Objective: The mitochondrion is known to be important to chondrocyte survival. This study was undertaken to analyze protein expression profiles in chondrocyte mitochondria that are affected by interleukin-1β (IL-1β).
Methods: Normal human chondrocytes were isolated from knee cartilage obtained at autopsy from subjects with no history of joint disease. Cells were incubated for 48 hours with or without IL-1β (5 ng/ml). Proteins were separated by 2-dimensional electrophoresis and stained with Sypro Ruby, Coomassie brilliant blue, or silver. Qualitative and quantitative analyses were carried out using PDQuest software. Proteins were identified by mass spectrometry using matrix-assisted laser desorption ionization-time-of-flight/time-of-flight technology. The proteomic results were validated by real-time polymerase chain reaction, Western blotting, and microscopy. Nitric oxide (NO) was quantified using Griess reagent.
Results: Comparative analysis revealed differential expression of signal transduction proteins that regulate cytoskeleton, transcription, metabolic, and stress-related pathways. In total extracts, dimethylarginine dimethylaminohydrolase 2 (DDAH-2) did not show any change in expression after stimulation with IL-1β. However, in mitochondrial extracts, DDAH-2 expression was significantly increased after exposure to IL-1β. Conventional immunofluorescence and confocal microscopy revealed the presence of DDAH-2 in the mitochondria of IL-1β-stimulated chondrocytes. These results were reproducible in cartilage explants treated with IL-1β. In addition, we demonstrated that inhibition of the expression of DDAH-2, as well as interruption of its translocation to the mitochondria, reduced the NO production induced by IL-1β. DDAH-2 protein expression was higher in osteoarthritic (OA) cartilage than in normal cartilage.
Conclusion: In the present study, the presence of DDAH-2 in normal human chondrocytes and cartilage was identified for the first time. DDAH-2 could play an important role in IL-1β-induced NO production and in OA pathogenesis.
Copyright © 2012 by the American College of Rheumatology.