Amelogenesis and Ion Transport

釉质生成和离子运输

• 批准号: 10193340
• 负责人: Michael Lansdell Paine
• 金额: $ 35.27万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-03-15 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10193340
• 关键词: Ameloblasts; Amelogenesis; Anions; Apical; Architecture; Bicarbonates; Binding; Carrier Proteins; Cell Polarity; Cell physiology; Cells; Chloride Ion; Chlorides; Clinic; Complex; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Cytoskeleton; Data; Defect; Dental Enamel; Disease; Distal; Dysmorphology; Enamel Formation; Enamel Organ; Epithelial Cells; Event; Extracellular Matrix; F-Actin; Family member; Family suidae; Gene Expression; Gene Expression Regulation; Gene Family; Hydroxyapatites; In Vitro; Inherited; Inorganic Phosphate Transporter; Ion Channel; Ion Transport; Ions; Knockout Mice; Lateral; Lead; Link; Longevity; Maturation-Stage Ameloblast; Mechanics; Membrane; Messenger RNA; MicroRNAs; Minerals; Modeling; Morphology; Movement; Mus; Mutant Strains Mice; Nature; Pathology; Pathway interactions; Process; Proteins; Protons; Regulation; Reporting; Role; Site; Sodium; Time; Tissues; apical membrane; base; biomineralization; calcium bicarbonate; cell type; cellular microvillus; extracellular; ezrin; in vivo; inorganic phosphate; member; mineralization; mutant; prevent; sodium-hydrogen exchanger regulatory factor; transcriptome

PROJECT SUMMARY / ABSTRACT During enamel formation (amelogenesis) the regulation of extracellular pH (pHe) is critical as shown by multiple mutant mice lines. Bicarbonate (HCO3-) export to the enamel matrix involves anion exchanger 2 (AE2) and members of the SLC26A gene family. AE2 is localized to the lateral membrane of maturation ameloblasts, while SLC26A1, 3, 4, 6 and 7 all localize to the apical/distal membrane of these same cells. In maturation ameloblasts SLC26A proteins colocalize with the cystic fibrosis transmembrane conductance regulator (CFTR) and function together to allow for the export of HCO3- and the bidirectional movements of chloride ions (Cl-) as part of the pHe regulatory process. In other polarized epithelial cell types of a secretory nature, SLC26A proteins and CFTR form a network with cytoskeletal filamentous actin (F-actin) at the apical pole dictating, to a large part, cell polarity and microvilli projections. NHERF1/SLC9A3R1 and Ezrin proteins are also involved with this network interaction. The pH-sensitive sodium dependent phosphate transporter (SLC34A2/NaPi2b), an inorganic phosphate (Pi) import channel also localizes to the apical pole of maturation ameloblasts, which may suggest a direct link between pHe regulation and Pi transport activities during enamel mineralization events. Data suggests that miRNAs, targeting the mRNAs of specific ion transport proteins, also influence ion transport and pHe regulation. Our prior whole transcriptome analysis of maturation and secretory enamel organ cells identify a potential role for miR-298 and miR-346 in the regulation of NHERF1, Slc26a1, Slc26a7 and Cftr. In this application we hypothesize that a “SLC26A/CFTR/NHERF1/NaPi2b network, directing ion movements directly related to HCO3- and Pi transport and pH regulation, while at the same time dictating apical membrane architecture, is critical for enamel maturation, and disruptions to this network result in enamel pathologies that will severely impact on enamel longevity.” We propose the following specific aims: 1) immunolocalization of NHERF1, EZRIN, CFTR and NaPi2b in maturation ameloblasts; 2) disrupt SLC26A/CFTR/NHERF1/NaPi2b network in enamel organ cells in vivo using miR-346 and miR-298 delivered directly at the site of mineralization; and; 3) investigate the nature of the NHERF1 and NaPi2b interactions in enamel organ cells in vitro and in vivo. In this study we anticipate that disrupting the SLC26A/CFTR network and NaPi2b activity, by directly targeting NHERF1 function, will result in a significant enamel dysmorphology. Findings from this study will have a significant impact on our understanding of the biomineralization process as it relates to enamel formation in all mammalian species. This study may also lead to strategies for handling inherited enamel defects in the clinic; and, in the long run help to prevent and alleviate the suffering of those afflicted with cystic fibrosis and other diseases.

项目摘要/摘要 在釉质形成(釉质形成)过程中，细胞外pH(Phe)的调节至关重要，如下所示 多个突变小鼠品系。碳酸氢盐(HCO3-)向牙釉质基质的输出涉及阴离子交换剂2(AE2) 和SLC26a基因家族的成员。AE2定位于成熟成釉细胞的侧膜， 而SLC26A1、3、4、6和7均定位于这些细胞的顶端/远端细胞膜。在成熟期 成釉细胞SLC26a蛋白与囊性纤维化跨膜电导调节因子共定位 (CFTR)和共同发挥作用，以允许HCO3-的出口和氯离子的双向移动 (CL-)作为PHE监管过程的一部分。在其他分泌性质的极化上皮细胞类型中， SLC26a蛋白和CFTR在顶端与细胞骨架丝状肌动蛋白(F-肌动蛋白)形成网络 这在很大程度上决定了细胞的极性和微绒毛的投射。NHERF1/SLC9A3R1和Ezrin蛋白是 也参与了这种网络互动。PH敏感的钠依赖磷酸盐转运蛋白 (SLC34A2/NaPi2b)，一种无机磷(PI)输入通道，也位于成熟的顶端 成釉细胞，这可能表明在釉质过程中Phe调节和PI转运活动之间存在直接联系 成矿事件。数据表明，针对特定离子运输蛋白的mRNAs的miRNAs， 也影响离子转运和Phe的调节。我们之前对成熟度和成熟度的完整转录组分析 分泌成釉细胞鉴定miR-298和miR-346在NHERF1调节中的潜在作用， Slc26a1、Slc26a7和CFtr。在本申请中，我们假设a“SLC26a/CFtr/NHERF1/NaPi2b 网络，直接与HCO3-和PI的运输和pH调节有关的离子运动，而在 同时决定顶膜结构，是牙釉质成熟的关键，而对此的破坏 网络会导致牙釉质病变，严重影响牙釉质的寿命。我们提出以下建议 特异性靶点：1)NHERF1、Ezrin、CFTR和NaPi2b在成釉细胞中的免疫定位； MiR-346和miR-298阻断成釉细胞SLC26a/CFTR/NHERF1/NaPi2b网络的实验研究 直接在矿化地点输送；以及；3)调查NHERF1和NaPi2b的性质 釉质器官细胞在体外和体内的相互作用。在这项研究中，我们预计会扰乱 SLC26a/cftr网络和NaPi2b活性，通过直接靶向NHERF1功能，将导致显著的 釉质畸形。这项研究的发现将对我们理解 生物矿化过程，因为它与所有哺乳动物的釉质形成有关。这项研究还可能 引导临床上处理遗传性釉质缺陷的策略；从长远来看，有助于预防和 减轻囊性纤维化和其他疾病患者的痛苦。