Regulation of Tenomodulin Expression Via Wnt/β-catenin Signaling in Equine Bone Marrow-derived Mesenchymal Stem Cells

Abstract

Tenomodulin has been recognized as a biomarker for tendon differentiation, and its gene expression is regulated by several transcription factors including Scleraxis and Mohawk. In this study, we found a novel regulatory mechanism of tenomodulin expression. Equine bone marrow-derived mesenchymal stem cells (BMSCs) in monolayer culture showed a low mRNA level of tenomodulin in comparison with the level in the tendon. When cultured in collagen gel containing a glycogen synthase kinase-3 (GSK-3) inhibitor (BIO), expression of tenomodulin in BMSCs increased up to the level in the tendon. Participation of GSK-3 in its gene expression was further demonstrated by a gene silencing experiment with small interference RNA corresponding to GSK-3, suggesting that Wnt/β-catenin signaling mediated expression of tenomodulin. These results were confirmed by nuclear translocation of β-catenin in BIO-treated BMSCs cultured in collagen gel. Under this culture condition, expression of tenomodulin-related transcription factors including Scleraxis and Mohawk was not affected, suggesting that Wnt/β-catenin signaling was independent from these transcription factors. Additionally, BIO strongly enhanced expression of type XIV collagen in collagen-embedded BMSCs up to the level in the tendon, and other tendon-related extracellular matrix components such as decorin and fibromodulin were also upregulated. Taken together, these results indicated that activation of Wnt/β-catenin signaling could induce differentiation of BMSCs into tenomodulin-expressing tendon cells in collagen gel.

Concise Review: Stem Cell Fate Guided By Bioactive Molecules for Tendon Regeneration

“…Another study of Wnt signaling involved the treatment of BMSCs with the glycogen synthase kinase‐3 (GSK‐3) inhibitor‐BIO. In 3D BIO‐containing collagen gel, BIO induced the expression of Tnmd , Col14a1 , Decorin ( Dcn ), and fibromodulin ( Fmod ), but not in monolayer BMSCs . The inhibition of GSK‐3 with siRNA corresponding to GSK‐3 also resulted in the upregulation of Tnmd , which suggested that Wnt/β‐catenin signaling mediated the expression of Tnmd .…”

“…The roles of TGF‐β signaling in tendon homeostasis and tendon development have been elucidated, while the roles of Wnt/β‐catenin signaling in tendon remain mostly elusive. Recently, a novel regulatory mechanism was found in equine BMSCs, suggesting that Wnt/β‐catenin signaling mediated the expression of Tnmd, whereas Scx and Mkx were not affected . During this process, tendon‐related ECM components were also upregulated, which showed that the activation of Wnt/β‐catenin signaling induced the tenogenic differentiation of BMSCs.…”

TENOgenic MODULating INsider factor: systematic assessment on the functions of tenomodulin gene

“…The use of gene silencing experiments was valuable for understanding why bone marrow-derived stem cells (BMDSCs) from horse, which in culture have very low amounts of TNMD compared to tendons, showed similar TNMD expression when cultured in collagen gels containing a glycogen synthase kinase-3 (GSK-3) inhibitor (Miyabara et al 2014). Application of inhibitor or small interference RNA corresponding to GSK-3α/β into BMDSCs resulted in nuclear translocation of β-catenin and this drove the expression of TNMD .…”

“…Application of inhibitor or small interference RNA corresponding to GSK-3α/β into BMDSCs resulted in nuclear translocation of β-catenin and this drove the expression of TNMD . Since the levels of Scx and Mkx were unaffected in the above conditions, it points out that the Wnt/β-catenin signalling works independent from these transcription factors (Miyabara et al 2014). …”

“…The figure is based on studies showing mostly correlations, indicated by the question marks, between TNMD expression or function to other genes and not a direct link in a common signalling cascade. Based on Alberton et al 2012; Alberton et al 2015; Brent et al 2005; Docheva et al 2005; Frolova et al 2014; Kimura et al 2008; Kyriakides et al 1998; Lejard et al 2011; Liu et al 2010; Mendias et al 2008; Miyabara et al 2014; Murchison et al 2007; Shukunami et al 2006. Abbreviations: Egr, early growth response protein; MMP, matrix metalloproteinase; Thbs, thrombospondin; VEGF, vascular endothelial growth factor.…”