Regulated expression of neurofibromin in migrating neural crest cells of avian embryos

Neurofibromatosis type 1 (NF1) is a common human genetic disease involving various neural crest (NC)-derived cell types, in particular, Schwann cells and melanocytes. The gene responsible for NF1 encodes the protein neurofibromin, which contains a domain with amino acid sequence homology to the ras-guanosine triphosphatase activating protein, suggesting that neurofibromin may play a role in intracellular signaling pathways regulating cellular proliferation or differentiation, or both. To determine whether neurofibromin plays a role in NC cell development, we used antibodies raised against human neurofibromin fusion proteins in western blot and immunocytochemical studies of early avian embryos. These antibodies specifically recognized the 235 kD chicken neurofibromin protein, which was expressed in migrating trunk and cranial NC cells of early embryos (E1.5 to E2), as well as in endothelial and smooth muscle cells of blood vessels and in a subpopulation of non-NC-derived cells in the dermamyotome. At slightly later stages (E3 to E5), neurofibromin immunostaining was observed in various NC derivatives, including dorsal root ganglia and peripheral nerves, as well as non-NC-derived cell types, including heart, skeletal muscle, and kidney. At still later stages (E7 to E9), neurofibromin immunoreactivity was found in almost all tissues in vivo. To determine whether the levels of neurofibromin changed during melanocyte and Schwann cell development, tissue culture experiments were performed. Cultured NC cells were found to express neurofibromin at early time points in culture, but the levels of immunoreactivity decreased as the cells underwent pigmentation. Schwann cells, on the other hand, continued to express neurofibromin in culture. These data suggest, therefore, that neurofibromin may play a role in the development of both NC cells and a variety of non-NC-derived tissues. © 1995 John Wiley & Sons, Inc.     

Angiogenic Expression Profile of Normal and Neurofibromin-Deficient Human Schwann Cells

Peripheral nerve sheath tumors from individuals with Neurofibromatosis Type 1 (NF1) are highly vascular and contain Schwann cells which are deficient in neurofibromin. This study examines the angiogenic expression profile of neurofibromin-deficient human Schwann cells relative to normal human Schwann cells, characterizing both pro-angiogenic and anti-angiogenic factors. Conditioned media from neurofibromin-deficient Schwann cell lines was pro-angiogenic as evidenced by its ability to stimulate endothelial cell proliferation and migration. Using gene array and protein array analysis, we found increased expression of pro-angiogenic factors and decreased expression of anti-angiogenic factors in neurofibromin-deficient Schwann cells relative to normal human Schwann cells. Neurofibromin-deficient Schwann cells also showed increased expression of several growth factor receptors and decreased expression of an integrin. We conclude that neurofibromin-deficient Schwann cells have dysregulated expression of pro-angiogenic factors, anti-angiogenic factors, growth factor receptors, and an integrin. These dysregulated molecules may contribute to the growth and progression of NF1 peripheral nerve sheath tumors.     

Poster Sessions BP02: Oligodendrocytes and Schwann Cells

Neurofibromatosis Type 1 tumors are highly vascularized and contain Schwann cells with hyperactivated Ras. In vitro , the NF1-derived neurofibromin deficient Schwann cells have an angiogenic profile, which favors angiogenesis and sustains the growth of the NF1-derived tumors. This study examined the relationship of the activation state of Ras as it related to the expression of angiogenic and antiangiogenic factors in both cultured NF1-derived Schwann cells and normal human Schwann cells. Western blot analysis of normal human Schwann cells revealed low expression of angiogenic vascular endothelial growth factor (VEGF) as well as low expression of the antiangiogenic pigment epithelium derived factor (PEDF). Relative to normal human Schwann cells, NF1-derived Schwann cells have increased RAS activity and a three-fold increase in VEGF expression. Surprisingly, PEDF was also expressed in the NF1-derived Schwann cells at approximately the same level as VEGF expression. Using a retroviral construct, we introduced the GAP-related domain of neurofibromin into the NF1-derived Schwann cells to reduce the level of activated Ras. Relative to the untreated NF1-derived Schwann cells the Schwann cells expressing the GAP-related domain expressed about one-half the VEGF but twice the PEDF. We conclude that decreasing the Ras activity in NF1-drived Schwann cells will not only decrease proliferation, but also slow tumor angiogenesis due to the decreased expression of angiogenic and increased expression of antiangiogenic factors.     

Poster Sessions BP02: Oligodendrocytes and Schwann Cells. RAS activation regulates angiogenic and anti‐angiogenic factor expression in NF1‐derived Schwann cells

Neurofibromatosis Type 1 tumors are highly vascularized and contain Schwann cells with hyperactivated Ras. In vitro, the NF1‐derived neurofibromin deficient Schwann cells have an angiogenic profile, which favors angiogenesis and sustains the growth of the NF1‐derived tumors. This study examined the relationship of the activation state of Ras as it related to the expression of angiogenic and antiangiogenic factors in both cultured NF1‐derived Schwann cells and normal human Schwann cells. Western blot analysis of normal human Schwann cells revealed low expression of angiogenic vascular endothelial growth factor (VEGF) as well as low expression of the antiangiogenic pigment epithelium derived factor (PEDF). Relative to normal human Schwann cells, NF1‐derived Schwann cells have increased RAS activity and a three‐fold increase in VEGF expression. Surprisingly, PEDF was also expressed in the NF1‐derived Schwann cells at approximately the same level as VEGF expression. Using a retroviral construct, we introduced the GAP‐related domain of neurofibromin into the NF1‐derived Schwann cells to reduce the level of activated Ras. Relative to the untreated NF1‐derived Schwann cells the Schwann cells expressing the GAP‐related domain expressed about one‐half the VEGF but twice the PEDF. We conclude that decreasing the Ras activity in NF1‐drived Schwann cells will not only decrease proliferation, but also slow tumor angiogenesis due to the decreased expression of angiogenic and increased expression of antiangiogenic factors.     

Single cell Ras-GTP analysis reveals altered Ras activity in a subpopulation of neurofibroma Schwann cells but not fibroblasts

Neurofibromatosis type 1 (NF1) is a common genetic disorder characterized by multiple neurofibromas, peripheral nerve tumors containing mainly Schwann cells and fibroblasts. The NF1 gene encodes neurofibromin, a tumor suppressor postulated to function in part as a Ras GTPase-activating protein. The roles of different cell types and of elevated Ras-GTP in neurofibroma formation are unclear. To determine which neurofibroma cell type has altered Ras-GTP regulation, we developed an immunocytochemical assay for active, GTP-bound Ras. In NIH 3T3 cells, the assay detected overexpressed, constitutively activated K-, N-, and Ha-Ras and insulin-induced endogenous Ras-GTP. In dissociated neurofibroma cells from NF1 patients, Ras-GTP was elevated in Schwann cells but not fibroblasts. Twelve to 62% of tumor Schwann cells showed elevated Ras-GTP, unexpectedly revealing neurofibroma Schwann cell heterogeneity. Increased basal Ras-GTP did not correlate with increased cell proliferation. Normal human Schwann cells, however, did not demonstrate elevated basal Ras activity. Furthermore, compared with cells from wild type littermates, Ras-GTP was elevated in all mouse Nf1(-/-) Schwann cells but never in Nf1(-/-) mouse fibroblasts. Our results indicate that Ras activity is detectably increased in only some neurofibroma Schwann cells and suggest that neurofibromin is not an essential regulator of Ras activity in fibroblasts.     

Prostaglandin E(2) metabolism is activated in Schwann cell lines derived from human NF1 malignant peripheral nerve sheath tumors

Malignant peripheral nerve sheath tumors (MPNSTs) are characteristic of Neurofibromatosis type 1 (NF1), a human genetic disorder affecting approximately 1 in 3000 individuals. The absence of neurofibromin in Schwann cells results in hyperactivation of Ras, which contributes to Schwann cell hyperplasia. However, additional intracellular abnormalities in Schwann cells might contribute to the malignancy. We now report that cell lines derived from MPNSTs secrete elevated levels of prostaglandin E(2) (PGE(2)), express higher levels of phosphorylated mitogen-activated protein kinase (MAPK), phosphorylated cytosolic phospholipaseA(2) (cPLA(2)) and cyclooxygenase 2 (COX-2) when compared to normal adult human Schwann cells (nhSCs). PCR analysis reveals that NF1 MPNST cell lines express mRNA for both EP2 and EP4 prostaglandin E2 receptors, whereas nhSCs express only the EP4 receptor. COX-2 inhibitors and PGE(2) receptor antagonists decrease the proliferation of MPNST cell lines. These results indicate that prostaglandin metabolism is activated in MPNSTs and might contribute to tumor growth in NF1.     

Voltage-Gated ion currents of schwann cells in cell culture models of human neurofibromatosis

K(+) (K) channels play a role in the proliferation of many cell types in normal cells and certain disease states. Several laboratories have studied K currents in cultured Schwann cells from models of the human diseases, neurofibromatosis type 1 (NF1) and neurofibromatosis type 2 (NF2). These diseases are characterized by the growth of Schwann cell tumors. In all cell culture NF models the K current properties differ in tumor-derived and normal Schwann cells. Depending on the model however, the type of K channel abnormality differs. K channels appear to play a role in the proliferation of Schwann cell cultures of these disease models, because a link has been established between K current blockade and the inhibition of Schwann cell proliferation in NF1 and NF2. Differences in the proliferation response of normal Schwann cells to K channel blockers suggest that in vitro regulation of proliferation in neoplastic and normal Schwann cells is complex.     

Reconstitution of the NF1 GAP-related domain in NF1-deficient human Schwann cells

Schwann cells derived from peripheral nerve sheath tumors from individuals with Neurofibromatosis Type 1 (NF1) are deficient for the protein neurofibromin, which contains a GAP-related domain (NF1-GRD). Neurofibromin-deficient Schwann cells have increased Ras activation, increased proliferation in response to certain growth stimuli, increased angiogenic potential, and altered cell morphology. This study examined whether expression of functional NF1-GRD can reverse the transformed phenotype of neurofibromin-deficient Schwann cells from both benign and malignant peripheral nerve sheath tumors. We reconstituted the NF1-GRD using retroviral transduction and examined the effects on cell morphology, growth potential, and angiogenic potential. NF1-GRD reconstitution resulted in morphologic changes, a 16-33% reduction in Ras activation, and a 53% decrease in proliferation in neurofibromin-deficient Schwann cells. However, NF1-GRD reconstitution was not sufficient to decrease the in vitro angiogenic potential of the cells. This study demonstrates that reconstitution of the NF1-GRD can at least partially reverse the transformation of human NF1 tumor-derived Schwann cells.     

Sensitivity of Malignant Peripheral Nerve Sheath Tumor Cells to TRAIL Is Augmented by Loss of NF1 through Modulation of MYC/MAD and Is Potentiated by Curcumin through Induction of ROS

Malignant peripheral nerve sheath tumor (MPNST) is a rare aggressive form of sarcoma often associated with the tumor syndrome neurofibromatosis type 1 (NF1). We investigated the effects of tumor necrosis factor-related apoptosis inducing ligand (TRAIL) on NF1 associated MPNST and determinants of TRAIL sensitivity. MPNST cell lines with complete neurofibromin deficiency were sensitive to apoptotic cell death induced by TRAIL whereas MPNST cells with retained neurofibromin expression or normal human Schwann cells were resistant. Increased sensitivity to TRAIL was associated with overexpression of death receptors, especially DR5. Re-expression of the GAP related domain of neurofibromin (NF1-GRD) suppressed DR5 expression and decreased sensitivity to TRAIL. We show that death receptor expression and TRAIL sensitivity critically depend on c-MYC and that c-MYC amounts are increased by MEK/ERK and PI3K/AKT signalling pathways which are suppressed by neurofibromin. Furthermore PI3K/AKT signalling strongly suppresses the MYC-antagonist MAD1 which significantly contributes to TRAIL sensitivity. Re-expression of the NF1-GRD decreased c-MYC and increased MAD1 amounts suggesting that neurofibromin influences TRAIL sensitivity at least in part by modulating the MYC/MAX/MAD network. The phytochemical curcumin further increased the sensitivity of neurofibromin deficient MPNST cells to TRAIL. This was presumably mediated by ROS, as it correlated with increased ROS production, was blocked by N-acetylcysteine and mimicked by exogenous ROS.     

Brain lipid binding protein in axon-Schwann cell interactions and peripheral nerve tumorigenesis

Loss of axonal contact characterizes Schwann cells in benign and malignant peripheral nerve sheath tumors (MPNST) from neurofibromatosis type 1 (NF1) patients. Tumor Schwann cells demonstrate NF1 mutations, elevated Ras activity, and aberrant epidermal growth factor receptor (EGFR) expression. Using cDNA microarrays, we found that brain lipid binding protein (BLBP) is elevated in an EGFR-positive subpopulation of Nf1 mutant mouse Schwann cells (Nf1(-/-) TXF) that grows away from axons; BLBP expression was not affected by farnesyltransferase inhibitor, an inhibitor of H-Ras. BLBP was also detected in EGFR-positive cell lines derived from Nf1:p53 double mutant mice and human MPNST. BLBP expression was induced in normal Schwann cells following transfection with EGFR but not H-Ras12V. Furthermore, EGFR-mediated BLBP expression was not inhibited by dominant-negative H-Ras, indicating that BLBP expression is downstream of Ras-independent EGFR signaling. BLBP-blocking antibodies enabled process outgrowth from Nf1(-/-) TXF cells and restored interaction with axons, without affecting cell proliferation or migration. Following injury, BLBP expression was induced in normal sciatic nerves when nonmyelinating Schwann cells remodeled their processes. These data suggest that BLBP, stimulated by Ras-independent pathways, regulates Schwann cell-axon interactions in normal peripheral nerve and peripheral nerve tumors.     

Neurofibromin 1 Is a miRNA Target in Neurons

Mutations of the neurofibromin 1 gene cause neurofibromatosis type 1, a disease in which learning and behavioral abnormalities are common. The disease is completely penetrant but shows variable phenotypic expression in patients. The repertoire of regulatory interactions utilized by neurons to control neurofibromin 1 expression is poorly understood. Here, we examined the contribution of microRNAs into this regulatory network. Using reporter assays, we provided evidence that miR-128 and to a lesser extent miR-137 and miR-103 reduced neurofibromin 1 reporter levels through specific binding to Nf1 3′-UTR. Mutations in all three predicted binding sites eliminated the reporter response. MiR-128 and miR-137, unlike miR-103 that showed a more ubiquitous expression, were predominantly expressed in brain with a distribution that resembled neurofibromin 1 expression in different tissues as well as during the course of neuronal development. In the nervous system, all three microRNAs showed highest expression in neurons and least in Schwann cells and astrocytes. Overexpression of miR-128 alone or with miR-103 and miR-137 significantly reduced endogenous neurofibromin 1 protein levels, while antisense inhibition of these microRNAs enhanced translation of endogenous neurofibromin 1 and reporter in primary cultures of hippocampal neurons. These findings revealed a significant additional mechanism by which neurofibromin 1 is regulated in neurons and implicated new candidates for the treatment of multifarious neurofibromatosis type 1 cognitive symptoms.     

Angiogenic and invasive properties of neurofibroma Schwann cells

Neurofibromas are benign tumors from patients with von Recklinghausen Neurofibromatosis (NF1) that are comprised primarily of Schwann cells. These Schwann cells are found both in association with axons and in the extracellular matrix that is prevalent in neurofibromas, and in which fibroblasts are also abundant. An unresolved question has been whether cells in neurofibromas are normal cells or are intrinsically abnormal. We have tested the hypothesis that cells in neurofibromas are abnormal and have shown that neurofibroma Schwann cells, unlike normal Schwann cells, promote angiogenesis in the chick chorioallantoic membrane model system, and invade basement membranes in this system. In contrast, neurofibroma fibroblasts neither promote angiogenic reactions nor invade basement membranes. When injected into nude mice, neurofibroma Schwann cells do not form progressive tumors. These results suggest that NF1 Schwann cells differ from normal Schwann cells, that they are preneoplastic, and that genetic and/or epigenetic changes in Schwann cells may be required for development of peripheral nerve tumors in NF1.     

Neurofibromatosis type I tumor suppressor neurofibromin regulates neuronal differentiation via its GTPase-activating protein function toward Ras

Neurofibromin, the neurofibromatosis type 1 (NF1) gene product, contains a central domain homologous to a family of proteins known as Ras-GTPase-activating proteins (Ras-GAPs), which function as negative regulators of Ras. The loss of neurofibromin function has been thought to be implicated in the abnormal regulation of Ras in NF1-related pathogenesis. In this study, we found a novel role of neurofibromin in neuronal differentiation in conjunction with the regulation of Ras activity via its GAP-related domain (GRD) in neuronal cells. In PC12 cells, time-dependent increases in the GAP activity of cellular neurofibromin (NF1-GAP) were detected after NGF stimulation, which were correlated with the down-regulation of Ras activity during neurite elongation. Interestingly, the NF1-GAP increase was due to the induction of alternative splicing of NF1-GRD type I triggered by the NGF-induced Ras activation. Dominant-negative (DN) forms of NF1-GRD type I significantly inhibited the neurite extension of PC12 cells via regulation of the Ras state. NF1-GRD-DN also reduced axonal and dendritic branching/extension of rat embryonic hippocampal neurons. These results demonstrate that the mutual regulation of Ras and NF1-GAP is essential for normal neuronal differentiation and that abnormal regulation in neuronal cells may be implicated in NF1-related learning and memory disturbance.     

Neurofibromatosis type 1 (NF1) tumor suppressor, neurofibromin, regulates the neuronal differentiation of PC12 cells via its associating protein, CRMP-2

Neurofibromatosis type 1 (NF1) tumor suppressor gene product, neurofibromin, functions in part as a Ras-GAP, a negative regulator of Ras. Neurofibromin is implicated in the neuronal abnormality of NF1 patients; however, the precise cellular function of neurofibromin has yet to be clarified. Using proteomic strategies, we identified a set of neurofibromin-associating cellular proteins, including axon regulator CRMP-2 (Collapsin response mediator protein-2). CRMP-2 directly bound to the C-terminal domain of neurofibromin, and this association was regulated by the manner of CRMP-2 phosphorylation. In nerve growth factor-stimulated PC12 cells, neurofibromin and CRMP-2 co-localized particularly on the distal tips and branches of extended neurites. Suppression of neurofibromin using NF1 small interfering RNA significantly inhibited this neurite outgrowth and up-regulated a series of CRMP-2 phosphorylations by kinases identified as CDK5, GSK-3b, and Rho kinase. Overexpression of the NF1-RAS-GAP-related domain rescued these NF1 small interfering RNA-induced events. Our results suggest that neurofibromin regulates neuronal differentiation by performing one or more complementary roles. First, neurofibromin directly regulates CRMP-2 phosphorylation accessibility through the complex formation. Also, neurofibromin appears to indirectly regulate CRMP-2 activity by suppressing CRMP-2-phosphorylating kinase cascades via its Ras-GAP function. Our study demonstrates that the functional association of neurofibromin and CRMP-2 is essential for neuronal cell differentiation and that lack of expression or abnormal regulation of neurofibromin can result in impaired function of neuronal cells, which is likely a factor in NF1-related pathogenesis.     

Expression of the neurofibromatosis type 2 gene in human tissues.

The neurofibromatosis Type 2 tumor suppressor gene is implicated in the hereditary tumor syndrome NF2, hallmarked by bilateral vestibular schwannomas, meningiomas, and ocular non-neoplastic features. The gene product has characteristics of a membrane cytoskeleton-linking protein but the mechanism of tumor suppression by the NF2 protein remains to be elucidated. The NF2 gene is widely expressed in mouse and rat tissues. In humans, most of the expression data have accumulated through Northern blot analysis, RT-PCR and, more recently, Western blot analysis, providing information on whole tissues and organs rather than on specific cell types. We report here an extensive survey of NF2 gene expression in human tissues using a combination of mRNA in situ hybridization (mRNA ISH) and immunohistochemistry (IH) with a panel of monoclonal antibodies (MAbs) supplemented by tissue immunoprecipitation experiments with affinity-purified polyclonal antibodies. Expression was observed in many different cell types, most of which appear functionally normal in individuals affected by NF2. Surprisingly, expression could not be consistently documented in Schwann cells and arachnoidal cells by IH or by mRNA ISH in formalin-fixed tissue. However, consistent immunostaining of Schwann cells was seen in frozen sections. (J Histochem Cytochem 47:1471-1479, 1999)     

Biofabrication of a three dimensional human-based personalized neurofibroma model

Neurofibromas are the most characteristic feature of neurofibromatosis type 1 (NF1), a multisystemic disorder caused by aberrations in the neurofibromin gene (NF1). Despite significant progress over the last several years in understanding this disease, a suitable in vitro model to better mimic neurofibroma formation and growth has yet to be described. There is therefore a need to establish an in vitro, three dimensional model that allows the incorporation of multicellular lineages and the modulation of the cellular microenvironment—known to be important for cellular crosstalk and distribution of soluble factors—to study neurofibroma biology and morphogenesis. A self-assembly approach was used to generate tissue-engineered skins (TES) in which patient-derived spheroids made of NF1-associated Schwann cells and fibroblasts were seeded. We describe the first in vitro three dimensional neurofibroma model—directly derived from NF1 patients presenting with histopathological features—having an ECM protein expression profile quite similar to that of a native tumor. We observed efficient incorporation, proliferation, and migration of spheroids within NF1-TES over time. This biotechnological approach could provide a unique tool for precision medicine targeting NF1 and for assessing the tumorigenic properties of each NF1 gene mutation linked to tumor formation.     

Aberrant cAMP Metabolism in NF1 Malignant Peripheral Nerve Sheath Tumor Cells

Malignant peripheral nerve sheath (MPNST) cell lines derived from patients with neurofibromatosis type 1 (NF!) were found to have basal cAMP levels which are two-fold higher than cAMP levels in normal human adult Schwann cells (nHSC). PCR analysis also revealed that normal adult human Schwann cells express mRNA for types Ill, IV, and IX adenylyl cyclase (AC) while NF1 MPNST cells express AC mRNA of types II, V, and VIII in addition to expressing all the isoforms of normal adult human Schwann cells. Further PCR analysis revealed that NF1 MPNST lines express mRNA for EP2 and EP4 prostaglandin receptors whereas nHSC only express mRNA for the EP2 receptor. Exogenous prostaglandins alone or in combination with PDGF BB induced greater increases in cAMP levels and proliferation of NF1 MPNST cells compared to nHSC. We conclude that aberrant cAMP signaling in NF1 MPNST cells contributes to tumor formation in NF1 patients.     

Alternative splicing of the neurofibromatosis type I pre-mRNA

NF1 (neurofibromatosis type?I) is a common genetic disease that affects one in 3500 individuals. The disease is completely penetrant but shows variable phenotypic expression in patients. NF1 is a large gene, and its pre-mRNA undergoes alternative splicing. The NF1 protein, neurofibromin, is involved in diverse signalling cascades. One of the best characterized functions of NF1 is its function as a Ras-GAP (GTPase-activating protein). NF1 exon 23a is an alternative exon that lies within the GAP-related domain of neurofibromin. This exon is predominantly included in most tissues, and it is skipped in CNS (central nervous system) neurons. The isoform in which exon 23a is skipped has 10?times higher Ras-GAP activity than the isoform in which exon 23a is included. Exon 23a inclusion is tightly regulated by at least three different families of RNA-binding proteins: CELF {CUG-BP (cytosine-uridine-guanine-binding protein) and ETR-3 [ELAV (embryonic lethal abnormal vision)-type RNA-binding protein]-like factor}, Hu and TIA-1 (T-cell intracellular antigen 1)/TIAR (T-cell intracellular antigen 1-related protein). The CELF and Hu proteins promote exon 23a skipping, while the TIA-1/TIAR proteins promote its inclusion. The widespread clinical variability that is observed among NF1 patients cannot be explained by NF1 mutations alone and it is believed that modifier genes may have a role in the variability. We suggest that the regulation of alternative splicing may act as a modifier to contribute to the variable expression in NF1 patients.     

Neurofibromin GTPase-activating protein-related domains restore normal growth in Nf1-/- cells

Members of the Ras superfamily of signaling proteins modulate fundamental cellular processes by cycling between an active GTP-bound conformation and an inactive GDP-bound form. Neurofibromin, the protein product of the NF1 tumor suppressor gene, and p120GAP are GTPase-activating proteins (GAPs) for p21(Ras) (Ras) and negatively regulate output by accelerating GTP hydrolysis on Ras. Neurofibromin and p120GAP differ markedly outside of their conserved GAP-related domains (GRDs), and it is therefore unknown if the respective GRDs contribute functional specificity. To address this question, we expressed the GRDs of neurofibromin and p120GAP in primary cells from Nf1 mutant mice in vitro and in vivo. Here we show that expression of neurofibromin GRD, but not the p120GAP GRD, restores normal growth and cytokine signaling in three lineages of primary Nf1-deficient cells that have been implicated in the pathogenesis of neurofibromatosis type 1 (NF1). Furthermore, utilizing a GAP-inactive mutant NF1 GRD identified in a family with NF1, we demonstrate that growth restoration is a function of NF1 GRD GAP activity on p21(Ras). Thus, the GRDs of neurofibromin and p120GAP specify nonoverlapping functions in multiple primary cell types.     

Neurofibromin can inhibit Ras-dependent growth by a mechanism independent of its GTPase-accelerating function.

The NF1 gene, which is altered in patients with type 1 neurofibromatosis, has been postulated to function as a tumor suppressor gene. The NF1 protein product neurofibromin stimulates the intrinsic GTPase activity of active GTP-bound Ras, thereby inactivating it. Consistent with a tumor suppressor function, we have found that the introduction of NF1 in melanoma cell lines that are deficient in neurofibromin inhibited their growth and induced their differentiation. In addition, overexpression of neurofibromin in NIH 3T3 cells was growth inhibitory but did not alter the level of GTP.Ras in the cells. Transformation by v-ras, whose protein product is resistant to GTPase stimulation by neurofibromin, was inhibited in a cell line overexpressing neurofibromin, while transformation by v-raf was not altered. The results demonstrate that NF1 is a tumor suppressor gene that can inhibit Ras-dependent growth by a regulatory mechanism that is independent of neurofibromin's ability to stimulate Ras GTPase.