Chunyan Jiang1,2,3,4,5, Yan Wang1,2,3,4, Mengqi Zhang1,2,3,4, Jin Xu6,7,8,9. 1. Department of Endocrinology, Shandong Provincial Hospital, Cheeloo College of Medicine, Shandong University, Jinan, Shandong, China. 2. Department of Endocrinology, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, Shandong, China. 3. Shandong Clinical Medical Center of Endocrinology and Metabolism, Jinan, Shandong, China. 4. Institute of Endocrinology and Metabolism, Shandong Academy of Clinical Medicine, Jinan, 250021, Shandong, China. 5. Department of Endocrinology, People's Hospital of Linyi, Linyi, Shandong, China. 6. Department of Endocrinology, Shandong Provincial Hospital, Cheeloo College of Medicine, Shandong University, Jinan, Shandong, China. slyyxujin@163.com. 7. Department of Endocrinology, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, Shandong, China. slyyxujin@163.com. 8. Shandong Clinical Medical Center of Endocrinology and Metabolism, Jinan, Shandong, China. slyyxujin@163.com. 9. Institute of Endocrinology and Metabolism, Shandong Academy of Clinical Medicine, Jinan, 250021, Shandong, China. slyyxujin@163.com.
Abstract
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.
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.
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