Role of Transcription Factor ERG in Skeletogenesis

描述（由申请人提供）：在肢体骨骼形成过程中，位于长骨原骺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，构成骨轴的软骨细胞是短暂的细胞，最终通过软骨内成骨被骨所取代。我们几年前开始了这个项目，以确定关节软骨细胞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转录因子ets家族，是包括fl -1在内的ets亚家族的一部分。在之前的资助期内，我们发现Erg与Gdf5和Wnt9a一起在滑膜关节发育早期表达，并且随着时间的推移其表达逐渐减少。为了测试其功能，我们建立了固定的Erg小鼠，并通过与Gdf5-Cre小鼠交配，在发育关节中有条件地消融Erg。出乎意料的是，肢体关节发育继续进行，ergg缺陷小鼠存活到成年。为了解释主要联合发育表型的缺失，我们询问了Fli-1是否共表达，事实上它是，并且可能补偿了Erg的缺失。为了测试Erg可能在出生后的作用，我们对2月龄ergg缺陷小鼠进行膝关节内侧副韧带（MCL）横断，以诱导实验性骨关节炎（OA）。引人注目的是，ergg缺陷小鼠比野生型小鼠更早出现严重的oa样缺陷。事实上，我们在未手术的7-11月龄ergd缺陷小鼠中观察到类似的严重oa样缺陷，而对照组小鼠则表现出轻微缺陷。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）， PTHrP也在发育中的关节中表达，稳定软骨细胞表型，并在软骨中过度表达时防止软骨细胞肥大（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，PTHrP基因启动子包含几个保守的反应性所需的结合位点。这些和其他数据导致我们的中心假设是Erg对关节软骨细胞的发育和长期稳定和功能至关重要，并与fl -1和PTHrP合作。我们的目标是：(1)揭示Erg和fl -1在关节形成和长期关节软骨稳定中的各自作用；(2)确定Erg和Fli-1如何调控PTHrP的表达；(3)确定转基因Erg表达是否保护关节免受手术诱导的OA。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。它将为关节软骨细胞的生物学和分子生物学提供全新的数据和见解，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，关节软骨细胞的功能可以在各种不利条件下恢复，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.