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: 7488565
• 负责人: Regis J O'Keefe
• 金额: $ 32.45万
• 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/7488565
• 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; Rate; Regulation; Role; Signal Transduction; Tamoxifen; Tissues; Transgenic Mice; Vascular Endothelial Growth Factors; Week; beta catenin; bone morphogenetic protein 2; concept; 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）：？ - 卡宁蛋白已成为内侧软骨骨形成的关键调节剂。但是，研究？ - 催他在生长和发育中的作用的能力受到所有与软骨相关的基因缺失发生的胚胎或早期致死性的严重限制，这会导致 - 钙蛋白功能的损失或增益。该提案定义了？ - catenin在产后生长和发育中的作用，并使用一种创新的方法，使我们能够在小鼠中暂时控制组织特定的条件基因缺失。初步数据表明，他莫昔芬向Col2a1-cre +/- ERT2转基因小鼠的递送导致组织特异性基因重组。 AIM 1表征了CRE聚集酶在鼠软骨中的时间表达，并评估了 - 卡丁蛋白功能在产后生长和发育中的作用。 AIM 1a由一组集中的实验组成，该实验在递送他莫昔芬后完成基因重组模式的表征，并使用COL2A1-CRE +/- ERT2优化其给药。 ROSA26R +/-小鼠。 AIM 1B检查-Catenin+/fiox（exon3）的表型； col2a1-cre +/- ERT2小鼠在2周时产后条件基因缺失会导致软骨中的组成性活性？-catenin的表达。 AIM 2表征了？ - 卡丁蛋白功能在产后生长和发展中的作用丧失，并使用了两个互补模型。 ICAT是一种具有竞争力结合的细胞内蛋白吗？ - 蛋白酶并抑制信号传导。初步数据表明，在COL2A1-ICAT转基因小鼠中软骨细胞的成熟和持续延迟，这些小鼠在整个发育和产后生长过程中都抑制了？ - 卡丁蛋白信号传导。 AIM 2a表征了Col2A1-ICAT转基因小鼠的表型。 AIM 2B表征了-Cateninfloxko/ Floxko的表型； col2a1-cre +/- ERT2小鼠在2周时产后条件基因缺失会导致软骨中 - 卡宁素表达的丧失。 AIM 3使用一种体外方法，并定义了与终末软骨细胞成熟期间VEGF基因表达相互依赖的机制。初步数据表明，VEGF的诱导同时需要BMP和-Catenin信号传导。 AIM 3A考察了软骨细胞中BMP-2，4和6表达的调节。 AIM 3B定义了BMP-2和？蛋白酶对VEGF基因的合作诱导。 AIM 3C定义了VEGF-A启动子上关键的SMAD - ？ - catenin的相互作用。总的来说，这些实验将确定一个概念，即通过？ - 卡宁蛋白调节关键成熟基因的基因需要存在BMP共同信号，以提高软骨细胞分化的速度。这些发现将建立？ - 蛋白蛋白及其与BMP的合作效应，是软骨细胞肥大的重要信号，内软骨骨形成的完成以及VEGF表达的调节。拟议的研究将提供有关软骨疾病在内的关键新见解，包括软骨增生，骨修复和骨关节炎。