CALM2介导YAP/TAZ调控牙上皮短暂扩增细胞命运的机制研究

Developmental defects of enamel (DDE), as one of the most common dental non-caries diseases, affects the quality of patients’ life. The formation of enamel has been shown to be tightly related to the fate of dental epithelial transit-amplifying cells (TA cells), as well as Ca2+ dependent signaling. In our previous study, we have identified that YAP/TAZ promote TA cells proliferation and inhibit precocious differentiation in mouse incisor, and the loss of YAP/TAZ could reduce the expression of calmodulin gene Calm2, the major regulator of Ca2+ dependent signaling with important function in the control of cell proliferation and differentiation. This let us to hypothesize that YAP/TAZ may regulate TA cells through CALM2 during amelogenesis. By genetically deleting YAP/TAZ in TA cells and ameloblast cells, we will use the mouse incisor as the model to characterize the phenotypes and address the role of YAP/TAZ in amelogenesis. At the same time, we will detect the CALM2 expression pattern during amelogenesis process and analyze the putative interaction of CALM2 and TEADs, known as the key YAP/TAZ-target transcription factors, to identify the mechanism of YAP/TAZ-mediated CALM2 regulation. In addition, we will focus on NFAT in TA cells when CALM2 expression is perturbed, followed by clarifying the influence on proliferation and differentiation of TA cells. Together, the results of these studies will reveal the mechanism of how YAP/TAZ mediate CALM2 to regulate proliferation and differentiation of TA cells during enamel development and provide a thorough understanding of the molecular processes of DDE, and in the long run the results will be useful for prevention and treatment of DDE in the future.

牙釉质发育缺陷是常见牙体硬组织疾病，影响患者生活质量。牙釉质形成与牙上皮短暂扩增细胞（transit-amplifying cells,TA cells）命运及钙转导信号密切相关。申请人前期研究发现YAP/TAZ调控小鼠切牙TA细胞增殖分化平衡，敲除YAP/TAZ导致钙调蛋白CALM2表达下降，提示YAP/TAZ可能通过CALM2调控TA细胞命运影响牙釉质形成。本项目拟构建TA细胞及成釉细胞条件性敲除YAP/TAZ基因小鼠模型，探索该模型中细胞分化与牙釉质形成的改变；检测CALM2在牙釉质形成过程中的表达变化，深入解析CALM2与YAP/TAZ关键结合转录因子TEADs之间的相互作用；通过靶向抑制CALM2表达，探索其通过NFAT调控TA细胞命运的作用。本项目的实施将阐明CALM2介导YAP/TAZ调控TA细胞命运，最终参与牙釉质发育的分子机制，为完善牙釉质发育缺陷的发病机制提供理论依据。

牙釉质作为人体最坚硬、矿化程度最高的组织，包绕在牙冠表面，行使切咬、美观以及保护等功能。其发育过程中，任何信号干扰或突变都可造成牙釉质发育缺陷。对于牙釉质形成过程中涉及的分子信号及其造成牙釉质发育缺陷的相关机制一直是口腔医学研究的热点。研究发现牙釉质形成与牙上皮短暂扩增（TA）细胞命运及钙转导信号密切相关，而YAP/TAZ和钙调蛋白Calm2在牙釉质形成过程中的表达具有时空特异性。本项目通过构建TA细胞及成釉细胞条件性敲除YAP/TAZ基因小鼠模型，发现YAP/TAZ调控小鼠切牙TA细胞增殖分化平衡，敲除YAP/TAZ后引起成釉细胞形态出现异常并且牙釉质导发育异常，同时检测到Calm2表达显著下降。进一步发现， YAP/TAZ关键结合转录因子TEADs与Calm2启动子区存在结合位点，并且干扰YAP/TAZ-TEADs结合后Calm2表达降低。特异性敲除Calm2后，牙上皮细胞增殖分化状态及牙釉质微观结构均发生改变，且NFAT表达水平受到抑制。体外细胞实验发现，过表达NFAT可一定程度上缓解Calm2缺失引起的牙上皮细胞增殖及分化相关蛋白下降的现象。综上所述，本项目研究结果发现YAP/TAZ可通过Calm2调控NFAT在牙釉质正常发育中发挥重要作用，为完善牙釉质发育缺陷的发病机制提供理论依据。