Siegfried Weller Research Institute, Department of Trauma and Reconstructive Surgery, Eberhard Karls University Tübingen, BG Trauma Center Tübingen, 72076 Tübingen, Germany.
Int J Mol Sci. 2019 Jun 14;20(12):2915. doi: 10.3390/ijms20122915.
It is well established that smoking has detrimental effects on bone integrity and is a preventable risk factor for metabolic bone disorders. Following orthopedic surgeries, smokers frequently show delayed fracture healing associated with many complications, which results in prolonged hospital stays. One crucial factor responsible for fracture repair is the recruitment and differentiation of mesenchymal stem cells (MSCs) at early stages, a mechanism mediated by transforming growth factor β (TGF-β). Although it is known that smokers frequently have decreased TGF-β levels, little is known about the actual signaling occurring in these patients. We investigated the effect of cigarette smoke on TGF-β signaling in MSCs to evaluate which step in the pathway is affected by cigarette smoke extract (CSE). Single-cell-derived human mesenchymal stem cell line (SCP-1 cells) were treated with CSE concentrations associated with smoking up to 20 cigarettes a day. TGF-β signaling was analyzed using an adenovirus-based reporter assay system. Primary cilia structure and downstream TGF-β signaling modulators (Smad2, Smad3, and Smad4) were analyzed by Western blot and immunofluorescence staining. CSE exposure significantly reduced TGF-β signaling. Intriguingly, we observed that protein levels of phospho-Smad2/3 (active forms) as well as nuclear translocation of the phospho-Smad3/4 complex decreased after CSE exposure, phenomena that affected signal propagation. CSE exposure reduced the activation of TGF-β modulators under constitutive activation of TGF-β receptor type I (ALK5), evidencing that CSE affects signaling downstream of the ALK5 receptor but not the binding of the cytokine to the receptor itself. CSE-mediated TGF-β signaling impaired MSC migration, proliferation, and differentiation and ultimately affected endochondral ossification. Thus, we conclude that CSE-mediated disruption of TGF-β signaling in MSCs is partially responsible for delayed fracture healing in smokers.
众所周知，吸烟对骨完整性有不利影响，是代谢性骨病的可预防危险因素。在骨科手术后，吸烟者经常表现出与许多并发症相关的骨折愈合延迟，这导致住院时间延长。负责骨折修复的一个关键因素是间充质干细胞（MSCs）在早期的募集和分化，这一机制是由转化生长因子 β（TGF-β）介导的。尽管已知吸烟者的 TGF-β水平经常降低，但对于这些患者中实际发生的信号通路知之甚少。我们研究了香烟烟雾对 MSC 中 TGF-β信号的影响，以评估香烟烟雾提取物（CSE）影响该通路的哪个步骤。用与每天吸烟 20 支香烟相关的 CSE 浓度处理单细胞衍生的人间充质干细胞系（SCP-1 细胞）。使用基于腺病毒的报告基因检测系统分析 TGF-β信号。通过 Western blot 和免疫荧光染色分析初级纤毛结构和下游 TGF-β信号调节剂（Smad2、Smad3 和 Smad4）。CSE 暴露显著降低了 TGF-β信号。有趣的是，我们观察到 CSE 暴露后磷酸化 Smad2/3（活性形式）的蛋白水平以及磷酸化 Smad3/4 复合物的核转位均降低，这些现象影响了信号传递。CSE 暴露降低了 TGF-β 受体 I（ALK5）组成性激活下 TGF-β调节剂的激活，表明 CSE 影响 ALK5 受体下游的信号传导，而不是细胞因子与受体本身的结合。CSE 介导的 TGF-β信号传导损害了 MSC 的迁移、增殖和分化，并最终影响软骨内骨化。因此，我们得出结论，CSE 介导的 MSC 中 TGF-β 信号传导的破坏部分导致了吸烟者骨折愈合延迟。
