TRANSCRIPTION FACTOR ERG AND SKELETOGENESIS

四肢、躯干和其他部位的骨骼形成涉及软骨内和膜内骨化。虽然在胚胎学、组织学和生化水平上对这些过程了解很多，但在分子水平上所知相对较少。在初步研究中，我们描述了禽类C-1-1的克隆，这是ets转录因子erg3的一种选择性剪接形式。我们发现C-1-1在发育中的鸡骨骼永久关节软骨细胞中表达，而erg3在增生前生长板软骨细胞中表达。病毒驱动的C-1-1的组成性表达阻碍软骨细胞成熟和软骨内成骨，而erg3的组成性表达促进成熟。这些和其他结果导致了我们的中心假设，即C-1-1和erg3参与骨骼形成和骨骼功能，并具有不同的作用。具体来说，我们提出(a)通过改变潜在下游效应物（包括印度刺猬、PTH-RP、PTH-RP受体或骨因子CBFA1）的表达来确定C-1-1或erg3的组成表达是否会对骨骼形成产生不同的影响，(b)确定反义策略或同源重组获得的基因消融对小鼠ERG抑制的发育后果。(c)确定c -1-1和erg3如何差异调节软骨细胞中的基因转录，以及它们是否可能通过与ets DNA基序和其他转录因子（如ets -2和AP-1复合物）的特定相互作用来调节。为了实现这些目标，我们将使用病毒表达载体、细胞培养、体内鸡胚操作、重组蛋白、蛋白蛋白和蛋白- dna相互作用测定、原位杂交和基因消融。该项目的结果将提供调节关节软骨、长骨和其他元素形成的分子机制的关键信息，并将指出分子缺陷可能是生长有机体和成人骨骼形成和骨骼功能的先天性和后天条件的基础。

Skeletogenesis in the limbs, trunk and other sites involves endochondral and intramembranous ossifications. While much is known about these processes at the embryological, histological and biochemical levels, relatively little is known at the molecular level. In Preliminary Studies, we describe the cloning of avian C-1-1, an alternatively spliced form of the ets transcription factor ERG-3. We find that C-1-1 is expressed in permanent articular chondrocytes while ERG-3 is expressed in prehypertrophic growth plate chondrocytes in the developing chick skeleton. Virally driven constitutive expression of C-1-1 blocks chondrocyte maturation and endochondral ossification, while constitutive expression of ERG-3 favors maturation. These and other results lead to our central hypothesis that C-1-1 and ERG-3 are involved and have distinct roles in skeletogenesis and skeletal function. Specifically, we propose (a) to determine whether constitutive expression of C-1-1 or ERG-3 affects skeletogenesis differentially by changing the expression of potential downstream effectors, including Indian hedgehog, PTH-RP, PTH-RP receptor or the bone factor CBFA1, (b) to determine the developmental consequences of ERG inhibition caused by antisense strategies or gene ablation obtained by homologous recombination in mice, and (c) to determine how C-1-1 and ERG-3 differentially regulate gene transcription in chondrocytes and whether they may do so by specific interactions with ets DNA motifs and other transcription factors such as ETS-2 and AP-1 complexes. To achieve these goals, we will use viral expression vectors, cell cultures, in vivo chick embryo manipulation, recombinant proteins, protein-protein and protein-DNA interaction assays, in situ hybridization and gene ablation. The results of the project will provide key information on molecular mechanisms regulating the formation of articular cartilage, long bones and other elements, and will point to molecular defects that may underlie congenital and acquired conditions of skeletogenesis and skeletal function in the growing organism and adult.