title: Phosphoproteome analysis of synoviocytes from patients with rheumatoid arthritis.
abstract: OBJECTIVE
  To explore disease-associated molecules in rheumatoid arthritis (RA), we comprehensively analyzed phosphoproteins purified from RA synoviocytes.
  
  
  METHODS
  Synoviocytes were obtained from three patients with RA and three patients with osteoarthritis (OA). Profiles of phosphoproteins purified from the synoviocytes were compared by two-dimensional differential gel electrophoresis (2D-DIGE) between the RA and OA groups. Protein spots with significantly different phosphorylation levels were identified by mass spectrometry. Recombinant protein of annexin A4 (ANXA4), one of the identified phosphoproteins, was transfected into synoviocytes from an OA patient to mimic RA synoviocytes and humoral factor secretion was compared between rANXA4-transfected and non-transfected synoviocytes under a tumor necrosis factor-α (TNFα)-stimulated condition.
  
  
  RESULTS
  In 2D-DIGE, 318 phosphoprotein spots were detected, of which 94 spots showed significantly different intensities between the two groups (P < 0.05). Among the 94 spots, 22 spots showed two-fold or higher intensity and one spot showed less than 1/2-fold intensity in the RA group compared to the OA group. From the 22 spots, 11 phosphoproteins were identified, which included kinases, carrier and chaperone proteins, cytoskeletal proteins, proteases and calcium-binding proteins. One of the identified calcium-binding proteins was ANXA4, an exocytosis-regulating protein. The transfected rANXA4 was found to be phosphorylated intracellularly, and secretion of chemokine (C-X-C motif) ligand 1 and interleukin-8 induced by TNFα stimulation was significantly suppressed by the transfection (P < 0.01).
  
  
  CONCLUSIONS
  The phosphoprotein profile of RA synoviocytes was different from that of OA synoviocytes. This difference would reflect the different pathophysiologies of the diseases. ANXA4 may be one of therapeutic targets in RA.
authors.affiliation: Research and Development, Clinical Department, LSI Medience Corporation, Tokyo, Japan.|Clinical Proteomics and Molecular Medicine, St. Marianna University Graduate School of Medicine, Kawasaki, Japan.
authors.forename: Masayoshi
authors.initials: M
authors.lastname: Katano
authors.affiliation: Disease Biomarker Analysis and Molecular Regulation, St. Marianna University Graduate School of Medicine, Kawasaki, Japan.
authors.forename: Manae S
authors.initials: MS
authors.lastname: Kurokawa
authors.affiliation: Department of Orthopaedic Surgery, Yokohama City University School of Medicine, Yokohama, Japan.
authors.forename: Kosuke
authors.initials: K
authors.lastname: Matsuo
authors.affiliation: Preventive Medical Center, Sanno Hospital Medical Center, Tokyo, Japan.
authors.forename: Kayo
authors.initials: K
authors.lastname: Masuko
authors.affiliation: Clinical Proteomics and Molecular Medicine, St. Marianna University Graduate School of Medicine, Kawasaki, Japan.
authors.forename: Naoya
authors.initials: N
authors.lastname: Suematsu
authors.affiliation: Clinical Proteomics and Molecular Medicine, St. Marianna University Graduate School of Medicine, Kawasaki, Japan.
authors.forename: Kazuki
authors.initials: K
authors.lastname: Okamoto
authors.affiliation: Department of Rheumatology, Nippon Medical School, Tokyo, Japan.
authors.forename: Toshikazu
authors.initials: T
authors.lastname: Kamada
authors.affiliation: Department of Orthopedic Surgery, International University of Health and Welfare, Tokyo, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Nakamura
authors.affiliation: Clinical Proteomics and Molecular Medicine, St. Marianna University Graduate School of Medicine, Kawasaki, Japan.
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Kato
mesh_terms: D000368:Aged; D017314:Annexin A4 / Q000235:genetics / Q000378:metabolism; D001172:Arthritis, Rheumatoid / Q000175:diagnosis / Q000235:genetics / Q000378:metabolism*; D015415:Biomarkers / Q000378:metabolism; D002478:Cells, Cultured; D054360:Chemokine CXCL1 / Q000378:metabolism; D005260:Female; D006801:Humans; D016209:Interleukin-8 / Q000378:metabolism; D008875:Middle Aged; D010003:Osteoarthritis / Q000175:diagnosis / Q000235:genetics / Q000378:metabolism*; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D040901:Proteomics / Q000379:methods*; D000070918:Synoviocytes / Q000187:drug effects / Q000378:metabolism*; D053719:Tandem Mass Spectrometry; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D057825:Two-Dimensional Difference Gel Electrophoresis
chemical_list: D017314:Annexin A4; D015415:Biomarkers; C475829:CXCL1 protein, human; C508606:CXCL8 protein, human; D054360:Chemokine CXCL1; D016209:Interleukin-8; D010750:Phosphoproteins; D014409:Tumor Necrosis Factor-alpha
keywords: annexin A4; phosphoprotein; rheumatoid arthritis; synoviocytes