Sulfation Attenuates Mitigating Effects of 25‐Hydroxycholesterol on Postmenopausal Bone Loss

Postmenopausal osteoporosis is a prevalent skeletal disorder with an unmet need for safer therapeutic strategies. While certain oxysterols show osteoinductive potential, the role of 25-hydroxycholesterol (25HC) in bone homeostasis remains unclear. Furthermore, 25HC can undergo sulfation, a largely underexplored chemical modification, to form 25HC3S. This study aimed to investigate the effects of 25HC and 25HC3S on bone loss and the underlying mechanisms. We found that treatment with 25HC, but not 25HC3S, ameliorated bone loss in an ovariectomized (OVX) mouse model of osteoporosis, as evidenced by increased bone mass and expression of type I collagen, alongside decreased osteoclast numbers. Transcriptomic analysis revealed that 25HC regulated genes related to cell cycle and Rho GTPase signaling in bone tissue and osteoblastic lineage cell cultures, while 25HC3S had attenuated effects. In vivo and in vitro experiments demonstrated that 25HC promoted pre-osteoblast proliferation and enhanced osteocyte survival. Mechanistically, 25HC inhibited Rho GTPase signaling in pre-osteoblasts but activated it in osteocytes, which was largely abolished by sulfation. Collectively, this study identifies 25HC as a protective regulator against postmenopausal bone loss and unveils sulfation as a critical switch that negates its benefit, supporting 25HC supplementation as promising therapeutic strategies for osteoporosis.