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

描述(申请人提供)：？-连环蛋白已成为软骨内骨形成的关键调节因子。然而，研究β-连环蛋白在生长发育中的作用的能力受到胚胎或早期致死的严重限制，所有软骨相关基因缺失都会导致β-连环蛋白功能的丧失或增加。这项建议定义了β-连环蛋白在出生后生长和发育中的作用，并使用了一种创新的方法，允许我们在小鼠身上暂时控制组织特异性条件性基因缺失。初步数据证实，将他莫昔芬传递给Col2a1-Cre/-ERT2转基因小鼠可导致组织特异性基因重组。目的1研究Cre重组酶在小鼠软骨中的时间表达特征，探讨其功能获得在出生后生长发育中的作用。目标1A由一组集中的实验组成，这些实验完成了他莫昔芬给药后基因重组模式的表征，并使用Col2a1-Cre/-ERT2；ROSA26R/-小鼠优化了给药。目的1B研究出生后2周条件性基因缺失导致软骨中具有结构性活性的β-连环蛋白表达的COL2a1-Cre/-ERT2小鼠的表型。目的2描述β-连环蛋白功能丧失对出生后生长发育的作用，并使用两个互补的模型。ICAT是一种细胞内蛋白，它竞争性地结合β-连环蛋白并抑制信号转导。初步数据显示，在Col2a1-ICAT转基因小鼠中，软骨细胞的成熟和奔跑延迟，在整个发育过程和出生后的生长过程中，β-连环蛋白信号都受到抑制。目的研究Col2a1-ICAT转基因小鼠的表型特征。目的2B研究出生后2周条件性基因缺失导致软骨中β-连环素表达缺失的β-cateninfloxKO/FloxKO；Col2a1-Cre/-ERT2小鼠的表型。目的采用体外实验方法，探讨软骨细胞终末成熟过程中β-连环蛋白和骨形态发生蛋白信号与血管内皮生长因子基因表达相互依赖的机制。初步数据显示，血管内皮生长因子的诱导需要骨形态发生蛋白和β-连环蛋白信号。目的研究β-连环蛋白对软骨细胞骨形态发生蛋白-2、4、6表达的调节作用。目的3B明确BMP-2和β-连环蛋白对血管内皮生长因子基因的协同诱导作用。AIM 3C定义了血管内皮生长因子-A启动子上关键的Smad-β-连环蛋白相互作用。总之，这些实验将确立这样一个概念，即β-连环蛋白对关键成熟相关基因的调节需要BMP共同信号的存在，以提高软骨细胞的分化率。这些发现将建立β-连环蛋白及其与骨形成蛋白的协同作用，作为软骨细胞肥大、软骨内骨形成完成和调节血管内皮生长因子表达的重要信号。拟议的研究将为软骨疾病提供关键的新见解，包括软骨发育不良、骨修复和骨关节炎。

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.