Mechanical injury suppresses autophagy regulators and pharmacologic activation of autophagy results in chondroprotection.
OBJECTIVE
Mechanical injury induces cell death in cartilage and triggers a remodeling process that ultimately can manifest as osteoarthritis. Autophagy is a process for turnover of intracellular organelles and macromolecules that protects cells during stress responses. This study was undertaken to determine changes in and functions of autophagy following mechanical injury to cartilage.


METHODS
Bovine and human cartilage explants were subjected to mechanical impact (40% strain for 500 msec). Cell viability, sulfated glycosaminoglycan (sGAG) release, and changes in the levels of the autophagy markers ULK1, beclin 1, and microtubule-associated protein 1 light chain 3 (LC3) were evaluated. Cartilage explants were treated with the mammalian target of rapamycin complex 1 (mTORC-1) inhibitor and the autophagy inducer rapamycin and tested for protective effects against mechanical injury. Explants were also treated with the cell death inducers nitric oxide and tumor necrosis factor α (TNFα) plus actinomycin D, and the proinflammatory cytokine interleukin-1α (IL-1α).


RESULTS
Mechanical injury induced cell death and loss of sGAG in a time-dependent manner. This was associated with significantly decreased ULK1, beclin 1, and LC3 expression in the cartilage superficial zone (P < 0.05) 48 hours after injury. The levels of LC3-II were increased 24 hours after injury but decreased at 48 and 96 hours. Rapamycin enhanced expression of autophagy regulators and prevented cell death and sGAG loss in mechanically injured explants. Rapamycin also protected against cell death induced by sodium nitroprusside and TNFα plus actinomycin D and prevented sGAG loss induced by IL-1α.


CONCLUSIONS
Our findings indicate that mechanical injury leads to suppression of autophagy, predominantly in the superficial zone where most of the cell death occurs. Pharmacologic inhibition of mTORC-1, at least in part by enhancement of autophagy, prevents cell and matrix damage, suggesting a novel approach for chondroprotection.
The Scripps Research Institute, La Jolla, California 92037, USA.
Beatriz
B
Caramés
Noboru
N
Taniguchi
Daisuke
D
Seino
Francisco J
FJ
Blanco
Darryl
D
D'Lima
Martin
M
Lotz
D000818:Animals; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism; D001343:Autophagy / Q000187:drug effects / Q000502:physiology*; D000071189:Autophagy-Related Protein-1 Homolog; D000071186:Beclin-1; D002358:Cartilage, Articular / Q000187:drug effects / Q000293:injuries* / Q000378:metabolism / Q000473:pathology*; D002417:Cattle; D016923:Cell Death / Q000187:drug effects; D019902:Chondrocytes / Q000187:drug effects / Q000378:metabolism / Q000473:pathology*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D020123:Sirolimus / Q000494:pharmacology
D051017:Apoptosis Regulatory Proteins; C491997:BECN1 protein, human; D000071186:Beclin-1; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D008869:Microtubule-Associated Proteins; D000071189:Autophagy-Related Protein-1 Homolog; D017346:Protein Serine-Threonine Kinases; C484754:ULK1 protein, human; D020123:Sirolimus