Hyperactivation of p21^ras and PI3-K Cooperate To Alter Murine and Human NF1 Haploinsufficient Osteoclast Functions.

Neurofibromatosis type 1 (NF1) is a common genetic disorder caused by mutations of the NF1 tumor suppressor gene that functions as a GTPase activating protein for Ras. Though nullizygous loss of NF1 is associated with the development of malignancies, haploinsufficient phenotypes are now being increasingly recognized to alter cell fates and functions in a number of tissues resulting in nonmalignant disease manifestations. Bone manifestations, including skeletal dysplasias, scoliosis, and osteoporosis occur in 30–60% of all NF1 patients and osteoporosis is an increasingly recognized health problem for women with NF1. 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. Using an established murine model of NF1 developed using homologous recombination, we found that Nf1+/− mice contain elevated numbers of multinucleated osteoclasts and osteoclast progenitors per femur in vivo. Both osteoclasts and osteoclast progenitors from Nf1+/− mice were hyperresponsive to limiting concentrations of M-CSF and RANKL, growth factors that are integral to osteoclast maturation and activation. M-CSF stimulated p21^ras-GTP and Akt phosphorylation was elevated in Nf1+/− osteoclasts associated with gains in function in survival and proliferation. Bone resorption by osteoclasts is linked to the migration and adherence of these cells to a local bone surface. Purified populations of Nf1+/− osteoclasts were initially placed in the upper chamber of a transwell coated with vitronectin and haptotaxis to M-CSF was determined. Nf1+/− osteoclasts had a 2–3 fold increase in migration as compared to syngeneic wildtype cells. A similar increase in adhesion of Nf1+/− osteoclasts to the integrin avb3 was also observed. Following adhesion, osteoclasts form a specialized cell-extracellular matrix to initiate degradation of bone matrix by secreting proteinases. Nf1+/− osteoclasts had significantly increased bone resorption as measured by scoring the number and area of individual bone resorbing “pits” on dentine slices and by scoring the total area of resorption. These collective increases in osteoclast function were validated in vivo by the observation that serum TRAP5b activity, a sensitive measure of osteoclast lytic activity was 2.5 fold higher in Nf1+/− mice as compared to WT mice. Furthermore, we performed ovariectomy, an established model of osteoporosis associated with an increase in osteoclast function. In two independent experiments, we found that Nf1+/− mice had a significantly greater reduction in bone mineral density following ovariectomy as compared to syngeneic wildtype mice. We hypothesized that hyperactivation of class1A-PI3-K may contribute to these gains in cellular function. We found that intercrossing Nf1+/− and Class1_A PI3-K deficient mice (p85a) restores elevated PI3-K activity, and Nf1+/− osteoclast functions to wildtype levels. Furthermore, in vitro differentiated osteoclasts from NF1 patients also display elevated Ras-PI3-K activity and increased lytic activity analogous to murine Nf1+/− osteoclasts. Collectively, we identify a novel cellular and biochemical NF1 haploinsufficient phenotype in osteoclasts that has potential implications in the pathogenesis of NF1 bone disease.