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The neurofibromatosis 2 (NF2) tumor suppressor gene encodes an intracellular membrane-associated protein, called merlin (or schwannomin), that belongs to the band 4.1 family of cytoskeleton-associated proteins. Inactivating NF2 mutations occur in several sporadic tumor types and have been linked to the NF2 disease, whose hallmark is the development of bilateral Schwann cell tumors (schwannomas) of the eighth cranial nerve. Two major alternatively spliced NF2 variants are expressed in normal tissues: 'NF2-17' lacking exon 16 and 'NF2-16' that contains exon 16 and encodes a merlin protein truncated at the C-terminus. We report that overexpression of NF2-17 in rat schwannoma cells inhibits their growth in vitro and in vivo, while NF2-16 fails to influence schwannoma growth. Tumor growth inhibition by merlin depends on an interdomain association occurring either in cis or in trans between the N- and C-termini. This association does not occur in the truncated NF2-16 protein nor in a mutant NF2-17 protein lacking C-terminal sequences. These data indicate that merlin has a unique mechanism of tumor suppression, inhibiting cell proliferation via self-association.

Neurofibromatosis 1 (NF1) is a common autosomal dominant disorder in which affected individuals develop benign and malignant tumors as well as non-tumor-related abnormalities, such as seizures and learning disabilities. Here, we report an NF1 isoform arising from the alternative splicing of exon 9a with predominant central nervous system (CNS) expression. Exon 9a expression is enriched in neurons of the forebrain, specifically septum, striatum, cortex, hippocampus and olfactory bulb with significantly less expression in brainstem, cerebellum and spinal cord. This pattern of NF1 exon 9a expression correlates with the postnatal maturation of these neurons and suggests a role for NF1 in neuronal differentiation.

Tumor suppressor genes encode proteins involved in growth regulation in differentiating and proliferating cells. Previous work from our laboratory has demonstrated that the neurofibromatosis 1 (NF1) tumor suppressor gene is dramatically upregulated in astrocytes stimulated with dibutyryl cyclic AMP and proinflammatory cytokines. To explore the possibility that the NF1 gene product, neurofibromin, plays a role in the reactive gliosis seen in response to cerebral ischemia, expression of NF1 was examined in both focal and global models of rat cerebral ischemia. In this report, we demonstrate the increased expression of both neurofibromin and glial fibrillary acidic protein (GFAP) in astrocytes surrounding areas of focal ischemia. Similar increases in neurofibromin and GFAP immunoreactivity were also observed in reactive astrocytes in the hippocampal region in a global model of ischemia. These results suggest a novel role for the NF1 tumor suppressor gene in growth regulatory pathways involved in cellular remodeling and in response to injury.

The neurofibromatosis type 1 (NF1) gene encodes a tumor-suppressor protein termed neurofibromin, which, in adults, is expressed predominantly in neurons, Schwann cells, and the adrenal medulla. Loss of NF1 gene expression has been reported in Schwann cell tumors (neurofibrosarcomas) from patients with NF1 as well as in malignant melanomas and neuroblastomas from patients without NF1. Previously, we demonstrated the lack of neurofibromin expression in six pheochromocytomas from patients with NF1, supporting the idea that neurofibromin might be an essential regulator of cell growth in these cells. To determine whether NF1 gene expression is similarly altered in pheochromocytomas from patients without NF1, we examined 20 pheochromocytomas for the presence of NF1 RNA and neurofibromin by reverse-transcribed polymerase chain reaction (RT-PCR) and immunohistochemistry, respectively. Reduced or absent NF1 gene expression was documented in 7 of these 20 tumors (35%) including 1 of 4 sporadic tumors, 3 of 10 tumors from patients with multiple endocrine neoplasia (MEN) 2A, 2 of 4 tumors from patients with MEN2B, and 1 of 2 tumors from patients with von Hippel-Lindau syndrome. In addition, most of these tumors expressed predominantly the type 1 NF1 isoform (75% type 1 NF1 isoform expression) as opposed to other neural crest-derived tissues such as adrenal gland and Schwann cells, which express predominantly type 2 NF1. This type 1 isoform predominance was also observed in the rat pheochromocytoma PC12 cell line, suggesting that this change in isoform expression may be associated with the genesis of these tumors.(ABSTRACT TRUNCATED AT 250 WORDS)

The neurofibromatosis 2 (NF2) gene product, merlin, encodes a 595 amino acid protein with sequence similarity to a family of proteins linking cell membrane proteins to the cytoskeleton. Two isoforms of merlin have been described which differ by the presence (type 2 merlin) or absence (type 1 merlin) of exon 16 sequences inserted into the extreme carboxyl terminus of the protein. To determine the role of this important negative growth regulator during normal embryonic development, the expression of these two merlin isoforms was examined at representative stages of rat embryogenesis and in adult tissues. Partial sequence analysis of the rat merlin gene demonstrated striking amino acid identity to the published mouse and human merlin gene sequences. In situ hybridization and RT-PCR analyses demonstrated that rat merlin is widely expressed during embryogenesis and early postnatal life in most tissues but becomes restricted to the brainstem, cerebellum, dorsal root ganglia, spinal cord, adrenal gland and testis in adult animals. The elucidation of the pattern of merlin gene expression in adult and embryonic tissues provides the foundations for future studies aimed at determining the function(s) of this protein during cell differentiation and embryonic development.

Neurofibromatosis 2 (NF2) is an inherited cancer syndrome resulting from mutations in the NF2 tumor suppressor gene. Analysis of NF2 mutations has revealed some general genotype-phenotype correlations. Severe disease has been associated with mutations that produce a premature termination while more mild disease has been associated with missense mutations. Here, we provide experimental proof for these genotype-phenotype correlations by demonstrating that nonsense mutations fail to produce stable merlin protein while missense mutations result in the generation of merlin proteins defective in negative growth regulation. This inability to suppress cell growth may result from defects in the function of merlin at several levels, including failure to form an intramolecular complex. Based on these findings, we propose a model for merlin growth suppression that provides a framework for analyzing NF2 patient mutations and merlin function.

The neurofibromatosis 1 (NF1) gene product, neurofibromin, is a tumor suppressor gene product capable of inhibiting the growth of cells in culture. If neurofibromin suppresses cell growth by arresting cells in G0 or G1, its expression might be regulated in a cell cycle-dependent fashion. In this study, we demonstrate that RAT-1A fibroblasts arrested in G0/G1 by serum starvation and then released to progress through the cell cycle do not demonstrate significant changes in NF1 expression. However, when arrested in G0/G1 by contact inhibition, NF1 expression in these cells is reversibly upregulated within 72 h, suggesting that NF1 expression is a late event associated with cell growth arrest which may contribute to the maintenance of the differentiated state.

The neurofibromatosis type 1 (NFl) gene encodes a tumor suppressor protein, termed neurofibromin, which is expressed pre- dominantly in neurons, Schwann cells, oligoden- drocytes, and leukocytes. There are at least three isoforms of neurofibromin produced by the alter- native use of exons 23a and 48a. Previously we described the identification of an NFl mRNA iso- form containing an additional 54 nucleotides from exon 48a (type 3 NFl) in human skeletal, cardiac and smooth muscle tissues by reverse-transcribed (RT)-PCR. To extend our initial observations, we have produced high titer chicken IgY antibodies which specifically recognize this muscle-specific neurofibromin isoform. An NF1 cDNA was gener- ated containing human exon 48a sequences and expressed as a fusion protein in bacteria. The muscle-specific neurofibromin antibodies de- tected this exon 48a fusion protein by Western im- munoblotting. Immunoprecipitation using these type 3 neurofibromin antibodies also specifically detected a 250 kDa protein in human and rat mus- cle tissues. Type 3 neurofibromin was found in rat heart and muscle, but not in liver, brain, kidney or spleen with levels of expression declining after postnatal day 7. Expression of total NFl RNA dur- ing rat embryonic development was detected at high levels in El5 heart, tongue, and limb bud. In addition, using type 2 neurofibromin-specific an- tibodies, the existence of a fourth isoform of neu- rofibromin (type 4 neurofibromin) containing both exon 23a and 48a sequences was demon- strated in rat heart muscle tissues. The identifica- tion of two muscle-specific isoforms of neurofibro- min expands our definition of this important tumor suppressor protein and suggests additional roles for neurofibromin in muscle development and differentiation. o 1995 WiIey-Liss, Inc.

The neurofibromatosis type 1 (NF1) gene encodes a tumor suppressor protein, neurofibromin, which is expressed at high levels in Schwann cells and other adult tissues. Loss of NF1 expression has been reported in Schwann cell tumors (neurofibrosarcomas) from patients with NF1 and its loss is associated with increased proliferation of these cells. In this report, we describe downregulation of NF1 expression in a single spinal schwannoma from an individual without clinical features of neurofibromatosis type 1 or 2. Barely detectable expression of NF1 RNA was found in this tumor by in situ hybridization using an NF1-specific riboprobe as well as by Northern blot and reverse-transcribed (RT)-PCR analysis. In Schwann cells cultured from this schwannoma, abundant expression of NF1 RNA could be detected by Northern blot and RT-PCR analysis. These results suggest that, in some tumors, expression of NF1 may be downregulated by factors produced within the tumor and may represent a novel mechanism for inactivating these growth suppressing genes and allowing for increased cell proliferation in tumors.

Summary Transcription of the antiphagocytic M protein in the group A streptococcus (Streptococcus pyogenes) is environmentally regulated in response to CO2 and requires Mry, a trans-acting positive regulatory protein. We have examined the role of Mry in environmental regulation by analysing the factors that regulate expression of the gene that encodes Mry (mry). By employing a strategy that utilizes integrational plasmids, it was found that expression of mry requires the participation of DNA sequences that extend 473 base pairs upstream of the Mry coding region. Transcription of mry, as analysed in S1 nuclease protection assays, is initiated from two separate promoters located within this extended regulatory region. Construction and analysis of transcriptional fusions between the mry promoters and a promoterless chloramphenicol acetyltransferase gene demonstrated that mry is autoregulated and environmentally regulated in response to the level of CO2. These data suggest a model for the regulation of virulence in S. pyogenes where positive transcriptional control of mry in response to environmental stimuli regulates the expression of the M protein.