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, 11 (1), 42

P53 and Parkin Co-Regulate Mitophagy in Bone Marrow Mesenchymal Stem Cells to Promote the Repair of Early Steroid-Induced Osteonecrosis of the Femoral Head

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P53 and Parkin Co-Regulate Mitophagy in Bone Marrow Mesenchymal Stem Cells to Promote the Repair of Early Steroid-Induced Osteonecrosis of the Femoral Head

Fei Zhang et al. Cell Death Dis.

Abstract

Survival and stemness of bone marrow mesenchymal stem cells (BMSCs) in osteonecrotic areas are especially important in the treatment of early steroid-induced osteonecrosis of the femoral head (ONFH). We had previously used BMSCs to repair early steroid-induced ONFH, but the transplanted BMSCs underwent a great deal of stress-induced apoptosis and aging in the oxidative-stress (OS) microenvironment of the femoral-head necrotic area, which limited their efficacy. Our subsequent studies have shown that under OS, massive accumulation of damaged mitochondria in cells is an important factor leading to stress-induced apoptosis and senescence of BMSCs. The main reason for this accumulation is that OS leads to upregulation of protein 53 (P53), which inhibits mitochondrial translocation of Parkin and activation of Parkin's E3 ubiquitin ligase, which decreases the level of mitophagy and leads to failure of cells to effectively remove damaged mitochondria. However, P53 downregulation can effectively reverse this process. Therefore, we upregulated Parkin and downregulated P53 in BMSCs. We found that this significantly enhanced mitophagy in BMSCs, decreased the accumulation of damaged mitochondria in cells, effectively resisted stress-induced BMSCs apoptosis and senescence, and improved the effect of BMSCs transplantation on early steroid-induced ONFH.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1. Osteogenesis of BMSCs in vivo.
a Magnetic resonance imaging (MRI) examination of the ONFH model. b H&E staining of the ONFH model. Lipopolysaccharide (LPS) and methylprednisolone were used to build the model; saline was used as control. c Observation of XACB and XACB/BMSCs via scanning electron microscopy (SEM). d BMSCs repair of ONFH. Control was treated with lesion debridement (LD) without BMSCs implantation. e H&E and Masson staining to detect new-bone formation and maturation. fi Western blot analysis of OPG, OCN, and Runx2 expression in osteonecrosis (n = 3). In (gi), data are presented as means ± standard deviation (SD). Statistical significances were calculated by Student’s t test. BMSCs = bone marrow mesenchymal stem cells, ONFH = osteonecrosis of the femoral head, XACB = xenogeneic antigen-extracted cancellous bone, XACB/BMSCs = tissue-engineered bone, OPG = osteoprotegerin, OCN = osteocalcin, Runx2 = runt-related transcription factor 2.
Fig. 2
Fig. 2. OS led to accumulation of damaged mitochondria.
a, b Mitochondrial-membrane potential (MMP) detected by JC-1 (n = 4). c, d MitoTracker Green analysis of mitochondrial content (n = 4). e Quantitative polymerase chain reaction (qPCR) analysis of mitochondrial deoxyribonucleic acid (mtDNA; n = 4). In (b), (d), and (e), data are presented as means ± SD. Statistical significances were calculated by Student’s t test. Data were compared with the control group: *P < 0.05. H2O2 was used to simulate OS; L-DMEM was used as control. OS = oxidative stress; DAPI = 4′,6-diamidino-2-phenylindole; Cytb: cytochrome b; ND1 = mitochondrially encoded 1,4-dihydronicotinamide adenine dinucleotide (NADH):ubiquinone oxidoreductase core subunit 1.
Fig. 3
Fig. 3. Accumulation of damaged mitochondria led to stress-induced apoptosis and senescence.
a, b 2′,7′-Dichlorofluorescin diacetate (DCFH-DA) detection of reactive oxygen species (ROS; n = 4). c, d TUNEL–DAPI detection of deoxyribonucleic acid (DNA) damage and apoptosis (n = 4). e, f Annexin V–FITC and propidium iodide (PI) detection of apoptosis (n = 4). g, h Detection of β-gal activity by β-gal staining (n = 4). ik Western blot analysis of P16 and P21 expression (n = 3). In (b), (d), (f), (h), and (jk), data are presented as means ± SD. Statistical significances were calculated by Student’s t test. Data were compared with the control group: *P < 0.05. H2O2 was used to simulate OS; L-DMEM was used as control. TUNEL = terminal deoxynucleotidyl transferase deoxyuridine-5′-triphosphate nick end labeling; DAPI = 4′,6-diamidino-2-phenylindole; FITC = fluorescein isothiocyanate; P16 = protein 16; P21 = protein 21; β-gal = β-galactosidase.
Fig. 4
Fig. 4. Enhancing mitophagy against stress-induced apoptosis and senescence in BMSCs.
a Observation of fluorescent protein by inverted fluorescence microscopy (n = 4). b qPCR analysis of Parkin mRNA expression (n = 4). c, d Western blot analysis of Parkin expression (n = 4). Non-transfected BMSCs as control. e Western blot analysis of Parkin mitochondrial translocation (n = 3). f Immunocoprecipitation (IP) and immunoblot (IB) analysis of VDAC1 ubiquitination level (n = 3). g Observation of mitophagy by transmission electron microscopy (TEM; n = 4). h, k MitoTracker Green analysis of mitochondrial content (n = 4). i, m Detection of β-gal activity by β-gal staining (n = 4). j, n Annexin V–FITC and PI detection of apoptosis (n = 4). l qPCR analysis of mitochondrial deoxyribonucleic acid (mtDNA; n = 4). o qPCR analysis of P53 mRNA expression (n = 4). p, q Western blot analysis of P53 expression (n = 4). In (p), A = BMSCs, B = H2O2 + BMSCs, C = H2O2 + BMSCs + Lv-EGFP, D = H2O2 + BMSCs + Lv-Parkin. In (b), (d), (ko), and (q), data are presented as means ± SD. Statistical significances were calculated by ANOVA and Student’s t test. In (b) and (d), data were compared with the control and Lv-EGFP groups separately; vs. control and Lv-EGFP: *P < 0.05. In (kn), data were compared with the Lv-EGFP group: *P < 0.05. In (o) and (q), data were compared with the BMSCs group: *P < 0.05. Lv-EGFP = lentiviral vector-encoded green fluorescent protein; Lv-Parkin-EGFP = lentiviral vector-encoded Parkin-enhanced green fluorescent protein; mRNA = messenger ribonucleic acid; Ub = ubiquitin; VDAC1 = voltage-dependent anion-selective channel 1; P53 = protein 53; GAPDH = glyceraldehyde 3-phosphate dehydrogenase.
Fig. 5
Fig. 5. Effects of mitophagy co-regulated by P53 and Parkin on stress-induced apoptosis and aging.
a Observation of fluorescent protein by inverted fluorescence microscopy (n = 4). b, c qPCR analysis of P53 and Parkin mRNA expression (n = 4). df Western blot analysis of P53 and Parkin expression (n = 4). g Observation of mitophagy by TEM (n = 3). h, m MitoTracker Green analysis of mitochondrial content (n = 3). i, n Annexin V–FITC and PI detection of apoptosis (n = 3). j, o Detection of β-gal activity by β-gal staining (n = 3). k, l qPCR analysis of mtDNA (n = 3). In (d), A = BMSCs, B = BMSCs/EGFP, C = BMSCs/shP53, D = BMSCs/Parkin, E = BMSCs/shP53/Parkin. In (b), (c), (e), (f), and (ko), data are presented as means ± SD. Statistical significances were calculated by ANOVA. In (b, c) and (e, f), data were compared with the BMSCs and BMSCs/EGFP groups: *P < 0.05. In (ko), data were compared with the BMSCs and BMSCs/EGFP groups: *P < 0.05; or with the BMSCs/shP53 and BMSCs/Parkin groups: #P < 0.05. shP53 = protein 53 short-hairpin ribonucleic acid.
Fig. 6
Fig. 6. P53 regulated mitophagy via the PINK1–Parkin pathway.
a P53 interacted with Parkin to inhibit Parkin mitochondrial translocation and mitochondrial-membrane protein ubiquitination (n = 3). b Observation of mitophagy by TEM (n = 3). c, d MitoTracker Green analysis of mitochondrial content (n = 3). e, f qPCR analysis of mtDNA (n = 3). In (df), data are presented as means ± SD. Statistical significances were calculated by ANOVA. Data were compared with the H2O2 EGFP and H2O2 Parkin groups: *P < 0.05. Data were compared with the H2O2+ EGFP and H2O2+ Parkin groups: #P < 0.05. PINK1 = phosphatase and tensin homolog (PTEN)-induced putative kinase protein 1.
Fig. 7
Fig. 7. P53 and Parkin co-regulated mitophagy in BMSCs to promote the repair of early steroid-induced ONFH.
a Gross specimens in which we observed repair of osteonecrosis (n = 3). b Masson staining for evaluating new-bone maturation (n = 3). c H&E staining for evaluating new-bone formation (n = 3). dg Western blot analysis of OCN, Runx2, and OPG expression (n = 3). Control was treated with lesion debridement (LD) without transplantation. In (eg), data are presented as means ± SD. Statistical significances were calculated by ANOVA. Data were compared with the control, XACB, and XACB/BMSCs groups: *P < 0.05. Data were compared with the XACB/BMSCs/Parkin and XACB/BMSCs/shP53 groups: #P < 0.05.
Fig. 8
Fig. 8. Hypothetical model of P53 and Parkin co-regulating mitophagy in BMSCs to interfere with cell stress-induced apoptosis and senescence.
Under OS, mitochondrial function is impaired and damaged mitochondria release excessive ROS, which can further damage the mitochondria, creating a vicious circle. Moreover, ROS can upregulate P53 expression via the DDR and mTOR pathways. P53 binds to Parkin and inhibits its mitochondrial translocation, the ubiquitination of mitochondrial outer-membrane proteins was inhibited. Eventually, the level of mitophagy decreased, and a large number of mitochondria accumulated in the cells, resulting in apoptosis and senescence of the cells. DDR = DNA damage response; mTOR = mammalian target of rapamycin.

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References

    1. Peng WX, Wang L. Adenovirus-mediated expression of BMP-2 and BFGF in bone marrow mesenchymal stem cells combined with demineralized bone matrix for repair of femoral head osteonecrosis in beagle dogs. Cell Physiol. Biochem. 2017;43:1648–1662. doi: 10.1159/000484026. - DOI - PubMed
    1. Liao H, et al. Bone mesenchymal stem cells co-expressing VEGF and BMP-6 genes to combat avascular necrosis of the femoral head. Exp. Ther. Med. 2018;15:954–962. - PMC - PubMed
    1. Zhang F, et al. Role of FGF-2 transfected bone marrow mesenchymal stem cells in engineered bone tissue for repair of avascular necrosis of femoral head in rabbits. Cell Physiol. Biochem. 2018;48:773–784. doi: 10.1159/000491906. - DOI - PubMed
    1. Fan L, et al. Transplantation of hypoxia preconditioned bone marrow mesenchymal stem cells enhances angiogenesis and osteogenesis in rabbit femoral head osteonecrosis. Bone. 2015;81:544–553. doi: 10.1016/j.bone.2015.09.005. - DOI - PubMed
    1. Xu J, et al. High density lipoprotein protects mesenchymal stem cells from oxidative stress-induced apoptosis via activation of the PI3K/Akt pathway and suppression of reactive oxygen species. Int. J. Mol. Sci. 2012;13:17104e20. - PMC - PubMed
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