Mechanisms of FGFR2 Signaling in Salivary Gland Branching Morphogenesis

FGFR2 信号在唾液腺分支形态发生中的机制

• 批准号: 8089478
• 负责人: JACOB V.P. ESWARAKUMAR
• 金额: $ 38.62万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8089478
• 关键词: Affect; BMP4; Binding Sites; Biological Models; Cell Differentiation process; Cell Line; Cells; Dental caries; Development; Docking; Dryness; Embryonic Development; Engineering; Epithelial; Epithelium; FGF1 gene; FGF10 gene; Fibroblast Growth Factor; Fibroblast Growth Factor Receptor 2; Fibronectins; Functional disorder; Gene Expression; Gene Targeting; Genetic; Genetic Models; Genetically Engineered Mouse; Goals; Histology; Investigation; Knock-in Mouse; Knockout Mice; Laminin; Light; Mediating; Mediator of activation protein; Mesenchyme; Methods; Molecular; Morphogenesis; Mus; Mutant Strains Mice; Mutate; Mutation; Natural regeneration; Oral; Organ Culture Techniques; Pathway Analysis; Pathway interactions; Patients; Pattern; Phenylalanine; Phosphotransferases; Play; Protein Isoforms; Protein Tyrosine Kinase; Protein Tyrosine Phosphatase; Proteins; Quality of life; Radiation therapy; Recombinants; Recruitment Activity; Role; Salivary Glands; Signal Pathway; Signal Transduction; Signaling Molecule; Speech; Stem cells; Structure; Submandibular gland; System; Testing; Tyrosine; Work; Xerostomia; base; chemotherapy; design; fibroblast growth factor receptor 2b; genome-wide; gland development; human FRS2 protein; insight; mRNA Expression; mouse model; mutant; mutant mouse model; novel; progenitor; public health relevance; receptor; research study; response; stem cell differentiation

DESCRIPTION (provided by applicant): Fibroblast growth factors (FGF) and their receptors (FGFR) play a central role in salivary gland branching morphogenesis. In mice, targeted disruption of Fgf10 or its receptor, Fgfr2b, causes salivary gland agenesis, demonstrating the essential role of FGF10-FGFR2b signaling pathways in salivary gland development. However, the lack of salivary glands in Fgf10 or Fgfr2b knockout mice precludes further investigation of branching morphogenesis in these mouse models. Therefore, it is necessary to develop an alternative approach to gain insights about the role of Fgfr2b intracellular signaling pathways in salivary gland branching morphogenesis. We have developed a novel mouse model system in which specific signaling pathways downstream of Fgfr2 have been abrogated by a knock-in mutation, without compromising the tyrosine kinase activity of the receptor to activate other signaling pathways. Our hypothesis is that Fgfr2b signaling via Frs21 is essential for salivary gland branching morphogenesis. In Aim 1, we will (1) analyze the branching morphogenesis of submandibular glands (SMGs) from different mutant mouse strains by histology and ex vivo organ culture; (2) use mesenchyme-free SMG cultures to investigate epithelial morphogenesis; and (3) use cell lines expressing the mutant and wild type receptors to evaluate the mechanism of epithelial transphosphorylation of Frs21 by Fgfr1b and Fgfr2b during branching morphogenesis. In Aim 2, we will determine the role of Frs21-mediated Grb2 and Shp2 signaling pathways in SMG branching morphogenesis. Our hypothesis is that Shp2 is the critical mediator of Fgfr2b signaling via Frs21 for SMG branching morphogenesis. We will test this hypothesis using two strains of genetically engineered mutant mice. In the first mutant mouse model, the four Grb2 binding sites are mutated to phenylalanine (4F), and thus cannot recruit Grb2. In the second mutant mouse model, the two Shp2 binding sites are mutated to phenylalanine (2F), and thus cannot recruit Shp2. We will perform (1) histology and ex vivo SMG organ culture to determine the role of Grb2 and Shp2 in branching morphogenesis; and (2) recombinant SMG culture to determine whether the mutations are cell autonomous or non-cell autonomous by culturing wild type epithelium with Grb2 or Shp2 mutant mesenchyme and vice versa. In addition, we propose two approaches to identify novel genetic pathways: a) genome-wide mRNA expression analysis in the epithelium and mesenchyme of the mutant SMGs, and b) expression-based pathway analysis of target genes in SMG. Collectively, this work will provide a detailed molecular picture of how FGF signaling, mediated by the Fgfr2b isoform and the docking protein Frs21, regulates salivary gland branching morphogenesis. The results of these studies may enable the design of novel methods for salivary gland regeneration using FGFR2b mediated pathways to regulate progenitor cell differentiation and morphogenesis. PUBLIC HEALTH RELEVANCE: Salivary gland dysfunction caused by mutations, radiotherapy or chemotherapy affect the quality of life of patients by causing oral dryness, dental caries, hampered speech, and xerostomia. The overall goal of this proposal is to obtain a comprehensive molecular picture of the signaling pathways that are activated in response to Fgfr2b stimulation, specifically the Frs21-mediated pathways essential for the development of salivary glands. We anticipate that our findings will shed new light on fundamental intracellular signaling pathways essential for epithelial morphogenesis of salivary glands, and will enable the design of novel methods for salivary gland regeneration using FGFR2b-mediated pathways to regulate progenitor/stem cell differentiation and morphogenesis.

描述（由申请人提供）：成纤维细胞生长因子（FGF）及其受体（FGFR）在唾液腺分支形态发生中起着核心作用。在小鼠中，有针对性的FGF10或其受体FGFR2B导致唾液腺发育不全，证明FGF10-FGFR2B信号通路在唾液腺发育中的重要作用。然而，FGF10或FGFR2B敲除小鼠缺乏唾液腺排除了这些小鼠模型中分支形态发生的进一步研究。因此，有必要开发一种替代方法来获得有关FGFR2B细胞内信号通路在唾液腺分支形态发生中的作用的见解。我们已经开发了一种新型的小鼠模型系统，其中FGFR2下游的特定信号通路被敲入突变所消除，而不会损害受体的酪氨酸激酶活性以激活其他信号通路。我们的假设是，通过FRS21信号传导对于唾液腺分支形态发生至关重要。在AIM 1中，（1）通过组织学和离体器官培养分析来自不同突变小鼠菌株的下颌下腺（SMG）的分支形态发生； （2）使用无间隙的SMG培养物研究上皮形态发生； （3）使用表达突变体和野生型受体的细胞系评估FGFR1B和FGFR2B在分支形态发生过程中FRS21上皮磷酸化的机理。在AIM 2中，我们将确定FRS21介导的GRB2和SHP2信号通路在SMG分支形态发生中的作用。我们的假设是，SHP2是通过FRS21发出SMG分支形态发生的FGFR2B信号传导的关键介体。我们将使用两种基因工程突变小鼠菌株检验该假设。在第一个突变小鼠模型中，四个GRB2结合位点突变为苯丙氨酸（4F），因此无法募集GRB2。在第二个突变小鼠模型中，将两个SHP2结合位点突变为苯丙氨酸（2F），因此无法募集SHP2。我们将执行（1）组织学和离体SMG器官培养，以确定GRB2和SHP2在分支形态发生中的作用； （2）重组SMG培养物，通过用GRB2或SHP2突变体间充质培养野生型上皮，以确定突变是细胞自主或非细胞自主的，反之亦然。此外，我们提出了两种鉴定新遗传途径的方法：a）在突变体SMG的上皮和间质中全基因组mRNA表达分析，b）基于SMG中靶基因的基于表达的途径分析。总的来说，这项工作将提供详细的分子图片，说明FGF信号传导如何由FGFR2B同工型和对接蛋白FRS21介导，可以调节唾液腺分支形态发生。这些研究的结果可能可以使用FGFR2B介导的途径来设计新的唾液腺再生方法，以调节祖细胞分化和形态发生。 公共卫生相关性：突变，放疗或化学疗法引起的唾液腺功能障碍，通过引起口腔干燥，龋齿，阻碍语音和静脉症来影响患者的生活质量。该提案的总体目标是获得响应于FGFR2B刺激而激活的信号通路的综合分子图，特别是FRS21介导的途径对于开发唾液腺的发展。我们预计我们的发现将为唾液腺上皮形态发生必不可少的基本细胞内信号传导途径提供新的启示，并将使用FGFR2B介导的途径来设计新方法的新方法，以调节祖细胞/干细胞的分化和形态生成。