies of the aggrecan gene promoter activity in chondrocytes and electromobility shift assays showed that Runx(s) regulate this gene promoter. Taken together, these studies suggest that Runx2 and Runx3 function as transcriptional regulators of aggrecan gene expression and are key regulators of cartilage development.