Modulation du Ca[indice supérieur 2+] nucléaire par l'ET-1 et le PAF dans les cellules cardiaques

Le Ca[indice supérieur 2+] joue un rôle important dans le contrôle des processus nucléaires tels la régulation de l'expression des gènes et la synthèse de l'ADN. Certaines études menées sur le Ca[indice supérieur 2+] nucléaire montrent qu'il existe un gradient calcique nucléo/cytosolique tandis que d'autres montrent une distribution égale dans les deux compartiments. En vue de ces contradictions, la microscopie confocale ainsi que la méthode de mesure du Ca[indice supérieur 2+] par un double marquage, à l'aide de la sonde calcique fluo-3 et le marqueur des acides nucléiques Syto-11, ont été utilisées afin d'étudier la régulation du Ca[indice supérieur 2+] nucléaire ainsi que le rôle du noyau comme tampon calcique durant l'excitation-contraction. Au repos, le niveau basal de l'intensité de fluorescence (Ca)[indice inférieur n] chez les myocytes d'origine embryonnaire de poulet est plus élevé que le (Ca)[indice inférieur c] suggérant qu'il existe un gradient calcique entre le noyau et le cytoplasme. De même, suite à la stimulation de ces cellules par l'ET-1 ou le PAF (récepteur-dépendent) ou par une dépolarisation soutenue avec une concentration élevée de K[indice supérieur +] (récepteur-indépendent), l'intensité de fluorescence du (Ca)[indice inférieur n] est plus élevée que celle du (Ca)[indice inférieur c]. Nos résultats montrent que l'augmentation nucléaire suit celle du cytoplasme mais elle est plus significative. Dans des cellules cardiaques perforées, le noyau répond à une augmentation du Ca[indice supérieur 2+] libre cytosolique en prenant une partie de ce Ca[indice supérieur 2+]. L'effet tampon maximal dans ces cellules cardiaques est de l'ordre de 1200 nM. Durant les oscillations calciques spontanées des cellules cardiaques d'origine foetale humaine et embryonnaire de poulet, la contribution nucléaire de la prise et de la relâche du Ca[indice supérieur 2+] cytosolique se produit d'une façon rythmique impliquant le rôle tampon du noyau durant l'excitation-contraction. L'apamine bloque l'amplitude des oscillations cytosoliques et nucléaires. --Résumé abrégé par UM

Voltage-gated Na+ Channel Activity Increases Colon Cancer Transcriptional Activity and Invasion Via Persistent MAPK Signaling

Functional expression of voltage-gated Na+ channels (VGSCs) has been demonstrated in multiple cancer cell types where channel activity induces invasive activity. The signaling mechanisms by which VGSCs promote oncogenesis remain poorly understood. We explored the signal transduction process critical to VGSC-mediated invasion on the basis of reports linking channel activity to gene expression changes in excitable cells. Coincidentally, many genes transcriptionally regulated by the SCN5A isoform in colon cancer have an over-representation of cis-acting sites for transcription factors phosphorylated by ERK1/2 MAPK. We hypothesized that VGSC activity promotes MAPK activation to induce transcriptional changes in invasion-related genes. Using pharmacological inhibitors/activators and siRNA-mediated gene knockdowns, we correlated channel activity with Rap1-dependent persistent MAPK activation in the SW620 human colon cancer cell line. We further demonstrated that VGSC activity induces downstream changes in invasion-related gene expression via a PKA/ERK/c-JUN/ELK-1/ETS-1 transcriptional pathway. This is the first study illustrating a molecular mechanism linking functional activity of VGSCs to transcriptional activation of invasion-related genes

Cross-Desensitization and Cointernalization of H1 and H2 Histamine Receptors Reveal New Insights into Histamine Signal Integration

G-protein coupled receptor (GPCR) signaling does not result from sequential activation of a linear pathway of proteins/enzymes, but rather from complex interactions of multiple, branched signaling routes, ie, signaling networks. In this work we present an exhaustive study of the crosstalk between H1 and H2 histamine receptors (H1R and H2R) in U937 cells and CHO transfected cells. By desensitization assays we demonstrated the existence of a cross-desensitization between both receptors independent of protein kinase A (PKA) or C (PKC). H1R agonist stimulation inhibited cell proliferation and induced apoptosis in U937 cells following treatment for 48h. H1R-induced antiproliferative and apoptotic response was inhibited by an H2R agonist suggesting that the crosstalk between both receptors modifies their function. Binding and confocal microscopy studies revealed cointernalization of both receptors upon treatment with the agonists. In order to evaluate potential heterodimerization of the receptors, sensitized emission FRET experiments were performed in HEK293T cells using H1R-CFP and H2R-YFP. To our knowledge these findings may represent the first demonstration of agonist-induced heterodimerization of the H1R and H2R. In addition, we also show that the inhibition of the internalization process did not prevent receptor cross-desensitization which was mediated by GRK2. Our study provides new insights into the complex signaling network mediated by histamine and further knowledge for the rational use of its ligands.Fil: Alonso, Maria Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina;Fil: Fernández, Natalia Brenda. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina; Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Farmacología. Cátedra de Química Medicinal; Argentina;Fil: Notcovich, Cintia Karina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina;Fil: Monczor, Federico. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Farmacología. Cátedra de Química Medicinal; Argentina; Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina;Fil: Simaan, May. National Institutes of Health; Estados Unidos de América;Fil: Baldi, Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina;Fil: Gutkind, J. Silvio. National Institutes of Health; Estados Unidos de América;Fil: Davio, Carlos Alberto. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Farmacología. Cátedra de Química Medicinal; Argentina; Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina;Fil: Shayo, Carina Claudia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina

Early warning of systemic risk in global banking: eigen-pair R number for financial contagion and market price-based methods

We analyse systemic risk in the core global banking system using a new network-based spectral eigen-pair method, which treats network failure as a dynamical system stability problem. This is compared with market price-based Systemic Risk Indexes, viz. Marginal Expected Shortfall, Delta Conditional Value-at-Risk, and Conditional Capital Shortfall Measure of Systemic Risk in a cross-border setting. Unlike paradoxical market price based risk measures, which underestimate risk during periods of asset price booms, the eigen-pair method based on bilateral balance sheet data gives early-warning of instability in terms of the tipping point that is analogous to the R number in epidemic models. For this regulatory capital thresholds are used. Furthermore, network centrality measures identify systemically important and vulnerable banking systems. Market price-based SRIs are contemporaneous with the crisis and they are found to covary with risk measures like VaR and betas

Somatic mutation of GRIN2A in malignant melanoma results in loss of tumor suppressor activity via aberrant NMDAR complex formation.

The ionotropic glutamate receptors (N-methyl-D-aspartate receptors (NMDARs)) are composed of large complexes of multi-protein subunits creating ion channels in the cell plasma membranes that allow for influx or efflux of mono- or divalent cations (e.g., Ca(2+)) important for synaptic transmissions, cellular migration, and survival. Recently, we discovered the high prevalence of somatic mutations within one of the ionotropic glutamate receptors, GRIN2A, in malignant melanoma. Functional characterization of a subset of GRIN2A mutants demonstrated a loss of NMDAR complex formation between GRIN1 and GRIN2A, increased anchorage-independent growth in soft agar, and increased migration. Somatic mutation of GRIN2A results in a dominant negative effect inhibiting the tumor-suppressive phenotype of wild-type (WT) GRIN2A in melanoma. Depletion of endogenous GRIN2A in melanoma cells expressing WT GRIN2A resulted in increased proliferation compared with control. In contrast, short-hairpin RNA depletion of GRIN2A in mutant cell lines slightly reduced proliferation. Our data show that somatic mutation of GRIN2A results in increased survival, and we demonstrate the functional importance of GRIN2A mutations in melanoma and the significance that ionotropic glutamate receptor signaling has in malignant melanoma

Inferring the Lifetime of Endosomal Protein Complexes by Fluorescence Recovery after Photobleaching

Cellular signal transduction is dynamic, with signaling proteins continually associating and dissociating into and from protein complexes. Here we present a fluorescence recovery after photobleaching technique to determine the lifetime of protein complexes on intracellular vesicles. We use Bayesian inference based on a model that includes the diffusion of cytosolic proteins and their interaction with membrane-bound receptors. Our analysis is general: we incorporate prior information on protein diffusion, measurement error in determining fluorescence intensities, corrections for photobleaching, and variation in the concentration of receptors between vesicles. We apply our method to the complexes formed on endosomes by G-protein-coupled receptors and the protein β-arrestin. The lifetime of these complexes determines the recycling rate of the receptors. We find in mammalian cells that the bradykinin type 2 receptor and β-arrestin2 complex has a lifetime of ∼2 min, while the angiotensin II type 1A receptor and β-arrestin2 complex has a lifetime of ∼6 min. As well as allowing quantitative comparisons between experiments, our method provides in vivo parameters for systems biology simulations of signaling networks