RESUMEN
Thrombotic thrombocytopenic purpura (TTP) is a life-threatening disease that presents with thrombocytopenia, disseminated thrombosis, hemolytic anemia, and organ dysfunction. The etiology of TTP has revealed that patients share a deficiency in plasma protease a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13), the enzyme responsible for cleaving ultra-large von Willebrand factor (VWF) multimers into nonthrombogenic fragments. Therefore, existing TTP mouse models were developed by targeted disruption of the ADAMTS13 gene. ADAMTS13(-/-) mice are mostly asymptomatic in the absence of a trigger, as redundant proteases appear to take on VWF processing. As an alternative approach to creating one such model, we devised a strategy based on the expression of a cleavage-resistant VWF mutant in mice. The creation of a disulfide bond within the A2 domain of VWF was found to render VWF multimers resistant to proteolysis by plasma proteases under flow. Furthermore, mice expressing the murine VWF/p.S1494C-p.A1534C mutant present with symptoms characteristics of acute TTP such as thrombocytopenia, red cell shredding, accumulation of VWF-rich thrombi in the microvasculature, and advanced TTP symptoms such as renal dysfunction and splenomegaly. Because this model appears to faithfully emulate the pathophysiology of TTP, it should prove most useful in the study of microangiopathic diseases and their treatment.
Asunto(s)
Púrpura Trombocitopénica Trombótica/genética , Púrpura Trombocitopénica Trombótica/metabolismo , Factor de von Willebrand/química , Factor de von Willebrand/genética , Secuencia de Aminoácidos , Animales , Disulfuros/química , Disulfuros/metabolismo , Células HEK293 , Humanos , Hidrólisis , Riñón/metabolismo , Riñón/fisiopatología , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Péptido Hidrolasas/metabolismo , Estructura Terciaria de Proteína , Púrpura Trombocitopénica Trombótica/patología , Factor de von Willebrand/metabolismoRESUMEN
Sorghum, Sorghum bicolor (L.) Moench, is the fifth most important cereal crop in the world. In this study, we identified the IgE production-suppressing activity of white sorghum bran extracts. White sorghum is one of the genotypes of sorghum. White sorghum bran extracts in 10 mM sodium phosphate buffer (pH 7.4) suppressed IgE production in human myeloma cell line U266. The extracts suppressed IgE production by decreasing mRNA transcription level of IgE, but they did not affect IgA or IgG production of mice splenocytes in vitro. Heat treatment and trypsin digestion did not affect IgE production-suppressing activity. The white sorghum bran extracts were fractionated by ultrafiltration, and the molecular weight of the active substance was estimated to be less than 1,000.
Asunto(s)
Inmunoglobulina E/biosíntesis , Mieloma Múltiple/metabolismo , Extractos Vegetales/farmacología , Sorghum/química , Animales , Secuencia de Bases , Línea Celular Tumoral , Cartilla de ADN , Ensayo de Inmunoadsorción Enzimática , Humanos , Inmunoglobulina E/genética , Ratones , Mieloma Múltiple/patología , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Bazo/efectos de los fármacos , Bazo/metabolismoRESUMEN
The antiangiogenic function of the tissue inhibitors of metalloproteinases (TIMPs) has been attributed to their matrix metalloproteinase inhibitory activity. Here we demonstrate that TIMP-1 but not Ala+TIMP-1 inhibits both basal and vascular endothelial growth factor (VEGF)-stimulated migration of human microvascular endothelial cells (hMVECs), suggesting that this effect is dependent on direct inhibition of matrix metalloproteinase (MMP) activity. In contrast, TIMP-2 and mutant Ala+TIMP-2, which is devoid of MMP inhibitory activity, block hMVEC migration in response to VEGF-A stimulation. TIMP-2 and Ala+TIMP-2 also suppress basal hMVEC migration via a time-dependent mechanism mediated by enhanced expression of RECK, a membrane-anchored MMP inhibitor, which, in turn, inhibits cell migration. TIMP-2 treatment of hMVECs increases the association of Crk with C3G, resulting in enhanced Rap1 activation. hMVECs stably expressing Rap1 have increased RECK expression and display reduced cell migration compared with those expressing inactive Rap1(38N). RECK-null murine embryo fibroblasts fail to demonstrate TIMP-2-mediated decrease in cell migration despite activation of Rap1. TIMP-2-induced RECK decreases cell-associated MMP activity. Anti-RECK antibody increases MMP activity and reverses the TIMP-2-mediated reduction in cell migration. The effects of TIMP-2 on RECK expression and cell migration were confirmed in A2058 melanoma cells. These results suggest that TIMP-2 can inhibit cell migration via several distinct mechanisms. First, TIMP-2 can inhibit cell migration after VEGF stimulation by direct inhibition of MMP activity induced in response to VEGF stimulation. Secondly, TIMP-2 can disrupt VEGF signaling required for initiation of hMVEC migration. Third, TIMP-2 can enhance expression of RECK via Rap1 signaling resulting in an indirect, time-dependent inhibition of endothelial cell migration.
Asunto(s)
Movimiento Celular/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Glicoproteínas de Membrana/biosíntesis , Inhibidor Tisular de Metaloproteinasa-2/farmacología , Línea Celular Tumoral , Endotelio Vascular/citología , Proteínas Ligadas a GPI , Humanos , Melanoma/metabolismo , Melanoma/patología , Glicoproteínas de Membrana/antagonistas & inhibidores , Inhibidor Tisular de Metaloproteinasa-1/farmacología , Factor A de Crecimiento Endotelial Vascular/farmacología , Proteínas de Unión al GTP rap1/metabolismoRESUMEN
Extracellular matrix (ECM) is known to play several important roles in vascular development, although the molecular mechanisms behind these remain largely unknown. RECK, a tumor suppressor downregulated in a wide variety of cancers, encodes a membrane-anchored matrix-metalloproteinase-regulator. Mice lacking functional Reck die in utero, demonstrating its importance for mammalian embryogenesis; however, the underlying causes of mid-gestation lethality remain unclear. Using Reck conditional knockout mice, we have now demonstrated that the lack of Reck in vascular mural cells is largely responsible for mid-gestation lethality. Experiments using cultured aortic explants further revealed that Reck is essential for at least two events in sprouting angiogenesis; (1) correct association of mural and endothelial tip cells to the microvessels and (2) maintenance of fibronectin matrix surrounding the vessels. These findings demonstrate the importance of appropriate cell-cell interactions and ECM maintenance for angiogenesis and the involvement of Reck as a critical regulator of these events.
Asunto(s)
Vasos Sanguíneos/metabolismo , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/metabolismo , Neovascularización Fisiológica/genética , Alelos , Animales , Matriz Extracelular/metabolismo , Femenino , Fibronectinas/metabolismo , Proteínas Ligadas a GPI/deficiencia , Silenciador del Gen , Ratones , Ratones Noqueados , Ratones Transgénicos , Microvasos/metabolismo , Mutación , Especificidad de Órganos/genéticaRESUMEN
The reversion-inducing-cysteine-rich protein with Kazal motifs (RECK) was initially isolated as a transformation-suppressor gene by expression cloning and found to encode a membrane-anchored regulator of the matrix metalloproteinases (MMPs). Experimental studies have shown that RECK can suppress tumour - invasion, metastasis and angiogenesis. However, the clinical impact of RECK remains unclear. To assess the clinical significance of RECK-expression in non-small cell lung cancer (NSCLC), a total of 171 patients with completely resected pathological stage (p-stage) I-IIIA NSCLC were retrospectively examined. Expression of RECK and vascular endothelial growth factor (VEGF) in tumour tissues was assessed by immunohistochemical staining (IHS). Intratumoural microvessel density (IMVD), a measurement of angiogenesis, was also determined by IHS using an anti-CD34 antibody. A significant inverse correlation between RECK-expression and tumour angiogenesis was documented; the mean IMVD in tumours with strong RECK-expression (157.1) was significantly lower than that observed in tumours with weak RECK-expression (194.5; P = 0.008). Interestingly, this inverse correlation was seen only when VEGF was strongly expressed, which suggests that RECK could suppress the angiogenesis induced by VEGF. The 5-year survival rate for patients with tumours with strong RECK-expression (75.8%) was significantly higher than that for patients with weakly expressing tumours (54.3%; P = 0.016). Subset analyses showed that the prognostic impact of RECK-status was evident in patients with either adenocarcinoma, poorly differentiated tumours, or p-stage IIIA disease. A multivariate analysis confirmed that reduced RECK-expression was an independent and significant factor in predicting a poor prognosis (P = 0.009; Hazard ratio (HR), 0.474 with a 95% Confidence interval (CI) of 0.271-0.830). In conclusion, RECK-status is a significant prognostic factor correlated with tumour angiogenesis in NSCLC patients.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Neoplasias Pulmonares/metabolismo , Glicoproteínas de Membrana/metabolismo , Apoptosis , Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Pulmón de Células no Pequeñas/cirugía , División Celular , Femenino , Proteínas Ligadas a GPI , Humanos , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/cirugía , Masculino , Persona de Mediana Edad , Análisis Multivariante , Periodo Posoperatorio , Pronóstico , Análisis de Supervivencia , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
The membrane-anchored metalloproteinase-regulator RECK has been characterized as a tumor suppressor. Here we report that mice with reduced Reck-expression show limb abnormalities including right-dominant, forelimb-specific defects in postaxial skeletal elements. The forelimb buds of low-Reck mutants have an altered dorsal ectoderm with reduced Wnt7a and Igf2 expression, and hypotrophy in two signaling centers (i.e., ZPA and AER) that are essential for limb outgrowth and patterning. Reck is abundantly expressed in the anterior mesenchyme in normal limb buds; mesenchyme-specific Reck inactivation recapitulates the low-Reck phenotype; and some teratogens downregulate Reck in mesenchymal cells. Our findings illustrate a role for Reck in the mesenchymal-epithelial interactions essential for mammalian development.
RESUMEN
The membrane-anchored matrix metalloproteinase-regulator RECK is often downregulated in various types of cancers; the levels of residual RECK in resected tumors often correlate with better prognosis. Forced expression of RECK in cancer cells suppresses tumor angiogenesis, invasion, and metastasis in xenograft models. RECK is therefore a promising marker for benignancy and a potential effector in cancer therapy. We established a cell line containing two transgene systems: (1) the secreted alkaline phosphatase (SEAP) gene fused to Reck promoter and (2) the HRAS(12V) oncogene driven by the Tet-off promoter system. This cell line exhibits transformed phenotype in regular medium and flat morphology with increased SEAP activity in the presence of doxycycline, allowing the assessment of RECK-inducing activity of chemicals in the contexts of both transformed and untransformed cells. Our pilot experiments with 880 known bioactive compounds detected 34 compounds that activate RECK promoter; among these, 10 were authentic anticancer drugs. Four selected compounds up-regulated endogenous RECK protein in several human cancer cell lines. The top-ranking compound, disulfiram, strongly suppressed spontaneous lung-metastasis of human fibrosarcoma cells in nude mice. Our data demonstrate the value of this screen in discovering effective cancer therapeutics.
Asunto(s)
Antineoplásicos/farmacología , Ensayos de Selección de Medicamentos Antitumorales/métodos , Proteínas Ligadas a GPI/genética , Regiones Promotoras Genéticas , Fosfatasa Alcalina/genética , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Disulfiram/farmacología , Doxiciclina/farmacología , Descubrimiento de Drogas , Regulación Neoplásica de la Expresión Génica , Humanos , Immunoblotting , Ratones , Metástasis de la Neoplasia , Pronóstico , Proteínas Proto-Oncogénicas p21(ras)/genética , RatasRESUMEN
Reck is a membrane-anchored glycoprotein identified as a transformation suppressor. Accumulating evidence indicates that Reck negatively regulates a wide spectrum of matrix metalloproteinases and is commonly down-regulated in a variety of malignant solid tumors. Physiological cues that regulate Reck expression, however, remained unknown. In this study, we found that Reck expression was up-regulated at high cell density, low serum, or after treatment with some kinase inhibitors, such as PP2 (Src inhibitor), LY294002 (PI3-kinase inhibitor), and PF573228 (FAK inhibitor), in mouse embryo fibroblasts. Curve fitting indicated that the levels of Reck protein and Reck mRNA are quadratic in the cell density. Other factors, including serum, extracellular matrix components (type I collagen and fibronectin), the kinase inhibitors, and some of their oncogenic targets (v-Src and PIK3CA mutants), modify the shape of the quadratic curve. Comparison of these modifications implicated Src in Reck down-regulation under sparse conditions, PI3-kinase in serum-induced Reck down-regulation, and FAK in Reck down-regulation at high cell density. Fibronectin and type I collagen down-regulated Reck, supporting the role of integrin-FAK signaling in Reck down-regulation at high cell density. Our study has revealed multiple signaling pathways impinging on Reck in cultured mouse embryo fibroblasts and sets a foundation for future studies to find effective Reck inducers of potential value in cancer therapy.
Asunto(s)
Fibroblastos/metabolismo , Glicoproteínas de Membrana/genética , Animales , Recuento de Células , Cromonas/farmacología , Embrión de Mamíferos/citología , Matriz Extracelular/metabolismo , Proteína-Tirosina Quinasas de Adhesión Focal/antagonistas & inhibidores , Proteínas Ligadas a GPI , Glicoproteínas de Membrana/metabolismo , Ratones , Morfolinas/farmacología , Células 3T3 NIH , Inhibidores de las Quinasa Fosfoinosítidos-3 , Pirimidinas/farmacología , Quinolonas/farmacología , Suero/metabolismo , Sulfonas/farmacología , Regulación hacia Arriba , Familia-src Quinasas/antagonistas & inhibidoresRESUMEN
Extracellular matrix (ECM) undergoes continuous remodeling during mammalian development. Although involvement of matrix metalloproteinases (MMPs) in ECM degradation has been well documented, how this process is regulated to allow proper ECM accumulation remains unclear. We previously showed the involvement of a membrane-anchored MMP regulator, RECK (reversion-inducing cysteine-rich protein with Kazal motifs), in vascular development in mice. Here we report that Reck mRNA can be detected in developing cartilage in E13.5 approximately 16.5 mouse embryos and is progressively upregulated during differentiation of a chondrogenic cell line ATDC5 in vitro. In the early phase of ATDC5 differentiation, RECK expression stays low, multiple MMPs are upregulated, and there is ECM degradation at the sites of cellular condensation. In the later phase, RECK is upregulated inside the expanding cartilaginous nodules where type II collagen is accumulated while active ECM degradation persists along the rim of the nodules. Constitutive RECK expression suppressed initial cellular condensation, whereas RECK knockdown suppressed the later ECM accumulation in the cartilaginous nodules. These results suggest that RECK expression at the right place (in the core of the nodules) and at the right time (only in the later phase) is important for proper chondrogenesis and that RECK, together with MMPs, plays a crucial role in regulating dynamic processes of tissue morphogenesis.
Asunto(s)
Diferenciación Celular/genética , Condrocitos/citología , Metaloproteinasas de la Matriz/metabolismo , Glicoproteínas de Membrana/fisiología , Animales , Cartílago/metabolismo , Línea Celular , Condrocitos/metabolismo , Colágeno/metabolismo , Matriz Extracelular/metabolismo , Técnica del Anticuerpo Fluorescente Indirecta , Proteínas Ligadas a GPI , Perfilación de la Expresión Génica , Silenciador del Gen , Immunoblotting , Hibridación in Situ , Metaloproteinasas de la Matriz/fisiología , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ratones , Mutación/genética , Mutación/fisiologíaRESUMEN
BACKGROUND: Reversion-inducing cysteine-rich protein with Kazal motifs (RECK) is a novel membrane-anchored matrix metalloproteinase inhibitor, and experimental studies have shown that RECK can suppress tumor progression through angiogenesis inhibition. We have already revealed that enhanced RECK expression is significantly correlated with a favorable prognosis in non-small-cell lung cancer (NSCLC). In this study, further analyses focused on pN2 disease were conducted to assess the clinical significance of RECK expression. METHODS: A total of 118 patients with completely resected pathologic stage IIIA N2 NSCLC were retrospectively examined. RECK expression in the primary tumor, along with involved N2 nodes, was examined immunohistochemically. RESULTS: RECK expression in the primary tumor was strong in 53 patients (44.9%) and was weak in the other 65 patients. The 5-year survival rate of patients with RECK-strong tumor (42.9%) was significantly higher than that of patients with RECK-weak tumor (23.1%; P = .017). Reduced RECK expression significantly correlated with a poor prognosis for patients with a single N2 node involved (P = .019), but not for patients with multiple N2 nodes involved (P = .440). A multivariate analysis confirmed that reduced RECK expression was an independent and significant factor to predict a poor prognosis (P = .031). RECK expression in involved N2 nodes was significantly higher than in primary tumors (P < .001). CONCLUSIONS: RECK status was a novel prognostic factor in pathologic stage IIIA N2 NSCLC.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Glicoproteínas de Membrana/biosíntesis , Anciano , Apoptosis , Biomarcadores de Tumor/análisis , Femenino , Proteínas Ligadas a GPI , Perfilación de la Expresión Génica , Humanos , Inmunohistoquímica , Metástasis Linfática , Masculino , Glicoproteínas de Membrana/fisiología , Persona de Mediana Edad , Pronóstico , Estudios Retrospectivos , Análisis de SupervivenciaRESUMEN
alpha-Synuclein-positive cytoplasmic inclusions are a pathological hallmark of several neurodegenerative disorders including Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy. Here we report that Sept4, a member of the septin protein family, is consistently found in these inclusions, whereas five other septins (Sept2, Sept5, Sept6, Sept7, and Sept8) are not found in these inclusions. Sept4 and alpha-synuclein can also be co-immunoprecipitated from normal human brain lysates. When co-expressed in cultured cells, FLAG-tagged Sept4 and Myc-tagged alpha-synuclein formed detergent-insoluble complex, and upon treatment with a proteasome inhibitor, they formed Lewy body-like cytoplasmic inclusions. The tagged Sept4 and alpha-synuclein synergistically accelerated cell death induced by the proteasome inhibitor, and this effect was further enhanced by expression of another Lewy body-associated protein, synphilin-1, tagged with the V5 epitope. Moreover, co-expression of the three proteins (tagged Sept4, alpha-synuclein, and synphilin-1) was sufficient to induce cell death. These data raise the possibility that Sept4 is involved in the formation of cytoplasmic inclusions as well as induction of cell death in alpha-synuclein-associated neurodegenerative disorders.