Novel Wnt Regulator NEL-Like Molecule-1 Antagonizes Adipogenesis and Augments Osteogenesis Induced by Bone Morphogenetic Protein 2

Abstract

The differentiation factor NEL-like molecule-1 (NELL-1) has been reported as osteoinductive in multiple in vivo preclinical models. Bone morphogenetic protein (BMP)-2 is used clinically for skeletal repair, but in vivo administration can induce abnormal, adipose-filled, poorquality bone. We demonstrate that NELL-1 combined with BMP2 significantly optimizes osteogenesis in a rodent femoral segmental defect model by minimizing the formation of BMP2-induced adipose-filled cystlike bone. In vitro studies using the mouse bone marrow stromal cell line M2-10B4 and human primary bone marrow stromal cells have confirmed that NELL-1 enhances BMP2-induced osteogenesis and inhibits BMP2-induced adipogenesis. Importantly, the ability of NELL-1 to direct BMP2-treated cells toward osteogenesis and away from adipogenesis requires intact canonical Wnt signaling. Overall, these studies establish the feasibility of combining NELL-1 with BMP2 to improve clinical bone regeneration and provide mechanistic insight into canonical Wnt pathway activity during NELL-1 and BMP2 osteogenesis. The novel abilities of NELL-1 to stimulate Wnt signaling and to repress adipogenesis may highlight new treatment approaches for bone loss in osteoporosis. NEL-like molecule-1 (NELL-1) is an osteoinductive growth factor first identified through its overexpression in pathologically fusing suture specimens from patients with craniosynostosis. 1,2 Transgenic Nell1-overexpressing mice recapitulate craniosynostosis-like phenotypes, exhibiting gross calvarial bone overgrowth and increased osteoblast differentiation. 3 Conversely, Nell1 deficiency severely disrupts bone growth, as mice with nonsense mutations in Nell1 die perinatally with major skeletal anomalies in the craniofacial complex, spine, and long bones. 4e6 Highlighting the central role of NELL-1 in skeletal development, NELL-1 mediates key downstream effects of the master osteogenic regulator runt-related transcription factor 2 (RUNX2) 7 and can partially rescue RUNX2 loss of function. 8 NELL-1 can also transiently activate mitogen-activated protein kinase signaling to induce RUNX2 phosphorylation and osteogenic differentiation. 9 Recently, we

NEL-Like Molecule-1 (Nell1) Is Regulated by Bone Morphogenetic Protein 9 (BMP9) and Potentiates BMP9-Induced Osteogenic Differentiation at the Expense of Adipogenesis in Mesenchymal Stem Cells

“…Mechanistically, Nell1 was shown to transduce osteogenic signals through the Ras-MAPK and Runx2 pathways [108] and Runx2 [109,110] although Nell1 was also found as a direct target of Osterix [111]. More recently, Nell1 was shown to activate canonical Wnt signaling [101]. Nonetheless, the cognate receptor(s) for Nell proteins have yet to be identified although Nell1 was shown to form oligomers and interact with heparin and integrins [105,[112][113][114][115].…”

“…Meanwhile, several studies revealed that exogenous Nell1 exhibits anti-adipogenic differentiation activity [99,100], probably through Nell1 activated canonical Wnt signaling pathway to repress BMP2-induced adipogenesis [101].…”

Inactivation of Nell-1 in Chondrocytes Significantly Impedes Appendicular Skeletogenesis

“…Western blot analysis was performed as previously described . Briefly, nuclear and cytoplasmic proteins were isolated using an NE‐PER Nuclear and Cytoplasmic Extraction Kit (Thermo Fisher Scientific, Waltham, MA, USA).…”

“…Western blot analysis was performed as previously described. (4,32) Briefly, nuclear and cytoplasmic proteins were isolated using an NE-PER Nuclear and Cytoplasmic Extraction Kit (Thermo Fisher Scientific, Waltham, MA, USA). Primary antibodies listed in Supplemental Table S2 were used at different concentrations for Western blot analyses.…”

NELL-1 induces Sca-1+ mesenchymal progenitor cell expansion in models of bone maintenance and repair

“…NELL-1 exerts regulatory effects on a broad array of mesenchymal cell types, including multipotent mesenchymal progenitor cells (MPCs) (4)(5)(6)(7), committed preosteoblast cells (4,8), odontoblasts (9), and chondrocytes (10)(11)(12). Mechanistically, NELL-1 binds to the cell surface heterodimer integrin α3β1 (13), resulting in several intracellular signaling changes that induce osteoblastogenic programming, including focal adhesion kinase (FAK) phosphorylation (14), increased mitogen-activated protein kinase (MAPK) signaling activity (15)(16)(17), and increased Wnt/β-catenin signaling activity (4,18). Despite the well-demonstrated pro-osteogenic effects of NELL-1 (19)(20)(21)(22)(23), there has been little understanding of the cellular mediators of its effects, nor the role of NELL-1 in the maintenance or expansion of MPC pools.…”