Essential SUMOylation Factors are Ubiquitously Expressed During Endochondral Ossification

Objectives: During organogenesis, post-translational modifications regulate cell migration, proliferation, differentiation, and apoptosis. SUMOylation is a ubiquitin-like post-translational modification that alters the target protein’s stability, activity, and cellular distribution. The sequential process of SUMOylation requires three enzymes. The small ubiquitin-related modifier (SUMO) substrates are activated by the E1 activating enzyme and transferred from E1 to Ubc9, the only E2 conjugating enzyme. Finally, the E3 ligases transfer the substrate from E2 to the target protein. Protein SUMOylation occurs during normal development, but the functional role of SUMOylation during skeletogenesis remains largely unknown. The objective of this study is to determine the expression of SUMOylation substrates and enzymes during skeletal development, specifically endochondral ossification.
Methods: Skeletal tissues from eviscerated/deskinned wild-type embryos were collected for RNA and protein isolation at day 12, 14, 16, 18, 19, newborn (NB), and 1-month stages and analyzed by real-time-PCR and western blotting. Spatio-temporal expression of selected SUMOylation pathway genes was evaluated by histologic sectioning and labeling of NB femurs using RNAScope and immunofluorescence.
Results: Skeletogenesis initiated at E11.5 progressively increases through embryonic and postnatal life. The SUMO1 expression decreased progressively, while SUMO2, Ubc9, and Pias1 were expressed consistently during skeletal development through 1 month of age. Sox9, Runx2, and Sp7 expression confirmed the progression of embryonic and postnatal bone development. The mRNA-sequencing performed on isolated chondrocytes from E18 pups showed the expression of all critical components of the SUMOylation pathway. Finally, we performed RNAScope experiments on the NB limb to uncover their spatial expression during chondrocyte differentiation. We find that the expression of SUMO2 progressively increases during chondrocyte differentiation, while Ubc9 and Pias1 expression is expressed consistently throughout the resting, proliferating, and hypertrophic chondrocytes.
Conclusions: The presence of all essential components of SUMOylation in chondrocytes indicates the involvement of SUMOylation during endochondral ossification.