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.

项目总结/摘要 关于指导分泌前细胞分化的转录调控机制知之甚少 成釉细胞（PAB）转化为分泌型成釉细胞（SAB）并分泌釉原蛋白。SAB负责 釉基质蛋白（EMP）（主要是釉原蛋白）的合成和分泌，以形成完整的厚度 釉质基质为了分泌，EMP被包装到分泌囊泡中，运输到Tomes'过程， SAB的特征性细胞质突起，然后外吞到釉质间隙中以引导羟基磷灰石 晶体生长我们已经发现了一种特殊的富含AT的序列结合蛋白1（SATB 1），它是一种基因组组织者， 调节染色质结构和基因表达，在PAB中高度表达，而在 SATB 1的缺失抑制了PAB向极化分泌型SAB的分化。Satb 1-/-小鼠中的SAB 缺乏细胞极性、顶端肌动蛋白丝组装和Tomes'突起形成。这些Satb 1-/-SAB显示 釉原蛋白分泌的主要缺陷，导致薄和低矿化的釉质。这些发现意味着 SATB 1可能通过调节釉原蛋白所需基因的表达来调控釉质形成 贩卖人口我们的初步研究发现突触孔蛋白（synaptoporin，SYNPR）和表皮生长因子受体（epidermal growth factor receptor，EGF） 途径底物8（EPS 8）作为SATB 1依赖性基因，可能介导釉原蛋白分泌， 贩卖人口肠微绒毛形成过程中肌动蛋白丝组装所需的EPS 8， 免疫定位在Tomes'过程中，并且发现SYNPR，一种囊泡成分，与 SAB细胞质内存在囊泡样结构，囊泡中大部分携带釉原蛋白。因此我们 假设SATB 1通过其靶基因调节釉原蛋白的分泌运输， 分泌囊泡的形成（通过SYNPR）和肌动蛋白丝组装/Tomes'过程的形成（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 突触孔蛋白是成釉细胞中含釉原蛋白的囊泡形成所必需的。通过免疫染色和 共聚焦显微镜成像野生型，Satb 1-/-和突触孔蛋白-/-小鼠SAB，我们将确定是否突触孔蛋白- 含有携带釉原蛋白的囊泡。同时，我们将进行SAB囊泡组成分析，以验证 结果。具体目标2：我们将研究SATB 1和EPS 8在顶端肌动蛋白丝组装中的作用 在Tomes突形成和釉原蛋白分泌中起重要作用。通过敲除和诱导性过表达 在3-D上使用人PAB原代细胞培养的策略，SATB 1调节EPS 8的机制， 将研究直接成釉细胞形态发生和蛋白质分泌。SATB 1如何调节染色质 并对Eps 8等靶基因位点的表观遗传状态进行研究。这些研究将 进一步了解成釉细胞如何分化形成釉质，以及SATB 1如何调控基因的表达， 表达和染色质结构的关键球员在这些过程中。这项研究的信息 可以提供一个洞察力，以帮助工程分泌成釉细胞再生釉质组织。