Friction-Induced Mitochondrial Dysregulation Contributes to Joint Deterioration in Prg4 Knockout Mice
Abstract
:1. Introduction
2. Results
2.1. Whole Joint COF of Prg4−/− Mice Following Intra-Articular PRG4 Compared to Sham Injected and Control Mice
2.2. Caspase-3 Activation of Prg4−/− Mice Following Intra-Articular PRG4
2.3. Peroxynitrite (PN) Assay in Prg4 Mutant Mice Femoral Head Cartilage
2.4. Superoxide (MitoSOX) Assay in Prg4 Mutant Mice Synoviocytes
2.5. Caspase-8 Activity in Chondrocytes and Synoviocytes from Prg4 Mutant Mice
2.6. Real Time PCR of mRNA Recovered from Cartilage of Prg4 Mutant Mouse Joints
2.7. Western Blots and Densitometry
2.8. Immunohistochemistry of Nitrosyl-Cys163 Caspase-3 Neoepitope in Cartilage from Prg4−/− Mouse Joints
3. Discussion
4. Materials and Methods
4.1. Prg4 Mutant Mice
4.2. Tribosupplementation In Vivo of Prg4−/− Mouse Knees
4.3. Whole Joint Coefficient of Friction (COF) Measurement
4.4. Tissue Harvest for Cells, Protein and mRNA
4.5. Quantitative Real-Time Polymerase Chain Reaction
4.6. Measurement of ROS and Peroxynitrite
4.7. Measurement of MitoSOX
4.8. Imaging of Synoviocytes Stained with MitoSOX
4.9. Caspase-8 Activity Quantification
4.10. Tissue Processing and Hematoxylin and Eosin Staining
4.11. Immunodetection of Caspase Proteases and Signal Transduction Pathways
4.12. Blot Imaging and Densitometric Analysis
4.13. Immunohistochemistry for Activated Caspase-3 and Nitrosylated Cys163 Caspase-3
4.14. Quantification of Active Caspase-3 and Nitrosylated Cys163 Caspase-3
4.15. Statistical Analyses
5. Conclusions
Acknowledgments
Author Contributions
Conflicts of Interest
Abbreviations
CACP | Camptodactyl-arthropathy-coxa vara-pericarditis |
ROS | Reactive oxygen species |
COF | Coefficient of friction |
OA | Osteoarthritis |
IA | Intra-articular |
HSL | Human synoviocyte lubricin (PRG4) |
PN | Peroxynitrite |
O−2 | Superoxide |
NO | Nitric oxide |
mRNA | Messenger RNA |
ONOO− | Peroxynitrite |
−OH | Hydroxyl radical |
DAPI | 4,6-Diamidino-2-phenylindole, dihydrochloride |
EDTA | Ethylenediaminetetraacetic acid |
EGTA | Ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid |
PMSF | Phenylmethylsulfonyl fluoride |
H&E | Hematoxylin and eosin |
Prg4 | Murine lubricin gene |
PRG4 | Purified lubricin protein (Proteoglycan 4) |
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Waller, K.A.; Zhang, L.X.; Jay, G.D. Friction-Induced Mitochondrial Dysregulation Contributes to Joint Deterioration in Prg4 Knockout Mice. Int. J. Mol. Sci. 2017, 18, 1252. https://doi.org/10.3390/ijms18061252
Waller KA, Zhang LX, Jay GD. Friction-Induced Mitochondrial Dysregulation Contributes to Joint Deterioration in Prg4 Knockout Mice. International Journal of Molecular Sciences. 2017; 18(6):1252. https://doi.org/10.3390/ijms18061252
Chicago/Turabian StyleWaller, Kimberly A., Ling X. Zhang, and Gregory D. Jay. 2017. "Friction-Induced Mitochondrial Dysregulation Contributes to Joint Deterioration in Prg4 Knockout Mice" International Journal of Molecular Sciences 18, no. 6: 1252. https://doi.org/10.3390/ijms18061252