Regulation of orofacial ectodermal polarity by the CLP protein, IRF6

CLP 蛋白 IRF6 对口面部外胚层极性的调节

• 批准号: 8525152
• 负责人: Emily Yin Chu
• 金额: $ 3.66万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-07-01 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8525152
• 关键词: Ablation; Adherens Junction; Adhesions; Affect; Ameloblasts; Apical; Area; Award; Basic Science; Behavior; Binding; Biological Assay; C-terminal; Cadherins; Cell Adhesion; Cell Adhesion Molecules; Cell physiology; Cleaved cell; Cleft Lip; Clinical; Co-Immunoprecipitations; Complex; Congenital Abnormality; Conjunctival Pterygium; Cytoplasm; Data; Defect; Dental Enamel; Dentists; Development; Electroporation; Embryo; Enamel Formation; Epithelial; Epithelial Cells; Epithelium; Exhibits; Face; Foundations; Gene Expression; General Population; Genes; Genetic; Human; Hypodontia; In Vitro; Incidence; Inherited; Interferon Regulatory Factor 2; Interferons; Laboratories; Lead; Lip structure; Live Birth; Maintenance; Mandible; Measures; Mentorship; Microarray Analysis; Modeling; Molecular Genetics; Molecular Profiling; Mus; Mutate; Mutation; NME1 gene; Neoplasm Metastasis; Nuclear; Nucleoside-Diphosphate Kinase; Oral; Palate; Patients; Phosphorylation; Play; Population; Post-Translational Protein Processing; Premaxillary palate; Primordium; Procedures; Protein Binding; Proteins; Public Health; Regulation; Reporting; Research; Research Project Grants; Role; Scientist; Sequence Homology; Shapes; Site-Directed Mutagenesis; Specificity; Staging; Surface; Syndrome; T-Cell Lymphoma; Tissues; Tooth Abnormalities; Tooth Germ; Tooth structure; Training; Transfection; Universities; Van der Woude syndrome; Variant; Washington; Yeasts; base; deciduous tooth; design; interest; keratinocyte; laser capture microdissection; mutant; nectin; novel; oral cavity epithelium; orofacial; palatal fusion; paralogous gene; protein protein interaction; public health relevance; research study; transcription factor; treatment strategy; yeast two hybrid system

DESCRIPTION (provided by applicant): Interferon regulatory factor 6 (IRF6) variants are associated with common isolated forms of cleft lip with or without clefting of the palate (CLP), as well as with dominantly inherited forms of CLP. Based on high sequence and structural homology with other IRFs, IRF6 has been predicted to act as a transcription factor, although studies in keratinocytes have found IRF6 primarily in the cytoplasm. Recent studies in the Cox laboratory have found that IRF6 binds, in a phosphorylation-enhanced manner, to NME2 (non-metastatic 2), which has been reported to interact with factors that activate the PAR (PARD3-PARD6) polarity complex. NME regulated PARD3 activation stimulates establishment of epithelial apical and basolateral domains (i.e. polarity), ultimately impacting epithelial cell adhesion, shape, and behavior. Thus, via protein-protein interactions, IRF6 is hypothesized to contribute to the regulation of epithelial polarity, which when disrupted, leads to CLP. Specific aims were developed to investigate the contribution of IRF6 to primary palate and tooth development. Using site-directed mutagenesis, reported VWS patient mutations will be introduced into yeast and mammalian expression constructs and subsequently compared with wild type IRF6 in their ability to interact with NME2 . Potential effects of the IRF6-NME2 interaction on epithelial polarity will be assessed by measuring activity of polarity effector proteins, assaying for nuclear:cytoplasmic ratios of polarity markers, and observing for epithelial morphological changes. To determine a relationship between IRF6-NME2 binding and primary palatal fusion, a novel in ovo procedure developed by the Cox laboratory will be used to specifically perturb IRF6/NME2 expression in embryonic chick orofacial epithelia, and effects on primary palatal development characterized. In human patients, CLP is often associated with tooth abnormalities, and in mice, IRF6 was detected in ameloblasts and has roles in tooth epithelial invagination. The tooth also presents another mechanism to investigate IRF6 contribution to epithelial polarity; ameloblasts are derived from the oral epithelium and exhibit observable polarity during the secretory stage. A murine model with IRF6 conditionally ablated in ameloblasts will be developed, and IRF6 epithelial polarity contribution will be evaluated based on changes in ameloblast polarity, enamel formation, and gene expression (laser capture microdissection followed by microarray analysis). Thus, these studies will also explore a possible relationship between CLP and tooth development. This proposed F30 project was developed to help provide a foundation for an aspiring dentist scientist and contribute to research centered on genetic and molecular causes of CLP. IRF6 has been targeted because of its prominent contribution to CLP incidence. Understanding regulatory mechanisms behind CLP will help identify susceptible populations, lower incidence, and develop treatment strategies. With strong institutional support from the University of Washington and excellent mentorship, we are confident that this F30 award will aid in the basic sciences and clinical training required to be a successful clinician scientist.

描述（由申请人提供）：干扰素调节因子 6 (IRF6) 变异体与常见分离形式的唇裂（伴或不伴腭裂 (CLP)）相关，如 以及显性遗传形式的 CLP。基于与其他 IRF 的高度序列和结构同源性，IRF6 被预测可充当转录因子，尽管对角质形成细胞的研究发现 IRF6 主要存在于细胞质中。 Cox 实验室最近的研究发现，IRF6 以磷酸化增强的方式与 NME2（非转移性 2）结合，据报道 NME2 与激活 PAR (PARD3-PARD6) 极性复合物的因子相互作用。 NME 调节的 PARD3 激活刺激上皮顶端和基底外侧域（即极性）的建立，最终影响上皮细胞粘附、形状和行为。因此，通过蛋白质-蛋白质相互作用，IRF6 被推测有助于上皮极性的调节，当上皮极性被破坏时，会导致 CLP。 制定了具体目标来研究 IRF6 对初级腭和牙齿发育的贡献。使用定点诱变，报告的 VWS 患者突变将被引入酵母和哺乳动物表达构建体中，随后与野生型 IRF6 与 NME2 相互作用的能力进行比较。 IRF6-NME2 相互作用对上皮极性的潜在影响将通过测量极性效应蛋白的活性、测定极性标记的核:细胞质比率以及观察上皮细胞极性来评估。 形态变化。为了确定 IRF6-NME2 结合与初级腭融合之间的关系，Cox 实验室开发的一种新型卵内程序将用于特异性干扰胚胎鸡口面部上皮细胞中的 IRF6/NME2 表达，并表征对初级腭发育的影响。 在人类患者中，CLP 通常与牙齿异常相关，而在小鼠中，在成釉细胞中检测到 IRF6，并且在牙齿上皮内陷中发挥作用。该牙齿还提供了另一种机制来研究IRF6对上皮极性的贡献；成釉细胞源自口腔上皮，在分泌阶段表现出可观察到的极性。将开发一种在成釉细胞中条件性消融 IRF6 的小鼠模型，并将根据成釉细胞极性、牙釉质形成和基因表达的变化（激光捕获显微切割，然后进行微阵列分析）来评估 IRF6 上皮极性的贡献。因此，这些研究还将探讨 CLP 和牙齿发育之间可能的关系。 开发这个拟议的 F30 项目是为了帮助为有抱负的牙医科学家奠定基础，并为以 CLP 的遗传和分子原因为中心的研究做出贡献。 IRF6 因其对 CLP 发病率的显着贡献而成为目标。了解 CLP 背后的监管机制将有助于识别易感人群、降低发病率并制定治疗策略。凭借华盛顿大学强有力的机构支持和出色的指导，我们相信 F30 奖项将有助于成为一名成功的临床科学家所需的基础科学和临床培训。