Role of Transcription Factor ERG in Skeletogenesis

描述(申请人提供)：在肢体骨骼形成过程中，位于长骨骨痂末端的软骨细胞发育成永久性关节软骨细胞，终生维持关节功能。相反，构成骨干的软骨细胞是暂时性细胞，最终通过软骨内成骨被骨取代。我们在几年前发起了这个项目，以确定关节软骨细胞的形成和长期表型稳定的分子机制，并专注于转录因子Erg。ERG属于调控几个关键过程的ETS转录因子家族，是包括Fli-1在内的ETS亚家族的一部分。在上一期的研究中，我们发现ERG在滑膜关节发育早期与Gdf5和Wnt9a一起表达，并且随着时间的推移其表达逐渐减弱。为了测试功能，我们建立了Flosed ERG小鼠，并通过与Gdf5-Cre小鼠交配来有条件地消融发育中的关节的ERG。出乎意料的是，肢体关节发育继续进行，ERG缺陷小鼠存活到成年。为了解释缺乏主要的联合发育表型，我们询问Fli-1是否共表达，事实上是的，并且可能补偿了ERG的缺失。为了测试ERG可能的出生后作用，我们对2月龄ERG缺陷小鼠进行了膝关节内侧副韧带(MCL)横断，以诱导实验性骨关节炎(OA)。令人惊讶的是，ERG缺陷小鼠比操作野生型小鼠更早出现严重的类骨关节炎缺陷。事实上，我们在7-11个月大的未手术的老年ERG缺陷小鼠中观察到了类似的严重的OA样缺陷，而对照组小鼠表现出轻微的缺陷。为了深入了解ERG如何维持关节软骨细胞的长期功能，我们重点研究了甲状旁腺激素相关蛋白(PTHrP)，它在发育中的关节中也有表达，当在软骨中过度表达时，稳定软骨细胞的表型并防止软骨细胞肥大(就像转基因ERG过表达一样)。我们发现ERG(以及Fli-1)刺激PTHrP的表达，并且PTHrP基因启动子含有几个保守的Ets结合位点，是反应所需的。这些数据和其他数据导致我们的中心假设是ERG对于关节软骨细胞的发育和长期稳定和功能是必不可少的，并与Fli-1和PTHrP协同完成这一任务。我们的目标是：(1)揭示ERG和Fli-1在关节形成和长期关节软骨稳定中各自的作用；(2)确定Erg和Fli-1如何调节PTHrP的表达；以及(3)确定转基因Erg表达是否保护关节免受手术诱导的骨关节炎的影响。这项工作将使用多种实验方法进行，包括转基因小鼠遗传学、显微外科手术和细胞表型表达。它将对关节软骨细胞的生物学和分子生物学产生根本性的新数据和见解，并将为创造未来的修复和再生疗法铺平道路，通过这种疗法，关节软骨细胞可以恢复受到各种不利条件影响的功能，包括骨关节炎和自然衰老。

DESCRIPTION (provided by applicant): During limb skeletogenesis, chondrocytes located at the epiphyseal ends of long bone anlagen develop into permanent articular chondrocytes that sustain joint function though life. Instead, the chondrocytes constituting the shaft are transient cells and are eventually replaced by bone via endochondral ossification. We initiated this project several years ago to identify the molecular mechanisms of formation and long-term phenotypic stabilization of articular chondrocytes and focused on transcriptional factor Erg. Erg belongs to the ets family of transcription factors that regulate several key processes, and is part of an ets subfamily that includes Fli-1. In the previous funding period, we showed that Erg is expressed during early synovial joint development along with Gdf5 and Wnt9a and its expression dwindles over time. To test function, we created floxed Erg mice and conditionally ablated Erg in developing joints by mating with Gdf5-Cre mice. Unexpectedly, limb joint development proceeded and the Erg-deficient mice survived to adulthood. To account for absence of a major joint developmental phenotype, we asked whether Fli-1 was co-expressed and in fact it was, and may have compensated for Erg absence. To test possible postnatal roles of Erg, we subjected 2 month-old Erg-deficient mice to knee's medial collateral ligament (MCL) transection to induce experimental osteoarthritis (OA). Strikingly, the Erg-deficient mice developed serious OA-like defects far sooner than operated wild type companions. Indeed, we observed similar severe OA-like defects in aging un-operated 7-11 month-old Erg-deficient mice, while control littermates displayed mild defects. To gain insights into how Erg maintains long-term articular chondrocyte function, we focused on parathyroid hormone-related protein (PTHrP) which is also expressed in developing joints, stabilizes the chondrocyte phenotype and prevents chondrocyte hypertrophy when over-expressed in cartilage (just as transgenic Erg over-expression does). We found that Erg (as well as Fli-1) stimulates PTHrP expression and the PTHrP gene promoter contains several conserved ets binding sites needed for responsiveness. These and other data lead to our central hypothesis is that Erg is essential for the development and long-term stabilization and function of articular chondrocytes and does so in cooperation with Fli-1 and PTHrP. Our Aims are: (1) To uncover the respective roles of Erg and Fli-1 in joint formation and long-term articular cartilage stabilization; (2) To determine how Erg and Fli-1 regulate PTHrP expression; and (3) To determine if transgenic Erg expression protects joints from surgically-induced OA. The work will be carried out using diverse experimental approaches that include transgenic mouse genetics, microsurgery and cell phenotypic expression. It will produce fundamentally new data and insights into the biology and molecular biology of articular chondrocytes and will pave the way to create future repair and regeneration therapies by which function can be restored in articular chondrocytes affected by a variety of adverse conditions including osteoarthritis and natural aging.