RESUMEN
The delivery of glucose and antioxidants is vital to maintain homeostasis and lens transparency. Here, we report a new mechanism whereby mechanically activated connexin (Cx) hemichannels serve as a transport portal for delivering glucose and glutathione (GSH). Integrin α6ß1 in outer cortical lens fiber activated by fluid flow shear stress (FFSS) induced opening of hemichannels. Inhibition of α6 activation prevented hemichannel opening as well as glucose and GSH uptake. The activation of integrin ß1, a heterodimeric partner of α6 in the absence of FFSS, increased Cx50 hemichannel opening. Hemichannel activation by FFSS depended on the interaction of integrin α6 and Cx50 C-terminal domain. Moreover, hemichannels in nuclear fiber were unresponsive owing to Cx50 truncation. Taken together, these results show that mechanically activated α6ß1 integrin in outer cortical lens fibers leads to opening of hemichannels, which transport glucose and GSH into cortical lens fibers. This study unveils a new transport mechanism that maintains metabolic and antioxidative function of the lens.
Asunto(s)
Antioxidantes/metabolismo , Proteínas Aviares/metabolismo , Conexinas/metabolismo , Glutatión/metabolismo , Integrina alfa6beta1/metabolismo , Cristalino/metabolismo , Mecanotransducción Celular , Animales , Proteínas Aviares/genética , Transporte Biológico Activo , Embrión de Pollo , Pollos , Conexinas/genética , Integrina alfa6beta1/genética , Dominios ProteicosRESUMEN
Integrins, the major receptors for cell-extracellular matrix (ECM) interactions, regulate multiple cell biological processes including adhesion, migration, proliferation and growth factor-dependent signaling. The principal laminin (LM) binding integrins α3ß1, α6ß1 and α6ß4 are usually co-expressed in cells and bind to multiple laminins with different affinities making it difficult to define their specific function. In this study, we generated kidney epithelial collecting duct (CD) cells that lack both the α3 and α6 integrin subunits. This deletion impaired cell adhesion and migration to LM-332 and LM-511 more than deleting α3 or α6 alone. Cell adhesion mediated by both α3ß1 and α6 integrins was PI3K independent, but required K63-linked polyubiquitination of Akt by the ubiquitin-modifying enzyme TRAF6. Moreover, we provide evidence that glial-derived neurotrophic factor (GDNF) and fibroblast growth factor 10 (FGF10)- mediated cell signaling, spreading and proliferation were severely compromised in double integrin α3/α6- but not single α3- or α6-null CD cells. Interestingly, these growth factor-dependent cell functions required both PI3K- and TRAF6-dependent Akt activation. These data suggest that expression of the integrin α3 or α6 subunit is sufficient to mediate GDNF- and FGF10-dependent spreading, proliferation and signaling on LM-511. Thus, our study shows that α3 and α6 containing integrins promote distinct functions and signaling by CD cells on laminin substrata.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Células Epiteliales/metabolismo , Matriz Extracelular/metabolismo , Integrina alfa3/metabolismo , Integrina alfa6/metabolismo , Laminina/metabolismo , Transducción de Señal , Animales , Adhesión Celular/efectos de los fármacos , Moléculas de Adhesión Celular/química , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Matriz Extracelular/química , Matriz Extracelular/efectos de los fármacos , Factor 10 de Crecimiento de Fibroblastos/farmacología , Eliminación de Gen , Regulación de la Expresión Génica , Factor Neurotrófico Derivado de la Línea Celular Glial/farmacología , Humanos , Integrina alfa3/genética , Integrina alfa3beta1/genética , Integrina alfa3beta1/metabolismo , Integrina alfa6/genética , Integrina alfa6beta1/genética , Integrina alfa6beta1/metabolismo , Integrina alfa6beta4/genética , Integrina alfa6beta4/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Túbulos Renales Colectores/citología , Túbulos Renales Colectores/metabolismo , Laminina/química , Ratones , Ratones Noqueados , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Cultivo Primario de Células , Unión Proteica , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Factor 6 Asociado a Receptor de TNF/genética , Factor 6 Asociado a Receptor de TNF/metabolismo , KalininaRESUMEN
Laminins regulate diverse cellular functions through interaction with integrins. Two regions of laminins-three laminin globular domains of the α chain (LG1-3) and the carboxyl-terminal tail of the γ chain (γ-tail)-are required for integrin binding, but it remains unclear how the γ-tail contributes to the binding. We determined the crystal structure of the integrin binding fragment of laminin-511, showing that the γ-tail extends to the bottom face of LG1-3. Electron microscopic imaging combined with biochemical analyses showed that integrin binds to the bottom face of LG1-3 with the γ1-tail apposed to the metal ion-dependent adhesion site (MIDAS) of integrin ß1. These findings are consistent with a model in which the γ-tail coordinates the metal ion in the MIDAS through its Glu residue.
Asunto(s)
Integrina alfa6beta1/química , Laminina/química , Sitios de Unión , Integrina alfa6beta1/genética , Integrina alfa6beta1/metabolismo , Iones/química , Laminina/genética , Laminina/metabolismo , Metales/química , Modelos Moleculares , Unión Proteica , Conformación Proteica , Multimerización de Proteína , Proteínas Recombinantes , Relación Estructura-ActividadRESUMEN
BACKGROUND: Angiogenesis, the process in which new blood vessels are formed from preexisting ones, is highly dependent on the presence of classical angiogenic factors. Recent evidence suggests that axonal guidance proteins and their receptors can also act as angiogenic regulators. Netrin, a family of laminin-like proteins, specifically Netrin-1 and 4, act via DCC/Neogenin-1 and UNC5 class of receptors to promote or inhibit angiogenesis, depending on the physiological context. METHODS: Mesenchymal stem cells secrete a broad set of classical angiogenic factors. However, little is known about the expression of non-canonical angiogenic factors such as Netrin-1. The aim was to characterize the possible secretion of Netrin ligands by Wharton's jelly-derived mesenchymal stem cells (WJ-MSC). We evaluated if Netrin-1 presence in the conditioned media from these cells was capable of inducing angiogenesis both in vitro and in vivo, using human umbilical vein endothelial cells (HUVEC) and chicken chorioallantoic membrane (CAM), respectively. In addition, we investigated if the RhoA/ROCK pathway is responsible for the integration of Netrin signaling to control vessel formation. RESULTS: The paracrine angiogenic effect of the WJ-MSC-conditioned media is mediated at least in part by Netrin-1 given that pharmacological blockage of Netrin-1 in WJ-MSC resulted in diminished angiogenesis on HUVEC. When HUVEC were stimulated with exogenous Netrin-1 assayed at physiological concentrations (10-200 ng/mL), endothelial vascular migration occurred in a concentration-dependent manner. In line with our determination of Netrin-1 present in WJ-MSC-conditioned media we were able to obtain endothelial tubule formation even in the pg/mL range. Through CAM assays we validated that WJ-MSC-secreted Netrin-1 promotes an increased angiogenesis in vivo. Netrin-1, secreted by WJ-MSC, might mediate its angiogenic effect through specific cell surface receptors on the endothelium, such as UNC5b and/or integrin α6ß1, expressed in HUVEC. However, the angiogenic response of Netrin-1 seems not to be mediated through the RhoA/ROCK pathway. CONCLUSIONS: Thus, here we show that stromal production of Netrin-1 is a critical component of the vascular regulatory machinery. This signaling event may have deep implications in the modulation of several processes related to a number of diseases where angiogenesis plays a key role in vascular homeostasis.
Asunto(s)
Membrana Corioalantoides/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Madre Mesenquimatosas/metabolismo , Neovascularización Fisiológica/efectos de los fármacos , Factores de Crecimiento Nervioso/farmacología , Proteínas Supresoras de Tumor/farmacología , Gelatina de Wharton/metabolismo , Animales , Bioensayo , Movimiento Celular , Embrión de Pollo , Membrana Corioalantoides/irrigación sanguínea , Membrana Corioalantoides/citología , Medios de Cultivo Condicionados/química , Medios de Cultivo Condicionados/farmacología , Relación Dosis-Respuesta a Droga , Regulación de la Expresión Génica , Células Endoteliales de la Vena Umbilical Humana/citología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Integrina alfa6beta1/genética , Integrina alfa6beta1/metabolismo , Células Madre Mesenquimatosas/citología , Factores de Crecimiento Nervioso/genética , Factores de Crecimiento Nervioso/metabolismo , Receptores de Netrina , Netrina-1 , Cultivo Primario de Células , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Transducción de Señal , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Gelatina de Wharton/citologíaRESUMEN
Laminins are a major constituent of the basement membranes of the kidney collecting system. Integrins, transmembrane receptors formed by non-covalently bound α and ß subunits, serve as laminin receptors, but their role in development and homeostasis of the kidney collecting system is poorly defined. Integrin α3ß1, one of the major laminin receptors, plays a minor role in kidney collecting system development, while the role of α6 containing integrins (α6ß1 and α6ß4), the other major laminin receptors, is unknown. Patients with mutations in α6 containing integrins not only develop epidermolysis bullosa, but also have abnormalities in the kidney collecting system. In this study, we show that selectively deleting the α6 or ß4 integrin subunits at the initiation of ureteric bud development in mice does not affect morphogenesis. However, the collecting system becomes dilated and dysmorphic as the mice age. The collecting system in both null genotypes was also highly susceptible to unilateral ureteric obstruction injury with evidence of excessive tubule dilatation and epithelial cell apoptosis. Mechanistically, integrin α6-null collecting duct cells are unable to withstand high mechanical force when adhered to laminin. Thus, we conclude that α6 integrins are important for maintaining the integrity of the kidney collecting system by enhancing tight adhesion of the epithelial cells to the basement membrane. These data give a mechanistic explanation for the association between kidney collecting system abnormalities in patients and epidermolysis bullosa.
Asunto(s)
Membrana Basal/metabolismo , Integrina alfa6beta1/genética , Integrina alfa6beta4/genética , Túbulos Renales Colectores/metabolismo , Laminina/genética , Obstrucción Ureteral/metabolismo , Animales , Apoptosis , Membrana Basal/patología , Adhesión Celular , Movimiento Celular , Proliferación Celular , Células Epiteliales/metabolismo , Células Epiteliales/patología , Fibrosis , Regulación de la Expresión Génica , Humanos , Integrina alfa6beta1/deficiencia , Integrina alfa6beta4/deficiencia , Túbulos Renales Colectores/patología , Laminina/metabolismo , Ratones , Ratones Noqueados , Unión Proteica , Transducción de Señal , Uréter/cirugía , Obstrucción Ureteral/patología , Obstrucción Ureteral/cirugíaRESUMEN
An in vitro system that supports primordial germ cells (PGCs) survival and proliferation is useful for enhancement of these cells and efficient transplantation in infertility disorders. One approach is cultivation of PGCs under proper conditions that allow self-renewal and proliferation of PGCs. For this purpose, we compared the effects of different concentrations of retinoic acid (RA), and the effect of PGCs co-culture (Co-C) with SIM mouse embryo-derived thioguanine- and ouabain-resistant (STO) cells on the proliferation of embryonic stem cells (ESCs)-derived PGCs. One-day-old embryoid body (EB) was cultured for 4 days in simple culture system in the presence of 5 ng/ml bone morphogenetic protein-4 (BMP4) (SCB group) for PGC induction. For PGC enrichment, ESCs-derived germ cells were cultured for 7 days in the presence of different doses (0-5 µM) of RA, both in the simple and STO Co-C systems. At the end of the culture period, viability and proliferation rates were assessed and expression of mouse vasa homologue (Mvh), α6 integrin, ß1 integrin, stimulated by retinoic acid 8 (Stra8) and piwi (Drosophila)-like 2 (Piwil2) was evaluated using quantitative PCR. Also, the inductive effects were investigated immunocytochemically with Mvh and cadherin1 (CDH1) on the selected groups. Immunocytochemistry/PCR results showed higher expression of Mvh, the PGC-specific marker, in 3 µM RA concentrations on the top of the STO feeder layer. Meanwhile, assessment of the Stra8 mRNA and CDH1 protein, the specific makers for spermatogonia, showed no significant differences between groups. Based on the results, it seems that in the presence of 3 µM RA on top of the STO feeder layer cells, the majority of the cells transdifferentiated into germ cells were PGCs.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Transdiferenciación Celular/efectos de los fármacos , Células Madre Embrionarias/efectos de los fármacos , Espermatogonias/citología , Tretinoina/farmacología , Proteínas Adaptadoras Transductoras de Señales/efectos de los fármacos , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Proteínas Argonautas/efectos de los fármacos , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Biomarcadores/metabolismo , Proteína Morfogenética Ósea 4/metabolismo , Proteínas Cdh1/efectos de los fármacos , Proteínas Cdh1/metabolismo , Diferenciación Celular , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Técnicas de Cocultivo , ARN Helicasas DEAD-box/efectos de los fármacos , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Células Madre Embrionarias/citología , Inmunohistoquímica , Integrina alfa6beta1/efectos de los fármacos , Integrina alfa6beta1/genética , Integrina alfa6beta1/metabolismo , Masculino , Ratones , Espermatogonias/metabolismo , TranscriptomaRESUMEN
Neutrophils need to penetrate the perivascular basement membrane for successful extravasation into inflamed tissue, but this process is incompletely understood. Recent findings have associated mammalian sterile 20-like kinase 1 (MST1) loss of function with a human primary immunodeficiency disorder, suggesting that MST1 may be involved in immune cell migration. Here, we have shown that MST1 is a critical regulator of neutrophil extravasation during inflammation. Mst1-deficient (Mst1-/-) neutrophils were unable to migrate into inflamed murine cremaster muscle venules, instead persisting between the endothelium and the basement membrane. Mst1-/- neutrophils also failed to extravasate from gastric submucosal vessels in a murine model of Helicobacter pylori infection. Mechanistically, we observed defective translocation of VLA-3, VLA-6, and neutrophil elastase from intracellular vesicles to the surface of Mst1-/- neutrophils, indicating that MST1 is required for this crucial step in neutrophil transmigration. Furthermore, we found that MST1 associates with the Rab27 effector protein synaptotagmin-like protein 1 (JFC1, encoded by Sytl1 in mice), but not Munc13-4, thereby regulating the trafficking of Rab27-positive vesicles to the cellular membrane. Together, these findings highlight a role for MST1 in vesicle trafficking and extravasation in neutrophils, providing an additional mechanistic explanation for the severe immune defect observed in patients with MST1 deficiency.
Asunto(s)
Factor de Crecimiento de Hepatocito/inmunología , Neutrófilos/inmunología , Proteínas Proto-Oncogénicas/inmunología , Vesículas Secretoras/inmunología , Migración Transendotelial y Transepitelial/inmunología , Músculos Abdominales/irrigación sanguínea , Músculos Abdominales/inmunología , Animales , Membrana Basal/inmunología , Transporte Biológico Activo/genética , Transporte Biológico Activo/inmunología , Mucosa Gástrica/química , Mucosa Gástrica/inmunología , Factor de Crecimiento de Hepatocito/genética , Humanos , Síndromes de Inmunodeficiencia/genética , Síndromes de Inmunodeficiencia/inmunología , Integrina alfa3beta1/genética , Integrina alfa3beta1/inmunología , Integrina alfa6beta1/genética , Integrina alfa6beta1/inmunología , Elastasa de Leucocito/genética , Elastasa de Leucocito/inmunología , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Ratones , Ratones Noqueados , Proteínas Proto-Oncogénicas/genética , Vesículas Secretoras/genética , Migración Transendotelial y Transepitelial/genética , Vénulas/inmunología , Proteínas de Transporte VesicularRESUMEN
The significance of matricellular proteins during development and cancer progression is widely recognized. However, how these proteins actively contribute to physiological development and pathological cancer progression is only partially elucidated. In this study, we investigated the role of the matricellular protein Cysteine-rich 61 (CYR61) in pancreatic islet development and carcinogenesis. Transgenic expression of CYR61 in ß cells (Rip1CYR mice) caused irregular islets morphology and distorted sorting of α cells, but did not alter islets size, number or vascularization. To investigate the function of CYR61 during carcinogenesis, we crossed Rip1CYR mice with Rip1Tag2 mice, a well-established model of ß cell carcinogenesis. Beta tumors in Rip1Tag2CYR mice were larger, more invasive and more vascularized compared to tumors in Rip1Tag2 mice. The effect of CYR61 on angiogenesis was fully abrogated by treating mice with the anti-VEGFR2 mAb DC101. Results from in vitro assays demonstrated that CYR61 modulated integrin α6ß1-dependent invasion and adhesion without altering its expression. Taken together, these results show that CYR61 expression in pancreatic ß cells interferes with normal islet architecture, promotes islet tumor growth, invasion and VEGF/VERGFR-2-dependent tumor angiogenesis. Taken together, these observations demonstrate that CYR61 acts as a tumor-promoting gene in pancreatic neuroendocrine tumors.
Asunto(s)
Proteína 61 Rica en Cisteína/genética , Islotes Pancreáticos/metabolismo , Tumores Neuroendocrinos/genética , Neoplasias Pancreáticas/genética , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Western Blotting , Línea Celular Tumoral , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Proteína 61 Rica en Cisteína/metabolismo , Progresión de la Enfermedad , Proteínas Activadoras de GTPasa/genética , Proteínas Activadoras de GTPasa/metabolismo , Integrina alfa6beta1/genética , Integrina alfa6beta1/metabolismo , Islotes Pancreáticos/irrigación sanguínea , Ratones Endogámicos C57BL , Ratones Transgénicos , Neovascularización Patológica/genética , Neovascularización Patológica/metabolismo , Neovascularización Patológica/prevención & control , Tumores Neuroendocrinos/irrigación sanguínea , Tumores Neuroendocrinos/metabolismo , Neoplasias Pancreáticas/irrigación sanguínea , Neoplasias Pancreáticas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Receptor 2 de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Receptor 2 de Factores de Crecimiento Endotelial Vascular/inmunología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Chondrosarcoma is a malignant tumor that produces cartilage matrix. The most lethal aspect is its metastatic property. We demonstrated that amphiregulin (AR) is significantly upregulated in highly aggressive cells. AR silencing markedly suppressed cell migration. Exogenous AR markedly increased cell migration by transactivation of α6ß1 integrin expression. A neutralizing α6ß1 integrin antibody can abolish AR-induced cell motility. Knockdown of AR inhibits metastasis of cells to the lung in vivo. Furthermore, elevated AR expression is positively correlated with α6ß1 integrin levels and higher grades in patients. These findings can potentially serve as biomarker and therapeutic approach for controlling chondrosarcoma metastasis.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Neoplasias Óseas/enzimología , Movimiento Celular/efectos de los fármacos , Condrosarcoma/enzimología , Familia de Proteínas EGF/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Integrina alfa6beta1/metabolismo , MAP Quinasa Quinasa 1/metabolismo , Factor de Transcripción AP-1/metabolismo , Quinasas raf/metabolismo , Proteínas ras/metabolismo , Anfirregulina , Animales , Anticuerpos/farmacología , Antineoplásicos/farmacología , Biomarcadores de Tumor/genética , Neoplasias Óseas/tratamiento farmacológico , Neoplasias Óseas/genética , Neoplasias Óseas/patología , Línea Celular Tumoral , Condrosarcoma/tratamiento farmacológico , Condrosarcoma/genética , Condrosarcoma/secundario , Relación Dosis-Respuesta a Droga , Familia de Proteínas EGF/genética , Familia de Proteínas EGF/farmacología , Regulación Neoplásica de la Expresión Génica , Humanos , Integrina alfa6beta1/antagonistas & inhibidores , Integrina alfa6beta1/genética , Neoplasias Pulmonares/enzimología , Neoplasias Pulmonares/prevención & control , Neoplasias Pulmonares/secundario , MAP Quinasa Quinasa 1/genética , Masculino , Ratones Desnudos , Clasificación del Tumor , Invasividad Neoplásica , Fosforilación , Interferencia de ARN , Transducción de Señal , Factores de Tiempo , Factor de Transcripción AP-1/genética , Transfección , Ensayos Antitumor por Modelo de Xenoinjerto , Quinasas raf/genética , Proteínas ras/genéticaRESUMEN
BACKGROUND: The growth properties and self-renewal capacity of embryonic stem (ES) cells are regulated by their immediate microenvironment such as the extracellular matrix (ECM). Integrins, a central family of cellular ECM receptors, have been implicated in these processes but their specific role in ES cell self-renewal remains unclear. RESULTS: Here we have studied the effects of different ECM substrates and integrins in mouse ES cells in the absence of Leukemia Inhibitory Factor (LIF) using short-term assays as well as long-term cultures. Removal of LIF from ES cell culture medium induced morphological differentiation of ES cells into polarized epistem cell-like cells. These cells maintained epithelial morphology and expression of key stemness markers for at least 10 passages in the absence of LIF when cultured on laminin, fibronectin or collagen IV substrates. The specific functional roles of α6-, αV- and ß1-integrin subunits were dissected using stable lentivirus-mediated RNAi methodology. ß1-integrins were required for ES cell survival in long-term cultures and for the maintenance of stem cell marker expression. Inhibition of α6-integrin expression compromised self-renewal on collagen while αV-integrins were required for robust ES cell adhesion on laminin. Analysis of the stemness marker expression revealed subtle differences between α6- and αV-depleted ES cells but the expression of both was required for optimal self-renewal in long-term ES cell cultures. CONCLUSIONS: In the absence of LIF, long-term ES cell cultures adapt an epistem cell-like epithelial phenotype and retain the expression of multiple stem cell markers. Long-term maintenance of such self-renewing cultures depends on the expression of ß1-, α6- and αV-integrins.
Asunto(s)
Integrina alfa6beta1/metabolismo , Integrina alfaV/metabolismo , Animales , Adhesión Celular , Diferenciación Celular , Línea Celular , Proliferación Celular , Supervivencia Celular , Colágeno/química , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Matriz Extracelular/metabolismo , Integrina alfa6beta1/antagonistas & inhibidores , Integrina alfa6beta1/genética , Integrina alfaV/química , Integrina alfaV/genética , Laminina/química , Factor Inhibidor de Leucemia/deficiencia , Ratones , Microscopía Fluorescente , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Factores de Transcripción/metabolismoRESUMEN
The mechanism by which angiogenic endothelial cells break the physical barrier of the vascular basement membrane and consequently sprout to form new vessels in mature tissues is unclear. Here, we show that the angiogenic endothelium is characterized by the presence of functional podosome rosettes. These extracellular-matrix-degrading and adhesive structures are precursors of de novo branching points and represent a key feature in the formation of new blood vessels. VEGF-A stimulation induces the formation of endothelial podosome rosettes by upregulating integrin α6ß1. In contrast, the binding of α6ß1 integrin to the laminin of the vascular basement membrane impairs the formation of podosome rosettes by restricting α6ß1 integrin to focal adhesions and hampering its translocation to podosomes. Using an ex vivo sprouting angiogenesis assay, transgenic and knockout mouse models and human tumour sample analysis, we provide evidence that endothelial podosome rosettes control blood vessel branching and are critical regulators of pathological angiogenesis.
Asunto(s)
Estructuras de la Membrana Celular/fisiología , Células Endoteliales/fisiología , Neoplasias/fisiopatología , Neovascularización Patológica/fisiopatología , Animales , Membrana Basal/metabolismo , Línea Celular Tumoral , Estructuras de la Membrana Celular/efectos de los fármacos , Estructuras de la Membrana Celular/metabolismo , Células Cultivadas , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Células HeLa , Humanos , Integrina alfa6beta1/genética , Integrina alfa6beta1/metabolismo , Laminina/metabolismo , Neoplasias Pulmonares/irrigación sanguínea , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/fisiopatología , Masculino , Metaloproteinasa 14 de la Matriz/metabolismo , Melanoma Experimental/irrigación sanguínea , Melanoma Experimental/metabolismo , Melanoma Experimental/fisiopatología , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Confocal , Microscopía Fluorescente , Neoplasias/irrigación sanguínea , Neoplasias/metabolismo , Neovascularización Patológica/metabolismo , Interferencia de ARN , Acetato de Tetradecanoilforbol/farmacología , Factor A de Crecimiento Endotelial Vascular/metabolismo , Factor A de Crecimiento Endotelial Vascular/farmacologíaRESUMEN
Cordycepin, also known as 3-deoxyadenosine, is an analogue of adenosine extracted from the traditional Chinese medicine "Dong Chong Xia Cao". Cordycepin is an active small molecular weight compound and is implicated in modulating multiple physiological functions including immune activation, anti-aging and anti-tumor effects. Several studies have indicated that cordycepin suppresses tumor progression. However, the signaling pathways involved in cordycepin regulating cancer cell motility, invasiveness and epithelial-mesenchymal transition (EMT) remain unclear. In this study, we found that cordycepin inhibits hepatocellular carcinoma (HCC) cell proliferation and migration/invasion. Treatment of cordycepin results in the increasing expression of epithelial marker, Ecadherin while no significant effect was found on N-cadherin α-catenin and ß-catenin. Furthermore, although the expression of focal adhesion kinase (FAK) was slightly reduced, the level of phosphorylated FAK was significantly reduced by the treatment of cordycepin. In addition, cordycepin significantly suppresses the expression of integrin α3, integrin α6 and integrin ß1 which are crucial interacting partners of FAK in regulating the focal adhesion complex. These results suggest cordycepin may contribute to EMT, antimigration/ invasion and growth inhibitory effects of HCC by suppressing E-cadherin and integrin/FAK signaling. Thus, cordycepin is a potential therapeutic or supplementary agent for preventing HCC tumor progression.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Carcinoma Hepatocelular/metabolismo , Desoxiadenosinas/farmacología , Transición Epitelial-Mesenquimal/efectos de los fármacos , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Integrina alfa3beta1/metabolismo , Integrina alfa6beta1/metabolismo , Neoplasias Hepáticas/metabolismo , Cadherinas/metabolismo , Carcinoma Hepatocelular/patología , Línea Celular Tumoral/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Humanos , Integrina alfa3beta1/genética , Integrina alfa6beta1/genética , Neoplasias Hepáticas/patología , Transducción de SeñalRESUMEN
Lysophosphatidic acid (LPA), a potent bioactive lipid found in atherosclerotic lesions, markedly induces smooth muscle cell (SMC) migration, which is an important process in atherogenesis. Therefore, understanding the mechanism of LPA-induced SMC migration is important. Several microarray databases suggest that the matricellular protein Cyr61 is highly induced by LPA. We hypothesized that Cyr61 mediates LPA-induced cell migration. Our data show that LPA induced temporal and spatial expression of Cyr61, which promptly accumulated in the cellular Golgi apparatus and then translocated to the extracellular matrix. Cyr61 antibody blockade and siRNA inhibition both diminished LPA-induced SMC migration, indicating a novel regulatory role of Cyr61. SMCs derived from LPA receptor 1 (LPA1) knock-out mice lack the ability of Cyr61 induction and cell migration, supporting the concept that LPA1 is required for Cyr61 expression and migration. By contrast, PPARγ was not found to be involved in LPA-mediated effects. Furthermore, focal adhesion kinase (FAK), a nonreceptor tyrosine kinase important for regulating cell migration, was activated by LPA at a late time frame coinciding with Cyr61 accumulation. Interestingly, knockdown of Cyr61 blocked LPA-induced FAK activation, indicating that an LPA-Cyr61-FAK axis leads to SMC migration. Our results further demonstrate that plasma membrane integrins α6ß1 and ανß3 transduced the LPA-Cyr61 signal toward FAK activation and migration. Taken together, these data reveal that de novo Cyr61 in the extracellular matrix bridges LPA and integrin pathways, which in turn, activate FAK, leading to cell migration. The current study provides new insights into mechanisms underlying cell migration-related disorders, including atherosclerosis, restenosis, and cancers.
Asunto(s)
Movimiento Celular/fisiología , Proteína 61 Rica en Cisteína/metabolismo , Integrina alfa6beta1/metabolismo , Integrina alfaVbeta3/metabolismo , Lisofosfolípidos/metabolismo , Miocitos del Músculo Liso/metabolismo , Animales , Células Cultivadas , Proteína 61 Rica en Cisteína/genética , Activación Enzimática/fisiología , Quinasa 1 de Adhesión Focal/genética , Quinasa 1 de Adhesión Focal/metabolismo , Integrina alfa6beta1/genética , Integrina alfaVbeta3/genética , Lisofosfolípidos/genética , Ratones , Ratones Noqueados , Miocitos del Músculo Liso/citología , PPAR gamma/genética , PPAR gamma/metabolismo , Receptores del Ácido Lisofosfatídico/genética , Receptores del Ácido Lisofosfatídico/metabolismoRESUMEN
Bone marrow-derived mesenchymal stem cells (BMSCs) facilitate the angiogenic response of endothelial cells (ECs) within three-dimensional (3D) matrices in vivo and in engineered tissues in vitro in part through paracrine mediators and by acting as stabilizing pericytes. However, the molecular interactions between BMSCs and nascent tubules during the process of angiogenesis are not fully understood. In this study, we have used a tractable 3D co-culture model to explore the functional role of the α6ß1 integrin adhesion receptor on BMSCs in sprouting angiogenesis. We report that knockdown of the α6 integrin subunit in BMSCs significantly reduces capillary sprouting, and causes their failure to associate with the nascent vessels. Furthermore, we demonstrate that the BMSCs with attenuated α6 integrin proliferate at a significantly lower rate relative to either control cells expressing non-targeting shRNA or wild type BMSCs; however, despite adding more cells to compensate for this deficit in proliferation, deficient sprouting persists. Collectively, our findings demonstrate that the α6 integrin subunit in BMSCs is important for their ability to stimulate vessel morphogenesis. This conclusion may have important implications in the optimization of cell-based strategies to promote angiogenesis.
Asunto(s)
Células de la Médula Ósea/citología , Médula Ósea/metabolismo , Células Endoteliales/citología , Integrina alfa6beta1/metabolismo , Células Madre Mesenquimatosas/metabolismo , Neovascularización Fisiológica , Diferenciación Celular , Células Cultivadas , Técnicas de Cocultivo/métodos , Células Endoteliales/fisiología , Humanos , Integrina alfa6beta1/genética , Células Madre Mesenquimatosas/citología , Neovascularización Fisiológica/fisiologíaRESUMEN
Mechanical forces are critical for normal fetal lung development. However, the mechanisms regulating this process are not well-characterized. We hypothesized that strain-induced release of HB-EGF and TGF-α is mediated via integrin-ADAM17/TACE interactions. Employing an in vitro system to simulate mechanical forces in fetal lung development, we showed that mechanical strain of fetal epithelial cells actives TACE, releases HB-EGF and TGF-α, and promotes differentiation. In contrast, in samples incubated with the TACE inhibitor IC-3 or in cells isolated from TACE knock-out mice, mechanical strain did not release ligands or promote cell differentiation, which were both rescued after transfection of ADAM17. Cell adhesion assay and co-immunoprecipitation experiments in wild-type and TACE knock-out cells using several TACE constructs demonstrated not only that integrins α6 and ß1 bind to TACE via the disintegrin domain but also that mechanical strain enhances these interactions. Furthermore, force applied to these integrin receptors by magnetic beads activated TACE and shed HB-EGF and TGF-α. The contribution of integrins α6 and ß1 to differentiation of fetal epithelial cells by strain was demonstrated by blocking their binding site with specific antibodies and by culturing the cells on membranes coated with anti-integrin α6 and ß1 antibodies. In conclusion, mechanical strain releases HB-EGF and TGF-α and promotes fetal type II cell differentiation via α6ß1 integrin-ADAM17/TACE signaling pathway. These investigations provide novel mechanistic information on how mechanical forces promote fetal lung development and specifically differentiation of epithelial cells. This information could be also relevant to other tissues exposed to mechanical forces.
Asunto(s)
Proteínas ADAM/metabolismo , Diferenciación Celular/fisiología , Células Epiteliales/metabolismo , Integrina alfa6beta1/metabolismo , Pulmón/embriología , Mucosa Respiratoria/embriología , Transducción de Señal/fisiología , Proteínas ADAM/genética , Proteína ADAM17 , Animales , Células Epiteliales/citología , Factor de Crecimiento Similar a EGF de Unión a Heparina , Integrina alfa6beta1/genética , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Pulmón/citología , Ratones , Ratones Noqueados , Unión Proteica , Mucosa Respiratoria/citología , Estrés Fisiológico/fisiología , Factor de Crecimiento Transformador alfa/genética , Factor de Crecimiento Transformador alfa/metabolismoRESUMEN
We describe a unique extracellular matrix (ECM) niche in the spleen, the marginal zone (MZ), characterized by the basement membrane glycoproteins, laminin α5 and agrin, that promotes formation of a specialized population of MZ B lymphocytes that respond rapidly to blood-borne antigens. Mice with reduced laminin α5 expression show reduced MZ B cells and increased numbers of newly formed (NF) transitional B cells that migrate from the bone marrow, without changes in other immune or stromal cell compartments. Transient integrin α6ß1-mediated interaction of NF B cells with laminin α5 in the MZ supports the MZ B-cell population, their long-term survival, and antibody response. Data suggest that the unique 3D structure and biochemical composition of the ECM of lymphoid organs impacts on immune cell fate.
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Linfocitos B/inmunología , Médula Ósea/inmunología , Movimiento Celular/inmunología , Matriz Extracelular/inmunología , Bazo/inmunología , Agrina/genética , Agrina/inmunología , Animales , Linfocitos B/citología , Movimiento Celular/genética , Supervivencia Celular/genética , Supervivencia Celular/inmunología , Matriz Extracelular/genética , Integrina alfa6beta1/genética , Integrina alfa6beta1/inmunología , Laminina/genética , Laminina/inmunología , Ratones , Ratones Noqueados , Bazo/citologíaRESUMEN
The characterization of cells with tumour initiating potential is significant for advancing our understanding of cancer and improving therapy. Aggressive, triple-negative breast cancers (TNBCs) are enriched for tumour-initiating cells (TICs). We investigated that hypothesis that VEGF receptors expressed on TNBC cells mediate autocrine signalling that contributes to tumour initiation. We discovered the VEGF receptor neuropilin-2 (NRP2) is expressed preferentially on TICs, involved in the genesis of TNBCs and necessary for tumour initiation. The mechanism by which NRP2 signalling promotes tumour initiation involves stimulation of the α6ß1 integrin, focal adhesion kinase-mediated activation of Ras/MEK signalling and consequent expression of the Hedgehog effector GLI1. GLI1 also induces BMI-1, a key stem cell factor, and it enhances NRP2 expression and the function of α6ß1, establishing an autocrine loop. NRP2 can be targeted in vivo to retard tumour initiation. These findings reveal a novel autocrine pathway involving VEGF/NRP2, α6ß1 and GLI1 that contributes to the initiation of TNBC. They also support the feasibility of NRP2-based therapy for the treatment of TNBC that targets and impedes the function of TICs.
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Comunicación Autocrina , Neoplasias de la Mama/metabolismo , Integrina alfa6beta1/metabolismo , Neuropilina-2/metabolismo , Factores de Transcripción/metabolismo , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/fisiopatología , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Integrina alfa6beta1/genética , Ratones , Ratones Endogámicos NOD , Ratones Transgénicos , Proteína Quinasa 7 Activada por Mitógenos/genética , Proteína Quinasa 7 Activada por Mitógenos/metabolismo , Células Madre Neoplásicas/metabolismo , Neuropilina-2/genética , Factores de Transcripción/genética , Proteína con Dedos de Zinc GLI1RESUMEN
The promising clinical effects of mesenchymal stromal/stem cells (MSCs) rely especially on paracrine and nonimmunogenic mechanisms. Delivery routes are essential for the efficacy of cell therapy and systemic delivery by infusion is the obvious goal for many forms of MSC therapy. Lung adhesion of MSCs might, however, be a major obstacle yet to overcome. Current knowledge does not allow us to make sound conclusions whether MSC lung entrapment is harmful or beneficial, and thus we wanted to explore MSC lung adhesion in greater detail. We found a striking difference in the lung clearance rate of systemically infused MSCs derived from two different clinical sources, namely bone marrow (BM-MSCs) and umbilical cord blood (UCB-MSCs). The BM-MSCs and UCB-MSCs used in this study differed in cell size, but our results also indicated other mechanisms behind the lung adherence. A detailed analysis of the cell surface profiles revealed differences in the expression of relevant adhesion molecules. The UCB-MSCs had higher expression levels of α4 integrin (CD49d, VLA-4), α6 integrin (CD49f, VLA-6), and the hepatocyte growth factor receptor (c-Met) and a higher general fucosylation level. Strikingly, the level of CD49d and CD49f expression could be functionally linked with the lung clearance rate. Additionally, we saw a possible link between MSC lung adherence and higher fibronectin expression and we show that the expression of fibronectin increases with MSC culture confluence. Future studies should aim at developing methods of transiently modifying the cell surface structures in order to improve the delivery of therapeutic cells.
Asunto(s)
Células de la Médula Ósea/citología , Trasplante de Células Madre de Sangre del Cordón Umbilical , Sangre Fetal/citología , Pulmón/citología , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/citología , Animales , Biomarcadores/metabolismo , Células de la Médula Ósea/metabolismo , Adhesión Celular , Diferenciación Celular , Femenino , Sangre Fetal/metabolismo , Expresión Génica , Semivida , Humanos , Infusiones Intravenosas , Integrina alfa4/genética , Integrina alfa4/metabolismo , Integrina alfa4beta1/genética , Integrina alfa4beta1/metabolismo , Integrina alfa6/genética , Integrina alfa6/metabolismo , Integrina alfa6beta1/genética , Integrina alfa6beta1/metabolismo , Marcaje Isotópico , Pulmón/inmunología , Pulmón/metabolismo , Células Madre Mesenquimatosas/metabolismo , Ratones , Ratones Desnudos , Proteínas Proto-Oncogénicas c-met/genética , Proteínas Proto-Oncogénicas c-met/metabolismo , Compuestos de Tecnecio , Trasplante HeterólogoRESUMEN
This review advances the hypothesis that the ability of integrins to engage their extracellular matrix ligands and signal can be regulated in tumor cells by vascular endothelial growth factor (VEGF), a major angiogenic factor that also has direct effects on the function of tumor cells. More specifically, we will discuss how neuropilins (NRPs), a distinct class of VEGF receptors, enable the function of specific integrins that contribute to tumor initiation and progression.
Asunto(s)
Transformación Celular Neoplásica/patología , Integrina alfa6beta1/metabolismo , Neovascularización Patológica/patología , Neuropilina-2/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Progresión de la Enfermedad , Activación Enzimática , Matriz Extracelular/metabolismo , Matriz Extracelular/patología , Quinasa 1 de Adhesión Focal/genética , Quinasa 1 de Adhesión Focal/metabolismo , Adhesiones Focales/metabolismo , Humanos , Integrina alfa6beta1/genética , Ligandos , Neovascularización Patológica/metabolismo , Neuropilina-2/genética , Unión Proteica , Mapeo de Interacción de Proteínas , Transducción de Señal , Microambiente Tumoral , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
BACKGROUND: Junctional epidermolysis bullosa with pyloric atresia (JEB-PA) is a rare autosomal recessive disease with blister formation within the lamina lucida due to mutations in the integrin ß4 (ITGB4) and α6 (ITGA6) genes. CASE REPORT: A female preterm infant, first child of healthy non-consanguineous parents, was born at 26 + 4 weeks of gestation by caesarean section, following polyhydramnion and abruption of placenta. She presented with extensive areas of denuded skin on both lateral sides of the head, neck and extremities. Auricles were hypoplastic. Abdominal ultrasound and X-ray were suggestive of pyloric atresia which was revised surgically on the 4th day of life. Further course was complicated by progressive skin detachment, sepsis, and renal insufficiency with fatal outcome at 18 days of age. Immunofluorescence mapping of cryopreserved skin showed junctional cleft formation with negative staining for integrin α6 and integrin ß4. Mutational analysis disclosed compound heterozygosity for two novel nonsense mutations in the ITGB4 gene: c.600dupC/p.F201fsX14 and c.2533C>T/p.Q845X. 2 subsequent pregnancies were terminated following prenatal diagnosis disclosing the same ITGB4 mutations, a 4th pregnancy was unaffected. CONCLUSION: We describe a case of lethal JEB-PA with negative immunoreactivity to integrin α6 and integrin ß4 predicting a poor outcome. Identification of compound heterozygosity for two novel ITGB4 mutations in the affected preterm infant permitted prenatal diagnosis and finally birth of a healthy sibling.