PINK1-mediated mitophagy contributes to glucocorticoid-induced cathepsin K production in osteocytes

Jun Yuan; You-Shui Gao; De-Lin Liu; Andrew Chi Pang Tai; Hong Zhou; John M Papadimitriou; Chang-Qing Zhang; Ming-Hao Zheng; Jun-Jie Gao

doi:10.1016/j.jot.2022.11.003

PINK1-mediated mitophagy contributes to glucocorticoid-induced cathepsin K production in osteocytes

J Orthop Translat. 2022 Nov 24:38:229-240. doi: 10.1016/j.jot.2022.11.003. eCollection 2023 Jan.

Authors

Jun Yuan^{1

2}, You-Shui Gao³, De-Lin Liu^{1

2}, Andrew Chi Pang Tai^{1

2}, Hong Zhou⁴, John M Papadimitriou⁵, Chang-Qing Zhang³, Ming-Hao Zheng^{1

2}, Jun-Jie Gao³

Affiliations

¹ Centre for Orthopaedic Translational Research, Medical School, The University of Western Australia, Nedlands, Western Australia, 6009, Australia.
² Perron Institute for Neurological and Translational Science, Nedlands, Western Australia, 6009, Australia.
³ Department of Orthopaedic Surgery, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai, 200233, China.
⁴ Bone Research Program, ANZAC Research Institute, The University of Sydney, Sydney, NSW, Australia.
⁵ Pathwest Laboratories and Faculty of Health and Medical Sciences, The University of Western Australia, Nedlands, WA, 6009, Australia.

Abstract

Background: Glucocorticoid (GC) is one of frequently used anti-inflammatory agents, but its administration is unfortunately accompanied with bone loss. Although sporadic studies indicated that osteocytes are subject to a series of pathological changes under GC stress, including overexpression of cathepsin K, the definite role of osteocytes in GC-induced bone loss remains largely unclear.

Methods: Gene expression of Ctsk and protein levels of cathepsin K were assessed in MLO-Y4 cell lines exposed to dexamethasone (Dex) of different time (0, 12, 24 hours) and dose (0, 10-8 and 10-6 M) courses by RT-qPCR and western blotting, respectively. Confocal imaging and immunostaining were then performed to evaluate the effects of osteocyte-derived cathepsin K on type I collagen in a primary osteocyte ex vivo culture system. MitoTracker Red was used to stain mitochondria for mitochondria morphology assessment and JC-1 assay was employed to evaluate the mitochondria membrane potential in MLO-Y4 cells following Dex treatment. Activation of PINK1-mediated mitophagy was evaluated by immunostaining of the PINK1 protein and CytoID assay. Mdivi-1 was used to inhibit mitophagy and siRNAs were used for the inhibition of Pink1 and Atg5.

Results: GC triggered osteocytes to produce excessive cathepsin K which in turn led to the degradation of type I collagen in the extracellular matrix in a primary osteocyte ex vivo culture system. Meanwhile, GC administration increased mitochondrial fission and membrane depolarization in osteocytes. Further, the activation of PINK1-mediated mitophagy was demonstrated to be responsible for the diminishment of dysfunctional mitochondria in osteocytes. Examination of relationship between mitophagy and cathepsin K production revealed that inhibition of mitophagy via knocking down Pink1 gene abolished the GC-triggered cathepsin K production. Interestingly, GC's activation effect towards cathepsin K via mitophagy was found to be independent on the canonical autophagy as this effect was not impeded when inhibiting the canonical autophagy via Atg5 suppression.

Conclusion: GC-induced PINK1-mediated mitophagy substantially modulates the production of cathepsin K in osteocytes, which could be an underlying mechanism by which osteocytes contribute to the extracellular matrix degradation during bone loss.

The translational potential of this article: Findings of the current study indicate a possible role of osteocyte mitophagy in GC-induced bone loss, which provides a potential therapeutic approach to alleviate GC-induced osteoporosis by targeting PINK1-mediated osteocytic mitophagy.

Keywords: CCCP, Carbonyl cyanide chlorophenylhydrazone; CTSK, cathepsin K; CTX, carboxyterminal telopeptide of type I collagen; Cathepsin K; Drp1, dynamin-related protein 1; GC, glucocorticoid; Glucocorticoid; MTR, MitoTracker Red; Mfn2, mitofusin-2; Mitophagy; Osteocyte; PINK1; PINK1, PTEN-induced putative kinase 1; TFEB, transcription factor EB; TRAP, tartrate-resistant acid phosphatase; ΔѰm, mitochondria membrane potential.