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Keywords:

  • Nf1;
  • osteoclasts;
  • mTOR

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHOD
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Individuals with nerofibromatosis Type 1 (NF1) frequently suffer a spectrum of bone pathologies, such as abnormal skeletal development (scoliosis, congenital bowing, and congenital pseudoarthroses, etc), lower bone mineral density with increased fracture risk. These skeletal problems may result, in part, from abnormal osteoclastogenesis. Enhanced RAS/PI3K activity has been reported to contribute to abnormal osteoclastogenesis in Nf1 heterozygous (Nf1+/−) mice. However, the specific downstream pathways linked to NF1 abnormal osteoclastogenesis have not been defined. Our aim was to determine whether mammalian target of rapamycin (mTOR) was a key effector responsible for abnormal osteoclastogenesis in NF1. Primary osteoclast-like cells (OCLs) were cultured from Nf1 wild-type (Nf1+/+) and Nf1+/− mice. Compared to Nf1+/+ controls, there were 20% more OCLs induced from Nf1+/− mice. Nf1+/− OCLs were larger and contained more nuclei. Hyperactive mTOR signaling was detected in Nf1+/− OCLs. Inhibition of mTOR signaling by rapamycin in Nf1+/− OCLs abrogated abnormalities in cellular size and number. Moreover, we found that hyperactive mTOR signaling induced abnormal osteoclastogenesis major through hyper-proliferation. Our research suggests that neurofibromin directly regulates osteoclastogenesis through mTOR signaling pathway. Inhibiting mTOR may represent a viable strategy to treat NF1 bone diseases. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 30:144–152, 2012

Neurofibromatosis Type 1 (NF1) is one of the most common autosomal dominant genetic disorders, affecting one in 3,500 individuals worldwide.1 NF1 is a tumor predisposition syndrome. The most common tumor pathologies in afflicted individuals are benign neurofibromas and malignant peripheral nerve sheath tumors (MPNSTs). Humans with NF1 may also suffer a spectrum of bone pathologies. Abnormal skeletal development occurs in 10–20% of NF1 individuals, resulting in short stature, scoliosis, congenital bowing, and congenital pseudoarthroses of long bones.2 Fractures of dysplastic bones occur in about 3% young NF1 children, especially boys.3 Orthopedic surgery to correct NF1 bone defects often fails due to non-union and pseudoarthroses of the healing bones.4 Several studies report lower bone mineral density (BMD), a known risk factor for fractures, in NF1 humans.5–7 Specifically, NF1 humans have a unique generalized skeletal dysplasia: when subjects with NF1 were separated in groups with and without a skeletal abnormality, those who did not have a skeletal abnormality still had statistically significant decreases in BMD compared with control subjects, although they were less pronounced than in those with osseous abnormalities.7 Unlike post-menopausal or male osteoporosis, NF1 osteopenia/osteoporosis has been diagnosed in children and adolescents, especially in those with skeletal abnormality; children with NF1 also have a general tendency toward osteopenia.7–9 These clinical data suggest an abnormal bone phenotype in NF1. Currently, there is limited treatment for NF1 bone pathologies.

Neurofibromin is expressed in osteoclasts.10 NF1 children have an increase in the urinary excretion of pyridinium crosslinks, reflecting increased bone resorption.11 High serum bone tartrate resistant acid phosphatase (TRAP) concentration and high serum calcium concentration have been reported to associate with lower BMD among the NF1 patients.12 Histomorphometric analysis reveals increased numbers of osteoclasts in biopsies from NF1 patients.13 Osteoclasts from NF1 patients are larger in size; their nuclei are more numerous; actin rings are more frequent; and the resorption pits are more numerous and larger.14 These data indicate that abnormal osteoclastogenesis in NF1 individuals, which may be in part responsible for the skeletal pathologies in NF1. However, the molecular mechanisms of abnormal osteoclastogenesis in NF1 are not quite clear.

Loss-of-function mutations in NF1 tumor suppressor gene underlie the disease. The gene product, neurofibromin acts as a Ras-GTPase activating protein (GAP). Neurofibromin converts RAS from its active GTP to its inactive GDP isoform. Loss-of-function of NF1 deregulates RAS signaling pathway, thus promoting abnormal cellular proliferation and tumorigenesis. Enhanced RAS activity has been reported in osteoclasts from Nf1+/− mice and NF1 patients. Initial therapies for NF1-associated tumors focused on the use of RAS inhibitors. RAS activation requires isoprenylation for membrane localization, which can be blocked by farnesyltransferase inhibitors. In preclinical studies, farnesyltransferase inhibitors can reduce cell proliferation dramatically both in NF1-deficient human and Nf1−/− mouse cells15–17; however, these agents have little effect on tumor growth in NF1 patients.18 The limited success of RAS inhibitors in NF1 clinical trials suggests that downstream pathways or other signaling pathways may play important roles in NF1 pathologies. Mammalian target of rapamycin (mTOR) is the downstream pathway for RAS. Recent research has shown that mTOR is constitutively activated in NF1-deficient primary cells (Nf1−/− mouse embryonic fibroblasts, Nf1−/− astrocytes, and NF1−/− Schwann cells), as well as in human NF1-associated tumors. NF1 tumor cells derived from NF1 patients are highly sensitive to the mTOR inhibitor, rapamycin.19, 20 These results suggest that mTOR pathway may be one of the critical downstream of NF1-related RAS signaling. mTOR also appears as an essential signaling pathway engaged in the stimulation of osteoclast survival.21 Tumor necrosis factor-a (TNF-a), receptor activator of nuclear factor kappa B ligand (RANKL) and macrophage-colony-stimulating factor (M-CSF) promote osteoclast survival by signaling through mTOR/S6K.22, 23 Furthermore, rapamycin, the inhibitor for mTOR, disturbs osteoclast differentiation and survival.21 Because mTOR is constitutively activated in Nf1 primary cells as well as in NF1 tumors,19, 20 we hypothesize that the mTOR pathway may be deregulated in Nf1-deficient osteoclasts and linked to abnormal osteoclastogenesis in NF1.

MATERIALS AND METHOD

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHOD
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Animals

Because Nf1−/− in mice proved lethal due to a defect in heart development, Nf1+/− mice were used to study NF1 osteoclastogenesis. Nf1+/− mice (strain name: B6.129S6-Nf1tm1Fcr/J) were obtained from The Jackson Laboratory (TJL, Bar Harbor, ME). Nf1+/+ mice from the same colony were used as control. These mice were originally developed by Dr. Brannan.24 All animal protocols were approved by the TJL Institutional Animal Care and Use Committee. Mice were housed under standard conditions of 12:12-h light/dark cycle, with water and food ad libitum (Purina Rodent lab Chow 5001, containing 0.95% Ca, 0.67% P, and vitamin D3 4,500 IU/kg). Mice were euthanized by CO2 inhalation; thoracic transection assured death.

Generation of Murine Osteoclast-Like Cells (OCLs)

Bone marrow cells were induced to differentiate into OCLs in the presence of RANKL and M-CSF. Age-matched Nf1+/− and Nf1+/+ male mice were used to obtain inducible OCLs when 10 weeks old. Diaphyseal marrow of the left tibia was flushed through a 21G needle to obtain bone marrow cells. Bone marrow cells from each genotype were pooled together (at least six mice from each genotype at each time). The pooled cells were plated in 24-well plates at a density of 1 × 106 cells/well for TRAP staining (n = 6 for each genotype) or in 12-well plates at a density of 2 × 106 cells/well for mTOR signaling study (n = 3 for each genotype). Cells were cultured in α-minimal essential medium (α-MEM; Invitrogen, Grand Island, NY) with 10% fetal calf serum (Invitrogen) and 1% penicillin–streptomycin solution (Invitrogen) for 8 days at 37°C with 5% CO2 in the presence of human soluble RANKL at 30 ng/ml (Chemicon, Temecula, CA) and recombinant mouse M-CSF at 25 ng/ml (R&D Systems, Minneapolis, MN). Medium was changed every 3 days. At the end of the culture period, OCLs were used for mTOR signaling study or TRAP staining.

TRAP Staining and Measurement

OCLs were fixed with 3.7% formalin for 10 min and stained for TRAP according to the manufacturer's instructions (Sigma, St. Louis, MO). TRAP+ multinucleated (containing three or more nuclei) cells were counted under microscope, and categorized as mature OCLs.25 Five fixed areas from each well were chosen as areas of interest. The cell surface area of OCLs within each area was analyzed by light microscopy and Osteomeasure software (OsteoMetrics, Inc., Atlanta, GA), as in our previous study.26

Real-Time qRT-PCR

OCLs were serum starved for 12 h. RNA was extracted from cells using RNeasy Mini kit (Qiagen, Inc., Valencia, CA). The TaqMan probes and primers for mouse mTOR and β-actin were designed using Primer Express software (Applied Biosystems, Foster City, CA). The primers and probes were designed to span at least one intron to eliminate non-specific signals due to genomic DNA amplification. RT and real-time quantitative PCR were performed in a one-step reaction using the TaqMan Gold RT-PCR kit and protocols provided by the manufacturer (Applied Biosystems). All primer sets were tested for specific amplification of mRNA by parallel analyses of controls that included omitting RT or template and resulted in no fluorescent signal detection. Each RNA sample was analyzed in triplicate, and each experiment was performed independently at least three times. The amplification of mTOR and β-actin was equally efficient, and the equation image method described by Livak and Schmittgen27 was used to analyze the data.

Western Blotting

The most commonly used readout for mTOR activation is phosphorylation of a well-characterized downstream substrate, ribosomal S6, at Ser240/244. Phosphorylation at this site depends on mTOR and is required for maximal S6 activation. To determine the activation of mTOR signal transduction, phosphorylated S6 was studied by Western Blotting in Nf1+/+ and Nf1+/− OCLs. OCLs were lysed in RIPA buffer. Protein quantitation was determined by a BCA protein assay kit (Pierce, Rockford, IL). 20 µg protein per lane was separated by 7.5% SDS–polyacrylamide gel electrophoresis (SDS–PAGE). The proteins were transferred to a polyvinylidine difluoride membrane (Millipore Corporation, Bedford, MA) in Mini Trans-Blot Cells (BioRad, Hercules, CA) according to the manufacturer's manual. The blots were blocked with blocking buffer (5% non-fat milk in TBS-T buffer). Blots were incubated overnight at 4°C with different first antibodies: phosphated S6 or total S6 (Cell Signaling, 1:1,000). The blots were then incubated for 1 h at room temperature with a horseradish peroxidase-conjugated secondary antibody: anti mouse IgG (Chemicon), diluted 1:10,000. All signals were visualized using ECL Plus chemiluminescence substrate (Amersham Pharmacia Biotech, Piscataway, NJ). TotalLab software (Phoretix, Durham, NC) was used to analyze bands. Total S6 was used as internal control. Experiments were repeated in triplicate.

Rapmaycin Treatment

For mTOR signaling study, OCLs were starved with serum-free medium for 12 h to avoid confounding effects of serum. Cells were pretreated with 20 nM rapamycin or vehicle control (dimethyl sulfoxide) for 1.5 h. OCLs were then stimulated with 25 ng/ml M-CSF for 10 or 30 min, phosphorylated S6 was studied by Western blotting. For OCL formation experiment, bone marrow cells were induced to differentiate into osteoclasts with the protocol listed above. Vehicle control (dimethyl sulfoxide) or 20 nM rapamycin was added to the medium at indicated time course. Medium was changed every 3 days. Cells were fixed and stained for TRAP at the end of the culture.

Statistical Analyses

Data were analyzed using JMP Statistical Discovery Software 8.0 (SAS Institute, Cary, NC). Multivariate analyses with pairwise comparisons were applied to detect significant interactions in multiple groups. If significant interactions were present, contrast t-tests were used to identify significant differences between individual pairs. If interactions were not statistically significant, one-way analysis of variance was used in combination with multiple pairwise comparisons by the Tukey–Kramer test. Statistical significance for all tests was set at p < 0.05. Data are expressed as mean ± standard error of the mean (SEM).

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHOD
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Abnormal OCL Formation in Nf1+/− Mice

TRAP staining analysis revealed a twofold increase in OCL number in the culture from Nf1+/− mice (Fig. 1A–C; p < 0.001). Nf1+/− OCLs were much larger, and exhibited increased surface area (Fig. 1B and D; p < 0.001). The average size of Nf1+/− OCLs was about fourfold larger than that of Nf1+/+ OCLs. Many giant osteoclasts (large size with nuclei over eight) were found in the cultures from Nf1+/− mice, while they were never found in the cultures from Nf1+/+ mice. These results demonstrate abnormal osteoclast formation with haploinsufficience in Nf1.

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Figure 1. Abnormal OCL formation in Nf1+/− mice. Bone marrow cells were induced to differentiate into osteoclasts in the presence of RANKL and M-CSF for 8 days. Cells were then fixed with 3.7% formalin and stained for TRAP. TRAP+ multinucleated (containing three or more nuclei) cells were classified as mature OCL cells. (A, B) Compared to Nf1+/+ culture, subjectively there appeared to be numerous giant OCLs (large size with much more nuclei) in Nf1+/− culture (Red arrows indicated mature OCLs). (C) The mature OCL numbers in Nf1+/− culture were significantly higher than that from Nf1+/+ culture. (D) The cell size of Nf1+/− mature OCL cells was significantly larger than that of Nf1+/+ controls (***p < 0.001, n = 6).

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Hyperactive mTOR Signaling in Nf1+/− OCLs

When mRNA expression was studied by real-time qRT-PCR, the level of mTOR mRNA was significant higher in Nf1+/− OCLs than that in Nf1+/+ OCLs (Fig. 2A; p < 0.001). Phosphorylated S6 is the readout for mTOR activation. Studied by Western blotting, elevated phosphorylated S6 signaling was detected in both Nf1+/+ and Nf1+/− OCL cells after M-CSF stimulation, with the highest level at 10 min after the stimulation (Fig. 2B and C). Compared to Nf1+/+ OCLs, Nf1+/− OCLs showed higher phosphorylated S6 signaling at baseline, as well as after the stimulation with M-CSF (Fig. 2B and C). Take together, these results indicated hyperactive mTOR signaling in Nf1+/− OCLs.

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Figure 2. Hyperactive mTOR signaling in Nf1+/− OCLs. OCLs were starved with serum-free medium for 12 h, mRNA expression of mTOR was determined by real-time qRT-PCR. OCLs were further stimulated with 25 ng/ml M-CSF for 10 or 30 min, phosphorylated S6 was studied by Western blotting. (A) Compared to Nf1+/+ OCLs, Nf1+/− OCLs showed higher mTOR mRNA expression (***p < 0.001, n = 3). (B) Results from Western blotting. (C) Bands from three independent experiments were analyzed with TotalLab software. Nf1+/− OCLs showed significantly higher phosphorylated S6 signaling at baseline and after M-CSF stimulation. Ribosomal S6 was used as internal control (total S6: total ribosomal S6; Pi-S6: phosphorylated ribosomal S6. a: compare to the same genotype at baseline, p < 0.05. n = 3; b: compared to Nf1+/+ with the same treatment, p < 0.05. n = 3).

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Rapamycin Inhibiting Hyperactive mTOR Signaling in Nf1+/− OCLs

Rapamycin is known as an effective inhibitor for mTOR signaling. We asked if rapamycin could block hyperactive mTOR signaling in Nf1+/− OCLs or not. Early study showed that the proliferation of MPNST cell lines was significantly inhibited by rapamycin at dose of 10 nM.28 A phase II study of rapamycin in NF1-related plexiform neurofibromas is ongoing, which aims to maintain rapamycin levels within the target range of 10–15 ng/ml (11–17 nM) in serum (http://www.druglib.com/trial/70/NCT00634270.html). Rapamycin has also been reported to inhibit mTOR signaling and reduce the generation of mature TRAP-positive OCLs, with maximal effects were observed at 10 nM or above.22 In our preliminary experiment, we found rapamycin at 30 nM or above induced significantly cell death in OCL culture (data not shown). On the basis of these data, we chose rapamycin at 20 nM to determine its effects in Nf1+/− OCLs. After treating with rapamycin, Nf1+/− OCLs demonstrated a similar level of phosphorylated S6 signaling to that of Nf1+/+ OCLs (Fig. 3A and B), both at the baseline and after stimulation with M-CSF. This result indicated rapamycin could restore hyperactive mTOR signaling in Nf1+/− OCLs.

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Figure 3. Rapamycin inhibiting hyperactive mTOR signaling in Nf1+/− OCL cells. OCLs from bone marrow cells were starved with serum-free medium for 12 h. Cells were pretreated with 20 nM rapamycin for 1.5 h. OCLs were then stimulated with 25 ng/ml M-CSF for 10 or 30 min, phosphorylated S6 was studied by Western blotting. Pretreatment with rapamycin restored phosphorylated S6 level in Nf1+/− OCLs to the similar level as that in Nf1+/+ OCLs. Panel A: bands from Western blotting; panel B: bands from three independent experiments were analyzed with TotalLab software. There was no difference in phosphorylated S6 level between Nf1+/+ OCLs and Nf1+/− OCLs (Total S6: total ribosomal S6; Pi-S6: phosphorylated ribosomal S6. a: compared to the same genotype at baseline, p < 0.05. n = 3).

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Blocking mTOR Restored Abnormal OCL Formation in Nf1+/− Mice

We further studied whether inhibiting mTOR signaling could correct the abnormal OCL formation in Nf1+/− mice, or not. Bone marrow cells from Nf1+/− and Nf1+/+ mice were induced to differentiate into OCLs in the presence of either vehicle DMSO or 20 nM rapamycin throughout the culture course to block mTOR signaling. Similar to our earlier results, significantly more OCLs and some giant osteoclasts were found in the cultures from Nf1+/− vehicle group (Fig. 4A–C). Rapamycin significantly decreased OCL formation in both Nf1+/+ and Nf1+/− mice. After rapamycin treatment, there were no significant differences in OCL number, size, and nuclei between Nf1+/+ and Nf1+/− mice (Fig. 4A–C).

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Figure 4. Blocking mTOR restoring abnormal OCL formation in Nf1+/− mice. Bone marrow cells were induced to differentiate into osteoclasts in the presence of RANKL and M-CSF plus vehicle control dimethyl sulfoxide (VEH) or 20 nM rapamycin for 8 days. Cells were then fixed and stained for TRAP. Nf1+/− mice show more OCLs with larger size and more nuclei when treated with VEH. Rapamycin significantly decreased OCL number and surface area in both Nf1+/+ and Nf1+/− culture. After rapamycin treatment, there was no difference in OCL number, size, and nuclei between Nf1+/+ and Nf1+/− mice. Panel A: TRAP staining for osteoclasts; panel B: OCL numbers; panel C: OCL surface area (a: compare to Nf1+/+ VEH, p < 0.05; b: compare to the same genotype treated with VEH, p < 0.05, n = 6).

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Hyperactive mTOR Signaling Induced Abnormal OCL Formation through Hyperproliferation

mTOR signaling may regulate osteoclast proliferation and/or survival. We further determined the pathological roles of hyperactive mTOR signaling in Nf1+/− OCL formation. Rapamycin was used either during the proliferative phase of culture (the first 4 days of OCL culture); or during the differentiating and maturing phases (the last 4–8 days of OCL culture); or through the entire culture period, to block mTOR signaling at different stages. Rapamycin reduced OCL formation equivalently in Nf1+/− and Nf1+/+ cell cultures during either the first 4 days of culture or the entire culture period (Fig. 5A–C). However, Nf1+/− mice showed more and larger OCLs with more nuclei when treated with vehicle DMSO or 20 nM rapamycin during later phase (the last 4–8 days of OCL culture; Fig. 5A–C). These results suggested that hyperactive mTOR signaling induced abnormal OCL formation in Nf1+/− mice mostly through hyperproliferation.

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Figure 5. Hyperactive mTOR signaling inducing abnormal OCL formation in early stage. Bone marrow cells were induced to differentiate into osteoclasts in the presence of RANKL and M-CSF plus 20 nM rapamycin (Rap) at indicated culture time. Nf1+/− mice showed more OCLs with larger size and more nuclei when treated with vehicle control dimethyl sulfoxide (VEH) or 20 nM Rapamycin at late stage (4–8 days). While Nf1+/− mice showed similar reduction in OCL formation as Nf1+/+ mice when treated with rapamycin during the entire culture course or during the early stage of cell culture (0–4 days) (a: compared to Nf1+/+ with the same treatment, p < 0.05; b: compare to the same genotype treated with VEH, p < 0.05, n = 6).

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHOD
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Osteoclasts are produced by the fusion of mononuclear precursors to form polykaryons, a critical step to perform resorption.29 Osteoclasts normally acquire up to eight nuclei before dying by apoptosis, probably as a result of exposure to the high extracellular concentration of calcium that occurs during bone resorption.30 The number of osteoclasts is a faithful index of bone resorption.31 Abnormal osteoclast formation is usually related to abnormal bone resorption, such as that in Paget's disease, where abundant hypernucleated osteoclasts correlate with extensive bone resorption.32 Here, we find that the number and size of osteoclasts, as well as the number of their nuclear profiles, increase in OCL culture from Nf1+/− mice at 10 weeks old. These results suggest that neurofibromin is required for ex vivo osteoclast formation and function in adult mice.

mTOR signaling is constitutively activated in neurofibromin deficient primary cells such as Nf1−/− mouse embryonic fibroblasts, Nf1−/− astrocytes, and NF1−/− Schwann cells, as well as in human NF1-associated tumors.19, 20 Our findings demonstrate that elevated mTOR signaling also occurs in primary Nf1+/− OCLs (Fig. 2). Elevated mTOR signaling in Nf1+/− OCLs may be due to abnormal activation of upstream signaling pathways. Indeed, our early research found that p21-GTP and Akt phosphorylation were elevated in Nf1+/− osteoclasts.33 Genetically restoring PI3K level corrected abnormal osteoclast formation and function in Nf1+/− mice.33 Because mTOR is the downstream target of PI3K/Akt, increased PI3K/Akt activity likely underlies hyperactive mTOR signaling in Nf1+/− osteoclasts. mTOR plays a critical role in cellular growth control (where cell growth refers to an increase in both cell size and cell number) by regulating cell translational machinery via effects on ribosomes and protein synthesis.34, 35 Since mTOR is activated in Nf1+/− osteoclasts, it is not surprise to detect giant osteoclasts and more osteoclast numbers in Nf1+/− mice (Fig. 1). Correcting osteoclast formation by rapamycin further confirms the critical role of mTOR in abnormal osteoclast formation in Nf1+/− mice (Fig. 4).

The molecular mechanisms under which mTOR regulates osteoclast formation and differentiation are still not quite clear. Abnormal osteoclast formation in Nf1+/− mice may be a consequence from hyper-proliferation and/or defective apoptosis. Our research demonstrates that inhibition of mTOR by rapamycin during the first 4-day culture, or during the entire 8-day culture period shows similar effect in reducing abnormal osteoclast formation (Fig. 5). These results suggest hyperactive mTOR signaling induces abnormal osteoclast formation mostly through hyper-proliferation in Nf1+/− mice. mTOR is a key regulator of cell proliferation, in part through regulation of cyclin D1.36 Cyclin D1 activates cyclin-dependent kinases 4 and 6 to regulate the passage of cells through the critical G1-S restriction point of the cell cycle.37, 38 Overexpression of cyclin D1 has been reported in NF1 Schwann cells as well as MPNSTs.39, 40 Inhibition of cyclin D1 expression inhibits growth of NF1 deficient tumor xenografts in mice.41 Based on these published data, we speculate that elevated mTOR may act via cyclin D1 to deregulate proliferation in Nf1+/− osteoclasts.

Published literatures show that rapamycin significantly inhibits proliferation in MC3T3-E1, bone marrow stromal cells (BMSCs), and ROS 17/2.8 (ROS) cells through reducing levels of cyclin A and D1 protein.42, 43 Some studies have demonstrated that rapamycin promotes osteogenic differentiation from mesenchymal stromal cells (MSCs),44 human embryonic stem cell (hESC),45 and ROS cells.43 On the other hand, one study reports rapamycin inhibits osteogenic differentiation in the early differentiating stage of MC3T3-E1 and BMSCs.42 These contradicting results indicate that rapamycin may play different roles during osteoblastic differentiation. We have found hyperactive RAS signaling in Nf1+/− osteoblasts.46 mTOR signaling might also function through osteoblasts to regulate osteoclastogensis in Nf1+/− mice. Further experiments are required to determine this possibility.

Rapamycin is an antifungal agent originally purified from Streptomyces hygroscopicus with potent immunosuppressive actions.47 Rapamycin is used in combinations with other drugs to prevent organ rejection after renal transplantation. Renal transplant patients who receive rapamycin daily for several years do not seem to have obligatory problems from sustained blockage of TOR. Among the side effects of rapamycin, there are no pronounced immunosuppressive problems (viral, bacterial, and fungal infections, Kaposi's sarcoma or osteoporosis).48 Ironically, it was assumed that rapamycin would predispose patients to osteoporosis and cancer, but instead rapamycin exhibited anti-cancer and bone-sparing activities.49, 50 Bone-resorbing osteoclasts depend on the TOR pathway for their formation, function, and survival. Rapamycin and other inhibitors of TOR inhibit osteoclast formation and activity, thus prevents the OVX-induced loss of cancellous bone.21 In the present study, we also find similar inhibition of osteoclast formation by rapamycin. Because Rapamycin and its derivatives inhibit mTOR-dependent mRNA translation both in osteoclasts and tumor cells, they are considered as bi-functional molecules affecting simultaneously bone and tumor metabolisms.51 Because mTOR signaling is constitutively activated in NF1-deficient Schwann cells and tumor cells,19, 20 rapamycin and its derivatives are in clinical trial for NF1 tumors in multicenter clinical trials. In the present study, we found that rapamycin can inhibit mTOR signaling in primary Nf1+/− OCLs and correct abnormal osteoclast formation in Nf1+/− mice. Our findings suggest that rapamycin may service as bi-functional molecules to treat NF1 individuals with osteoporosis/osteopenia.

In summary, our research suggests that neurofibromin directly regulates osteoclastogenesis through mTOR signal transduction. Inhibiting mTOR may represent a viable strategy to target NF1 bone diseases.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHOD
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

This work was supported by grants to Dr. Xijie Yu from National Natural Science Foundation of China (No. 30872632; No. 81072190), the Irving Oil Company, Canada, and US Dept Defense Telemedicine and Technology Research Command Award W81XWH-07-2-0116 (PI: JM Hock). Some experiments were performed by Dr X Yu and Ms J Ma while employed at the Maine Institute for Human Genetics & Health. We thank Ms. Ning Liu, Maine Institute for Human Genetics and Health, for her technical support.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHOD
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES