Department of Oral and Maxillofacial Surgery, The Affiliated Hospital of Stomatology, Zhejiang University, Hangzhou, Zhejiang 310006, PR China.
Int J Mol Med. 2012 Nov;30(5):1119-25. doi: 10.3892/ijmm.2012.1107. Epub 2012 Aug 23.
Recent studies have suggested that synovium-derived mesenchymal stem cells (SMSCs) may be promising candidates for tissue engineering and play an important role in cartilage regeneration. However, the mechanisms of SMSC chondrogenesis remain to be identified and characterized. The aim of this study was to evaluate the activation of the RhoA/Rho kinase (ROCK) pathway, as well as the manner by which it may contribute to chondrogenesis and the actin cytoskeletal organization of rat temporomandibular SMSCs in response to transforming growth factor-β1 (TGF-β1). Primary isolated SMSCs were treated with TGF-β1, and their actin organization was examined by fluorescein isothiocyanate-phalloidin staining. The specific biochemical inhibitors, C3 transferase, Y27632 and SB431542, were employed to evaluate the function of RhoA/ROCK and Smads. The effect of C3 transferase and Y27632 on the gene expression of chondrocyte-specific markers was evaluated by quantitative real-time polymerase chain reaction. To examine the effect of Y27632 on Smad2/3 phosphorylation induced by TGF-β1, western blot analysis was also performed. The stimulation of TGF-β1 in SMSCs resulted in the activation of the RhoA/ROCK pathway and concomitantly induced cytoskeletal reorganization, which was specifically blocked by C3 transferase and Y27632. The TGF-β-induced gene expression of Sox9, type I collagen, type II collagen and aggrecan was also inhibited by both C3 transferase and Y27632, at different levels. Y27632 treatment reduced the phosphorylation of Smad2/3 in a concentration-dependent manner. These results demonstrate the RhoA/ROCK activation regulates chondrocyte-specific gene transcription and cytoskeletal organization induced by TGF-β1 by interacting with the Smad pathway. This may have significant implications for the successful utilization of SMSCs as a cell source for articular cartilage tissue engineering.
最近的研究表明，滑膜衍生间充质干细胞（SMSCs）可能是组织工程有前途的候选物，并在软骨再生中发挥重要作用。然而，SMSC 软骨生成的机制仍有待确定和表征。本研究旨在评估 RhoA/Rho 激酶（ROCK）通路的激活，以及该通路在转化生长因子-β1（TGF-β1）刺激下对大鼠颞下颌 SMSC 软骨生成和肌动蛋白细胞骨架组织的作用。原代分离的 SMSCs 用 TGF-β1 处理，并用荧光异硫氰酸荧光素鬼笔环肽染色检查肌动蛋白组织。使用特定的生化抑制剂 C3 转移酶、Y27632 和 SB431542 来评估 RhoA/ROCK 和 Smads 的功能。通过定量实时聚合酶链反应评估 C3 转移酶和 Y27632 对软骨细胞特异性标记物基因表达的影响。为了研究 Y27632 对 TGF-β1 诱导的 Smad2/3 磷酸化的影响，还进行了 Western blot 分析。TGF-β1 刺激 SMSCs 导致 RhoA/ROCK 通路的激活，并同时诱导细胞骨架重排，这一过程可被 C3 转移酶和 Y27632 特异性阻断。C3 转移酶和 Y27632 也不同程度地抑制 TGF-β 诱导的 Sox9、I 型胶原、II 型胶原和聚集蛋白聚糖的基因表达。Y27632 处理以浓度依赖的方式降低 Smad2/3 的磷酸化。这些结果表明，RhoA/ROCK 激活通过与 Smad 通路相互作用调节 TGF-β1 诱导的软骨细胞特异性基因转录和细胞骨架组织。这对于成功利用 SMSCs 作为关节软骨组织工程的细胞来源具有重要意义。
