title: Dimethylarginine dimethylaminohydrolase 2, a newly identified mitochondrial protein modulating nitric oxide synthesis in normal human chondrocytes.
abstract: 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.
  
  
  CONCLUSIONS
  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.
authors: Osteoarticular and Aging Research Laboratory, INIBIC-Complejo Hospitalario Universitario A Coruña, A Coruña, Spain.
authors: Berta
authors: B
authors: Cillero-Pastor
authors: Jesús
authors: J
authors: Mateos
authors: Carlos
authors: C
authors: Fernández-López
authors: Natividad
authors: N
authors: Oreiro
authors: Cristina
authors: C
authors: Ruiz-Romero
authors: Francisco J
authors: FJ
authors: Blanco
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000581:Amidohydrolases / Q000235:genetics / Q000378:metabolism*; D002358:Cartilage, Articular / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D019902:Chondrocytes / Q000187:drug effects / Q000201:enzymology*; D015971:Gene Expression Regulation, Enzymologic; D006710:Homocysteine / Q000494:pharmacology; D006801:Humans; D011163:Hydroxymethylbilane Synthase / Q000235:genetics / Q000378:metabolism; D053583:Interleukin-1beta / Q000494:pharmacology; D008875:Middle Aged; D008928:Mitochondria / Q000187:drug effects / Q000201:enzymology*; D024101:Mitochondrial Proteins / Q000235:genetics / Q000378:metabolism*; D009569:Nitric Oxide / Q000096:biosynthesis*; D020370:Osteoarthritis, Knee / Q000201:enzymology; D020543:Proteome / Q000032:analysis; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D055815:Young Adult
chemical_list: D053583:Interleukin-1beta; D024101:Mitochondrial Proteins; D020543:Proteome; D006710:Homocysteine; D009569:Nitric Oxide; D011163:Hydroxymethylbilane Synthase; D000581:Amidohydrolases; C059999:dimethylargininase