Tissue engineered and regenerative approaches for treating tendon injuries are challenged by the limited information on the cellular signaling pathways driving tenogenic differentiation of stem cells. Members of the transforming growth factor (TGF) β family, particularly TGFβ2, play a role in tenogenesis, which may proceed via Smad-mediated signaling. However, recent evidence suggests some aspects of tenogenesis may be independent of Smad signaling, and other pathways potentially involved in tenogenesis are understudied. Here, we examined the role of Akt/mTORC1/P70S6K signaling in early TGFβ2-induced tenogenesis of mesenchymal stem cells (MSCs) and evaluated TGFβ2-induced tenogenic differentiation when Smad3 is inhibited. Mouse MSCs were treated with TGFβ2 to induce tenogenesis, and Akt or Smad3 signaling was chemically inhibited using the Akt inhibitor, MK-2206, or the Smad3 inhibitor, SIS3. Effects of TGFβ2 alone and in combination with these inhibitors on the activation of Akt signaling and its downstream targets mTOR and P70S6K were quantified using western blot analysis, and cell morphology was assessed using confocal microscopy. Levels of the tendon marker protein, tenomodulin, were also assessed. TGFβ2 alone activated Akt signaling during early tenogenic induction. Chemically inhibiting Akt prevented increases in tenomodulin and attenuated tenogenic morphology of the MSCs in response to TGFβ2. Chemically inhibiting Smad3 did not prevent tenogenesis, but appeared to accelerate it. MSCs treated with both TGFβ2 and SIS3 produced significantly higher levels of tenomodulin at 7 days and morphology appeared tenogenic, with localized cell alignment and elongation. Finally, inhibiting Smad3 did not appear to impact Akt signaling, suggesting that Akt may allow TGFβ2-induced tenogenesis to proceed during disruption of Smad3 signaling. These findings show that Akt signaling plays a role in TGFβ2-induced tenogenesis and that tenogenesis of MSCs can be initiated by TGFβ2 during disruption of Smad3 signaling. These findings provide new insights into the signaling pathways that regulate tenogenic induction in stem cells.

中文翻译：

---

Akt信号传导被TGFβ2激活并影响间充质干细胞的腱鞘诱导

用于组织肌腱损伤的组织工程和再生方法受到驱动干细胞肌腱分化的细胞信号通路的有限信息的挑战。转化生长因子（TGF）β家族的成员，特别是TGFβ2，在肌腱发生中起作用，其可能通过Smad介导的信号传导进行。但是，最近的证据表明，腱鞘生成的某些方面可能独立于Smad信号传导，并且尚未充分研究可能参与腱鞘生成的其他途径。在这里，我们检查了Akt / mTORC1 / P70S6K信号在早期TGFβ2诱导的间充质干细胞（MSCs）腱鞘发生中的作用，并评估了当Smad3被抑制时TGFβ2诱导的腱鞘分化。用TGFβ2处理小鼠MSC以诱导肌腱形成，并且使用Akt抑制剂MK-2206或Smad3抑制剂SIS3化学抑制Akt或Smad3信号传导。使用蛋白质印迹分析定量了单独的TGFβ2以及与这些抑制剂联合使用对Akt信号及其下游靶标mTOR和P70S6K活化的影响，并使用共聚焦显微镜评估了细胞形态。还评估了肌腱标记蛋白Tenomodulin的水平。单独的TGFβ2在早期肌腱诱导期间激活了Akt信号传导。化学抑制Akt阻止了响应于TGFβ2的MSC的腱糖调节蛋白的增加和腱鞘形态的减弱。化学抑制Smad3不能阻止肌腱形成，但似乎可以加速肌腱形成。用TGFβ2和SIS3处理的MSC在7天时产生的Tenomodulin水平明显升高，并且形态表现为腱变性，具有局部细胞排列和延伸。最后，抑制Smad3似乎不影响Akt信号传导，表明Akt可能允许TGFβ2诱导的肌腱发生在Smad3信号传导中断期间继续进行。这些发现表明，Akt信号传导在TGFβ2诱导的肌腱形成中起作用，并且MSC的肌腱形成可以在Smad3信号传递的破坏期间由TGFβ2引发。这些发现为调节干细胞肌腱诱导的信号传导途径提供了新的见解。这些发现表明，Akt信号传导在TGFβ2诱导的肌腱形成中起作用，并且MSC的肌腱形成可以在Smad3信号传递的破坏期间由TGFβ2引发。这些发现为调节干细胞肌腱诱导的信号传导途径提供了新的见解。这些发现表明，Akt信号传导在TGFβ2诱导的肌腱形成中起作用，并且MSC的肌腱形成可以在Smad3信号传递的破坏期间由TGFβ2引发。这些发现为调节干细胞肌腱诱导的信号传导途径提供了新的见解。