Overexpression of Nell-1, a Craniosynostosis-associated Gene, Induces Apoptosis in Osteoblasts During Craniofacial Development

Background: Craniosynostosis (CS), one of the most common congenital craniofacial deformities, is the premature closure of cranial sutures. Previously, we reported NELL-1 as a novel molecule overexpressed during premature cranial suture closure in patients with CS. Nell-1 overexpression induced calvarial overgrowth and resulted in premature suture closure in a rodent model. On a cellular level, Nell-1 is suggested to be sufficient for osteoblast differentiation. Objectives: To test the hypothesis that Nell-1 plays a role in osteoblast apoptosis. Methods: Osteoblasts were transfected with Nell-1 adenovirus or Nell-1 protein were added at different dosages. Nell-1 overexpression transgenic mice were examined at various developmental stages. Histology, , Annexin V and propidium iodide staining, and In situ TUNEL were used. Results: In this report we further showed that overexpression of Nell-1 induced apoptosis along with modulation of apoptosis related genes. The induction of apoptosis by Nell-1 was observed only in osteoblast cells and not in NIH3T3 or primary fibroblasts. The CS mouse model overexpressing Nell-1 showed increased levels of apoptosis in the calvaria and was also characterized by a severely distorted cranial base with prematurely apoptotic chondrocytes. A higher incidence of exencephaly was also seen in these mice where the protruding brain tissue and craniofacial skeleton, including the bone and hypertrophic cartilage, had drastically increased levels of apoptosis. Conclusion: we demonstrate that Nell-1 expression modulates calvarial osteoblast differentiation and apoptosis pathways. Nell-1 overexpression disrupts these pathways resulting in neurocranial and chondrocranial anomalies such as premature suture closure, exencephaly and cranial base malformation. This research is supported by the Wunderman Family Foundation, March of Dimes Birth Defect Foundation #6-FY02-163, NIH/NIDCR RO3 DE 014649-01, and NIH/NIDCR K23DE00422.