Project Summery/Abstract This Competitive Revision Application is in response to Notice Number (NOT-OD-09-058) and Notice Title: NIH Announces the Availability of Recovery Act Funds for Competitive Revision Applications. We previously identified osteoinductive molecule, long Nell-1 [LNell-1;a protein strongly expressed in neural tissue encoding epidermal growth factor (EGF)-like domain] from prematurely fusing sutures in craniosynostosis (CS) patients (Many of these studies were performed before identification of the short Nell-1, SNell-1 isoform. For clarification, we will use """"""""LNell-1"""""""" in place of Nell-1 when we are certain that the long form is involved. Meanwhile, Nell-1 can refer to both LNell-1 and SNell-1). LNell-1 is a secretory protein with a signal peptide, an NH2-terminal thrombospondin-1 (TSP1-N)-like module, five chordin-like cysteine-rich (CR) domains, and EGF-like domains. The long term goal of this Competitive Revision and parent grant proposal is to understand the mechanisms behind excessive bone growth in CS patients, so that we can harness and channel the molecules responsible into clinical therapies to regenerate bone. Towards this end, we have worked continuously to better understand the molecular regulation and function of LNell-1 so as to build a strong scientific foundation for therapeutic LNell-1 application. Our progress report and preliminary data for the parent grant Competitive Renewal demonstrate that: 1) runt-related transcription factor 2 (Runx2) regulates LNell-1 through cis-acting element 2 (OSE2) response elements (REs);2) LNell-1 increases osteoblastic differentiation and can partially compensate for Runx2 haploinsufficiency;and 3) Nell-1 deletion animals demonstrate impaired chondrocyte hypertrophy with delayed bone formation as well as decreased Runx2 and increased osterix expression. For this one-year Competitive Revision we propose to significantly accelerate the tempo of our current investigations on mechanistic regulation of Nell-1 by a concentrated focus on identification of LNell-1 receptors and/or binding proteins (R/BPs). To accomplish this objective, we have obtained preliminary T7 phage display and mass spectrometry (MS)-based proteomic data demonstrating LNell-1 binding to membranous and cytosolic proteins. These data have led to the hypothesis that Nell-1 exerts its function in osteochondral differentiation by serving as a ligand to cell specific and potentially novel receptors/binding proteins. In order to identify LNell-1 R/BPs, three aims representing a logical extension of the parent proposal will be undertaken:
Aim A) Candidate receptor approach to examine interaction of LNell-1 with integrin family. Structurally, the TSP1-N-like domain of LNell-1 shares a high degree of similarity with TSP1-N domain where integrin binding sites reside.
Aim B) LNell-1 R/BP identification using a T7 phage display system.
Aim C) MS-based functional proteomics on the membranous protein fraction binding. This Competitive Revision will also stimulate the economy by enabling hiring of additional staff and increasing hours of current part-time staff.
TO PUBLIC HEALTH STATEMENT Nell-1, a cause of excessive bone growth in craniosynostosis patients has shown significant promise as a potent bone forming agent in preclinical studies. A better understanding of how and why Nell-1 works to form bone can accelerate the development of clinical therapies to help patients grow better and faster bone.
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