Department of Molecular Genetics, Dental Research Institute and BK21 Program, School of Dentistry, Seoul National University, Seoul 08826, Korea.
Department of Pharmacology & Dental Therapeutics, School of Dentistry, Seoul National University, Seoul 08826, Korea.
Int J Mol Sci. 2017 Nov 9;18(11):2380. doi: 10.3390/ijms18112380.
Cementum is a mineralized layer on the tooth's root surface and facilitates the biomechanical anchoring of fibrous connective tissues as a part of tooth-supportive complexes. Previously, we observed that OCCM30 cementoblasts cultured on fibrin matrices underwent apoptosis due to fibrin degradation through the expression of proteases. Here, we demonstrated that OCCM30 on fibrin matrices (OCCM30-fibrin) enhanced canonical Wnt signaling, which directed to plasminogen expression. The OCCM30-fibrin showed higher levels of Wnt3a expression, nuclear translocation of β-catenin, and T-cell factor (TCF) optimal motif (TOP) reporter activity than the cells on tissue culture dishes (OCCM30-TCD), indicating that the OCCM30-fibrin enhanced canonical Wnt/β-catenin signaling. Also, OCCM30-fibrin expressed biomineralization-associated markers at higher levels than OCCM30-TCD, of which levels were further increased with LiCl, a Wnt signaling activator. The OCCM30 cementoblasts simultaneously showed that high levels of plasminogen, a critical component of fibrinolysis, were expressed in the OCCM30-fibrin. Activation of canonical Wnt signaling with LiCl treatment or with forced lymphoid enhancer factor 1 (LEF1)-expression increased the expression of plasminogen. On the contrary, the inhibition of canonical Wnt signaling with siRNAs against Wnt3a or β-catenin abrogated fibrin-enhanced plasminogen expression. Furthermore, there are three conserved putative response elements for the LEF1/β-catenin complex in the plasminogen proximal promoter regions (-900 to +54). Site-directed mutations and chromatin immunoprecipitation indicated that canonical Wnt signaling directed plasminogen expression. Taken together, this study suggests that fibrin-based materials can modulate functional periodontal formations in controlling cementoblast differentiation and fibrin degradation.
牙骨质是牙齿根部表面的矿化层，有助于纤维结缔组织作为牙齿支持复合体的一部分进行生物力学锚固。以前，我们观察到在纤维蛋白基质上培养的 OCCM30 成牙骨质细胞由于纤维蛋白降解而通过表达蛋白酶而发生凋亡。在这里，我们证明了纤维蛋白基质上的 OCCM30（OCCM30-纤维蛋白）增强了经典的 Wnt 信号通路，该信号通路指导纤溶酶原的表达。OCCM30-纤维蛋白表现出比在组织培养板上的细胞（OCCM30-TCD）更高水平的 Wnt3a 表达、β-连环蛋白核易位和 T 细胞因子（TCF）最佳基序（TOP）报告活性，表明 OCCM30-纤维蛋白增强了经典的 Wnt/β-连环蛋白信号通路。此外，OCCM30-纤维蛋白表达的生物矿化相关标志物的水平高于 OCCM30-TCD，并且在用 Wnt 信号激活剂 LiCl 处理时其水平进一步增加。OCCM30 成牙骨质细胞同时表明，在 OCCM30-纤维蛋白中高表达纤溶酶原，这是纤维溶解的关键组成部分。用 LiCl 处理或强制淋巴增强因子 1（LEF1）表达激活经典的 Wnt 信号会增加纤溶酶原的表达。相反，用针对 Wnt3a 或 β-连环蛋白的 siRNA 抑制经典的 Wnt 信号会阻断纤维蛋白增强的纤溶酶原表达。此外，在纤溶酶原近端启动子区域（-900 至+54）中存在三个保守的潜在 LEF1/β-连环蛋白复合物反应元件。定点突变和染色质免疫沉淀表明，经典的 Wnt 信号指导纤溶酶原的表达。总之，这项研究表明纤维蛋白基材料可以调节功能性牙周形成，以控制成牙骨质细胞分化和纤维蛋白降解。
