PINK1/Parkin-mediated mitophagy inhibits osteoblast apoptosis induced by advanced oxidation protein products

Abstract

Osteoblast apoptosis plays an important role in age-related bone loss and osteoporosis. Our previous study revealed that advanced oxidation protein products (AOPPs) could induce nicotinamide adenine dinucleotide phosphate oxidase (NOX)-derived reactive oxygen species (ROS) production, cause mitochondrial membrane potential (ΔΨm) depolarization, trigger the mitochondria-dependent intrinsic apoptosis pathway, and lead to osteoblast apoptosis and ultimately osteopenia and bone microstructural destruction. In this study, we found that AOPPs also induced mitochondrial ROS (mtROS) generation in osteoblastic MC3T3-E1 cells, which was closely related to NOX-derived ROS, and aggravated the oxidative stress condition, thereby further promoting apoptosis. Removing excessive ROS and damaged mitochondria is the key factor in reversing AOPP-induced apoptosis. Here, by in vitro studies, we showed that rapamycin further activated PINK1/Parkin-mediated mitophagy in AOPP-stimulated MC3T3-E1 cells and significantly alleviated AOPP-induced cell apoptosis by eliminating ROS and damaged mitochondria. Our in vivo studies revealed that PINK1/Parkin-mediated mitophagy could decrease the plasma AOPP concentration and inhibit AOPP-induced osteoblast apoptosis, thus ameliorating AOPP accumulation-related bone loss, bone microstructural destruction and bone mineral density (BMD) loss. Together, our study indicated that therapeutic strategies aimed at upregulating osteoblast mitophagy and preserving mitochondrial function might have potential for treating age-related osteoporosis.

Fig. 1. AOPPs induced mtROS production in MC3T3-E1 cells.

A MitoSOX results showed that AOPPs (200 μg/mL) significantly increased mtROS levels in MC3T3-E1 cells. Pretreatment with apocynin, MitoTEMPO or rapamycin significantly reduced AOPP-induced mtROS production. Apocynin or MitoTEMPO alone also decreased mtROS levels in MC3T3-E1 cells, while rapamycin alone had no significant effect on mtROS levels. However, 3-MA not only elevated the mtROS level when administered alone but also further increased AOPP-induced mtROS generation (n = 6 for each group). B DCFH-DA results showed that the intracellular ROS production induced by 200 μg/mL AOPPs could be decreased by pretreatment with apocynin, MitoTEMPO and rapamycin. 3-MA further increased the intracellular ROS generation induced by AOPPs. Apocynin, MitoTEMPO, rapamycin and 3-MA had no significant effect on intracellular ROS production compared to the control group (n = 6 for each group). Data are presented as the mean ± SD. *P < 0.05 versus control, ^#P < 0.05 versus AOPPs.

Fig. 2. AOPPs induced mitophagy in MCET3-E1 cells.

A–G AOPP treatment significantly increased PINK1, Parkin, LC3A/B-II, Atg12-Atg5, and Beclin-1 expression while decreasing p62 expression in a concentration-dependent manner. H–K AOPP treatment significantly increased the phosphorylation of AMPK and ULK1 but inhibited the phosphorylation of mTOR in a concentration-dependent manner. L, M The expression level of LC3A/B-II in AOPPs+BafA1 was significantly higher than AOPPs group. N Representative confocal microscope images using LysoTracker Red revealed that AOPPs induced lysosome generation in MC3T3-E1 cells in a concentration-dependent manner (scale bar = 20 μm). O Representative confocal microscope images showed that AOPPs induced TOM20 degradation in a concentration-dependent manner (scale bar = 20 μm). P Representative confocal microscope images showed that AOPPs induced HSP60 degradation in a concentration-dependent manner (scale bar = 50 μm). Q Representative TEM images showing autophagosomes (white arrow) and mitophagy (red arrow, M: mitochondria) in MC3T3-E1 cells after AOPP treatment (scale bar = 500 nm). All experiments were repeated for at least three times. Data are presented as the mean ± SD. *P < 0.05 versus control.

Fig. 3. PINK1/Parkin-mediated mitophagy inhibited AOPP-induced MC3T3-E1 cell apoptosis.

A–D AOPP treatment significantly increased PINK1, Parkin, and LC3A/B-II expression, and this tendency was further upregulated by rapamycin or inhibited by 3-MA. E–H AOPPs increased the phosphorylation of AMPK and ULK1 while decreasing mTOR phosphorylation. Rapamycin decreased the phosphorylation of mTOR but had no significant effect on AMPK or ULK1 phosphorylation. Rapamycin combined with AOPPs further decreased the phosphorylation of mTOR and increased the phosphorylation ULK1, but had no significant effect on AMPK phosphorylation. 3-MA significantly decreased the AOPP-induced phosphorylation of AMPK and ULK1. I The MitoTracker Green/LysoTracker Red fluorescence results showed that AOPP stimulation significantly increased mitophagy (white arrow), and this tendency could be further enhanced by rapamycin or inhibited by 3-MA (scale bar = 10 μm). J Co-localization of mitochondrial protein COX IV and LAMP1 by immunofluorescence staining showed AOPP stimulation significantly increased mitophagy, and this tendency could be further enhanced by rapamycin or inhibited by 3-MA (scale bar = 10 μm). K The JC-1 results showed that rapamycin could significantly reverse the AOPP-induced ΔΨm decrease, but 3-MA further exacerbated the damaging effect of AOPPs on mitochondria (scale bar = 100 μm). L, M Flow cytometry results showed that AOPP-induced MC3T3-E1 cell apoptosis could be inhibited by rapamycin but further exacerbated by 3-MA. Rapamycin or 3-MA alone had no significant effect on the cell apoptotic rate. N–R The AOPP-induced expression of cleaved PARP, BAX, cleaved Caspase-3 and cytochrome c was significantly inhibited by rapamycin but further upregulated by 3-MA. However, rapamycin or 3-MA alone had no significant effect on apoptotic protein expression compared with the control group. All experiments were repeated for at least three times. Data are presented as the mean ± SD. *P < 0.05 versus control. ^#P < 0.05 versus the AOPPs group. Clvd PARP, cleaved PARP; Clvd Cas-3 cleaved Caspase-3; Cyt-C cytochrome c.

Fig. 4. PINK1 knockdown aggravated AOPP-induced osteoblast apoptosis.

A, B PINK1 knockdown by shRNA had no significant effect on the expression of cleaved PARP, BAX, cleaved Caspase-3, or cytochrome c in MC3T3-E1 cells. C, D Flow cytometry results showed that PINK1 knockdown further increased the cell apoptotic rate when stimulated with AOPPs, and the application of rapamycin did not reverse this trend. E–I Rapamycin failed to inhibit the upregulation of cleaved PARP, BAX and cytochrome c in AOPP-stimulated shPINK1 MC3T3-E1 cells. All experiments were repeated for at least three times. Data are presented as the mean ± SD. *^#†P < 0.05 between connected groups. Clvd PARP cleaved PARP; Clvd Cas-3 cleaved Caspase-3; Cyt-C cytochrome c.

Fig. 5. Plasma AOPP and B-ALP concentrations in each group.

A The intraperitoneal injection of AOPPs daily for 16 weeks markedly increased the plasma AOPP level, while rapamycin intervention significantly inhibited this trend. B The intraperitoneal injection of AOPPs daily for 16 weeks markedly decreased the plasma B-ALP level, while rapamycin intervention significantly inhibited this trend. Data are presented as the mean ± SD, *P < 0.05 versus control, ^#P < 0.05 versus AOPPs, n = 8.

Fig. 6. Rapamycin further upregulated PINK1/Parkin-mediated mitophagy in AOPP-stimulated mice.

A, B Western blot analysis showed that AOPP accumulation upregulated the expression of PINK1, Parkin, LC3A/B-II and Atg12-Atg5 in tibial bone tissues, which was further upregulated by additional rapamycin treatment. C, D Immunofluorescent staining results showed that both AOPPs and rapamycin could increase the expression of PINK1 and Parkin in osteoblasts, while the highest expression level was observed in the AOPPs+Rapamycin group (scale bar = 50 μm). E Representative TEM images showing mitophagy (marked as MP) in the AOPPs, AOPPs+Rapamycin, and Rapamycin groups (scale bar = 500 nm). Data are presented as the mean ± SD, *P < 0.05 versus control, ^#P < 0.05 versus AOPPs, n = 8.

Fig. 7. Mitophagy inhibited AOPP-induced apoptosis of osteoblast in mice.

A, B Western blot results of the tibia bone tissue showed that rapamycin significantly decreased the AOPP-induced upregulation of cleaved PARP, BAX, cleaved Caspase-3, and cytochrome c. C TUNEL assay results showed that rapamycin significantly inhibited AOPP-induced osteoblast apoptosis in vivo (scale bar = 50 μm). D Immunohistochemical results showed that rapamycin inhibited the AOPP-induced expression of BAX and cleaved Caspase-3 in osteoblasts in vivo (scale bar = 50 μm). Data are presented as the mean ± SD, *P < 0.05 versus control, ^#P < 0.05 versus AOPPs, n = 8. Clvd PARP cleaved PARP; Clvd Cas-3 cleaved Caspase-3; Cyt-C cytochrome c.

Fig. 8. Mitophagy ameliorated osteopenia and bone microstructural destruction induced by AOPP accumulation.

A–G Micro-CT evaluation revealed that chronic AOPP loading significantly decreased BV/TV, Tb. N, and Tb. Th, while increasing Tb. Sp and SMI in the proximal tibias of mice. This finding was further confirmed by representative three-dimensional reconstructions (Scale bar = 500 μm). In addition, we also found that AOPP stimulation decreased the bone mineral density (BMD) in the trabeculae of the proximal tibias. Notably, rapamycin alleviated the deterioration of bone microstructure and the decrease in BMD caused by AOPP treatment. Data are presented as the mean ± SD, *P < 0.05 versus control, ^#P < 0.05 versus AOPPs, n = 8.