Abstract
Introduction
Modulation of signaling pathways, which involves tendon development, regeneration, or homeostasis, is one of the potential modalities to facilitate proper regeneration of the injured tendon. Authors have previously reported that activation of Wnt/beta-catenin signaling suppressed the expression of tenogenic genes (i.e. Scleraxis (Scx), Mohawk (Mkx), Tenomodulin (Tnmd)) in rat primary tendon-derived cells (TDCs) and SCX-transduced human mesenchymal stem cells (hMSC-Scx cells), as a tendon progenitor cell line (kindly provided Dr. Docheva). The roles of TGF-beta signaling in tenogenesis have been elucidated. The purpose of the study was to evaluate the effect of TGF-beta signaling on tenogenic genes and relationship between both two signalings in rat TDCs and hMSC-Scx cells.
Materials and Methods
Cell cultures
TDCs were harvested from the Achilles tendons of 6 week-old SD rats and the 3rd passage TDCs were used. To evaluate the effect of TGF-beta signaling, TGF-beta 1 protein and SD208 (TGF-beta receptor inhibitor) were utilized. To assess the effect of Wnt/beta-catenin signaling on TGF-beta signaling, BIO (Wnt/b-catenin activator) was utilized.
Quantative RT-PCR
TDCs and hMSC-Scx cells were cultured with TGF-beta1 or SD208. Total RNA was isolated from the cells and cDNA was synthesized. Expression of Axin2, as a target gene for Wnt/beta-catenin signaling, as well as tenogenic genes was quantified and normalized by Gapdh.
Western blotting
Each cells were cultured with BIO. Whole cell lysates were used for immunoblotting with antibodies against beta-actin and phosphorylated Smad2/Smad3 (p-Smad2/3), which indicates activation of TGF-beta signaling. Band intensity was quantified and normalized by beta-actin.
Statistical analysis
Two groups were compared by unpaired Student”s t-test. Multiple groups were analyzed by one-way analysis of variance (ANOVA). (P <0.05)
Results
Scx expression was increased by TGF-beta1 and decreased by SD208 in a dose-dependent manner in TDCs. However, TGF-beta1 and SD208 showed no significant effect on Mkx and Tnmd expression in TDCs and hMSC-Scx cells. BIO treatment decreased p-Smad2/3 proteins, while TGF-beta1 and SD208 had no effect on Axin2 expression in both cells.
Discussion
Activation of TGF-b signaling induced Scx expression, independent of Wnt/beta-catenin signaling. Activation of Wnt/beta-catenin signaling suppressed p-Smad2/3 amounts for TGF-beta signaling and further Scx expressions. Taken together, activation of Wnt/beta-catenin signaling antagonizes Scx expression induced by TGF-beta signaling. Identification of compounds, which control Wnt/beta-catenin and/or TGF-beta signaling, may lead to developments of novel therapeutic options to facilitate regeneration of injured tendons.
