Effect of Compressive Force on TGF-β1/2 Signaling Pathway in MC3T3-E1 Cells

Abstract

Transforming growth factor (TGF)-β are strongly associated with osteoblast differentiation. Mechanical stress including compressive force (CF) also involve in osteoblast differentiation via the alteration of the expression of bone-specific transcription factors, namely Runx2 and Osterix. However, the role of TGF-βs in mediating the effects of CF on osteoblasts remains unclear. In the present study, we examined the effects of CF on the expression of TGF-β1, TGF-β2, TGF-β type 1 and 2 receptor (Tβr1 and Tβr2), and Runx2 and Osterix in osteoblasts. We also investigated the effects of CF on the phosphorylation of Smad2, Smad3 and p38; which were located on the downstream of Tβrs. Effect of Tβr inhibitor (LY2109761) on expression of Runx2 and Osterix, and phosphorylation of Smad2, Smad3 and p38 were additionally examined. Cultured MC3T3-E1 osteoblast-like cells were subjected, or not, to continuous CF (1.0 g/ cm 2 or 2.0 g/cm 2 ) for 1-9 h. TGF-β1, TGF-β2, Tβr1, Tβr2, Runx2 and Osterix expression were measured by real-time polymerase chain reaction and Western blot analysis. Phosphorylation levels of Smad2, Smad3 and p38 were determined by Western blot analysis. The mRNA and protein expression of TGF-β1 and TGF-β2 were significantly increased by 1.0 g/cm 2 , but not 2.0 g/cm 2 , CF for 3-6 h, relative to control cells; Tβr1 and Tβr2 expression were unaffected by either CF condition. The 1.0 g/cm 2 CF also increased the phosphorylation of Smad2, Smad3 and p38 and the expression of Runx2 and Osterix, and these increases were attenuated by pretreatment with LY2109761. The present findings indicate that 1.0 g/cm 2 CF can induce bone-specific transcription factors via autocrine action of CF-induced TGF-β signaling in osteoblasts.

Mimicking Bone Extracellular Matrix: From BMP‐2‐Derived Sequences to Osteogenic‐Multifunctional Coatings

“…As a results of such interaction, the activated BMPR-I recruits BMPR-II, forming the BMP-induced signaling complex, and thus, activation of Smad-independent signaling pathways occurs, triggering overexpression of Osterix and ALP. [11,[92][93][94][95] In a previous work, we demonstrated that the RGD-DWIVA peptide activated Smad-independent signaling through the p38 pathway, [59] which is in agreement with the high expression of Osterix and ALP genes observed at days 4 and 7 in the present study.…”

Protease-degradable hydrogels with multifunctional biomimetic peptides for bone tissue engineering

“…Such discrepancies reflect how critical is the translation of in vitro testing from 2D to 3D environments, in which cell behavior may substantially vary (Duval et al, 2017;Mirbagheri et al, 2019;Jensen and Teng, 2020). Moreover, the overexpression of ALP and Osterix genes is paramount in our system, as they are involved in the Smadindependent pathway (Wei et al, 2015;Sano et al, 2017) [pathway triggered by our biomimetic peptides (Oliver-Cervelló et al, 2021)]. This is in agreement with the high expression of ALP at day 7 and Osterix at day 14 for the cRGD-DWIVA.…”

“…Moreover, the overexpression of ALP and Osterix genes is paramount in our system, as they are involved in the Smad-independent pathway ( Wei et al, 2015 ; Sano et al, 2017 ) [pathway triggered by our biomimetic peptides ( Oliver-Cervelló et al, 2021 )]. This is in agreement with the high expression of ALP at day 7 and Osterix at day 14 for the cRGD-DWIVA.…”