Mechanistic Role of NELL-1 in Premature Suture Closure

NELL-1 在缝线过早闭合中的机制作用

• 批准号: 6485869
• 负责人: KANG TING
• 金额: $ 7.63万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-01 至 2004-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6485869
• 关键词: 3T3 cells; DNA footprinting; binding sites; cell cycle; craniosynostosis; developmental genetics; developmental neurobiology; fibroblast growth factor; gel mobility shift assay; gene expression; gene interaction; genetic promoter element; genetically modified animals; homeobox genes; in situ hybridization; laboratory mouse; neural crest; northern blottings; osteoblasts; polymerase chain reaction; protein structure function; secretory protein; site directed mutagenesis; transcription factor; transforming growth factors

DESCRIPTION: (provided by applicant) We have isolated a novel gene, NELL-1, that is highly expressed during premature suture fusion in craniosynostosis (CS) patients. Our initial structure analysis demonstrated that NELL-1 is a secretory protein with a signal peptide, an NH2-terminal thrombospondin (TSP)-like module, five von Willebrand factor C domains, and six epidermal growth factor-like domains. Our preliminary data indicated preferential NELL-1 expression in neural crest origin cranial osteoblasts and during cranial suture closure. We have shown that NELL-1 over-expression in rat primary calvarial osteoblasts (FRCC) and MC3T3 osteoblast cell cultures significantly enhances osteoblast mineralization through increased osteoblast differentiation. Microarrays show up-regulation of osteoblast differentiation marker like osteopontin and osteocalcin. To further delineate the in vivo effects of NELL-1, we have constructed NELL-1 over-expression transgenic mice in which NELL-1 expression is regulated by the general CMV promoter. Initial morphologic analysis of the CMV transgenic mice demonstrate multiple cranial abnormalities including obliteration of ventricles, premature closure of cranial sutures, and deformities of cranial bones. CBFA1 has recently been shown to play an important role in FGFR1 induced CS. TGF-beta1 and FGF2, which affect CBFA1 expression and induce cranial suture closure, induce NELL-1 expression in FRCC cells. IGF 1 & 2, PDGF, VEFG, and BMP2, on the other hand, have no effect on NELL-1 expression. NELL-1?s predicted promoter sequence contains a highly conserved OSE2 (CBFA1 binding site) sequence and a MSX2 binding sequence. CBFA1 can induce NELL-1 over-expression in osteoblasts. We have provided evidence that NELL-1 over-expression induces dysregulation of bone formation/modeling processes in the cranial suture, resulting in premature suture closure. We also suggest that NELL-1 may be a common down-steam target/effector of several known CS candiate genes like FGFRs, MSX-2, and TGF-beta, in causing local premature suture closure. In this proposal, we hypothesize that NELL-1 is a down-stream effector/target of TGFbeta and FGF in osteoblast-like cells with CBFA1 and MSX2 as its direct transcriptional regulators. Furthermore, CBFA1 and MSX2 may synergistically induce NELL-1. We will examine the interactions of known CS candidate genes like FGFs, FGFRs, TGF-betas, and MXS-2 with NELL-1. In addition, we will characterize NELL-1?s promoter to understand its promoter regulatory mechanisms. Since NELL-1 is preferentially/specifically expressed in neural crest origin calvarial osteoblasts, we will identify neural crest and/or osteoblast specific transcription factor. Finally, we will integrate NELL-1 into the suture closure pathway with these known candidate genes. In the future, we will analyze NELL-1?s down-stream mechanism to further complete the premature closure mechanism in CS.

描述：(申请人提供)我们已经分离出一个新的基因Nell-1， 在颅缝早闭的早期缝合融合过程中高表达 (CS)患者。我们的初步结构分析表明，Nell-1是一种 含有信号肽的分泌蛋白，一种NH2末端的血栓反应蛋白 (TSP)样模块，5个von Willebrand因子C结构域和6个表皮 生长因子样域。我们的初步数据显示Nell-1优先 神经沟来源的颅骨成骨细胞在颅骨缝合过程中的表达 结案了。我们已经证明Nell-1在大鼠原代颅骨中过表达 成骨细胞(FRCC)和MC3T3成骨细胞培养显著增强 通过增加成骨细胞分化促进成骨细胞矿化。 基因芯片显示成骨细胞分化标志物如 骨桥蛋白和骨钙素。为了进一步描述其在体内的作用 Nell-1，我们构建了Nell-1过表达转基因小鼠 Nell-1的表达受CMV启动子的调控。初始形态 巨细胞病毒转基因小鼠多发颅骨异常的分析 包括脑室闭塞，颅缝过早闭合，以及 颅骨畸形。CBFA1最近被证明扮演着一个 在FGFR1诱导CS中的重要作用。影响CBFA1的转化生长因子-β1和成纤维细胞生长因子2 FRCC表达及诱导颅缝闭合，诱导Nell-1表达 细胞。另一方面，IGF 1和2、PDGF、VEFG和BMP2对 Nell-1的表达。Nell-1？S预测的启动子序列含有高度 保守的OSE2(CBFA1结合位点)序列和MSX2结合序列。CBFA1 可诱导Nell-1在成骨细胞中过度表达。我们已经提供了证据 Nell-1过表达诱导骨形成/建模失调 颅缝中的突起，导致缝合过早关闭。我们也 提示Nell-1可能是几个已知的常见的下行目标/效应器 CS候选FGFRs、MSX-2和转化生长因子-β等基因导致局部早产 缝合缝合。在这个方案中，我们假设NELL-1是一个下游 CBFA1和MSX2诱导成骨样细胞表达转化生长因子β和成纤维细胞生长因子的效应/靶点 作为它的直接转录调节因子。此外，CBFA1和MSX2可以 协同诱导Nell-1。我们将研究已知CS的相互作用 与Nell-1相关的候选基因，如FGFs、FGFRs、转化生长因子-β和MXS-2。在……里面 此外，我们还将对nell-1？S启动子进行表征，以了解其启动子。 监管机制。由于Nell-1优先/专门在 神经沟起源于颅骨成骨细胞，我们将鉴定神经沟和/或 成骨细胞特异性转录因子。最后，我们将整合NELL-1 与这些已知的候选基因一起进入缝合闭合途径。在 未来，我们将分析内尔-1？S的下行机制，进一步完成 CS的早期闭合机制。