The Role of Ubiquitin Conjugating Enzyme (Ubc9) in Endochondral Ossification - Post-translational modifications play a crucial role in cell proliferation, differentiation, and tissue development. SUMOylation is a post-translational modification that alters stability, activity, protein interactions and cellular distribution of target proteins. The sequential process of SUMOylation requires three enzymes. The small ubiquitin-related modifiers (Sumo) are initially activated by the E1 enzyme and transferred from E1 to Ubc9, the only E2 conjugating enzyme. Finally, the E3 ligases transfer Sumo from E2 to the target protein. Abnormal SUMOylation has been associated with cancers, neurodegenerative and skeletal disorders, including rheumatoid arthritis and Paget’s disease of bone. SUMOylation occurs during embryonic development but the functional role of SUMOylation during skeletogenesis remains unknown. The development of skeletal tissue in mice is first noted at embryonic day 11.5 and increases through embryonic and postnatal life. We found that the level of Runx2 increased by 13-fold from E12 to birth. A similar progressive expression was noted for Sp7 gene. Sumo1 expression was progressively decreased from E12 to newborn stage. The vital substrate Sumo2, however showed a constitutive expression throughout embryonic skeletal development. These data indicate the availability of Sumo substrate during skeletogenesis. The evolutionarily conserved Ube2i gene encodes the only conjugating enzyme in vertebrates, Ubc9, that is required for SUMOylation. Our data shows that the levels of Ubc9 mRNA remain stable during embryonic skeletogenesis. Our mRNA sequencing confirmed the presence of all Sumo substrates, E1, E2 and critical E3 ligases in chondrocyte and during endochondral ossification. Based on these data, we hypothesize that Ubc9 is required for differentiation of chondrocytes during embryonic and post-natal skeletogenesis. In this study we aim to elucidate the role of Ubc9 mediated SUMOylation during endochondral ossification. In Aim 1 we will uncover the requirement of Ubc9 mediated SUMOylation during endochondral ossification. We will first establish the expression pattern of the of essential components of the SUMOylation pathway in the growth plate chondrocyte by RNA and protein analysis. To uncover how SUMOylation regulates chondrocyte differentiation and endochondral ossification, the Ubc9 gene will be deleted in resting chondrocytes using Col2a-Cre transgenic mice. In Aim 2, will investigate the role of SUMOylation in hypertrophic chondrocyte mediated cartilage turnover and bone formation by conditionally deleting Ubc9 in hypertrophic chondrocytes using Col10-Cre mice. Completion of this proposed study will contribute to the field by providing novel mouse models for studying SUMOylation during endochondral ossification. Additionally, elucidating the role of Ubc9 during chondrogenesis will provide critical knowledge about the role of SUMOylation during skeletal cell differentiation, which could lead to new therapeutics for many skeletal disorders.
