Zou Ting, Dissanayaka Waruna Lakmal, Jiang Shan, Wang Shuai, Heng Boon Chin, Huang Xiaojing, Zhang Chengfei
Endodontology, Faculty of Dentistry, The University of Hong Kong, Hong Kong, China.
Department of Endodontics and Operative Dentistry, School and Hospital of Stomatology, Fujian Medical University, Fuzhou, Fujian, China.
J Endod. 2017 Feb;43(2):297-305. doi: 10.1016/j.joen.2016.10.019. Epub 2016 Dec 24.
To investigate the roles of semaphorin 4D (Sema4D)/plexin-B1 signaling on the angiogenic potential and osteo-/odontogenic differentiation of human dental pulp stem cells (DPSCs) and to uncover the corresponding molecular mechanisms.
DPSCs were treated with Sema4D (10 μg/mL) for different time durations. Osteo-/odontogenic differentiation was assessed by quantifying alkaline phosphatase activity, mineralized nodule formation, and osteo-/odontogenic gene (ALP, Col1A1, BSP, RUNX2, and DSPP) and protein (Col1A1 and DSPP) expression. Involvement of the Sema4D/plexin-B1 signaling pathway was analyzed by Western blot analysis. Additionally, angiogenic gene and protein expression was assessed by reverse-transcription polymerase chain reaction and enzyme-linked immunosorbent assay. In vitro endothelial tube formation assay on Matrigel (BD Biosciences, San Jose, CA) was performed to evaluate the angiogenic inductive potential of the Sema4D-treated DPSCs conditioned medium. Results were analyzed using 1-way analysis of variance and the Student t test.
Sema4D significantly inhibited ALP activity and mineralized nodule formation of DPSCs. Furthermore, Sema4D-treated DPSCs displayed marked down-regulation in the expression of osteo-/odontogenic genes (ALP, Col1A1, BSP, RUNX2, and DSPP) as well as proteins (Col1A1 and DSPP). Elevated levels of plexin-B1 and downstream RhoA protein expression together with phosphorylated plexin-B1 confirmed the involvement of Sema4D/plexin-B1 signaling. Protein expression of ErbB2 was up-regulated, and Met was slightly down-regulated. Furthermore, Sema4D-treated DPSCs exhibited enhanced expression of vascular endothelial growth factor at both the messenger RNA and protein level. Accordingly, the conditioned medium of Sema4D-treated DPSCs promoted the formation of vessel-like structures as shown by the Matrigel assay.
Sema4D markedly enhances the angiogenic potential but suppresses osteo-/odontogenic differentiation of DPSCs. Sema4D/plexin-B signaling was activated via the RhoA-mediated pathway.
研究信号素4D（Sema4D）/丛状蛋白-B1信号通路在人牙髓干细胞（DPSC）血管生成潜能及成骨/成牙潜能分化中的作用，并揭示相应的分子机制。
用Sema4D（10μg/mL）处理DPSC不同时间。通过定量碱性磷酸酶活性、矿化结节形成以及成骨/成牙基因（碱性磷酸酶、I型胶原蛋白α1链、骨桥蛋白、Runx2和牙本质涎磷蛋白）和蛋白质（I型胶原蛋白α1链和牙本质涎磷蛋白）表达来评估成骨/成牙潜能分化。通过蛋白质免疫印迹分析来分析Sema4D/丛状蛋白-B1信号通路的参与情况。此外，通过逆转录聚合酶链反应和酶联免疫吸附测定来评估血管生成基因和蛋白质表达。在基质胶（BD生物科学公司，加利福尼亚州圣何塞）上进行体外内皮管形成试验，以评估经Sema4D处理的DPSC条件培养基的血管生成诱导潜能。使用单因素方差分析和学生t检验分析结果。
Sema4D显著抑制DPSC的碱性磷酸酶活性和矿化结节形成。此外，经Sema4D处理的DPSC在成骨/成牙基因（碱性磷酸酶、I型胶原蛋白α1链、骨桥蛋白、Runx2和牙本质涎磷蛋白）以及蛋白质（I型胶原蛋白α1链和牙本质涎磷蛋白）的表达上表现出明显下调。丛状蛋白-B1和下游RhoA蛋白表达水平升高以及磷酸化丛状蛋白-B1证实了Sema4D/丛状蛋白-B1信号通路的参与。ErbB2的蛋白表达上调，而Met略有下调。此外，经Sema4D处理的DPSC在信使核糖核酸和蛋白质水平上均表现出血管内皮生长因子表达增强。因此，如基质胶试验所示，经Sema4D处理的DPSC条件培养基促进了血管样结构的形成。
Sema4D显著增强DPSC的血管生成潜能，但抑制其成骨/成牙潜能分化。Sema4D/丛状蛋白-B信号通路通过RhoA介导的途径被激活。
