Dimethylarginine dimethylaminohydrolase 2, a newly identified mitochondrial protein modulating nitric oxide synthesis in normal human chondrocytes

• Published in: Arthritis & rheumatology (Hoboken, N.J.) Vol. 64; no. 1; pp. 204 - 212
• Main Authors: Cillero-Pastor, Berta, Mateos, Jesús, Fernández-López, Carlos, Oreiro, Natividad, Ruiz-Romero, Cristina, Blanco, Francisco J.
• Format: Journal Article
• Language: English
• Published: Hoboken Wiley Subscription Services, Inc., A Wiley Company 01.01.2012; Wiley; Wiley Subscription Services, Inc
• Subjects: Adolescent; Adult; Aged; Amidohydrolases - genetics; Amidohydrolases - metabolism; Beta; Biological and medical sciences; Cartilage, Articular - drug effects; Cartilage, Articular - metabolism; Cells, Cultured; Chondrocytes - drug effects; Chondrocytes - enzymology; Desorption; Diseases of the osteoarticular system; Gene Expression Regulation, Enzymologic; Homocysteine - pharmacology; Humans; Hydroxymethylbilane Synthase - genetics; Hydroxymethylbilane Synthase - metabolism; Interleukin-1beta - pharmacology; Mass spectrometry; Medical sciences; Microscopy; Middle Aged; Mitochondria; Mitochondria - drug effects; Mitochondria - enzymology; Mitochondrial Proteins - genetics; Mitochondrial Proteins - metabolism; Nitric Oxide - biosynthesis; Osteoarthritis, Knee - enzymology; Protein expression; Proteins; Proteome - analysis; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Young Adult; Human; Chondrocyte; Protein synthesis; Nitric oxide; Rheumatology
• ISSN: 0004-3591; 2326-5191; 1529-0131; 1529-0131; 2326-5205
• DOI: 10.1002/art.30652

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.