Implication of granulocyte-macrophage colony-stimulating factor induced neutrophil gelatinase-associated lipocalin in pathogenesis of rheumatoid arthritis revealed by proteome analysis

Introduction In rheumatoid arthritis (RA), synovial fluid (SF) contains a large number of neutrophils that contribute to the inflammation and destruction of the joints. The SF also contains granulocyte-macrophage colony-stimulating factor (GM-CSF), which sustains viability of neutrophils and activat...
Ausführliche Beschreibung

Introduction In rheumatoid arthritis (RA), synovial fluid (SF) contains a large number of neutrophils that contribute to the inflammation and destruction of the joints. The SF also contains granulocyte-macrophage colony-stimulating factor (GM-CSF), which sustains viability of neutrophils and activates their functions. Using proteomic surveillance, we here tried to elucidate the effects of GM-CSF on neutrophils. Methods Neutrophils stimulated by GM-CSF were divided into four subcellular fractions: cytosol, membrane/organelle, nuclei, and cytoskeleton. Then, proteins were extracted from each fraction and digested by trypsin. The produced peptides were detected using matrix-assisted laser desorption ionisation-time-of-flight mass spectrometry (MALDI-TOF MS). Results We detected 33 peptide peaks whose expression was upregulated by more than 2.5-fold in GM-CSF stimulated neutrophils and identified 11 proteins out of the 33 peptides using MALDI-TOF/TOF MS analysis and protein database searches. One of the identified proteins was neutrophil gelatinase-associated lipocalin (NGAL). We confirmed that the level of NGAL in SF was significantly higher in patients with RA than in those with osteoarthritis. We next addressed possible roles of the increased NGAL in RA. We analysed proteome alteration of synoviocytes from patients with RA by treatment with NGAL in vitro. We found that, out of the detected protein spots (approximately 3,600 protein spots), the intensity of 21 protein spots increased by more than 1.5-fold and the intensity of 10 protein spots decreased by less than 1 to 1.5-fold as a result of the NGAL treatment. Among the 21 increased protein spots, we identified 9 proteins including transitional endoplasmic reticulum ATPase (TERA), cathepsin D, and transglutaminase 2 (TG2), which increased to 4.8-fold, 1.5-fold and 1.6-fold, respectively. Two-dimensional electrophoresis followed by western blot analysis confirmed the upregulation of TERA by the NGAL treatment and, moreover, the western blot analysis showed that the NGAL treatment changed the protein spots caused by post-translational modification of TERA. Furthermore, NGAL cancelled out the proliferative effects of fibroblast growth factor (FGF)-2 and epidermal growth factor (EGF) on chondrocytes from a patient with RA and proliferative effect of FGF-2 on chondrosarcoma cells. Conclusions Our results indicate that GM-CSF contributes to the pathogenesis of RA through upregulation of NGAL in neutrophils, followed by induction of TERA, cathepsin D and TG2 in synoviocytes. NGAL and the upregulated enzymes may therefore play an important role in RA. Ausführliche Beschreibung

Autor*in:	Katano, Masayoshi [verfasserIn] Okamoto, Kazuki Arito, Mitsumi Kawakami, Yuki Kurokawa, Manae S Suematsu, Naoya Shimada, Sonoko Nakamura, Hiroshi Xiang, Yang Masuko, Kayo Nishioka, Kusuki Yudoh, Kazuo Kato, Tomohiro

Format:	E-Artikel
Sprache:	Englisch

Erschienen:	2009

Schlagwörter:	Rheumatoid Arthritis Synovial Fluid Protein Spot Proliferative Effect Chondrosarcoma Cell

Anmerkung:	© Katano et al.; licensee BioMed Central Ltd. 2009. This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (

Übergeordnetes Werk:	Enthalten in: Arthritis Research & Therapy - London : BioMed Central, 1999, 11(2009), 1 vom: 08. Jan.
Übergeordnetes Werk:	volume:11 ; year:2009 ; number:1 ; day:08 ; month:01

Links:	Volltext

DOI / URN:	10.1186/ar2587

Katalog-ID:	SPR03082978X