The catabolic pathway mediated by Toll-like receptors in human osteoarthritic chondrocytes
The catabolic pathway mediated by Toll-like receptors in human osteoarthritic chondrocytes
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DOI：
10.1002/art.21951
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发表时间：
2006-07-01
影响因子：
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通讯作者：
Kim, Ho-Youn
中科院分区：
文献类型：
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作者：
Kim, Hyun Ah;Cho, Mi-La;Kim, Ho-Youn
Objective.研究Toll样受体（TLR）配体在人骨关节炎（OA）软骨细胞中介导的分解代谢途径。通过免疫组织化学分析OA和非OA关节软骨中TLR的存在。通过逆转录-聚合酶链反应分析白细胞介素-1（IL-1）和肿瘤坏死因子α（TNF α）对TLR信使RNA（mRNA）的调节。为了刺激TLR-2和TLR-4，分别用金黄色葡萄球菌肽聚糖和脂多糖（LPS）处理软骨细胞。用酶联免疫吸附试验检测基质金属蛋白酶（MMPs）1、3、13和前列腺素E-2（PGE（2））的产生。通过Griess反应分析一氧化氮（NO）的产生。通过蛋白质印迹或固相激酶测定来评估环氧化酶2蛋白的调节和MAPK（p38、ERK和JNK）的磷酸化。NF-κ B活化通过电泳迁移率改变分析来评估。TLRs 2和4在OA软骨病变区表达上调。用IL-1、TNF α、肽聚糖和LPS处理培养的软骨细胞均显著上调TLR-2 mRNA表达。用TLR配体处理软骨细胞后，MMPs 1、3和13以及NO和PGE（2）的产生显著增加。软骨外植体与TLR配体的长期培养也导致蛋白聚糖和11型胶原降解产物的释放显著增加。TLR配体处理导致所有3种MAPK的磷酸化和NF-κ B的活化。我们发现TLR在OA软骨病变中增加。TLR-2和TLR-4配体强烈诱导软骨细胞的分解代谢反应。调节TLR介导的信号传导作为治疗策略将需要详细阐明所涉及的信号传导途径。
Objective. To examine the catabolic pathways mediated by Toll-like receptor (TLR) ligands in human osteoarthritic (OA) chondrocytes.Methods. The presence of TLRs in OA and non-OA articular cartilage was analyzed by immunohistochemistry. The regulation of TLR messenger RNA (mRNA) by interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF alpha) was analyzed by reverse transcription-polymerase chain reaction. For stimulation of TLR-2 and TLR-4, chondrocytes were treated with Staphylococcus aureus peptidoglycan and lipopolysaccharides (LPS), respectively. Production of matrix metalloproteinases (MMPs) 1, 3, and 13 and prostaglandin E-2 (PGE(2)) was evaluated by enzyme-linked immunosorbent assay. Production of nitric oxide (NO) was analyzed by the Griess reaction. Regulation of cyclooxygenase 2 protein and phosphorylation of MAPKs (p38, ERK, and JNK) were evaluated by Western blotting or solid-phase kinase assay. NF-kappa B activation was evaluated by electrophoretic mobility shift assay.Results. Expression of TLRs 2 and 4 was upregulated in lesional areas of OA cartilage. Treatment with IL-1, TNF alpha, peptidoglycan, and LPS all significantly up-regulated TLR-2 mRNA expression in cultured chondrocytes. Production of MMPs 1, 3, and 13 and of NO and PGE(2) was significantly increased after treating chondrocytes with either of the TLR ligands. Prolonged culture of cartilage explants with TLR ligands also led to a significant increase in the release of proteoglycan and type 11 collagen degradation product. Treatment with TLR ligands led to phosphorylation of all 3 MAPKs and activation of NF-kappa B.Conclusion. We found that TLRs are increased in OA cartilage lesions. TLR-2 and TLR-4 ligands strongly induce catabolic responses in chondrocytes. Modulation of TLR-mediated signaling as a therapeutic strategy would require detailed elucidation of the signaling pathways involved.