NEL-2 GENE IN BONE FORMATION & CRANIAL SUTURE CLOSURES

申请人建议研究NEL-2，一种分离于颅缝融合(CS)的新基因，在骨形成和过早缝合中的功能。CS大约每3000名婴儿中就有一名受到影响，它是与颅骨畸形相关的颅缝过早闭塞。我们已经分离和鉴定了在一种流行类型的CS患者的早期缝合融合部位表达上调的cDNAs，非家族性单侧冠状突触(UCS)。该基因全长cDNA的核苷酸序列与鸡NEL基因有约61%的同源性，因此命名为人NEL-2。鸡的NEL和人的NEL-2都由6个类EGF重复序列组成。我们证明，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，沿缝旁骨缘和新形成骨的浓缩间充质细胞内。人的NEL-2在胎脑中特异表达，但在胎肺、肾或肝脏中不表达。我们还发现，大鼠NEL-2在大鼠颅骨成骨祖细胞中表达，而在大鼠胫骨和成纤维细胞培养中几乎不表达。构建了表达大鼠NEL-2的哺乳动物表达载体，并将其导入MC3T3细胞。与对照相比，矿化增加了大约2.5倍。此外，大鼠NEL-2过表达可诱导BMP-2基因表达。我们的数据提示NEL-2基因在颅膜内骨和神经组织中优先表达，可能与膜性骨的形成有关。因此，我们推测NEL-2基因产物的过度表达导致了颅膜骨的过度生产。此外，颅缝过早闭合，如颅缝融合(CS)和非家族性单侧冠状融合(UCS)，可能是由于颅膜骨过度生成和NEL-2过度表达所致。为了验证这一假说，我们提出了以下具体目标：1)检测NEL-2在非家族性颈椎病患者成骨细胞和体外不同成骨阶段的表达；2)测定NEL-2在体外对骨形成的影响；3)检测NEL-2对CS患者颅骨成骨细胞成骨形成的影响；4)表征NEL-2的表达调控；以及5)研究NEL-2过表达对大鼠颅缝闭合的影响。

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.