MOLECULAR MECHANISMS FOR EXTRACELLULAR PH CONTROL DURING AMELOGENESIS

釉质形成过程中细胞外 pH 值控制的分子机制

基本信息

批准号：
7631397
负责人：
Michael Lansdell Paine
金额：
$ 40.69万
依托单位：
UNIVERSITY OF SOUTHERN CALIFORNIA
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-09-17 至 2011-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7631397
关键词：
Acid-Base Equilibrium Acids Affect Age Ameloblasts Amelogenesis Amelogenesis Imperfecta Animals Anion Exchangers (Proteins)Anions Apical Applications Grants Awareness Bicarbonates Biological Assay Blindness Case Study Cataract Cell membrane Cells Characteristics Childhood Chloride Channels Clinic Code Communication Cystic Fibrosis Transmembrane Conductance Regulator Data Defect Dental Dental Care Dental Enamel Dental caries Dentists Dentition Diagnosis Documentation Enamel Formation Equilibrium Exposure to Eye Eye diseases Face Failure Gene Expression Genes Glaucoma Hardness Hereditary Disease Human Image Immunofluorescence Immunologic In Situ Hybridization Inborn Genetic Diseases Incidence Indium Inherited Keratopathy Kidney Kidney Diseases Knockout Mice Label Life Location Maturation-Stage Ameloblast Mechanics Membrane Messenger RNA Metabolic Diseases Microscopic Molecular Molecular Profiling Mus Mutation Pathology Patients Phenotype Physicians Physiologic pulse Physiological Play Population Process Promoter Regions Protein Isoforms Proteins Proximal Renal Tubular Acidosis Publications Publishing RNA Renal function Reporter Reporting Resolution Role Sodium-Bicarbonate Symporters Staging Structure Syndrome System Tetanus Helper Peptide Tooth Diseases Tooth structure Transcript Transgenic Animals apical membrane base basolateral membrane cell type cost extracellular in vivo infant animal meetings mutant public health relevance spatiotemporal trait treatment planning

项目摘要

DESCRIPTION (provided by applicant): Amelogenesis imperfecta (AI) is a group of diverse inherited disorders most often considered as a single trait and not often associated with syndromes or metabolic disorders. However, there have been documented case reports in which AI has been identified in patients with inherited proximal renal tubular acidosis. These reports identify enamel anomalies directly related to this condition of pH control affecting kidney function. We have initiated studies to better understand what role transcellular bicarbonate (HCO3-) transport plays in acid/base transport and pH control during enamel formation. We have found that two proteins involved in acid/base transport are expressed in polarized ameloblasts during amelogenesis. These two proteins are the anion exchanger (AE2) and the electrogenic sodium bicarbonate cotransporter (NBCe1). Our preliminary data shows that NBCe1 is expressed at the basolateral membrane of secretory ameloblasts, whereas AE2 is expressed at the apical (secretory) membrane. These data are the first evidence that AE2 and NBCe1 are expressed in ameloblasts, in vivo, in a polarized fashion, thereby providing a mechanism for ameloblast transcellular bicarbonate secretion in the process of enamel formation and maturation. The hypothesis of this proposal is that "the spatiotemporal expression profiles of AE2 and NBCe1 in ameloblast cells are highly regulated and are responsive to changing extracellular pH conditions, and any failure in proper AE2 and NBCe1 activity will result in disruptions to the enamel prismatic structure". Five specific aims are proposed to investigate this hypothesis. These are: 1) will define the expression profiles for AE2 and NBCe1 by in situ hybridization; 2) will establish the spatial location of this AE2 and an associated chloride channel (CFTR) within the secretory face of ameloblasts using immunogold labeling and high resolution TEM; 3) will develop transgenic animal lines in which AE2 and NBCe1 silencing is achieved following the animals exposure to doxycyclin; 4) will be a radiographic and microscopic documentation of enamel defects in animals with mutations in the AE2 and NBCe1 gene loci; and 5) will identify pH-responsive elements in the promoter regions of AE2 and NBCe1 using an appropriate reporter assay. At the conclusion of this study there will be a greater understanding of dental disease that results from disruptions to HCO3- and acid/base transport. A raised awareness of dental disease in patients with kidney disease should result greater interactions between physicians and dentists, and result in more appropriate and comprehensive treatment plans that are initiated immediately after diagnosis, and carried out in a more cost-effective manner. PUBLIC HEALTH RELEVANCE: Patients with hereditary kidney and eye disease, where the cause is directly related to mutations in either the AE2 or NBCe1 proteins, frequently have dental anomalies including poorly mineralized enamel. These dental defects result in a higher incidence of dental decay starting at a very early age, and because of this these patients require significantly greater dental care that a normal population. AE2 and NBCe1 are both bicarbonate transporters involved in maintaining intracellular and extracellular pH. Once complete, this proposal will better define the molecular mechanism available to dental cells to maintain correct pH balance during the stages of tooth formation.

描述（由申请人提供）：Amelogenesis implfecta（AI）是一组最常见的遗传性疾病，通常被视为单一特征，并且通常与综合症或代谢性疾病有关。但是，已经有记录的病例报告，其中遗传性肾小管酸中毒的患者已经鉴定出AI。这些报告确定了与这种影响肾脏功能的pH控制状况直接相关的搪瓷异常。我们已经开始研究，以更好地了解在牙釉质形成期间在酸/碱基转运和pH控制中的跨细胞碳酸氢盐（HCO3-）转运作用。我们发现，在脱纤维细胞中，两种参与酸/碱基转运的蛋白在青年成年中表达。这两种蛋白质是阴离子交换剂（AE2）和碳酸氢钠共转运蛋白（NBCE1）。我们的初步数据表明，NBCE1在分泌成成细胞的基底外侧膜上表达，而AE2在顶端（分泌）膜上表示。这些数据是第一个证据表明，AE2和NBCE1以两极分化的方式在体内表达，从而在搪瓷形成和成熟过程中提供了一种无素细胞跨细胞碳酸氢盐分泌的机制。该提案的假设是“熟细胞细胞中AE2和NBCE1的时空表达谱受到高度调节，并且对改变细胞外pH条件的反应有响应，并且适当的AE2和NBCE1活性的任何失败都会导致对搪瓷棱镜结构的破坏” 。提出了五个具体目标来研究这一假设。这些是：1）将通过原位杂交定义AE2和NBCE1的表达曲线； 2）将使用免疫金标签和高分辨率TEM建立该AE2和相关的氯化物通道（CFTR）的空间位置； 3）将开发转基因动物品系，在这种动物暴露于多西环蛋白后，AE2和NBCE1沉默。 4）将是AE2和NBCE1基因基因座突变的动物搪瓷缺陷的射线照相和显微镜文献； 5）将使用适当的记者测定法确定AE2和NBCE1启动子区域中的pH响应元件。在这项研究的结论结束时，将对牙齿疾病有更多的了解，这是由于对HCO3和酸/碱转运而导致的牙齿疾病。对肾脏疾病患者牙齿疾病的认识提高，应导致医生和牙医之间的更多相互作用，并导致更合适，更全面的治疗计划，这些治疗计划在诊断后立即启动，并以更具成本效益的方式进行。公共卫生相关性：遗传性肾脏和眼病的患者，原因与AE2或NBCE1蛋白的突变直接相关，经常患有牙齿异常，包括矿物质不良。这些牙齿缺陷会导致牙齿衰减的发生率从很小的时候开始，因此这些患者需要比正常人群更大的牙齿护理。 AE2和NBCE1都是碳酸氢盐转运蛋白，参与维持细胞内和细胞外pH。完成后，该建议将更好地定义牙科细胞可用的分子机制，以在牙齿形成阶段保持正确的pH平衡。