Nf1 Haploinsufficiency Alters Myeloid Lineage Commitment and Function, Leading to Deranged Skeletal Homeostasis

Abstract

While nullizygous loss of NF1 leads to myeloid malignancies, haploinsufficient loss of NF1 (Nf1) has been shown to contribute to osteopenia and osteoporosis which occurs in approximately 50 percent of neurofibromatosis type 1 (NF1) patients. Bone marrow mononuclear cells of haploinsufficient NF1 patients and Nf1+/- mice exhibit increased osteoclastogenesis and accelerated bone turnover, however the culprit hematopoietic lineages responsible for perpetuating these osteolytic manifestations have yet to be elucidated. Here we demonstrate that conditional inactivation of a single Nf1 allele within the myeloid progenitor cell population (Nf1-LysM) is necessary and sufficient to promote multiple osteoclast gain-in-functions, resulting in enhanced osteoclastogenesis and accelerated osteoclast bone lytic activity in response to pro-resorptive challenge in vivo. Surprisingly, mice conditionally Nf1 heterozygous in mature, terminally differentiated osteoclasts (Nf1-Ctsk) do not exhibit any of these skeletal phenotypes, indicating a critical requirement for Nf1 haploinsufficiency at a more primitive/progenitor stage of myeloid development in perpetuating osteolytic activity. We further identified p21Ras dependent hyper-phosphorylation of Pu.1 within the nucleus of Nf1 haploinsufficent myelomoncytic osteoclast precursors, providing a novel therapeutic target for the potential treatment of NF1 associated osteolytic manifestations.

Constitutional bone impairment in Noonan syndrome

“…Low bone mineral density was reported in NF1 [Brunetti‐Pierri et al, ; Dulai et al, ; Stevenson et al, ; Yilmaz et al, ; Armstrong et al, ]. Moreover, recent studies demonstrated in vitro bone cell anomalies consisting in increased proliferation, decreased osteoblasts differentiation, and excessive osteoclasts resorption activity in Nf1 +/− mices; NF1 haploinsufficiency determines the hyperactivation of the Ras/MAPK signaling cascade, also observed in the gain‐of‐function mutations of the other Rasopathies [Alanne et al, ; Heervä et al, ; Sharma et al, ; Rhodes et al, ]. High levels of urinary resorption markers were found in patients with Ras/MAPK disorders consistent with increased osteoclast activity [Stevenson et al, ].…”

“…In the last years, numerous studies explored the effects of the Ras/MAPK pathway deregulation on the skeletal development and bone metabolism [Yu et al, ; Stevenson et al, ; Stevenson and Yang, ; Choudhry et al, ; Petramala et al, ; Rhodes et al, ]. Low bone mineral density was reported in NF1 [Brunetti‐Pierri et al, ; Dulai et al, ; Stevenson et al, ; Yilmaz et al, ; Armstrong et al, ].…”

“…Prenatal features might include enlarged nuchal translucency, polyhydramnios, hydrothorax, cystic hygroma, and multiple effusions [Baldassarre et al, , ; Croonen et al, ; Gaudineau et al, ; Myers et al, ]. Short stature and skeletal dysmorphisms are almost constant and several studies hypothesized that the Ras pathway may regulate bone metabolism and homeostasis [Yu et al, ; Stevenson et al, ; Stevenson and Yang, ; Choudhry et al, ; Petramala et al, ; Rhodes et al, ]. However, excepting NF1 which has been largely explored both from a clinical and biochemical viewpoint, data on skeletal mineralization, bone metabolism and fracture rate are scanty in the other Rasopathies .…”

Tet2 Regulates Osteoclast Differentiation by Interacting with Runx1 and Maintaining Genomic 5-Hydroxymethylcytosine (5hmC)

“…Flow cytometric analyses on bone marrow derived macrophages (CD11b + ) from heterozygous Tet2:nGFP knock-in mice [11] revealed that all macrophages expressed high level of GFP reporter, which correlates with Tet2 protein level ( Figure 1 B). We then examined the effects of Tet2 loss on osteoclast differentiation in vitro after the stimulation of macrophages with receptor activator for nuclear factor κ B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) and counted the tartrate-resistant acid phosphatase (TRACP) positive multinucleated osteoclasts [21] . Surprisingly, the Tet2 −/− macrophage cultures contained significantly lower number of TRACP + multinucleated osteoclasts compared to WT macrophage cultures ( P < 0.001, Student’s t test) ( Figure 1 C and D).…”

“…MEL cells were maintained in RPMI1640 supplemented with l-glutamine, 10% FBS, and 1% penicillin–streptomycin. Murine macrophages and osteoclasts were obtained from in vitro culture of bone marrow mononuclear cells (BMMNCs) as described previously [21] . To generate macrophages, BMMNCs were cultured in alpha minimum essential medium (α-MEM) supplemented with 10% FBS and macrophage colony-stimulating factor (M-CSF; 20 ng/ml, PeproTech, Rocky Hill, NJ) for 3 days.…”