Cholesterol inhibits autophagy in RANKL-induced osteoclast differentiation through activating the PI3K/AKT/mTOR signaling pathway.

Background: A dysregulated balance between bone formation and bone resorption controlled by osteoblast and osteoclast will lead to osteoporosis. Cholesterol (CHO) is a crucial factor leading to osteoporosis, and autophagy appears to involve it. Therefore, we aimed to study the molecular mechanism of autophagy in CHO-induced osteoclasts differentiation.

Methods: Nuclear factor-κ B ligand as a receptor activator was used to induce osteoclasts differentiation of murine macrophage RAW264.7 treated with CHO, PI3-kinase inhibitor (LY294002), and Rapamycin (RAPA), respectively. Western blot assay was used to detect the expression of TRAP/ACP5 and the proteins involved in autophagy and the PI3K/AKT/mTOR signaling pathway. In addition, TRAP staining, bone resorption assay, and F-actin immunofluorescence were performed to evaluate the ability of osteoclast formation. Transmission electron microscopy and immunofluorescence were also executed to observed the expression of LC3B, and autophagosome.

Results: When RAW264.7 was treated with 20 μg/mL CHO for 5 consecutive days, It exhibited the optimal osteoclast activity. In addition, CHO could inhibit autophagy and activate the PI3K/AKT/mTOR signaling pathway. Moreover, the effects of CHO on osteoclast differentiation and autophagy could partially be reversed by LY294002 and RAPA.

Conclusion: Therefore, our results demonstrated that CHO could inhibit autophagy during osteoclast differentiation by activating the PI3K/AKT/mTOR signaling pathway. These findings provided important theoretical basis for CHO in bone resorption and formation.