Amelogenesis and Ion Transport

釉质生成和离子运输

• 批准号: 10372197
• 负责人: Michael Lansdell Paine
• 金额: $ 38.8万
• 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/10372197
• 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 在牙釉质形成（ameleogeny）期间，细胞外pH（PHE）的调节至关重要，如 多个突变小鼠线。碳酸氢盐（HCO3-）导出到搪瓷基质涉及阴离子交换器2（AE2） 和SLC26A基因家族的成员。 AE2位于成熟的横向膜成熟细胞， 而SLC26A1、3、4、6和7都位于这些相同细胞的顶部/远端膜上。在成熟 成成木细胞SLC26A蛋白与囊性纤维化跨膜电导量调节剂共定位 （CFTR）并共同发挥功能，以允许出口HCO3-和氯离子的双向运动 （Cl-）作为PHE调节过程的一部分。在其他分泌性质的其他极化上皮细胞类型中， SLC26A蛋白和CFTR形成一个网络，该网络在顶极处具有细胞骨架丝状肌动蛋白（F-肌动蛋白） 在很大程度上决定了细胞极性和微绒毛项目。 NHERF1/SLC9A3R1和EZRIN蛋白是 还参与了此网络交互。 pH敏感的磷酸磷酸钠转运蛋白 （SLC34A2/NAPI2B），无机磷酸盐（PI）进口通道也定位于成熟的顶极 木材细胞，这可能暗示PHE法规与PI运输活动之间的直接联系 矿化事件。数据表明，针对特定离子转运蛋白mRNA的miRNA， 还影响离子运输和PHE调节。我们先前对成熟和成熟和 秘书搪瓷器官细胞确定了miR-298和miR-346在NHERF1调节中的潜在作用， SLC26A1，SLC26A7和CFTR。在此应用中，我们假设“ SLC26A/CFTR/NHERF1/NAPI2B 网络，指导离子运动与HCO3和PI传输和pH调节直接相关，而在 同时决定顶膜结构，对于搪瓷成熟至关重 网络结果会导致牙釉质的病理，会严重影响搪瓷寿命。 具体目的：1）成熟成熟的nherf1，ezrin，cftr和napi2b的免疫定位； 2） 使用miR-346和miR-298，在体内破坏搪瓷器官细胞中的SLC26A/CFTR/NHERF1/NHERF1/NHERF1/NAPI2B网络 直接在矿化现场交付；和; 3）研究NHERF1和NAPI2B的性质 体外和体内搪瓷器官细胞中的相互作用。在这项研究中，我们预计会破坏 SLC26A/CFTR网络和NAPI2B活动直接针对NHERF1函数，将导致重要 搪瓷畸形学。这项研究的发现将对我们对 与所有哺乳动物物种中牙釉质形成有关的生物矿化过程。这项研究也可能 导致处理诊所中遗传搪瓷缺陷的策略；而且，从长远来看，有助于预防和 减轻患有囊性纤维化和其他疾病的人的苦难。