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

描述（由申请人提供）：连环蛋白已成为软骨内骨形成的关键调节剂。然而，研究的作用的能力？-连环蛋白在生长和发育中受到胚胎或早期致死性的严重限制，这种致死性发生在所有软骨相关基因缺失中，导致？catenin功能该提案确定了？-连环蛋白在出生后生长和发育中的作用，并使用一种创新的方法，使我们能够暂时控制小鼠中的组织特异性条件基因缺失。初步数据证实，将他莫昔芬递送至Col 2a 1-Cre+/-ERT 2转基因小鼠导致组织特异性基因重组。目的1描述Cre重组酶在小鼠软骨中的时间表达，并评估Cre重组酶在软骨中的作用。catenin在出生后生长发育中的作用。目的1A由一组集中的实验组成，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Col 2a 1-Cre+/-ERT 2; ROSA 26 R +/-小鼠优化了其给药。目的1B检查？- catenin+/fox（exon3）; Col 2a 1-Cre+/-ERT 2小鼠，其中出生后2周的条件性基因缺失导致组成型活性？软骨中的连环蛋白。目标2描述了？-连环蛋白功能丧失对出生后生长发育的影响，并采用两种互补模型。ICAT是一种细胞内蛋白，竞争性结合？catenin并抑制信号传导。初步数据显示，Col 2a 1-ICAT转基因小鼠的软骨细胞成熟和发育延迟，在整个发育过程中和出生后的生长过程中的连环蛋白信号传导。目的对Col 2a 1-ICAT转基因小鼠进行表型鉴定。目标2B描述了？-的表型特征在出生后2周条件性基因缺失导致？-ERT 2缺失的连环蛋白敲除/缺失敲除; Col 2a 1-Cre+/-ERT 2小鼠中，软骨中的连环蛋白表达。目标3使用体外方法，并定义了？在终末软骨细胞成熟过程中catenin和BMP信号对VEGF基因表达的影响。初步数据显示，VEGF的诱导需要BMP和？连环蛋白信号传导。目的3A研究骨形态发生蛋白-2，4和6在软骨细胞中表达的调节。连环蛋白。目的3B确定BMP-2和β-CD协同诱导VEGF基因表达。连环蛋白。目标3C定义了关键Smad-？- VEGF-A启动子上的连环蛋白相互作用。总之，这些实验将建立的概念，调控关键成熟相关基因的？连环蛋白需要BMP辅助信号的存在，该辅助信号用于提高软骨细胞分化的速率。调查结果将确定？连环蛋白及其与BMPs的协同作用是软骨细胞肥大、软骨内骨形成完成和VEGF表达调节的重要信号。拟议的研究将提供关于软骨疾病的重要新见解，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.