Disease-associated TNFRSF11B variants differentially disrupt osteoprotegerin secretion and function.

Abstract

Osteoprotegerin (OPG), encoded by TNFRSF11B, plays a critical role in suppressing osteoclastogenesis as a secreted decoy receptor for RANKL. Loss-of-function mutations in TNFRSF11B cause juvenile Paget's disease and related bone metabolic disorders; however, the mechanisms by which individual mutations impair OPG secretion and function remain incompletely understood. Here, we quantitatively analyzed the effects of disease-associated TNFRSF11B variants on OPG secretion and osteoclastogenesis-inhibitory activity. Wild-type and variant OPG proteins were expressed as Gaussia luciferase fusion constructs to measure extracellular secretion, and functional activity was evaluated using a mouse osteoclastogenesis assay. The cysteine-substituted variants C65F and C87Y exhibited severe secretion defects, whereas the T76P and D182del variants showed reductions in both secretion and inhibitory activity. The F117L variant showed normal secretion but markedly reduced inhibitory activity, indicating a secretion-independent functional defect. In contrast, the truncation variants D323fs and R333Ter exhibited reduced secretion while retaining substantial anti-RANKL activity. Further analysis revealed that endoplasmic reticulum-associated degradation (ERAD) contributes to the regulation of OPG secretion. Proteasome inhibition and knockdown of the ERAD components HRD1 and SEL1L increased extracellular levels of wild-type OPG as well as those of D323fs and R333Ter variants. Collectively, our results define mutation-specific mechanisms underlying OPG dysfunction and identify ERAD as a regulator of OPG secretion.