Individuals with neurofibromatosis type 1 (NF1) have a high incidence of osteoporosis and osteopenia. However, understanding of the cellular and molecular basis of these sequelae is incomplete. Osteoclasts are specialized myeloid cells that are the principal bone-resorbing cells of the skeleton. We found that Nf1 +/-mice contain elevated numbers of multinucleated osteoclasts. Both osteoclasts and osteoclast progenitors from Nf1 +/-mice were hyperresponsive to limiting concentrations of M-CSF and receptor activator of NF-κB ligand (RANKL) levels. M-CSF-stimulated p21 ras -GTP and Akt phosphorylation was elevated in Nf1 +/-osteoclasts associated with gains of function in survival, proliferation, migration, adhesion, and lytic activity. These gains of function are associated with more severe bone loss following ovariectomy as compared with that in syngeneic WT mice. Intercrossing Nf1 +/-mice and mice deficient in class 1 A PI3K (p85α) restored elevated PI3K activity and Nf1 +/-osteoclast functions to WT levels. Furthermore, in vitro-differentiated osteoclasts from NF1 patients also displayed elevated Ras/PI3K activity and increased lytic activity analogous to those in murine Nf1 +/-osteoclasts. Collectively, our results identify a what we believe to be a novel cellular and biochemical NF1-haploinsufficient phenotype in osteoclasts that has potential implications for the pathogenesis of NF1 bone disease. IntroductionNeurofibromatosis type 1 (NF1) is a common, pandemic genetic disorder that is characterized by a range of both malignant and nonmalignant manifestations and is caused by mutations in the NF1 gene. Neurofibromin, the protein encoded by NF1, functions as a GTPase-activating protein (GAP) for Ras. Though loss of both alleles of NF1 in tumorigenic cells is consistent with NF1 being a tumor suppressor gene, recent genetic evidence in murine models has indicated that nullizygous loss of Nf1 in the tumorigenic cells of plexiform neurofibromas (1) and optic gliomas (2) is necessary but not sufficient for tumor progression, though tumors occur with high penetrance when lineages of the tumor microenvironment are haploinsufficient at Nf1. These in vivo data provide evidence that haploinsufficiency of Nf1 in at least a subset of lineages has a role in the malignant manifestations of NF1 and also suggest a potential role for haploinsufficiency of Nf1 (NF1) in the nonmalignant pathogenesis of NF1.Three recent reports, including a controlled trial using WHO criteria for osteoporosis and osteopenia, have provided evidence that NF1 patients have a significantly higher incidence of osteoporosis and osteopenia (3-5). Bone homeostasis is maintained by balancing skeletal matrix formation and resorption. Osteoclasts are specialized cells derived from the myeloid monocyte/macro-

Skeletal abnormalities including scoliosis, tibial dysplasia, sphenoid wing dysplasia, and decreased bone mineral density (BMD) are associated with neurofibromatosis type 1 (NF1). We report the cellular phenotype of NF1 human-derived osteoclasts and compare the in vitro findings with the clinical phenotype.Functional characteristics (e.g. osteoclast formation, migration, adhesion, resorptive capacity) and cellular mechanistic alterations (e.g. F-actin polymerization, MAPK phosphorylation, RhoGTPase activity) from osteoclasts cultured from peripheral blood of individuals with NF1 (N=75) were assessed. Osteoclast formation was compared to phenotypic, radiologic, and biochemical data.NF1 osteoprogenitor cells demonstrated increased osteoclast forming capacity. Human NF1-derived osteoclasts demonstrated increased migration, adhesion, and in vitro bone resorption. These activities coincided with increased actin belt formation and hyperactivity in MAPK and RhoGTPase pathways. Although osteoclast formation was increased, no direct correlation of osteoclast formation with BMD, markers of bone resorption, or the clinical skeletal phenotype was observed suggesting that osteoclast formation in vitro cannot directly predict NF1 skeletal phenotypes.While NF1 haploinsufficiency produces a generalized osteoclast gain-in-function and may contribute to increased bone resorption, reduced BMD, and focal skeletal defects associated with

Neurofibromatosis type I (NF1) is a congenital disorder resulting from loss-of-function of the tumor suppressor gene, NF1, a GTPase-activating protein for p21ras. Fifty percent of NF1 patients have osseous manifestations including a high incidence of osteoporosis. Osteoclasts are specialized macrophage/monocyte lineage-derived cells that resorb bone and NF1 haploinsufficient osteoclasts have abnormal Ras-dependent bone resorption. Ras-regulated functions are in part mediated via the activation of small Rho family of GTPases including the Rac-GTPases. In the present study, we demonstrate that the Rho-GTPase Rac1 is a crucial Ras-mediated effector in Nf1 haploinsufficient (+/-) osteoclasts. Nf1+/- mice were intercrossed with conditional Rac1(flox/flox)Mxcre+ (Rac1-/-) mice to generate Nf1+/-; Rac1-/- mice. Genetic disruption of Rac1 restored the pathological increase in osteoclast progenitor cells in Nf1+/- mice and was sufficient to correct the increased Nf1+/- osteoclast motility and osteoclast belt formation, an f-actin structure observed in mature osteoclasts critical for bone resorption and lytic activity. Finally, we demonstrate that Nf1+/-; Rac1-/- osteoclasts have normalized Erk activation compared with Nf1+/- osteoclasts, a biochemical function critical for osteoclast formation, actin organization and motility. Collectively, these data demonstrate that Rac1 critically contributes to increased osteoclast function induced by haploinsufficiency of Nf1 and implicate Rac1 as a rational therapeutic target for osteoporosis.

Intracellular signals involved in the maturation and function of osteoclasts are poorly understood. Here, we demonstrate that osteoclasts express multiple regulatory subunits of class I A phosphatidylinositol 3-kinase (PI3-K) although the expression of the full-length form of p85␣ is most abundant. In vivo, deficiency of p85␣ results in a significantly greater number of trabeculae and significantly lower spacing between trabeculae as well as increased bone mass in both males and females compared to their sex-matched wild-type controls. Consistently, p85␣؊/؊ osteoclast progenitors show impaired growth and differentiation, which is associated with reduced activation of Akt and mitogen-activated protein kinase extracellular signal-regulated kinase 1 (Erk1)/Erk2 in vitro. Furthermore, a significant reduction in the ability of p85␣ ؊/؊ osteoclasts to adhere to as well as to migrate via integrin ␣v␤3 was observed, which was associated with reduced bone resorption. Microarray as well as quantitative real-time PCR analysis of p85␣؊/؊ osteoclasts revealed a significant reduction in the expression of several genes associated with the maturation and migration of osteoclasts, including microphathalmia-associated transcription factor, tartrate-resistant acid phosphatase, cathepsin K, and ␤3 integrin. Restoring the expression of the full-length form of p85␣ but not the version with a deletion of the Src homology-3 domain restored the maturation of p85␣ ؊/؊ osteoclasts to wild-type levels. These results highlight the importance of the full-length version of the p85␣ subunit of class I A PI3-K in controlling multiple aspects of osteoclast functions.Osteoclasts (OCs) are derived from precursors of monocyte/ macrophage lineage, whose growth and maturation are mainly dependent on two osteoblast/stromal cell-derived cytokines, including macrophage colony stimulating factor (M-CSF) and receptor activator of NF-B ligand (RANKL) (22,30,35,63). The critical role for these two cytokines in OC growth and differentiation has been further illustrated by studying mice lacking the expression of 64). These mice show severe osteopetrosis and lack mature OCs. M-CSF and RANKL regulate OC progenitor (OCp) growth and function in part by regulating the expression of several OC genes, including tartrate-resistant acid phosphatase (TRAP), cathepsin K, calcitonin receptor, and integrin ␤3 (15, 29). Stimulation of OC precursors by RANKL and M-CSF results in the activation of a number of signaling molecules, including Gab2, Grb2, Vav, Src homology-2 (SH2)-containing inositol-5-