Involvement of Integrin Beta1 and FAK in NELL1-Induced Osteoblastic Differentiation

Objectives: NELL1 is a human craniosynostosis associated protein excessively expressed at the sutures of non-syndromic unilateral coronal synostosis, causing the premature fusion of the growing cranial bone fronts. The preferential expression of NELL1 in the osteoblasts of cranial sutures and its overexpression in craniosynostosis, combined with its role in osteogenic differentiation and bone formation of osteoblastic cells, warrant the investigation of the molecular mechanisms of NELL1. The purpose of this study is to identify the NELL-1 receptors and signaling pathways that would provide an insight into the biologic mechanisms and potential clinical applications of NELL1.

Methods: Utilizing His-tagged NELL1, we have shown that NELL1 binds to Saos-2 cells via flow cytometric analysis. Pull-down, Immunoprecipitation, and Far Western assays, independently and conjunctively, followed by Mass Spectrometry were performed to identify the receptor of NELL1 and confirmed the interaction via Pull-down and Co-Immunoprecipitation assays. To elucidate the intracellular signaling cascade evoked by NELL1, activation of focal adhesion kinase (FAK) was examined by anti-phospho-FAK antibody upon binding of NELL1 to the receptor.

Results: Among the potential binding proteins of NELL1 that are found, we have shown the interaction of NELL1 proteins with integrin beta1 on Saos-2 cell surface. The phosphorylation of FAK increased in a dose dependent manner of added NELL1 proteins.

Conclusions: These findings suggest that upon binding to integrin beta1 on Saos-2 cells, NELL1 transduces an osteogenic signal through the activation of FAK associated with the Ras-MAPK cascade, and leads to the osteogenic differentiation. The discovery of the NELL1 receptor and its signaling pathway is of profound clinical importance as once the mechanism is clarified, the specific targets for pharmacologic and genetic mechanisms that control NELL1 signal transduction may potentially resolve craniosynostosis, reducing the probability of multiple surgical procedures and the side-effects resulting from such interventions.

Funding: DE 016107-01