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Involvement of BMPs/smad signaling pathway in mechanical response in osteoblasts

机译：BMP / smad信号通路参与成骨细胞机械反应的过程

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Background/Aims: Mechanical strain plays an important role in osteoblasts differentiation and bone formation but the underlying mechanism remains unclear. The aim of this study was to determine whether Bone Morphogenetic Proteins (BMPs)/Smad signaling pathway is involved in mechanical response in osteoblasts. Methods: MC3T3-E1 cells were exposed to mechanical strain via a four-point bending system. mRNA levels and protein levels of BMP-2, BMP-4, Smad1, Smad5, Smurf1, and Smurf2 were assessed using RT-PCR and immunoblotting. Protein levels of BMP-2 and BMP-4 in the culture medium were also determined using Enzyme-linked Immunosorbent Assay (ELISA). Pretreatment with Noggin and transfection with Smad4 siRNA were carried out to block the BMPs/Smad signaling pathway and MG132 was used to inhibit the proteasome pathway. Results: We found that mechanical strain enhanced alkaline phosphatase (ALP) expression and activated BMPs/Smad signaling pathway. Mechanical strain induced expression of ALP was attenuated by Noggin and by Smad4 siRNA. The protein levels of Smad1 and Smad5, but not their mRNA levels, were up-regulated by mechanical strain. This finding could be explained by the down-regulation of Smurf1. The protein degradation of Smad might be inhibited by mechanical strain through down-regulation of Smuf1 expression. The addition of MG132 further enhanced the mechanical strain induced activation of Smad proteins and the increased expression of ALP. Conclusions: Mechanical strain might promote osteoblasts differentiation through BMPs/Smad signaling pathway. The strain causes a drop in Smurf1 levels, leading to accumulation of Smad proteins and, subsequently, to enhanced BMPs/Smad signaling.
机译：背景/目的：机械应变在成骨细胞分化和骨形成中起着重要作用，但其潜在机制仍不清楚。这项研究的目的是确定成骨细胞的机械反应是否涉及骨形态发生蛋白（BMPs）/ Smad信号通路。方法：通过四点弯曲系统使MC3T3-E1细胞受到机械应变。使用RT-PCR和免疫印迹法评估BMP-2，BMP-4，Smad1，Smad5，Smurf1和Smurf2的mRNA水平和蛋白质水平。还使用酶联免疫吸附测定法（ELISA）测定了培养基中BMP-2和BMP-4的蛋白水平。用Noggin预处理并用Smad4 siRNA转染来阻断BMPs / Smad信号通路，并用MG132抑制蛋白酶体通路。结果：我们发现机械应变增强了碱性磷酸酶（ALP）的表达并激活了BMP / Smad信号通路。机械应变诱导的ALP表达被Noggin和Smad4 siRNA减弱。 Smad1和Smad5的蛋白质​​水平，但不是其mRNA水平，是由机械应变上调的。 Smurf1的下调可以解释这一发现。 Smad的蛋白降解可能通过下调Smuf1表达而受到机械应变的抑制。 MG132的加入进一步增强了机械应变诱导的Smad蛋白的活化和增加的ALP表达。结论：机械应变可能通过BMPs / Smad信号通路促进成骨细胞分化。该菌株导致Smurf1水平下降，导致Smad蛋白积聚，进而导致BMP / Smad信号增强。

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