Dataset Information

Injurious Loading of Articular Cartilage Compromises Chondrocyte Respiratory Function.

ABSTRACT: To determine whether repeatedly overloading healthy cartilage disrupts mitochondrial function in a manner similar to that associated with osteoarthritis (OA) pathogenesis.We exposed normal articular cartilage on bovine osteochondral explants to 1 day or 7 consecutive days of cyclic axial compression (0.25 MPa or 1.0 MPa at 0.5 Hz for 3 hours) and evaluated the effects on chondrocyte viability, ATP concentration, reactive oxygen species (ROS) production, indicators of oxidative stress, respiration, and mitochondrial membrane potential.Neither 0.25 MPa nor 1.0 MPa of cyclic compression caused extensive chondrocyte death, macroscopic tissue damage, or overt changes in stress-strain behavior. After 1 day of loading, differences in respiratory activities between the 0.25 MPa and 1.0 MPa groups were minimal; however, after 7 days of loading, respiratory activity and ATP levels were suppressed in the 1.0 MPa group relative to the 0.25 MPa group, an effect prevented by pretreatment with 10 mM N-acetylcysteine. These changes were accompanied by increased proton leakage and decreased mitochondrial membrane potential, as well as by increased ROS formation, as indicated by dihydroethidium staining and glutathione oxidation.Repeated overloading leads to chondrocyte oxidant-dependent mitochondrial dysfunction. This mitochondrial dysfunction may contribute to destabilization of cartilage during various stages of OA in distinct ways by disrupting chondrocyte anabolic responses to mechanical stimuli.

SUBMITTER: Coleman MC

PROVIDER: S-EPMC4767543 | biostudies-literature | 2016 Mar

REPOSITORIES: biostudies-literature

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Injurious Loading of Articular Cartilage Compromises Chondrocyte Respiratory Function.

Coleman Mitchell C MC Ramakrishnan Prem S PS Brouillette Marc J MJ Martin James A JA

Arthritis & rheumatology (Hoboken, N.J.) 20160301 3

<h4>Objective</h4>To determine whether repeatedly overloading healthy cartilage disrupts mitochondrial function in a manner similar to that associated with osteoarthritis (OA) pathogenesis.<h4>Methods</h4>We exposed normal articular cartilage on bovine osteochondral explants to 1 day or 7 consecutive days of cyclic axial compression (0.25 MPa or 1.0 MPa at 0.5 Hz for 3 hours) and evaluated the effects on chondrocyte viability, ATP concentration, reactive oxygen species (ROS) production, indicato ...[more]

PMID: 26473613

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Project description:Epigenome alteration in chondrocytes correlates with osteoarthritis (OA) development. H3K27me3 demethylase UTX regulates tissue homeostasis and deterioration, while its role was not yet studied in articulating joint tissue in situ. We now uncovered that increased UTX and H3K27me3 expression in articular chondrocytes positively correlated with human knee OA. Forced UTX expression upregulated the H3K27me3 enrichment at transcription factor Sox9 promoter, inhibiting key extracellular matrix molecules collagen II, aggrecan, and glycosaminoglycan in articular chondrocytes. Utx overexpression in knee joints aggravated the signs of OA, including articular cartilage damage, synovitis, osteophyte formation, and subchondral bone loss in mice. Chondrocyte-specific Utx knockout mice developed thicker articular cartilage than wild-type mice and showed few gonarthrotic symptoms during destabilized medial meniscus- and collagenase-induced joint injury. In vitro, Utx loss changed H3K27me3-binding epigenomic landscapes, which contributed to mitochondrial activity, cellular senescence, and cartilage development. Insulin-like growth factor 2 (Igf2) and polycomb repressive complex 2 (PRC2) core components Eed and Suz12 were, among others, functional target genes of Utx. Specifically, Utx deletion promoted Tfam transcription, mitochondrial respiration, ATP production and Igf2 transcription but inhibited Eed and Suz12 expression. Igf2 blockade or forced Eed or Suz12 expression increased H3K27 trimethylation and H3K27me3 enrichment at Sox9 promoter, compromising Utx loss-induced extracellular matrix overproduction. Taken together, UTX repressed articular chondrocytic activity, accelerating cartilage loss during OA. Utx loss promoted cartilage integrity through epigenetic stimulation of mitochondrial biogenesis and Igf2 transcription. This study highlighted a novel noncanonical role of Utx, in concert with PRC2 core components, in controlling H3K27 trimethylation and articular chondrocyte anabolism and OA development.

Project description:The degradation of proteoglycan was examined in cultured slices of pig articular cartilage. Pig leucocyte catabolin (10 ng/ml) was used to stimulate the chondrocytes and induce a 4-fold increase in the rate of proteoglycan loss from the matrix for 4 days. Material in the medium of both control and depleted cultures was mostly a degradation product of the aggregating proteoglycan. It was recovered as a very large molecule slightly smaller than the monomers extracted with 4M-guanidinium chloride and lacked a functional hyaluronate binding region. The size and charge were consistent with a very limited cleavage or conformational change of the core protein near the hyaluronate binding region releasing the C-terminal portion of the molecule intact from the aggregate. The 'clipped' monomer diffuses very rapidly through the matrix into the medium. The amount of proteoglycan extracted with 4M-guanidinium chloride decreased during culture from both the controls and depleted cartilage, and the average size of the molecules initially remained the same. However, the proportion of molecules with a smaller average size increased with time and was predominant in explants that had lost more than 70% of their proteoglycan. All of this material was able to form aggregates when mixed with hyaluronate, and glycosaminoglycans were the same size and charge as normal, indicating either that the core protein had been cleaved in many places or that larger molecules were preferentially released. A large proportion of the easily extracted and non-extractable proteoglycan remained in the partially depleted cartilage and the molecules were the same size and charge as those found in the controls. There was no evidence of detectable glycosidase activity and only very limited sulphatase activity. A similar rate of breakdown and final distribution pattern was found for newly synthesized proteoglycan. Increased amounts of latent neutral metalloproteinases and acid proteinase activities were present in the medium of depleted cartilage. These were not thought to be involved in the breakdown of proteoglycan. Increased release of proteoglycan ceased within 24h of removal of the catabolin, indicating that the effect was reversible and persisted only while the stimulus was present.

Project description:Cartilage is essential throughout vertebrate life. It starts developing in embryos when osteochondroprogenitor cells commit to chondrogenesis, activate a pancartilaginous program to form cartilaginous skeletal primordia, and also embrace a growth-plate program to drive skeletal growth or an articular program to build permanent joint cartilage. Various forms of cartilage malformation and degeneration diseases afflict humans, but underlying mechanisms are still incompletely understood and treatment options suboptimal. The transcription factor SOX9 is required for embryonic chondrogenesis, but its postnatal roles remain unclear, despite evidence that it is down-regulated in osteoarthritis and heterozygously inactivated in campomelic dysplasia, a severe skeletal dysplasia characterized postnatally by small stature and kyphoscoliosis. Using conditional knockout mice and high-throughput sequencing assays, we show here that SOX9 is required postnatally to prevent growth-plate closure and preosteoarthritic deterioration of articular cartilage. Its deficiency prompts growth-plate chondrocytes at all stages to swiftly reach a terminal/dedifferentiated stage marked by expression of chondrocyte-specific (Mgp) and progenitor-specific (Nt5e and Sox4) genes. Up-regulation of osteogenic genes (Runx2, Sp7, and Postn) and overt osteoblastogenesis quickly ensue. SOX9 deficiency does not perturb the articular program, except in load-bearing regions, where it also provokes chondrocyte-to-osteoblast conversion via a progenitor stage. Pathway analyses support roles for SOX9 in controlling TGFβ and BMP signaling activities during this cell lineage transition. Altogether, these findings deepen our current understanding of the cellular and molecular mechanisms that specifically ensure lifelong growth-plate and articular cartilage vigor by identifying osteogenic plasticity of growth-plate and articular chondrocytes and a SOX9-countered chondrocyte dedifferentiation/osteoblast redifferentiation process.

Dataset Information

Injurious Loading of Articular Cartilage Compromises Chondrocyte Respiratory Function.

Publications

Injurious Loading of Articular Cartilage Compromises Chondrocyte Respiratory Function.

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