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

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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.

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Abbreviations

CHO:

Cholesterol

OPG:

Osteoprotegerin

NFATc1:

Nuclear factor of activated T-cells, c1

CTSK:

Cathepsin K

RANKL:

Receptor activator of nuclear factor kappa-B ligand

Acp5:

Acid phosphatase 5

RAPA:

Rapamycin

PI3K:

Phosphatidylinositol 3-kinase

mTOR:

Mammalian target of rapamycin

BMD:

Bone mineral density

TRAP staining:

Tartrate-resistant acid phosphatase

AKT:

Protein kinase B

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Acknowledgements

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Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

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Authors and Affiliations

  1. Department of Endocrinology, Shandong Provincial Hospital, Cheeloo College of Medicine, Shandong University, Jinan, Shandong, China

    Chunyan Jiang, Yan Wang, Mengqi Zhang & Jin Xu

  2. Department of Endocrinology, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, Shandong, China

    Chunyan Jiang, Yan Wang, Mengqi Zhang & Jin Xu

  3. Shandong Clinical Medical Center of Endocrinology and Metabolism, Jinan, Shandong, China

    Chunyan Jiang, Yan Wang, Mengqi Zhang & Jin Xu

  4. Institute of Endocrinology and Metabolism, Shandong Academy of Clinical Medicine, Jinan, 250021, Shandong, China

    Chunyan Jiang, Yan Wang, Mengqi Zhang & Jin Xu

  5. Department of Endocrinology, People’s Hospital of Linyi, Linyi, Shandong, China

    Chunyan Jiang

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  1. Chunyan Jiang
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  2. Yan Wang
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Contributions

JX conceived and designed the experiments. CJ performed the experiments and YW and MZ analyzed the data. JX and CJ wrote the paper. All authors read and approved the final manuscript.

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Correspondence to Jin Xu.

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Chunyan Jiang, Yan Wang, Mengqi Zhang, Jin Xu declare that there are no conflicts of interest.

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Jiang, C., Wang, Y., Zhang, M. et al. Cholesterol inhibits autophagy in RANKL-induced osteoclast differentiation through activating the PI3K/AKT/mTOR signaling pathway. Mol Biol Rep 49, 9217–9229 (2022). https://doi.org/10.1007/s11033-022-07747-w

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