Background and purpose
The sonic hedgehog pathway (Shh) plays a central role in maintaining stem cell function and behaviour in various processes related to self-renewal and tissue regeneration. However, the therapeutic effect of Shh on mouse embryonic stem cells (mESCs) has not yet been clearly elucidated. Thus, we investigated the effect of Shh on the regulation of mESC behaviour as well as the effect of Shh-pretreated mESCs in skin wound healing. Experimental approach: The underlying mechanisms of Shh signalling pathway in growth and motility of mESCs were investigated using Western blot analysis, a cell proliferation assay and cell migration assay. In addition, the effect of Shh-pretreated mESCs in skin wound healing was determined using a mouse excisional wound splinting model. Key
Purpose
The sonic hedgehog pathway (Shh) plays a central role in maintaining stem cell function and behaviour in various processes related to self-renewal and tissue regeneration. However, the therapeutic effect of Shh on mouse embryonic stem cells (mESCs) has not yet been clearly elucidated. Thus, we investigated the effect of Shh on the regulation of mESC behaviour as well as the effect of Shh-pretreated mESCs in skin wound healing. Experimental approach: The underlying mechanisms of Shh signalling pathway in growth and motility of mESCs were investigated using Western blot analysis, a cell proliferation assay and cell migration assay. In addition, the effect of Shh-pretreated mESCs in skin wound healing was determined using a mouse excisional wound splinting model. Key
Results
Shh disrupted the adherens junction through proteolysis by activating MMPs. In addition, the release of β-catenin from adherens junctions mediated by Shh led to cell cycle-dependent mESC proliferation. Shh-mediated Gli1 expression led to integrin β1 up-regulation, followed by FAK and Src phosphorylation. Furthermore, among the Rho-GTPases, Rac1 and Cdc42 were activated in a Shh-dependent manner while F-actin expression was suppressed by Rac1 and Cdc42 siRNA transfection. Consistent with the in vitro results, the skin wound healing assay revealed that Shh-treated mESCs increased angiogenesis and skin wound repair compared to that in Shh-treated mESCs transfected with integrin β1 siRNA in vivo. Conclusions and implications: Our results imply that Shh induces adherens junction disruption and integrin β1-dependent F-actin formation by a mechanism involving FAK/Src and Rac1/Cdc42 signalling pathways in mESCs.