RESUMEN
BACKGROUND: Glioblastoma multiforme (GBM) is the most malignant central nervous system tumor. Alkylating agent, temozolomide (TMZ), is currently the first-line chemotherapeutic agent for GBM. However, the sensitivity of GBM cells to TMZ is affected by many factors. And, several clinic trials, including co-administration of TMZ with other drugs, have failed in successful treatment of GBM. We have previously reported that Netrin-4 (NTN4), a laminin-like axon guidance protein, plays a protective role in GBM cell senescence upon TMZ-triggered DNA damage. However, the master regulator of NTN4 needs further elucidation. Epidermal growth factor/Epidermal growth factor receptor (EGF/EGFR) can modulate the expression of various extracellular matrix related molecules, and prevent DNA damage in GBM cells. In this study, we investigated the relationship between EGF/EGFR signaling and NTN4, and explored their effect on therapeutic efficacy in GBM cells upon TMZ treatment. METHODS: Co-expression analysis were performed by using the RNA sequencing data from NIH 934 cell lines and from single cell RNA sequencing data of GBM tumor. The co-expressing genes were used for GO enrichment and signaling pathway enrichment. mRNA expression of the target genes were quantified by qPCR, and cell senescence were investigated by Senescence-Associated Beta-Galactosidase Staining. Protein phosphorylation were observed and analyzed by immunoblotting. The RNA sequencing data and clinical information of TMZ treated patients were extracted from TCGA-glioblastoma project, and then used for Kaplan-Meier survival analysis. RESULTS: Analysis of RNA sequencing data revealed a potential co-expression relationship between NTN4 and EGFR. GO enrichment of EGFR-correlated genes indicated that EGFR regulates GBM cells in a manner similar to that in central nervous system development and neural cell differentiation. Pathway analysis suggested that EGFR and its related genes contribute to cell adhesion, extracellular matrix (ECM) organization and caspase related signaling. We also show that EGF stimulates NTN4 expression in GBM cells and cooperates with NTN4 to attenuate GBM cell senescence induced by DNA damage, possibly via AKT and ERK. Clinical analysis showed that co-expression of EGFR and NTN4 significantly predicts poor survival in TMZ-treated GBM patients. CONCLUSIONS: This study indicates that EGF/EGFR regulates and cooperates with NTN4 in DNA damage resistance in GBM. Therefore, our findings provide a potential therapeutic target for GBM.
Asunto(s)
Neoplasias Encefálicas/metabolismo , Daño del ADN/fisiología , Factor de Crecimiento Epidérmico/biosíntesis , Glioblastoma/metabolismo , Netrinas/biosíntesis , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Senescencia Celular/fisiología , Factor de Crecimiento Epidérmico/genética , Receptores ErbB/biosíntesis , Receptores ErbB/genética , Glioblastoma/genética , Glioblastoma/patología , Humanos , Netrinas/genética , Regulación hacia Arriba/fisiologíaRESUMEN
Glioblastoma multiforme is an aggressively invasive human brain cancer, which lacks effective treatment. The axonal guidance protein, netrin-1, is overexpressed in glioblastoma tumor biopsies. In Matrigel invasion assays we observed that experimental overexpression of netrin-1 increased cell invasiveness and its downregulation decreased invasiveness. Using tandem affinity purification and mass spectrometry protein identification we found that netrin-1 forms a complex with both Notch2 and Jagged1. Recombinant netrin-1 colocalized with Jagged1 and Notch2 at the cell surface and was also present in the intracellular vesicles with Jagged1, but not with Notch2. Netrin-1 activated Notch signaling and subsequent glioblastoma cell invasion. Interestingly, the recombinant central domain of netrin-1 counteracted the effects of the full-length netrin-1: it inhibited glioblastoma cell invasion and Notch activation by retaining the Notch signaling complex at the cell surface. This finding may give rise to therapeutic applications. These results reveal a new mechanism leading to glioblastoma cell invasion, in which netrin-1 activates Notch signaling.
Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Glioblastoma/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas de la Membrana/metabolismo , Factores de Crecimiento Nervioso/metabolismo , Receptor Notch2/metabolismo , Transducción de Señal , Proteínas Supresoras de Tumor/metabolismo , Proteínas de Unión al Calcio/genética , Línea Celular Tumoral , Glioblastoma/tratamiento farmacológico , Glioblastoma/genética , Glioblastoma/patología , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Proteína Jagged-1 , Proteínas de la Membrana/genética , Invasividad Neoplásica , Factores de Crecimiento Nervioso/genética , Factores de Crecimiento Nervioso/farmacología , Netrina-1 , Estructura Terciaria de Proteína , Receptor Notch2/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Proteínas Serrate-Jagged , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/farmacologíaRESUMEN
PURPOSE: To explore factors related to pathogenesis of rhegmatogenous retinal detachment (RRD) and development of proliferative vitreoretinopathy (PVR), vitreous levels of angiopoietin-1 and -2 (Ang-1 and -2), previously undefined in RRD, transforming growth factor-(TGF) ß1, vascular endothelial growth factor (VEGF), erythropoietin (EPO) and proteolytic mediators of extracellular matrix remodelling (MMP-2 and -9) were compared in eyes with RRD and eyes with idiopathic macular hole or pucker. METHODS: Vitreous samples were collected from 117 eyes with RRD (study group) and 40 eyes with macular hole or pucker (control group). Growth factors were measured by ELISA and matrix metalloproteinases (MMPs) by gelatin zymography. RESULTS: The mean vitreous concentrations of Ang-2, MMP-2, and MMP-9 were higher (all p < 0.01), whereas concentration of VEGF was lower (p = 0.01) in eyes with RRD relative to controls. Logistic regression analysis identified Ang-2 concentration as a novel marker of RRD (p = 0.0001, OR 48.7). Ang-1, EPO, and total TGF-ß1 levels were not significantly different between the groups. However, TGF-ß1 and MMP-2 were increased in eyes with total RRD compared to those with local RRD (p ≤ 0.05). In eyes with PVR, no differences were observed in any studied marker as compared with non-PVR eyes. CONCLUSIONS: Current results reveal Ang-2 as a key factor upregulated in RRD. It may co-operate with fibrosis-associated factors and contribute to vascular complications such as breakdown of blood-eye barrier and PVR development.
Asunto(s)
Angiopoyetina 2/metabolismo , Biomarcadores/metabolismo , Desprendimiento de Retina/metabolismo , Cuerpo Vítreo/metabolismo , Anciano , Angiopoyetina 1/metabolismo , Ensayo de Inmunoadsorción Enzimática , Eritropoyetina/metabolismo , Femenino , Humanos , Masculino , Metaloproteinasas de la Matriz/metabolismo , Persona de Mediana Edad , Estudios Prospectivos , Desprendimiento de Retina/diagnóstico , Factor de Crecimiento Transformador beta1/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Glioblastoma is the most common brain cancer. Ephrins and their Eph receptors play important roles in the development of central nervous system and the regulation of cancer cell migration and invasion. In a search for the Eph receptor complexes, we used tandem affinity purification based interaction screening with tagged ephrins A1, A3 and A4 combined with protein identification by mass-spectrometry in U251MG glioblastoma cells. Ephrins bound to Eph receptors, mainly to EphA2 in these cells. Integrin α3 was identified in protein complexes with ephrin-As. Soluble ephrin-A1 colocalised with integrin α3 at the cell surface, and was rapidly endocytosed by the cells. However, integrin α3 did not colocalise with internalised ephrin-A1, whereas EphA2 receptor did. In U251MG cells, integrin α3 colocalised with EphA2 receptor at the cell edges and protrusions. Sites of EphA2-integrin α3 colocalisation were positive for vinculin, focal adhesion kinase and phosphotyrosine, that is, markers for cell adhesion and active signalling. The interaction between ephrin-As, Eph receptors and integrin α3 is plausibly important for the crosstalk between Eph and integrin signalling pathways at the membrane protrusions and in the migration of brain cancer cells.
Asunto(s)
Neoplasias Encefálicas/metabolismo , Extensiones de la Superficie Celular/metabolismo , Efrinas/metabolismo , Glioblastoma/metabolismo , Integrina alfa3/metabolismo , Receptor EphA2/metabolismo , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Membrana Celular/metabolismo , Adhesiones Focales/metabolismo , Glioblastoma/patología , Humanos , Espacio Intracelular/metabolismo , Ligandos , Unión Proteica , Transporte de Proteínas , Seudópodos/metabolismo , Solubilidad , Factores de TiempoRESUMEN
Tumor cells use membrane type 1 matrix metalloproteinase (MT1-MMP) for invasion and metastasis. However, the signaling mechanisms that underlie MT1-MMP regulation in cancer have remained unclear. Using a systematic gain-of-function kinome screen for MT1-MMP activity, we have here identified kinases that significantly enhance MT1-MMP activity in tumor cells. In particular, we discovered an MT1-MMP/FGF receptor-4 (FGFR4) membrane complex that either stimulates or suppresses MT1-MMP and FGFR4 activities, depending on a tumor progression-associated polymorphism in FGFR4. The FGFR4-R388 allele, linked to poor cancer prognosis, increased collagen invasion by decreasing lysosomal MT1-MMP degradation. FGFR4-R388 induced MT1-MMP phosphorylation and endosomal stabilization, and surprisingly, the increased MT1-MMP in return enhanced FGFR4-R388 autophosphorylation. A phosphorylation-defective MT1-MMP was stabilized on the cell surface, where it induced simultaneous FGFR4-R388 internalization and dissociation of cell-cell junctions. In contrast, the alternative FGFR4-G388 variant down-regulated MT1-MMP, and the overexpression of MT1-MMP and particularly its phosphorylation-defective mutant vice versa induced FGFR4-G388 degradation. These results provide a mechanistic basis for FGFR4-R388 function in cancer invasion.
Asunto(s)
Metaloproteinasa 14 de la Matriz/metabolismo , Polimorfismo Genético , Receptor Tipo 4 de Factor de Crecimiento de Fibroblastos/genética , Endosomas/enzimología , Estabilidad de Enzimas , Humanos , Lisosomas/enzimología , Fosforilación , Transducción de SeñalRESUMEN
The inhalation of asbestos fibers is considered to be highly harmful, and lead to fibrotic and/or malignant disease. Epithelial-to-mesenchymal transition (EMT) is a common pathogenic mechanism in asbestos associated fibrotic (asbestosis) and malignant lung diseases. The characterization of molecular pathways contributing to EMT may provide new possibilities for prognostic and therapeutic applications. The role of asbestos as an inducer of EMT has not been previously characterized. We exposed cultured human lung epithelial cells to crocidolite asbestos and analyzed alterations in the expression of epithelial and mesenchymal marker proteins and cell morphology. Asbestos was found to induce downregulation of E-cadherin protein levels in A549 lung carcinoma cells in 2-dimensional (2D) and 3D cultures. Similar findings were made in primary small airway epithelial cells cultured in 3D conditions where the cells retained alveolar type II cell phenotype. A549 cells also exhibited loss of cell-cell contacts, actin reorganization and expression of α-smooth muscle actin (α-SMA) in 2D cultures. These phenotypic changes were not associated with increased transforming growth factor (TGF)-ß signaling activity. MAPK/Erk signaling pathway was found to mediate asbestos-induced downregulation of E-cadherin and alterations in cell morphology. Our results suggest that asbestos can induce epithelial plasticity, which can be interfered by blocking the MAPK/Erk kinase activity.
Asunto(s)
Células Epiteliales Alveolares/efectos de los fármacos , Asbesto Crocidolita/toxicidad , Células Epiteliales/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Alveolos Pulmonares/citología , Actinas/biosíntesis , Células Epiteliales Alveolares/metabolismo , Células Epiteliales Alveolares/patología , Cadherinas/biosíntesis , Línea Celular Tumoral , Células Epiteliales/metabolismo , Transición Epitelial-Mesenquimal/efectos de los fármacos , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , FN-kappa B/antagonistas & inhibidores , Alveolos Pulmonares/metabolismo , Fibrosis Pulmonar , Factor de Crecimiento Transformador beta/biosíntesisRESUMEN
Idiopathic pulmonary fibrosis (IPF) is a progressive disease of unknown cause. The pathogenesis of the disease is characterized by fibroblast accumulation and excessive transforming growth factor-ß (TGF-ß) activation. Although TGF-ß activation is a complex process involving various protein interactions, little is known of the specific routes of TGF-ß storage and activation in human lung. Here, we have systematically analyzed the expression of specific proteins involved in extracellular matrix targeting and activation of TGF-ß. Latent TGF-ß-binding protein (LTBP)-1 was found to be significantly upregulated in IPF patient lungs. LTBP-1 expression was especially high in the fibroblastic foci, in which P-Smad2 immunoreactivity, indicative of TGF-ß signaling activity, was less prominent. In cultured primary lung fibroblasts and epithelial cells, short-interfering-RNA-mediated downregulation of LTBP-1 resulted in either increased or decreased TGF-ß signaling activity, respectively, suggesting that LTBP-1-mediated TGF-ß activation is dependent on the cellular context in the lung. Furthermore, LTBP-1 was shown to colocalize with fibronectin, fibrillin-1 and fibrillin-2 proteins in the IPF lung. Fibrillin-2, a developmental gene expressed only in blood vessels in normal adult lung, was found specifically upregulated in IPF fibroblastic foci. The TGF-ß-activating integrin ß8 subunit was expressed at low levels in both control and IPF lungs. Alterations in extracellular matrix composition, such as high levels of the TGF-ß storage protein LTBP-1 and the re-appearance of fibrillin-2, probably modulate TGF-ß availability and activation in different pulmonary compartments in the fibrotic lung.
Asunto(s)
Fibrosis Pulmonar Idiopática/metabolismo , Pulmón/metabolismo , Pulmón/patología , Factor de Crecimiento Transformador beta1/metabolismo , Adulto , Anciano , Estudios de Casos y Controles , Células Cultivadas , Regulación hacia Abajo/genética , Células Epiteliales/metabolismo , Células Epiteliales/patología , Fibrilina-1 , Fibrilina-2 , Fibrilinas , Fibroblastos/metabolismo , Fibroblastos/patología , Fibronectinas/metabolismo , Humanos , Integrinas/metabolismo , Proteínas de Unión a TGF-beta Latente/genética , Proteínas de Unión a TGF-beta Latente/metabolismo , Proteínas de Microfilamentos/metabolismo , Persona de Mediana Edad , Fosforilación , Transporte de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteína Smad2/metabolismo , Regulación hacia Arriba/genéticaRESUMEN
Proteolytic fragments of various components of the extracellular matrix exhibit antiangiogenic activity via interaction with endothelial cell surface integrins. Kalluri and coworkers (this issue of Cancer Cell) use gene-targeted mice to show a physiological role for a carboxy-terminal fragment of collagen IV in the regulation of tumor angiogenesis.
Asunto(s)
Colágeno Tipo IV/fisiología , Neoplasias/irrigación sanguínea , Neovascularización Patológica/metabolismo , Animales , Matriz Extracelular/fisiología , Humanos , Metaloproteinasa 9 de la Matriz/fisiología , Ratones , Ratones NoqueadosRESUMEN
Our previous studies of chromosome 14 transfer into tumorigenic esophageal squamous cell carcinoma (ESCC) cell line, SLMT, suggested the existence of tumor suppressor genes on chromosome 14. Gene expression profiling of microcell hybrids and the tumor segregants identified an interesting gene, LTBP-2 (latent transforming growth factor ß binding protein 2), which has been analyzed here for its role in ESCC. LTBP-2 maps to 14q24 and encodes a secreted protein, which is a component of the extracellular matrix microfibrils. LTBP-2 expression was downregulated in ESCC cell lines and tumor tissues. Promoter hypermethylation was found to be involved in LTBP-2 inactivation. Functional studies indicated its tumor-suppressive roles in ESCC. In the in vitro colony formation and Matrigel three-dimensional culture assays, LTBP-2 decreased the colony-forming abilities of ESCC cell lines. LTBP-2 expression was associated with reduction of cell migrating and invasive abilities. LTBP-2 could also reduce the tube-forming ability of endothelial cells. Moreover, LTBP-2 induced tumor suppression in in vivo nude mouse assays. Tissue microarray immunohistochemical staining analysis indicated that LTBP-2 expression is reduced in tumor tissues when compared to normal tissues, and LTBP-2 expression correlated significantly with the survival of ESCC patients. Thus, LTBP-2 appears to play an important role in ESCC.
Asunto(s)
Carcinoma de Células Escamosas/metabolismo , Neoplasias Esofágicas/metabolismo , Proteínas de Unión a TGF-beta Latente/metabolismo , Animales , Carcinoma de Células Escamosas/mortalidad , Línea Celular Tumoral , Metilación de ADN , Regulación hacia Abajo , Neoplasias Esofágicas/mortalidad , Femenino , Genes Supresores de Tumor , Humanos , Proteínas de Unión a TGF-beta Latente/genética , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Trasplante de Neoplasias , Ensayo de Tumor de Célula MadreRESUMEN
Several members of the matrix metalloproteinase (MMP) family function in various processes of innate immunity, particularly in controlling leukocyte influx. Epilysin (MMP-28) is expressed in numerous tissues and, in adult mice, it has the highest expression in lung, where it is detected in bronchial epithelial cells (Clara cells). Epilysin is also expressed by bone marrow-derived macrophages, but not by alveolar macrophages, suggesting that its expression by macrophages is dependent on localization and differentiation. To assess the role of this MMP, we generated epilysin-null (Mmp28(-/-)) mice. Although epilysin is constitutively expressed in normal tissues, Mmp28(-/-) mice have no overt phenotype. However, using a murine model of Pseudomonas aeruginosa pneumonia, we found that Mmp28(-/-) mice had an early increase in macrophage recruitment into the lungs, as well as enhanced bacterial clearance and reduced pulmonary neutrophilia, which we predicted were due to accelerated macrophage influx. Macrophage depletion in WT and Mmp28(-/-) mice confirmed a role for macrophages in clearing P. aeruginosa and regulating neutrophil recruitment. Furthermore, we observed that macrophages derived from Mmp28(-/-) mice migrated faster than did wild-type cells to bronchoalveolar lavage fluid from P. aeruginosa-treated mice of either genotype. These observations indicate that epilysin functions as an intrinsic negative regulator of macrophage recruitment by retarding the chemotaxis of these cells.
Asunto(s)
Inhibición de Migración Celular/inmunología , Quimiotaxis de Leucocito/inmunología , Macrófagos Alveolares/inmunología , Metaloproteinasas de la Matriz Secretadas/fisiología , Neumonía Bacteriana/inmunología , Infecciones por Pseudomonas/inmunología , Animales , Células de la Médula Ósea/enzimología , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/patología , Inhibición de Migración Celular/genética , Células Cultivadas , Quimiotaxis de Leucocito/genética , Femenino , Mediadores de Inflamación/metabolismo , Mediadores de Inflamación/fisiología , Macrófagos Alveolares/enzimología , Macrófagos Alveolares/patología , Masculino , Metaloproteinasas de la Matriz Secretadas/deficiencia , Metaloproteinasas de la Matriz Secretadas/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neutrófilos/enzimología , Neutrófilos/inmunología , Neutrófilos/patología , Neumonía Bacteriana/enzimología , Neumonía Bacteriana/patología , Infecciones por Pseudomonas/enzimología , Infecciones por Pseudomonas/patología , Factores de TiempoRESUMEN
Transforming growth factor (TGF)-beta is secreted and targeted into the extracellular matrix (ECM) in association with one of the latent TGF-beta binding proteins (LTBPs). Activation of these latent complexes is an important regulatory step in TGF-beta signaling. LTBPs target the growth factor into the ECM and expose it to activating mechanisms. Disruption of LTBP-4 gene causes severe developmental abnormalities in both humans and mice. Transcripts for two N-terminally distinct LTBP-4 variants, LTBP-4S (short) and -4L (long), have been identified. In the current work, we have characterized differences in the expression, processing, and ECM targeting of these LTBP-4 variants. Heart and skeletal muscle displayed expression of both variants, while liver expressed mainly LTBP-4L and lung as well as small intestine LTBP-4S. This tissue-specific expression pattern was found to originate from control of transcription by two independent promoters. Furthermore, LTBP-4S and -4L proteins were secreted and processed differently. During secretion, LTBP-4L was complexed with TGF-beta1, whereas the majority of LTBP-4S was secreted in a free form. In addition, LTBP-4S was incorporated into the ECM, while full-length LTBP-4L was not readily detectable in the ECM. These data suggest that LTBP-4 functions are modified by tissue-specific expression of the two N-terminally distinct variants, which in addition exhibit significant differences in cellular processing and targeting, that is, this provides a basis for understanding molecular diversity in LTBP-4 structure and function.
Asunto(s)
Fibroblastos/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Proteínas de Unión a TGF-beta Latente/genética , Animales , Secuencia de Bases , Células Cultivadas , Secuencia Conservada , Humanos , Proteínas de Unión a TGF-beta Latente/química , Proteínas de Unión a TGF-beta Latente/metabolismo , Ratones , Datos de Secuencia Molecular , Regiones Promotoras Genéticas/genética , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Procesamiento Proteico-Postraduccional , Análisis de Secuencia de ADN , Sitio de Iniciación de la TranscripciónRESUMEN
The components of the extracellular matrix (ECM) and their differential expression patterns play important roles in tissue formation. The deposition of latent TGF-beta binding proteins (LTBPs) to the ECM exhibit distinct distribution profiles. We have analyzed here the temporal and spatial ECM association of latent TGF-beta binding protein LTBP-2 in cultured human embryonic lung fibroblasts. We found that LTBP-2 was not assembled to the ECM until by confluency of cultures following the deposition of fibronectin (FN) and fibrillin-1. In 5-day-old cultures LTBP-2 was rapidly secreted from cells and it subsequently associated with the ECM as shown by metabolic labeling and immunoprecipitation. LTBP-2 colocalized transiently with fibronectin and failed to assemble to the ECM of FN deficient mouse fibroblasts. Analysis of different cultured human cell lines revealed partial colocalization of LTBP-2 and fibrillin-1 in the ECM of fibroblasts, MG-63 osteosarcoma cells and human vascular endothelial cells. Silencing of fibrillin-1 expression by lentiviral shRNAs profoundly disrupted the deposition of LTBP-2. Current results suggest that LTBP-2 is not an element of the provisional ECM of fibroblasts but is more likely a component of more mature ECM and indicate that matrix association of LTBP-2 depends on a pre-formed fibrillin-1 network.
Asunto(s)
Matriz Extracelular/metabolismo , Proteínas de Unión a TGF-beta Latente/metabolismo , Proteínas de Microfilamentos/metabolismo , Animales , Línea Celular , Línea Celular Tumoral , Células Cultivadas , Embrión de Mamíferos/citología , Células Endoteliales/metabolismo , Fibrilina-1 , Fibrilinas , Fibroblastos/metabolismo , Fibronectinas/metabolismo , Expresión Génica/genética , Humanos , Proteínas de Unión a TGF-beta Latente/genética , Ratones , Ratones Noqueados , Proteínas de Microfilamentos/genética , Unión Proteica/fisiología , Interferencia de ARNRESUMEN
High biological activity of the transforming growth factor (TGF)-beta-Smad pathway characterizes the malignant phenotype of malignant gliomas and confers poor prognosis to glioma patients. Accordingly, TGF-beta has become a novel target for the experimental treatment of these tumors. TGF-beta is processed by furin-like proteases (FLP) and secreted from cells in a latent complex with its processed propeptide, the latency-associated peptide (LAP). Latent TGF-beta-binding protein 1 (LTBP-1) covalently binds to this small latent TGF-beta complex (SLC) and regulates its function, presumably via interaction with the extracellular matrix (ECM). We report here that the levels of LTBP-1 protein in vivo increase with the grade of malignancy in gliomas. LTBP-1 is associated with the ECM as well as secreted into the medium in cultured malignant glioma cells. The release of LTBP-1 into the medium is decreased by the inhibition of FLP activity. Gene-transfer mediated overexpression of LTBP-1 in glioma cell lines results in an increase inTGF-beta activity. Accordingly, Smad2 phosphorylation as an intracellular marker of TGF-beta activity is enhanced. Conversely, LTBP-1 gene silencing reduces TGF-beta activity and Smad2 phosphorylation without affecting TGF-beta protein levels. Collectively, we identify LTBP-1 as an important modulator of TGF-beta activation in glioma cells, which may contribute to the malignant phenotype of these tumors.
Asunto(s)
Neoplasias Encefálicas/metabolismo , Glioma/metabolismo , Proteínas de Unión a TGF-beta Latente/metabolismo , Transducción de Señal , Proteína Smad2/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Astrocitoma/metabolismo , Línea Celular Tumoral , Proliferación Celular , Matriz Extracelular/metabolismo , Regulación Neoplásica de la Expresión Génica , Glioblastoma/metabolismo , Humanos , Immunoblotting , Inmunohistoquímica , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenotipo , Fosforilación , Reacción en Cadena de la Polimerasa , Regulación hacia ArribaRESUMEN
Transforming growth factor (TGF)-betas are powerful cytokines that are secreted as inactive (latent) precursors into the extracellular space. To exert their pleiotropic functions, latent TGF-betas require activation. This requisite restricts TGF-beta signaling to tissues that express TGF-beta-activating proteins such as the adhesion molecule alphavbeta6 integrin. Recent work has uncovered the molecular mechanism by which alphavbeta6 integrin activates latent TGF-beta. Latent-TGF-beta-binding protein 1 has been identified as being the major component of this process, and the integrin-interacting region has been mapped to a poorly conserved sequence stretch called the hinge region.
Asunto(s)
Antígenos de Neoplasias/fisiología , Integrinas/fisiología , Péptidos y Proteínas de Señalización Intracelular/fisiología , Factor de Crecimiento Transformador beta/fisiología , Animales , Antígenos de Neoplasias/metabolismo , Humanos , Integrinas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de Unión a TGF-beta Latente , Modelos Biológicos , Isoformas de Proteínas/fisiología , Estrés Mecánico , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
We have analyzed the effects of latent TGF-beta binding protein 2 (LTBP-2) and its fragments on lung fibroblast adhesion. Quantitative cell adhesion assays indicated that fibroblasts do not adhere to full-length LTBP-2. Interestingly, LTBP-2 had dominant disrupting effects on the morphology of fibroblasts adhering to fibronectin (FN). Fibroblasts plated on LTBP-2 and FN substratum exhibited less adherent morphology and displayed clearly decreased actin stress fibers than cells plated on FN. These cells formed, instead, extensive membrane ruffles. LTBP-2 had no effects on cells adhering to collagen type I. Fibroblasts adhered weakly to the NH2-terminal fragment of LTBP-2. Unlike FN, this fragment did not augment actin stress fiber formation. Interestingly, the adhesion-mediating and cytoskeleton-disrupting effects were localized to the same NH2-terminal proline-rich region of LTBP-2. LTBP-2 and its antiadhesive fragment bound to FN in vitro, and the antiadhesive fragment associated with the extracellular matrix FN fibrils. These observations reveal a potentially important role for LTBP-2 as an antiadhesive matrix component.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Portadoras/metabolismo , Fibroblastos/metabolismo , Fibronectinas/metabolismo , Citoesqueleto de Actina/metabolismo , Animales , Sitios de Unión/efectos de los fármacos , Sitios de Unión/fisiología , Células CHO , Células COS , Proteínas Portadoras/farmacología , Adhesión Celular/efectos de los fármacos , Adhesión Celular/fisiología , Moléculas de Adhesión Celular/metabolismo , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Tamaño de la Célula/efectos de los fármacos , Tamaño de la Célula/fisiología , Extensiones de la Superficie Celular/efectos de los fármacos , Extensiones de la Superficie Celular/metabolismo , Cricetinae , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/fisiología , Matriz Extracelular/efectos de los fármacos , Matriz Extracelular/metabolismo , Fibroblastos/efectos de los fármacos , Adhesiones Focales/metabolismo , Humanos , Proteínas de Unión a TGF-beta Latente , Unión Proteica/fisiología , Estructura Terciaria de Proteína/efectos de los fármacos , Estructura Terciaria de Proteína/fisiología , Fibras de Estrés/metabolismoRESUMEN
Disruption of latent TGF-beta binding protein (LTBP)-4 expression in the mouse leads to abnormal lung development and colorectal cancer. Lung fibroblasts from these mice produced decreased amounts of active TGF-beta, whereas secretion of latent TGF-beta was significantly increased. Expression and secretion of TGF-beta2 and -beta3 increased considerably. These results suggested that TGF-beta activation but not secretion would be severely impaired in LTBP-4 -/- fibroblasts. Microarrays revealed increased expression of bone morphogenic protein (BMP)-4 and decreased expression of its inhibitor gremlin. This finding was accompanied by enhanced expression of BMP-4 target genes, inhibitors of differentiation 1 and 2, and increased deposition of fibronectin-rich extracellular matrix. Accordingly, increased expression of BMP-4 and decreased expression of gremlin were observed in mouse lung. Transfection of LTBP-4 rescued the -/- fibroblast phenotype, while LTBP-1 was inefficient. Treatment with active TGF-beta1 rescued BMP-4 and gremlin expression to wild-type levels. Our results indicate that the lack of LTBP-4-mediated targeting and activation of TGF-beta1 leads to enhanced BMP-4 signaling in mouse lung.
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Proteínas Adaptadoras Transductoras de Señales/fisiología , Proteínas Morfogenéticas Óseas/metabolismo , Transducción de Señal , Factor de Crecimiento Transformador beta/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Northern Blotting , Proteína Morfogenética Ósea 4 , Diferenciación Celular , ADN Complementario/metabolismo , Electroforesis en Gel de Poliacrilamida , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Humanos , Inmunohistoquímica , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas de Unión a TGF-beta Latente , Pulmón/metabolismo , Ratones , Ratones Transgénicos , Microscopía Fluorescente , Análisis de Secuencia por Matrices de Oligonucleótidos , Oligonucleótidos/química , Fenotipo , ARN/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transfección , Regulación hacia ArribaRESUMEN
Targeting of transforming growth factor beta (TGF-beta) to the extracellular matrix (ECM) by latent TGF-beta binding proteins (LTBPs) regulates the availability of TGF-beta for interactions with endothelial cells during their quiescence and activation. However, the mechanisms which release TGF-beta complexes from the ECM need elucidation. We find here that morphological activation of endothelial cells by phorbol 12-myristate 13-acetate (PMA) resulted in membrane-type 1 matrix metalloproteinase (MT1-MMP) -mediated solubilization of latent TGF-beta complexes from the ECM by proteolytic processing of LTBP-1. These processes required the activities of PKC and ERK1/2 signaling pathways and were coupled with markedly increased MT1-MMP expression. The functional role of MT1-MMP in LTBP-1 release was demonstrated by gene silencing using lentiviral short-hairpin RNA as well as by the inhibition with tissue inhibitors of metalloproteinases, TIMP-2 and TIMP-3. Negligible effects of TIMP-1 and uPA/plasmin system inhibitors indicated that secreted MMPs or uPA/plasmin system did not contribute to the release of LTBP-1. Current results identify MT1-MMP-mediated proteolytic processing of ECM-bound LTBP-1 as a mechanism to release latent TGF-beta from the subendothelial matrix.
Asunto(s)
Células Endoteliales/metabolismo , Matriz Extracelular/metabolismo , Proteínas de Unión a TGF-beta Latente/metabolismo , Metaloproteinasa 14 de la Matriz/fisiología , Factor de Crecimiento Transformador beta1/metabolismo , Forma de la Célula/fisiología , Células Cultivadas , Células Endoteliales/citología , Células Endoteliales/enzimología , Matriz Extracelular/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Humanos , Metaloproteinasa 14 de la Matriz/genética , Metaloproteinasa 14 de la Matriz/metabolismo , Inhibidores de la Metaloproteinasa de la Matriz , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Proteína Quinasa C/metabolismo , Procesamiento Proteico-Postraduccional/efectos de los fármacos , ARN Interferente Pequeño/farmacología , Transducción de Señal/fisiologíaRESUMEN
Latent transforming growth factor (TGF)-beta binding proteins are extracellular matrix (ECM) proteins involved in the regulation of TGF-beta sequestration and activation. In this study, we have identified binding domains in LTBP-4, which mediate matrix targeting and cell adhesion. LTBP-4 was found to possess heparin binding activity, especially in its N-terminal region. The C-terminal domain of LTBP-4 supported fibroblast adhesion, a property reduced by soluble heparin. In addition, we found that LTBP-4 binds directly to fibronectin (FN), which was indispensable for the matrix assembly of LTBP-4. The FN binding sites were also located in the N-terminal region. Interestingly, heparin was able to reduce the binding of LTBP-4 to FN. In fibroblast cultures, LTBP-4 colocalized first with FN and subsequently with fibrillin-1, pointing to a role for FN in the early assembly of LTBP-4. In FN -/- fibroblasts, LTBP-mediated ECM targeting was disturbed, resulting in increased TGF-beta activity. These results revealed new molecular interactions which are evidently important for the ECM targeting, but which also are evidence of novel functions for LTBP-4 as an adhesion molecule.
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Comunicación Celular , Matriz Extracelular/metabolismo , Fibronectinas/metabolismo , Heparina/metabolismo , Proteínas de Unión a TGF-beta Latente/metabolismo , Proteínas de Unión a TGF-beta Latente/fisiología , Animales , Sitios de Unión/fisiología , Células CHO , Adhesión Celular , Células Cultivadas , Cricetinae , Cricetulus , Fibroblastos/metabolismo , Humanos , Proteínas de Unión a TGF-beta Latente/química , Ratones , Visón , Modelos Biológicos , Unión Proteica , Estructura Terciaria de Proteína/fisiologíaRESUMEN
RATIONALE: Members of the transforming growth factor (TGF)-beta superfamily, including TGF-betas and bone morphogenetic proteins (BMPs), are essential for the maintenance of tissue homeostasis and regeneration after injury. We have observed that the BMP antagonist, gremlin, is highly up-regulated in idiopathic pulmonary fibrosis (IPF). OBJECTIVES: To investigate the role of gremlin in the regulation of BMP signaling in pulmonary fibrosis. METHODS: Progressive asbestos-induced fibrosis in the mouse was used as a model of human IPF. TGF-beta and BMP expression and signaling activities were measured from murine and human fibrotic lungs. The mechanism of gremlin induction was analyzed in cultured lung epithelial cells. In addition, the possible therapeutic role of gremlin inhibition was tested by administration of BMP-7 to mice after asbestos exposure. MEASUREMENTS AND MAIN RESULTS: Gremlin mRNA levels were up-regulated in the asbestos-exposed mouse lungs, which is in agreement with the human IPF biopsy data. Down-regulation of BMP signaling was demonstrated by reduced levels of Smad1/5/8 and enhanced Smad2 phosphorylation in asbestos-treated lungs. Accordingly, analyses of cultured human bronchial epithelial cells indicated that asbestos-induced gremlin expression could be prevented by inhibitors of the TGF-beta receptor and also by inhibitors of the mitogen-activated protein kinase kinase/extracellular signal-regulated protein kinase pathways. BMP-7 treatment significantly reduced hydroxyproline contents in the asbestos-treated mice. CONCLUSIONS: The TGF-beta and BMP signaling balance is important for lung regenerative events and is significantly perturbed in pulmonary fibrosis. Rescue of BMP signaling activity may represent a potential beneficial strategy for treating human pulmonary fibrosis.
Asunto(s)
Proteínas Morfogenéticas Óseas/fisiología , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Fibrosis Pulmonar/fisiopatología , Factor de Crecimiento Transformador beta/fisiología , Animales , Amianto/efectos adversos , Proteína Morfogenética Ósea 7 , Línea Celular Tumoral , Modelos Animales de Enfermedad , Regulación hacia Abajo , Humanos , Ratones , Fibrosis Pulmonar/inducido químicamente , Fibrosis Pulmonar/metabolismo , Transducción de Señal , Factor de Crecimiento Transformador beta1/fisiología , Regulación hacia ArribaRESUMEN
Chronic obstructive pulmonary disease (COPD) is characterized by inflammatory immune response, emphysematous destruction of alveolar structures and obstruction in the small conducting airways. Transforming growth factor (TGF)-beta is involved in the maintenance of normal lung tissue homeostasis as a regulator of extracellular proteolysis, tissue repair and inflammatory functions. This study was undertaken to characterize TGF-beta signaling in pathologically distinct areas of COPD lungs. Using Smad2 phosporylation (P-Smad2) as an indicator of TGF-beta signaling activity we analyzed COPD patient tissues and controls by immunohistochemistry. Emphysematous lung showed significantly reduced P-Smad2 immunoreactivity both in the alveolar and bronchiolar epithelium, which is evidence of reduced TGF-beta signaling activity. On the contrary, in the thickened peribronchial areas there was an increase in the amount of P-Smad2 positive cells. Isolated COPD lung fibroblasts also displayed increased TGF-beta signaling and target gene expression suggesting that the fibroblasts are characteristic to the small airway disease phenotype. Our results indicate that TGF-beta signaling activity is differentially regulated in distinct areas of COPD lung and likely contributes to both emphysematous development and small airway obstruction.