Evaluating the Role of Wnt/β-Catenin Signaling in Tenogenic Stem Cell Differentiation
Additional Funding Sources
The project described was supported by the Arnold and Mabel Beckman Foundation through a Beckman Scholars Program award to the University of Idaho.
Presentation Date
7-2020
Abstract
Tendon injuries are common, and tendons possess poor self-healing capacity, resulting in long-term dysfunction. Few treatment options exist, motivating the need to differentiate stem cells towards the tendon lineage (tenogenesis) for new regenerative strategies. Transforming growth factor (TGF)β2 has been explored as a regulator of tenogenesis in mesenchymal stem cells (MSCs) and has been found to impact levels of both β-catenin and the cell-cell junction protein, N-cadherin. β-catenin is a dual-role protein that may link cadherins to the actin cytoskeleton or translocate into the nucleus, where it facilitates canonical Wnt signaling. Based on these findings, we hypothesized that β-catenin regulates tenogenesis in MSCs via Wnt/β-catenin signaling and cadherin interactions. To test this hypothesis, murine MSCs were treated with TGFβ2, 6-bromoindirubin-3'-oxime (6BIO); an inactivator of the β-catenin regulation complex that allows intracellular β-catenin accumulation, TGFβ2+6BIO, or vehicle controls. Western blotting or immunofluorescence staining at 1, 3, 7, and 14 day (d) timepoints were used to evaluate β-catenin and N-cadherin protein levels and localization. Western blot analysis showed that TGFβ2 treatment significantly decreased cellular levels of β-catenin at 3d, but levels were maintained with TGFβ2+6BIO. TGFβ2+6BIO altered tenogenic cell morphology. TGFβ2 globally decreased N-cadherin levels, and was not impacted by 6BIO. Immunofluorescence imaging demonstrated low levels of β-catenin in early timepoints, and an increase in nuclear translocation in 7d and 14d timepoints. β-catenin nuclear translocation appears to be regulated by TGFβ2-induced tenogenesis in MSCs. These findings will improve our understanding of the β-catenin interactions in tenogenesis to improve tendon regeneration strategies.
Evaluating the Role of Wnt/β-Catenin Signaling in Tenogenic Stem Cell Differentiation
Tendon injuries are common, and tendons possess poor self-healing capacity, resulting in long-term dysfunction. Few treatment options exist, motivating the need to differentiate stem cells towards the tendon lineage (tenogenesis) for new regenerative strategies. Transforming growth factor (TGF)β2 has been explored as a regulator of tenogenesis in mesenchymal stem cells (MSCs) and has been found to impact levels of both β-catenin and the cell-cell junction protein, N-cadherin. β-catenin is a dual-role protein that may link cadherins to the actin cytoskeleton or translocate into the nucleus, where it facilitates canonical Wnt signaling. Based on these findings, we hypothesized that β-catenin regulates tenogenesis in MSCs via Wnt/β-catenin signaling and cadherin interactions. To test this hypothesis, murine MSCs were treated with TGFβ2, 6-bromoindirubin-3'-oxime (6BIO); an inactivator of the β-catenin regulation complex that allows intracellular β-catenin accumulation, TGFβ2+6BIO, or vehicle controls. Western blotting or immunofluorescence staining at 1, 3, 7, and 14 day (d) timepoints were used to evaluate β-catenin and N-cadherin protein levels and localization. Western blot analysis showed that TGFβ2 treatment significantly decreased cellular levels of β-catenin at 3d, but levels were maintained with TGFβ2+6BIO. TGFβ2+6BIO altered tenogenic cell morphology. TGFβ2 globally decreased N-cadherin levels, and was not impacted by 6BIO. Immunofluorescence imaging demonstrated low levels of β-catenin in early timepoints, and an increase in nuclear translocation in 7d and 14d timepoints. β-catenin nuclear translocation appears to be regulated by TGFβ2-induced tenogenesis in MSCs. These findings will improve our understanding of the β-catenin interactions in tenogenesis to improve tendon regeneration strategies.