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Phosphoproteome analysis of synoviocytes from patients with rheumatoid arthritis
Author(s) -
Katano Masayoshi,
Kurokawa Manae S.,
Matsuo Kosuke,
Masuko Kayo,
Suematsu Naoya,
Okamoto Kazuki,
Kamada Toshikazu,
Nakamura Hiroshi,
Kato Tomohiro
Publication year - 2017
Publication title -
international journal of rheumatic diseases
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.795
H-Index - 41
eISSN - 1756-185X
pISSN - 1756-1841
DOI - 10.1111/1756-185x.12997
Subject(s) - phosphoprotein , transfection , microbiology and biotechnology , tumor necrosis factor alpha , medicine , difference gel electrophoresis , phosphorylation , kinase , recombinant dna , proteome , proteomics , immunology , chemistry , biology , cell culture , biochemistry , gene , genetics
Aim To explore disease‐associated molecules in rheumatoid arthritis ( RA ), we comprehensively analyzed phosphoproteins purified from RA synoviocytes. Method 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 ( ANXA 4), one of the identified phosphoproteins, was transfected into synoviocytes from an OA patient to mimic RA synoviocytes and humoral factor secretion was compared between rANXA 4‐transfected and non‐transfected synoviocytes under a tumor necrosis factor‐α ( TNF α)‐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 ANXA 4, an exocytosis‐regulating protein. The transfected rANXA 4 was found to be phosphorylated intracellularly, and secretion of chemokine (C‐X‐C motif) ligand 1 and interleukin‐8 induced by TNF α stimulation was significantly suppressed by the transfection ( P < 0.01). Conclusion The phosphoprotein profile of RA synoviocytes was different from that of OA synoviocytes. This difference would reflect the different pathophysiologies of the diseases. ANXA 4 may be one of therapeutic targets in RA .