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: 6651152
• 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 2005-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6651152
• 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：（由申请人提供）我们已经隔离了一个新型基因Nell-1， 这在颅突式中的早产缝合融合期间高度表达 （CS）患者。我们的初始结构分析表明NELL-1是 分泌蛋白具有信号肽，NH2末端血小板素 （TSP）类似模块，五个von Willebrand因子C域和六个表皮 生长因子样域。我们的初步数据表明优先Nell-1 在神经rest起源颅骨成骨细胞和颅骨缝合线中的表达 关闭。我们已经表明，大鼠原发性颅骨中的NELL-1过表达 成骨细胞（FRCC）和MC3T3成骨细胞培养物显着增强 成骨细胞通过增加成骨细胞分化而矿化。 微阵列显示成骨细胞分化标记的上调 骨桥和骨钙素。进一步描述了体内影响 NELL-1，我们已经构建了Nell-1过表达的转基因小鼠，其中 NELL-1表达受一般CMV启动子的调节。最初的形态学 CMV转基因小鼠的分析表明多种颅异常 包括心室的闭塞，过早关闭颅骨缝合线和 颅骨的畸形。 CBFA1最近已显示出 FGFR1诱导的CS中的重要作用。 TGF-BETA1和FGF2，影响CBFA1 表达并诱导颅缝合线闭合，在FRCC中诱导NELL-1表达 细胞。另一方面，IGF 1和2，PDGF，VEFG和BMP2对 NELL-1表达。 Nell-1的预测启动子序列包含高度 保守的OSE2（CBFA1结合位点）序列和MSX2结合序列。 CBFA1 可以在成骨细胞中诱导NELL-1过表达。我们提供了证据 NELL-1的过表达诱导骨形成/建模的失调 颅骨缝合线中的过程，导致缝合线过早闭合。我们也是 表明NELL-1可能是几个已知的常见下降目标/效应子 FGFR，MSX-2和TGF-beta等CS糖果基因导致局部过早 缝合线闭合。在此提案中，我们假设Nell-1是下游 TGFBETA和FGF的效应子/靶标在成骨细胞样细胞中具有CBFA1和MSX2 作为其直接的转录调节器。此外，CBFA1和MSX2可能 协同诱导Nell-1。我们将检查已知CS的相互作用 NELL-1的候选基因，例如FGFS，FGFR，TGF-BETA和MXS-2。在 此外，我们将以Nell-1的启动子来了解其发起人 监管机制。由于Nell-1优先/在 神经rest起源钙质成骨细胞，我们将确定神经rest和/或 成骨细胞特异性转录因子。最后，我们将整合Nell-1 使用这些已知候选基因进入缝合线闭合途径。在 未来，我们将分析Nell-1的下游机制，以进一步完成 CS中的过早闭合机制。