Membrane type-1 matrix metalloproteinase is induced following cyclic compression of in vitro grown bovine chondrocytes
Membrane type-1 matrix metalloproteinase is induced following cyclic compression of in vitro grown bovine chondrocytes
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DOI：
10.1016/j.joca.2007.04.006
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发表时间：
2007-11-01
影响因子：
7
通讯作者：
Kandel, R. A.
中科院分区：
文献类型：
--
作者：
De Croos, J. N. A.;Jang, B.;Kandel, R. A.
目的：目的：探讨膜型基质金属蛋白酶（MT 1-MMP）对体外培养的软骨细胞周期性压迫的反应及其调控机制。研究方法：在开始培养的3天内，将循环压缩（30 min，1 kPa，1 Hz）施加于在生物可降解基质上生长的牛软骨细胞（6-9月龄动物）。使用牛关节软骨细胞进行荧光素酶测定以证明MT 1-MMP的机械敏感性。半定量逆转录聚合酶链反应（RT-PCR）和蛋白质印迹分析被用来建立响应于循环压缩的基因和蛋白质上调的时间过程。通过电泳迁移率变动分析、RT-PCR和western blot分析来评估MT 1-MMP的调节。同样，MT 1-MMP诱饵寡核苷酸和细胞外信号调节激酶1/2（ERK 1/2）药理学抑制剂被用来进一步表征MT 1-MMP调节。结果：在循环压缩后，MT 1-MMP的基因表达迅速和短暂的增加。在刺激后2小时内检测到蛋白水平升高，6小时后恢复至基线水平。在循环压缩，磷酸化的丝裂原活化蛋白激酶ERK 1/2显着增加。随后是转录因子的基因和蛋白表达增加;早期生长因子-1（Egr-1）和与MT 1-MMP启动子结合的Egr-1。用诱饵MT 1-MMP寡核苷酸阻断Egr-1 DNA结合，下调MT 1-MMP基因表达。ERK 1/2抑制剂U 0126还降低了Egr-1 DNA与MT 1-MMP启动子序列的结合活性以及随后MT 1-MMP的转录。结论：体外循环压缩软骨细胞通过ERK 1/2依赖性激活Egr-1结合上调MT 1-MMP。机械刺激激活的调控通路的描述将进一步加深我们对影响组织重塑的机制的理解。(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: To determine if membrane type-1 matrix metal loproteinase (MT1 -MMP) will respond to cyclic compression of chondrocytes grown in vitro and the regulatory mechanisms underlying this response. Methods: Cyclic compression (30 min, 1 kPa, 1 Hz) was applied to bovine chondrocytes (6-9-month-old animals) grown on top of a biodegradable substrate within 3 days of initiating culture. Luciferase assays using bovine articular chondrocytes were undertaken to demonstrate the mechanosensitivity of MT1 -MMP. Semi-quantitative reverse-transcription polymerase chain reaction (RT-PCR) and western blot analysis were used to establish the time course of gene and protein upregulation in response to cyclic compression. The regulation of MT1-MMP was assessed by electrophoretic mobility shift assays, RT-PCR and western blot analysis. As well, an MT1 -MMP decoy oligonucleotide and an extracellular signal-regulated kinase 1/2 (ERK1/2) pharmacological inhibitor were utilized to further characterize MT1-MMP regulation. Results: After cyclic compression, MT1 -MMP showed a rapid and transient increase in gene expression. Elevated protein levels were detected within 2 h of stimulation which returned to baseline by 6 h. During cyclic compression, phosphorylation of the mitogen activated protein kinase ERK1/2 increased significantly. This was followed by increased gene and protein expression of the transcription factor; early growth factor-1 (Egr-1) and Egr-1 binding to the MT1 -MMP promoter. Blocking Egr-1 DNA binding with a decoy MT1 -MMP oligonucleotide, downregulated MT1-MMP gene expression. The ERK1/2 inhibitor U0126 also reduced Egr-1 DNA binding activity to MT1-MMP promoter sequences and subsequent transcription of MT1 -MMP. Conclusions: These data suggest that cyclic compression of chondrocytes in vitro upregulates MT1-MMP via ERK1/2 dependent activation of Egr-1 binding. Delineation of the regulatory pathways activated by mechanical stimulation will further our understating of the mechanisms influencing tissue remodeling. (c) 2007 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.