All ETDs from UAB

Advisory Committee Chair

Amjad Javed

Advisory Committee Members

Louise T Chow

Mohammad Q Hassan

Rosa A Serra

Hengbin Wang

Document Type

Dissertation

Date of Award

2014

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

Runx2 and Sp7 transcription factors are essential for skeletogenesis. Deletion of either gene in mice results in failure of bone tissue development. However, underlying mechanisms responsible for a surprisingly similar phenotype by two distinctly unrelated proteins remain unknown. Sp7 is a Runx2 downstream target gene and is not expressed in Runx2 null mice. Thus, the Runx2 null model represents a compound phenotype of loss of both proteins. In contrast, normal levels of Runx2 mRNA are noted in Sp7 null mice. The failure of Runx2 to promote bone formation in Sp7 null mice suggests that Sp7 is required for Runx2 function in osteoblasts. Here we have investigated the mechanism underlying functional incompetency of Runx2 in the Sp7 null model. We uncovered that despite normal levels of Runx2 mRNA in skeletal tissue of Sp7 null mice Runx2 protein is barely detected. These data strongly suggest that during skeletogenesis, Sp7 regulates stability or turnover of Runx2 protein. This hypothesis was experimentally tested in osteoblasts that endogenously express both Runx2 and Sp7 proteins. Osteoblasts depleted for Sp7 using Sp7-specific siRNA also showed a robust decrease in Runx2 protein. Change in Runx2 protein was not due to transcriptional regulation of Runx2 by Sp7, as levels of Runx2 mRNA remained unchanged between control and Sp7 siRNA treated osteoblasts. The involvement of Sp7 in regulating Runx2 protein stability was further confirmed by co-expression studies in cell that lack endogenous Runx2 and Sp7. HeLa cells were co-transfected with increasing amounts of Sp7 expression vector and a fixed amount of Runx2 expression vector. Interestingly, a dose dependent increase in Sp7 protein also caused a progressive increase of Runx2 protein. Blockage of De novo protein synthesis revealed that Sp7 significantly extend the half-life of Runx2 protein. Co-immunoprecipitation (Co-IP) studies revealed that endogenous Runx2 and Sp7 proteins form a molecular complex in osteoblasts. Reciprocal Co-IP studies confirmed physical association between Runx2 and Sp7 proteins. Moreover, MEK mediated phosphorylation of Runx2 and Sp7 proteins is required for physical interaction. Deletion mutagenesis has identified that the runt homology domain of Runx2 and the proline rich transactivation domain of Sp7 are involved in their molecular interaction. Physical association of Sp7 with Runx2 is essential for stability of Runx2 protein. Deletion mutant form of Sp7 protein (288-428), which does not interact with Runx2, fails to stabilize Runx2 protein. We next tested whether Sp7 regulates the stability of Runx2 protein, by preventing ubiquitination mediated proteasomal degradation of Runx2. Our results showed that Sp7 protect Runx2 protein from Smurf1 mediated ubiquitination and proteasomal degradation. We further demonstrated that Sp7 protein lacking the Runx2 interaction domain fail to prevent Smurf1 mediated degradation of Runx2 protein. Finally, the effects of Runx2-Sp7 molecular interaction on regulation of mature osteoblasts, osteocytes and hypertrophic chondrocytes marker genes were assessed by promoter-reporter assays. The cell phenotype-restricted genes osteocalcin (OC), sclerostin (SOST) and type X collagen (ColX) promoters were activated by both Runx2 and Sp7. Interestingly, co-expression of Runx2 and Sp7 resulted in a synergistic activation of OC, SOST and ColX gene promoters. Runx2-Sp7 physical association is essential for synergistic activation of OC promoter. Furthermore, during embryonic skeletogenesis expression of mature osteoblast marker genes such as OC, DMP1, SOST and mineralization of bone tissues coincides with high levels of both Runx2 and Sp7. Finally, inhibition of phosphorylation of Runx2 and Sp7 proteins abrogated synergistic activation of the osteocalcin gene promoter. Thus, multiple lines of evidence demonstrate that Sp7 regulates the stability and osteogenic function of Runx2 protein.

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