Wednesday, January 15, 2025
1/15/2025
ABSTRACT: Development of skeleton in mammals is an exceedingly complex process. Completion of both endochondral and intramembranous ossification entails a highly intricate but well-coordinated process of patterning, cell fate commitment, differentiation, growth, and remodeling. These events are specified by a coordinated temporal and spatial pattern of gene expression. At first, secreted morphogens such as hedgehog, bone morphogenetic proteins, wingless proteins, and others, signal to key transcription factors to specify gene expression. Runx2 is an essential transcription factor for both chondrocyte and osteoblast differentiation. Runx2 gene deletion results in embryonic lethality due to a complete failure of bone formation. In humans, mutation of the Runx2 gene causes cleidocranial dysplasia, a dominantly inherited skeletal disorder. Another master regulator of skeletogenesis is the Specificity protein-7 (Sp7). Sp7 belongs to the Sp subgroup of the Krüppel-like family of transcription factors characterized by three zinc-finger DNA- binding domains. Deletion of Sp7 gene results in failure of osteoblasts, and bone formation. In humans, mutation of the Sp7 gene is linked with the recessive form of osteogenesis imperfecta, skeletal fragility and delayed tooth eruption. However, very little is known about the underlying molecular mechanism for the surprisingly similar phenotype from the two seemingly unrelated proteins. Runx2 is required for the expression of Sp7, as mice with targeted disruption of the Runx2 gene completely lack expression of Sp7. In sharp contrast, the Runx2 expression is normal in the skeletal cells of Sp7 null animals. The functional incompetency of Runx2 in the Sp7 null mice suggests that Sp7 presence is obligatory for completion of the Runx2 osteogenic activity. It is important to note that the observation of Runx2 expression in Sp7 null mice is limited to only RNA, determined by in situ hybridization of embryonic tissues. Our data show that despite normal levels of Runx2 mRNA, Runx2 protein is highly unstable in skeletal tissues of Sp7 null mice. We further demonstrate that Runx2 and Sp7 proteins form a molecular complex and their transcriptional activity is regulated by unique posttranslational modifications. Our findings strongly suggest that in skeletal cells, Sp7 acts as a molecular rheostat and is necessary for functional stability and turnover of Runx2 protein. Our experiment will assess endogenous levels of Runx2 protein in Sp7 null background and by a regulated and selective gene reconstitution in osteoprogenitor cells. The goal of this application is to identify and define a) spatial and temporal organization and assembly of Runx2 and Sp7 regulatory complexes for the formation and/or maintenance of osteoblasts and b) mechanisms supporting the stable complex formation and retention of competency for skeletal gene expression. Knowledge obtained from this study will provide molecular insights into components of a bone regulatory complex that can be targeted for innovative therapy to improve cartilage and bone formation and repair.
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