Modulation of TGF-β signaling by proinflammatory cytokines in articular chondrocytes
Modulation of TGF-β signaling by proinflammatory cytokines in articular chondrocytes
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DOI：
10.1016/j.joca.2007.04.011
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发表时间：
2007-12-01
影响因子：
7
通讯作者：
Jimenez, S. A.
中科院分区：
文献类型：
--
作者：
Roman-Blas, J. A.;Stokes, D. G.;Jimenez, S. A.
目的：关节软骨的正常结构和功能是合成代谢和分解代谢过程之间精确平衡的相互作用的结果。生长因子的转化生长因子-β（TGF-β）家族通常发挥合成代谢或修复反应;相反，促炎细胞因子如白介素1 β（IL-1 β）和肿瘤坏死因子-α（TNF-α）发挥强烈的分解代谢作用。最近的证据表明，IL-1 β、TNF-α和TGF-β信号通路具有拮抗关系。本研究的目的是确定通过IL-1 β或TNF-α信号通路调节关节软骨细胞对TGF-β的反应是否通过调节母体对DPP的活性和可用性而发生。（果蝇）人同源物（Smad）蛋白。将从骨关节炎（CA）患者膝关节分离的人关节软骨细胞或正常牛软骨细胞在聚乙烯中悬浮培养。在用单独的IL-1 β、单独的TNF-α或IL-10随后TGF-β处理之前6小时，用10%胎牛血清培养基或无血清培养基培养（甲基丙烯酸2-羟乙酯）包被的培养皿。通过电泳迁移率变动分析（EMSA）检测核提取物的核因子-κ B（NF-κ B）和Smad 3/4脱氧核糖核酸（DNA）结合。还将核提取物置于TranSignal蛋白/DNA阵列（Panomics，红杉城，CA）中，使得能够同时半定量评估54种不同转录因子的DNA结合活性。Western blot法检测细胞核内Smad 2/3磷酸化蛋白和Smad 7总蛋白的表达。结果：TNF-α或IL-1 β处理后，细胞内Smad 3/4的DNA结合活性下调46%（P < 0. 05），IL-1 β处理后，细胞内Smad 7、Ⅱ型胶原α 1（COL 2A 1）、聚集蛋白聚糖（aggrecan）和SRY相关的高迁移率族蛋白盒基因9（SOX-9）mRNA表达下调42%（P < 0. 05）。EMSA分析显示，在用IL-1 β或TNF-α处理的人关节软骨细胞中，Smad 3/4 DNA结合活性一致降低。TGF-β诱导的Smad 3/4 DNA结合活性和Smad 2/3磷酸化在人OA和牛软骨细胞中用IL-1 β预处理后也减少。实时荧光定量PCR和Western blot分析表明，IL-1 β部分逆转TGF-β刺激的Smad 7 mRNA和蛋白水平在TGF-β处理的人OA细胞。相反，TGF-β刺激的COL 2A 1，聚集蛋白聚糖，SOX-9 mRNA水平被废除IL-1 β。结论：IL-1 β或TNF-α对Smad 3/4 DNA结合活性在人关节软骨细胞，以及TGF-β诱导的刺激Smad 3/4 DNA结合活性和Smad 2/3磷酸化在人OA和牛关节软骨细胞产生抑制作用。IL-10部分逆转TGF-β刺激的Smad 7 mRNA或蛋白水平的增加，表明Smad 7可能不参与关节软骨细胞中IL-1 β或TNF-α诱导的TGF-β信号传导的抑制。IL-1 β或TNF-α和TGF-β信号通路之间的平衡对于维持关节软骨稳态至关重要，其破坏可能在OA的发病机制中起重要作用。(c)2007年国际骨关节炎研究学会。由爱思唯尔有限公司出版。保留所有权利。
Objective: The normal structure and function of articular cartilage are the result of a precisely balanced interaction between anabolic and catabolic processes. The transforming growth factor-beta (TGF-beta) family of growth factors generally exerts an anabolic or repair response; in contrast, proinflammatory cytokines such as interleukin 1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) exert a strong catabolic effect. Recent evidence has shown that IL-1 beta, and TNF-alpha, and the TGF-beta signaling pathways share an antagonistic relationship. The aim of this study was to determine whether the modulation of the response of articular chondrocytes to TGF-beta by IL-1 beta or TNF-alpha signaling pathways occurs through regulation of activity and availability of mothers against DPP (Drosophila) human homologue (Smad) proteins.Methods: Human articular chondrocytes isolated from knee joints from patients with osteoarthritis (CA) or normal bovine chondrocytes were cultured in suspension in poly-(2-hydroxyethyl methacrylate)-coated dishes with either 10% fetal bovine serum media or serum-deprived media 6 h before treatment with IL-1 beta alone, TNF-alpha alone or IL-10 followed by TGF-beta. Nuclear extracts were examined by electrophoretic mobility-shift assays (EMSA) for nuclear factor-kappa B (NF-kappa B) and Smad3/4 deoxyribonucleic acid (DNA) binding. Nuclear extracts were also subjected to the TranSignal Protein/DNA array (Panomics, Redwood City, CA) enabling the simultaneous semiquantitative assessment of DNA-binding activity of 54 different transcription factors. Nuclear phospho-Smad2/3 and total Smad7 protein expression in whole cell lysates were studied by Western blot. Cytoplasmic Smad7, type II collagen alpha 1 (COL2A1), aggrecan and SRY-related high mobility group-Box gene 9 (SOX-9) mRNA expression were measured by real-time polymerase chain reaction (PCR).Results: The DNA-binding activity of Smad3/4 in the TranSignal Protein/DNA array was downregulated by TNF-alpha (46%) or IL-1 beta treatment (42%). EMSA analysis showed a consistent reduction in Smad3/4 DNA-binding activity in human articular chondrocytes treated with IL-1 beta or TNF-alpha. TGF-beta-induced Smad3/4 DNA-binding activity and Smad2/3 phosphorylation were also reduced following pretreatment with IL-1 beta in human OA and bovine chondrocytes. Real-time PCR and Western blot analysis showed that IL-1 beta partially reversed the TGF-beta stimulation of Smad7 mRNA and protein levels in TGF-beta-treated human OA cells. In contrast, TGF-beta-stimulated COL2A1, aggrecan, and SOX-9 mRNA levels were abrogated by IL-1 beta.Conclusions: IL-1 beta or TNF-alpha exerted a suppressive effect on Smad3/4 DNA-binding activity in human articular chondrocytes, as well as on TGF-beta-induced stimulation of Smad3/4 DNA-binding activity and Smad2/3 phosphorylation in human OA and bovine articular chondrocytes. IL-1 0 partially reversed the increase in TGF-beta-stimulated Smad7 mRNA or protein levels suggesting that Smad7 may not be involved in the suppression of TGF-beta signaling induced by IL-1 beta or TNF-alpha in articular chondrocytes. The balance between the IL-1 beta or TNF-alpha and the TGF-beta signaling pathways is crucial for maintenance of articular cartilage homeostasis and its disruption likely plays a substantial role in the pathogenesis of OA. (c) 2007 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.