Furumatsu Takayuki, Maehara Ami, Ozaki Toshifumi
Department of Orthopaedic Surgery, Okayama University Graduate School, 2-5-1 Shikata-cho, Kita-ku, Okayama, Japan.
Department of Orthopaedic Surgery, Okayama University Graduate School, 2-5-1 Shikata-cho, Kita-ku, Okayama, Japan.
J Orthop Sci. 2016 Jul;21(4):524-529. doi: 10.1016/j.jos.2016.02.013. Epub 2016 Apr 22.
Proper functioning of the meniscus depends on the composition and organization of its fibrocartilaginous extracellular matrix. We previously demonstrated that the avascular inner meniscus has a more chondrocytic phenotype compared with the outer meniscus. Inhibition of the Rho family GTPase ROCK, the major regulator of the actin cytoskeleton, stimulates the chondrogenic transcription factor Sry-type HMG box (SOX) 9-dependent α1(II) collagen (COL2A1) expression in inner meniscus cells. However, the crosstalk between ROCK inhibition, SOX9, and other transcription modulators on COL2A1 upregulation remains unclear in meniscus cells. The aim of this study was to investigate the role of SOX9-related transcriptional complex on COL2A1 expression under the inhibition of ROCK in human meniscus cells.
Human inner and outer meniscus cells were prepared from macroscopically intact lateral menisci. Cells were cultured in the presence or absence of ROCK inhibitor (ROCKi, Y27632). Gene expression, collagen synthesis, and nuclear translocation of SOX9 and Smad2/3 were analyzed.
Treatment of ROCKi increased the ratio of type I/II collagen double positive cells derived from the inner meniscus. In real-time PCR analyses, expression of SOX9 and COL2A1 genes was stimulated by ROCKi treatment in inner meniscus cells. ROCKi treatment also induced nuclear translocation of SOX9 and phosphorylated Smad2/3 in immunohistological analyses. Complex formation between SOX9 and Smad3 was increased by ROCKi treatment in inner meniscus cells. Chromatin immunoprecipitation analyses revealed that association between SOX9/Smad3 transcriptional complex with the COL2A1 enhancer region was increased by ROCKi treatment.
This study demonstrated that ROCK inhibition stimulated SOX9/Smad3-dependent COL2A1 expression through the immediate nuclear translocation of Smad3 in inner meniscus cells. Our results suggest that ROCK inhibition can stimulates type II collagen synthesis through the cooperative activation of Smad3 in inner meniscus cells. ROCKi treatment may be useful to promote the fibrochondrocytic healing of the injured inner meniscus.
半月板的正常功能取决于其纤维软骨细胞外基质的组成和组织结构。我们之前证明，与外侧半月板相比，无血管的内侧半月板具有更多的软骨细胞表型。肌动蛋白细胞骨架的主要调节因子Rho家族GTP酶ROCK的抑制作用，可刺激内侧半月板细胞中软骨形成转录因子Sry型HMG盒（SOX）9依赖的α1（II）型胶原蛋白（COL2A1）的表达。然而，在半月板细胞中，ROCK抑制、SOX9和其他转录调节因子之间关于COL2A1上调的相互作用仍不清楚。本研究的目的是探讨在人半月板细胞中，ROCK抑制作用下SOX9相关转录复合物对COL2A1表达的作用。
从肉眼完整的外侧半月板制备人内侧和外侧半月板细胞。细胞在有或无ROCK抑制剂（ROCKi，Y27632）的情况下进行培养。分析基因表达、胶原蛋白合成以及SOX9和Smad2/3的核转位情况。
ROCKi处理增加了源自内侧半月板的I/II型胶原蛋白双阳性细胞的比例。在实时PCR分析中，ROCKi处理刺激了内侧半月板细胞中SOX9和COL2A1基因的表达。在免疫组织学分析中，ROCKi处理还诱导了SOX9和磷酸化Smad2/3的核转位。ROCKi处理增加了内侧半月板细胞中SOX9和Smad3之间的复合物形成。染色质免疫沉淀分析显示，ROCKi处理增加了SOX9/Smad3转录复合物与COL2A1增强子区域的结合。
本研究表明，ROCK抑制通过Smad3在内侧半月板细胞中的直接核转位，刺激了SOX9/Smad3依赖的COL2A1表达。我们的结果表明，ROCK抑制可通过内侧半月板细胞中Smad3的协同激活来刺激II型胶原蛋白的合成。ROCKi处理可能有助于促进受损内侧半月板的纤维软骨愈合。
