NF1 is a common, pandemic, autosomal-dominant genetic disorder that affects 1 in 3,000 individuals. The detection of somatic mutations in the residual normal NF1
allele within the cancers of individuals with NF1 is consistent with NF1
functioning as a tumor-suppressor gene. However evidence in selected lineages (2
) now indicates that analogous to recent discoveries in p53 (32
) and p27 (33
), gene dosage effects of NF1
alter cell fates and functions. The most recent studies using Nf1+/–
cells have focused on the role of NF1
haploinsufficiency in lineages of the tumor microenvironment of plexiform neurofibromas and optic gliomas (2
). However, a phenotype in long-term learning in Nf1+/–
mice, similar to a spatial-visual discoordination observed in NF1 patients, has been established (34
). The high frequency of nonmalignant manifestations in NF1 patients, including cerebrovascular disease (35
), learning deficits, and osseous abnormalities, such as osteoporosis, suggest the importance of NF1 haploinsufficiency in multiple lineages. Recognition of the cellular and biochemical underpinnings of these physical findings is important in identifying specific molecular therapies and in disease treatment and prevention.
Many skeletal diseases occur as a consequence of a skeletal imbalance that favors bone resorption (36
). In response to extracellular signals, macrophage progenitors differentiate into monocytes that evaginate from the marrow and circulate in the peripheral blood. Recent discoveries have demonstrated that M-CSF together with signals from a family of biologically related tumor necrosis factor receptor–like proteins (particularly RANKL and its decoy ligand osteoprotegerin) control the induction of osteoclast differentiation and are key to the physiologic and pathophysiologic responses of osteoclasts (38
). Common chronic diseases such as arthritis or menopause functionally lead to an increase in production of M-CSF and RANKL, leading to increased osteoclast activation and consequent bone loss (36
). Our results demonstrating that, at limiting concentrations of M-CSF and RANKL, murine (Nf1+/–
) and human NF1 monocytes have an increased potential to mature into multinucleated osteoclasts and an increased ability to adhere to bone and form polykaryons that lead to increased bone lysis are consistent with previous findings relating to other pathological causes of osteoporosis and osteopenia. Data demonstrating an increased number of multinucleated osteoclasts in Nf1+/–
mice, an increase in TRAP5b in the serum of Nf1+/–
mice, and an increase in bone resorption following OVX provide in vivo support of this concept.
The increases in osteoclast numbers and lytic activity reported here in murine and human NF1 heterozygous cells is consistent with recent initial studies showing that NF1 patients have decreased bone density (3
). Previous work by Yu et al. (40
) found that Nf1+/–
mice also had a trend toward reduced total bone as compared with WT controls, though the difference was not statistically significant. There are established species-specific differences in skeletal development and in the weight forces between quadripedal mice and bipedal humans that influence skeletal density (41
). These species differences could be important when considering bone remodeling disease manifestations in preclinical models. Additionally, even in chronic diseases, osteoporosis frequently develops over the course of decades as opposed to 2–6 months, a typical duration of experiments using murine models. Future studies to evaluate measurements of bone remodeling in NF1 patients and epidemiologic studies to evaluate the risks of bone fractures in young and elderly adults with NF1 are also indicated.
Recent studies have emphasized the role of phospholipids in cytoskeletal reorganization, as well as cell survival and proliferation (42
in particular is a phospholipid that is a known modulator of these cellular functions. Myeloid progenitors, macrophages, and osteoclasts from SHIP–/–
mice are exquisitely hypersensitive to multiple growth factors, including M-CSF. Strains of mice with complementary mutations influencing osteoclast differentiation and function include op/op
), which have an inactivating mutation in the M-CSF receptor, and Gab2–/–
), which contain a disruption of the Grb-2
–associated binder adaptor protein that renders them unable to mediate RANKL-induced activation of Akt, JNK, and NF-κB. Consequently, both op/op
mice and Grb2–/–
mice have profound osteopetrosis. Data in Nf1+/–
mice provide evidence for the first time to our knowledge that support the concept that subtle regulations of Ras activity in osteoclasts lead to osteoclasts and osteoclast precursors being hypersensitive to growth factor signals that modulate osteoclast functions in vitro and in vivo.
The paradigm observed in Nf1+/–
osteoclasts is similar to that in studies using Nf1+/–
mast cells, where limiting concentrations of kit ligand led to activation of a receptor in the same growth factor family as M-CSF (c-kit receptor tyrosine kinase). Previous studies in mast cells that were WT or haploinsufficient at the Nf1
locus have positioned the small RhoGTPase Rac as a key downstream effector of class I A
). Preferential inhibition of Nf1+/–
osteoclast migration by a low concentration of the PI3K inhibitor (5 μM, LY294002) as compared with a Mek inhibitor (50 μM, PD98059) (85% vs. 20% mean reduction) is consistent with these results. Further, in preliminary studies in osteoclasts, we have also identified Rac2 as a class 1A
PI3K effector that modulates the migration, adhesion, and lytic activity of osteoclasts that are heterozygous or WT at the Nf1
locus (our unpublished observations). These data suggest that alterations of the Ras/PI3K/Rac signaling pathway may ultimately be critical in osteoclast development and function. The availability of conditional knockout mice will also allow an understanding of the role of Nf1+/–
osteoclast function in other skeletal manifestations in the context of nullizygous loss of Nf1
in selected mesenchymal cell populations.
Though bisphosphonates have been the cornerstone of nonspecific agents utilized to treat osteoporosis since the 1960s, recent work has begun to focus on more specific targeted therapies (6
). Currently available molecular targets to Ras itself, such as farnesyltransferase inhibitors, have been disappointing, since farnesylation is not sufficient to inhibit posttranslational modification of K-ras and N-ras, the 2 most prevalent isoforms in myeloid lineages. Data in osteoclasts in this genetic model provide genetic, cellular, and biochemical support for further evaluation of experimental compounds to inhibit class1A
PI3K activity that are currently in development.