Molecular Genetics of Craniofacial Development and Disease

颅面发育和疾病的分子遗传学

• 批准号: 7967081
• 负责人: Ashok Kulkarni
• 金额: $ 34.02万
• 依托单位: NATIONAL INSTITUTE OF DENTAL & CRANIOFACIAL RESEARCH
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7967081
• 关键词: Affect; Aging; Ameloblasts; Amelogenesis Imperfecta; Animals; Area; Basement membrane; Breeding; Candidate Disease Gene; Cell physiology; Cells; Cementoblast; Chondroitin Sulfate Proteoglycan; Chondroitin Sulfates; Cleaved cell; Code; Collaborations; Collagen Type I; Communities; Coupled; DSPP gene; Defect; Dental Cementum; Dental Enamel; Dental Enamel Hypoplasia; Dental Pulp; Dentin; Dentin Formation; Dentinogenesis; Dentinogenesis Imperfecta; Dentition; Development; Disease; Epithelial; Extracellular Matrix; Extracellular Matrix Proteins; Extramural Activities; Future; Gene Mutation; Gene Targeting; Genes; Genomics; Glycoproteins; Goals; Head and neck structure; Hereditary Disease; Human; Immune response; Inflammation; Inherited; Japan; Kidney; Knockout Mice; Laboratories; Labyrinth; Length; Maintenance; Messenger RNA; Metabolic; Minerals; Molecular; Molecular Genetics; Mus; Mutation; Odontoblasts; Odontogenic Tumors; Oral mucous membrane structure; Pain; Patients; Peptides; Periodontal Ligament; Phase; Phenotype; Phosphoproteins; Play; Process; Protein Isoforms; Proteins; Proteoglycan; Proteomics; Pulp Chambers; RNA Splicing; Recovery; Reporting; Research; Research Personnel; Resolution; Rodent; Role; Salivary Glands; School Dentistry; Signal Pathway; Signal Transduction; Structure; Tissues; Tokyo; Tooth Germ; Tooth structure; Transgenic Mice; Transgenic Organisms; Translating; Universities; Variant; Width; X-Ray Computed Tomography; amelogenin; biglycan; biomineralization; bone; bone metabolism; craniofacial; decorin; density; dentin sialoprotein; effective therapy; functional genomics; high risk; insight; leucine-rich amelogenin peptide; migration; mineralization; mouse Dspp protein; mouse model; osteoclastogenesis; promoter; rhoB p20 GDI; spatiotemporal; tumor growth; vector

Our research focuses on discovering and characterizing cellular processes that affect key components of craniofacial structures and their functions. Our efforts are concentrated on studying processes that regulate mineralization of teeth, trigger inflammation in teeth and salivary glands, promote tumor growth in the head-and-neck region, and cause painful conditions. We have used a variety of molecular approaches, such as conventional and conditional gene targeting coupled with genomic and proteomic analysis, to specifically investigate molecular roles of amelogenins and dentin sialophosphoprotein in tooth mineralization, TGF- signaling in inflammation of teeth and salivary glands. To pursue our research inquiries, which include some high-risk projects, we have extensively collaborated with many leading researchers in the intramural and extramural communities. Our present studies will not only contribute to a greater understanding of the molecular roles of the candidate genes in development and disease, but should also aid in future efforts to find more effective treatments for many disorders. Tooth Development and Disease Enamel Studies: Functional and enduring dentition is critical to the survival of many species and, not surprisingly, many of the genes involved in tooth formation are highly conserved throughout the animal kingdom. Amelogenin is one such gene that codes for multiple spliced variants of amelogenin in ameloblasts and is important for normal enamel development. Human amelogenin gene mutations result in amelogenesis imperfecta (AI), which is the most common hereditary condition affecting enamel and is characterized by thin and/or poorly mineralized enamel. Our earlier studies demonstrated that amelogenin null (Amel-/-) mice have an enamel phenotype that appears similar to that observed in human AI, confirming the important role of amelogenins in normal development of enamel. Subsequently, we demonstrated that 2 amelogenin isoforms, a full-length M180 amelogenin and a leucine-rich amelogenin peptide (LRAP), are expressed in cementum, and that their absence in aging Amel-/- mice is associated with cementum defects. We have also reported the functions of the amelogenin isoforms in osteoclastogenesis, and in the proliferation and migration of cementoblast/periodontal ligament (CM/PDL) cells. Our results provide strong evidence that LRAP inhibits osteoclastogenesis. In collaboration with Dr. Yoshi Yamada, we developed ameloblastin knockout mice that have severe enamel hypoplasia, detached ameloblasts, and odontogenic tumors of epithelial origin. In order to search for synergistic roles of amelogenin and ameloblastin in enamel development, we generated amelogenin and ameloblastin double-null (Amel-/-/Ambn-/-) mice. These mice showed more severe enamel defects than Amel-/- mice or Ambn-/- mice. SEM analysis showed that enamel structure was completely lost, and that the ameloblast layer was irregular and detached from the basement membrane in Amel-/-/Ambn-/- mice. Proteomic analysis revealed the presence of an increased level of Rho-GDI only in Amel-/-/Ambn-/- tooth buds, suggesting the synergistic effects of amelogenin and ameloblastin. We also initiated studies to analyze amelogenin functions in bone metabolism, by generating transgenic mice that express M180 and LRAP in bones. Our initial analysis revealed that amelogenins may play a role in the maintenance of bone metabolism. We have phased out this project and given all the mouse lines to Dr. Naoto Haruyama, who generated these mice in my laboratory and recently returned to Tokyo University School of Dentistry in Japan. In collaboration with extramural researchers, we also continued to study amelogenin mutations, further characterize the Amel-/- phenotype, and extend our focus to interactions between amelogenin and MMP20. As mentioned above, we have phased out all the amelogenin projects, but we will continue to collaboratively (A) delineate functions of amelogenin spliced variants found in humans and rodents, particularly for their roles in osteoclastogenesis and signaling, and (B) analyze LRAP function in osteoclastogenesis. Dentin Studies: Dentin sialophosphoprotein (DSPP) forms a major constituent of dentin extracellular matrix proteins, and is believed to play an important role in the mineralization process that forms mature dentin. Several mutations have been identified in the DSPP gene of patients with dentinogenesis imperfecta. DSPP is predominantly expressed in dentin-producing odontoblasts, and transiently expressed in enamel-producing ameloblasts. Low levels have also been detected in several other tissues like bone, inner ear, salivary glands, and kidneys. To gain insight into the molecular role of DSPP in dentinogenesis, we previously generated DSPP-/- mice. The structural tooth defects observed in these mice were enlarged pulp chambers, increased width of predentin zone, hypomineralization, pulp exposure, irregular mineralization front, and a lack of uniform coalescence of calcospherites in the dentin. The levels of the proteoglycans biglycan and decorin were increased in the widened predentin zone and in the void spaces among the calcospherites in the null dentin. These enhanced levels correlated well with the regions defective in mineralization, and further indicated that these molecules may adversely affect the dentin mineralization process by interfering with the coalescence of calcospherites. However, type I collagen levels remained unaffected in the null teeth. In order to understand the molecular mechanism underlying this phenotype, we first examined whether the elevated levels of biglycan and decorin were causative factors, or were elevated as a consequence of DSPP deficiency. Towards this goal, we have generated 2 mouse models: DSPP-/-;biglycan-/- and DSPP-/-;decorin-/-. Detailed analysis of these 2 mouse models indicates that the deficiency of decorin, but not biglycan, rescues the enlarged predentin phenotype of DSPP-/- mice. Increased levels of decorin in DSPP-/- predentin contribute to abnormal enlargement of predentin. The DSPP mRNA is translated into a single protein, DSPP, which is cleaved into 3 peptides: dentin sialoprotein (DSP), dentin glycoprotein (DGP), and dentin phosphoprotein (DPP). We constructed transgenic vectors that express DSP/DGP under the control of the DSPP promoter and generated independent transgenic mouse lines that express DSP/DGP in odontoblasts and preameloblasts. Two of these lines were bred with DSPP-/- mice to establish 2 mouse lines that express DSP/DGP at low and high levels in the DSPP null background (DPPcKO). DPPcKO teeth show a partial rescue of the DSPP null phenotype with more normal predentin width, an absence of irregular unmineralized areas in dentin, and less frequent pulp exposure. Micro-computed tomography (micro-CT) analysis of DPPcKO molars confirmed this partial rescue with significant recovery of the dentin volume, but with no improvement in the dentin mineral density. In addition, a dramatic decrease of chondroitin sulfate proteoglycan in dentin was observed in DSPP null mice that was largely restored in DPPcKO mice, which suggests that DSP is a major chondroitin sulfate chain proteoglycan in dentin. These results indicate the distinct roles of DSP and DPP in dentin mineralization, with DSP possibly regulating initiation of dentin mineralization, and DPP perhaps playing a role in the maturation of mineralized dentin.

我们的研究重点是发现和表征影响颅面结构及其功能的关键组成部分的细胞过程。我们的努力集中在研究调节牙齿矿物质，触发牙齿和唾液腺炎症的过程上，促进头颈区域的肿瘤生长并导致痛苦的疾病。 我们已经使用了多种分子方法，例如靶向基因组和蛋白质组学分析的常规和条件基因，以特异性研究氨基蛋白蛋白和乙蛋白唾液磷酸蛋白在牙齿矿化中的分子作用，TGF-在牙齿炎症中的TGF-信号传导。为了进行我们的研究询问，包括一些高风险项目，我们与壁内和壁外社区的许多主要研究人员进行了广泛的合作。我们目前的研究不仅将有助于对候选基因在发育和疾病中的分子作用有更多了解，而且还应有助于将来努力为许多疾病寻找更有效的治疗方法。 牙齿发育和疾病 搪瓷研究：功能性和持久的牙列对许多物种的生存至关重要，毫不奇怪，许多参与牙齿形成的基因在整个动物界都高度保守。 氨基蛋白蛋白是一种基因，它代码为阿素细胞中多种氨基蛋白蛋白的剪接变体编码，对于正常的搪瓷发育很重要。人氨基蛋白蛋白基因突变会导致无eleogenesofic imperfecta（AI），这是影响牙釉质的最常见的遗传条件，其特征是薄和/或矿物质不良的搪瓷。我们的较早研究表明，氨基蛋白蛋白null（amel - / - ）小鼠具有与人AI中观察到的牙釉质表型相似的牙釉质表型，这证实了amelogenins在搪瓷正常发育中的重要作用。随后，我们证明了2种蛋白质蛋白同工型，全长M180 amelogen蛋白和富含亮氨酸的杏仁素肽（LRAP），在水泥中表达，并且它们在衰老的Amel-小鼠中的缺失与骨质缺陷有关。我们还报道了阿米他蛋白同工型在破骨细胞生成以及牙骨质细胞/牙周韧带（CM/PDL）细胞的增殖和迁移中的功能。我们的结果提供了有力的证据表明LRAP抑制破骨细胞生成。 与Yoshi Yamada博士合作，我们开发了具有严重的搪瓷发育不全，分离的amellasts和上皮起源的牙源性肿瘤，开发了成纤维细胞敲除小鼠。 为了搜索氨基蛋白蛋白和阿素细胞蛋白在搪瓷发育中的协同作用，我们产生了amelogenin和amelogoentin double-Null（amel-/ - / - /Ambn - / - ）小鼠。这些小鼠表现出比Amel - / - 小鼠或AMBN - / - 小鼠更严重的搪瓷缺陷。 SEM分析表明，搪瓷结构完全丢失，而成成布层是不规则的，并且与Amel - / - /Ambn - / - 小鼠中的地下膜分离。 蛋白质组学分析表明，仅在Amel - / - /Ambn - / - 牙齿芽中存在升高的Rho-GDI水平，这表明氨基蛋白蛋白和杏仁糖蛋白的协同作用。 我们还通过产生表达M180和LRAP的转基因小鼠来分析骨代谢中的氨基蛋白蛋白功能的研究。我们的最初分析表明，氨基蛋白酶可能在维持骨代谢中发挥作用。我们已经淘汰了该项目，并将所有鼠标线都送给了Naoto Haruyama博士，Naoto Haruyama博士在我的实验室中产生了这些老鼠，最近回到了日本东京大学牙科学院。我们与校外研究人员合作，我们还继续研究氨基蛋白酶突变，进一步描述了Amel - / - 表型，并将我们的重点扩展到氨基蛋白酶和MMP20之间的相互作用。如上所述，我们已经淘汰了所有氨基蛋白蛋白项目，但是我们将继续进行协作（a）划定在人和啮齿动物中发现的氨基蛋白蛋白剪接变体的功能，尤其是对于它们在骨质栓塞和信号传导和信号传导中的作用，以及（b）分析lrap在骨质塑料中的功能。 牙本质研究：牙本质唾液磷蛋白（DSPP）形成了牙本质外基质基质蛋白的主要成分，并且据信在形成成熟牙本质的矿化过程中起着重要作用。 牙抑素发生不完美的患者的DSPP基因中已经发现了几种突变。 DSPP主要在产生牙本质的牙本质细胞中表达，并在产生搪瓷的成成布中瞬时表达。 在骨，内耳，唾液腺和肾脏等其他几个组织中也发现了低水平。为了深入了解DSPP在牙本质发生中的分子作用，我们以前产生了DSPP - / - 小鼠。 在这些小鼠中观察到的结构牙齿缺陷是果室增大，牙素区域的宽度增加，低矿物质化，果肉暴露，不规则的矿化前局部以及牙本质中骨磷酸盐的均匀合并。 蛋白聚糖大型群蛋白蛋白酶的水平和装饰蛋白在较宽的preventin区和无牙本质中的骨磷岩中的空隙中升高。 这些增强的水平与矿化中有缺陷的区域息息相关，进一步表明，这些分子可能通过干扰骨磷酸盐的聚结，从而对牙本质矿化过程产生不利影响。 但是，I型胶原蛋白水平在空牙齿中仍然不受影响。 为了理解该表型的分子机制，我们首先检查了大型群体和装饰水平升高是导致因素，还是由于DSPP缺乏症而被升高。 为了实现这一目标，我们生成了2种鼠标型号：DSPP - / - ; Biglycan - / - 和DSPP - / - ; DecorIn-/ - / - 。对这两种小鼠模型的详细分析表明，Decorin的缺乏（但不是Biglycan）挽救了DSPP - / - 小鼠的放大predentin表型。 DSPP - / - predentin中的Decorin水平升高会导致presentin异常增大。 DSPP mRNA被翻译成单个蛋白DSPP，该蛋白被裂解为3种肽：牙本质唾液蛋白（DSP），牙本质糖蛋白（DGP）和牙本质磷酸蛋白（DPP）。我们构建了在DSPP启动子的控制下表达DSP/DGP的转基因矢量，并生成了独立的转基因小鼠系，这些小鼠线在odontoblasts和preamelolboplasts中表达DSP/DGP。其中两条线与DSPP - / - 小鼠育成，以在DSPP无效背景（DPPCKO）中以低水平和高水平表达DSP/DGP的2种小鼠线。 DPPCKO牙齿显示出对DSPP NULL表型的部分营救，具有更正常的预易蛋白宽度，牙本质中没有不规则的非矿化区域以及较少频繁的纸浆暴露。 DPPCKO磨牙的微型计算机断层扫描（Micro-CT）分析证实了这种部分营救，并显着恢复了牙本质体积，但牙本质矿物质密度没有改善。此外，在DSPP NULL小鼠中观察到了牙本质中硫酸软骨蛋白蛋白聚糖的巨大减少，该小鼠在DPPCKO小鼠中已在很大程度上恢复了，这表明DSP是牙本质中硫酸盐蛋白链的主要硫酸盐链蛋白聚糖。这些结果表明，DSP和DPP在牙本质矿化中的不同作用，DSP可能调节牙本质矿化的起始，而DPP可能在矿化牙本质的成熟中起作用。