Wang Dan, Cai Jing, Zeng Zhaobin, Gao Xue, Shao Xi, Ding Yuanjun, Feng Xue, Jing Da
Laboratory of Tissue Engineering, Faculty of Life Sciences, Northwest University, Xi'an, China.
Department of Biomedical Engineering, Fourth Military Medical University, Xi'an, China.
J Cell Physiol. 2020 Dec 2. doi: 10.1002/jcp.30184.
Mechanical stretch is known to promote osteoblast differentiation in vitro and accelerate bone regeneration in vivo, whereas the relevant mechanism remains unclear. Recent studies have shown the importance of reciprocal interactions between mammalian target of rapamycin (mTOR) and nuclear factor kappa B (NF-κB; two downstream molecules of Akt) in the regulation of tumor cells. Thus, we hypothesize that mTOR and NF-κB as well as their interconnection play a critical role in mediating stretch-induced osteogenic differentiation in osteoblasts. We herein found that mechanical stretch (10% elongation at six cycles/min) significantly promoted the expression of osteoblast differentiation-related markers (including ALP, BMP2, Col1α, OCN, and Runx2) in osteoblast-like MG-63 cells, accompanied by increased mTOR phosphorylation and NF-κB p65 phosphorylation and nuclear translocation. Blockade of mTOR by antagonist or small interfering RNA suppressed osteogenesis-related gene expression in response to mechanical stretch, whereas inhibition of NF-κB further increased stretch-induced osteoblast differentiation. Moreover, inhibition of mTOR decreased the phosphorylation of NF-κB, and blockade of NF-κB reduced the mTOR activation in MG63 cells under mechanical stretch. Coinhibition of mTOR and NF-κB abolishes the alteration of osteogenic differentiation induced by single mTOR or NF-κB inhibition under mechanical stretch, which is equivalent to the noninhibition level for osteoblasts under mechanical stretch. The expression levels of osteogenic differentiation in osteoblasts after inhibition of Akt were similar to those after co-inhibition of mTOR and NF-κB under mechanical stretch. This study for the first time reveals the reciprocal interconnection between mTOR and NF-κB in osteoblasts under mechanical stretch and indicates that mTOR and NF-κB as well as their interactions play a key role in the regulation of cellular homeostasis of osteoblasts in response to mechanical stretch. These findings are helpful for enriching our basic knowledge of the molecular mechanisms of osteoblast mechanotransduction, and also providing insight into the clinical therapeutic modality associated with mechanical stretch (e.g., distraction osteogenesis).
已知机械拉伸可在体外促进成骨细胞分化，并在体内加速骨再生，但其相关机制仍不清楚。最近的研究表明，雷帕霉素哺乳动物靶蛋白（mTOR）与核因子κB（NF-κB；Akt的两个下游分子）之间的相互作用在肿瘤细胞调控中具有重要意义。因此，我们推测mTOR和NF-κB及其相互联系在介导成骨细胞拉伸诱导的成骨分化中起关键作用。我们在此发现，机械拉伸（以每分钟6个循环的速度伸长10%）显著促进了成骨样MG-63细胞中成骨细胞分化相关标志物（包括碱性磷酸酶、骨形态发生蛋白2、I型胶原α1链、骨钙素和Runx2）的表达，同时mTOR磷酸化以及NF-κB p65磷酸化和核转位增加。用拮抗剂或小干扰RNA阻断mTOR可抑制机械拉伸诱导的成骨相关基因表达，而抑制NF-κB则进一步增强拉伸诱导的成骨细胞分化。此外，抑制mTOR可降低NF-κB的磷酸化，阻断NF-κB可减少机械拉伸下MG63细胞中mTOR的激活。同时抑制mTOR和NF-κB可消除机械拉伸下单独抑制mTOR或NF-κB所诱导的成骨分化改变，这与机械拉伸下成骨细胞的非抑制水平相当。在机械拉伸下，抑制Akt后成骨细胞中的成骨分化表达水平与同时抑制mTOR和NF-κB后的水平相似。本研究首次揭示了机械拉伸下成骨细胞中mTOR与NF-κB之间的相互联系，并表明mTOR和NF-κB及其相互作用在响应机械拉伸时对成骨细胞的细胞稳态调节中起关键作用。这些发现有助于丰富我们对成骨细胞机械转导分子机制的基础知识，也为深入了解与机械拉伸相关的临床治疗方式（如牵张成骨）提供了思路。
