Implication of granulocyte-macrophage colony-stimulating factor induced neutrophil gelatinase-associated lipocalin in pathogenesis of rheumatoid arthritis revealed by proteome analysis.
Implication of granulocyte-macrophage colony-stimulating factor induced neutrophil gelatinase-associated lipocalin in pathogenesis of rheumatoid arthritis revealed by proteome analysis.
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DOI：
10.1186/ar2587
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发表时间：
2009
影响因子：
4.9
通讯作者：
Kato T
中科院分区：
文献类型：
--
作者：
Katano M;Okamoto K;Arito M;Kawakami Y;Kurokawa MS;Suematsu N;Shimada S;Nakamura H;Xiang Y;Masuko K;Nishioka K;Yudoh K;Kato T
在类风湿性关节炎（RA）中，滑液（SF）含有大量的中性粒细胞，这些中性粒细胞有助于关节的炎症和破坏。SF还含有粒细胞-巨噬细胞集落刺激因子（GM-CSF），维持中性粒细胞的活力并激活其功能。利用蛋白质组学监测，我们试图阐明GM-CSF对中性粒细胞的影响。GM-CSF刺激的中性粒细胞分为四个亚细胞部分：细胞质、膜/细胞器、细胞核和细胞骨架。然后，从每个馏分中提取蛋白质并用胰蛋白酶消化。采用基质辅助激光解吸电离飞行时间质谱法（MALDI-TOF MS）对制备的多肽进行检测。我们在GM-CSF刺激的中性粒细胞中检测到33个表达上调超过2.5倍的肽峰，并通过MALDI-TOF/TOF质谱分析和蛋白质数据库搜索鉴定了33个肽中的11个蛋白。其中一种鉴定的蛋白质是中性粒细胞明胶酶相关脂钙蛋白（NGAL）。我们证实RA患者SF中NGAL水平明显高于骨关节炎患者。接下来，我们探讨了NGAL增加在RA中的可能作用。我们分析了NGAL在体外治疗RA患者滑膜细胞的蛋白质组改变。我们发现，在检测到的蛋白斑点（约3600个蛋白斑点）中，由于NGAL处理，21个蛋白斑点的强度增加了1.5倍以上，10个蛋白斑点的强度下降了不到1到1.5倍。在增加的21个蛋白点中，我们鉴定出过渡性内质网atp酶（TERA）、组织蛋白酶D和谷氨酰胺转酶2 （TG2）等9个蛋白，分别增加了4.8倍、1.5倍和1.6倍。双向电泳和western blot分析证实了NGAL处理对TERA的上调作用，并且western blot分析显示NGAL处理改变了TERA翻译后修饰引起的蛋白斑点。此外，NGAL抵消了成纤维细胞生长因子(FGF)-2和表皮生长因子（EGF）对RA患者软骨细胞的增殖作用以及FGF-2对软骨肉瘤细胞的增殖作用。我们的研究结果表明，GM-CSF通过上调中性粒细胞中的NGAL，随后诱导滑膜细胞中的TERA、组织蛋白酶D和TG2，参与RA的发病机制。因此，NGAL和上调酶可能在RA中起重要作用。
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. 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). 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. 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.
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影响因子：
4.8
作者：
Lee, JM;Kim, YS;Kim, SY
通讯作者：
Kim, SY
DOI：
10.1016/j.bbrc.2006.02.171
发表时间：
2006-05-05
影响因子：
3.1
作者：
Chiou, M;Xu, Y;Longaker, MT
通讯作者：
Longaker, MT
影响因子：
5
作者：
BLASER, J;TRIEBEL, S;TSCHESCHE, H
通讯作者：
TSCHESCHE, H
影响因子：
15.9
作者：
Dang, Pham My-Chan;Stensballe, Allan;El-Benna, Jamel
通讯作者：
El-Benna, Jamel
DOI：
10.1006/clin.1996.0025
发表时间：
1996-02-01
期刊：
CLINICAL IMMUNOLOGY AND IMMUNOPATHOLOGY
影响因子：
--
作者：
Gruber, BL;Sorbi, D;Arbeit, LA
通讯作者：
Arbeit, LA