Mitochondrial Dysregulation of Osteoarthritic Human Articular Chondrocytes Analyzed by Proteomics

Abstract

Mitochondria are involved in many cellular processes; mitochondrial dysfunctions have been associated with apoptosis, aging, and a number of pathological conditions, including osteoarthritis (OA). Mitochondrial proteins are attractive targets for the study of metabolism of the chondrocyte, the unique cell type present in mature cartilage, and its role in tissue degradation. Using a proteomics approach based on two-dimensional DIGE and MALDI-TOF/ TOF mass spectrometric identification of mitochondriaenriched protein fractions from human articular chondrocytes, we analyzed mitochondrial protein changes that are characteristic of OA chondrocytes. A total of 73 protein forms were unambiguously identified as significantly altered in OA; 23 of them have been previously described as mitochondrial. An extensive statistical and cluster analysis of the data revealed a mitochondrial protein profile characteristic for OA. This pattern includes alterations in energy production, maintenance of mitochondrial membrane integrity, and free radical detoxification. Real time PCR, Western blot, and immunohistofluorescence assays confirmed a significant decrease of the major mitochondrial antioxidant protein manganese-superoxide dismutase (SOD2) in the superficial layer of OA cartilage. As possible outputs for this antioxidant deficiency, we found an increase of intracellular reactive oxygen species generation in OA chondrocytes and also verified an OA-dependent increase in the mitochondrial tumor necrosis factor-␣ receptor-associated protein 1 (TRAP1), a chaperone with a reported reactive oxygen species antagonist role. Our results describe the differences between the mitochondrial protein profiles of normal and OA chondrocytes, demonstrating that mitochondrial dysregulation occurs in cartilage cells during OA and highlighting redox imbalance as a key factor in OA The mitochondrion is one of the most complex and important organelles found in eukaryotic cells and carries out a wide variety of biochemical processes. Mitochondria are critical subcellular organelles responsible for energy production through the coupling of respiration to the generation of ATP. Mitochondria consist of four components: an outer membrane, an intermembrane space, an inner membrane, and a matrix. These components all function in concert to convert pyruvate and fatty acids to acetyl CoA, which is metabolized by the citric acid cycle to produce NADH. High energy electrons from NADH are then passed to oxygen by means of the respiratory chain in the inner membrane, producing ATP by a chemiosmotic process. Transcription and translation take place in mitochondria, which also actively import proteins and metabolites from the cytosol, influence programmed cell death, and respond to cellular signals such as oxidative stress (1). In addition to their central role in energy metabolism, mitochondria are involved in many cellular processes; mitochondrial dysfunctions have been associated with apoptosis, aging, and a number of pathological conditions, including Parkinson d...

Oxidative Stress Inhibits Insulin-like Growth Factor-I Induction of Chondrocyte Proteoglycan Synthesis through Differential Regulation of Phosphatidylinositol 3-Kinase-Akt and MEK-ERK MAPK Signaling Pathways

“…The hypothesis that this imbalance in signaling may result from the excessive levels of ROS that have been observed in OA cartilage (14,17,18) was supported by the findings that induction of oxidative stress in normal chondrocytes with tBHP reproduced the changes seen in OA cells, whereas treatment of OA cells with anti-oxidants improved the balance of Akt to ERK phosphorylation and improved the anabolic response of the cells to IGF-I.…”

“…Oxidative Stress Inhibits IGF-I-mediated IRS-1-Akt Signaling but Activates MEK-ERK-We hypothesized that increased levels of endogenous ROS due to oxidative stress, previously shown to be present in OA cells (11,14,17,18), might act to inhibit IGF-I signaling. To test this, we induced oxidative stress in normal human chondrocytes by incubating cultures for 30 min with 250 M tBHP.…”

Ascorbic acid provides protection for human chondrocytes against oxidative stress

“…Reactive oxygen species (ROS), another major determinant of stress, including hydrogen peroxide (H 2 O 2 ), hypochlorite ion, hydroxyl radical and superoxide anion, are involved in normal intracellular transduction and degenerative cellular processes. Increased ROS production is considered to be an important cause of chondrocyte dysfunction and articular cartilage degradation, which leads to the pathogenesis of OA and the aging of cartilage (7)(8)(9)(10). Chondrocytes and cartilage tissues exhibit efficient protective strategies to minimize damage induced by increased ROS production, including H 2 O 2 (7,11).…”

Modulation of inflammation and oxidative stress in canine chondrocytes

“…Its main function is to protect cells and the ECM against the effects of superoxide anion (18). SOD2 has been shown to be down-regulated in OA while SOD3 is decreased in human OA and a mouse model of OA (16,22,23). Recently Scott et al found that all three SOD family members (SOD1, SOD2, and SOD3) were down regulated in diseased cartilage.…”

“…MCP-1 recruits monocytes into areas of high inflammation, but the exact signaling pathways still remain unclear. There is evidence to suggest the involvement of prostaglandin E2 (PGE 2 ) in the attraction of monocytes to the site of inflammation (22). It is produced by a variety of cell types either at the time of or after the induction by oxidative stress, inflammation (cytokines) growth factors (17), and tumor necrosis factor alpha (TNF-α), which have been shown to upregulate MCP-1 expression in sensory neurons (23)(24)(25).…”