Cytoskeletal tension modulates MMP-1 gene expression from tenocytes on micropillar substrates.
Cytoskeletal tension modulates MMP-1 gene expression from tenocytes on micropillar substrates.
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DOI：
10.1016/j.jbiomech.2012.11.056
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发表时间：
2013-03
影响因子：
2.4
通讯作者：
Eijiro Maeda;M. Sugimoto;T. Ohashi
中科院分区：
文献类型：
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作者：
Eijiro Maeda;M. Sugimoto;T. Ohashi
肌动蛋白细胞骨架与肌球蛋白II聚集在一起，产生细胞内的细胞骨架张力，作为细胞牵引力被诱导到细胞附着基质上。细胞骨架张力与细胞功能密切相关。本研究探讨了肌腱细胞骨架张力与细胞代谢平衡之间的定量关系。利用微机械加工技术，用聚二甲基硅氧烷制备微柱基底，通过改变微柱高度具有三种不同的基底弹性值（6、18和33 kPa）。牛腱细胞在这些微柱基底上孵育24小时后，测定细胞牵引力。还用qPCR分析来自微柱上的腱细胞的I型胶原蛋白（合成代谢标志物）和MMP-1（分解代谢标志物）的基因表达。此外，使用抑制剂blebbistatin研究了肌球蛋白II活性抑制对腱细胞细胞骨架张力和代谢的影响。结果表明，肌腱细胞在33 kPa基质上的细胞牵引力明显大于6 kPa基质上的细胞牵引力。这与33 kPa基质上MMP-1 mRNA表达显著降低有关。补充blebbistatin后，细胞牵引力显著降低，且呈剂量依赖性。事实上，与相应的对照组相比，用10μM blebbistatin处理的肌腱细胞的牵引力显著更小，MMP-1 mRNA的表达更高。因此，腱细胞对基质硬度的反应与细胞内张力和分解代谢基因表达的改变有关。另一方面，腱细胞的抗张性，作为衡量I型胶原mRNA的表达，没有改变与基板刚度。
Actin cytoskeletons, aggregated with myosin II, generate intracellular cytoskeletal tension, which is induced to cell attaching substrate as cell traction forces. It is thought that cytoskeletal tension links closely to cell functions. The present study examined quantitative relationships between cytoskeleton tension and the balance of cell metabolism of tenocytes. Using micromachining techniques, micropillar substrates were prepared with polydimethylsiloxane, having three different values of substrate elasticity (6, 18 and 33kPa) by changing the micropillar height. After 24h incubation of bovine tenocytes on these micropillar substrates, cell traction forces were determined. Gene expressions for type I collagen (anabolic marker) and MMP-1 (catabolic marker) from tenocytes on micropillars were also analyzed with qPCR. In addition, effects of an inhibition of myosin II activity on tenocyte cytoskeletal tension and metabolism were examined using the inhibitor, blebbistatin. It was exhibited that cell traction forces were significantly larger in tenocytes on 33kPa substrates compared to those on 6kPa substrates. This was associated with significant lower expression of MMP-1 mRNA on 33kPa substrates. Cell traction forces were decreased significantly by the supplementation of blebbistatin in a dose-dependent manner. Indeed, there were significant smaller traction forces and higher expression for MMP-1 mRNA from tenocytes treated with 10μM blebbistatin compared to their corresponding controls. Accordingly, tenocyte responses to substrate stiffness are associated with alterations in intracellular tension and catabolic gene expression. On the other hand, tenocyte anabolism, as measured by type I collagen mRNA expression, was not altered with substrate stiffness.