Vesicle Trafficking and Osteoblast Function

囊泡运输和成骨细胞功能

基本信息

批准号：
10709486
负责人：
JOY Y WU
金额：
$ 17.12万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-09-23 至 2024-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10709486
关键词：
Acceleration Adult Affect Age Biological Assay Biology CRISPR/Cas technology Cells Collagen Collagen Type I Data Fibroblasts Fracture Future Gene Deletion Gene Expression Gene Mutation Genes Golgi Apparatus Green Fluorescent Proteins Human Hydroxylation Impairment Knockout Mice Measures Methods Modification Monitor Mus Nodule Ontology Osteoblasts Osteogenesis Osteoporosis Pharmaceutical Preparations Pluripotent Stem Cells Post-Translational Protein Processing Production Protocols documentation Qualifying Rattus Reporter Role Small Interfering RNA Source Techniques Vesicle Woman bone conditional knockout directed differentiation embryonic stem cell endoplasmic reticulum stress gene transplantation for gene therapy in vitro Assay in vivo innovation insight interest knock-down men mineralization new therapeutic target novel osteoblast differentiation promoter skeletal disorder small hairpin RNA subcutaneous therapeutic target trafficking transcriptome sequencing

项目摘要

Project Summary Osteoporosis is a disease of skeletal fragility that causes fractures in 50% of women and 25% of men over age 50. The most commonly prescribed anti-resorptive osteoporosis medications cannot cure osteoporosis, while currently available bone-building anabolic osteoporosis medications are limited by waning efficacy. There is still a great unmet need for safe and sustained approaches to increasing bone formation. We have shown that 2.3-kb rat type I collagen promoter-driven green fluorescent protein (Col2.3GFP) is highly expressed in mature osteoblasts. We isolated Col2.3GFP(hi) osteoblasts from bones, cultured bone chips, and directed differentiation of embryonic stem cells, and by RNA-sequencing identified 593 genes that are enriched in mature osteoblasts. Gene ontology (GO) analysis identified ER-to-Golgi vesicle trafficking as the most highly enriched GO term. Our preliminary data reveal that transient knockdown in MC3T3 osteoblasts of several vesicle trafficking genes results in increased mineralized nodule formation and accelerated osteoblast marker expression. Our central hypothesis is that disruption of vesicle trafficking impairs bone formation due to collagen overmodification and hypermineralization. We will leverage several innovative methods: direct reprogramming of fibroblasts to derive induced osteoblasts, CRISPR/Cas9 gene editing to delete vesicle trafficking genes in induced osteoblasts, Col2.3GFP as a cell-autonomous osteoblast reporter, and subcutaneous transplantation of gene-edited osteoblasts to assess in vivo bone formation. We screened vesicle trafficking genes with transient siRNA knockdown, and selected 9 genes (Preb, Stx5a, Rab2a, Gosr2, Bet1, Ramp1, Arf4, Cog6, Pacs1) whose knockdown increased mineralized nodule formation, osteoblast marker expression, and ER stress. In Aim 1 we will determine whether disruption of vesicle trafficking increases mineralization due to collagen overmodification. We will perform permanent knockdown by CRISPR/Cas9 gene editing of each gene in mouse and human induced osteoblasts with the Col2.3GFP osteoblast reporter and assess osteoblast marker expression and mineralized nodule formation. We will measure collagen production and post-translational modification by prolyl hydroxylation, and determine whether inhibition of collagen overmodification can restore osteoblast function. In Aim 2 we will determine whether disruption of vesicle trafficking impairs bone formation in vivo. We will examine bone formation in vivo by subcutaneous transplantation of gene-edited mouse and human iOBs. We will determine whether inhibition of collagen overmodification restores bone formation in vivo. Successful completion of these aims will identify novel genes involved in osteogenesis as potential therapeutic targets for the treatment of osteoporosis, and will provide mechanistic insights into the role of vesicle trafficking machinery in osteoblast function.

项目摘要骨质疏松症是一种骨骼脆弱性的疾病，会导致50％的女性和25％以上的男性骨折 50。最常见的抗敏感性骨质疏松症药物无法治愈骨质疏松症，而当前可用的骨建造合成代谢骨质疏松药物受到降低功效的限制。还有对安全和持续的骨骼形成方法的巨大需求。我们已经显示了 2.3-kb大鼠I型胶原蛋白启动子驱动的绿色荧光蛋白（Col2.3GFP）在成熟的成骨细胞。我们从骨骼，培养的骨芯片中分离了Col2.3GFP（HI）成骨细胞，并定向胚胎干细胞的分化，并通过RNA测序鉴定出593个基因，这些基因富含成熟成骨细胞。基因本体论（GO）分析确定ER到高尔基囊泡运输是最高度富集的去学期。我们的初步数据表明，几个囊泡的MC3T3成骨细胞中的瞬时敲低运输基因导致矿化结节形成增加并加速成骨细胞标记表达。我们的中心假设是，囊泡运输的破坏会损害由于胶原蛋白过度修饰和高矿化。我们将利用几种创新方法：直接重编程成纤维细胞以得出诱导的成骨细胞，CRISPR/CAS9基因编辑以删除囊泡诱导成骨细胞中的运输基因，Col2.3GFP作为细胞自治成骨细胞记者和基因编辑的成骨细胞的皮下移植以评估体内骨形成。我们筛选了囊泡具有瞬时siRNA敲低的运输基因，并选择了9个基因（PERB，STX5A，RAB2A，GOSR2，BET1， RAMP1，ARF4，COG6，PACS1）的敲低矿化结节形成，成骨细胞标记表达和ER应力。在AIM 1中，我们将确定囊泡运输的破坏是否增加由于胶原蛋白过度变化而引起的矿化。我们将通过CRISPR/CAS9基因执行永久敲低用COL2.3GFP成骨细胞记者的小鼠和人类诱导的成骨细胞编辑每个基因的编辑评估成骨细胞标记表达和矿化结节形成。我们将测量胶原蛋白生产和通过羟基化羟基化的翻译后修饰，并确定是否抑制胶原蛋白过度修饰可以恢复成骨细胞功能。在AIM 2中，我们将确定囊泡的破坏贩运会损害体内骨形成。我们将通过皮下检查体内骨形成基因编辑的小鼠和人IOB的移植。我们将确定是否抑制胶原蛋白过度修饰可在体内恢复骨形成。这些目标的成功完成将确定新的基因参与骨生成，作为治疗骨质疏松症的潜在治疗靶标，将提供囊泡运输机制在成骨细胞功能中的作用的机械洞察力。