Kong Xiangwei, Liu Yan, Ye Ruidong, Zhu Bin, Zhu Yuan, Liu Xianghui, Hu Chenghu, Luo Hailang, Zhang Yongjie, Ding Yin, Jin Yan
Department of Orthodontics, School of Stomatology, Fourth Military Medical University, Xi'an, China; Research and Development Center for Tissue Engineering, Fourth Military Medical University, Xi'an, China; Department of Stomatology, Nanjing Bayi Hospital, Nanjing, China.
Biochim Biophys Acta. 2013 Nov;1830(11):5119-29. doi: 10.1016/j.bbagen.2013.07.027. Epub 2013 Aug 2.
The fate and differentiation of mesenchymal stem cells (MSCs) depend on various microenvironmental cues. In chronic inflammatory bone disease, bone regeneration is inhibited. The present study therefore sought to identify the underlying molecule mechanisms.
We isolated periodontal ligament stem cells (PDLSCs), a new population of MSCs, from the periodontal ligament tissues of periodontitis patients and healthy controls (p-PDLSCs and h-PDLSCs). The secretion of inflammatory cytokines, like TNF-α, IL-1β, IL-6 and IL-8, after LPS stimulation was measured by ELISA. The expressions of p-GSK3β and GSK3β in two types of PDLSCs were detected by Western blot. TOPFlash was used to assay the Tcf/Lef transcriptional activity. Knockdown of GSK3β by siRNA and over-expression of GSK3β by adenoviruses were performed to confirm the role of GSK3β in the impaired osteogenic differentiation of PDLSCs under inflammatory microenvironment.
We demonstrated that p-PDLSCs displayed impaired osteogenic capacity than h-PDLSCs. Upon inflammatory stimulation, monocytes, but not PDLSCs, released inflammatory cytokines among which TNF-α directly act on PDLSCs and suppressed their osteogenic differentiation. TNF-α induced the phosphorylation of GSK3β, the deactivated form of GSK3β, which increased nuclear β-catenin and Lef-1 accumulation, and eventually reduced the Runx2-associated osteogenesis in PDLSCs. Over-expression of GSK3β rescued osteogenesis in TNF-α-stimulated PDLSCs, whereas inactivation of GSK3β was sufficient to liberate the β-catenin/Lef-1/Runx2 pathway.
GSK3β plays an obligatory role in the TNF-α-mediated inhibition of osteogenesis in MSCs.
The strategy to target GSK3β may provide a potential approach to bone regeneration in inflammatory microenvironments.
间充质干细胞（MSCs）的命运和分化取决于多种微环境信号。在慢性炎症性骨病中，骨再生受到抑制。因此，本研究旨在确定其潜在的分子机制。
我们从牙周炎患者和健康对照者的牙周韧带组织中分离出牙周韧带干细胞（PDLSCs），这是一种新的MSCs群体（p-PDLSCs和h-PDLSCs）。通过ELISA检测脂多糖（LPS）刺激后炎性细胞因子如肿瘤坏死因子-α（TNF-α）、白细胞介素-1β（IL-1β）、白细胞介素-6（IL-6）和白细胞介素-8（IL-8）的分泌情况。通过蛋白质免疫印迹法检测两种类型PDLSCs中磷酸化糖原合成酶激酶3β（p-GSK3β）和糖原合成酶激酶3β（GSK3β）的表达。使用TOPFlash检测Tcf/Lef转录活性。通过小干扰RNA（siRNA）敲低GSK3β以及通过腺病毒过表达GSK3β，以证实GSK3β在炎性微环境下对PDLSCs成骨分化受损中的作用。
我们证明p-PDLSCs的成骨能力比h-PDLSCs受损。在炎性刺激下，单核细胞而非PDLSCs释放炎性细胞因子，其中TNF-α直接作用于PDLSCs并抑制其成骨分化。TNF-α诱导GSK3β（GSK3β的失活形式）磷酸化，这增加了细胞核内β-连环蛋白和淋巴样增强因子-1（Lef-1）的积累，并最终降低了PDLSCs中与Runx2相关的成骨作用。过表达GSK3β可挽救TNF-α刺激的PDLSCs中的成骨作用，而GSK3β的失活足以释放β-连环蛋白/Lef-1/Runx2信号通路。
GSK3β在TNF-α介导的MSCs成骨抑制中起关键作用。
靶向GSK3β的策略可能为炎性微环境下的骨再生提供一种潜在方法。
