RESUMEN
Glioblastoma is the most aggressive primary brain tumor in adults. Although the rapid recurrence of glioblastomas after treatment is a major clinical challenge, the relationships between tumor growth and intracerebral spread remain poorly understood. We have identified the cofilin phosphatase chronophin (gene name: pyridoxal phosphatase, PDXP) as a glial tumor modifier. Monoallelic PDXP loss was frequent in four independent human astrocytic tumor cohorts and increased with tumor grade. We found that aberrant PDXP promoter methylation can be a mechanism leading to further chronophin downregulation in glioblastomas, which correlated with shorter glioblastoma patient survival. Moreover, we observed an inverse association between chronophin protein expression and cofilin phosphorylation levels in glioma tissue samples. Chronophin-deficient glioblastoma cells showed elevated cofilin phosphorylation, an increase in polymerized actin, a higher directionality of cell migration, and elevated in vitro invasiveness. Tumor growth of chronophin-depleted glioblastoma cells xenografted into the immunodeficient mouse brain was strongly impaired. Our study suggests a mechanism whereby the genetic and epigenetic alterations of PDXP resulting in altered chronophin expression may regulate the interplay between glioma cell proliferation and invasion.
Asunto(s)
Neoplasias Encefálicas/enzimología , Neoplasias Encefálicas/patología , Glioblastoma/enzimología , Glioblastoma/patología , Fosfoproteínas Fosfatasas/metabolismo , Animales , Neoplasias Encefálicas/genética , Línea Celular Tumoral , Proliferación Celular/fisiología , Metilación de ADN , Femenino , Glioblastoma/genética , Xenoinjertos , Humanos , Ratones , Ratones Endogámicos NOD , Invasividad Neoplásica , Fosfoproteínas Fosfatasas/genética , Regiones Promotoras GenéticasRESUMEN
Cell adhesion molecules are key players in transsynaptic communication, precisely coordinating presynaptic differentiation with postsynaptic specialization. At glutamatergic synapses, their retrograde signaling has been proposed to control presynaptic vesicle clustering at active zones. However, how the different types of cell adhesion molecules act together during this decisive step of synapse maturation is largely unexplored. Using a knockout approach, we show that two synaptic adhesion systems, N-cadherin and neuroligin-1, cooperate to control vesicle clustering at nascent synapses. Live cell imaging and fluorescence recovery after photobleaching experiments at individual synaptic boutons revealed a strong impairment of vesicle accumulation in the absence of N-cadherin, whereas the formation of active zones was largely unaffected. Strikingly, also the clustering of synaptic vesicles triggered by neuroligin-1 overexpression required the presence of N-cadherin in cultured neurons. Mechanistically, we found that N-cadherin acts by postsynaptically accumulating neuroligin-1 and activating its function via the scaffolding molecule S-SCAM, leading, in turn, to presynaptic vesicle clustering. A similar cooperation of N-cadherin and neuroligin-1 was observed in immature CA3 pyramidal neurons in an organotypic hippocampal network. Moreover, at mature synapses, N-cadherin was required for the increase in release probability and miniature EPSC frequency induced by expressed neuroligin-1. This cooperation of two cell adhesion systems provides a mechanism for coupling bidirectional synapse maturation mediated by neuroligin-1 to cell type recognition processes mediated by classical cadherins.
Asunto(s)
Cadherinas/fisiología , Moléculas de Adhesión Celular Neuronal/fisiología , Vesículas Sinápticas/fisiología , Receptores de Activinas Tipo II/genética , Receptores de Activinas Tipo II/metabolismo , Animales , Secuencia de Bases , Región CA3 Hipocampal/fisiología , Cadherinas/deficiencia , Cadherinas/genética , Moléculas de Adhesión Celular Neuronal/genética , Células Cultivadas , Células Madre Embrionarias/fisiología , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Técnicas In Vitro , Ratones , Ratones Noqueados , Ratones Transgénicos , Potenciales Postsinápticos Miniatura , Neuronas/fisiología , Células Piramidales/fisiología , Interferencia de ARN , ARN Interferente Pequeño/genética , Ratas , Ratas Wistar , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Pasteurella multocida toxin (PMT) is a highly potent mitogen for a variety of cell types. PMT has been shown to induce various cellular signaling processes, and it has been suggested to function through the heterotrimeric G-proteins G(q)/G(11). To analyze the role of G(q)/G(11) in the action of PMT, we have studied the effect of the toxin in Galpha(q)/Galpha(11) double-deficient fibroblasts as well as in fibroblasts lacking only Galpha(q) or Galpha(11). Interestingly, formation of inositol phosphates in response to PMT was exclusively dependent on Galpha(q) but not on the closely related Galpha(11). Although Galpha(q)/Galpha(11) double-deficient and Galpha(q)-deficient cells did not respond with any production of inositol phosphates to PMT, PMT was still able to induce various other cellular effects in these cells, including the activation of Rho, the Rho-dependent formation of actin stress fibers and focal adhesions, as well as the stimulation of c-Jun N-terminal kinase and extracellular signal-regulated kinase. These data show that PMT leads to a variety of cellular effects that are mediated only in part by the heterotrimeric G-protein G(q).
Asunto(s)
Proteínas Bacterianas , Toxinas Bacterianas/farmacología , Proteínas de Unión al GTP Heterotriméricas/fisiología , Pasteurella multocida/metabolismo , Animales , Células Cultivadas , Subunidades alfa de la Proteína de Unión al GTP Gq-G11 , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Ratones , FosforilaciónRESUMEN
Neuropeptides like galanin produced and released by small cell lung cancer (SCLC) cells are considered principal mitogens in these tumors. We identified the galanin receptor type 2 (GALR2) as the only galanin receptor expressed in H69 and H510 cells. Photoaffinity labeling of G proteins in H69 cell membranes revealed that GALR2 activates G proteins of three subfamilies: G(q), G(i), and G(12). In H69 cells, galanin-induced Ca2+ mobilization was pertussis toxin-insensitive. While phorbol ester-induced extracellular signal-regulated kinase (ERK) activation required protein kinase C (PKC) activity, preincubation of H69 cells with the PKC-inhibitor GF109203X had no effect on galanin-dependent ERK activity. A rise of the intracellular calcium concentration was necessary and sufficient to mediate galanin-induced ERK activation. In support of G(i) coupling, stimulation of GALR2 expressed in HEK293 cells inhibited isoproterenol-induced cAMP accumulation and raised cAMP levels in COS-7 cells when coexpressed with a chimeric G alpha(S)-G alpha(i) protein In H69 cells, galanin activated the monomeric GTPase RhoA and induced stress fiber formation in Swiss 3T3 cells expressing GALR2. Thus, we provide the first direct evidence that in SCLC the mitogenic neuropeptide galanin, interacting with GALR2, simultaneously activates multiple classes of G proteins and signals through the G(q) phospholipase C/calcium sequence and a G(12)/Rho pathway. Oncogene (2000) 19, 4199 - 4209
Asunto(s)
Señalización del Calcio/fisiología , Carcinoma de Células Pequeñas/patología , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/fisiología , Proteínas de Unión al GTP/fisiología , Proteínas de Unión al GTP Heterotriméricas/fisiología , Neoplasias Pulmonares/patología , Proteínas de Neoplasias/fisiología , Receptores de Neuropéptido/fisiología , 1-Metil-3-Isobutilxantina/farmacología , Células 3T3/efectos de los fármacos , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Animales , Células COS/efectos de los fármacos , Señalización del Calcio/efectos de los fármacos , Carcinoma de Células Pequeñas/metabolismo , Adhesión Celular , AMP Cíclico/fisiología , Inhibidores Enzimáticos/farmacología , Subunidades alfa de la Proteína de Unión al GTP G12-G13 , Subunidades alfa de la Proteína de Unión al GTP Gq-G11 , Galanina/metabolismo , Galanina/farmacología , Humanos , Indoles/farmacología , Fosfatos de Inositol/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Isoproterenol/farmacología , Neoplasias Pulmonares/metabolismo , Maleimidas/farmacología , Ratones , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas de Neoplasias/antagonistas & inhibidores , Toxina del Pertussis , Fosfatidilinositol Diacilglicerol-Liasa , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Receptor de Galanina Tipo 2 , Receptores de Galanina , Receptores de Neuropéptido/efectos de los fármacos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/fisiología , Transfección , Células Tumorales Cultivadas/metabolismo , Fosfolipasas de Tipo C/metabolismo , Factores de Virulencia de Bordetella/farmacología , Quinasas Asociadas a rho , Proteína de Unión al GTP rhoA/fisiologíaRESUMEN
Receptor-induced vascular smooth muscle cell contraction is mediated by dual regulation of myosin light chain (MLC(20)) phosphorylation through Ca(2+)-dependent stimulation of myosin light chain kinase and Rho/Rho-kinase-mediated inhibition of myosin phosphatase. Although myosin light chain kinase regulation is initiated by the coupling of receptors to G proteins of the G(q) family, G(q) and G(11), it is not known how receptors regulate the Rho/Rho-kinase-mediated pathway. In vascular smooth muscle cells, receptor-mediated MLC(20) phosphorylation and cell contraction was blocked by inhibitors of each of the pathways. Receptors of various vasocontractors were found to couple to G(q)/G(11) and G(12)/G(13), and constitutively active forms of G alpha(12) and G alpha(13) induced a pronounced contraction of vascular smooth muscle cells that could be blocked by C3 exoenzyme, by inhibition of Rho-kinase, and by stable analogues of cGMP and cAMP. Receptor-mediated smooth muscle cell contraction was strongly inhibited by dominant-negative forms of G alpha(12) and G alpha(13). These data indicate that a G(12)/G(13)-mediated Rho/Rho-kinase-dependent pathway operates in smooth muscle cells and that dual regulation of MLC(20) phosphorylation by vasocontractors is initiated by the dual coupling of their receptors to G proteins of the G(q) and G(12) families.
Asunto(s)
Endotelina-1/farmacología , Subunidades alfa de la Proteína de Unión al GTP Gi-Go , Proteínas de Unión al GTP Heterotriméricas/fisiología , Músculo Liso Vascular/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Proto-Oncogénicas/fisiología , Receptores de Endotelina/efectos de los fármacos , Vasoconstricción/fisiología , Vasoconstrictores/farmacología , Proteína de Unión al GTP rhoA/fisiología , Animales , Señalización del Calcio/fisiología , Bovinos , Subunidad alfa de la Proteína de Unión al GTP Gi2 , Subunidades alfa de la Proteína de Unión al GTP G12-G13 , Péptidos y Proteínas de Señalización Intracelular , Modelos Biológicos , Músculo Liso Vascular/citología , Músculo Liso Vascular/fisiología , Cadenas Ligeras de Miosina/metabolismo , Quinasa de Cadena Ligera de Miosina/metabolismo , Fosforilación , Procesamiento Proteico-Postraduccional , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Receptor de Endotelina A , Receptores de Endotelina/fisiología , Transducción de Señal/fisiología , Vasoconstricción/efectos de los fármacos , Quinasas Asociadas a rhoRESUMEN
The ubiquitously expressed heterotrimeric guanine nucleotide-binding proteins (G-proteins) G12 and G13 have been shown to activate the small GTPase Rho. Rho stimulation leads to a rapid remodeling of the actin cytoskeleton and subsequent stress fiber formation. We investigated the involvement of G12 or G13 in stress fiber formation induced through a variety of Gq/G11-coupled receptors. Using fibroblast cell lines derived from wild-type and Galphaq/Galpha11-deficient mice, we show that agonist-dependent activation of the endogenous receptors for thrombin or lysophosphatidic acid and of the heterologously expressed bradykinin B2, vasopressin V1A, endothelin ETA, and serotonin 5-HT2C receptors induced stress fiber formation in either the presence or absence of Galphaq/Galpha11. Stress fiber assembly induced through the muscarinic M1 and the metabotropic glutamate subtype 1alpha receptors was dependent on Gq/G11 proteins. The activation of the Gq/G11-coupled endothelin ETB and angiotensin AT1A receptors failed to induce stress fiber formation. Lysophosphatidic acid, B2, and 5-HT2C receptor-mediated stress fiber formation was dependent on Galpha13 and involved epidermal growth factor (EGF) receptors, whereas thrombin, ETA, and V1A receptors induced stress fiber accumulation via Galpha12 in an EGF receptor-independent manner. Our data demonstrate that many Gq/G11-coupled receptors induce stress fiber assembly in the absence of Galphaq and Galpha11 and that this involves either a Galpha12 or a Galpha13/EGF receptor-mediated pathway.
Asunto(s)
Actinas/metabolismo , Citoesqueleto/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go , Proteínas de Unión al GTP/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Receptores de Superficie Celular/metabolismo , Animales , Calcio/metabolismo , AMP Cíclico/metabolismo , Receptores ErbB/metabolismo , Fibroblastos , Subunidad alfa de la Proteína de Unión al GTP Gi2 , Proteínas de Unión al GTP/genética , Ligandos , Lisofosfolípidos/fisiología , Ratones , Ratones Noqueados , Microinyecciones , Proteínas Proto-Oncogénicas/genética , Quinazolinas , Transducción de Señal , Trombina/farmacología , Tirfostinos/farmacologíaRESUMEN
Heterotrimeric G proteins of the Gq class have been implicated in signaling pathways regulating cardiac growth under physiological and pathological conditions. Knockout mice carrying inactivating mutations in both of the widely expressed G alpha q class genes, G alpha q and G alpha 11, demonstrate that at least two active alleles of these genes are required for extrauterine life. Mice carrying only one intact allele [G alpha q(-/+);G alpha 11(-/-) or G alpha q(-/-);G alpha 11(-/+)] died shortly after birth. These mutants showed a high incidence of cardiac malformation. In addition, G alpha q(-/-);G alpha 11(-/+) newborns suffered from craniofacial defects. Mice lacking both G alpha q and G alpha 11 [G alpha q(-/-);G alpha 11(-/-)] died at embryonic day 11 due to cardiomyocyte hypoplasia. These data demonstrate overlap in G alpha q and G alpha 11 gene functions and indicate that the Gq class of G proteins plays a crucial role in cardiac growth and development.
Asunto(s)
Anomalías Craneofaciales/embriología , Anomalías Craneofaciales/genética , Proteínas de Unión al GTP/genética , Cardiopatías Congénitas/embriología , Cardiopatías Congénitas/genética , Miocardio/patología , Alelos , Animales , Animales Recién Nacidos/genética , Anomalías Craneofaciales/patología , Cruzamientos Genéticos , Proteínas de Unión al GTP/deficiencia , Dosificación de Gen , Cardiopatías Congénitas/mortalidad , Cardiopatías Congénitas/patología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Ratones NoqueadosRESUMEN
Lysophosphatidic acid (LPA) utilizes a G-protein-coupled receptor to activate the small GTP-binding protein Rho and to induce rapid remodeling of the actin cytoskeleton. We studied the signal transduction from LPA receptors to Rho activation. Analysis of the G-protein-coupling pattern of LPA receptors by labeling activated G-proteins with [alpha-32P]GTP azidoanilide revealed interaction with proteins of the Gq, Gi, and G12 subfamilies. We could show that in COS-7 cells, expression of GTPase-deficient mutants of Galpha12 and Galpha13 triggered Rho activation as measured by increased Rho-GTP levels. In Swiss 3T3 cells, incubation with LPA or microinjection of constitutively active mutants of Galpha12 and Galpha13 induced formation of actin stress fibers and assembly of focal adhesions in a Rho-dependent manner. Interestingly, the LPA-dependent cytoskeletal reorganization was suppressed by microinjected antibodies directed against Galpha13, whereas Galpha12-specific antibodies showed no inhibition. The tyrosine kinase inhibitor tyrphostin A 25 and the epidermal growth factor (EGF) receptor-specific tyrphostin AG 1478 completely blocked actin stress fiber formation caused by LPA or activated Galpha13 but not the effects of activated Galpha12. Also, expression of the dominant negative EGF receptor mutant EGFR-CD533 markedly prevented the LPA- and Galpha13-induced actin polymerization. Coexpression of EGFR-CD533 and activated Galpha13 in COS-7 cells resulted in decreased Rho-GTP levels compared with expression of activated Galpha13 alone. These data indicate that in Swiss 3T3 cells, G13 but not G12 is involved in the LPA-induced activation of Rho. Moreover, our results suggest an involvement of the EGF receptor in this pathway.
Asunto(s)
Receptores ErbB/metabolismo , Proteínas de Unión al GTP/metabolismo , Receptores de Superficie Celular/metabolismo , Receptores Acoplados a Proteínas G , Transducción de Señal , Tirfostinos , Células 3T3 , Animales , Células COS , Membrana Celular/metabolismo , Inhibidores Enzimáticos/farmacología , Receptores ErbB/antagonistas & inhibidores , Lisofosfolípidos/farmacología , Ratones , Nitrilos/farmacología , Receptores del Ácido LisofosfatídicoRESUMEN
Gbetagamma dimers of heterotrimeric G proteins have been shown to be important for the translocation of cytosolic proteins to membranes. The involvement of Gbetagamma in those signaling processes mediated by small GTP-binding proteins of the Rho family was studied using purified proteins. We showed specific binding of bovine brain Gbetagamma to immobilized GST-Rho fusion proteins. In addition, brain Gbetagamma, but not transducin Gbetagamma, was able to inhibit GTPgammaS binding to GST-Rho in a concentration-dependent manner. GTPgammaS binding to GST-Rac was also decreased by brain Gbetagamma whereas nucleotide binding to GST-Cdc42 was not changed. We conclude that Gbetagamma dimers may participate in the process of membrane attachment and/or other regulations of Rho and Rac.
Asunto(s)
Proteínas de Unión al GTP/metabolismo , Nucleótidos de Guanina/metabolismo , Animales , Biopolímeros , Bovinos , Unión Proteica , Transducción de SeñalRESUMEN
A new quantitative assay for the study of tumour cell invasion in vitro is described. Employing the novel fluorescent dye YO-PRO-1, cells that penetrate Matrigel-coated transwells are counted on the basis of dye-bound cellular nucleic acid content. Following transmigration, the cells in the lower compartments are lysed by freezing in water. After a brief incubation with YO-PRO-1, nucleic acid or DNA content is measured as fluorescence intensity in 96-well microplates and quantitated by a cell- or DNA-calibration curve. Using standard curves, a linear relationship between fluorescence intensity and cell number was found in the range tested (from 100 to 80 000 cells). The mean relative intra- and inter-assay variability of the cell quantitation in this range was 3.5 and 4.2%, respectively. When applied to Matrigel invasion studies, as few as 400 cells could be counted. The quantitation could be performed within 3 h. HCT 116, MDA MB 231 and HT 29 cells were investigated as examples of tumour cells with different invasive abilities in the 48-h Matrigel invasion assay. Using YO-PRO-1, 6.5 +/- 0.6% invasive HCT 116 cells and 52.6 +/- 4.5% MDA MB 231 cells (percentage of the inoculated cell population) were measured. HT 29 cells were practically non-invasive. These results were confirmed by visual scoring of DAPI-stained nuclei. In conclusion, the main advantages of the assay are its sensitive, reproducible and rapid quantitation of tumour cell invasion in vitro and the applicability to extended sample numbers by measuring in 96-well microplates.