RUNX2 AND PPAR-GAMMA IN CHONDROCYTE FUNCTION

描述（由申请人提供）：人们早就知道关节软骨细胞终生保持稳定的表型，而生长板软骨细胞调节其表型，经历成熟和肥大，并被骨取代。然而，软骨细胞如何能够维持或调节其表型以发挥这些根本不同的功能仍然很大程度上不清楚。Runx2和PPARgamma是近年来备受关注的两个转录因子。Runx2在软骨细胞肥大和骨化过程中上调，是必需的；PPARgamma在人关节软骨中表达，具有抗炎作用。我们现在发现这两种因子在骺软骨细胞和未成熟生长板软骨细胞中都有表达。Runx2过表达加速成熟，而过表达显性阴性（DN） Runx2阻滞成熟并导致软骨细胞去分化。后者也见于从Runx2-/-小鼠胚胎中分离的软骨细胞。相反，PPARgamma过表达阻碍成熟，而DN-PPARgamma过表达促进成熟。另外的体外和体内数据表明，Runx2和PPARgamma的表达是紧密协调的，这两个基因在软骨细胞中具有相互依赖的作用。我们的中心假设是：(a)稳定的软骨细胞表型需要持续且相对低的Runx2和PPARgamma表达；(b) Runx2上调和PPARgamma下调导致软骨细胞成熟和肥大。我们将通过分析Runx2和PPARgamma在关节和生长板软骨和细胞中的产前和产后表达和活性模式来验证这些假设。我们将确定在体外和体内对Runx2和PPARgamma的表达和活性进行实验操作的后果，包括在产前和/或产后对软骨细胞中的每个基因进行条件消融。将测试Runx2和PPARgamma调控的分子机制，包括可能的直接蛋白质-蛋白质相互作用，导致复合物具有改变的反式激活特性。该项目的结果将为软骨细胞表型的分子调控提供新的重要见解，并将为改善骨骼和关节自然衰老和病理状态下的细胞和组织功能提供新的方法。

DESCRIPTION (provided by applicant): It has long been known that articular chondrocytes maintain a stable phenotype through life, whereas growth plate chondrocytes modulate their phenotype, undergo maturation and hypertrophy, and are replaced by bone. However, it is still largely unclear how chondrocytes are able to maintain or modulate their phenotype to exert those fundamentally distinct functions. Runx2 and PPARgamma are two transcription factors that have received a great deal of attention recently. Runx2 is up-regulated in, and required for, chondrocyte hypertrophy and ossification, and PPARgamma is expressed in human articular cartilage and exerts anti-inflammatory roles. We have now found that both factors are expressed in epiphyseal and immature growth plate chondrocytes. Runx2 over-expression accelerates maturation, whereas over-expression of a dominant-negative (DN) Runx2 blocks maturation and leads to chondrocyte de-differentiation. The latter is seen also in chondrocytes isolated from Runx2-/- mouse embryos. In contrast, PPARgamma over-expression blocks maturation, while DN-PPARgamma over-expression favors maturation. Additional in vitro and in vivo data indicate that expression of Runx2 and PPARgamma is tightly coordinated and that the two genes have interdependent roles in chondrocytes. Our central hypotheses are: (a) Continuous and relatively low expression of Runx2 and PPARgamma is needed for a stable chondrocyte phenotype; and (b) Runx2 up-regulation and PPARgamma down-regulation lead to chondrocyte maturation and hypertrophy. We will test these hypotheses by analyzing pre-natal and post-natal patterns of expression and activity of Runx2 and PPARgamma in articular and growth plate cartilages and cells. We will determine consequences of experimental manipulation of Runx2 and PPARgamma expression and activity in vitro and in vivo, including conditional ablation of each gene in chondrocytes at prenatal and/or postnatal points. Molecular mechanisms of regulation of Runx2 and PPARgamma will be tested, including possible direct protein-protein interactions resulting in complexes with altered trans-activating properties. The results of the project will shed novel and important insights into the molecular regulation of chondrocyte phenotype, and will suggest new ways to improve cell and tissue function during natural aging and pathological conditions of the skeleton and joints.