Osterix Regulate Runx2 Protein Stability and Function During Bone Formation

Introduction: Targeted disruption of Osterix (Osx) and Runx2 genes results in complete lack of bone development. Molecular reasons for surprisingly similar phenotype from two unrelated proteins remain unknown. As a downstream molecule, Osx is not expressed in Runx2-null mice. Osx-null mice, however fail to form bone, despite normal levels of Runx2 mRNA.  Objective: To identify molecular mechanisms for functional incompetency of Runx2 in Osx-null model. Methods: Genetic, cellular and biochemical approaches were used. Results: Osteoblasts that endogenously express both proteins were transfected with multiple siRNA to specifically knock-down Osx. We noted a dose dependent inhibition of Osx protein. To our surprise, a concomitant reduction of Runx2 protein was also noted. Changes in Runx2 protein were not related to transcriptional regulation of Runx2 by Osx, as siRNA treatment only decreased Osx mRNA but the levels of Runx2 mRNA remain unchanged. Thus regulation of Runx2 protein by Osx is post-transcriptional. We next confirmed that Osx is necessary for stability of Runx2-protein, by co-expression studies. HeLa cells were transfected with same amount of Runx2 and increasing concentration of Osx plasmid. We observed a dose dependent increase in Osx but surprisingly a progressive increase in Runx2 protein 15-fold was also noted. To understand how Osx stabilize Runx2-protein, we performed Co-immunoprecipitation studies. Endogenous Osx forms a molecular complex with Runx2-protein in osteoblasts.  Both ubiquitination and degradation of Runx2 protein by smurf1 was strongly inhibited by the presence of Osx. Runx2-Osx physical interaction is an integral component of this process, as mutant Osx protein that lack Runx2 interaction, fail to block Runx2 degradation by smurf1. Runx2 and Osx functionally cooperate to induce osteoblast genes. Finally, we demonstrate Runx2 protein is highly unstable in both intramembranous and endochondral bones of Osx-null mice. Conclusion: Osterix regulate Runx2 protein stability and function through molecular interaction in bone cells.