C/EBP Regulation of Dentinogenesis

C/EBP 牙本质发生的调节

基本信息

批准号：
7585801
负责人：
JOHN R HARRISON
金额：
$ 18.5万
依托单位：
UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-04-01 至 2011-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7585801
关键词：
Ablation Acceleration CCAAT-Enhancer-Binding Proteins Cell Culture Techniques Cells Characteristics Dental Dental Pulp Dental crowns Dentin Dentin Dysplasia Dentinogenesis Dentistry Development Dimerization Dominant-Negative Mutation Electron Microscopy Embryo Engineered Gene Exhibits Future Gene Expression Genes Harvest Histology In Situ Hybridization Incisor Introns Kidney Knockout Mice Leucine Zippers Messenger RNA Modeling Morphology Mus Mutant Strains Mice Mutation Nude Mice Odontoblasts Odontogenesis Osteogenesis Pathway interactions Phenotype Plant Roots Play Production Protein Family Protein Isoforms Proteins Regulation Role Secondary to Side Signal Pathway Signal Transduction Signal Transduction Pathway Sphingolipids Sphingomyelinase Sphingomyelins Stem cells Structure Surface Testing Thick Tissue Engineering Tissues Tooth structure Transcriptional Regulation Transgenes Transgenic Mice Transgenic Model Transgenic Organisms Transplantation Tubular formation Visual Wild Type Mouse bZIP Domain base capsule density dentin matrix protein 1 factor C gene therapy inhibitor/antagonist loss of function mineralization mouse development mouse model novel osteoblast differentiation overexpression postnatal promoter public health relevance regenerative stem three-dimensional modeling transcription factor transgene expression

项目摘要

DESCRIPTION (provided by applicant): Transgenic (TG) mice with overexpression of p20C/EBP¿, a naturally occurring dominant negative C/EBP¿ isoform, showed evidence of dentin dysplasia occurring Preferentially on the lingual side of the incisors and in the molar roots. This was characterized by reduced thickness and disrupted tubular structure of the dentin. Odontoblasts in this region were small and did not assume their characteristic elongated, polarized morphology. C/EBP¿ null mice, in contrast, exhibit a tooth phenotype characterized by a spatial acceleration of incisor differentiation that appears to involve both lingual and labial surfaces. Based on a strikingly similar dentin phenotype between our TG model and fro/fro mutant mice with a mutation in the smpd3 gene that encodes neutral sphingomyelinase 2, we hypothesize a novel role for sphingolipid signaling acting through C/EBP¿ in the regulation of odontoblast differentiation. The following aims are proposed: 1) To assess the temporal and spatial effects of C/EBP¿ loss-of-function on tooth development using C/EBP¿ TG and knockout mice. Tooth development will be assessed in these models by histology, electron microscopy, and immunohistochemical localization of endogenous C/EBP and its dimerization partner Nrf1. 2) To assess cell autonomy of the odontoblast phenotype using ex vivo dental pulp cultures and heterologous pulp transplantation. Dental pulp cultures established from wild type (WT), TG and null mice will be examined for the tempo and extent of odontoblast differentiation by assessing expression of Col1a1-promoter-driven GFP, dental sialophosphoprotein and dentin matrix protein-1 mRNA, along with the degree of mineralization of the culture. Results will be corroborated by assessing odontoblast differentiation and dentin production in dental pulp transplanted under the kidney capsule of nude mice. 3) To determine the role of the sphingomyelin signal transduction pathway on odontogenesis and the requirement for C/EBP¿ function. The role of the sphingomyelin signaling pathway on odontogenesis will be assessed by testing the effects of pharmacological activators and inhibitors of the pathway on the rate and extent of odontoblast differentiation in dental pulp cultures derived from WT and mutant mice. Public Health Relevance: Understanding the transcriptional regulation of dentinogenesis will provide an essential underpinning for future tissue engineering and gene therapy approaches to reparative and regenerative dentistry using dental pulp stem/progenitor cells. The current proposal is focused on the role of the leucine zipper transcription factor C/EBP¿ and the sphingomyelin signaling pathway in odontoblast differentiation. These novel, possibly interconnected pathways may have translational implications for tooth reregeneration, elucidating the mechanisms of odontoblast lineage commitment and differentiation, and the specification of highly specialized crown or root odontoblast phenotypes.

描述（应用程序提供）：具有P20C/EBP€的过表达的转基因（TG）小鼠，一种天然存在的显性负C/EBP？同工，显示出牙本质发育不良的证据优先在门牙的舌侧和摩尔根部。其特征是厚度减小和牙本质的管状结构破坏。该地区的牙糖细胞很小，没有假定其特征性的延长，两极化的形态。相比之下，c/eBp¿无效的小鼠暴露了一种牙齿表型，其特征是门牙分化的空间加速度，似乎涉及舌表面和唇表面。基于我们的TG模型与FRO/FRO突变小鼠之间具有突变的SMPD3基因中的突变的牙本质表型，该基因编码中性鞘磷脂酶2的SMPD3基因中，我们假设在ODONTOBAST分化的调节中，通过C/EBP作用的鞘脂信号的新作用。提出了以下目的：1）评估使用C/EBP¿TG和基因敲除小鼠c/ebp？c/eBP的暂时和空间效应。在这些模型中，将通过组织学，电子显微镜和内源性C/EBP及其二聚化伴侣NRF1的免疫组织化学定位来评估牙齿发育。 2）使用离体牙髓培养物和异源纸浆移植来评估牙糖细胞表型的细胞自主。通过评估COL1A1-促进剂驱动的GFP的表达，牙磷酸蛋白和牙本质Matrix Protin-1 mRNA，以及培养的矿化程度，将检查由野生型（WT），TG和NULL小鼠建立的牙纸浆培养物，以评估COL1A1-促进剂驱动的GFP，猪磷酸蛋白和牙本质磷脂蛋白-1 mRNA，以及培养培养的程度，从而检查ODONTOBAST分化的节奏和程度。通过评估在裸鼠肾囊下移植的牙髓中的牙本质细胞分化和牙本质产生来证实结果。 3）确定鞘磷脂信号转导途径在牙肠发生和C/EBP函数的需求中的作用。鞘磷脂信号通路在牙生物发生上的作用将通过测试途径的药物激活剂和抑制剂对源自WT和突变小鼠的牙浆培养物的效果和程度的抑制剂的影响来评估。公共卫生相关性：了解牙齿生成的转录调节将为未来的组织工程和基因治疗方法提供必要的基础，以使用牙浆茎/祖细胞来修复和再生牙科。当前的建议集中在亮氨酸拉链转录因子C/EBP和鞘磷脂信号通路中的作用上。这些新颖的，可能的相互联系的途径可能对牙齿的重量产生了转化的影响，阐明了牙本质细胞谱系承诺和分化的机制，以及高度专业的牙冠或根源圆形细胞表型的规范。