Hyperactivation of p21ras and PI3K cooperate to alter murine and human neurofibromatosis type 1–haploinsufficient osteoclast functions
Feng-Chun Yang, Shi Chen, Alexander G. Robling, Xijie Yu, Todd D. Nebesio, Jincheng Yan, Trent Morgan, Xiaohong Li, Jin Yuan, Janet Hock, David A. Ingram, D. Wade Clapp
Feng-Chun Yang, Shi Chen, Alexander G. Robling, Xijie Yu, Todd D. Nebesio, Jincheng Yan, Trent Morgan, Xiaohong Li, Jin Yuan, Janet Hock, David A. Ingram, D. Wade Clapp
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Research Article Bone biology

Hyperactivation of p21ras and PI3K cooperate to alter murine and human neurofibromatosis type 1–haploinsufficient osteoclast functions

Abstract

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 p21ras-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 1A 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.

Authors

Feng-Chun Yang, Shi Chen, Alexander G. Robling, Xijie Yu, Todd D. Nebesio, Jincheng Yan, Trent Morgan, Xiaohong Li, Jin Yuan, Janet Hock, David A. Ingram, D. Wade Clapp

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Figure 7

Genetic disruption of p85α restores M-CSF responsiveness of Nf1+/– osteoclasts.

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Genetic disruption of p85α restores M-CSF responsiveness of Nf1+/– osteo...
(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.