RESUMO
AIMS: The aim of this study was to assess associations between neurological biomarkers and distal sensorimotor polyneuropathy (DSPN). MATERIALS AND METHODS: Cross-sectional analyses were based on 1032 participants aged 61-82 years from the population-based KORA F4 survey, 177 of whom had DSPN at baseline. The prevalence of type 2 diabetes was 20%. Prospective analyses used data from 505 participants without DSPN at baseline, of whom 125 had developed DSPN until the KORA FF4 survey. DSPN was defined based on the examination part of the Michigan Neuropathy Screening Instrument. Serum levels of neurological biomarkers were measured using proximity extension assay technology. Associations between 88 biomarkers and prevalent or incident DSPN were estimated using Poisson regression with robust error variance and are expressed as risk ratios (RR) and 95% CI per 1-SD increase. Results were adjusted for multiple confounders and multiple testing using the Benjamini-Hochberg procedure. RESULTS: Higher serum levels of CTSC (cathepsin C; RR [95% CI] 1.23 (1.08; 1.39), pB-H = 0.044) and PDGFRα (platelet-derived growth factor receptor A; RR [95% CI] 1.21 (1.08; 1.35), pB-H = 0.044) were associated with prevalent DSPN in the total study sample. CDH3, JAM-B, LAYN, RGMA and SCARA5 were positively associated with DSPN in the diabetes subgroup, whereas GCP5 was positively associated with DSPN in people without diabetes (all pB-H for interaction <0.05). None of the biomarkers showed an association with incident DSPN (all pB-H>0.05). CONCLUSIONS: This study identified multiple novel associations between neurological biomarkers and prevalent DSPN, which may be attributable to functions of these proteins in neuroinflammation, neural development and myelination.
Assuntos
Biomarcadores , Humanos , Biomarcadores/sangue , Masculino , Feminino , Idoso , Estudos Transversais , Pessoa de Meia-Idade , Estudos Prospectivos , Idoso de 80 Anos ou mais , Polineuropatias/sangue , Polineuropatias/epidemiologia , Polineuropatias/diagnóstico , Polineuropatias/etiologia , Seguimentos , Neuropatias Diabéticas/epidemiologia , Neuropatias Diabéticas/sangue , Neuropatias Diabéticas/diagnóstico , Neuropatias Diabéticas/etiologia , Prognóstico , Diabetes Mellitus Tipo 2/epidemiologia , Diabetes Mellitus Tipo 2/sangue , PrevalênciaRESUMO
The Ras-Erk pathway is frequently overactivated in human tumors. Neurofibromatosis types 1 and 2 (NF1, NF2) are characterized by multiple tumors of Schwann cell origin. The NF1 tumor suppressor neurofibromin is a principal Ras-GAP accelerating Ras inactivation, whereas the NF2 tumor suppressor merlin is a scaffold protein coordinating multiple signaling pathways. We have previously reported that merlin interacts with Ras and p120RasGAP. Here, we show that merlin can also interact with the neurofibromin/Spred1 complex via merlin-binding sites present on both proteins. Further, merlin can directly bind to the Ras-binding domain (RBD) and the kinase domain (KiD) of Raf1. As the third component of the neurofibromin/Spred1 complex, merlin cannot increase the Ras-GAP activity; rather, it blocks Ras binding to Raf1 by functioning as a 'selective Ras barrier'. Merlin-deficient Schwann cells require the Ras-Erk pathway activity for proliferation. Accordingly, suppression of the Ras-Erk pathway likely contributes to merlin's tumor suppressor activity. Taken together, our results, and studies by others, support targeting or co-targeting of this pathway as a therapy for NF2 inactivation-related tumors.
Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Neurofibromina 1/metabolismo , Neurofibromina 2/metabolismo , Proteínas Repressoras/metabolismo , Células de Schwann/patologia , Proteínas ras/metabolismo , Animais , Células Cultivadas , MAP Quinases Reguladas por Sinal Extracelular/genética , Neurofibromina 1/genética , Neurofibromina 2/genética , Ratos , Proteínas Repressoras/genética , Células de Schwann/metabolismo , Proteínas ras/genéticaRESUMO
Astrocytes are increasingly being recognized as contributors to physiological brain function and behavior. Astrocytes engage in glia-synaptic interactions through peripheral astrocyte processes, thus modulating synaptic signaling, for example, by handling glutamate removal from the synaptic cleft and (re)provision to axonal terminals. Peripheral astrocyte processes are ultrafine membrane protrusions rich in the membrane-to-actin cytoskeleton linker Ezrin, an essential component of in vitro filopodia formation and in vivo peripheral astrocyte process motility. Consequently, it has been postulated that Ezrin significantly contributes to neurodevelopment as well as astrocyte functions within the adult brain. However, while Ezrin has been studied in vitro within cultured primary astrocytes, in vivo studies on the role of Ezrin in astrocytes remain to be conducted and consequences of its depletion to be studied. Here, we investigated consequences of Ezrin deletion in the mouse brain starting from early neuronal specification. While Ezrin knockout did not impact prenatal cerebral cortex development, behavioral phenotyping depicted reduced exploratory behavior. Starting with postnatal appearance of glia cells, Ezrin was verified to remain predominantly expressed in astrocytes. Proteome analysis of Ezrin deficient astrocytes revealed alterations in glutamate and ion homeostasis, metabolism and cell morphology - important processes for synaptic signal transmission. Notably, Ezrin deletion in astrocytes provoked (GFAP) glial fibrillary acidic protein upregulation - a marker of astrocyte activation and reactive astrogliosis. However, this spontaneous, reactive astrogliosis exhibited proteome changes distinct from ischemic-induced reactive astrogliosis. Moreover, in experimental ischemic stroke, Ezrin knockout mice displayed reduced infarct volume, indicating a protective effect of the Ezrin deletion-induced changes and astrogliosis.
Assuntos
Astrócitos , Gliose , Animais , Astrócitos/metabolismo , Proteínas do Citoesqueleto , Feminino , Proteína Glial Fibrilar Ácida/metabolismo , Gliose/metabolismo , Ácido Glutâmico/metabolismo , Camundongos , Camundongos Knockout , Fenótipo , Gravidez , Proteoma/metabolismo , Regulação para CimaRESUMO
Repeated injections of psychomotor stimulants like amphetamine (AMPH) to rodents can induce behavioral sensitization, which represents a long-lasting craving that is usually observed in human addicts. Behavioral sensitization is characteristically maintained for a long duration, accompanied by structural plasticity in some brain areas involved in reward circuitry. For example, it increased dendritic spine densities in the nucleus accumbens (NAcc), which is considered to reflect neurophysiological changes at this site, leading to addictive behaviors. The ezrin, radixin, and moesin (ERM) proteins regulate spine maturity by modifying their phosphorylation at the C-terminal region. We previously showed that ERM phosphorylation is reduced by AMPH in the NAcc core, suggesting that ERM-mediated spine changes at this site might be associated with AMPH sensitization. To test this hypothesis, we administered AMPH to rats according to a sensitization development schedule, with lentivirus encoding a phosphomimetic pseudo-active mutant of radixin (Rdx T564D) in the NAcc core, and examined dendritic spines at this site. We found that compared to acute AMPH, AMPH sensitization increased thin spine density with a similar ratio of filopodia-like to mature thin spines. However, with Rdx T564D, the density of thin spines increased, with augmented filopodia-like thin spines, resulting in no AMPH sensitization. These results indicate that Rdx T564D forces thin spines to immaturity and thereby inhibits AMPH sensitization, for which an increase in mature thin spines is normally necessary. These findings provide significant clues to our understanding of the role of dendritic spines in mediating the development of psychomotor stimulant addiction.
Assuntos
Anfetamina , Estimulantes do Sistema Nervoso Central , Anfetamina/farmacologia , Animais , Encéfalo , Estimulantes do Sistema Nervoso Central/farmacologia , Núcleo Accumbens , RatosRESUMO
Osteosarcoma is the most common type of pediatric bone tumor. Despite great advances in chemotherapy during the past decades, the survival rates of osteosarcoma patients remain unsatisfactory. Drug resistance is one of the main reasons, leading to treatment failure and poor prognosis. Previous reports correlated expression of cluster of differentiation 44 (CD44) with drug resistance and poor survival of osteosarcoma patients, however the underlying mechanisms are poorly defined. Here, we investigated the role of CD44 in the regulation of drug chemoresistance, using osteosarcoma cells isolated from mice carrying a mutation of the tumor suppressor neurofibromatosis type 2 (Nf2) gene. CD44 expression was knocked-down in the cells using CRISPR/Cas9 approach. Subsequently, CD44 isoforms and mutants were re-introduced to investigate CD44-dependent processes. Sensitivity to doxorubicin was analyzed in the osteosarcoma cells with modified CD44 expression by immunoblot, colony formation- and WST-1 assay. To dissect the molecular alterations induced by deletion of Cd44, RNA sequencing was performed on Cd44-positive and Cd44-negative primary osteosarcoma tissues isolated from Nf2-mutant mice. Subsequently, expression of candidate genes was evaluated by quantitative reverse transcription PCR (qRT-PCR). Our results indicate that CD44 increases the resistance of osteosarcoma cells to doxorubicin by up-regulating the levels of multidrug resistance (MDR) 1 protein expression, and suggest the role of proteolytically released CD44 intracellular domain, and hyaluronan interactions in this process. Moreover, high throughput sequencing analysis identified differential regulation of several apoptosis-related genes in Cd44-positive and -negative primary osteosarcomas, including p53 apoptosis effector related to PMP-22 (Perp). Deletion of Cd44 in osteosarcoma cells led to doxorubicin-dependent p53 activation and a profound increase in Perp mRNA expression. Overall, our results suggest that CD44 might be an important regulator of drug resistance and suggest that targeting CD44 can sensitize osteosarcoma to standard chemotherapy.
Assuntos
Neoplasias Ósseas , Osteossarcoma , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Animais , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/genética , Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Doxorrubicina/farmacologia , Doxorrubicina/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Receptores de Hialuronatos/genética , Receptores de Hialuronatos/metabolismo , Camundongos , Osteossarcoma/tratamento farmacológico , Osteossarcoma/genética , Osteossarcoma/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Merlin is a versatile tumor suppressor protein encoded by the NF2 gene. Several lines of evidence suggest that Merlin exerts its tumor suppressor activity, at least in part, by forming an inhibitory complex with cluster of differentiation 44 (CD44). Consistently, numerous NF2 mutations in cancer patients are predicted to perturb the interaction of Merlin with CD44. We hypothesized that disruption of the Merlin-CD44 complex through loss of Merlin, unleashes putative tumor- or metastasis-promoting functions of CD44. To evaluate the relevance of the Merlin-CD44 interaction in vivo, we compared tumor growth and progression in Cd44-positive and Cd44-negative Nf2-mutant mice. Heterozygous Nf2-mutant mice were prone to developing highly metastatic osteosarcomas. Importantly, while the absence of the Cd44 gene had no effect on the frequency of primary osteosarcoma development, it strongly diminished osteosarcoma metastasis formation in the Nf2-mutant mice. In vitro assays identified transendothelial migration as the most prominent cellular phenotype dependent on CD44. Adhesion to endothelial cells was blocked by interfering with integrin α4ß1 (very late antigen-4, VLA-4) on osteosarcoma cells and CD44 upregulated levels of integrin VLA-4 ß1 subunit. Among other putative functions of CD44, which may contribute to the metastatic behavior, the passage through the endothelial cells also appears to be critical in vivo, as CD44 significantly promoted formation of lung metastasis upon intravenous injection of osteosarcoma cells into immunocompromised mice. Altogether, our results strongly suggest that CD44 plays a metastasis-promoting role in the absence of Merlin.
Assuntos
Neoplasias Ósseas/genética , Receptores de Hialuronatos/metabolismo , Neoplasias Pulmonares/genética , Neurofibromina 2/genética , Osteossarcoma/genética , Animais , Neoplasias Ósseas/patologia , Osso e Ossos/patologia , Adesão Celular/genética , Linhagem Celular Tumoral/transplante , Proliferação de Células/genética , Modelos Animais de Doenças , Progressão da Doença , Humanos , Receptores de Hialuronatos/genética , Pulmão/patologia , Neoplasias Pulmonares/secundário , Masculino , Camundongos , Camundongos Knockout , Osteossarcoma/secundárioRESUMO
Schwannomatosis is the third form of neurofibromatosis and characterized by the occurrence of multiple schwannomas. The most prominent symptom is chronic pain. We aimed to test whether pain in schwannomatosis might be caused by small-fiber neuropathy. Twenty patients with schwannomatosis underwent neurological examination and nerve conduction studies. Levels of pain perception as well as anxiety and depression were assessed by established questionnaires. Quantitative sensory testing (QST) and laser-evoked potentials (LEP) were performed on patients and controls. Whole-body magnetic resonance imaging (wbMRI) and magnetic resonance neurography (MRN) were performed to quantify tumors and fascicular nerve lesions; skin biopsies were performed to determine intra-epidermal nerve fiber density (IENFD). All patients suffered from chronic pain without further neurological deficits. The questionnaires indicated neuropathic symptoms with significant impact on quality of life. Peripheral nerve tumors were detected in all patients by wbMRI. MRN showed additional multiple fascicular nerve lesions in 16/18 patients. LEP showed significant faster latencies compared to normal controls. Finally, IENFD was significantly reduced in 13/14 patients. Our study therefore indicates the presence of small-fiber neuropathy, predominantly of unmyelinated C-fibers. Fascicular nerve lesions are characteristic disease features that are associated with faster LEP latencies and decreased IENFD. Together these methods may facilitate differential diagnosis of schwannomatosis.
Assuntos
Fibras Nervosas/patologia , Neoplasias do Sistema Nervoso/etiologia , Neuralgia/patologia , Neurilemoma/complicações , Neurofibromatoses/complicações , Neoplasias Cutâneas/complicações , Adulto , Idoso , Dor Crônica , Feminino , Humanos , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Mutação , Neoplasias do Sistema Nervoso/diagnóstico por imagem , Neuralgia/etiologia , Neoplasias do Sistema Nervoso Periférico/diagnóstico por imagem , Neoplasias do Sistema Nervoso Periférico/etiologia , Fatores de Transcrição/genética , Imagem Corporal TotalRESUMO
The neurofibromatosis type 1 (NF1) tumor suppressor gene is one of the most frequently mutated genes in human tumors. Research on the NF1 proteins has been partially hindered by the difficulties in cloning and propagating the full-length coding cDNAs. We have now established a condition for propagating the natural open reading frames (ORFs) and have assembled the ORFs for human NF1 type 1 and 2 isoforms. Furthermore, we were able to eliminate the cDNA cloning toxicity by introducing a mini-intron. These NF1 minigenes were expressed similarly to the intronless version and could be used to purify full-length NF1 proteins. The NF1 isoforms expressed from the minigenes showed Ras-GAP activity in vivo and in vitro, while the type 1 was more potent. Our constructs expand currently available full-length NF1 constructs and should be valuable tools in expediting the understanding of NF1, particularly the isoform-specific functions and regulation.
Assuntos
Neurofibromatose 1/genética , Neurofibromina 1/genética , Isoformas de Proteínas/genética , Sequência de Aminoácidos/genética , Animais , Clonagem Molecular , DNA Complementar/genética , Regulação da Expressão Gênica/genética , Genes Supressores de Tumor , Humanos , Íntrons/genética , Mamíferos , Mutação , Neurofibromatose 1/patologia , Fases de Leitura AbertaRESUMO
Heat shock factor 1 (HSF1) generally exhibits its properties under stress conditions. In tumors, HSF1 has a pleiotropic feature in regulating growth, survival, and aggressiveness of cancer cells. In this study, we found HSF1 was increased in colorectal cancer (CRC) and had a positive correlation with shorter disease-free survival (DFS). Knockdown of HSF1 in CRC cells attenuated their growth while inhibiting mTOR activation and glutamine metabolism. HSF1 inhibited the expression of microRNA137 (MIR137), which targeted GLS1 (glutaminase 1), thus stimulating GLS1 protein expression to promote glutaminolysis and mTOR activation. HSF1 bound DNA methyltransferase DNMT3a and recruited it to the promoter of lncRNA MIR137 host gene (MIR137HG), suppressing the generation of primary MIR137. The chemical inhibitor of HSF1 also reduced cell growth, increased apoptosis, and impaired glutamine metabolism in vitro. Moreover, both chemical inhibition and genetic knockout of HSF1 succeeded in increasing MIR137 expression, reducing GLS1 expression, and alleviating colorectal tumorigenesis in azoxymethane (AOM)/dextran sulfate sodium (DSS) mice. In conclusion, HSF1 expression was increased and associated with poor prognosis in CRC. By recruiting DNMT3a to suppress the expression of MIR137 that targets GLS1 mRNA, HSF1 stimulated GLS1-dependent mTOR activation to promote colorectal carcinogenesis. Therefore, targeting HSF1 to attenuate glutaminolysis and mTOR activation could be a promising approach for CRC treatment.
Assuntos
Carcinogênese/genética , Neoplasias Colorretais/genética , Epigênese Genética/genética , Glutaminase/genética , Fatores de Transcrição de Choque Térmico/genética , Serina-Treonina Quinases TOR/genética , Animais , Apoptose/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , DNA (Citosina-5-)-Metiltransferases/genética , Regulação Neoplásica da Expressão Gênica/genética , Resposta ao Choque Térmico/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Regiões Promotoras Genéticas/genética , Transdução de Sinais/genéticaRESUMO
Protein kinases, including the serine/threonine kinase Akt, mediate manifold bioactivities of vitamin A, although the mechanisms behind the sustained kinase activation are diffuse. To investigate the role of cellular lipids as targetable factors in Akt signaling, we combined mass spectrometry-based lipidomics with immunologic detection of Akt (Ser473) phosphorylation. A screening campaign revealed retinol (vitamin A alcohol) and all-trans retinoic acid (vitamin A acid) (RA) as hits that time-dependently (≥24 h) deplete phosphatidylcholine-bound polyunsaturated fatty acids (PUFA-PCs) from NIH-3T3 mouse fibroblasts while inducing Akt activation (EC50 ≈ 0.1-1 µM). Other mitogenic and stress-regulated kinases were hardly affected. Organized in a coregulated phospholipid subcluster, PUFA-PCs compensated for the RA-induced loss of cellular PUFA-PCs and diminished Akt activation when supplemented. The counter-regulation of phospholipids and Akt by RA was mimicked by knockdown of lysophosphatidylcholine acyltransferase-3 or the selective retinoid X receptor (RXR) agonist bexarotene and prevented by the selective RXR antagonist Hx531. Treatment of mice with retinol decreased the tissue ratio of PUFA-PC and enhanced basal Akt activation preferentially in brain, which was attributed to astrocytes in dissociated cortical cultures. Together, our findings show that RA regulates the long-term activation of Akt by changes in the phospholipid composition.-Pein, H., Koeberle, S. C., Voelkel, M., Schneider, F., Rossi, A., Thürmer, M., Loeser, K., Sautebin, L., Morrison, H., Werz, O., Koeberle, A. Vitamin A regulates Akt signaling through the phospholipid fatty acid composition.
Assuntos
Ácidos Graxos/metabolismo , Fosfolipídeos/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tretinoína/farmacologia , Vitamina A/metabolismo , Animais , Proliferação de Células/efeitos dos fármacos , Camundongos , Fosforilação , Receptores X de Retinoides/metabolismo , Tretinoína/metabolismo , Vitamina A/farmacologiaRESUMO
Organized and hosted by the Children's Tumor Foundation (CTF), the Neurofibromatosis (NF) conference is the premier annual gathering for clinicians and researchers interested in neurofibromatosis type 1 (NF1), neurofibromatosis type 2 (NF2), and schwannomatosis (SWN). The 2016 edition constituted a blend of clinical and basic aspects of NF research that helped in clarifying different advances in the field. The incorporation of next generation sequencing is changing the way genetic diagnostics is performed for NF and related disorders, providing solutions to problems like genetic heterogeneity, overlapping clinical manifestations, or the presence of mosaicism. The transformation from plexiform neurofibroma (PNF) to malignant peripheral nerve sheath tumor (MPNST) is being clarified, along with new management and treatments for benign and premalignant tumors. Promising new cellular and in vivo models for understanding the musculoskeletal abnormalities in NF1, the development of NF2 or SWN associated schwannomas, and clarifying the cells that give rise to NF1-associated optic pathway glioma were presented. The interaction of neurofibromin and SPRED1 was described comprehensively, providing functional insight that will help in the interpretation of pathogenicity of certain missense variants identified in NF1 and Legius syndrome patients. Novel promising imaging techniques are being developed, as well as new integrative and holistic management models for patients that take into account psychological, social, and biological factors. Importantly, new therapeutic approaches for schwannomas, meningiomas, ependymomas, PNF, and MPNST are being pursued. This report highlights the major advances that were presented at the 2016 CTF NF conference.
Assuntos
Neurilemoma/diagnóstico , Neurilemoma/etiologia , Neurofibromatoses/diagnóstico , Neurofibromatoses/etiologia , Neurofibromatose 1/diagnóstico , Neurofibromatose 1/etiologia , Neurofibromatose 2/diagnóstico , Neurofibromatose 2/etiologia , Neoplasias Cutâneas/diagnóstico , Neoplasias Cutâneas/etiologia , Animais , Gerenciamento Clínico , Modelos Animais de Doenças , Estudos de Associação Genética , Predisposição Genética para Doença , Humanos , Técnicas de Diagnóstico Molecular , Neurilemoma/terapia , Neurofibromatoses/terapia , Neurofibromatose 1/terapia , Neurofibromatose 2/terapia , Neoplasias Cutâneas/terapia , Pesquisa Translacional BiomédicaRESUMO
Schwannomatosis is a genetic disorder characterized by the occurrence of multiple peripheral schwannomas. Segmental schwannomatosis is diagnosed when schwannomas are restricted to 1 extremity and is thought to be caused by genetic mosaicism. We studied 5 patients with segmental schwannomatosis through microstructural magnetic resonance neurography and mutation analysis of NF2, SMARCB1, and LZTR1. In 4 of 5 patients, subtle fascicular nerve lesions were detected in clinically unaffected extremities. Two patients exhibited LZTR1 germline mutations. This appears contrary to a simple concept of genetic mosaicism and suggests more complex and heterogeneous mechanisms underlying the phenotype of segmental schwannomatosis than previously thought. Ann Neurol 2016;80:625-628.
Assuntos
Extremidade Inferior/diagnóstico por imagem , Neurilemoma/diagnóstico por imagem , Neurilemoma/genética , Neurofibromatoses/diagnóstico por imagem , Neurofibromatoses/genética , Nervos Periféricos/diagnóstico por imagem , Neoplasias Cutâneas/diagnóstico por imagem , Neoplasias Cutâneas/genética , Fatores de Transcrição/genética , Extremidade Superior/diagnóstico por imagem , Adulto , Análise Mutacional de DNA , Feminino , Mutação em Linhagem Germinativa , Humanos , Extremidade Inferior/inervação , Plexo Lombossacral/diagnóstico por imagem , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Extremidade Superior/inervaçãoRESUMO
Ectodomain shedding of transmembrane precursor proteins generates numerous life-essential molecules, such as epidermal growth factor receptor ligands. This cleavage not only releases the regulatory growth factor, but it is also the required first step for the subsequent processing by γ-secretase and the release of gene regulatory intracellular fragments. Signaling within the cell modifies the cytoplasmic tails of substrates, a step important in starting the specific and regulated cleavage of a large number of studied substrates. Ectodomain cleavage occurs, however, on the outside of the plasma membrane and is carried out by membrane-bound metalloproteases. How the intracellular domain modification communicates with the ectodomain of the substrate to allow for cleavage to occur is unknown. Here, we show that homodimerization of a cluster-of-differentiation-44 or of pro-neuregulin-1 monomers represents an essential pre-condition for their regulated ectodomain cleavage. Both substrates are associated with their respective metalloproteases under both basal or cleavage-stimulated conditions. These interactions only turn productive by specific intracellular signal-induced intracellular domain modifications of the substrates, which in turn regulate metalloprotease access to the substrates' ectodomain and cleavage. We propose that substrate intracellular domain modification induces a relative rotation or other positional change of the dimerization partners that allow metalloprotease cleavage in the extracellular space. Our findings fill an important gap in understanding substrate-specific inside-out signal transfer along cleaved transmembrane proteins and suggest that substrate dimerization (homo- or possibly heterodimerization) might represent a general principle in ectodomain shedding.
Assuntos
Receptores de Hialuronatos/metabolismo , Neuregulina-1/metabolismo , Proteínas ADAM/química , Proteínas ADAM/genética , Proteínas ADAM/metabolismo , Proteína ADAM10 , Proteína ADAM17 , Secretases da Proteína Precursora do Amiloide/química , Secretases da Proteína Precursora do Amiloide/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Bovinos , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Células Cultivadas , Células HEK293 , Humanos , Receptores de Hialuronatos/química , Receptores de Hialuronatos/genética , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Modelos Biológicos , Mutagênese Sítio-Dirigida , Células NIH 3T3 , Neuregulina-1/química , Neuregulina-1/genética , Multimerização Proteica , Estrutura Terciária de Proteína , Proteólise , Ratos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Especificidade por SubstratoRESUMO
Schwannomas are predominantly benign nerve sheath neoplasms caused by Nf2 gene inactivation. Presently, treatment options are mainly limited to surgical tumor resection due to the lack of effective pharmacological drugs. Although the mechanistic understanding of Nf2 gene function has advanced, it has so far been primarily restricted to Schwann cell-intrinsic events. Extracellular cues determining Schwann cell behavior with regard to schwannoma development remain unknown. Here we show pro-tumourigenic microenvironmental effects on Schwann cells where an altered axonal microenvironment in cooperation with injury signals contribute to a persistent regenerative Schwann cell response promoting schwannoma development. Specifically in genetically engineered mice following crush injuries on sciatic nerves, we found macroscopic nerve swellings in mice with homozygous nf2 gene deletion in Schwann cells and in animals with heterozygous nf2 knockout in both Schwann cells and axons. However, patient-mimicking schwannomas could only be provoked in animals with combined heterozygous nf2 knockout in Schwann cells and axons. We identified a severe re-myelination defect and sustained macrophage presence in the tumor tissue as major abnormalities. Strikingly, treatment of tumor-developing mice after nerve crush injury with medium-dose aspirin significantly decreased schwannoma progression in this disease model. Our results suggest a multifactorial concept for schwannoma formation-emphasizing axonal factors and mechanical nerve irritation as predilection site for schwannoma development. Furthermore, we provide evidence supporting the potential efficacy of anti-inflammatory drugs in the treatment of schwannomas.
Assuntos
Axônios/patologia , Neurilemoma/patologia , Células de Schwann/patologia , Nervo Isquiático/patologia , Microambiente Tumoral/fisiologia , Animais , Camundongos Transgênicos , Bainha de Mielina/patologia , Neurilemoma/genética , Neurofibromatose 2/genética , Microambiente Tumoral/genéticaRESUMO
Receptor tyrosine kinases participate in several signaling pathways through small G proteins such as Ras (rat sarcoma). An important component in the activation of these G proteins is Son of sevenless (SOS), which catalyzes the nucleotide exchange on Ras. For optimal activity, a second Ras molecule acts as an allosteric activator by binding to a second Ras-binding site within SOS. This allosteric Ras-binding site is blocked by autoinhibitory domains of SOS. We have reported recently that Ras activation also requires the actin-binding proteins ezrin, radixin, and moesin. Here we report the mechanism by which ezrin modulates SOS activity and thereby Ras activation. Active ezrin enhances Ras/MAPK signaling and interacts with both SOS and Ras in vivo and in vitro. Moreover, in vitro kinetic assays with recombinant proteins show that ezrin also is important for the activity of SOS itself. Ezrin interacts with GDP-Ras and with the Dbl homology (DH)/pleckstrin homology (PH) domains of SOS, bringing GDP-Ras to the proximity of the allosteric site of SOS. These actions of ezrin are antagonized by the neurofibromatosis type 2 tumor-suppressor protein merlin. We propose an additional essential step in SOS/Ras control that is relevant for human cancer as well as all physiological processes involving Ras.
Assuntos
Proteínas do Citoesqueleto/metabolismo , Guanosina Difosfato/metabolismo , Sistema de Sinalização das MAP Quinases , Neurofibromina 2/metabolismo , Proteína Oncogênica p21(ras)/metabolismo , Proteínas Son Of Sevenless/metabolismo , Animais , Proteínas do Citoesqueleto/genética , Guanosina Difosfato/genética , Humanos , Camundongos , Células NIH 3T3 , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Neurofibromina 2/genética , Proteína Oncogênica p21(ras)/genética , Proteínas Son Of Sevenless/genéticaRESUMO
Gain-of-function alterations in several components and modulators of the Ras-MAPK pathway lead to dysregulation of the pathway and cause a broad spectrum of autosomal dominant developmental disorders, collectively known as RASopathies. These findings demonstrate the importance of tight multilevel Ras regulation to safeguard signaling output and prevent aberrant activity. We have recently identified ezrin as a novel regulatory element required for Ras activation. Homozygosity mapping and exome sequencing have now revealed the first presumably disease-causing variant in the coding gene EZR in two siblings with a profound intellectual disability. Localization and membrane targeting of the altered ezrin protein appeared normal but molecular modeling suggested protein interaction surfaces to be disturbed. Functional analysis revealed that the altered ezrin protein is no longer able to bind Ras and facilitate its activation. Furthermore, expression of the altered ezrin protein in different cell lines resulted in abnormal cellular processes, including reduced proliferation and neuritogenesis, thus revealing a possible mechanism for its phenotype in humans. To our knowledge, this is the first report of an autosomal recessively inherited loss-of-function mutation causing reduced Ras activity and thus extends and complements the pathogenicity spectrum of known Ras-MAPK pathway disturbances.
Assuntos
Proteínas do Citoesqueleto/genética , Deficiência Intelectual/genética , Proteínas ras/metabolismo , Animais , Estudos de Casos e Controles , Proliferação de Células , Criança , Consanguinidade , Análise Mutacional de DNA , Exoma , Estudos de Associação Genética , Homozigoto , Humanos , Masculino , Camundongos , Mutação de Sentido Incorreto , Células NIH 3T3 , Linhagem , Ligação Proteica , Transporte ProteicoRESUMO
Brain-invasive growth of a subset of meningiomas is associated with less favorable prognosis. The molecular mechanisms causing invasiveness are only partially understood, however, the expression of matrix metalloproteinases (MMPs) has been identified as a contributing factor. We have previously found that loss of density enhanced phosphatase-1 (DEP-1, also designated PTPRJ), a transmembrane protein-tyrosine phosphatase, promotes meningioma cell motility and invasive growth in an orthotopic xenotransplantation model. We have now analyzed potential alterations of the expression of genes involved in motility control, caused by DEP-1 loss in meningioma cell lines. DEP-1 depleted cells exhibited increased expression of mRNA encoding MMP-9, and the growth factors EGF and FGF-2. The increase of MMP-9 expression in DEP-1 depleted cells was also readily detectable at the protein level by zymography. MMP-9 upregulation was sensitive to chemical inhibitors of growth factor signal transduction. Conversely, MMP-9 mRNA levels could be stimulated with growth factors (e.g. EGF) and inflammatory cytokines (e.g. TNFα). Increase of MMP-9 expression by DEP-1 depletion, or growth factor/cytokine stimulation qualitatively correlated with increased invasiveness in vitro scored as transmigration through matrigel-coated membranes. The studies suggest induction of MMP-9 expression promoted by DEP-1 deficiency, or potentially by growth factors and inflammatory cytokines, as a mechanism contributing to meningioma brain invasiveness.
Assuntos
Regulação Neoplásica da Expressão Gênica/genética , Glioma/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Análise de Variância , Linhagem Celular Tumoral , Citocinas/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioma/patologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/genética , Neurofibromatose 2/genética , Neurofibromatose 2/metabolismo , RNA Mensageiro/metabolismo , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores/deficiência , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores/genética , TransfecçãoRESUMO
Axonal surface proteins encompass a group of heterogeneous molecules, which exert a variety of different functions in the highly interdependent relationship between axons and Schwann cells. We recently revealed that the tumour suppressor protein merlin, mutated in the hereditary tumour syndrome neurofibromatosis type 2, impacts significantly on axon structure maintenance in the peripheral nervous system. We now report on a role of neuronal merlin in the regulation of the axonal surface protein neuregulin 1 important for modulating Schwann cell differentiation and myelination. Specifically, neuregulin 1 type III expression is reduced in sciatic nerve tissue of neuron-specific knockout animals as well as in biopsies from seven patients with neurofibromatosis type 2. In vitro experiments performed on both the P19 neuronal cell line and primary dorsal root ganglion cells demonstrate the influence of merlin on neuregulin 1 type III expression. Moreover, expression of ERBB2, a Schwann cell receptor for neuregulin 1 ligands is increased in nerve tissue of both neuron-specific merlin knockout animals and patients with neurofibromatosis type 2, demonstrating for the first time that axonal merlin indirectly regulates Schwann cell behaviour. Collectively, we have identified that neuronally expressed merlin can influence Schwann cell activity in a cell-extrinsic manner.
Assuntos
Neuregulina-1/fisiologia , Neurofibromina 2/fisiologia , Neurônios/fisiologia , Receptor ErbB-2/biossíntese , Células de Schwann/metabolismo , Transdução de Sinais/fisiologia , Adulto , Idoso , Animais , Linhagem Celular , Células Cultivadas , Feminino , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Pessoa de Meia-Idade , Neurofibromatose 2/metabolismo , Neurofibromatose 2/patologia , Neurônios/patologia , Células de Schwann/patologiaRESUMO
The neurofibromatoses (NF) are autosomal dominant genetic disorders that encompass the rare diseases NF1, NF2, and schwannomatosis. The NFs affect more people worldwide than Duchenne muscular dystrophy and Huntington's disease combined. NF1 and NF2 are caused by mutations of known tumor suppressor genes (NF1 and NF2, respectively). For schwannomatosis, although mutations in SMARCB1 were identified in a subpopulation of schwannomatosis patients, additional causative gene mutations are still to be discovered. Individuals with NF1 may demonstrate manifestations in multiple organ systems, including tumors of the nervous system, learning disabilities, and physical disfigurement. NF2 ultimately can cause deafness, cranial nerve deficits, and additional severe morbidities caused by tumors of the nervous system. Unmanageable pain is a key finding in patients with schwannomatosis. Although today there is no marketed treatment for NF-related tumors, a significant number of clinical trials have become available. In addition, significant preclinical efforts have led to a more rational selection of potential drug candidates for NF trials. An important element in fueling this progress is the sharing of knowledge. For over 20 years the Children's Tumor Foundation has convened an annual NF Conference, bringing together NF professionals to share novel findings, ideas, and build collaborations. The 2012 NF Conference held in New Orleans hosted over 350 NF researchers and clinicians. This article provides a synthesis of the highlights presented at the conference and as such, is a "state-of-the-field" for NF research in 2012.
Assuntos
Neurilemoma/etiologia , Neurofibromatoses/etiologia , Neurofibromatose 1/etiologia , Neurofibromatose 2/etiologia , Neoplasias Cutâneas/etiologia , Humanos , Neurilemoma/genética , Neurilemoma/terapia , Neurofibromatoses/genética , Neurofibromatoses/terapia , Neurofibromatose 1/genética , Neurofibromatose 1/terapia , Neurofibromatose 2/genética , Neurofibromatose 2/terapia , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/terapiaRESUMO
Preconditioning with lipopolysaccharide (LPS) induces neuroprotection against subsequent cerebral ischemic injury, mainly involving innate immune pathways. Microglia are resident immune cells of the central nervous system (CNS) that respond early to danger signals through memory-like differential reprogramming. However, the cell-specific molecular mechanisms underlying preconditioning are not fully understood. To elucidate the distinct molecular mechanisms of preconditioning on microglia, we compared these cell-specific proteomic profiles in response to LPS preconditioning and without preconditioning and subsequent transient focal brain ischemia and reperfusion, - using an established mouse model of transient focal brain ischemia and reperfusion. A proteomic workflow, based on isolated microglia obtained from mouse brains by cell sorting and coupled to mass spectrometry for identification and quantification, was applied. Our data confirm that LPS preconditioning induces marked neuroprotection, as indicated by a significant reduction in brain infarct volume. The established brain cell separation method was suitable for obtaining an enriched microglial cell fraction for valid proteomic analysis. The results show a significant impact of LPS preconditioning on microglial proteome patterns by type I interferons, presumably driven by the interferon cluster regulator proteins signal transducer and activator of transcription1/2 (STAT1/2).