釉质发育不全致病基因FAM83H在釉质形成中的功能研究

To date, FAM83H gene is a major contributor to the etiology of AI. The function of FAM83H during the formation of enamel has not been revealed and no reports focused on it. Until now, all identified mutations are located in the C-terminal of FAM83H and caused hypocalcified AI, which indicates that the C-terminal of FAM83H is important for the function of ameloblasts and enamel mineralization. The present studies and our preliminary studies indicated the following hypothesis：FAM83H combining with CK1α, keratin and amelogenin, functions as a complex. On one hand, it regulates the function of the complex; on the other hand, it upstream regulates CK1α and affects the regulation homeostasis between CK1α and β-catenin, and then plays roles on ameloblast adjunction, polarization and enamel mineralization. The present project plans to perform the following studies: (1)with mouse ameloblast cell line LS8, study the effects of expression alteration of Fam83h, specific Fam83h mutations, and the CK1α regulation on: the formation of the complex, the expression and function of the proteins, the function of ameloblasts and enamel formation as well as signaling pathway for mineralization; (2)with mouse models, study the effects of the CK1α regulation alteration and Fam83h mutations on: ameloblast adjunction and polarization, spatio-temporal expression of the proteins, signaling pathway for mineralization, enamel formation; (3) rescue on cell and mouse models, explore the potential intervention targets. The project aims to reveal the function of FAM83H during enamel formation and molecular mechanism of AI resulting from FAM83H mutations, and to provide new way for prevention and control of the disease.

FAM83H (F)是目前发现的常显釉质发育不全的主要基因，其在釉质形成中的具体功能不明确，未见文献报道。所有F突变为集中于C端的突变，表明其C端结构与成釉细胞功能及釉质矿化密切相关。现有研究及我们前期研究提示，F与釉原蛋白等多个蛋白结合形成复合体，一方面调控复合体功能，对釉质形成发挥重要作用；另一方面，上游调节CK1α，影响CK1α与β-catenin调节平衡，对成釉细胞连接和极化及釉质矿化起重要作用。本项目拟进行以下研究：选用小鼠成釉细胞系，研究F特定突变、表达变化及CK1α调节，对复合体形成及各蛋白表达和功能，对成釉细胞功能和釉质形成及矿化信号通路的影响；构建小鼠CK1α调节及肾囊膜F突变牙胚移植模型，研究突变对复合体各蛋白时空表达、成釉细胞连接和极化、矿化信号通路及釉质形成的影响；并对细胞和动物模型进行挽救实验，寻找干预靶点。阐明F在釉质形成中的功能及突变致病的分子机制。

FAM83H基因是常染色体显性遗传釉质钙化不全(ADHCAI)的主要致病基因。然而，FAM83H的功能及致病机制尚待进一步阐明。为探寻FAM83H功能，本项目选取50种脊椎动物，对FAM83H进行进化分析；构建了Fam83h突变的LS8细胞，通过矿化实验、IP、WB、高通量测序、IHC、IF实验，检测矿化过程中CK1α和β-catenin的表达定位、突变细胞中信号分子的变化，以及Fam83h、CK1α、CK14、Amelx的相互作用。进一步还构建了Fam83h突变小鼠，通过micro-CT检测了釉质的改变。进化分析揭示了FAM83H分化过程，提示FAM83H的C端含有对釉质发育重要的保守区域，在进化过程中产生了一定的选择作用。Fam83h突变改变了Fam83h、CK1α和β-catenin在LS8中的表达定位，抑制矿化。Wnt信号通路抑制剂挽救了Fam83h突变细胞中被抑制的矿化。Fam83h突变可通过激活Wnt信号通路抑制成釉细胞矿化。另外，Fam83h突变的LS8细胞的差异表达基因聚类于发育功能及信号传导通路。且LS8细胞中Fam83h、CK1α、CK14、Amelx可形成四聚体；在Fam83h突变细胞中，未检测到四聚体。Fam83h突变小鼠中，纯合子小鼠上颌磨牙牙冠高度低于野生型和杂合子小鼠，提示Fam83h突变小鼠磨牙咬合面的磨耗更为严重。这些结果表明Fam83h突变可能通过影响釉原蛋白的功能及成釉细胞的分化影响釉质发育。同时，本项目对环境因素导致的釉质发育缺陷也进行了研究，探索了节律基因Per2对釉质发育的影响。检测了成釉细胞系ALC细胞和c57鼠牙胚发育的节律变化。利用IHC、IF、矿化实验和WB检测了节律紊乱对amelx、信号分子和骨架蛋白的影响。结果显示ALC细胞中Per2节律性表达、节律紊乱影响了小鼠切牙生长、釉基质形成、CK14、F-actin的表达，并减少了节律基因Per2、PPARγ及磷酸化Akt和β-catenin表达。ALC细胞中敲减Per2抑制细胞矿化、PPARγ表达及Akt和β-catenin的磷酸化水平。在ALC-Per2-sh细胞中过表达PPARγ逆转了Akt磷酸化和β-catenin磷酸化水平及矿化分化。这些结果表明Per2通过PPARγ/Akt/β-catenin信号轴调控釉质发育。以上研究为釉质发育异常的防治提供了新思路。