Toll-like receptors and chondrocytes - The lipopolysaccharide-induced decrease in cartilage matrix synthesis is dependent on the presence of Toll-like receptor 4 and antagonized by bone morphogenetic protein 7
Toll-like receptors and chondrocytes - The lipopolysaccharide-induced decrease in cartilage matrix synthesis is dependent on the presence of Toll-like receptor 4 and antagonized by bone morphogenetic protein 7
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DOI：
10.1002/art.22637
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发表时间：
2007-06-01
影响因子：
--
通讯作者：
Smolen, J. S.
中科院分区：
文献类型：
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作者：
Bobacz, K.;Sunk, I. G.;Smolen, J. S.
目标。目的探讨Toll样受体(TLRs)1-9在人关节软骨中的表达，以及脂多糖(LPS)诱导的TLR-4活化对人关节软骨细胞生物合成活性和基质生成的影响。用逆转录聚合酶链式反应(RT-PCR)检测人关节软骨中TLR-1~9的表达，用Western blotting和免疫组织化学方法检测TLR-4的表达。关节软骨细胞分离自人供体和野生型或TLR-4(-/-)小鼠。软骨细胞单层培养在不加或不加骨形态发生蛋白7(BMP-7)和IL-1受体拮抗剂(IL-LRA)的情况下，分别与白细胞介素1β(IL-1β)和脂多糖(LPS)共同孵育。用S-35-硫酸盐掺入法测定硫酸氨基葡聚糖的新合成。RT-PCR法检测软骨标志物和IL-1、6的内源性基因表达。免疫印迹法、RT-PCR法和SGAG合成法检测p38或p44/42激酶(ERK-1/2)在脂多糖介导的TLR-4信号转导中的作用。在人关节软骨细胞的信使RNA(MRNA)水平上发现TLR1-9。在蛋白质水平上也观察到TLR-4的存在。在小鼠和人关节软骨细胞中，但在TLR-4(-/-)小鼠的软骨细胞中，脂多糖刺激导致总蛋白多糖合成减少。TLR-4激活后IL-1β的mRNA表达增加，而聚集素和11型胶原的表达显著降低。BMP-7和IL-1ra的存在可拮抗内毒素的抗合成代谢作用。阻断p38，但不阻断ERK-1/2，可抑制内毒素介导的IL-1β基因表达，并抑制内毒素对基质生物合成的负面影响。这些数据表明，在人类关节软骨中存在TLRs。内毒素对软骨生物合成活性的抑制作用依赖于TLR-4的存在，至少部分由IL-1β上调所控制，并由p38激酶介导。这些体外数据表明，TLR-4在关节软骨细胞中具有抗合成代谢作用，可能会阻碍各种关节疾病的软骨修复。
Objective. To assess the presence of Toll-like receptors (TLRs) 1-9 in human articular cartilage, and to investigate the effects of lipopolysaccharide (LPS)induced activation of TLR-4 on biosyntlietic activity and matrix production by human articular chondrocytes.Methods. TLRs 1-9 were assessed in human articular cartilage by reverse transcription-polymerase chain reaction (RT-PCR); TLR-4 was also analyzed by Western blotting and immunohistochemistry. Articular chondrocytes were isolated from human donors and from wild-type or TLR-4(-/-) mice. Chondrocyte monolayer cultures were incubated with interieukin-1 beta (IL-1 beta) and LPS in the absence or presence of bone morphogenetic protein 7 (BMP-7) and IL-1 receptor antagonist (IL-lRa). Neosynthesis of sulfated glycosaminoglycans (sGAG) was measured by S-35-sulfate incorporation. Endogenous gene expression of cartilage markers as well as IL-1,6 was examined using RT-PCR. The involvement of p38 kinase or p44/42 kinase (ERK-1/2) in LPS-mediated TLR-4 signaling was investigated by immunoblotting, RT-PCR, and sGAG synthesis.Results. TLRs 1-9 were found on the messenger RNA (mRNA) level in human articular chondrocytes. The presence of TLR-4 was also observed on the protein level. In murine and human articular chondrocytes, but not in chondrocytes derived from TLR-4(-/-) mice, stimulation with LPS resulted in a decrease in total proteoglycan synthesis. IL-1 beta mRNA expression was increased by TLR-4 activation, whereas expression of aggrecan and type 11 collagen was significantly decreased. The presence of BMP-7 and IL-1Ra antagonized the anti-anabolic effects of LPS. Blocking of p38, but not ERK-1/2, resulted in inhibition of both LPS-mediated IL-1 beta gene expression and the negative effects of LPS on matrix biosynthesis.Conclusion. These data demonstrate the presence of TLRs in human articular cartilage. The suppressive effects of LPS on cartilage biosynthetic activity are dependent on the presence of TLR-4, are governed, at least in part, by an up-regulation of IL-1 beta, and are mediated by p38 kinase. These in vitro data indicate an anti-anabolic effect of TLR-4 in articular chondrocytes that may hamper cartilage repair in various joint diseases.