Genomic approaches to the study of HIV‐1 acquisition.

Host genome studies are increasingly available for the study of infectious disease susceptibility. Current technologies include large-scale genotyping, genome-wide screens such as transcriptome and silencing (silencing RNA) studies, and increasingly, the possibility to sequence complete genomes. These approaches are of interest for the study of individuals who remain uninfected despite documented exposure to human immunodeficiency virus type 1. The main limitation remains the ascertainment of exposure and establishing large cohorts of informative individuals. The pattern of enrichment for CCR5 Δ32 homozygosis should serve as the standard for assessing the extent to which a given cohort (of white subjects) includes a large proportion of exposed uninfected individuals.

Monoubiquitination and Activity of the Paracaspase MALT1 Requires Glutamate 549 in the Dimerization Interface.

The mucosa-associated lymphoid tissue protein-1 (MALT1, also known as paracaspase) is a protease whose activity is essential for the activation of lymphocytes and the growth of cells derived from human diffuse large B-cell lymphomas of the activated B-cell subtype (ABC DLBCL). Crystallographic approaches have shown that MALT1 can form dimers via its protease domain, but why dimerization is relevant for the biological activity of MALT1 remains largely unknown. Using a molecular modeling approach, we predicted Glu 549 (E549) to be localized within the MALT1 dimer interface and thus potentially relevant. Experimental mutation of this residue into alanine (E549A) led to a complete impairment of MALT1 proteolytic activity. This correlated with an impaired capacity of the mutant to form dimers of the protease domain in vitro, and a reduced capacity to promote NF-κB activation and transcription of the growth-promoting cytokine interleukin-2 in antigen receptor-stimulated lymphocytes. Moreover, this mutant could not rescue the growth of ABC DLBCL cell lines upon MALT1 silencing. Interestingly, the MALT1 mutant E549A was unable to undergo monoubiquitination, which we identified previously as a critical step in MALT1 activation. Collectively, these findings suggest a model in which E549 at the dimerization interface is required for the formation of the enzymatically active, monoubiquitinated form of MALT1.

Effects of epigenetic regulatory elements on telomeric gene expression

Transgene expression in eukaryotic cells strongly depends on the locus of integration in the host genome and often results in limited transcription level because of unfavorable chromatin structure at the integration site. Epigenetic regulators are DNA sequences which are believed to act on the chromatin structure and may protect transgenes from this so-called position effect. Despite being extensively used to increase transgene expression, the mechanism of action of many of these elements remains largely unknown. Here we evaluated different epigenetic regulatory DNA elements for their ability to protect transgene transcription at telomeres, a defined chromatin environment associated to low or inconsistent expression caused by the Telomere Position Effect (TPE). For the assessment of the effects of epigenetic regulators at telomeres, a novel dual reporter system had to be designed. Telomeric integration of the newly-developed dual reporter system carrying different epigenetic regulators showed that MARs (Matrix Attachment Regions), a UCOE (Ubiquitous Chromatin-Opening Element) or the chicken cHS4 insulator have strong barrier activity which prevented TPE from spreading toward the centromere, resulting in stable and in some cases increased expression of a telomeric-distal reporter gene. In addition, MARs and STAR element 40 resulted in an increase of cells expressing the telomeric-proximal reporter gene, suggesting also an anti-silencing effect. Chromatin immunoprecipitation assays revealed that at telomeres MARs actively promote the deposition of euchromatic histone marks, especially acetylation of both histone H3 and H4, which might be involved in MARs' barrier and transcriptional activator activities. Differently, the chromatin in proximity of the UCOE element was depleted of several repressive chromatin marks, such as trimethylated lysine 9 and lysine 27 on histone H3 and trimethylated lysine 20 of histone H4, possibly favoring the preservation of an open chromatin structure at the integration site. We conclude that epigenetic regulatory elements that may be used to enhance and sustain transgene expression have all a specific epigenetic signature which might be at the basis of their mechanism of action, and that a combination of different classes of epigenetic regulators might be advantageous when high levels of protein expression are required. - L'expression des transgènes dans les cellules eucaryotes est fortement influencée par leur site d'intégration dans le génome. En effet, une structure chromatinienne défavorable au niveau du site d'intégration peut fortement limiter le degré d'expression d'un transgène. Il existe toutefois des séquences d'ADN qui, en agissant sur la structure de la chromatine, permettent de limiter cet effet de position et, par conséquent, de promouvoir l'expression soutenue d'un transgène. Ces éléments génomiques, connus comme régulateurs épigénétiques, sont largement utilisés dans plusieurs domaines où une expression élevée et soutenue est requise, malgré un mode de fonctionnement parfois méconnu. Dans cette étude, j'ai évalué la capacité de différents régulateurs épigénétiques à protéger la transcription de transgènes au niveau des télomères, régions chromatiniennes bien définies qui ont été associées à un fort effet de silençage, connu comme «effet de position télomérique». Pour cela, un nouveau système à deux gènes rapporteurs a été développé. Lorsque des MARs (Matrix Attachment Regions, séquences d'ADN pouvant s'associer à la matrice nucléaire), un UCOE (Ubiquitous Chromatin-Opening Element, élément pouvant ouvrir la chromatine) ou l'isolateur génétique cHS4 (dérivé du locus de la β-globine de poulet) sont placés entre les deux gènes rapporteurs, une forte activité barrière bloquant la propagation de la chromatine répressive télomérique est observée, résultant en un plus grand nombre de cellules exprimant le gène télomérique-distal. D'autre part, une augmentation du nombre de cellules exprimant le gène télomérique-proximal, observée en présence des éléments MAR et STAR 40 (Stabilizing Anti-Repressor element 40, un élément pouvant prévenir la répression génique), suggère aussi un faible effet anti-silençage pour ces éléments. Des expériences d'immunoprécipitation de la chromatine démontrent qu'au télomère, les MARs favorisent l'assemblage de marqueurs de la chromatine active, surtout l'acétylation des histones H3 et H4, qui pourraient être à la base de l'activité barrière et de celle d'activateur transcriptionel. Différemment, la chromatine à proximité de l'élément UCOE est particulièrement pauvre en marqueurs de la chromatine silencieuse, comme la trimethylation des lysines 9 et 27 de l'histone H3, ainsi que la trimethylation de la lysine 20 de l'histone H4. Cela suggère que UCOE pourrait préserver une structure chromatinienne ouverte au site d'intégration, favorisant l'expression des gènes à sa proximité. En conclusion, les régulateurs épigénétiques analysés lors de cette étude ont tous montré une signature épigénétique spécifique qui pourrait être à la base de leurs mécanismes de fonctionnement, suggérant aussi qu'une utilisation d'éléments épigénétiques de classe différente dans un même vecteur d'expression pourrait être avantageuse lorsque de hauts et soutenus niveaux d'expression sont nécessaires.

Genome organization and gene expression shape the transposable element distribution in the Drosophila melanogaster euchromatin.

The distribution of transposable elements (TEs) in a genome reflects a balance between insertion rate and selection against new insertions. Understanding the distribution of TEs therefore provides insights into the forces shaping the organization of genomes. Past research has shown that TEs tend to accumulate in genomic regions with low gene density and low recombination rate. However, little is known about the factors modulating insertion rates across the genome and their evolutionary significance. One candidate factor is gene expression, which has been suggested to increase local insertion rate by rendering DNA more accessible. We test this hypothesis by comparing the TE density around germline- and soma-expressed genes in the euchromatin of Drosophila melanogaster. Because only insertions that occur in the germline are transmitted to the next generation, we predicted a higher density of TEs around germline-expressed genes than soma-expressed genes. We show that the rate of TE insertions is greater near germline- than soma-expressed genes. However, this effect is partly offset by stronger selection for genome compactness (against excess noncoding DNA) on germline-expressed genes. We also demonstrate that the local genome organization in clusters of coexpressed genes plays a fundamental role in the genomic distribution of TEs. Our analysis shows that-in addition to recombination rate-the distribution of TEs is shaped by the interaction of gene expression and genome organization. The important role of selection for compactness sheds a new light on the role of TEs in genome evolution. Instead of making genomes grow passively, TEs are controlled by the forces shaping genome compactness, most likely linked to the efficiency of gene expression or its complexity and possibly their interaction with mechanisms of TE silencing.

Different internalization properties of the alpha1a- and alpha1b-adrenergic receptor subtypes: the potential role of receptor interaction with beta-arrestins and AP50.

The internalization properties of the alpha1a- and alpha1b-adrenergic receptors (ARs) subtypes transiently expressed in human embryonic kidney (HEK) 293 cells were compared using biotinylation experiments and confocal microscopy. Whereas the alpha1b-AR displayed robust agonist-induced endocytosis, the alpha1a-AR did not. Constitutive internalization of the alpha1a-AR was negligible, whereas the alpha1b-AR displayed significant constitutive internalization and recycling. We investigated the interaction of the alpha1-AR subtypes with beta-arrestins 1 and 2 as well as with the AP50 subunit of the clathrin adaptor complex AP2. The results from both coimmunoprecipitation experiments and beta-arrestin translocation assays indicated that the agonistinduced interaction of the alpha1a-AR with beta-arrestins was much weaker than that of the alpha1b-AR. In addition, the alpha1a-AR did not bind AP50. The alpha1b-AR mutant M8, lacking the main phosphorylation sites in the receptor C tail, was unable to undergo endocytosis and was profoundly impaired in binding beta-arrestins despite its binding to AP50. In contrast, the alpha1b-AR mutant DeltaR8, lacking AP50 binding, bound beta-arrestins efficiently, and displayed delayed endocytosis. RNA interference showed that beta-arrestin 2 plays a prominent role in alpha1b-AR endocytosis. The findings of this study demonstrate differences in internalization between the alpha1a- and alpha1b-AR and provide evidence that the lack of significant endocytosis of the alpha1a-AR is linked to its poor interaction with beta-arrestins as well as with AP50. We also provide evidence that the integrity of the phosphorylation sites in the C tail of the alpha1b-AR is important for receptor/beta-arrestin interaction and that this interaction is the main event triggering receptor internalization.

The role of Bmi1 in CNS development and in retinal degeneration

SUMMARY : The present work addresses several aspects of cell cycle regulation, cell fate specification and cell death in the central nervous system (CNS), specifically the cortex and the retina. More precisely, we investigated the role of Bmi1, a polycomb family gene required for stem cell proliferation and self-renewal, in the development of the cerebral cortex, as well as in the genesis of the retina. These data, together with studies published during the last two decades concerning cell cycle re-activation in apoptotic neurons in the CNS, raised the question of a possible link between regulation of the cell cycle during development and during retinal degeneration. 1. The effects of Bmi1 loss in the cerebral cortex : Consistently with our and others' observations on failure of Bmi9-/- stem cells to proliferate and self-renew in vitro, the Bmi9-/- cerebral cortex presented slight defects in proliferation in stem/progenitor cells compartments in vivo. This was in accordance with the pattern of Bmi1 expression in the developing forebrain. The modest proliferation defects, compared to the drastic consequences of Bmi9 loss in vitro, suggest that cell-extrinsic mechanisms may partially compensate for Bmi1 deletion in vivo during cortical histogenesis. Nevertheless, we observed a decreased proliferating activity in neurogenic regions of the adult telencephalon, more precisely in the subventricular zone, showing that Bmi1 controls neural stem/progenitor proliferation during adulthood in vivo. Our data also highlight an increased production of astrocytes at birth, and a generalized gliosis in the adult Bmi9-/- brain. Importantly, glial progenitors and astrocytes retained the ability to proliferate in the absence of Bmi1. 2. The effects of Bmi1 loss in the retina : The pattern of expression of Bmi1 during development and in the adult retina suggests a role for Bmi1 in cell fate specification and differentiation rather than in proliferation. While the layering and the global structure of the retina appear normal in Bmi1 /adult mice, immunohistochemìcal analysis revealed defects in the three major classes of retinal interneurons, namely: horizontal, bipolar and amacrine cells. Electroretinogram recordings in Bmi9-/- mice are coherent with the defects observed at the histological level, with a reduced b-wave and low-profile oscillatory potentials. These results show that Bmi1 controls not only proliferation, but also cell type generation, as previously observed in the cerebellum. 3. Cell cycle events and related neuroprotective strategies in retinal degeneration : In several neurodegenerative disorders, neurons re-express cell cycle proteins such as cyclin dependent kinases (Cdks) prior to apoptosis. Here, we show for the first time that this is also the case during retinal degeneration. Rd1 mice carry a recessive defect (Pdeóbrd/rd) that causes retinal degeneration and serves as a model of retinitis pigmentosa. We found that photoreceptors express Cdk4 and Cdk2, and undergo DNA synthesis prior to cell death. To interfere with the reactivation of Cdk-related pathways, we deleted E2fs or Brni1, which normally allow cell cycle progression. While deleting E2f1 (downstream of Cdk4/6) in Rd1 mice provides only temporary protection, knocking out Bmi1 (upstream of Cdks) leads to an extensive neuroprotective effect, independent of p16ink4a or p19arf, two tumor suppressors regulated by Bmi1. Analysis of Cdks and the DNA repair-related protein Ligase IV showed that Bmi1 acts downstream of DNA repair events and upstream of Cdks in this neurodegenerative mechanism. Expression of Cdks during an acute model of retinal degeneration, light damage-induced photoreceptor death, points to a role for Bmi1 and cell cycle proteins in retinal degeneration. Considering the similarity with the cell cycle-related apoptotic pathway observed in other neurodegenerative diseases, Bmi1 is a possible general target to prevent or delay neuronal death. RESUME : Ce travail aborde plusieurs aspects de la régulation du cycle cellulaire, de la spécification du devenir des cellules et de la mort cellulaire dans le système nerveux centrale (SNC), plus particulièrement dans le cortex cérébral et dans la rétine. Nous nous sommes intéressés au gène Bmi1, appartenant à la famille polycomb et nécessaire à la prolifération et au renouvellement des cellules souches. Nous avons visé à disséquer son rôle dans le développement du cortex et de la rétine. Ces données, ainsi qu'une série de travaux publiés au cours des deux dernières décennies concernant la réactivation du cycle cellulaire dans les neurones en voie d'apoptose dans le SNC, nous ont ensuite poussé à chercher un lien entre la régulation du cycle cellulaire pendant le développement et au cours de la dégénérescence rétinienne. 1. Les effets de l'inactivation de Bmi1 dans le cortex cérébral : En accord avec l'incapacité des cellules souches neurales in vitro à proliférer et à se renouveler en absence de Bmi1, le cortex cérébral des souris Bmi1-/- présente de légers défauts de prolifération dans les compartiments contenant les cellules souches neurales. Ceci est en accord avec le profil d'expression de Bmi1 dans le télencéphale. Les conséquences de la délétion de Bmi1 sont toutefois nettement moins prononcées in vivo qu'in vitro ; cette différence suggère l'existence de mécanismes pouvant partiellement compenser l'absence de Bmi1 pendant la corticogenèse. Néanmoins, l'observation d'une réduction de la prolifération dans la zone sous-ventriculaire, la zone majeure de neurogenèse dans le télencéphale adulte, montre que Bmi1 contrôle la prolifération des cellules souche/progénitrices neurales chez la souris adulte. Nos résultats démontrent par ailleurs une augmentation de la production d'astrocytes à la naissance ainsi qu'une gliose généralisée à l'état adulte chez les souris Bmi1-/-. Les progéniteurs gliaux et les astrocytes conservent donc leur capacité à proliférer en absence de Bmi1. 2. Les effets de l'inactivation de Bmi1 dans la rétine : Le profil d'expression de Bmi1 pendant fe développement ainsi que dans la rétine adulte suggère un rôle de Bmi1 dans la spécification de certains types cellulaires et dans la différentiation plutôt que dans la prolifération. Alors que la structure et la lamination de la rétine semblent normales chez les souris Bmi1-/-, l'analyse par immunohistochimie amis en évidence des défauts au niveau des trois classes d'interneurones rétiniens (les cellules horizontales, bipolaires et amacrines). Les électrorétinogrammes des souris Bmi1-/- sont cohérents avec les défauts observés au niveau histologique et montrent une réduction de l'onde « b » et des potentiels oscillatoires. Ces résultats montrent que Bmi1 contrôle la génération de certaines sous-populations de neurones, comme démontré auparavant au niveau de cervelet. 3. Réactivation du cycle cellulaire et stratégies théraoeutiaues dans les dégénérescences rétiniennes : Dans plusieurs maladies neurodégénératives, les neurones ré-expriment des protéines du cycle cellulaire telles que les kinases cycline-dépendantes (Cdk) avant d'entrer en apoptose. Nous avons démontré que c'est aussi le cas dans les dégénérescences rétiniennes. Les souris Rd1 portent une mutation récessive (Pde6brd/rd) qui induit une dégénérescence de la rétine et sont utilisées comme modèle animal de rétinite pigmentaire. Nous avons observé que les photorécepteurs expriment Cdk4 et Cdk2, et entament une synthèse d'ADN avant de mourir par apoptose. Pour interférer avec la réactivation les mécanismes Cdk-dépendants, nous avons inactivé les gènes E2f et Bmi1, qui permettent normalement la progression du cycle cellulaire. Nous avons mis en évidence que la délétion de E2f1 (en aval de Cdk4/6) dans les souris Rd1 permet une protection transitoire des photorécepteurs. Toutefois, l'inactivation de Bmi1 (en amont des Cdk) est corrélée à une neuroprotection bien plus durable et ceci indépendamment de p16ink4a et p19arf, deux suppresseurs de tumeurs normalement régulés par Bmi1. L'analyse des Cdk et de la ligase IV (une protéine impliquée dans les mécanismes de réparation de l'ADN) a montré que Bmi1 agit en aval des événements de réparation de l'ADN et en amont des Cdk dans la cascade apoptotique dans les photorécepteurs des souris Rd1. Nous avons également observé la présence de Cdk dans un modèle aigu de dégénérescence rétinienne induit par une exposition des animaux à des niveaux toxiques de lumière. Nos résultats suggèrent donc un rôle général de Bmi1 et des protéines du cycle cellulaire dans les dégénérescences de la rétine. Si l'on considère la similarité avec les événements de réactivation du cycle cellulaire observés dans d'autres maladies neurodégénératives, Bmi1 pourrait être une cible thérapeutique générale pour prévenir la mort neuronale.

Analysis of LEDGF/p75 expression regulation

Background To replicate, retroviruses must insert DNA copies of their RNA genomes into the host genome. This integration process is catalyzed by the viral integrase protein. The site of viral integration has been shown to be non-random and retrovirus-specific. LEDGF/p75, a splice variant encoded by PSIP1 gene and described as a general transcription coactivator, was identified as a tethering factor binding both to chromatin and to lentiviral integrases, thereby affecting integration efficiency as well as integration site selection. LEDGF/p75 is still a poorly characterized protein, and its cellular endogenous function has yet to be fully determined. In order to start unveiling the roles of LEDGF/p75 in the cell, we started to investigate the mechanisms involved in the regulation of LEDGF/p75. Materials and methods To identify PSIP1 minimal promoter and associated regulatory elements, we cloned a region starting 5 kb upstream the transcription start site (TSS, +1 reference position) to the ATG start codon (+816), as well as systematic truncations, in a plasmid containing the firefly luciferase reporter gene. These constructs were co-transfected into HEK293 cells with a plasmid encoding the Renilla luciferase under the pTK promoter as an internal control for transfection efficiency. Both luciferase activities were assessed by luminescence as an indicator of promoter activity. Results Luciferase assays identified regions -76 to +1 and +1 to +94 as two independent minimal promoters showing respectively a 3.7x and 2.3x increase in luciferase activity. These two independent minimal promoters worked synergistically increasing luciferase activity up to 16.3x as compared to background. Moreover, we identified five regulatory blocks which modulated luciferase activity depending on the DNA region tested, three enhancers (- 2007 to -1159, -284 to -171 and +94 to +644) and two silencers (-171 to -76 and +796 to +816). However, the silencing effect of the region -171 to -76 is dependent on the presence of the +94 to +644 region, ruling out the enhancer activity of the latter. Computational analysis of PSIP1 promoter revealed the absence of TATA box and initiator (INR) sequences, classifying this promoter as nonconventional. TATA-less and INR-less promoters are characterized by multiple Sp1 binding sites, involved in the recruitment of the RNA pol II complex. Consistent with this, PSIP1 promoter contains multiple putative Sp1 binding sequences in regions -76 to +1 and +1 to +94.

CNOT3 is a modifier of PRPF31 mutations in retinitis pigmentosa with incomplete penetrance.

Heterozygous mutations in the PRPF31 gene cause autosomal dominant retinitis pigmentosa (adRP), a hereditary disorder leading to progressive blindness. In some cases, such mutations display incomplete penetrance, implying that certain carriers develop retinal degeneration while others have no symptoms at all. Asymptomatic carriers are protected from the disease by a higher than average expression of the PRPF31 allele that is not mutated, mainly through the action of an unknown modifier gene mapping to chromosome 19q13.4. We investigated a large family with adRP segregating an 11-bp deletion in PRPF31. The analysis of cell lines derived from asymptomatic and affected individuals revealed that the expression of only one gene among a number of candidates within the 19q13.4 interval significantly correlated with that of PRPF31, both at the mRNA and protein levels, and according to an inverse relationship. This gene was CNOT3, encoding a subunit of the Ccr4-not transcription complex. In cultured cells, siRNA-mediated silencing of CNOT3 provoked an increase in PRPF31 expression, confirming a repressive nature of CNOT3 on PRPF31. Furthermore, chromatin immunoprecipitation revealed that CNOT3 directly binds to a specific PRPF31 promoter sequence, while next-generation sequencing of the CNOT3 genomic region indicated that its variable expression is associated with a common intronic SNP. In conclusion, we identify CNOT3 as the main modifier gene determining penetrance of PRPF31 mutations, via a mechanism of transcriptional repression. In asymptomatic carriers CNOT3 is expressed at low levels, allowing higher amounts of wild-type PRPF31 transcripts to be produced and preventing manifestation of retinal degeneration.

MAR elements and transposons for improved transgene integration and expression.

Reliable and long-term expression of transgenes remain significant challenges for gene therapy and biotechnology applications, especially when antibiotic selection procedures are not applicable. In this context, transposons represent attractive gene transfer vectors because of their ability to promote efficient genomic integration in a variety of mammalian cell types. However, expression from genome-integrating vectors may be inhibited by variable gene transcription and/or silencing events. In this study, we assessed whether inclusion of two epigenetic control elements, the human Matrix Attachment Region (MAR) 1-68 and X-29, in a piggyBac transposon vector, may lead to more reliable and efficient expression in CHO cells. We found that addition of the MAR 1-68 at the center of the transposon did not interfere with transposition frequency, and transgene expressing cells could be readily detected from the total cell population without antibiotic selection. Inclusion of the MAR led to higher transgene expression per integrated copy, and reliable expression could be obtained from as few as 2-4 genomic copies of the MAR-containing transposon vector. The MAR X-29-containing transposons was found to mediate elevated expression of therapeutic proteins in polyclonal or monoclonal CHO cell populations using a transposable vector devoid of selection gene. Overall, we conclude that MAR and transposable vectors can be used to improve transgene expression from few genomic transposition events, which may be useful when expression from a low number of integrated transgene copies must be obtained and/or when antibiotic selection cannot be applied.

Identification of novel elements of the Drosophila blisterome sheds light on potential pathological mechanisms of several human diseases.

Main developmental programs are highly conserved among species of the animal kingdom. Improper execution of these programs often leads to progression of various diseases and disorders. Here we focused on Drosophila wing tissue morphogenesis, a fairly complex developmental program, one of the steps of which - apposition of the dorsal and ventral wing sheets during metamorphosis - is mediated by integrins. Disruption of this apposition leads to wing blistering which serves as an easily screenable phenotype for components regulating this process. By means of RNAi-silencing technique and the blister phenotype as readout, we identify numerous novel proteins potentially involved in wing sheet adhesion. Remarkably, our results reveal not only participants of the integrin-mediated machinery, but also components of other cellular processes, e.g. cell cycle, RNA splicing, and vesicular trafficking. With the use of bioinformatics tools, these data are assembled into a large blisterome network. Analysis of human orthologues of the Drosophila blisterome components shows that many disease-related genes may contribute to cell adhesion implementation, providing hints on possible mechanisms of these human pathologies.

The GTPase RalA regulates different steps of the secretory process in pancreatic beta-cells.

BACKGROUND: RalA and RalB are multifuntional GTPases involved in a variety of cellular processes including proliferation, oncogenic transformation and membrane trafficking. Here we investigated the mechanisms leading to activation of Ral proteins in pancreatic beta-cells and analyzed the impact on different steps of the insulin-secretory process. METHODOLOGY/PRINCIPAL FINDINGS: We found that RalA is the predominant isoform expressed in pancreatic islets and insulin-secreting cell lines. Silencing of this GTPase in INS-1E cells by RNA interference led to a decrease in secretagogue-induced insulin release. Real-time measurements by fluorescence resonance energy transfer revealed that RalA activation in response to secretagogues occurs within 3-5 min and reaches a plateau after 10-15 min. The activation of the GTPase is triggered by increases in intracellular Ca2+ and cAMP and is prevented by the L-type voltage-gated Ca2+ channel blocker Nifedipine and by the protein kinase A inhibitor H89. Defective insulin release in cells lacking RalA is associated with a decrease in the secretory granules docked at the plasma membrane detected by Total Internal Reflection Fluorescence microscopy and with a strong impairment in Phospholipase D1 activation in response to secretagogues. RalA was found to be activated by RalGDS and to be severely hampered upon silencing of this GDP/GTP exchange factor. Accordingly, INS-1E cells lacking RalGDS displayed a reduction in hormone secretion induced by secretagogues and in the number of insulin-containing granules docked at the plasma membrane. CONCLUSIONS/SIGNIFICANCE: Taken together, our data indicate that RalA activation elicited by the exchange factor RalGDS in response to a rise in intracellular Ca2+ and cAMP controls hormone release from pancreatic beta-cell by coordinating the execution of different events in the secretory pathway.

Role of mTOR, Bad, and Survivin in RasGAP Fragment N-Mediated Cell Protection.

Partial cleavage of p120 RasGAP by caspase-3 in stressed cells generates an N-terminal fragment, called fragment N, which activates an anti-apoptotic Akt-dependent survival response. Akt regulates several effectors but which of these mediate fragment N-dependent cell protection has not been defined yet. Here we have investigated the role of mTORC1, Bad, and survivin in the capacity of fragment N to protect cells from apoptosis. Neither rapamycin, an inhibitor of mTORC1, nor silencing of raptor, a subunit of the mTORC1 complex, altered the ability of fragment N from inhibiting cisplatin- and Fas ligand-induced death. Cells lacking Bad, despite displaying a stronger resistance to apoptosis, were still protected by fragment N against cisplatin-induced death. Fragment N was also able to protect cells from Fas ligand-induced death in conditions where Bad plays no role in apoptosis regulation. Fragment N expression in cells did neither modulate survivin mRNA nor its protein expression. Moreover, the expression of cytoplasmic survivin, known to exert anti-apoptotic actions in cells, still occurred in UV-B-irradiated epidermis of mouse expressing a caspase-3-resistant RasGAP mutant that cannot produce fragment N. Additionally, survivin function in cell cycle progression was not affected by fragment N. These results indicate that, taken individually, mTOR, Bad, or Survivin are not required for fragment N to protect cells from cell death. We conclude that downstream targets of Akt other than mTORC1, Bad, or survivin mediate fragment N-induced protection or that several Akt effectors can compensate for each other to induce the pro-survival fragment N-dependent response.

Rôle de la MGMT et implications cliniques dans les tumeurs cérébrales [Role of MGMT and clinical applications in brain tumours]

Glioblastoma multiforme is the most common and most malignant primary brain tumour with a dismal prognosis. The advent of new chemotherapies with alkylating agents crossing the blood-brain barrier, like temozolomide, have permitted to notably ameliorate the survival of a subgroup of patients. Improved outcome was associated with epigenetic silencing of the MGMT (O6-methylguanin methyltransferase) gene by promotor methylation, thereby blocking its repair capability, thus rendering the alkylating agents more effective. This particularity can be tested by methylation specific PCR on resected tumour tissue, best on fresh frozen biopsies, and allows identification of patients more susceptible to respond favourably to the treatment.

ATP release due to Thy-1-integrin binding induces P2X7-mediated calcium entry required for focal adhesion formation.

Thy-1, an abundant mammalian glycoprotein, interacts with αvβ3 integrin and syndecan-4 in astrocytes and thus triggers signaling events that involve RhoA and its effector p160ROCK, thereby increasing astrocyte adhesion to the extracellular matrix. The signaling cascade includes calcium-dependent activation of protein kinase Cα upstream of Rho; however, what causes the intracellular calcium transients required to promote adhesion remains unclear. Purinergic P2X7 receptors are important for astrocyte function and form large non-selective cation pores upon binding to their ligand, ATP. Thus, we evaluated whether the intracellular calcium required for Thy-1-induced cell adhesion stems from influx mediated by ATP-activated P2X7 receptors. Results show that adhesion induced by the fusion protein Thy-1-Fc was preceded by both ATP release and sustained intracellular calcium elevation. Elimination of extracellular ATP with Apyrase, chelation of extracellular calcium with EGTA, or inhibition of P2X7 with oxidized ATP, all individually blocked intracellular calcium increase and Thy-1-stimulated adhesion. Moreover, Thy-1 mutated in the integrin-binding site did not trigger ATP release, and silencing of P2X7 with specific siRNA blocked Thy-1-induced adhesion. This study is the first to demonstrate a functional link between αvβ3 integrin and P2X7 receptors, and to reveal an important, hitherto unanticipated, role for P2X7 in calcium-dependent signaling required for Thy-1-stimulated astrocyte adhesion.

The chemokine receptor CXCR4 strongly promotes neuroblastoma primary tumour and metastatic growth, but not invasion.

Neuroblastoma (NB) is a heterogeneous, and particularly malignant childhood neoplasm in its higher stages, with a propensity to form metastasis in selected organs, in particular liver and bone marrow, and for which there is still no efficient treatment available beyond surgery. Recent evidence indicates that the CXCR4/CXCL12 chemokine/receptor axis may be involved in promoting NB invasion and metastasis. In this study, we explored the potential role of CXCR4 in the malignant behaviour of NB, using a combination of in vitro functional analyses and in vivo growth and metastasis assessment in an orthotopic NB mouse model. We show here that CXCR4 overexpression in non-metastatic CXCR4-negative NB cells IGR-NB8 and in moderately metastatic, CXCR4 expressing NB cells IGR-N91, strongly increased tumour growth of primary tumours and liver metastases, without altering the frequency or the pattern of metastasis. Moreover shRNA-mediated knock-down experiments confirmed our observations by showing that silencing CXCR4 in NB cells impairs in vitro and almost abrogates in vivo growth. High levels of CXCL12 were detected in the mouse adrenal gland (the primary tumour site), and in the liver suggesting a paracrine effect of host-derived CXCL12 on NB growth. In conclusion, this study reveals a yet unreported NB-specific predominant growth and survival-promoting role of CXCR4, which warrants a critical reconsideration of the role of CXCR4 in the malignant behaviour of NB and other cancers.