Integrated single-cell and experimental analyses implicate GPRC5B in macrophage senescence-related alterations in postmenopausal osteoporosis

Abstract Background: Post-menopausal osteoporosis (PMO) is characterized by senescence, estrogen deficiency, immune dysregulation and progressive bone loss, yet the molecular alterations involved in PMO remain incompletely understood. Methods: Single-cell and transcriptomic analyses were integrated with machine learning and experimental validation. Ovariectomized mouse models, primary bone marrow-derived macrophages and RAW264.7 cells were used to evaluate gene expression, senescence-associated changes, macrophage polarization and related signaling pathways. Results: Single-cell RNA sequencing revealed substantial remodeling of the bone marrow microenvironment in PMO and identified APOE-positive macrophages as a dominant subset with elevated KRAS-related signaling. Integrated co-expression and machine-learning analyses yielded GPRC5B as a PMO-associated candidate gene that was consistently upregulated across multiple datasets. In vivo and in vitro , GPRC5B knockdown attenuated senescence-associated changes in macrophages, reduced inflammatory activation, promoted M2-like features and improved phagocytic clearance of apoptotic osteoblasts. Mechanistically, GPRC5B was associated with DLG1/Kv1.3-related signaling, and rescue experiments supported the involvement of this axis in the observed macrophage phenotypes. In PMO mice, GPRC5B knockdown was accompanied by improved bone phenotypes, and this effect was enhanced by DLG1 inhibition. Drug-signature screening and docking analysis further identified Doramectin as a candidate compound of interest, and Doramectin treatment was associated with reduced GPRC5B expression under estrogen-deprived conditions. Conclusions: GPRC5B is a macrophage-enriched, PMO-associated candidate identified through integrated transcriptomic analyses and supported by preliminary functional experiments. These findings suggest that GPRC5B may participate in macrophage-related alterations in PMO and warrant further mechanistic and translational investigation.