Advisor(s)
Amjad Javed
Committee Member(s)
Nabiha Yusuf
Rosa Serra
Russell Taichman
Xu Feng
School
Joint Health Sciences (Interdisciplinary)
Document Type
Dissertation
Department (new version)
Joint Health Sciences
Date of Award
1-6-2025
Abstract
The bone matrix synthesis by osteoblast during skeletogenesis is regulated by several transcription factors and signaling pathways. Runx2 and Sp7 are master regulators of osteoblast differentiation and bone formation. Deletion of both genes in mice results in the complete failure of ossification. In humans, Sp7 and Runx2 genes mutations cause osteogenesis imperfecta and cleidocranial dysplasia, respectively. The progression of osteoblast differentiation requires a coordinated and temporal expression of Runx2 and Sp7 genes. Posttranslational modifications regulate the cellular levels and function of Runx2 and Sp7 proteins. Ubiquitination controls protein turnover through targeted degradation. The multi-subunit SCF-complex is the largest class of E3-ligases that regulate the ubiquitination of target proteins. Rbx1 and Rbx2, are the only E3- ligase within the SCF complex. The role of Rbx1 or Rbx2 in regulating the turnover of Runx2 and Sp7 protein is unknown. We show that Rbx1/Rbx2 are expressed during osteoblast differentiation. In-silico analysis identified multiple evolutionarily conserved lysines in the Sp7 and Runx2 proteins that are likely targeted for ubiquitination. Three lysines in the Sp7 protein, K55, K227, and K229 were identified as the potential ubiquitination targets. On the other hand, iv six lysines, K202, K225, K240, K245, K365, and K366, in the Runx2 protein can be ubiquitinated. In-situ immunofluorescence and biochemical fractionation revealed that Rbx1 and Rbx2 enzymes are present in the nucleus and cytoplasm of osteoblast. Rbx1 and Rbx2 both target Sp7 protein for degradation. In sharp contrast, only the Rbx2 targets the Runx2 protein for degradation. Co-immunoprecipitation experiments showed a physical interaction between Rbx1 and Rbx2 and Sp7 protein. Experiments involving biochemical inhibitors showed that RING-box E3-ligase promotes ubiquitination and proteasomal-mediated degradation of Sp7 protein. A panel of deletion and point mutants identified that lysine-55 is targeted by Rbx1 and Rbx2 enzymes to ubiquitinate the Sp7 protein. The gene ablation approach was used to establish the biological significance of Sp7 protein turnover by the Rbx2 enzyme. Deleting the Rbx2 gene in osteoprogenitors significantly increases the level of endogenous Sp7 protein and enhances osteoblast differentiation and matrix mineralization. Our data demonstrate that Rbx2 inhibits osteoblast differentiation by promoting the turnover of Sp7 and Runx2 proteins.
ProQuest ID
Recommended Citation
Sikandar, Abeera, "Role Of Ring-Box E3 Ligases In Runx2 And Sp7 Protein Turnover During Osteoblast Differentiation" (2025). All ETDs from UAB. 7322.
https://digitalcommons.library.uab.edu/etd-collection/7322
