Background/Purpose: Mitochondria not only ‘power’ the cell, but are also essential for maintaining physiological levels of prooxidants (ROS) which is critical for homeostasis. Damaged and dysfunctional mitochondria are hallmarks of aging-related diseases including osteoarthritis (OA) but how they contribute to the pathogenesis of OA is not clear. Here we investigated the mechanism of mitochondrial dysfunction-induced expression of IL-6 and MMP-3, -9 and -13 in human normal and OA chondrocytes.

Methods:

OA cartilage samples were obtained from donors who underwent total joint replacement surgery at Crystal Clinic, Akron, OH. Normal knee cartilage samples were from post mortem donors via NDRI. Mitochondrial dysfunction in chondrocytes was induced by treatment with IL-1β or CCCP (Carbonyl cyanide m-chlorophenyl hydrazone). JC-1 staining of chondrocytes was used to measure the loss of mitochondrial membrane potential. Mitochondrial ROS levels were analyzed by MitoSOX probe. Chondrocytes treated to induce mitochondrial damage were harvested and (1) RNA was prepared for measuring gene expression by TaqMan assays; and (2) total lysate was prepared for protein expression analysis by immunoblotting for OA signature genes. Culture supernatants were used to determine the levels of secreted IL-6 and MMP-13 protein by ELISA. ATP levels in normal and OA chondrocytes treated with CCCP or IL-1β were measured by Mitochondrial ToxGlo Assay. Activation of p38-MAPK and cFos was determined by Western immunoblotting using antibodies validated for the total and phosphorylated proteins. Activation of the NF-kB and AP1 signaling pathways was analyzed by reporter assays, immunoblotting for IkBa degradation and nuclear translocation of cFos using immunofluorescence staining. Nucleofection was used to transfect chondrocytes.

Results:

IL-1β or CCCP treatment caused a significant loss of mitochondrial membrane potential and production of mitochondrial ROS in chondrocytes as measured by JC-1 and MitoSOX staining respectively. OA chondrocytes compared to normal chondrocytes had significantly low ATP levels and treatment with IL-1β or CCCP resulted in reduced production of ATP in normal chondrocytes. Both normal and OA chondrocytes with damaged mitochondria showed increased mRNA expression of IL-6 and MMP-3, -9 and -13 and the phosphorylation of p38MAPK, cFos and nuclear translocation of cFos. In normal or OA chondrocytes treated with CCCP or H₂O₂, activation of NFkB as determined by immunoblotting for Ikβα degradation, was not observed. Also, in normal or OA chondrocytes transfected with either AP1 or NFkB activity reporter vectors and then treated with CCCP, activation of AP1 but not of NF-kB was observed. Treatment of chondrocytes with damaged/dysfunctional mitochondria with mitochondrial ROS inhibitor Mito-TEMPO, inhibited the AP1 activation and suppressed the expression of IL-6, MMP-3 -9 and -13 mRNA and protein.

Conclusion: Here we show for the first time that mitochondrial damage/dysfunction contributes to the pathogenesis of OA via p38 MAPK/AP1 activation and not through the activation of NF-kB pathway. This knowledge may help in designing novel therapies for the management of OA.

Supported by USPHS/NIH grants

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/mitochondrial-ros-activate-the-p38-mapkap1-pathway-to-induce-the-expression-of-matrix-metalloproteases-and-il-6-in-human-chondrocytes/