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Neurofibromatosis type 1 (NF1) is an autosomal dominant disease caused by mutations in the NF1 tumor suppressor gene, which affect approximately 1 out of 3000 individuals. Patients with NF1 suffer from a range of malignant and nonmalignant manifestations such as plexiform neurofibromas and skeletal abnormalities. We previously demonstrated that Nf1 haploinsufficiency in mesenchymal stem/progenitor cells (MSPCs) results in impaired osteoblastic differentiation, which may be associated with the skeletal manifestations in NF1 patients. Here we sought to further ascertain the role of Nf1 in modulating the migration and adhesion of MSPCs of the Nf1 haploinsufficient (Nf1(+/-)) mice. Nf1(+/-) MSPCs demonstrated increased nuclear-cytoplasmic ratio, increased migration, and increased actin polymerization as compared to wild-type (WT) MSPCs. Additionally, Nf1(+/-) MSPCs were noted to have significantly enhanced cell adhesion to fibronectin with selective affinity for CH271 with an overexpression of its complimentary receptor, CD49e. Nf1(+/-) MSPCs also showed hyperactivation of phosphoinositide 3-kinase (PI3-K) and mitogen activated protein kinase (MAPK) signaling pathways when compared to WT MSPCs, which were both significantly reduced in the presence of their pharmacologic inhibitors, LY294002 and PD0325901, respectively. Collectively, our study suggests that both PI3-K and MAPK signaling pathways play a significant role in enhanced migration and adhesion of Nf1 haploinsufficient MSPCs.

X-linked hypophosphatemic rickets (XLH) is a dominantly inherited disorder characterized by renal phosphate wasting, aberrant vitamin D metabolism, and abnormal bone mineralization. XLH is caused by inactivating mutations in PHEX (phosphate-regulating gene with homologies to endopeptidases on the X chromosome). In this study, we sequenced the PHEX gene in subjects from 26 kindreds who were clinically diagnosed with XLH. Sequencing revealed 18 different mutations, of which thirteen have not been reported previously. In addition to deletions, splice site mutations, and missense and nonsense mutations, a rare point mutation in the 3'-untranslated region (3'-UTR) was identified as a novel cause of XLH. In summary, we identified a wide spectrum of mutations in the PHEX gene. Our data, in accord with those of others, indicate that there is no single predominant PHEX mutation responsible for XLH.

Neurofibromas are the clinical hallmark of neurofibromatosis Type 1 (NF1), a genetic disorder caused by mutations of the NF1 tumor suppressor gene, which encodes neurofibromin that functions as a GTPase activating protein (GAP) for Ras. During pregnancy, up to 50% of existing neurofibromas enlarge and as many as 60% of new neurofibromas appear for the first time. Lysophosphatidic acid (LPA) is a prototypic lysophospholipid that modulates cell migration and survival of Schwann cells (SCs) and is made in increasing concentrations throughout pregnancy. We addressed the influence of LPA on the biochemical and cellular functions of SCs with a homozygous mutation of the murine homologue of the NF1 gene (Nf1-/-). LPA promoted F-actin polymerization and increased migration and survival of Nf1-/- SCs as compared to wild type (WT) SCs. Furthermore, LPA induced a higher level of Ras-GTP and Akt phosphorylation in Nf1-/- SCs as compared to WT cells. Pharmacologic inhibition or siRNA for the p85beta regulatory subunit of Class I A PI3-K significantly reduced LPA-induced Schwann cell survival and migration. Introduction of NF1-GRD reconstitution was sufficient to normalize the LPA-mediated motility of Nf1-/- SCs. As LPA modulates excessive cell survival and motility of Nf1-/- SCs, which are the tumorigenic cells in NF1, targeting PI3-K may be a potential therapeutic approach in diminishing the development and progression of neurofibromas in pregnant women with NF1., ((c) 2007 Wiley-Liss, Inc.)

Mutations in the NF1 tumor suppressor gene cause neurofibromatosis type 1, a pandemic autosomal dominant genetic disorder with an incidence of 1:3000. Individuals with NF1 have a variety of malignant and non-malignant manifestations, including skeletal manifestations, such as osteoporosis, scoliosis and short statures. However, the mechanism(s) underlying the osseous manifestations in NF1 are poorly understood. In the present study, utilizing Nf1 haploinsufficient (+/-) mice, we demonstrate that Nf1+/- mesenchymal stem/progenitor cells (MSPC) have increased proliferation and colony forming unit-fibroblast (CFU-F) capacity compared with wild-type (WT) MSPC. Nf1+/- MSPC also have fewer senescent cells and have a significantly higher telomerase activity compared with WT MSPC. Nf1+/- MSPC have impaired osteoblast differentiation as determined by alkaline phosphatase staining, and confirmed by single CFU-F replating assays. The impaired osteoblast differentiation in Nf1+/- MSPC is consistent with the reduced expression of osteoblast markers at the mRNA level, including osteocalcin and osteonectin. Importantly, re-expression of the full-length NF1 GTPase activating related domain (NF1 GAP-related domain) is sufficient to restore the impaired osteoblast differentiation in Nf1+/- MSPC. Taken together, our results suggest that neurofibromin plays a crucial role in modulating MSPC differentiation into osteoblasts, and the defect in osteoblast differentiation may contribute at least in part to the osseous abnormalities seen in individuals with NF1.

Neurofibromas are common tumors found in neurofibromatosis type 1 (NF1) patients. These complex tumors are composed of Schwann cells, mast cells, fibroblasts and perineurial cells embedded in collagen that provide a lattice for tumor invasion. Genetic studies demonstrate that in neurofibromas, nullizygous loss of Nf1 in Schwann cells and haploinsufficiency of Nf1 in non-neuronal cells are required for tumorigenesis. Fibroblasts are a major cellular constituent in neurofibromas and are a source of collagen that constitutes approximately 50% of the dry weight of the tumor. Here, we show that two of the prevalent heterozygous cells found in neurofibromas, mast cells and fibroblasts interact directly to contribute to tumor phenotype. Nf1+/- mast cells secrete elevated concentrations of the profibrotic transforming growth factor-beta (TGF-beta). In response to TGF-beta, both murine Nf1+/- fibroblasts and fibroblasts from human neurofibromas proliferate and synthesize excessive collagen, a hallmark of neurofibromas. We also establish that the TGF-beta response occurs via hyperactivation of a novel Ras-c-abl signaling pathway. Genetic or pharmacological inhibition of c-abl reverses fibroblast proliferation and collagen synthesis to wild-type levels. These studies identify a novel molecular target to inhibit neurofibroma formation.