Department of Orthopaedic Surgery, Kinki University Faculty of Medicine, Osaka, Japan.
Inflamm Res. 2012 Oct;61(10):1093-100. doi: 10.1007/s00011-012-0500-4. Epub 2012 Jun 12.
Excessive mechanical stress on the cartilage causes the degradation of the matrix, leading to the osteoarthritis (OA). Matrix metalloproteinases 13 (MMP13) is a major catalytic enzyme in OA and p38 plays an important role in its induction. However, precise pathway inducing p38 activation has not been elucidated. We hypothesized here that the small GTPase Rho and its effector ROCK might function in upper part of the mechanical stress-induced matrix degeneration pathway.
Bovine metacarpal phalangeal articular cartilage explants were loaded with 1 MPa dynamic compression for 6 h with or without a ROCK specific inhibitor Y27632 or/and a p38 specific inhibitor SB202190. Then p38 phosphorylation and MMP13 expression were assessed by western blot or/and quantitative RT-PCR. Rho-activity was measured by pull-down assay using glutathione S-transferase fusion protein of Rho binding domain.
Cyclic compression caused Rho activation, p38 phosphorylation and MMP13 expression. Both Y27632 and SB202190 were found to block the mechanical stress-enhanced p38 phosphorylation and subsequent MMP13 expression.
The present results show that p38 phosphorylation and MMP13 expression are regulated by Rho/ROCK activation, and support the potential novel pathway that Rho/ROCK is in the upper part of the mechanical stress-induced matrix degeneration cascade in cartilage comprised of p38 and MMP13.
软骨承受过度的机械压力会导致基质降解，从而引发骨关节炎（OA）。基质金属蛋白酶 13（MMP13）是 OA 的主要催化酶，p38 在其诱导中起着重要作用。然而，精确的诱导 p38 激活的途径尚未阐明。我们假设小 GTPase Rho 及其效应物 ROCK 可能在上部机械压力诱导的基质退化途径中发挥作用。
用 1 MPa 的动态压缩对牛掌指关节软骨标本加载 6 小时，同时或不使用 ROCK 特异性抑制剂 Y27632 和/或 p38 特异性抑制剂 SB202190。然后通过使用 Rho 结合域谷胱甘肽 S-转移酶融合蛋白的下拉测定法测量 Rho 活性，并通过 Western blot 或定量 RT-PCR 评估 p38 磷酸化和 MMP13 的表达。
周期性压缩导致 Rho 激活、p38 磷酸化和 MMP13 表达。发现 Y27632 和 SB202190 均可阻断机械应力增强的 p38 磷酸化及随后的 MMP13 表达。
本研究结果表明，p38 磷酸化和 MMP13 表达受 Rho/ROCK 激活调节，支持 Rho/ROCK 位于 p38 和 MMP13 组成的软骨机械应力诱导基质退化级联反应上游的潜在新途径。
