RESUMEN
Interactions between tumorigenic cells and their surrounding microenvironment are critical for tumor progression yet remain incompletely understood. Germline mutations in the NF1 tumor suppressor gene cause neurofibromatosis type 1 (NF1), a common genetic disorder characterized by complex tumors called neurofibromas. Genetic studies indicate that biallelic loss of Nf1 is required in the tumorigenic cell of origin in the embryonic Schwann cell lineage. However, in the physiologic state, Schwann cell loss of heterozygosity is not sufficient for neurofibroma formation and Nf1 haploinsufficiency in at least one additional nonneoplastic lineage is required for tumor progression. Here, we establish that Nf1 heterozygosity of bone marrow-derived cells in the tumor microenvironment is sufficient to allow neurofibroma progression in the context of Schwann cell Nf1 deficiency. Further, genetic or pharmacologic attenuation of c-kit signaling in Nf1+/- hematopoietic cells diminishes neurofibroma initiation and progression. Finally, these studies implicate mast cells as critical mediators of tumor initiation.
Asunto(s)
Neurofibroma/metabolismo , Neurofibromina 1/metabolismo , Proteínas Proto-Oncogénicas c-kit/metabolismo , Animales , Benzamidas , Médula Ósea/fisiopatología , Trasplante de Médula Ósea , Preescolar , Genes de Neurofibromatosis 1 , Humanos , Mesilato de Imatinib , Mastocitos/metabolismo , Ratones , Ratones Endogámicos C57BL , Neurofibroma/tratamiento farmacológico , Neurofibroma/genética , Neurofibroma/patología , Neurofibroma Plexiforme/tratamiento farmacológico , Neurofibroma Plexiforme/metabolismo , Piperazinas/uso terapéutico , Pirimidinas/uso terapéutico , Células de Schwann/metabolismoRESUMEN
Schwannomas are common, highly morbid and medically untreatable tumors that can arise in patients with germ line as well as somatic mutations in neurofibromatosis type 2 (NF2). These mutations most commonly result in the loss of function of the NF2-encoded protein, Merlin. Little is known about how Merlin functions endogenously as a tumor suppressor and how its loss leads to oncogenic transformation in Schwann cells (SCs). Here, we identify nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB)-inducing kinase (NIK) as a potential drug target driving NF-κB signaling and Merlin-deficient schwannoma genesis. Using a genomic approach to profile aberrant tumor signaling pathways, we describe multiple upregulated NF-κB signaling elements in human and murine schwannomas, leading us to identify a caspase-cleaved, proteasome-resistant NIK kinase domain fragment that amplifies pathogenic NF-κB signaling. Lentiviral-mediated transduction of this NIK fragment into normal SCs promotes proliferation, survival, and adhesion while inducing schwannoma formation in a novel in vivo orthotopic transplant model. Furthermore, we describe an NF-κB-potentiated hepatocyte growth factor (HGF) to MET proto-oncogene receptor tyrosine kinase (c-Met) autocrine feed-forward loop promoting SC proliferation. These innovative studies identify a novel signaling axis underlying schwannoma formation, revealing new and potentially druggable schwannoma vulnerabilities with future therapeutic potential.
Asunto(s)
Neurilemoma/genética , Neurofibromatosis 2/genética , Neurofibromina 2/genética , Proteínas Serina-Treonina Quinasas/genética , Animales , Comunicación Autocrina/genética , Carcinogénesis/genética , Caspasa 1/genética , Proliferación Celular/genética , Modelos Animales de Enfermedad , Regulación Neoplásica de la Expresión Génica , Factor de Crecimiento de Hepatocito/genética , Humanos , Ratones , Terapia Molecular Dirigida , FN-kappa B/genética , Neurilemoma/complicaciones , Neurilemoma/tratamiento farmacológico , Neurilemoma/patología , Neurofibromatosis 2/complicaciones , Neurofibromatosis 2/tratamiento farmacológico , Neurofibromatosis 2/patología , Complejo de la Endopetidasa Proteasomal/genética , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-met/genética , Células de Schwann , Transducción de Señal/genética , Quinasa de Factor Nuclear kappa BRESUMEN
BACKGROUND: Neurofibromatosis type 1 (NF1) is a common genetic disorder characterized by plexiform neurofibromas (pNF), which are thought to be congenital tumors that arise in utero and enlarge throughout life. Genetic studies in murine models delineated an indispensable role for the stem cell factor (SCF)/c-kit pathway in pNF initiation and progression. A subsequent phase 2 clinical trial using imatinib mesylate to inhibit SCF/c-kit demonstrated tumor shrinkage in a subset of preexisting pNF; however, imatinib's role on preventing pNF development has yet to be explored. PROCEDURE: We evaluated the effect of imatinib dosed at 10-100 mg/kg/day for 12 weeks to one-month-old Nf1flox/flox ;PostnCre(+) mice, prior to onset of pNF formation. To determine durability of response, we then monitored for pNF growth at later time points, comparing imatinib- with vehicle-treated mice. We assessed gross and histopathological analysis of tumor burden. RESULTS: Imatinib administered preventatively led to a significant decrease in pNF number, even at doses as low as 10 mg/kg/day. Tumor development continued to be significantly inhibited after cessation of imatinib dosed at 50 and 100 mg/kg/day. In the cohort of treated mice that underwent prolonged follow-up, the size of residual tumors was significantly reduced as compared with age-matched littermates that received vehicle control. CONCLUSIONS: Early administration of imatinib inhibits pNF genesis in vivo, and effects are sustained after discontinuation of therapy. These findings may guide clinical use of imatinib in young NF1 patients prior to the substantial development of pNF.
Asunto(s)
Mesilato de Imatinib/administración & dosificación , Neoplasias Experimentales/prevención & control , Neurofibroma Plexiforme/prevención & control , Neurofibromatosis 1/prevención & control , Animales , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Ratones , Ratones Transgénicos , Neoplasias Experimentales/genética , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/patología , Neurofibroma Plexiforme/genética , Neurofibroma Plexiforme/metabolismo , Neurofibroma Plexiforme/patología , Neurofibromatosis 1/genética , Neurofibromatosis 1/metabolismo , Neurofibromatosis 1/patologíaRESUMEN
Neurofibromatosis type 2 (NF2) is an autosomal dominant genetic disorder resulting from germline mutations in the NF2 gene. Bilateral vestibular schwannomas, tumors on cranial nerve VIII, are pathognomonic for NF2 disease. Furthermore, schwannomas also commonly develop in other cranial nerves, dorsal root ganglia and peripheral nerves. These tumors are a major cause of morbidity and mortality, and medical therapies to treat them are limited. Animal models that accurately recapitulate the full anatomical spectrum of human NF2-related schwannomas, including the characteristic functional deficits in hearing and balance associated with cranial nerve VIII tumors, would allow systematic evaluation of experimental therapeutics prior to clinical use. Here, we present a genetically engineered NF2 mouse model generated through excision of the Nf2 gene driven by Cre expression under control of a tissue-restricted 3.9kbPeriostin promoter element. By 10 months of age, 100% of Postn-Cre; Nf2(flox/flox) mice develop spinal, peripheral and cranial nerve tumors histologically identical to human schwannomas. In addition, the development of cranial nerve VIII tumors correlates with functional impairments in hearing and balance, as measured by auditory brainstem response and vestibular testing. Overall, the Postn-Cre; Nf2(flox/flox) tumor model provides a novel tool for future mechanistic and therapeutic studies of NF2-associated schwannomas.
Asunto(s)
Moléculas de Adhesión Celular/genética , Ganglios Espinales/patología , Neurofibromatosis 2/genética , Neurofibromina 2/genética , Neuroma Acústico/fisiopatología , Nervio Vestibulococlear/patología , Animales , Modelos Animales de Enfermedad , Exones , Audición , Humanos , Ratones , Ratones Transgénicos , Mutación , Neurofibromatosis 2/complicaciones , Neurofibromatosis 2/fisiopatología , Neuroma Acústico/genética , Neuroma Acústico/patologíaRESUMEN
Fanconi anemia is a complex heterogeneous genetic disorder with a high incidence of bone marrow failure, clonal evolution to acute myeloid leukemia and mesenchymal-derived congenital anomalies. Increasing evidence in Fanconi anemia and other genetic disorders points towards an interdependence of skeletal and hematopoietic development, yet the impact of the marrow microenvironment in the pathogenesis of the bone marrow failure in Fanconi anemia remains unclear. Here we demonstrated that mice with double knockout of both Fancc and Fancg genes had decreased bone formation at least partially due to impaired osteoblast differentiation from mesenchymal stem/progenitor cells. Mesenchymal stem/progenitor cells from the double knockout mice showed impaired hematopoietic supportive activity. Mesenchymal stem/progenitor cells of patients with Fanconi anemia exhibited similar cellular deficits, including increased senescence, reduced proliferation, impaired osteoblast differentiation and defective hematopoietic stem/progenitor cell supportive activity. Collectively, these studies provide unique insights into the physiological significance of mesenchymal stem/progenitor cells in supporting the marrow microenvironment, which is potentially of broad relevance in hematopoietic stem cell transplantation.
Asunto(s)
Médula Ósea/patología , Microambiente Celular , Anemia de Fanconi/patología , Animales , Huesos/anomalías , Huesos/fisiopatología , Linaje de la Célula , Anemia de Fanconi/fisiopatología , Proteína del Grupo de Complementación C de la Anemia de Fanconi/genética , Hematopoyesis , Trasplante de Células Madre Hematopoyéticas , Células Madre Mesenquimatosas/patología , Ratones , Ratones NoqueadosRESUMEN
ASXL1 is mutated/deleted with high frequencies in multiple forms of myeloid malignancies, and its alterations are associated with poor prognosis. De novo ASXL1 mutations cause Bohring-Opitz syndrome characterized by multiple congenital malformations. We show that Asxl1 deletion in mice led to developmental abnormalities including dwarfism, anophthalmia, and 80% embryonic lethality. Surviving Asxl1(-/-) mice lived for up to 42 days and developed features of myelodysplastic syndrome (MDS), including dysplastic neutrophils and multiple lineage cytopenia. Asxl1(-/-) mice had a reduced hematopoietic stem cell (HSC) pool, and Asxl1(-/-) HSCs exhibited decreased hematopoietic repopulating capacity, with skewed cell differentiation favoring granulocytic lineage. Asxl1(+/-) mice also developed mild MDS-like disease, which could progress to MDS/myeloproliferative neoplasm, demonstrating a haploinsufficient effect of Asxl1 in the pathogenesis of myeloid malignancies. Asxl1 loss led to an increased apoptosis and mitosis in Lineage(-)c-Kit(+) (Lin(-)c-Kit(+)) cells, consistent with human MDS. Furthermore, Asxl1(-/-) Lin(-)c-Kit(+) cells exhibited decreased global levels of H3K27me3 and H3K4me3 and altered expression of genes regulating apoptosis (Bcl2, Bcl2l12, Bcl2l13). Collectively, we report a novel ASXL1 murine model that recapitulates human myeloid malignancies, implying that Asxl1 functions as a tumor suppressor to maintain hematopoietic cell homeostasis. Future work is necessary to clarify the contribution of microenvironment to the hematopoietic phenotypes observed in the constitutional Asxl1(-/-) mice.
Asunto(s)
Mutación , Síndromes Mielodisplásicos/genética , Proteínas Represoras/genética , Proteínas Represoras/fisiología , Animales , Apoptosis , Células de la Médula Ósea/citología , Diferenciación Celular , Linaje de la Célula , Proliferación Celular , Cruzamientos Genéticos , Modelos Animales de Enfermedad , Eliminación de Gen , Proteínas Fluorescentes Verdes/metabolismo , Hematopoyesis , Células Madre Hematopoyéticas/citología , Homeostasis , Homocigoto , Humanos , Ratones , Ratones Transgénicos , Mitosis , Síndromes Mielodisplásicos/metabolismo , FenotipoRESUMEN
p21-Activated kinase 2 (Pak2), a serine/threonine kinase, has been previously shown to be essential for hematopoietic stem cell (HSC) engraftment. However, Pak2 modulation of long-term hematopoiesis and lineage commitment remain unreported. Using a conditional Pak2 knockout mouse model, we found that disruption of Pak2 in HSCs induced profound leukopenia and a mild macrocytic anemia. Although loss of Pak2 in HSCs leads to less efficient short- and long-term competitive hematopoiesis than wild-type cells, it does not affect HSC self-renewal per se. Pak2 disruption decreased the survival and proliferation of multicytokine stimulated immature progenitors. Loss of Pak2 skewed lineage differentiation toward granulocytopoiesis and monocytopoiesis in mice as evidenced by (a) a three- to sixfold increase in the percentage of peripheral blood granulocytes and a significant increase in the percentage of granulocyte-monocyte progenitors in mice transplanted with Pak2-disrupted bone marrow (BM); (b)Pak2-disrupted BM and c-kit(+) cells yielded higher numbers of more mature subsets of granulocyte-monocyte colonies and polymorphonuclear neutrophils, respectively, when cultured in the presence of granulocyte-macrophage colony-stimulating factor. Pak2 disruption resulted, respectively, in decreased and increased gene expression of transcription factors JunB and c-Myc, which may suggest underlying mechanisms by which Pak2 regulates granulocyte-monocyte lineage commitment. Furthermore, Pak2 disruption led to (a) higher percentage of CD4(+) CD8(+) double positive T cells and lower percentages of CD4(+) CD8(-) or CD4(-) CD8(+) single positive T cells in thymus and (b) decreased numbers of mature B cells and increased numbers of Pre-Pro B cells in BM, suggesting defects in lymphopoiesis.
Asunto(s)
Diferenciación Celular , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/enzimología , Quinasas p21 Activadas/metabolismo , Anemia Macrocítica/patología , Animales , Apoptosis , Proliferación Celular , Supervivencia Celular , Eliminación de Gen , Regulación de la Expresión Génica , Hematopoyesis , Leucopenia/patología , Linfopoyesis , Ratones Noqueados , Células Mieloides/patología , Fenotipo , Factores de Transcripción/metabolismo , Quinasas p21 Activadas/deficienciaRESUMEN
PURPOSE: Plexiform neurofibromas (pNF) are pathognomonic nerve and soft tissue tumors of neurofibromatosis type I (NF1), which are highly resistant to conventional chemotherapy and associated with significant morbidity/mortality. Disruption of aberrant SCF/c-Kit signaling emanating from the pNF microenvironment induced the first ever objective therapeutic responses in a recent phase 2 trial. Sunitinib malate is a potent, highly selective RTK inhibitor with activity against c-Kit, PDGFR, and VEGFR, which have also been implicated in the pathogenesis of these lesions. Here, we evaluate the efficacy of sunitinib malate in a preclinical Krox20;Nf1(flox/-) pNF murine model. EXPERIMENTAL DESIGN: Proliferation, ß-hexosaminidase release (degranulation), and Erk1/2 phosphorylation were assessed in sunitinib treated Nf1(+/-) mast cells and fibroblasts, respectively. Krox20;Nf1(flox/-) mice with established pNF were treated sunitinib or PBS-vehicle control for a duration of 12 weeks. pNF metabolic activity was monitored by serial [(18)F]DG-PET/CT imaging. RESULTS: Sunitinib suppressed multiple in vitro gain-in-functions of Nf1(+/-) mast cells and fibroblasts and attenuated Erk1/2 phosphorylation. Sunitinib treated Krox20;Nf1(flox/-) mice exhibited significant reductions in pNF size, tumor number, and FDG uptake compared to control mice. Histopathology revealed reduced tumor cellularity and infiltrating mast cells, markedly diminished collagen deposition, and increased cellular apoptosis in sunitinib treated pNF. CONCLUSIONS: Collectively, these results demonstrate the efficacy of sunitinib in reducing tumor burden in Krox20;Nf1(flox/-) mice. These preclinical findings demonstrate the utility of inhibiting multiple RTKs in pNF and provide insights into the design of future clinical trials.
Asunto(s)
Antineoplásicos/farmacología , Indoles/farmacología , Neurofibroma Plexiforme/patología , Pirroles/farmacología , Microambiente Tumoral/efectos de los fármacos , Animales , Western Blotting , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Femenino , Fibroblastos/efectos de los fármacos , Fibroblastos/patología , Masculino , Mastocitos/efectos de los fármacos , Mastocitos/patología , Ratones , Ratones Transgénicos , Tomografía de Emisión de Positrones , SunitinibRESUMEN
Neurofibromatosis type 1 (NF1) is a common genetic disorder affecting 1 in 3500 individuals. Patients with NF1 are predisposed to debilitating skeletal manifestations, including osteopenia/osteoporosis and long bone pseudarthrosis (nonunion fracture). Hyperactivation of the Ras/mitogen-activated protein kinase (MAPK) pathway in NF1 is known to underlie aberrant proliferation and differentiation in cell lineages, including osteoclast progenitors and mesenchymal stem cells (MSCs) also known as osteoblast progenitors (pro-OBLs). Our current study demonstrates the hyper Ras/MAPK as a critical pathway underlying the pathogenesis of NF1-associated fracture repair deficits. Nf1-deficient pro-OBLs exhibit Ras/MAPK hyperactivation. Introduction of the NF1 GTPase activating-related domain (NF1 GAP-related domain) in vitro is sufficient to rescue hyper Ras activity and enhance osteoblast (OBL) differentiation in Nf1(-/-) pro-OBLs and NF1 human (h) MSCs cultured from NF1 patients with skeletal abnormalities, including pseudarthrosis or scoliosis. Pharmacologic inhibition of mitogen-activated protein kinase kinase (MEK) signaling with PD98059 partially rescues aberrant Erk activation while enhancing OBL differentiation and expression of OBL markers, osterix and osteocalcin, in Nf1-deficient murine pro-OBLs. Similarly, MEK inhibition enhances OBL differentiation of hMSCs. In addition, PD98059 rescues aberrant osteoclast maturation in Nf1 haploinsufficient bone marrow mononuclear cells (BMMNCs). Importantly, MEK inhibitor significantly improves fracture healing in an NF1 murine model, Col2.3Cre;Nf1(flox/-). Collectively, these data indicate the Ras/MAPK cascade as a critical pathway in the pathogenesis of bone loss and pseudarthrosis related to NF1 mutations. These studies provide evidence for targeting the MAPK pathway to improve bone mass and treat pseudarthrosis in NF1.
Asunto(s)
Proteínas Quinasas Activadas por Mitógenos/metabolismo , Neurofibromatosis 1/metabolismo , Neurofibromina 1/deficiencia , Seudoartrosis/fisiopatología , Transducción de Señal/fisiología , Proteínas ras/metabolismo , Animales , Linaje de la Célula , Células Cultivadas , Modelos Animales de Enfermedad , Flavonoides/farmacología , Humanos , Ratones , Ratones Transgénicos , Neurofibromatosis 1/genética , Neurofibromatosis 1/patología , Neurofibromina 1/genética , Neurofibromina 1/metabolismo , Osteoblastos/fisiología , Osteoclastos/efectos de los fármacos , Osteoclastos/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Seudoartrosis/tratamiento farmacológico , Seudoartrosis/genética , Seudoartrosis/patología , Transducción de Señal/efectos de los fármacos , Células Madre/efectos de los fármacos , Células Madre/metabolismo , Fracturas de la Tibia/fisiopatologíaRESUMEN
Off-line gamma-ray spectrometry was used to accurately measure the Cumulative fission product yields (CFPYs) of fission products in the 235U (n, f) reaction induced by 2.8 MeV neutrons. The 2.8 MeV quasi-monoenergetic neutron beam was produced by the CPNG-600 Cockcroft Walton accelerator at the China Institute of Atomic Energy ï¼CIAEï¼and the gamma spectra were measured by the HPGe γ-ray Spectrometer. After fully considering and revising the sources of uncertainty, high-precision CFPYs of 4 fission products were obtained. This study has important applications in reactor design and operation and is conducive to the establishment of an evaluated nuclear database.
RESUMEN
Dyslexia is a disorder characterized by an impaired ability to understand written and printed words or phrases. Epidemiological longitudinal data show that dyslexia is highly prevalent, affecting 10-20% of the population regardless of gender. This study aims to provide a detailed overview of research status and development characteristics of dyslexia from types of articles, years, countries, institutions, journals, authors, author keywords, and highly cited papers. A total of 9,166 publications have been retrieved from the Social Sciences Citation Index (SSCI) and Science Citation Index Expanded (SCI-E) from 2000 to 2021. The United States of America, United Kingdom, and Germany were the top three most productive countries in terms of the number of publications. China, Israel, and Japan led the Asia research on dyslexia. University of Oxford had the most publications and won first place in terms of h-index. Dyslexia was the most productive journal in this field and Psychology was the most used subject category. Keywords analysis indicated that "developmental dyslexia," "phonological awareness," children and fMRI were still the main research topics. "Literacy," "rapid automatized naming (RAN)," "assessment," "intervention," "meta-analysis," "Chinese," "executive function," "morphological awareness," "decoding," "dyscalculia," "EEG," "Eye tracking," "rhythm," "bilingualism," and "functional connectivity" might become the new research hotspots.
Asunto(s)
Bibliometría , Dislexia , Asia , Niño , China , Humanos , Reino Unido , Estados UnidosRESUMEN
Despite the high prevalence and significant morbidity of spinal anomalies in neurofibromatosis type 1 (NF1), the pathogenesis of these defects remains largely unknown. Here, we present two murine models: Nf1flox/-;PeriCre and Nf1flox/-;Col.2.3Cre mice, which recapitulate spinal deformities seen in the human disease. Dynamic histomorphometry and microtomographic studies show recalcitrant bone remodeling and distorted bone microarchitecture within the vertebral spine of Nf1flox/-;PeriCre and Nf1flox/-;Col2.3Cre mice, with analogous histological features present in a human patient with dystrophic scoliosis. Intriguingly, 36-60% of Nf1flox/-;PeriCre and Nf1flox/-;Col2.3Cre mice exhibit segmental vertebral fusion anomalies with boney obliteration of the intervertebral disc (IVD). While analogous findings have not yet been reported in the NF1 patient population, we herein present two case reports of IVD defects and interarticular vertebral fusion in patients with NF1. Collectively, these data provide novel insights regarding the pathophysiology of dystrophic spinal anomalies in NF1, and provide impetus for future radiographic analyses of larger patient cohorts to determine whether IVD and vertebral fusion defects may have been previously overlooked or underreported in the NF1 patient population.
Asunto(s)
Modelos Animales de Enfermedad , Neurofibromatosis 1/patología , Columna Vertebral/patología , Animales , Remodelación Ósea , Vértebras Cervicales/patología , Niño , Femenino , Humanos , Ratones , Ratones Transgénicos , Escoliosis/patología , Vértebras Torácicas/patología , Adulto JovenRESUMEN
Although nullizygous loss of NF1 leads to myeloid malignancies, haploinsufficient loss of NF1 (Nf1) has been shown to contribute to osteopenia and osteoporosis which occurs in approximately 50% of neurofibromatosis type 1 (NF1) patients. Bone marrow mononuclear cells of haploinsufficient NF1 patients and Nf1(+/-) mice exhibit increased osteoclastogenesis and accelerated bone turnover; however, the culprit hematopoietic lineages responsible for perpetuating these osteolytic manifestations have yet to be elucidated. Here we demonstrate that conditional inactivation of a single Nf1 allele within the myeloid progenitor cell population (Nf1-LysM) is necessary and sufficient to promote multiple osteoclast gains-in-function, resulting in enhanced osteoclastogenesis and accelerated osteoclast bone lytic activity in response to proresorptive challenge in vivo. Surprisingly, mice conditionally Nf1 heterozygous in mature, terminally differentiated osteoclasts (Nf1-Ctsk) do not exhibit any of these skeletal phenotypes, indicating a critical requirement for Nf1 haploinsufficiency at a more primitive/progenitor stage of myeloid development in perpetuating osteolytic activity. We further identified p21Ras-dependent hyperphosphorylation of Pu.1 within the nucleus of Nf1 haploinsufficient myelomonocytic osteoclast precursors, providing a novel therapeutic target for the potential treatment of NF1 associated osteolytic manifestations.
Asunto(s)
Haploinsuficiencia , Células Progenitoras Mieloides , Neurofibromina 1 , Osteoclastos , Osteólisis , Osteoporosis , Animales , Humanos , Ratones , Ratones Transgénicos , Células Progenitoras Mieloides/metabolismo , Células Progenitoras Mieloides/patología , Neurofibromina 1/genética , Neurofibromina 1/metabolismo , Osteoclastos/metabolismo , Osteoclastos/patología , Osteólisis/genética , Osteólisis/metabolismo , Osteólisis/patología , Osteoporosis/genética , Osteoporosis/metabolismo , Osteoporosis/patología , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Transactivadores/genética , Transactivadores/metabolismoRESUMEN
Anisakid larvae are regarded as an important hazard in marine products, and demand is growing for on-line nondestructive analytical techniques for effective monitoring of these parasites. A UV fluorescent imaging was developed for detection of the third-stage anisakid larvae in marine fishes, and different processing methods were investigated and optimized based on principal component and gray value analyses. Using cod fillets as samples, the efficiency of the developed technique was evaluated, and the overall detection ratio was greater than 80 % . These results indicate a promising application of UV fluorescent imaging as an effective and nondestructive technique for identification of anisakid larvae in fishery products.
Asunto(s)
Anisakis/aislamiento & purificación , Contaminación de Alimentos/análisis , Parasitología de Alimentos , Gadiformes/parasitología , Animales , Seguridad de Productos para el Consumidor , Fluorescencia , Humanos , Larva , Análisis de Componente Principal , Rayos UltravioletaRESUMEN
Mitosis is controlled by a network of kinases and phosphatases. We screened a library of small interfering RNAs against a genome-wide set of phosphatases to comprehensively evaluate the role of human phosphatases in mitosis. We found four candidate spindle checkpoint phosphatases, including the tumor suppressor CDKN3. We show that CDKN3 is essential for normal mitosis and G1/S transition. We demonstrate that subcellular localization of CDKN3 changes throughout the cell cycle. We show that CDKN3 dephosphorylates threonine-161 of CDC2 during mitotic exit and we visualize CDC2(pThr-161) at kinetochores and centrosomes in early mitosis. We performed a phosphokinome-wide mass spectrometry screen to find effectors of the CDKN3-CDC2 signaling axis. We found that one of the identified downstream phosphotargets, CKß phosphorylated at serine 209, localizes to mitotic centrosomes and controls the spindle checkpoint. Finally, we show that CDKN3 protein is down-regulated in brain tumors. Our findings indicate that CDKN3 controls mitosis through the CDC2 signaling axis. These results have implications for targeted anticancer therapeutics.
Asunto(s)
Proteínas Inhibidoras de las Quinasas Dependientes de la Ciclina/fisiología , Fosfatasas de Especificidad Dual/fisiología , Mitosis/fisiología , Proteína Quinasa CDC2 , Centrosoma/metabolismo , Centrosoma/ultraestructura , Ciclina B/metabolismo , Proteínas Inhibidoras de las Quinasas Dependientes de la Ciclina/análisis , Proteínas Inhibidoras de las Quinasas Dependientes de la Ciclina/metabolismo , Quinasas Ciclina-Dependientes , Fosfatasas de Especificidad Dual/análisis , Fosfatasas de Especificidad Dual/metabolismo , Células HeLa , Humanos , Cinetocoros/metabolismo , Cinetocoros/ultraestructura , Espectrometría de Masas , Mitosis/genética , Fosforilación , Interferencia de ARN , Transducción de SeñalRESUMEN
Dysregulated transforming growth factor beta (TGF-ß) signaling is associated with a spectrum of osseous defects as seen in Loeys-Dietz syndrome, Marfan syndrome, and Camurati-Engelmann disease. Intriguingly, neurofibromatosis type 1 (NF1) patients exhibit many of these characteristic skeletal features, including kyphoscoliosis, osteoporosis, tibial dysplasia, and pseudarthrosis; however, the molecular mechanisms mediating these phenotypes remain unclear. Here, we provide genetic and pharmacologic evidence that hyperactive TGF-ß1 signaling pivotally underpins osseous defects in Nf1(flox/-) ;Col2.3Cre mice, a model which closely recapitulates the skeletal abnormalities found in the human disease. Compared to controls, we show that serum TGF-ß1 levels are fivefold to sixfold increased both in Nf1(flox/-) ;Col2.3Cre mice and in a cohort of NF1 patients. Nf1-deficient osteoblasts, the principal source of TGF-ß1 in bone, overexpress TGF-ß1 in a gene dosage-dependent fashion. Moreover, Nf1-deficient osteoblasts and osteoclasts are hyperresponsive to TGF-ß1 stimulation, potentiating osteoclast bone resorptive activity while inhibiting osteoblast differentiation. These cellular phenotypes are further accompanied by p21-Ras-dependent hyperactivation of the canonical TGF-ß1-Smad pathway. Reexpression of the human, full-length neurofibromin guanosine triphosphatase (GTPase)-activating protein (GAP)-related domain (NF1 GRD) in primary Nf1-deficient osteoblast progenitors, attenuated TGF-ß1 expression levels and reduced Smad phosphorylation in response to TGF-ß1 stimulation. As an in vivo proof of principle, we demonstrate that administration of the TGF-ß receptor 1 (TßRI) kinase inhibitor, SD-208, can rescue bone mass deficits and prevent tibial fracture nonunion in Nf1(flox/-) ;Col2.3Cre mice. In sum, these data demonstrate a pivotal role for hyperactive TGF-ß1 signaling in the pathogenesis of NF1-associated osteoporosis and pseudarthrosis, thus implicating the TGF-ß signaling pathway as a potential therapeutic target in the treatment of NF1 osseous defects that are refractory to current therapies.
Asunto(s)
Huesos/anomalías , Huesos/metabolismo , Neurofibromatosis 1/metabolismo , Neurofibromatosis 1/patología , Transducción de Señal , Factor de Crecimiento Transformador beta1/metabolismo , Animales , Huesos/patología , Diferenciación Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Modelos Animales de Enfermedad , Dosificación de Gen , Haploinsuficiencia , Humanos , Integrasas/metabolismo , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Células Madre Mesenquimatosas/metabolismo , Ratones , Ratones Transgénicos , Modelos Biológicos , Neurofibromina 1/deficiencia , Neurofibromina 1/genética , Osteoblastos/metabolismo , Osteoblastos/patología , Proteínas Serina-Treonina Quinasas/metabolismo , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Proteínas Smad/metabolismo , Proteínas ras/metabolismoRESUMEN
Skeletal abnormalities including osteoporosis and osteopenia occur frequently in both pediatric and adult neurofibromatosis type 1 (NF1) patients. NF1 (Nf1) haploinsufficient osteoclasts and osteoclast progenitors derived from both NF1 patients and Nf1(+/-) mice exhibit increased differentiation, migration, and bone resorptive capacity in vitro, mediated by hyperactivation of p21(Ras) in response to limiting concentrations of macrophage-colony stimulating factor (M-CSF). Here, we show that M-CSF binding to its receptor, c-Fms, results in increased c-Fms activation in Nf1(+/) (-) osteoclast progenitors, mediating multiple gain-in-functions through the downstream effectors Erk1/2 and p90RSK. PLX3397, a potent and selective c-Fms inhibitor, attenuated M-CSF mediated Nf1(+/-) osteoclast migration by 50%, adhesion by 70%, and pit formation by 60%. In vivo, we administered PLX3397 to Nf1(+/-) osteoporotic mice induced by ovariectomy (OVX) and evaluated changes in bone mass and skeletal architecture. We found that PLX3397 prevented bone loss in Nf1(+/-)-OVX mice by reducing osteoclast differentiation and bone resorptive activity in vivo. Collectively, these results implicate the M-CSF/c-Fms signaling axis as a critical pathway underlying the aberrant functioning of Nf1 haploinsufficient osteoclasts and may provide a potential therapeutic target for treating NF1 associated osteoporosis and osteopenia.
Asunto(s)
Factor Estimulante de Colonias de Macrófagos/metabolismo , Neurofibromina 1/metabolismo , Osteoclastos/metabolismo , Receptor de Factor Estimulante de Colonias de Macrófagos/metabolismo , Transducción de Señal , Animales , Resorción Ósea/tratamiento farmacológico , Resorción Ósea/genética , Resorción Ósea/metabolismo , Resorción Ósea/patología , Adhesión Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Inhibidores Enzimáticos/uso terapéutico , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Eliminación de Gen , Humanos , Ratones , Ratones Endogámicos C57BL , Neurofibromina 1/genética , Osteoclastos/citología , Osteoclastos/efectos de los fármacos , Osteoclastos/patología , Osteoporosis/tratamiento farmacológico , Osteoporosis/genética , Osteoporosis/metabolismo , Osteoporosis/patología , Receptor de Factor Estimulante de Colonias de Macrófagos/antagonistas & inhibidores , Proteínas Quinasas S6 Ribosómicas 90-kDa/metabolismoRESUMEN
The extracellular signal-regulated kinases (ERK1 and 2) are widely-expressed and they modulate proliferation, survival, differentiation, and protein synthesis in multiple cell lineages. Altered ERK1/2 signaling is found in several genetic diseases with skeletal phenotypes, including Noonan syndrome, Neurofibromatosis type 1, and Cardio-facio-cutaneous syndrome, suggesting that MEK-ERK signals regulate human skeletal development. Here, we examine the consequence of Erk1 and Erk2 disruption in multiple functions of osteoclasts, specialized macrophage/monocyte lineage-derived cells that resorb bone. We demonstrate that Erk1 positively regulates osteoclast development and bone resorptive activity, as genetic disruption of Erk1 reduced osteoclast progenitor cell numbers, compromised pit formation, and diminished M-CSF-mediated adhesion and migration. Moreover, WT mice reconstituted long-term with Erk1(-/-) bone marrow mononuclear cells (BMMNCs) demonstrated increased bone mineral density as compared to recipients transplanted with WT and Erk2(-/-) BMMNCs, implicating marrow autonomous, Erk1-dependent osteoclast function. These data demonstrate Erk1 plays an important role in osteoclast functions while providing rationale for the development of Erk1-specific inhibitors for experimental investigation and/or therapeutic modulation of aberrant osteoclast function.
Asunto(s)
Resorción Ósea/metabolismo , Diferenciación Celular , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Osteoclastos/metabolismo , Fosfatasa Ácida/metabolismo , Animales , Western Blotting , Densidad Ósea , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Trasplante de Médula Ósea , Resorción Ósea/genética , Resorción Ósea/patología , Adhesión Celular , Movimiento Celular , Células Cultivadas , Colágeno Tipo I/metabolismo , Femenino , Humanos , Isoenzimas/metabolismo , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Osteoclastos/citología , Péptidos/metabolismo , Células Madre/citología , Células Madre/metabolismo , Fosfatasa Ácida Tartratorresistente , Microtomografía por Rayos XRESUMEN
Neurofibromatosis type 1 (NF1) is a common autosomal dominant genetic disorder caused by mutation of the NF1 tumor suppressor gene. Spinal deformities are common skeletal manifestations in patients with NF1. To date, the mechanism of vertebral abnormalities remains unclear because of the lack of appropriate animal models for the skeletal manifestations of NF1. In the present study, we report a novel murine NF1 model, Nf1(flox/-);Col2.3Cre(+) mice. These mice display short vertebral segments. In addition, a significant reduction in cortical and trabecular bone mass of the vertebrae was observed in Nf1(flox/-);Col2.3Cre(+) mice as measured by dual-energy X-ray absorptiometry (DEXA) and peripheral quantitative computed tomography (pQCT). Peak stress and peak load were also significantly reduced in Nf1(flox/-);Col2.3Cre(+) mice as compared to controls. Furthermore, the lumbar vertebrae showed enlargement of the inter-vertebral canal, a characteristic feature of lumbar vertebrae in NF1 patients. Finally, histologic analysis demonstrated increased numbers of osteoclasts and decreased numbers of osteoblasts in the vertebrae of Nf1(flox/-);Col2.3Cre(+) mice in comparison to controls. In summary, Nf1(flox/-);Col2.3Cre(+) mice demonstrate multiple structural and functional abnormalities in the lumbar vertebrae which recapitulate the dystrophic vertebral changes in NF1 patients. This novel murine model provides a platform to understand the cellular and molecular mechanisms underlying the pathogenesis of spinal deficits in NF1 patients.
Asunto(s)
Modelos Animales de Enfermedad , Neurofibromatosis 1/patología , Columna Vertebral/patología , Absorciometría de Fotón , Animales , Fenómenos Biomecánicos , Genes de Neurofibromatosis 1 , Ratones , Ratones Noqueados , Neurofibromatosis 1/genética , Tomografía Computarizada por Rayos XRESUMEN
Germline mutations in the NF1 tumor suppressor gene cause neurofibromatosis type 1 (NF1), a complex genetic disorder with a high predisposition of numerous skeletal dysplasias including short stature, osteoporosis, kyphoscoliosis, and fracture non-union (pseudoarthrosis). We have developed murine models that phenocopy many of the skeletal dysplasias observed in NF1 patients, including reduced bone mass and fracture non-union. We also show that the development of these skeletal manifestations requires an Nf1 haploinsufficient background in addition to nullizygous loss of Nf1 in mesenchymal stem/progenitor cells (MSCs) and/or their progenies. This is replicated in two animal models of NF1, PeriCre(+);Nf1(flox/-) and Col2.3Cre(+);Nf1(flox/-) mice. Adoptive transfer experiments demonstrate a critical role of the Nf1+/- marrow microenvironment in the impaired fracture healing in both models and adoptive transfer of WT bone marrow cells improves fracture healing in these mice. To our knowledge, this is the first demonstration of a non-cell autonomous mechanism in non-malignant NF1 manifestations. Collectively, these data provide evidence of a combinatory effect between nullizygous loss of Nf1 in osteoblast progenitors and haploinsufficiency in hematopoietic cells in the development of non-malignant NF1 manifestations.