NEL-2 GENE IN BONE FORMATION & CRANIAL SUTURE CLOSURES

申请人提出研究NEL-2（一种在颅缝早闭（CS）中分离的新基因）在骨形成和过早缝合中的功能。 大约每3,000名婴儿中就有一名患有CS，这是一种与颅畸形相关的颅缝过早闭塞。 我们已经分离并鉴定了其表达在具有流行类型的CS（非家族性单侧冠状骨结合（UCS））的患者的过早缝合融合部位中上调的cDNA。该基因的全长cDNA的核苷酸序列与鸡NEL基因具有约61%的同源性，因此将该cDNA命名为人NEL-2。 鸡NEL和人NEL-2均由6个EGF样重复序列组成。 我们证明了人NEL-2信息主要定位于成骨前沿的间充质细胞和成骨细胞中，沿着缝旁骨边缘和新形成骨的凝聚间充质细胞内。 人NEL-2在胎儿脑中特异性表达，但在胎儿肺、肾或肝中不表达。 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物中基本不表达。 构建表达大鼠NEL-2的哺乳动物表达载体并转染MC 3 T3细胞系。 与对照组相比，矿化增加约2.5倍。此外，大鼠NEL-2的过表达诱导BMP-2基因表达。 我们的数据表明，NEL-2基因优先表达在颅内膜内骨和神经组织，它可能与膜骨形成。 因此，我们推测NEL-2基因产物的过度表达诱导了颅膜骨的过度生成。 此外，颅缝早闭，如在颅缝早闭（CS）和非家族性单侧冠状缝早闭（UCS）中所见，可能是由于颅膜骨的过度生成和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）确定NEL-2在非家族性UCS患者的成骨细胞中以及在体外骨形成的不同阶段的表达; 2）确定NEL-2对体外骨形成的作用; 3）确定NEL-2对来自CS患者的人颅骨成骨细胞中骨形成的作用; 4）表征NEL-2表达的调节;和5）研究NEL-2对大鼠颅缝闭合的过表达作用。

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.