Multipotent mesenchymal stromal cells (MSC) represent a promising therapeutic tool for tendon regeneration. They are capable of tenogenic differentiation, which is one of the mechanisms contributing to the regenerative effect of MSC after transplantation into tendon lesions and is furthermore required for tissue engineering approaches. Tenogenic differentiation is most typically induced by growth factors such as transforming growth factor (TGF)-β3, which involves intracellular activation of smad signalling . On the other hand, tenogenic differentiation was shown to be induced by mechanical stimuli and extracellular matrix (ECM) components, for which rho/ROCK signalling was demonstrated as essential . In addition, previous studies demonstrated an alteration of TGF-β3-induced tenogenic differentiation during culture on tendon ECM . This suggests that there is an interaction between the different tenogenic induction mechanisms and their signalling pathways. Phosphorylation of the smad2/3 molecule at different phosphorylation sites represents a possible interface. While TGF-β3 is known to phosphorylate the carboxy-terminal region of smad2/3 and thereby activate it, some studies described an inhibitory phosphorylation of the linker region of smad2/3 by the rho/ROCK pathway. Here, the interplay of rho/ROCK and TGF-β3/smad signalling in tenogenic differentiation were investigated, with the smad2/3 molecules as possible interface.
Primary human adipose-derived MSC were cultured as monolayers, on equine tendon-derived decellularized scaffolds or on 3D collagen I gels. ROCK was inhibited by adding Y-27632 (10 µM) to the culture medium 2 h before further stimulation, then tenogenic differentiation was induced by adding TGF-β3 (10 ng/ml). Control cells were cultured accordingly, without Y-27632 and/or without TGF-β3. MSC were analyzed at different time points by real-time RT-PCR, immunofluorescence and western blot analysis.
TGF-β3 stimulation caused an increase in gene expression of the tendon marker scleraxis under all culture conditions. The strongest upregulation occurred when TGF-β3 and ROCK inhibition were combined . Translocation of smad2/3 and scleraxis to the nucleus were observed accordingly by immunofluorescence staining. Furthermore, preliminary western blot analyses indicated a reduced linker region smad (S245/250/255) phosphorylation upon ROCK inhibition. ROCK inhibition was confirmed by disruption of the actin cytoskeleton.
In conclusion, the results showed that ROCK inhibition promotes the TGF-β3/smad2/3 axis, which might due to reduced inhibitory phosphorylation of the smad molecule in the linker region.
1. Nakao, A. et al., The EMBO Journal, vol. 16, no. 17, pp. 5353–5362 (1997)
2. Maharam, E. et al., Bone research, vol. 3, no. 1, p. 15015, (2015)
3. Roth, S. P. et al., Cell Transplantation, vol. 27, no. 10, pp. 1434–1450 (2018)
4. Melzer, M. et al., Stem Cells International, 8, 2021:8284690 (2021)