ATP Antagonizes Thrombin-Induced Signal Transduction through 12(S)-HETE and cAMP

In this study we have investigated the role of extracellular ATP on thrombin induced-platelet aggregation (TIPA) in washed human platelets. ATP inhibited TIPA in a dose-dependent manner and this inhibition was abolished by apyrase but not by adenosine deaminase (ADA) and it was reversed by extracellular magnesium. Antagonists of P2Y1 and P2Y12 receptors had no effect on this inhibition suggesting that a P2X receptor controlled ATP-mediated TIPA inhibition. ATP also blocked inositol phosphates (IP1, IP2, IP3) generation and [Ca2+]i mobilization induced by thrombin. Thrombin reduced cAMP levels which were restored in the presence of ATP. SQ-22536, an adenylate cyclase (AC) inhibitor, partially reduced the inhibition exerted by ATP on TIPA. 12-lipoxygenase (12-LO) inhibitors, nordihidroguaretic acid (NDGA) and 15(S)-hydroxy-5,8,11,13- eicosatetraenoic acid (15(S)-HETE), strongly prevented ATP-mediated TIPA inhibition. Additionally, ATP inhibited the increase of 12(S)-hydroxy-5,8,10,14-eicosatetraenoic acid (12(S)-HETE) induced by thrombin. Pretreatment with both SQ- 22536 and NDGA almost completely abolished ATP-mediated TIPA inhibition. Our results describe for the first time that ATP implicates both AC and 12-LO pathways in the inhibition of human platelets aggregation in response to agonists.Fil: Burzaco, Jaione. UNIVERSIDAD DEL PAIS VASCO;Fil: Conde, Manuel. UNIVERSIDAD DEL PAIS VASCO;Fil: Parada, Luis Antonio. Consejo Nacional de Invest.cientif.y Tecnicas. Centro Cientifico Tecnol.conicet - Salta. Instituto de Patologia Experimental;Fil: Zugaza, José L.. UNIVERSIDAD DEL PAIS VASCO;Fil: Dehaye, Jean-Paul. UNIVERSIDAD DEL PAIS VASCO;Fil: Marino, Aída. UNIVERSIDAD DEL PAIS VASCO

PLAGL1 gene function during hepatoma cells proliferation

Hepatocellular carcinoma develops as a multistep process, in which cell cycle deregulation is a central feature, resulting in unscheduled proliferation. The PLAGL1 gene encodes a homonym zinc finger protein that is involved in cell-proliferation control. We determined the genomic profile and the transcription and expression level of PLAGL1, simultaneously with that of its molecular partners p53, PPARγ and p21, in cell-lines derived from patients with liver cancer, during in vitro cell growth. Our investigations revealed that genomic and epigenetic changes of PLAGL1 are also present in hepatoma cell-lines. Transcription of PLAGL1 in tumor cells is significantly lower than in normal fibroblasts, but no significant differences in terms of protein expression were detected between these two cell-types, indicating that there is not a direct relationship between the gene transcriptional activity and protein expression. RT-PCR analyses on normal fibroblasts, used as control, also showed that PLAGL1 and p53 genes transcription occurs as an apparent orchestrated process during normal cells proliferation, which gets disturbed in cancer cells. Furthermore, abnormal trafficking of the PLAGL1 protein may occur in hepatocarcinogenesis.Fil: Vega Benedetti, Ana Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; ArgentinaFil: Saucedo, Cinthia Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; ArgentinaFil: Zavattari, Patrizia. University of Cagliari; ItaliaFil: Vanni, Roberta. University of Cagliari; ItaliaFil: Royo, Felix. CIC BioGUNE CIBERehd; EspañaFil: LLavero, Freancisco. Universidad del País Vasco; España. IKERBASQUE; EspañaFil: Zugaza, José L.. Universidad del País Vasco; España. IKERBASQUE; EspañaFil: Parada, Luis Antonio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; Argentin

Cell Signaling in Neurodegeneration

Neurodegenerative diseases are characterized by the progressive loss of specific subsets of neurons [...

The Human Serotonin 5-HT4 Receptor Regulates Secretion of Non-amyloidogenic Precursor Protein

The serotonin 5-HT4 receptor has recently gained a lot of attention for its functional roles in central processes such as memory and cognition. In this study, we show that activation of the human 5-HT4 (h5-HT4) receptor stimulates the secretion of the non-amyloidogenic soluble form of the amyloid precursor protein (sAPPα). 5-HT enhanced the level of secreted sAPPα in a time- and dose-dependent manner in Chinese hamster ovary cells stably expressing the h5-HT4(e) receptor isoform. The increase was inhibited by the selective 5-HT4 receptor antagonist, GR113808. The 5-HT4 selective agonists, prucalopride and renzapride, also increased secreted sAPPα in IMR32 human neuroblastoma cells. The stimulatory effect of 5-HT was mimicked by forskolin, a direct activator of adenylyl cyclase, and 8-bromo-cAMP, a membrane-permeant cAMP analogue. On the contrary, inhibition of protein kinase A (PKA) by H89 potentiated the 5-HT-induced increase in both secreted and cellular sAPPα. This phenomenon involves a novel PKA-independent stimulatory process that overcomes a PKA-dependent inhibitory one. Finally, activation of the h5-HT 4(e) receptor did not modify extracellular amyloid β-protein in Chinese hamster ovary cells transfected with the human APP695. Given the neuroprotective and enhancing memory effects of sAPPα, our results may open a new avenue for the treatment of Alzheimer's disease.Peer Reviewe

Amyloid β oligomers induce Ca2+ dysregulation and neuronal death through activation of ionotropic glutamate receptors

El pdf sel artículo es la versión pre-print.-- et al.Amyloid beta (Aβ) oligomers accumulate in brain tissue of Alzheimer disease patients and are related to pathogenesis. The precise mechanisms by which Aβ oligomers cause neurotoxicity remain unresolved. In this study, we investigated the role of ionotropic glutamate receptors on the intracellular Ca2+ overload caused by Aβ. Using rat cortical neurons in culture and entorhinal-hippocampal organotypic slices, we found that Aβ oligomers significantly induced inward currents, intracellular Ca2+ increases and apoptotic cell death through a mechanism requiring NMDA and AMPA receptor activation. The massive entry of Ca2+ through NMDA and AMPA receptors induced by Aβ oligomers caused mitochondrial dysfunction as indicated by mitochondrial Ca2+ overload, oxidative stress and mitochondrial membrane depolarization. Importantly, chronic treatment with nanomolar concentration of Aβ oligomers also induced NMDA- and AMPA receptor-dependent cell death in entorhinal cortex and hippocampal slice cultures. Together, these results indicate that overactivation of NMDA and AMPA receptor, mitochondrial Ca2+ overload and mitochondrial damage underlie the neurotoxicity induced by Aβ oligomers. Hence, drugs that modulate these events can prevent from Aβ damage to neurons in Alzheimer's disease. © 2009 Elsevier Ltd. All rights reserved.This work was supported by CIBERNED and by grants from Ministerio de Educación y Ciencia, Gobierno Vasco and Universidad del País Vasco.Peer Reviewe

Inverted signaling hierarchy between RAS and RAC in T-lymphocytes

In order to generate coherent biological responses to extracellular stimuli, cells have established synergistic and antagonistic crosstalk between pathways with similar or opposing functions, respectively. Two routes cooperating in the generation of mitogenic and cytoskeletal functions are those induced by Ras and Rho/Rac GTPases. In these signaling interactions, Rho/Rac proteins have been always placed in a downstream position respect to Ras in all cell systems analysed so far. In this report, we describe that such signaling hierarchy does not apply to T-lymphocytes. Thus, we show that both Rac1 GDP/GTP exchange factors such as Vav and constitutively active versions of Rac1 can promote the effective stimulation of the Ras pathway in T-lymphocytes. The molecular link for this new type of pathway interconnectivity is RasGRP1, a diacylglycerol-dependent GDP/GTP exchange factor for Ras that translocates to the plasma membrane in a Vav- and Rac1-dependent manner. The effect of the Vav/Rac1 pathway on the Ras pathway is highly dependent on the activity of phospliolipase C-γ, the key cellular supplier of intracellular diacylglycerol. Signaling experiments suggest that this crosstalk represents a signaling strategy used by the T-cell receptor to promote robust biological responses of both the Rac/Rho and Ras pathways upon antigen engagement.We thank M Blázquez for technical assistance. This work was supported by the US National Cancer Institute (CA7373501), the Programa General del Conocimiento of the Spanish Ministry of Science and Technology (PM99-0093), and a grant from the Ministry of Education and Culture of the Autonomous Government of Castilla- León (SA051/02). MJC and JLZ are investigators of the Ramón y Cajal Program (Spanish Ministry of Science and Technology) associated to the University of Salamanca. The Centro de Investigación del Cáncer is supported by endowments from the CSIC, University of Salamanca, Castilla-León Autonomous Government, the Spanish National Network of Cancer Centers (Carlos III Institute, Spanish Ministry of Health), the Foundation for Cancer Research of Salamanca (FICUS), and the Solórzano and Moraza Foundations.Peer Reviewe

Global conformational rearrangements during the activation of the GDP/GTP exchange factor Vav3

Activation of Rho/Rac GTPases during cell signaling requires the participation of GDP/GTP exchange factors of the Dbl family. Although the structure of the catalytic core of Dbl proteins has been established recently, the molecular changes that the full-length proteins experience during normal or oncogenic conditions of stimulation are still unknown. Here, we have used single-particle electron microscopy to solve the structures of the inactive (unphosphorylated), active (phosphorylated), and constitutively active (N-terminally deleted) versions of the exchange factor Vav3. Comparison of these forms has revealed the interdomain interactions maintaining the inactive Vav3 state and the dynamic changes that the overall Vav3 structure undergoes upon tyrosine phosphorylation. We have also found that the conformations of phosphorylated Vav3 and N-terminally deleted Vav3 are distinct, indicating that the acquisition of constitutive activity by exchange factors is structurally more complex than the mere elimination of inhibitory interactions between structural domains. © 2005 European Molecular Biology Organization | All Rights Reserved.This work was supported by the US National Cancer Institute (5 RO1 CA073735-07) to XRB, the Spanish Ministry of Education and Science (MES) Biomedicine Program to OL (SAF2002-01715) and XRB (SAF2003-00028), and an MES Action on Genomics and Proteomics (GEN2003-20239-C06-00) to both XRB and OL.Peer Reviewe

Vav mediates Ras stimulation by direct activation of the GDP/GTP exchange factor Ras GRP1

Here we describe a new signaling cross-talk between the Vav/Rac1 and Ras pathways that is established through the stimulation of RasGRP1, an exchange factor for Ras subfamily GTPases. This interaction is crucial for Ras activation in lymphoid cells, since this GTPase cannot become activated in the absence of Vav proteins. The activation of RasGRP1 requires both the generation of diacylglycerol via phospho lipase C-γ and the induction of actin polymerization, two responses induced by Vav and Rac1 that facilitate the translocation of RasGRP1 to juxtamembrane areas of the cell. Consistent with this, the cross-talk can be activated by tyrosine-phosphorylated wild-type Vav, oncogenic Vav and constitutively active Rac1. Conversely, Ras activation can be blocked in lymphocytes and ectopic systems using inhibitors affecting either phospholipase C-γ or F-actin polymerization. These results indicate that a relay mechanism exists in lymphoid and other cells helping in the generation of robust signaling responses by the Rac/Rho and Ras pathways upon receptor engagement

Combined fluorescent-chromogenic in situ hybridization for identification and laser microdissection of interphase chromosomes.

Chromosome territories constitute the most conspicuous feature of nuclear architecture, and they exhibit non-random distribution patterns in the interphase nucleus. We observed that in cell nuclei from humans with Down Syndrome two chromosomes 21 frequently localize proximal to one another and distant from the third chromosome. To systematically investigate whether the proximally positioned chromosomes were always the same in all cells, we developed an approach consisting of sequential FISH and CISH combined with laser-microdissection of chromosomes from the interphase nucleus and followed by subsequent chromosome identification by microsatellite allele genotyping. This approach identified proximally positioned chromosomes from cultured cells, and the analysis showed that the identity of the chromosomes proximally positioned varies. However, the data suggest that there may be a tendency of the same chromosomes to be positioned close to each other in the interphase nucleus of trisomic cells. The protocol described here represents a powerful new method for genome analysis