Autophagy Prevents Osteocyte Cell Death under Hypoxic Conditions

Hypoxia occurs under important clinical conditions such as cancers, heart disease, and ischemia. However, the relationship between hypoxia and autophagy in osteocytes is still unclear. The objective of the present study was to uncover the regulatory mechanisms that prevent regulated cell death, such as apoptosis, necrosis, and autophagy, under hypoxia. MLO-Y4 cells, a mouse osteocyte cell line, were exposed to various O₂ partial pressures (PO₂). Subsequently, the cells underwent apoptosis, autophagy, autophagic cell death, and/or necrosis, and thereby we designated PO₂ = 2% as a representative hypoxic condition. Immunofluorescence staining showed an increase of LC3 and a decrease of p62 in MLO-Y4 cells exposed to hypoxia, indicating the induction of autophagy. We then hypothesized that β-estradiol (E2) and vitamin D play an important role in apoptosis and autophagy of osteocytes under hypoxia. 1,25α-dihydroxyvitamin D3 (VitD) protected MLO-Y4 cells from cell death and induced autophagy. However, E2 showed little effect. Finally, Western blotting for phosphorylated mTOR and Akt was carried out in order to investigate the altered autophagy signaling pathways affected by the addition of VitD and E2. However, neither E2 nor VitD were capable of recovering the decreased phosphorylation of those factors. Our results indicated that the effects of VitD on autophagy under hypoxia were dependent on the Akt and mTOR pathways. Thus, the results of the present study showed that VitD suppresses osteocyte cell death in an mTOR pathway-dependent manner in hypoxic conditions. This suggests the potential of VitD as a therapeutic intervention for diseases in which the cell death of osteocytes mainly occurs via hypoxia.