Phosphoproteome analysis of synoviocytes from patients with rheumatoid arthritis
Phosphoproteome analysis of synoviocytes from patients with rheumatoid arthritis
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DOI：
10.1111/1756-185x.12997
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发表时间：
2017-06-01
影响因子：
2.5
通讯作者：
Kato, Tomohiro
中科院分区：
文献类型：
--
作者：
Katano, Masayoshi;Kurokawa, Manae S.;Kato, Tomohiro
目的：为探讨类风湿关节炎（RA）的疾病相关分子，我们对从RA滑膜细胞中纯化的磷蛋白进行了全面分析。用双向差异凝胶电泳（2D-DIGE）比较RA组和OA组滑膜细胞磷蛋白的电泳图谱。通过质谱鉴定具有显著不同磷酸化水平的蛋白质点。将膜联蛋白A4（ANXA 4）的重组蛋白（一种鉴定的磷蛋白）转染到来自OA患者的滑膜细胞中以模拟RA滑膜细胞，并在肿瘤坏死因子-α（TNF-α）刺激下比较rANXA 4转染和未转染的滑膜细胞之间的体液因子分泌。（TNF α）-刺激条件。2D-DIGE共检测到318个磷酸化蛋白斑点，其中94个斑点的强度在两组间有显著性差异（P < 0.05）。在94个斑点中，与OA组相比，RA组中22个斑点显示两倍或更高的强度，一个斑点显示小于1/2倍的强度。从22个点中鉴定出11个磷蛋白，包括激酶、载体和伴侣蛋白、细胞骨架蛋白、蛋白酶和钙结合蛋白。其中一种已鉴定的钙结合蛋白是ANXA 4，一种胞吐调节蛋白。转染rANXA 4后，RA滑膜细胞内磷酸化水平明显升高，TNF α诱导的趋化因子配体1（C-X-C motif）和白细胞介素8（IL-8）的分泌明显减少（P < 0.01）。这种差异反映了疾病的不同病理生理学。ANXA 4可能是RA治疗的靶点之一。
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 (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-alpha (TNF alpha)-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 TNF alpha 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. ANXA4 may be one of therapeutic targets in RA.