Department of Stomatology, Qilu Hospital, and Institute of Stomatology, Shandong University, Jinan, Shandong, China.
Department of Periodontology, School of Stomatology, Shandong University, Jinan, Shandong, China.
J Periodontal Res. 2018 Feb;53(1):66-72. doi: 10.1111/jre.12488. Epub 2017 Aug 31.
The majority of experiments show that tumor necrosis factor-alpha (TNF-α) inhibits osteogenic differentiation of mesenchymal stem cells and pre-osteoblasts by activated nuclear factor-kappaB (NF-κB) signaling. However, the underlying mechanisms by which NF-κB signaling inhibits osteogenic differentiation are not fully understood. The aim of the present study was to investigate whether EphB4 signaling inhibition mediates the effects of TNF-α-activated NF-κB signaling on osteogenic differentiation of pre-osteoblasts.
Murine MC3T3-E1 pre-osteoblasts were treated with 10 ng/mL of TNF-α. NF-κB inhibitor, pyrrolidine dithiocarbamate, was used to achieve NF-κB signaling inhibition. EphB4 signaling was activated using ephrinB2-fc. The mRNA expressions of runt related transcription factor 2 (Runx2), bone sialoprotein (BSP) and EphB4 were determined using reverse transcription-polymerase chain reaction. The protein levels of Runx2, BSP, Col Ia1, osteopontin, EphB4, p-NF-κB p65 and NF-κB p65 were evaluated using western blot assays. Alkaline phosphatase (ALP) activity in MC3T3-E1 cells was evaluated by ALP activity kit, and mineral nodule formation was evaluated by Alizarin Red S staining.
TNF-α inhibited EphB4 expression, while it suppressed Runx2, BSP expression from gene and protein levels as well as ALP activity and mineral nodule formation in MC3T3-E1 cells. Activation of EphB4 signaling by ephrinB2-fc promoted osteogenic differentiation of MC3T3-E1 cells, whereas TNF-α impaired the osteogenic differentiation enhanced by ephrinB2-fc. Pyrrolidine dithiocarbamate blocked the activation of NF-κB signaling induced by TNF-α, while it prevented the downregulation of Runx2, BSP and EphB4, induced by TNF-α.
TNF-α inhibits osteogenic differentiation of pre-osteoblasts by downregulation of EphB4 signaling via activated NF-κB signaling pathway.
大多数实验表明，肿瘤坏死因子-α（TNF-α）通过激活核因子-κB（NF-κB）信号抑制间充质干细胞和成骨前体细胞的成骨分化。然而，NF-κB 信号抑制成骨分化的潜在机制尚不完全清楚。本研究旨在探讨 EphB4 信号抑制是否介导 TNF-α 激活的 NF-κB 信号对成骨前体细胞成骨分化的影响。
用 10ng/mL TNF-α处理小鼠 MC3T3-E1 成骨前体细胞。使用 NF-κB 抑制剂吡咯烷二硫代氨基甲酸盐（pyrrolidine dithiocarbamate）实现 NF-κB 信号抑制。使用 EphrinB2-fc 激活 EphB4 信号。采用逆转录聚合酶链反应（reverse transcription-polymerase chain reaction）测定 runt 相关转录因子 2（Runx2）、骨涎蛋白（BSP）和 EphB4 的 mRNA 表达。采用 Western blot 检测 Runx2、BSP、Col Ia1、骨桥蛋白（osteopontin）、EphB4、p-NF-κB p65 和 NF-κB p65 的蛋白水平。通过碱性磷酸酶（ALP）活性试剂盒评估 MC3T3-E1 细胞中的 ALP 活性，通过茜素红 S 染色评估矿化结节形成。
TNF-α抑制 EphB4 表达，同时抑制 MC3T3-E1 细胞中 Runx2、BSP 的基因和蛋白水平表达以及 ALP 活性和矿化结节形成。EphrinB2-fc 激活 EphB4 信号促进 MC3T3-E1 细胞的成骨分化，而 TNF-α则削弱 EphrinB2-fc 增强的成骨分化。吡咯烷二硫代氨基甲酸盐阻断 TNF-α诱导的 NF-κB 信号激活，同时防止 TNF-α诱导的 Runx2、BSP 和 EphB4 的下调。
TNF-α 通过激活的 NF-κB 信号通路下调 EphB4 信号，抑制成骨前体细胞的成骨分化。
