The use of genetic models to define the role of beta-catenin in post-natal growth

使用遗传模型来定义 β-连环蛋白在产后生长中的作用

• 批准号: 7913043
• 负责人: Regis J O'Keefe
• 金额: $ 32.12万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-01 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7913043
• 关键词: Binding; Bone Regeneration; Cartilage; Cartilage Diseases; Chondrocytes; Data; Degenerative polyarthritis; Development; Embryo; Gene Deletion; Gene Expression; Genes; Genetic Models; Genetic Recombination; Growth; Growth and Development function; Hypertrophy; In Vitro; Modeling; Mus; Osteogenesis; Pattern; Phenotype; Proteins; Regulation; Role; Signal Transduction; Tamoxifen; Tissues; Transgenic Mice; Vascular Endothelial Growth Factors; beta catenin; bone morphogenetic protein 2; chondrodysplasia; gain of function; innovation; insight; loss of function; promoter; recombinase; research study

DESCRIPTION (provided by applicant): ?-catenin has emerged as a critical regulator of endochondral bone formation. However, the ability to study the role of ?-catenin in growth and development is severely limited by the embryonic or early lethality that occurs in all cartilage-related gene deletions that result in loss or gain of ?-catenin function. This proposal defines the role of ?-catenin in post-natal growth and development and uses an innovative approach that permits us to temporally control tissue specific conditional gene deletion in mice. Preliminary data establish that delivery of tamoxifen to Col2a1-Cre+/-ERT2 transgenic mice results in tissue specific gene recombination. Aim 1 characterizes the temporal expression of Cre-recombinase in murine cartilage and assesses the role of ?-catenin gain of function on post-natal growth and development. Aim 1A consists of a focused set of experiments that complete a characterization of the pattern of gene recombination following delivery of tamoxifen and optimizes its administration using Col2a1-Cre+/-ERT2; ROSA26R +/- mice. Aim 1B examines the phenotype of ?-catenin+/fiox(exon3); Col2a1-Cre+/-ERT2 mice in which post-natal conditional gene deletion at 2 weeks results in expression of a constitutively active ?-catenin in cartilage. Aim 2 characterizes the role of ?-catenin loss of function on post-natal growth and development and uses two complementary models. ICAT is an intracellular protein that competitively binds ?-catenin and inhibits signaling. Preliminary data show delayed chondrocyte maturation and runting in Col2a1-ICAT transgenic mice, which have inhibition of ?-catenin signaling throughout development and during post-natal growth. Aim 2A characterizes the phenotype of Col2a1-ICAT transgenic mice. Aim 2B characterizes the phenotype of ?-cateninfloxKO/ floxKO; Col2a1-Cre+/-ERT2 mice in which post-natal conditional gene deletion at 2 weeks results in loss of ?-catenin expression in cartilage. Aim 3 uses an in vitro approach and defines mechanisms involved in the interdependence of ?-catenin and BMP signaling on VEGF gene expression during terminal chondrocyte maturation. Preliminary data show that induction of VEGF requires both BMP and ?-catenin signaling. Aim 3A examines regulation of BMP-2, 4, and 6 expressions in chondrocytes by ?-catenin. Aim 3B defines the cooperative induction of the VEGF gene by BMP-2 and ?-catenin. Aim 3C defines critical Smad-?-catenin interactions on the VEGF-A promoter. Altogether, these experiments will establish the concept that regulation of key maturation associated genes by ?-catenin requires the presence of BMP co-signals that act to enhance the rate of chondrocyte differentiation. The findings will establish ?-catenin and its cooperative effects with BMPs as an essential signal for chondrocyte hypertrophy, completion of endochondral bone formation, and regulation of VEGF expression. The proposed studies will provide critical new insights regarding cartilage diseases including chondrodysplasia, bone repair, and osteoarthritis.

描述（由申请人提供）：β-连环蛋白已成为软骨内骨形成的关键调节剂。然而，研究β-连环蛋白在生长和发育中的作用的能力受到胚胎或早期致死性的严重限制，这种死亡发生在所有软骨相关基因缺失中，导致β-连环蛋白功能的丧失或获得。该提案定义了β-连环蛋白在产后生长和发育中的作用，并使用了一种创新方法，使我们能够暂时控制小鼠组织特异性条件基因缺失。初步数据表明，将他莫昔芬递送至 Col2a1-Cre+/-ERT2 转基因小鼠会导致组织特异性基因重组。目标 1 表征小鼠软骨中 Cre 重组酶的时间表达，并评估 β-连环蛋白功能获得对出生后生长和发育的作用。目标 1A 由一组重点实验组成，这些实验完成了他莫昔芬递送后基因重组模式的表征，并使用 Col2a1-Cre+/-ERT2 优化了其给药； ROSA26R +/- 小鼠。目标 1B 检查 β-catenin+/fiox(exon3) 的表型； Col2a1-Cre+/-ERT2 小鼠，其中出生后 2 周条件性基因缺失导致软骨中组成型活性 β-连环蛋白的表达。目标 2 描述了 β-连环蛋白功能丧失对产后生长和发育的作用，并使用了两种互补模型。 ICAT 是一种细胞内蛋白质，可竞争性结合 β-连环蛋白并抑制信号传导。初步数据显示，Col2a1-ICAT 转基因小鼠的软骨细胞成熟和发育延迟，在整个发育过程和出生后生长过程中抑制 β-连环蛋白信号传导。目标 2A 表征 Col2a1-ICAT 转基因小鼠的表型。目标 2B 表征 β-cateninfloxKO/ floxKO 的表型； Col2a1-Cre+/-ERT2 小鼠，其中出生后 2 周条件性基因缺失导致软骨中 β-连环蛋白表达丧失。目标 3 使用体外方法并定义了终末软骨细胞成熟过程中 β-连环蛋白和 BMP 信号对 VEGF 基因表达相互依赖性的机制。初步数据表明，VEGF 的诱导需要 BMP 和 β-连环蛋白信号传导。目标 3A 检查 β-连环蛋白对软骨细胞中 BMP-2、4 和 6 表达的调节。目标 3B 定义了 BMP-2 和 β-连环蛋白对 VEGF 基因的协同诱导。目标 3C 定义了 VEGF-A 启动子上关键的 Smad-β-连环蛋白相互作用。总之，这些实验将建立这样的概念：β-连环蛋白对关键成熟相关基因的调节需要 BMP 协同信号的存在，从而增强软骨细胞的分化率。研究结果将确定β-连环蛋白及其与 BMP 的协同作用，作为软骨细胞肥大、软骨内骨形成完成和 VEGF 表达调节的重要信号。拟议的研究将为软骨疾病（包括软骨发育不良、骨修复和骨关节炎）提供重要的新见解。