Haploinsufficiency of Nf1 in Myeloid Lineages Contributes to Neurofibromatosis Type 1 Associated Skeletal Deficits

Abstract 830

Myelopoiesis influences multiple physiological processes including skeletal remodeling. Osteoclasts (OCs) are specialized myeloid cells responsible for bone resorption. Dysregulated myeloid differentiation and OC bone lytic activity can lead not only to hematopoietic deficits, but also to skeletal diseases such as osteoporosis and osteopetrosis. Haploinsufficient loss of the Nf1 tumor suppressor gene leads to Ras mediated cytokine hypersensitivity in multiple myeloid lineages, including marrow-derived myeloid progenitor cells, mast cells, and OCs. Aside from neoplasia, patients with the genetic disorder neurofibromatosis type 1 (NF1) exhibit a spectrum of osseous defects including osteoporosis and pseudarthrosis – severe non-union fractures that respond poorly to treatment and often require amputation. Detailed investigation of the cellular and molecular mechanisms underlying NF1 pathological bone repair is needed to identify targeted therapies.

In a series of adoptive bone marrow transfer experiments, we recently established that Nf1 haploinsufficiency in the hematopoietic system plays a critical role in the pathogenesis of tibial fracture non-union in the NF1 murine model. Since Nf1^+/− OCs exhibit multiple gain-in-fuctions, we reasoned that haploinsufficient loss of Nf1 in myeloid OC progenitors is required for the genesis of osseous phenotypes. To test this hypothesis, we generated Nf1^f/+;LysMCre⁺ mice harboring conditional inactivation of a single Nf1 allele in myeloid lineages. Here, we report that Nf1^f/+;LysMCre⁺ mice exhibit increased frequency of osteoclast progenitors (CFU-M), enhanced osteoclastogenesis, and accelerated bone resorption following ovariectomy induced resorptive stress in vivo. Interestingly, recombination of the floxed Nf1 allele under CtskCre, restricted to mature osteoclasts, did not recapitulate these phenotypes in analogous experiments. These data provide direct genetic evidence that haploinsufficiency of Nf1 in myeloid progenitors, but not terminally differentiated OCs alone, is required to expand the pool of OC progenitors, thereby inducing increased bone resorption in vivo.

To further investigate the contribution of Nf1 hapolinsufficient myeloid cells in a NF1 psuedarthrosis mouse model, we transplanted bone marrow (BM) cells from WT and Nf1^f/+;LysMCre⁺ mice and monitored tibial fracture repair after stable hemapoietic reconstitution. Compared to WT BM cells, mice reconstituted with Nf1^f/+;LysMCre BM cells exhibited deficient cortical bridging and significantly reduced callus bone volume fraction (BV/TV) as determined by micro-computed tomography (μCT), indicating substantial deficits in fracture repair. Histological analysis revealed fibrous infiltrates and excessive osteoclast numbers within the fracture callus. To determine the molecular mechanisms by which Nf1 status affects the proliferation and commitment of OC progenitors, we considered transcription factors that are important for early myeloid differentiation. Here, we show that Nf1^+/− OC gain-in functions are associated with increased nuclear levels of phosphorylated Pu.1. These data implicate Ras as a molecular switch regulating macrophage and OC development by controlling both the expression and post-translational modification of the critical myeloid transcription factor Pu.1. In sum, our genetic and bone marrow transplantation studies further demonstrate that Nf1^+/− OCs and their progenitors are the culprit hematopoietic cell lineage responsible for the pathogenesis of NF1 non-union fracture. Thus, therapies targeting the myeloid lineage, especially OCs and their progenitors, may be required for the treatment of NF1 skeletal deficits.