Retinoic-Acid-Induced Downregulation of the 67 KDa Laminin Receptor Correlates with Reduced Biological Aggressiveness of Human Neuroblastoma Cells

16 pages (217-232), 6 figuresThe authors are indebted to Dr. S. Menard (Milan, Italy) for the gift of the antibody against 37LRP. C. M. R. L. was supported by Programa de Personal Técnico de Apoyo (PTA-2003-02-00207; Ministry of Education and Science, Spain). This work was supported by grants from the Spanish former Ministry of Education and Science and Ministry of Science and Innovation (SAF2003-00311, SAF2006–00647 and SAF2007–60780) and Generalitat Valenciana (GRUPOS 03/15 and ACOMP 09/212) (to D. B.) , and Instituto de Salud Carlos III (RD20-102 to S. N.).Peer reviewe

Estudio de la expresión génica durante la diferenciaciónde células de neuroblastoma SH-SY5Y inducida con Acido Retinoico

Póster original, presentado al XXXI Congreso de la Sociedad Española de Bioquímica y Biología Molecular. Bilbao, del 10 al 13 de Septiembre de 2008.El Ácido Retinoico (RA) actúa como molécula reguladora en la diferenciación de las células SH-SY5Y. En este proceso de diferenciación inducida por RA se ponen en marcha no sólo mecanismos genómicos clásicos sino también mecanismos extra-genómicos de acción rápida. Concretamente se han identificado la activación rápida de la ruta de señalización de la PI3K/AKT, una activación que resulta ser necesaria para la inducción de la diferenciación neuronal y que tiene lugar a través de la interacción del Receptor del Ácido Retinoico (RAR) con la subunidad reguladora de la PI3K (López-Carballo G. et al., J. Biol. Chem. 277, 25297-25304, 2002; Masiá S. et al., Mol Endocrinol. 21(10): 2391-402, 2007). Los cambio en la expresión génica producidos por integración de mecanismos de acción del RA tanto genómicos como extra-genómicos han sido estudiados mediante la tecnología de microarrays. Durante este proceso de diferenciación de las células SH-SY5Y con RA, hemos identificado 140 genes distribuidos en tres patrones de expresión génica claramente definidos: genes de expresión rápida, mediana y tardía. El análisis funcional de los genes de cada patrón revelan que: los genes de expresión rápida participan especialmente en procesos de procesamiento de RNA y de apoptosis, los de expresión intermedia están implicados principalmente en procesos de desarrollo, diferenciación y metabolismo del RA, y finalmente los genes de expresión tardía participan fundamentalmente en metabolismo, especialmente de lípidos y en quimiotaxis. Con el objetivo de establecer posibles puntos de interacción entre la activación de vías de señal por RA (acciones extra-genómicas del RA) y la activación transcripcional regulada por los receptores de RA (acciones genómicas) se ha estudiado el efecto de los inhibidores de les principales rutas de señalización sobre la expresión génica. Concretamente, este estudio nos ha permitido identificar que la activación de la PI3K/AKT ejerce un efecto inhibidor en la expresión de algunos genes de expresión intermedia y tardía, entre ellos, RARB. Finalmente, para obtener una visión más completa de todos los mecanismos de regulación que desencadena el Ácido Retinoico durante la diferenciación neuronal, hemos iniciado el estudio de la expresión de microRNAs (miRNAs). Los miRNAs son moléculas pequeñas (18-22 nt) de RNA de cadena sencilla no codificantes que regulan la traducción de genes específicos. Estas moléculas se expresan de forma diferencial en distintos tejidos y momentos del desarrollo, y controlan procesos en la célula como proliferación, diferenciación y apoptosis. Por este motivo es importante analizar los cambios de expresión de estas moléculas durante la diferenciación neuronal inducida por Ácido Retinoico, y tratar de identificar sus genes diana. En un estudio preliminar con un panel de miRNAs seleccionados hemos indentificado un grupo de 5 miRNAs inducidos por RA.S Meseguer es becario predoctoral I3P-CSIC, N Ruiz es técnico titulado superior de investigación I3P-CSIC. Financiado por el Plan Nacional de I+D+i, P. N. de Biomedicina (proyectos SAF2006-00647 y SAF2007-60780).Peer reviewe

Estudi de la Expressió gènica durant la diferenciació de cel.lules de neuroblastoma SH-SY5Y induida amb acid retinoic

Póster original presentado a la I Jornada conjunta GenProt de la Redes Catalana y Valenciana de Genómica y Proteómica, marzo de 2008.L’Àcid Retinoic (RA) actua com a molècula reguladora en la diferenciació de les cèl·lules SH-SY5Y. En aquest procés de diferenciació induït per RA es posen en marxa no sols mecanismes genòmics clàssics sinó a més a més mecanismes extra-genòmics d’acció ràpida. Concretament s’ha identificat l’activació ràpida de la ruta de senyalització de la PI3K/AKT, una activació que resulta ser necessària per la inducció de la diferenciació neuronal i que té lloc a través de la interacció del Receptor d’Àcid Retinoic (RAR) amb la subunitat reguladora de la PI3K (López-Carballo G. et al., J. Biol. Chem. 277, 25297-25304, 2002;Masiá S et al., Mol Endocrinol. 21(10): 2391-402, 2007). La integració d’ambos mecanismes d’acció del RA, genòmics i extra-genòmics suposa un canvi en l’expressió gènica que pot estudiar-se mitjançant la tecnologia de microarrays. Durant aquest procés de diferenciació de les cèl·lules SH-SY5Y amb RA, hem identificat 140 gens distribuïts en tres patrons d’expressió gènica clarament definits: gens d’expressió ràpida, intermèdia i tardana. L’anàlisi funcional dels gens de cada patró revelen que: els gens d’expressió ràpida participen especialment en processos de processament de RNA i d’apoptosis, els d’expressió intermèdia estan implicats principalment en processos de desenvolupament, diferenciació i metabolisme del RA, i finalment els gens d’expressió tardana participen fonamentalment en metabolisme, especialment de lípids i en quimiotaxis. Per tal d’establir possibles punts d’interacció entre l’activació de vies de senyal per RA (accions extra-genòmiques del RA) i l’activació transcripcional regulada pels receptors de RA (accions genòmiques) s’ha estudiat l’efecte dels inhibidors de les principals rutes de senyalització sobre l’expressió gènica. Concretament, en aquest estudi ens ha permès identificar que l’activació de la PI3K/AKT exerceix un efecte inhibidor en l’expressió d’alguns gens d’expressió intermèdia i tardana, entre ells, RARB.Este trabajo ha sido financiado por. SAF-2006-00647, SAF-2007-60780. Programa Nacional de Biomedicina, Ministeri d’Educació i CiènciaPeer reviewe

Análisis de los cambios en la fosforilación de proteínas inducidos por Acido Retinoico en células de Neuroblastoma

Póster original presentado XXVIII Congreso Nacional de la Sociedad Española de Bioquímica y Biología Molecular, celebrado en Zaragoza, septiembre, 2005El Acido Retinoico (RA), la forma activa de la vitamina A, induce en células de neuroblastoma SH-SY5Y la diferenciación neural. Los efectos del RA están mediados por su Receptor RAR, que pertenece a la superfamilia de los Receptores Nucleares de Hormonas. Además de sus acciones transcripcionales clásicas regulando la expresión de genes concretos, el RA actúa también de un modo extra-genómico, modulando la actividad de rutas de transducción de señal. El tratamiento con RA activa, entre otras la vía de señalización, PI3K/Akt, cuya activación por RA es un requisito para la diferenciación neural (López-Carballo G. et al., J. Biol. Chem. 277, 25297-25304, 2002). Estamos interesados en conocer cuáles son las dianas de estas acciones extra-genómicas del RA en células de neuroblastoma, y que influencia pueden tener sobre la respuesta transcripcional a RA. Para ello hemos iniciado un abordaje proteómico, basado en el hecho de que el resultado final de estas acciones extra-genómicas debe ser un cambio en los patrones de fosforilación de proteínas específicas. Para ello realizamos un enriquecimiento en proteínas fosforiladas a partir de extractos nucleares o celulares totales de células control y tratadas con RA, mediante cromatografía de afinidad. Estos extractos enriquecidos en proteínas fosforiladas son separados mediante electroforesis bidimensional (IEF/SDS-PAGE), y los geles son analizados y comparados informáticamente para identificar las bandas expresadas diferencialmente en ambas condiciones. Finalmente, las manchas identificadas son aisladas del gel, digeridas con tripsina y analizadas mediante espectrometría de masas (MALDI-TOF y LC/MS/MS).E. J. Laserna es becario FPI de la Generalitat Valenciana Financiado por el PNI+D+I (SAF2003-00311).Peer reviewe

Rapid, non-genomic actions of Retinoic Acid on Phosphatidyl-inositol-3-Kinase signalling pathway mediated by the Retinoic Acid Receptor

Póster original presentado EMBO Conference on Nuclear Receptors: Structure and Function in Health and Disease, celebrado en Italia en 2007Retinoic Acid (RA) treatment of SH-SY5Y neuroblastoma cells results in activation of phosphatidyl-inositol-3-kinase (PI3K) signalling pathway, and this activation is required for RA-induced differentiation. RA activates PI3K and ERK1/2 MAP Kinase signalling pathways through a rapid, non-genomic mechanism that does not require new gene transcription or newly synthesized proteins. Activation of PI3K by RA appears to involve the classical nuclear receptor RAR, on the basis of the pharmacological profile of the activation, loss and gain of function experiments with MEF-RAR(αβγ)L-/L- null cells, and the physical association between liganded RAR and PI3K kinase activity. Ligand binding differentially regulated the association of RAR with the two subunits of PI3K. Immunoprecipitation experiments performed in SH-SY5Y cells showed stable association between RARα and p85, the regulatory subunit of PI3K, independently of the presence of RA. In contrast, ligand administration increased the association of p110, the catalytic subunit of PI3K, to this complex. The intracellular localization of RAR resulted to be relevant for PI3K activation. A chimerical RAR receptor fusing c-Src myristylation domain to the N-terminal of RARα (Myr-RARα) was targeted to plasma membrane. Transfection of Myr-RARα to MEF-RAR(αβγ)L-/L- null cells and COS-7 cells results in strong activation of PI3K signalling pathway, although both in the absence as well in the presence of RA. Our results suggest a mechanism in which ligand binding to RAR would play a major role in the assembly and intracellular location of a signalling complex involving RAR and the subunits of PI3K. Next we have investigated the consequences of the activation of signalling pathways by RA in neuroblastoma cells. Downstream targets of the PI3K/Akt signalling pathway, like mTOR or p70S6K, are activated upon RA addition. In addition we show that RA addition rapidly increased phosphorylation of several Akt kinase target proteins in the nucleus, identified by an antibody recognizing the phosphorylated Akt motif. We have analyzed possible nuclear protein targets for RA-induced phosphorylation, with the aim of finding functional links between non-genomic actions and classical transcriptional actions mediated by RAR. We have found that RA treatment rapidly results in phosphorylation of chromatin proteins (core histone H3), as well as proteins involved in transcriptional activation (transcription factor CREB). In a second approach, nuclear phospho-proteins from control and RA-treated neuroblastoma cells were purified by affinity chromatography and analysed by 2D-electrophoresis. The differentially expressed spots were identified by Mass Spectrometry. We have found that RA treatment induces phosphorylation of chromatin proteins (HMGB1 and Histone H1.5) and proteins involved in the processing and transport of mRNA (PABP, hnRNP-K, hnRNP-C1/C2, Nucleophosmin).E. J. Laserna was supported by a FPI fellowship from Generalitat Valenciana.Peer reviewe

Non-genomic actions of retinoic acid induce pi3k signaling pathway and phosphorylation of nuclear proteins

Póster original presentado Bregenz Summer School on Endocrinology: Nuclear Receptors in Health and Disease, celebrado en Bregenz (Austria), 2007Retinoic acid (RA), the active form of vitamin A, induces neuroblastoma cells SH-SY5Y to differentiate. In addition to its classical transcriptional actions regulating the expression of specific genes, RA acts in an extra-genomic way, modulating the activity of relevant signalling cascades. In particular, RA treatment of SH-SY5Y neuroblastoma cells results in activation of phosphatidyl-inositol-3-kinase (PI3K) signaling pathway, and this activation is required for RA-induced differentiation (Lopez-Carballo G. et al., J. Biol. Chem. 277, 25297-25304, 2002). RA activates PI3K and also ERK1/2 MAP Kinase signaling pathways through a rapid, non-genomic mechanism that does not require new gene transcription or newly synthesized proteins. Activation of PI3K by RA appears to involve the classical nuclear receptor RAR, on the basis of the pharmacological profile of the activation, loss and gain of function experiments with MEF-RAR(αβγ)L-/L- null cells, and the physical association between liganded RAR and PI3K kinase activity. Immunoprecipitation experiments performed in SH-SY5Y cells showed stable association between RARα and p85, the regulatory subunit of PI3K, independently of the presence of RA. In contrast, ligand administration increased the association of p110, the catalytic subunit of PI3K, to this complex. The intracellular localization of RAR resulted to be relevant for PI3K activation. A chimerical RAR receptor fusing c-Src myristylation domain to the N-terminal of RARα (Myr-RARα) was targeted to plasma membrane. Transfection of Myr-RARα to MEF-RAR(αβγ)L-/L- null cells and COS-7 cells results in strong activation of PI3K signalling pathway. Our results suggest a mechanism in which ligand binding to RAR would play a major role in the assembly and intracellular location of a signalling complex involving RAR and the subunits of PI3K( Masiá S. et al., Mol. Endrocinol., in press, 2007). Next, we have investigated the consequences of the activation of signalling pathways by RA in neuroblastoma cells. Western blots using an antibody which specifically recognizes the phosphorylation motif of Akt have demonstrated that Akt activation in response to RA treatment produced a rapid phosphorylation of downstream substrates. Moreover, RA treatment resulted in rapid phosphorylation of well-known targets of Akt, such as mTOR and the ribosomal kinase p70S6, and other proteins, as CREB and histone H3, which are likely targets of RA non-genomic actions according to the scientific literature. In a second approach, nuclear phospho-proteins from control and RA-treated neuroblastoma cells were purified by affinity chromatography and analysed by 2D-electrophoresis. The differentially expressed spots were identified by Mass Spectrometry. We have found that RA treatment induces phosphorylation of chromatin proteins (HMGB1 and Histone H1.5) and proteins involved in the processing and transport of mRNA (PABP, hnRNP-K, hnRNP-C1/C2, Nucleophosmin). Finally, the observed RA-induced phosphorylation changes in these proteins were confirmed in 2-D western blots and immunoprecipitation experiments.Supported by grants SAF2003-00311 and SAF2006-00647 from D. G. Investigación (MEC, Spain). E. J. Laserna was supported by a FPI fellowship from Generalitat Valenciana. S. Masiá was supported by a FPI fellowship (MEC) and a training contract from Programa de Personal Técnico de Apoyo (MEC/Fondo Social Europeo).Peer reviewe

Antithrombin activity and outcomes in patients undergoing cardiac surgery with cardiopulmonary bypass

14 páginas, 1 figura, 1 tablaBackground: We recently reported prospective results from a cohort of patients scheduled for elective cardiac surgery with cardiopulmonary bypass (CPB) in which most baseline clinical parameters of patients and surgery outcomes failed to demonstrate relationships with post-CPB antithrombin (AT) activity. Objective and Methods: In this extension study, a larger sample size (250 patients) was analyzed following general linear models. Patients’ demographic and pre-CPB clinical data as well as pre/post-CPB AT activity and outcomes were collected. Results: There was a significant decrease of post-CPB AT activity (95.6±13.7% to 64.6±12.1%; p<0.001). Univariate and multivariate analyses revealed that a decrease of ~1% post-CPB AT activity may be expected per 3 years increase in patient’s age. Univariate analysis showed that post-CBP AT activity was inversely related to the need for transfusions,acute renal failure and occurrence of any complication (re-intervention, low cardiac output,arrhythmia, lung dysfunction, stroke, acute renal failure, mesenteric ischemia and re-hospitalization; P<0.05). Multivariate analysis adjusted for age and pre-CPB AT did not show statistical significance. Odds ratio (OR) <1 was observed in most outcomes (0.8 in average), which suggested a reduction of the probability for an increase of 10% in post-CBP AT. Conclusions: Our results confirm the role of low postsurgery AT activity influencing outcomes in patients undergoing CPB.Peer reviewe

Rapid, non-genomic actions of Retinoic Acid on Phosphatidyl-Inositol-3-Kinase

17 páginas, 9 figuras.Retinoic Acid (RA) treatment of SH-SY5Y neuroblastoma cells results in activation of phosphatidyl-inositol-3-kinase (PI3K) signaling pathway, and this activation is required for RA-induced differentiation. Here we show that RA activates PI3K and ERK1/2 MAP Kinase signaling pathways through a rapid, non-genomic mechanism that does not require new gene transcription or newly synthesized proteins. Activation of PI3K by RA appears to involve the classical nuclear receptor RAR, on the basis of the pharmacological profile of the activation, loss and gain of function experiments with MEF-RAR(αβγ)L-/L- null cells, and the physical association between liganded RAR and PI3K kinase activity. The association of RAR with the two subunits of PI3K was differentially regulated by the ligand. Immunoprecipitation experiments performed in SH-SY5Y cells showed stable association between RARα and p85, the regulatory subunit of PI3K, independently of the presence of RA. In contrast, ligand administration increased the association of p110, the catalytic subunit of PI3K, to this complex. The intracellular localization of RAR resulted to be relevant for PI3K activation. A chimerical RAR receptor fusing c-Src myristylation domain to the N-terminal of RARα (Myr-RARα) was targeted to plasma membrane. Transfection of Myr-RARα to MEF-RAR(αβγ)L-/Lnull cells and COS-7 cells results in strong activation of PI3K signaling pathway, although both in the absence as well in the presence of RA. Our results support a mechanism in which ligand binding to RAR would play a major role in the assembly and intracellular location of a signaling complex involving RAR and the subunits of PI3K.This work was supported by grants of the Spanish former Ministry of Science and Technology and Ministry of Education and Science [SAF2003-00311 and SAF2006-00647 (to D.B.)]; SAF2004-07131 (to A.R.d.L.), European Commission [QLK3-2002-02029 “Anticancer Retinoids” (to A.R.d.L.)], Generalitat Valenciana [GRUPOS 03/15 and ACOMP 06/212 (to D.B.)] and Fondo Europeo de Desarrollo Regional.Peer reviewe

Activation of the phosphatidylinositol 3-kinase/Akt signaling pathway by retinoic acid is required for neural differentiation of SH-SY5Y human neuroblastoma cells

8 pages, 6 figures.--PMID: 12000752 [PubMed]Retinoic acid (RA) induces neural differentiation of SH-SY5Y neuroblastoma cells. We show that the mRNA levels of the differentiation-inhibiting basic helix-loop-helix transcription factors ID1, ID2, and ID3 are down-regulated during RA-induced differentiation of SH-SY5Y cells. The levels of ID proteins decreased in parallel to the observed transcriptional repression. The expression of other basic helix-loop-helix genes changed during RA-induced differentiation: expression of neuroblast-specific ASCL1 (HASH-1) gene was promptly reduced after RA treatment, whereas expression of differentiation-promoting genes NEUROD6 (NEX-1, HATH-2) and NEUROD1 was increased. Treatments with 12-O-tetradecanoylphorbol-13-acetate, another inducer of neuroblastoma cell differentiation, also resulted in coordinated down-regulation of ID gene expression, underscoring the role of ID genes in differentiation. Down-regulation of ID gene expression by RA involves a complex mechanism because full transcriptional repression required newly synthesized proteins and signaling by phosphatidylinositol 3-kinase (PI3K). RA treatment activates the PI3K/Akt signaling pathway, resulting in increased PI3K activity in extracts from RA-treated cells and a rapid increase in phosphorylation of Akt in Ser-473. Inhibition of PI3K by LY294002 impaired RA-induced differentiation, as assessed by morphological and biochemical criteria. We propose that RA, by activating the PI3K/Akt signaling pathway, plays an important role in the regulation of neuronal cell survival.This work has been supported by grants of the Spanish Ministry of Education and Culture (PM96-0073) and Ministry of Science and Technology (PM1999-0112) to D.B. G. L.-C. was the recipient of a pre-doctoral fellowship (FPI) from the Consellería de Educación y Cultura de la Generalitat Valenciana. L.M. was the recipient of a fellowship from the Bancaixa-CSIC Program. S. M. was the recipient of a pre-doctoral fellowship (FPI) from the Ministry of Science and Technology.Peer reviewe