Regulation of enamel matrix protein secretion in ameloblasts

成釉细胞釉质基质蛋白分泌的调节

• 批准号: 10192703
• 负责人: Yan Zhang
• 金额: $ 37.64万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-07-01 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10192703
• 关键词: 3-Dimensional; AT Rich Sequence; ATAC-seq; Address; Ameloblasts; Apical; Architecture; Binding Proteins; Binding Sites; Biological; Bundling; Cell Culture System; Cell Lineage; Cell Polarity; Cells; ChIP-seq; Characteristics; Chromatin; Chromatin Structure; Crystallization; Cytoplasm; Cytosol; Defect; Dental Enamel; EPS8 gene; Ectopic Expression; Electron Microscope; Enamel Formation; Engineering; Epidermal Growth Factor Receptor Pathway Substrate 8; Epigenetic Process; Epithelial Cells; Gene Expression; Genes; Genetic Transcription; Genome; Growth; Human; Hydroxyapatites; Impairment; Incisor; Intestines; Laboratories; Length; Mass Spectrum Analysis; Mediating; Microarray Analysis; Microfilaments; Minerals; Molecular; Morphogenesis; Mus; Natural regeneration; Phenotype; Physiological; Primary Cell Cultures; Process; Protein Secretion; Proteins; Regulation; Research; Resolution; Role; Secretory Vesicles; Structure; Synaptic Vesicles; System; Testing; Thick; Thinness; Time; Tissues; Tooth structure; Vesicle; Western Blotting; Wild Type Mouse; amelogenin; cell regeneration; cellular microvillus; chromatin immunoprecipitation; enamel matrix proteins; experimental study; genomic locus; histone modification; insight; knock-down; microscopic imaging; overexpression; recruit; small hairpin RNA; trafficking; transcription factor; transcriptome sequencing; transmission process; vesicle transport

Project Summary/Abstract Little is known about the transcriptional regulatory mechanisms directing the differentiation of presecretory ameloblasts (PAB) into secretory ameloblasts (SAB) and amelogenin secretion. SABs are responsible for the synthesis and secretion of enamel matrix proteins (EMPs) (primarily amelogenins) to form the full thickness of enamel matrix. For secretion, EMPs are packaged into secretory vesicles, transported to Tomes' process, the characteristic cytoplasmic projections of SAB, then exocytosed into the enamel space to direct hydroxyapatite crystal growth. We have found that special AT-rich sequence binding protein 1 (SATB1), a genome organizer which regulates chromatin architecture and gene expression, is highly expressed in PAB, and that in the absence of SATB1, differentiation of PAB to the polarized secretory SAB is inhibited. SABs in Satb1-/- mice lack cell polarity, apical actin filament assembly, and Tomes' process formation. These Satb1-/- SABs display major defects in amelogenin secretion, resulting in thin and hypomineralized enamel. These findings imply that SATB1 may govern enamel formation by regulating the expression of genes required for amelogenin trafficking. Our preliminary studies identified synaptoporin (SYNPR) and epidermal growth factor receptor pathway substrate 8 (EPS8) as SATB1-dependent genes that potentially mediate amelogenin secretion and trafficking. EPS8, required for actin filament assembly in the formation of intestinal microvilli, was immunolocalized in Tomes' processes, and SYNPR, a vesicle component, was found associated with the vesicle-like structures in the cytoplasm of SAB, where the most of vesicles carry amelogenins. Thus, we hypothesize that SATB1 regulates amelogenin secretory trafficking through its target genes associated with the formation of secretory vesicles (via SYNPR) and the actin filament assembly/Tomes' process formation (via EPS8). To address this hypothesis we propose two specific aims: Specific Aim 1: We will determine whether synaptoporin is necessary for amelogenin-containing vesicle formation in ameloblasts. By immunostaining and confocal microscope imaging of wt, Satb1-/- and Synaptoporin-/- mouse SAB, we will determine if synaptoporin- containing vesicles carry amelogenins. In parallel, we will perform SAB vesicle composition analysis to verify the results. Specific Aim 2: We will investigate the roles of SATB1 and EPS8 on apical actin filament assembly in Tomes' process formation and amelogenin secretion. By knockdown and inducible overexpression strategies using the human PAB primary cell culture on 3-D, mechanisms by which SATB1 regulates EPS8 to direct ameloblast morphogenesis and protein secretion will be investigated. How SATB1 regulates chromatin and epigenetic statuses of the loci of Eps8 and other target genes will also be studied. These studies will advance our understanding how ameloblasts differentiate to form enamel, and how SATB1 regulates gene expression and chromatin structure for key players in these processes. The information from this research may provide an insight to help engineer secretory ameloblasts to regenerate enamel tissues.

项目摘要/摘要 关于指导预偏分化的转录调节机制知之甚少 成纤维细胞（PAB）成分泌的成成成成成组织（SAB）和氨基蛋白素分泌。 SABS负责 牙釉质基质蛋白（EMP）（主要是氨基蛋白）的合成和分泌，形成了完整的厚度 搪瓷矩阵。为了分泌，EMP被包装成分泌的囊泡，运输到Tomes的过程， SAB的特征性细胞质投影，然后外旋转在牙釉质空间中以直接羟基磷灰石 晶体生长。我们发现特殊的富富序列结合蛋白1（SATB1），一种基因组组织者 调节染色质结构和基因表达，在PAB中高度表达 缺乏SATB1，抑制了PAB与偏振分泌SAB的分化。 SATB1 - / - 小鼠中的SABS 缺乏细胞极性，根尖肌动蛋白丝组装和TOMES的过程形成。这些SATB1 - / - SABS显示 氨基蛋白蛋白分泌的主要缺陷，导致薄和低矿物化的搪瓷。这些发现暗示着 SATB1可以通过调节氨基蛋白素所需的基因表达来控制搪瓷的形成 贩运。我们的初步研究确定了突触蛋白（SYNPR）和表皮生长因子受体 途径底物8（EPS8）作为依赖SATB1的基因，可能介导amelogen蛋白分泌和 贩运。肌动蛋白丝组件在形成肠道微绒毛中所需的EPS8是 在tomes的过程中进行免疫定位，并且发现与囊泡成分Synpr相关 SAB的细胞质中的囊泡状结构，其中大部分囊泡携带蛋白蛋白。因此，我们 假设SATB1通过其靶基因与与 分泌囊泡的形成（通过SYNPR）和肌动蛋白细丝组件/Tomes的过程形成（通过 EPS8）。为了解决这一假设，我们提出了两个具体目标：具体目标1：我们将确定是否确定是否是否 突触蛋白对于含蛋白蛋白的囊泡形成是必需的。通过免疫染色和 WT，SATB1 - / - 和突触蛋白 - / - 小鼠SAB的共聚焦显微镜成像，我们将确定突触突触蛋白 - 是否是否 含有囊泡携带蛋白蛋白。同时，我们将执行SAB囊泡组成分析以验证 结果。具体目标2：我们将研究SATB1和EPS8在顶端肌动蛋白丝组件上的作用 在TOMES的过程形成和氨基蛋白蛋白分泌中。通过敲低和诱导的过表达 使用人类PAB原代细胞培养的策略在3-D的机制上进行了调节EPS8的机制 将研究直接的成成细胞形态发生和蛋白质分泌。 SATB1如何调节染色质 EPS8基因座和其他靶基因的表观遗传状态也将进行研究。这些研究会 促进我们理解成成木如何区分形成搪瓷的方式，以及SATB1如何调节基因 在这些过程中，主要参与者的表达和染色质结构。这项研究的信息 可以提供一个见识，以帮助工程师分泌的成成细胞再生牙釉质组织。