NEL-2 GENE IN BONE FORMATION & CRANIAL SUTURE CLOSURES

申请人建议研究NEL-2的功能，这是一种在颅缝闭合（CS）中分离的新基因，在骨形成和过早缝合关闭中的功能。大约每3000名婴儿中就有1名患有CS，这是一种与颅畸形相关的颅缝过早闭塞症。我们已经分离并鉴定了cDNA，其表达在非家族性单侧冠状关节闭锁（UCS）患者的早期缝合融合部位上调。该基因全长cDNA序列与鸡nel基因的同源性约为61%，因此被命名为cDNA human nel -2。鸡的nel和人的nel -2都包含6个egf样重复序列。我们证明了人类NEL-2信息主要定位于成骨前沿的间充质细胞和成骨细胞，沿准骨边缘和新形成骨的凝聚间充质细胞内。人类NEL-2在胎儿脑中特异性表达，而在胎儿肺、肾和肝中不表达。我们还发现，大鼠NEL-2在大鼠颅骨骨祖细胞中表达，而在大鼠胫骨和成纤维细胞培养中基本缺失。构建表达大鼠NEL-2的哺乳动物表达载体，并将其转染MC3T3细胞系。与对照组相比，矿化度大约增加了2.5倍。此外，过表达大鼠NEL-2可诱导BMP-2基因表达。我们的数据表明，NEL-2基因在颅膜内骨和神经组织中优先表达，它可能与膜性骨的形成有关。因此，我们假设il -2基因产物的过度表达诱导了颅膜骨的过度生成。此外，颅缝闭合过早，如颅缝闭塞（CS）和非家族性单侧冠状口闭塞（UCS），可能是由于颅膜骨过度生成和NEL-2的过度表达。为了验证这一假设，我们提出了以下具体目标：1)在体外测定非家族性UCS患者和不同骨形成阶段成骨细胞中NEL-2的表达；2)体外测定NEL-2对骨形成的影响；3)测定NEL-2对CS患者人颅成骨细胞成骨形成的影响；4)表征NEL-2的表达调控；5)研究NEL-2过表达对大鼠颅骨缝合闭合的影响。

The applicant proposes to investigate the function of NEL-2, a novel gene isolated in craniosynostosis (CS), in bone formation and premature suture closure. CS, which affects roughly one of every 3,000 infants, is the premature obliteration of cranial sutures in association with cranial dysmorphism. We have isolated and identified cDNA whose expression is upregulated in the premature suture fusion sites of patients with a prevalent type of CS, non-familial unilateral coronal synostosis (UCS). The nucleotide sequence of the full length cDNA of this gene has approximately 61 percent homology with the chicken nel gene, and thus has been named this cDNA human NEL-2. Both chicken nel and human NEL-2 consist of 6 EGF-like repeats. We demonstrated that the human NEL-2 messages are primarily localized in the mesenchymal cells and osteoblasts at the osteogenic front, along the parasutural bone margins and within the condensing mesenchymal cells of the newly formed bone. Human NEL-2 is specifically expressed in fetal brain but not in fetal lung, kidney or liver. We have also shown that rat NEL-2 is expressed in rat calvarial osteoprogenitor cells and is largely absent in rat tibia and fibroblast cell culture. Mammalian expression vectors expressing rat NEL-2 were constructed and transfected into MC3T3 cell line. An approximate two and a half-fold increase in mineralization was seen as compared to the control. In addition, overexpression of rat NEL-2 induced BMP-2 gene expression. Our data suggest that the NEL-2 gene is preferentially expressed in cranial intramembranous bone and neural tissue, and it may be associated with membranous bone formation. Therefore, we hypothesize that the overexpression of the NEL-2 gene product induces the overproduction of cranial membranous bone. In addition premature cranial suture closure, as seen in craniosynostosis (CS) and non-familial unilateral coronal synostosis (UCS), may be due to the overproduction of cranial membranous bone and the overexpression of NEL-2. To test this hypothesis, we propose the following specific aims: 1) Determining expression of NEL-2 in osteoblasts of non-familial UCS patients and at different stages of bone formation in vitro; 2) Determining the effect of NEL-2 on bone formation in vitro; 3) Determining the effect of NEL-2 on bone formation in human calvaria osteoblastic cells from CS patients; 4) Characterizing the regulation of NEL-2 expression; and 5) investigating the overexpression effect of NEL-2 on the rat cranial suture closure.