G-quadruplex located in the 5’UTR of the BAG-1 mRNA affects both its cap-dependent and cap-independent translation through global secondary structure maintenance

Abstract: The anti-apoptotic BAG-1 protein isoforms are known to be overexpressed in colorectal tumors and are considered to be potential therapeutic targets. The isoforms are derived fromalternative translation initiations occuring at four in-frame start codons of a single mRNA transcript. Its 5'UTR also contains an internal ribosome entry site (IRES) regulating the capindependent translation of the transcript. An RNA Gquadruplex (rG4) is located at the 5'end of the BAG- 1 5'UTR, upstream of the known cis-regulatory elements. Herein, we observed that the expression of BAG-1 isoforms is post-transcriptionally regulated in colorectal cancer cells and tumors, and that stabilisation of the rG4 by small molecules ligands reduces the expression of endogenous BAG-1 isoforms. We demonstrated a critical role for the rG4 in the control of both cap-dependent and independent translation of the BAG-1 mRNA in colorectal cancer cells. Additionally, we found an upstream ORF that also represses BAG-1 mRNA translation. The structural probing of the complete 5'UTR showed that the rG4 acts as a steric block which controls the initiation of translation at each start codon of the transcript and also maintains the global 5'UTR secondary structure required for IRES-dependent translation

The MEK2-binding tumor suppressor hDlg is recruited by E-cadherin to the midbody ring

Background: The human homologue of the Drosophila Discs-large tumor suppressor protein, hDlg, is a multi-domain cytoplasmic protein that localizes to the membrane at intercellular junction sites. At both synaptic junctions and epithelia cell-cell junctions, hDlg is known to recruit several signaling proteins into macromolecular complexes. hDlg is also found at the midbody, a small microtubule-rich structure bridging the two daughter cells during cytokinesis, but its function at this site is not clear. Results: Here we describe the interaction of hDlg with the activated form of MEK2 of the canonical RAF/MEK/ERK pathway, a protein that is found at the midbody during cytokinesis. We show that both proteins localize to a sub-structure of the midbody, the midbody ring, and that the interaction between the PDZ domains of hDlg and the C-terminal portion of MEK2 is dependent on the phosphorylation of MEK2. Finally, we found that E-cadherin also localizes to the midbody and that its expression is required for the isoform-specific recruitment of hDlg, but not activated MEK2, to that structure. Conclusion: Our results suggest that like at other cell-cell junction sites, hDlg is part of a macromolecular complex of structural and signaling proteins at the midbody.Molecular and Cellular Biolog

The activation of neuronal NO synthase is mediated by Gâ protein βγ subunit and the tyrosine phosphatase SHPâ 2

In CHO cells we had found that CCK positively regulated cell proliferation via the activation of a soluble guanylate cyclase. Here we demonstrate that CCK stimulated a nitric oxide synthase (NOS) activity. The production of NO was involved in the proliferative response elicited by CCK regarding the inhibitory effect of NOS inhibitors Lâ NAME and αâ guanidinoglutaric acid. We identified the NOS activated by the peptide as the neuronal isoform: the expression of the C415A neuronal NOS mutant inhibited both CCKâ induced stimulation of NOS activity and cell proliferation. These two effects were also inhibited after expression of the C459S tyrosine phosphatase SHPâ 2 mutant and the βARKl (495â 689) sequestrant peptide, indicating the requirement of activated SHPâ 2 and Gâ βγ subunit. Kinetic analysis (Western blot after coimmunoprecipitation and specific SHPâ 2 activity) revealed that in response to CCKâ treatment, SHPâ 2 associated to Gâ β1 subunit, became activated, and then dephosphorylated the neuronal NOS through a direct association. These data demonstrate that the neuronal NOS is implicated in proliferative effect evoked by CCK. A novel growth signaling pathway is described, involving the activation of neuronal NOS by dephosphorylation of tyrosyl residues.â Cordelier, P., Estève, J.â P., Rivard, N., Marletta, M., Vaysse, N., Susini, C., Buscail, L. The activation of neuronal no synthase is mediated by Gâ protein βγ subunit and the tyrosine phosphatase SHPâ 2. FASEB J. 13, 2037â 2050 (1999)Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154447/1/fsb2fasebj13142037.pd

The serine protease inhibitor serpinE2 is a novel target of ERK signaling involved in human colorectal tumorigenesis

<p>Abstract</p> <p>Background</p> <p>Among the most harmful of all genetic abnormalities that appear in colorectal cancer (CRC) development are mutations of KRAS and its downstream effector BRAF as they result in abnormal extracellular signal-related kinase (ERK) signaling. In a previous report, we had shown that expression of a constitutive active mutant of MEK1 (caMEK) in normal rat intestinal epithelial cells (IECs) induced morphological transformation associated with epithelial to mesenchymal transition, growth in soft agar, invasion and metastases in nude mice. Results from microarrays comparing control to caMEK-expressing IECs identified the gene encoding for serpinE2, a serine protease inhibitor, as a potential target of activated MEK1.</p> <p>Results</p> <p>1- RT-PCR and western blot analyses confirmed the strong up-regulation of serpinE2 expression and secretion by IECs expressing oncogenic MEK, Ras or BRAF. 2- Interestingly, serpinE2 mRNA and protein were also markedly enhanced in human CRC cells exhibiting mutation in <it>KRAS </it>and <it>BRAF</it>. 3- RNAi directed against serpinE2 in caMEK-transformed rat IECs or in human CRC cell lines HCT116 and LoVo markedly decreased foci formation, anchorage-independent growth in soft agarose, cell migration and tumor formation in nude mice. 4- Treatment of CRC cell lines with U0126 markedly reduced <it>serpinE2 </it>mRNA levels, indicating that expression of <it>serpinE2 </it>is likely dependent of ERK activity. 5- Finally, Q-PCR analyses demonstrated that mRNA levels of serpinE2 were markedly increased in human adenomas in comparison to healthy adjacent tissues and in colorectal tumors, regardless of tumor stage and grade.</p> <p>Conclusions</p> <p>Our data indicate that serpinE2 is up-regulated by oncogenic activation of Ras, BRAF and MEK1 and contributes to pro-neoplastic actions of ERK signaling in intestinal epithelial cells. Hence, serpinE2 may be a potential therapeutic target for colorectal cancer treatment.</p

Schizophrénie, approche spécialisée et continuité de soins. Le programme spécifique d’intervention Premier-Épisode de l’Hôtel-Dieu de Lévis

La schizophrénie est une maladie complexe à caractère évolutif. Reposant sur un cadre conceptuel d'orientation cognitive, le programme spécifique d'intervention Premier épisode de l'Hôtel-Dieu de Lévis fournit une évaluation complète et standardisée au plan individuel et familial. Puis sont rendues disponibles différentes modalités de traitement, selon une approche individuelle (psycho-éducation, psychothérapie) et de groupe (intervention psychologique au plan cognitif ou Integrated Psychological Therapy, de Brenner). L'intervention psycho-éducative familiale est également offerte aux familles. Les structures et la démarche décrites s'harmonisent avec celles qui étaient en place avant la création du programme, ce qui offre une continuité de soins. Le cadre conceptuel sous-jacent et les modalités du fonctionnement du programme sont aussi présentés.Schizophrenia is a complex illness with an evolutive character. Based on a conceptual framework of cognitive orientation, the specific intervention program First Episode of Hôtel-Dieu in Lévis includes a complete and standardized assessment to an individualized and family plan. Different methods of treatment acording to an individualized approach (psyhco-education, psychotherapy) as well as group therapy (psychological intervention at the cognitive level or Brenner's Integrated psychological therapy) are then proposed. Psycho-education intervention for families is also offered. Structures and different steps described here, harmonize with those already in place before the program's creation thus offering a continuity in care. The underlying conceptual framework and the different methods of functioning of the program are also presented.La esquizofrenia es una enfermedad compleja da caracter evolu-tivo. El programa especifico de intervention Primer episodio de Hôtel-Dieu de Lévis que reposa en un marco conceptual de orientaciôn cognoscitiva, ofrece una evaluaciôn compléta y estandarizada a nivel individual y familiar. Ademâs se ofrecen diferentes modalidades de tratamiento, segûn un enfoque individual (psicoeducaciôn, psicoterapia) y de grupo (intervenciôn psicolôgica a nivel cognoscitivo o Integrated psychological therapy, de Brenner). Igualmente se le ofrece a las fami-lias, la Intervenciôn psioeducativa familiar. Las estructuras y los pasos a seguir que se describen se armonizan a las que ya existian antes de la creation del programa, Io que ofrece una continuidad de tratamiento. Son presentados, el marco conceptual subyacente y las modalidades del fucionamiento des programa

RDR2 Partially Antagonizes the Production of RDR6-Dependent siRNA in Sense Transgene-Mediated PTGS

Background: RNA-DEPENDENT RNA POLYMERASE6 (RDR6) and SUPPRESSOR of GENE SILENCING 3 (SGS3) are required for DNA methylation and post-transcriptional gene silencing (PTGS) mediated by 21-nt siRNAs produced by sense transgenes (S-PTGS). In contrast, RDR2, but not RDR6, is required for DNA methylation and TGS mediated by 24-nt siRNAs, and for cellto-cell spreading of IR-PTGS mediated by 21-nt siRNAs produced by inverted repeat transgenes under the control of a phloem-specific promoter. Principal Findings: In this study, we examined the role of RDR2 and RDR6 in S-PTGS. Unlike RDR6, RDR2 is not required for DNA methylation of transgenes subjected to S-PTGS. RDR6 is essential for the production of siRNAs by transgenes subjected to S-PTGS, but RDR2 also contributes to the production of transgene siRNAs when RDR6 is present because rdr2 mutations reduce transgene siRNA accumulation. However, the siRNAs produced via RDR2 likely are counteractive in wildtype plants because impairement of RDR2 increases S-PTGS efficiency at a transgenic locus that triggers limited silencing, and accelerates S-PTGS at a transgenic locus that triggers efficient silencing. Conclusions/Significance: These results suggest that RDR2 and RDR6 compete for RNA substrates produced by transgenes subjected to S-PTGS. RDR2 partially antagonizes RDR6 because RDR2 action likely results in the production of counteractiv

The PTEN Phosphatase Controls Intestinal Epithelial Cell Polarity and Barrier Function: Role in Colorectal Cancer Progression

The PTEN phosphatase acts on phosphatidylinositol 3,4,5-triphosphates resulting from phosphatidylinositol 3-kinase (PI3K) activation. PTEN expression has been shown to be decreased in colorectal cancer. Little is known however as to the specific cellular role of PTEN in human intestinal epithelial cells. The aim of this study was to investigate the role of PTEN in human colorectal cancer cells.Caco-2/15, HCT116 and CT26 cells were infected with recombinant lentiviruses expressing a shRNA specifically designed to knock-down PTEN. The impact of PTEN downregulation was analyzed on cell polarization and differentiation, intercellular junction integrity (expression of cell-cell adhesion proteins, barrier function), migration (wound assay), invasion (matrigel-coated transwells) and on tumor and metastasis formation in mice. Electron microscopy analysis showed that lentiviral infection of PTEN shRNA significantly inhibited Caco-2/15 cell polarization, functional differentiation and brush border development. A strong reduction in claudin 1, 3, 4 and 8 was also observed as well as a decrease in transepithelial resistance. Loss of PTEN expression increased the spreading, migration and invasion capacities of colorectal cancer cells in vitro. PTEN downregulation also increased tumor size following subcutaneous injection of colorectal cancer cells in nude mice. Finally, loss of PTEN expression in HCT116 and CT26, but not in Caco-2/15, led to an increase in their metastatic potential following tail-vein injections in mice.Altogether, these results indicate that PTEN controls cellular polarity, establishment of cell-cell junctions, paracellular permeability, migration and tumorigenic/metastatic potential of human colorectal cancer cells

Régulation de la croissance pancréatique : mécanismes d'action de la cholécystokinine et de la somatostatine

Synthétisée par des cellules endocrines de la muqueuse intestinale, la cholécystokinine (CCK) est une hormone dont la libération est stimulée par la présence de nutriments dans la lumière intestinale. Les principales actions de la CCK consistent en la contraction de la vésicule biliaire et en la sécrétion d'enzymes digestives à partir des cellules acineuses pancréatiques. Il est bien connu que la CCK administrée par voie sous­cutanée, favorise aussi la croissance du pancréas. Cette action trophique de la CCK sur le pancréas peut être contrecarrée par la somatostatine (SS), reconnue comme agent antiprolifératif du pancréas. Cependant, les mécanismes d'action de ces deux hormones dans la régulation de la croissance pancréatique restent à élucider. Dans un premier temps, nous avons tenté de définir le rôle de la CCK endogène dans la régulation de la croissance pancréatique normale et stimulée par la diversion du suc pancréatique. Nous avons démontré que la CCK endogène était responsable de l'effet trophique causé par la diversion du suc pancréatique sur le pancréas en établissant l'effet inhibiteur du MK 329, un antagoniste spécifique de la CCK, sur cette croissance stimulée par la diversion du suc pancréatique. Nous avons aussi tenté d'établir une corrélation entre la sécrétion de protéines et le développement de la croissance pancréatique. Nous avons effectivement démontré qu'une sécrétion soutenue élevée en protéines était associée au développement de la croissance pancréatique. Nous avons aussi confirmé l'effet antitrophique de la SS et tenté de déterminer les mécanismes d'action de cette hormone au cours de l'inhibition de la croissance pancréatique normale. La SS pourrait réduire la croissance pancréatique indirectement en inhibant la libération d'agents trophiques pour le pancréas tels que la CCK et l'IGF-1. Nous avons aussi tenté de définir les mécanismes cellulaires déclenchés dans le pancréas lorsque la croissance de celui-ci est stimulée par la CCK et inhibée par la SS. Depuis quelques années, nos connaissances sur les mécanismes intracellulaires qui gouvernent la prolifération cellulaire ont beaucoup progressé. La phosphorylation de résidus tyrosines de certaines protéines cellulaires jouent un rôle prédominant aussi bien dans la prolifération cellulaire normale que dans la tumorigénicité. De plus, la production d'acide phosphatidique suite à l'activation de la phospholipase D (PLD), de même que l'activation de la phosphatidyl inositol 3-kinase (PI 3- kinase) sont des réactions cellulaires déclenchées en réponse à une stimulation mitogénique. Notre hypothèse était donc que la CCK favorise la croissance pancréatique en stimulant une ou des activité(s) tyrosine kinase (Tyr-k) tandis que la SS réduit la croissance pancréatique en stimulant directement une ou des activité(s) tyrosine phosphatase (PTP) dans la cellule acineuse pancréatique. Nous avons donc démontré que la caeruléine stimulait des activités Tyr-k membranaires et cytosoliques dans la cellule acineuse pancréatique. Nous avons aussi démontré que la CCK médiait son effet trophique par l'occupation de son récepteur de haute affinité en reproduisant les effets de la caeruléine avec le JMV-180. De plus, l'effet stimulateur marqué d'une pancréatectomie sur les activités Tyr-k suggère, en effet, que des Tyr-ks sont probablement impliquées dans les processus de régénération et de croissance du pancréas. La caeruléine, le carbachol et la diversion du suc pancréatique stimulent une activité PTP dans les membranes de grains de zymogène qui pourrait être impliquée dans le processus de la sécrétion. De plus, la stimulation transitoire des PTPs des membranes basolatérales et du cytosol de même que l'inhibition des activités Tyr-k, PI 3-kinase et PLD, pourrait être associée à l'effet antiprolifératif du SMS 201-995 sur le pancréas. De plus, nous avons aussi mis en évidence l'implication de protéines G, de la phospholipase C (PLC) et du calcium dans l'activation des Tyr-ks et de la PLD par la caeruléine. En résumé, toutes ces études ont permis de confirmer l'effet trophique de la CCK et l'effet antitrophique de la SS sur le pancréas. Nos résultats suggèrent que la CCK induit la croissance pancréatique en stimulant des Tyr-ks, la PLD et la PI 3-kinase tandis que la SS inhibe la croissance pancréatique en stimulant une activité PTP et en inhibant les activités Tyr-k, PI 3-kinase et PLD