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.
