Akt signaling is activated by TGFβ2 and impacts tenogenic induction of mesenchymal stem cells

Background: 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.

Methods: 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.

Results: 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.

Conclusions: 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.