Exogenous vitamin C regulates osteoclast differentiation kinetics, cytoskeletal maturation, and bone resorption through ERK1/2-DAPK1-caspase-3 signaling.

Abstract

Vitamin C (VC) plays essential but incompletely defined roles in osteoclast (OC) development. We examined how exogenous VC and AKR1A-dependent endogenous VC regulate OC differentiation, cytoskeletal organization, resorptive activity, and apoptosis. Murine bone marrow-derived macrophages (BMDMs) from wild-type and AKR1A-deficient mice, together with RAW264.7 cells, were used to assess the effects of exogenous and endogenous VC on OC differentiation, actin ring formation, resorption, apoptotic signaling, and ERK1/2 activation. AKR1A1 deficiency altered osteoclastogenic kinetics, delaying early differentiation, impairing actin ring formation and early resorption, yet promoting late accumulation of large multinucleated OCs. VC exhibited stage-dependent effects: it was required to initiate osteoclastogenesis but, when supplemented at later stages, suppressed OC maturation and enhanced apoptosis. In both primary cells and RAW264.7 cultures, VC dose-dependently promoted peripheral F-actin ring assembly and increased matrix resorption. Mechanistically, VC enhanced ERK1/2 phosphorylation in mature OCs, selectively increased reactive oxygen species, and activated the DAPK1-caspase-3 axis, including nuclear localization of phospho-DAPK1 and cleaved caspase-3. Pharmacologic ERK inhibition suppressed OC formation and resorptive function. VC acts as a stage-dependent modulator of OC fate, linking ERK1/2-associated cytoskeletal maturation and resorptive activity to DAPK1-caspase-3-mediated apoptotic termination during bone remodeling.