The Nf1 tumor suppressor regulates mouse skin wound healing, fibroblast proliferation, and collagen deposited by fibroblasts.
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Neurofibromatosis type 1 patients develop peripheral nerve tumors (neurofibromas) composed mainly of Schwann cells and fibroblasts, in an abundant collagen matrix produced by fibroblasts. Trauma has been proposed to trigger neurofibroma formation. To test if loss of the neurofibromatosis type 1 gene (Nf1) compromises fibroblast function in vivo following trauma, skin wounding was performed in Nf1 knockout mice. The pattern and amount of collagen-rich granulation bed tissue, manufactured by fibroblasts, was grossly abnormal in 60% of Nf1+/- wounds. Nf1 mutant fibroblasts showed cell autonomous abnormalities in collagen deposition in vitro that were not mimicked by Ras activation in fibroblasts, even though some Nf1 effects are mediated through Ras. Nf1+/- skin wound fibroblasts also proliferated past the normal wound maturation phase; this in vivo effect was potentiated by muscle injury. In vitro, Nf1+/- fibroblasts showed higher proliferation in 10% serum than Nf1+/+ fibroblasts. Macrophage-conditioned media or epidermal growth factor potentiated Nf1+/- fibroblast proliferation in vitro, demonstrating abnormal response of mutant fibroblasts to wound cytokines. Thus Nf1 is a key regulator of fibroblast responses to injury, and Nf1 mutation in mouse fibroblasts causes abnormalities characteristic of human neurofibromas. (+info)
Site-specific DNA methylation in the neurofibromatosis (NF1) promoter interferes with binding of CREB and SP1 transcription factors.
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Tumour suppressor genes and growth regulatory genes are frequent targets for methylation defects that can result in aberrant expression and mutagenesis. We have established a methylation map of the promoter region of the neurofibromatosis (NF1) gene and demonstrated functional sensitivity for methylation at specific sites for the SP1 and CRE binding (CREB) proteins in the NF1 regulatory region. We evaluated the methylation status of CpG dinucleotides within five promoter subregions in the human and mouse homologues of the neurofibromatosis (NF1) genes. Three 5' subregions were found to be consistently methylated in all the tissues analysed. In contrast, DNA methylation was absent in the vicinity of the transcription start site bounded by SP1 recognition sequences. Gelshift assays showed that methylation specifically inhibits the CREB transcription factor from binding to its recognition site at the NF1 transcription start site. Furthermore, SP1 elements within the NF1 promoter are methylation sensitive, particularly when methylation is present on the antisense strand. We propose that for NF1 as with several other tumour suppressor genes, CpG methylation occurs in a complex, site-specific manner with the maintenance of a methylation-free promoter region bounded by SP1 binding sites that allow an accessible promoter to be retained. When these SP1 boundaries are breached, methylation can sweep in, rendering the promoter inaccessible for specific methylation-sensitive transcription factors and leading to a loss of functional integrity of the methylation-free CpG island. (+info)
Haploinsufficiency for the neurofibromatosis 1 (NF1) tumor suppressor results in increased astrocyte proliferation.
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Individuals affected with neurofibromatosis 1 (NF1) harbor increased numbers of GFAP-immunoreactive cerebral astrocytes and develop astrocytomas that can lead to blindness and death. Mice heterozygous for a targeted Nf1 mutation (Nf1+/-) were employed as a model for the human disease to evaluate the hypothesis that reduced NF1 protein (neurofibromin) expression may confer a growth advantage for astrocytes, such that inactivation of only one NF1 allele is sufficient for abnormal astrocyte proliferation. Here, we report that Nf17+/- mice have increased numbers of cerebral astrocytes and increased astrocyte proliferation compared to wild-type littermates. Intriguingly, primary Nf1+/- astrocyte cultures failed to demonstrate a cell-autonomous growth advantage unless they were cocultured with C17 neuronal cells. This C17 neuronal cell-induced Nf1+/- increase in proliferation was blocked by MEK inhibition (PD98059), suggesting a p21-ras-dependent effect. Furthermore, mice heterozygous for a targeted mutation in another GAP molecule, p120-GAP, demonstrated no increases in cerebral astrocyte number. These findings suggest that reduced NF1 expression results in a cell context-dependent increase in astrocyte proliferation that may be sufficient for the development of astrocytic growth abnormalities in patients with NF1. (+info)
Mouse models of tumor development in neurofibromatosis type 1.
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Neurofibromatosis type 1 (NF1) is a prevalent familial cancer syndrome resulting from germ line mutations in the NF1 tumor suppressor gene. Hallmark features of the disease are the development of benign peripheral nerve sheath tumors (neurofibromas), which can progress to malignancy. Unlike humans, mice that are heterozygous for a mutation in Nf1 do not develop neurofibromas. However, as described here, chimeric mice composed in part of Nf1-/- cells do, which demonstrates that loss of the wild-type Nf1 allele is rate-limiting in tumor formation. In addition, mice that carry linked germ line mutations in Nf1 and p53 develop malignant peripheral nerve sheath tumors (MPNSTs), which supports a cooperative and causal role for p53 mutations in MPNST development. These two mouse models provide the means to address fundamental aspects of disease development and to test therapeutic strategies. (+info)
Mouse tumor model for neurofibromatosis type 1.
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Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder characterized by increased incidence of benign and malignant tumors of neural crest origin. Mutations that activate the protooncogene ras, such as loss of Nf1, cooperate with inactivating mutations at the p53 tumor suppressor gene during malignant transformation. One hundred percent of mice harboring null Nf1 and p53 alleles in cis synergize to develop soft tissue sarcomas between 3 and 7 months of age. These sarcomas exhibit loss of heterozygosity at both gene loci and express phenotypic traits characteristic of neural crest derivatives and human NF1 malignancies. (+info)
Absence of APOBEC-1 mediated mRNA editing in human carcinomas.
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The transgene expression of the catalytic subunit APOBEC-1 of the apo B mRNA editing enzyme-complex can cause hepatocellular carcinoma in mice and rabbits. It has been proposed that aberrant editing of mRNA may represent a novel oncogenic principle. This investigation aimed to define whether such aberrant hyperediting mediated by APOBEC-1 occurs in human carcinomas. Editing and hyperediting of apo B, NAT1 or NF1 mRNA was not identified in any of 28 resected tumor specimens, including hepatocellular, bile duct, gastric, colorectal, pancreatic adeno- and neuroendocrine, lung adeno-, medullary thyroid and breast carcinoma, soft tissue sarcoma and neuroblastoma. In most types of carcinoma, significant levels for full-length APOBEC-1 mRNA could not be detected. Low level expression of APOBEC-1 was found in colorectal and gastric carcinoma where most of the APOBEC-1 mRNA is inactivated by alternate splicing. The 'auxiliary' components of the apo B mRNA editing enzyme-complex are missing in many tumors including colorectal and gastric carcinoma, but are highly expressed in hepatocellular, lung adeno- and breast carcinoma all of which lack APOBEC-1. Taken together, either APOBEC-1 or the 'auxiliary' components of the apo B mRNA editing enzyme-complex or both are missing in human carcinomas resulting in the absence of mRNA editing. Currently, there is no evidence that aberrant editing mediated by APOBEC-1 contributes to the tumorigenesis of natural human carcinomas. (+info)
Mutations affecting mRNA splicing are the most common molecular defects in patients with neurofibromatosis type 1.
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Neurofibromatosis type 1 (NF1) is one of the most common inherited disorders in humans and is caused by mutations in the NF1 gene. To date, the majority of the reported NF1 mutations are predicted to result in protein truncation, but very few studies have correlated the causative NF1 mutation with its effect at the mRNA level. We have applied a whole NF1 cDNA screening methodology to the study of 80 unrelated NF1 patients and have identified 44 different mutations, 32 being novel, in 52 of these patients. Mutations were detected in 87% of the familial cases, but in 51% of the sporadic ones. At least 15 of the 80 NF1 patients (19%) had recurrent mutations. The study shows that in 50% of the patients in whom the mutations were identified, these resulted in splicing alterations. Most of the splicing mutations did not involve the conserved AG/GT dinucleotides of the splice sites. One frameshift, two nonsense and two missense mutations were also responsible for alterations in mRNA splicing. The location and type of mutation within the NF1 gene, and its putative effect at the protein level, do not indicate any relationship to any specific clinical feature of NF1. The high proportion of aberrant spliced transcripts detected in NF1 patients stresses the importance of studying mutations at both the genomic and RNA level. It is possible that part of the clinical variability in NF1 could be due to mutations affecting mRNA splicing, which is the most common molecular defect in NF1. (+info)
Genetic and biochemical evidence that haploinsufficiency of the Nf1 tumor suppressor gene modulates melanocyte and mast cell fates in vivo.
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Neurofibromatosis type 1 (NF1) is a common autosomal-dominant disorder characterized by cutaneous neurofibromas infiltrated with large numbers of mast cells, melanocyte hyperplasia, and a predisposition to develop malignant neoplasms. NF1 encodes a GTPase activating protein (GAP) for Ras. Consistent with Knudson's "two hit" model of tumor suppressor genes, leukemias and malignant solid tumors in NF1 patients frequently demonstrate somatic loss of the normal NF1 allele. However, the phenotypic and biochemical consequences of heterozygous inactivation of Nf1 are largely unknown. Recently neurofibromin, the protein encoded by NF1, was shown to negatively regulate Ras activity in Nf1-/- murine myeloid hematopoietic cells in vitro through the c-kit receptor tyrosine kinase (dominant white spotting, W). Since the W and Nf1 locus appear to function along a common developmental pathway, we generated mice with mutations at both loci to examine potential interactions in vivo. Here, we show that haploinsufficiency at Nf1 perturbs cell fates in mast cells in vivo, and partially rescues coat color and mast cell defects in W(41) mice. Haploinsufficiency at Nf1 also increased mast cell proliferation, survival, and colony formation in response to Steel factor, the ligand for c-kit. Furthermore, haploinsufficiency was associated with enhanced Ras-mitogen-activated protein kinase activity, a major downstream effector of Ras, via wild-type and mutant (W(41)) c-kit receptors. These observations identify a novel interaction between c-kit and neurofibromin in vivo, and offer experimental evidence that haploinsufficiency of Nf1 alters both cellular and biochemical phenotypes in two cell lineages that are affected in individuals with NF1. Collectively, these data support the emerging concept that heterozygous inactivation of tumor suppressor genes may have profound biological effects in multiple cell types. (+info)