Mechanical Unloading Inhibits Osteoblast Differentiation via Downregulation of OGT-Mediated O-GlcNAcylation.

Prolonged spaceflight and sustained bed rest induce mechanical unloading, leading to disuse osteoporosis and an elevated risk of fractures. Although post-translational modifications are increasingly recognized as key contributors to the pathogenesis of disuse bone loss, the functional role and underlying molecular mechanisms of O-linked N-acetylglucosaminylation (O-GlcNAcylation) remain poorly understood. Here, we demonstrate that mechanical unloading via 2D clinorotation downregulates the levels of O-GlcNAc transferase (OGT) and global protein O-GlcNAcylation in MC3T3-E1 cells, whereas osteogenic induction elicits the opposite effect. Both small interfering RNA (siRNA) targeting OGT and pharmacological inhibition using OSMI-1 recapitulated unloading-induced deficits, significantly impairing osteogenic differentiation and matrix mineralization. Conversely, OGT overexpression or inhibition of O-GlcNAcase (OGA) with Thiamet-G enhanced these processes. Importantly, OGT re-expression partially reversed the deficits caused by mechanical unloading. Notably, exogenous elevation of global O-GlcNAcylation levels via Thiamet-G treatment even after OGT knockdown also partially restored osteogenic capacity. Together, these findings establish the OGT/O-GlcNAcylation axis as a critical regulator of unloading-induced suppression of osteogenesis and identify it as a promising therapeutic target for disuse osteoporosis.