Ye Xin, Huang Haiyun, Zhao Ning, Zhang Jin, Yang Pishan
School of Stomatology, Shandong University, 44-1 West Wen Hua Road, Jinan, Shandong Province, 250012, China.
Shandong Provincial Key Laboratory of Oral Tissue Regeneration, Shandong University, Jinan, China.
In Vitro Cell Dev Biol Anim. 2016 Dec;52(10):1026-1033. doi: 10.1007/s11626-016-0068-3. Epub 2016 Jul 11.
Tumor necrosis factor-alpha (TNF-α) inhibits osteogenic differentiation of murine bone marrow stromal cells, and transcription factor Runx2 serves as an essential regulation target in the process. The underlying mechanism may involve the regulation of Runx2 expression and the Runx2 activity in downstream gene transcription, which has not been fully elucidated. In this study, ST2 murine bone marrow-derived stromal cells were treated with bone morphogenetic protein-2 (BMP-2) and/or TNF-α in osteogenic medium, and the expression of Runx2 was estimated. Cells were transfected with Runx2, p65, inhibitor of κBα (IκBα), 9.0 kb bone sialoprotein (BSP) promoter-luciferase or osteoblast-specific cis-acting element 2 (OSE2)-luciferase reporter vectors, and then real time-PCR and dual luciferase analysis were used to investigate the effect of TNF-α on Runx2-activated osteogenic gene transcription and the molecular mechanism. We found that TNF-α inhibited BMP-2-induced osteogenic marker expression and both the spontaneous and BMP-2-induced Runx2 expression. TNF-α stimulation or overexpression of nuclear factor-kappa B (NF-κB) p65 subunit repressed the Runx2-activated BSP and osteocalcin (OC) transcriptions. The Runx2-induced 9.0 kb BSP promoter activity was attenuated by TNF-α or p65, while the OSE2 activity was not affected. Besides, blockage of NF-κB by IκBα overexpression eliminated these inhibitory effects of TNF-α on Runx2 signaling. These results suggest that in murine bone marrow stromal cells undergoing osteogenic differentiation, TNF-α and it activated NF-κB pathway inhibit the expression of Runx2 gene, and suppress the Runx2-mediated osteogenic gene transcription via the 9.0 kb BSP promoter.
肿瘤坏死因子-α（TNF-α）抑制小鼠骨髓基质细胞的成骨分化，转录因子Runx2在此过程中作为重要的调控靶点。其潜在机制可能涉及Runx2表达的调控以及下游基因转录中Runx2的活性调控，而这尚未完全阐明。在本研究中，将ST2小鼠骨髓来源的基质细胞在成骨培养基中用骨形态发生蛋白-2（BMP-2）和/或TNF-α处理，并评估Runx2的表达。细胞用Runx2、p65、κBα抑制剂（IκBα）、9.0 kb骨唾液酸蛋白（BSP）启动子-荧光素酶或成骨细胞特异性顺式作用元件2（OSE2）-荧光素酶报告载体转染，然后用实时PCR和双荧光素酶分析来研究TNF-α对Runx2激活的成骨基因转录的影响及其分子机制。我们发现TNF-α抑制BMP-2诱导的成骨标志物表达以及自发和BMP-2诱导的Runx2表达。TNF-α刺激或核因子-κB（NF-κB）p65亚基的过表达抑制了Runx2激活的BSP和骨钙素（OC）转录。TNF-α或p65减弱了Runx2诱导的9.0 kb BSP启动子活性，而OSE2活性未受影响。此外，IκBα过表达对NF-κB的阻断消除了TNF-α对Runx2信号的这些抑制作用。这些结果表明，在经历成骨分化的小鼠骨髓基质细胞中，TNF-α及其激活的NF-κB途径抑制Runx2基因的表达，并通过9.0 kb BSP启动子抑制Runx2介导的成骨基因转录。
