Arthritis and rheumatism · 2012
Dimethylarginine dimethylaminohydrolase 2, a newly identified mitochondrial protein modulating nitric oxide synthesis in normal human chondrocytes.
СтатьяПоддержка исследования: вне правительства СШАНазвание и аннотация: Перевод в очереди
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Cillero-Pastor Berta, Mateos Jesús, Fernández-López Carlos, Oreiro Natividad, Ruiz-Romero Cristina, Blanco Francisco J
Аннотация
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.
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.
Предметные рубрики MeSH
AdolescentAdultAgedAmidohydrolasesCartilage, ArticularCells, CulturedChondrocytesGene Expression Regulation, EnzymologicHomocysteineHumansHydroxymethylbilane SynthaseInterleukin-1betaMiddle AgedMitochondriaMitochondrial ProteinsNitric OxideOsteoarthritis, KneeProteomeSpectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationYoung Adult