MBD3 D246-dependent Runx2–NuRD association promotes Runx2 deacetylation, osteoblast dysfunction, and glucocorticoid-induced osteoporosis

Chromatin remodeling complexes are important regulators of osteoblast fate, yet the epigenetic mechanisms governing Runx2 activity during glucocorticoid-induced osteoporosis (GIOP) remain incompletely understood. Here, we identify methyl CpG binding domain protein 3 (MBD3), a core component of the nucleosome remodeling and deacetylase complex, as an epigenetic regulator associated with glucocorticoid-induced bone loss. MBD3 was markedly upregulated in bone tissues from rats with GIOP and disrupted osteoblast homeostasis by suppressing osteogenic differentiation and promoting caspase-dependent apoptosis, without affecting cell proliferation or cell cycle progression. Mechanistically, MBD3 associated with Runx2 and increased the detectable association of HDAC1 with Runx2, accompanied by reduced Runx2 acetylation, altered Runx2 chromatin occupancy, and impaired osteogenic activity. Truncation mapping localized the Runx2-associated region of MBD3 to its C-terminal 216 to 279 amino acid segment, whereas site-directed mutagenesis identified D246 as a functionally important residue contributing to MBD3 and Runx2 association, HDAC1 association with Runx2, Runx2 deacetylation, and osteoblast dysfunction. In vitro, the D246K mutation markedly attenuated the inhibitory effects of MBD3 on osteogenic differentiation and reduced MBD3-induced osteoblast apoptosis. In vivo, MBD3 knockdown restored trabecular acetyl lysine signals and Runx2-associated osteogenic activity, reduced osteoblast apoptosis, and ameliorated trabecular bone deterioration in rats with GIOP. Pharmacological rescue experiments further supported the involvement of HDAC-dependent deacetylation, as Exifone, which has been reported to enhance HDAC1 activity, weakened the bone-protective effects of MBD3 depletion, whereas the pan-HDAC inhibitor SAHA partially improved glucocorticoid-induced bone defects. Collectively, these findings identify an MBD3-associated mechanism regulating Runx2 deacetylation and osteoblast dysfunction during GIOP and highlight the functional importance of MBD3 D246 as a potential target for further investigation.