Effect of compressive force on TGF-β signaling pathway in MC3T3-E1 cells

Objectives: Bone remodeling is induced by therapeutic mechanical stress that occurs during orthodontic tooth movement. A higher activity of bone remodeling is required to achieve effective tooth movement. Transforming growth factor (TGF)-βs expression is strongly expressed in osteoblasts during bone development. However, the mechanism linkage between mechanical stress and TGF-βs signaling pathway in osteoblasts remains unclear. The aim of this study was to identify effects of the compressive force (CF) on TGF-β signaling path ways in MC3T3-E1 cells.
Methods: MC3T3-E1 osteoblast-like cells were cultured in α-MEM containing 10% (v/v) heat-inactivated FBS at 37 °C in a humidified atmosphere of 95 % air and 5 % CO₂. The cells were loaded by placing a lead weight with 1.0 or 2.0 g/cm² CF for 1, 3, and 6, and 9 hr. Unloaded cells were used as controls. The expressions of TGF-β1, TGF-β2 and their receptors (TβRI/II) were measured using real-time PCR and Western blot analysis. Intracellular mediators of TGF-β signaling, p-Smad2 and p-p38 were analyzed by Western blot analysis, and Runx2 and Osterix were analyzed by RT-PCR. We also examined the inhibitory effect of TβRI/II using specific inhibitor (LY2109761) on downstream signal expressions.
Results: Loading the osteoblast cells with 1.0 g/cm² CF for 3 hr significantly increased the expressions of TGF-β1 and TGF-β2 comparing with control. There was no significantly difference TGF-βs and receptors expressions between loading cells with 2.0 g/cm² CF and control. Regarding downstream expressions, the loading with 1.0 g/cm² CF for 6hr was induced p-Smad2, p-p38, Runx2, and Osterix expressions. The phosphorylation of Smad2 and p-38 were significantly reduced when the the loading with 1.0 g/cm² CF for 6hr osteoblasts were cultured with an inhibitor of TβRI/II. Interestingly, Runx2 and Osterix expressions were also significantly influenced by the inhibitor.
Conclusions: This study demonstrates that 1.0 g/cm² CF may stimulate osteogenetic activity via increasing TGF-βs