Mechanistic Role of NELL-1 in Premature Suture Closure

描述：（由申请人提供）我们分离了一种新的基因，NELL-1， 在颅缝早闭的过早缝合融合中高度表达 (CS)患者我们的初步结构分析表明，NELL-1是一个 一种带有信号肽的分泌蛋白，一种NH2-末端血小板反应蛋白 （TSP）样模块，5个von Willebrand因子C结构域和6个表皮 生长因子样结构域。我们的初步数据表明，优先NELL-1 在神经嵴起源的颅骨成骨细胞和颅骨缝中的表达 结束我们已经证明，NELL-1在大鼠原代颅骨中过表达， 成骨细胞（FRCC）和MC3T3成骨细胞培养物显著增强了成骨细胞（FRCC）和MC3T3成骨细胞培养物的生长。 通过增加成骨细胞分化的成骨细胞矿化。 微阵列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 NELL-1的转基因小鼠，我们构建了NELL-1过表达转基因小鼠， NELL-1表达受通用CMV启动子调控。初始形态学 对CMV转基因小鼠的分析表明， 包括脑室闭塞，颅缝过早闭合， 颅骨畸形。CBFA1最近被证明可以发挥作用， 在FGFR1诱导的CS中起重要作用。TGF-β 1和FGF2，影响CBFA 1 表达并诱导颅缝闭合，诱导FRCC中NELL-1表达 细胞另一方面，IGF 1和2、PDGF、VEFG和BMP 2对骨形成没有影响。 NELL-1表达。NELL-1？s预测的启动子序列含有高度 保守的OSE 2（CBFA 1结合位点）序列和MSX 2结合序列。CBFA1 可诱导成骨细胞中NELL-1的过度表达。我们提供了证据 NELL-1过表达诱导骨形成/建模的失调 颅缝中的突起，导致缝过早闭合。我们也 这表明NELL-1可能是几种已知靶/效应物的共同下游靶/效应物 CS候选基因如FGFR、MSX-2和TGF-β，在引起局部早产中的作用 缝合闭合。在这个建议中，我们假设NELL-1是一个下游， CBFA 1和MSX 2在成骨细胞样细胞中TGF β和FGF的效应物/靶点 作为其直接转录调节因子。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 候选基因如FGF、FGFR、TGF-β和MXS-2与NELL-1。在 此外，我们将表征NELL-1？s启动子来了解它的启动子 监管机制。由于NELL-1优先/特异性表达于 神经嵴起源颅骨成骨细胞，我们将确定神经嵴和/或 成骨细胞特异性转录因子。最后，我们将集成NELL-1 这些已知的候选基因进入缝合通路。在 未来，我们将分析NELL-1？的下游机制，以进一步完善 CS中的过早闭合机制。

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.