Amelogenesis and Ion Transport

釉质生成和离子运输

基本信息

批准号：
10570193
负责人：
Michael Lansdell Paine
金额：
$ 39.19万
依托单位：
UNIVERSITY OF SOUTHERN CALIFORNIA
依托单位国家：
美国
项目类别：
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/10570193
关键词：
Ameloblasts Amelogenesis Anions Apical Architecture Bicarbonates Binding Calcium Carrier Proteins Cell Polarity Cell physiology Cells Chloride Ion Chlorides Clinic Compensation 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 Link Longevity Maturation-Stage Ameloblast Mechanics Membrane Messenger RNA MicroRNAs Modeling Morphology Movement Mus Mutant Strains Mice Nature Pathology Pathway interactions Process Proteins Protons Regulation Reporting Role Site Sodium Time Tissues apical membrane biomineralization cell type cellular microvillus extracellular ezrin in vivo inorganic phosphate member mineralization mutant prevent sodium-hydrogen exchanger regulatory factor transcriptome

项目摘要

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.

项目概要/摘要在牙釉质形成（釉质生成）过程中，细胞外 pH (pHe) 的调节至关重要，如下所示：多个突变小鼠系向牙釉质基质输出碳酸氢盐（HCO3-）涉及阴离子交换剂2（AE2）。 SLC26A 基因家族的成员位于成熟成釉细胞的侧膜，而SLC26A1、3、4、6和7在成熟时都定位于这些相同细胞的顶/远端膜。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) 并共同作用以允许 HCO3- 的输出和氯离子的双向移动 (Cl-) 作为 pHe 调节过程的一部分，在其他具有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) 形成网络在很大程度上，细胞极性和微绒毛投射由 NHERF1/SLC9A3R1 和 Ezrin 蛋白决定。 pH 敏感的钠依赖性磷酸盐转运蛋白也参与了这种网络相互作用。 (SLC34A2/NaPi2b)，一种无机磷酸盐 (Pi) 输入通道，也定位于成熟的顶极成釉细胞，这可能表明 pHe 调节与牙釉质期间 Pi 转运活动之间存在直接联系数据表明，针对特定离子转运蛋白 mRNA 的 miRNA，也影响离子传输和 pHe 调节。分泌釉质器官细胞确定 miR-298 和 miR-346 在 NHERF1 调节中的潜在作用， Slc26a1、Slc26a7 和 Cftr 在本应用中，我们采用“SLC26A/CFTR/NHERF1/NaPi2b”。网络，指导与 HCO3- 和 Pi 运输以及 pH 调节直接相关的离子运动，同时在同时决定顶膜结构，对于牙釉质成熟至关重要，而对其的破坏网络会导致牙釉质病变，从而严重影响牙釉质寿命。” 具体目标：1) NHERF1、EZRIN、CFTR 和 NaPi2b 在成熟成釉细胞中的免疫定位；2) 使用 miR-346 和 miR-298 破坏体内釉质器官细胞中的 SLC26A/CFTR/NHERF1/NaPi2b 网络 3) 研究 NHERF1 和 NaPi2b 的性质在这项研究中，我们预计会破坏釉质器官细胞的体外和体内相互作用。通过直接靶向 NHERF1 功能，SLC26A/CFTR 网络和 NaPi2b 活性将产生显着的这项研究的结果将对我们对牙釉质畸形的理解产生重大影响。这项研究也可能涉及生物矿化过程，因为它与所有哺乳动物物种的牙釉质形成有关。制定临床处理遗传性牙釉质缺陷的策略；从长远来看，有助于预防和治疗遗传性牙釉质缺陷；减轻囊性纤维化和其他疾病患者的痛苦。