PMC

(A) Ras activity in WT and Nf1^+/– osteoclasts was measured at the indicated times following stimulation with M-SCF. A representative blot is shown (left panel). The relative increase in Ras activity in Nf1^+/– osteoclasts compared with WT osteoclasts in 4 independent experiments is indicated (right panel). Data represent mean ± SEM. *P < 0.01, Ras activity of Nf1^+/– osteoclasts versus WT osteoclasts. (B) Akt phosphorylation was measured at 0 and 2 minutes following stimulation with M-CSF. Results from 1 of 4 independent experiments are shown (left panel), and the relative increase (mean ± SEM; n = 4) in Akt phosphorylation in Nf1^+/– osteoclasts compared with WT osteoclasts is indicated (right panel). **P < 0.01, Akt phosphorylation of Nf1^+/– osteoclasts versus WT osteoclasts. (C) Erk phosphorylation was measured at the indicated levels following stimulation with M-CSF. A representative Western blot (left panel) is shown, and the relative mean increase (mean ± SEM; n = 4) in Erk phosphorylation in Nf1^+/– osteoclasts is indicated (right panel). ^†P < 0.01, Nf1^+/– osteoclasts versus WT osteoclasts.

(A) Osteoclasts were transduced with retroviral constructs expressing a selectable marker and the GRDs of NF1; the GRDs containing a point mutation that inactivates GAP activity (R1276P); or a construct encoding only the reporter transgene (Pac). M-CSF–mediated migration following antibiotic selection of transduced cells was measured. Results represent the mean ± SEM of 3 replicates from 1 of 4 independent experiments with similar results. *P < 0.01, Nf1^+/– osteoclasts transduced with the Pac construct versus WT osteoclasts or Nf1^+/– osteoclasts expressing the GRD or 1276 constructs. (B) Ras activity of osteoclasts at basal levels and 5 minutes following stimulation with M-CSF are shown. Genotypes and stimuli are indicated.

Data in B, C, and E represent the mean ± SEM of 5 independent experiments. (A) Clonogenic assays were established to determine the number of CFU-Ms per femur. Representative TRAP^– CFU-M (left panel) and TRAP⁺ CFU-M (right panel). (B) Total CFU-Ms per femur from Nf1^+/– or WT mice. *P < 0.01, Nf1^+/– CFU-Ms versus WT CFU-Ms by Student’s t test. (C) TRAP⁺ CFU-Ms per femur from mice of the indicated genotypes. *P < 0.01, Nf1^+/– versus WT. (D) Representative photomicrographs (magnification, ×10) of WT and Nf1^+/– distal femoral metaphyses following TRAP staining. Arrows indicate selected osteoclasts. (E) Average size of osteoclasts from Nf1^+/– and WT mice. Ten high-power fields per experimental mouse were scored. **P < 0.05, Nf1^+/– versus WT osteoclasts.

(A) Haptotaxis of WT and Nf1^+/– osteoclasts in response to M-CSF. A representative photomicrograph (magnification, ×20) from 1 of 5 experiments. (B) Quantitative evaluation of migration in response to M-CSF. Results represent the mean ± SEM of 5 experiments. *P < 0.01, Nf1^+/– versus WT by Student’s t test. (C) Photomicrographs (magnification, ×10) of M-CSF–stimulated osteoclast adhesion to αVβ3. Genotypes and length of adhesion are indicated. (D) Quantitative evaluation (mean ± SEM; n = 5) of osteoclast adhesion 10–60 minutes following incubation with M-CSF. Genotypes and length of adhesion are indicated. *P < 0.01, Nf1^+/– versus WT osteoclasts by Student’s t test. (E) M-CSF–mediated F-actin polymerization. Osteoclasts were stimulated with 10 ng/ml M-CSF and fixed at the time points indicated. WT and Nf1^+/– cells were examined in triplicate. Results are expressed as mean channel fluorescence (MCF). **P < 0.001, Nf1^+/– osteoclasts versus WT osteoclasts by Student’s t test.

(A) MNCs from Nf1^+/– and p85α^–/– intercrossed fetal liver cells were stimulated with 0.1–50 ng/ml of M-CSF and 50 ng/ml RANKL. TRAP⁺ cells from 3 replicate wells/concentration were calculated. Results represent the mean ± SEM of 4 experiments. Statistical analyses were conducted using ANOVA. *P < 0.01, Nf1^+/– osteoclasts versus WT osteoclasts; **P < 0.001, p85α^–/– versus WT osteoclasts; ^#P < 0.01, Nf1^+/– versus Nf1^+/–p85α^–/– osteoclasts. (B) Representative photographs (magnification, ×20) of osteoclasts from the 4 experimental groups. (C) M-CSF–mediated migration of Nf1^+/– and p85α intercrossed osteoclasts. Results represent the mean ± SEM of 4 experiments. *P < 0.01, WT versus other experimental groups using ANOVA. **P < 0.001, p85α^–/– versus WT osteoclasts; ^#P < 0.01, Nf1^+/– versus Nf1^+/– p85α^–/– osteoclasts. (D) Evaluation of M-CSF–mediated adhesion. Results represent the mean ± SEM of 4 experiments. *P < 0.01, number of Nf1^+/– osteoclasts versus WT osteoclasts; **P < 0.001, number of p85α^–/– versus WT osteoclasts; ^#P < 0.01, number of Nf1^+/– versus Nf1^+/–p85α^–/– osteoclasts. (E) Representative photomicrographs (magnification, ×10) of bone pits from the 4 F₂ genotypes. (F) Osteoclast survival was evaluated by determining the annexin V–negative/PI-negative population. ^†P < 0.01, survival of Nf1^+/– versus WT and Nf1^+/– versus Nf1^+/–p85α^–/– cells; **P < 0.01 survival of p85α^–/– versus WT osteoclasts using ANOVA. (G) Akt phosphorylation of the 4 F₂ genotypes at basal levels and following M-CSF stimulation. Genotypes and length of stimulation are indicated.

(A) Increase in the number of CFU-Ms per femur in response to M-CSF. Data represent the mean ± SEM of 5 experiments. *P < 0.01, Nf1^+/– versus WT CFU-Ms by Student’s t test. (B and C) Evaluation of the ability of Nf1^+/– and WT BMMNCs to form osteoclasts in response to M-CSF (B) and RANKL (C). Data represent the mean ± SEM of TRAP⁺ cells/5 × 10⁴ LDMNCs from 5 experiments. ^#P < 0.01, Nf1^+/– versus WT TRAP⁺ cells. (D) Osteoclast DNA content of cultured WT osteoclast (left panel) and Nf1^+/– osteoclasts (right panel) was determined using fluorescence cytometry following PI staining. (E) Representative photomicrographs (magnification, ×20) of Nf1^+/– and WT osteoclasts in liquid culture. Arrows indicate selected osteoclasts. (F) Osteoclast survival was determined by calculating annexin V– and PI-negative cell populations. ^##P < 0.05 and **P < 0.01, Nf1^+/– versus WT osteoclasts.

(A) Neurofibromin levels in unaffected controls and NF1 patients. (B) Number of osteoclasts formed from culture of 1 × 10⁵ peripheral blood LDMNCs following M-SCF and RANKL stimulation for 14 days (left panel). Data represent the mean ± SEM for 5 individuals with NF1 and 5 unaffected sex- and age-matched controls. Representative photomicrographs (magnification, ×20) from the indicated genotypes (right panel) are shown. (C) Evaluation of M-CSF– and PI3K-mediated migration of osteoclasts from NF1 patients and controls. The genotypes and addition of M-CSF and the PI3K inhibitor LY294002 are indicated. Data represent the mean ± SEM of 4 experiments. *P < 0.01, osteoclasts from NF1 patients versus unaffected controls; **P < 0.01, number of osteoclasts that migrated following preincubation in 5 μM of LY294002 versus osteoclasts preincubated in vehicle. (D) Pit formation of osteoclasts. Osteoclasts from NF1 patients and unaffected controls were cultured on bone sections in the presence of M-CSF and LY294002 or the vehicle control for 14 days. (E) Akt phosphorylation in M-CSF–stimulated osteoclasts. The genotype, stimulus, inhibitor, and length of stimulation are indicated.

(A) Osteoclasts were incubated on bone sections and stained with toluidine blue at the end of culture. The resorbed bone area stained dark blue. Representative photomicrographs (magnification, ×10) of the indicated genotypes are shown. (B) Quantitative evaluation of bone resorption. Results represent the mean area ± SEM of 5 independent experiments. *P < 0.01, WT versus Nf1^+/– bone resorption. (C) The areas of individual bone resorption pits of the 2 genotypes are represented by individual symbols. The horizontal line represents the mean area of each genotype. *P < 0.01, WT versus Nf1^+/–. (D) ELISA of serum TRAP5b activity. Results represent mean ± SEM (n = 6) using age- and sex-matched controls. Genotypes are indicated. ^#P < 0.05, Nf1^+/– versus WT. (E) OVX-induced reduction (percent change compared with sham-operated animals) in BMD among adult female WT and Nf1^+/– mice. Data represent the mean ± SEM (n = 7) of the indicated genotypes. *P < 0.01, WT versus Nf1^+/– OVX mice by Student’s t test. (F) Osteoclast number per femur. Data represent the mean ± SEM (n = 7) of the indicated genotypes and treatment groups generated following ex vivo culture. ^##P < 0.01, WT OVX versus WT sham; **P < 0.01, Nf1^+/– sham versus WT sham; and ^†P < 0.01, Nf1^+/– OVX versus Nf1^+/– sham and Nf1^+/– OVX versus WT OVX mice using ANOVA.