Role of Transcription Factor ERG in Skeletogenesis

描述（由申请人提供）：在肢体骨骼发生过程中，位于长骨原基骨骺端的软骨细胞发育成永久性关节软骨细胞，终身维持关节功能。相反，构成骨干的软骨细胞是瞬时细胞，并最终通过软骨内骨化被骨取代。几年前，我们启动了这个项目，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ets转录因子家族，并且是包括Fli-1的ets亚家族的一部分。在之前的资助期间，我们发现Erg在早期滑膜关节发育过程中与Gdf 5和Wnt 9a一起沿着表达，并且其表达随着时间的推移而减少。为了测试功能，我们创建了floxed Erg小鼠，并通过与Gdf 5-Cre小鼠交配在发育中的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，而缺乏ERG的小鼠存活到成年。为了解释主要关节发育表型的缺失，我们询问Fli-1是否共表达，事实上它是共表达的，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个月大的Erg缺陷小鼠进行膝内侧副韧带（MCL）横断以诱导实验性骨关节炎（OA）。引人注目的是，Erg缺陷小鼠比野生型小鼠更快地出现严重的OA样缺陷。事实上，我们在7-11月龄的未手术的Erg缺陷小鼠中观察到类似的严重OA样缺陷，而对照组的同窝小鼠显示出轻度缺陷。为了深入了解Erg如何维持长期的关节软骨细胞功能，我们专注于甲状旁腺相关蛋白（PTHrP），该蛋白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过度表达一样）。我们发现，Erg（以及Fli-1）刺激PTHrP的表达和PTHrP基因启动子包含几个保守的ets结合位点所需的响应。这些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至关重要，并且与Fli-1和PTHrP合作。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的各自作用;（2）确定Erg和Fli-1如何调节PTHrP表达;和（3）确定转基因Erg表达是否保护关节免受关节炎诱导的OA。这项工作将使用不同的实验方法进行，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产生全新的数据和见解，并为创造未来的修复和再生疗法铺平道路，通过这些疗法，可以恢复受各种不良条件影响的关节软骨细胞的功能，包括骨关节炎和自然衰老。 公共卫生相关性：关节软骨对关节功能、不受阻碍的身体运动和生活质量至关重要，但会受到包括骨关节炎在内的常见疾病的影响，并在自然衰老过程中变得无功能。该项目将继续阐明组织正常维持其功能的机制，从而产生可用于创建新的关节修复和再生疗法的重要信息。

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. PUBLIC HEALTH RELEVANCE: Articular cartilage is essential for joint function, unhindered body movement and quality of life, but is affected by common diseases including osteoarthritis and becomes non-functional during natural aging. This project will continue to clarify mechanisms by which the tissue normally maintains its function and will thus generate important information that can be used to create novel joint repair and regeneration therapies.