Zfp28 and Mesenchymal Stem Cell Differentiation

Zfp28 和间充质干细胞分化

• 批准号: 7095408
• 负责人: THOMAS Martin HERING
• 金额: $ 19.24万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-05-15 至 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7095408
• 关键词: cartilage development; cell differentiation; cell line; chondroitin sulfates; chromatin immunoprecipitation; collagen; developmental genetics; gene deletion mutation; gene expression; genetic manipulation; genetic promoter element; genetic transcription; genetically modified animals; laboratory mouse; mesenchyme; microarray technology; protein structure function; proteoglycan; reporter genes; stem cells; transfection

DESCRIPTION (provided by applicant): Although there is much interest in the clinical use of mesenchymal stem cells for repair and regeneration of skeletal tissues, the molecular mechanisms controlling differentiation are not well understood. Differentiation of mesenchymal cells into chondrocytes requires the coordinated expression of specific regulatory genes, of which few have been characterized. Zfp28, a candidate chondrogenic regulator, is a Cys2His2 zinc finger protein (ZFP) having N-terminal KRAB-A and B domains. Most KRAB zinc finger proteins have been shown to function as transcriptional repressors. Zfp28 is expressed in a limited subset of adult tissues, and is expressed in mouse embryos at the onset of skeletogenesis. Zfp28 is expressed in the chondrogenic murine ATDC5 cell line in a differentiation stage-specific manner. Overexpression of Zfp28 results in nuclear translocation, downregulation of Col2a1 mRNA, and inhibition of chondrogenic differentiation. Coexpression of Zfp28 results in downregulation of Col2a1 promoter activity in ATDC5 cells. The Zfp28 gene resides on mouse chromosome 7 (Mm7) within a cluster of imprinted zinc-finger genes, some of which are expressed in limb development. ZFP28, the human ortholog of Zfp28 is on 19q13.4, in a region having synteny with the region of Mm7 containing Zfp28. As zinc finger proteins have been shown to be highly divergent between species, conservation of the Zfp28 sequence between human and mouse suggests an important function. We hypothesize that Zfp28 functions as a transcriptional repressor of genes expressed during chondrogenesis. Specific aims to test this hypothesis are: (1) To determine whether Zfp28 is required for chondrogenesis in vivo. We will produce Zfp28 conditional knockout mice to determine the relationship of this gene to skeletal morphogenesis. The skeletal phenotype of the embryos will be analyzed for gross morphology, histology and gene expression. (2) to examine the role of Zfp28 in chondrogenic differentiation in vitro. We will analyze chondrogenic differentiation following manipulation of Zfp28 expression in ATDC5 cells or in the conditionally immortalized mesenchymal progenitor cell line BMC9. We will study the effect of Zfp28 expression upon transcription from collagen II and XI, and aggrecan promoter-reporter constructs. We will identify Zfp28 target genes by coupled chromatin immunoprecipitation and mouse promoter microarray analysis. This project will result in a better understanding of the molecular events regulating the differentiation of mesenchymal stem cells toward chondrocytes. This knowledge may accellerate progress toward novel therapies to repair or regenerate damaged or osteoarthritic cartilage.

描述(申请人提供)：尽管临床上使用间充质干细胞修复和再生骨骼组织很有兴趣，但控制分化的分子机制还不是很清楚。间充质细胞向软骨细胞的分化需要特定调控基因的协调表达，但目前还很少有这种基因的特征。Zfp28是一种具有N端KRAB-A和B结构域的Cys2His2锌指蛋白(ZFP)，是一种候选的软骨形成调控因子。大多数KRAB锌指蛋白已被证明是转录抑制蛋白。Zfp28在一组有限的成体组织中表达，在小鼠骨骼发育开始时在胚胎中表达。Zfp28在软骨小鼠ATDC5细胞系中以分化阶段特异的方式表达。Zfp28过表达导致核转位、COL2a1基因表达下调、抑制软骨细胞分化。Zfp28共表达导致ATDC5细胞中COL2a1启动子活性下调。Zfp28基因位于小鼠7号染色体(MM7)上，位于一组印迹锌指基因中，其中一些基因在肢体发育中表达。Zfp28，Zfp28的人类直系同源基因位于19q13.4，与含有Zfp28的MM7区域同源。由于锌指蛋白已被证明在不同物种之间存在高度差异，人类和小鼠之间Zfp28序列的保守表明了一个重要的功能。我们假设Zfp28作为软骨形成过程中表达基因的转录抑制因子发挥作用。验证这一假说的具体目的是：(1)确定Zfp28是否是体内软骨形成所必需的。我们将制造Zfp28条件性基因敲除小鼠，以确定该基因与骨骼形态发生的关系。将对胚胎的骨骼表型进行大体形态、组织学和基因表达分析。(2)探讨Zfp28在体外向软骨细胞分化中的作用。我们将分析Zfp28在ATDC5细胞或条件永生化间充质祖细胞系BMC9中表达后的软骨分化。我们将研究Zfp28的表达对II型和XI型胶原转录的影响，以及aggrecan启动子-报告结构。我们将通过染色质免疫沉淀和小鼠启动子微阵列分析来鉴定Zfp28靶基因。该项目将有助于更好地理解调节间充质干细胞向软骨细胞分化的分子事件。这一知识可能会加速修复或再生受损或骨关节炎软骨的新疗法的进展。