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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
321

Characterization of initiation of DNA replication in HCT116 Ku80+- Cells

Sibani, Sahar January 2005 (has links)
DNA replication initiates at origins with the origin recognition complex (ORC) binding and activating initiation. Although ORC specifically binds origins in vivo, it possesses no sequence specific DNA binding activity in vitro. Thus, it is thought to interact with another protein that recruits it to the origin. The heterodimeric Ku (Ku70/Ku80) protein is one candidate. Ku is an abundant nuclear protein that has recently been implicated in the initiation of DNA replication, but the mechanism by which it performs this function is yet unknown. Here, using haploinsufficient human HCT116 Ku80 +/- cells that express 25-40% Ku80 protein, the origin activity and origin-association of other replication proteins were examined in a Ku80-deficient environment. Ku80 was not found to influence the expression of replication proteins, except for Ku70, its partner, which displayed a reduction of its protein expression to 64% of wildtype cells. Ku80+/- cells displayed a prolongation of G1 phase by 2 hours, but normal progression through S-phase. Furthermore, the activity of the early-activated replication origins of the lamin B2, beta-globin and c-myc loci decreased by 4.5-, 3.4-, and 4.3-fold, respectively. This was associated with a 2.1-, 1.5- and 1.7-fold decrease in the association of Ku80 with these origins, respectively, and a 1.5-, 2.3- and 2.5-fold decrease in that of Ku70, respectively. Moreover, the origin-association of three of the six subunits of the ORC complex, Orc-3, -4, and -6 was significantly decreased in Ku80 +/- cells, indicating decreased ORC complex formation at the origins. In contrast, the association of Orc-2 was not influenced by Ku80 deficiency, nor was the association of Ku80 with origins affected by Orc2 deficiency. Overall, these results suggest that Ku binds origins to allow assembly of the ORC complex, which is necessary for origin licensing and activation. / Ku80-/- murine embryonic fibroblasts (MEFs) are viable but display a prolonged doubling time of 43 hours vs. 23 hours for wildtype cells. To analyze the effect of Ku80 absence on DNA replication, the late-firing murine adenosine deaminase origin was fine-mapped by nascent strand abundance analysis. Two origins were identified, mAdA-1 and mAdA-C, with 34- and 29-fold greater nascent strand abundance relative to non-origin DNA. These origins were able to replicate episomally when transiently transfected into MEF cells. In Ku80-/- cells, in situ and episomal DNA replication assay of both origins indicated that Ku80 did not influence their DNA replication activity.
322

Characterization of granulin gene expression in wounds and myelogenous leukemic cells

Ong, Colin Heng Piew, 1968- January 2005 (has links)
Progranulin (PC-cell derived growth factor, acrogranin, granulin-epithelin precursor) is a secreted glycoprotein with growth promoting activities for diverse cell types and is the precursor for the 6 kDa granulin peptides. These peptides were initially identified in inflammatory cells i.e. neutrophils; and this led to the hypothesis that the grn(granulin ) gene and its products may be regulated during, and function in, wound repair. In response to tissue injury, the grn gene is induced in endothelial cells and fibroblasts. Expression was observed in epithelial and inflammatory cells with higher expression in the inflammatory cells in the early phases of wound healing. This is the first evidence that demonstrates grn gene regulation in an adult physiological state. Given the high expression of the grn gene in hematopoietic cells, particularly the cells of the myeloid lineage, and that these cells are a source of granulin peptides, it was important to understand the signals that regulate progranulin transcript levels in these cells. For this investigation, two myelogenous cell lines, U937 (human histiocytic lymphoma) and HL-60 (human promyelocytic), which are models for monocytic and granulocytic maturation were chosen. Both these lineage related cell lines displayed differential regulation of the progranulin mRNA. The two cell lines were responsive to all three of the differentiation agents, investigated; all-trans retinoic acid (ATRA), dimethylsulphoxide (DMSO) and phorbol-12-myristate-13-acetate (PMA) with the exception of PMA-treated HL-60 cells which exhibit no change in the progranulin transcript levels. As differentiation occurs, the levels of progranulin transcript are up-regulated. However, progranulin does not stimulate the differentiation of these cells as assessed by the expression of the surface marker, CD11b which suggests that the increased levels of progranulin transcript is likely to be a characteristic of matured cells (inf
323

Transcriptional regulation by the estrogen-related receptors

Tremblay, Annie January 2010 (has links)
The Estrogen-Related receptors (ERRα, ERRβ and ERRγ) are ubiquitous, constitutively active, orphan nuclear receptors and little is known concerning post-translational modifications affecting their transcriptional activity. We observed that the conserved phosphorylation-dependent sumoylation motif (PDSM) within the N-terminal domain of the ERRs represses their transcriptional activity on compound promoters via a synergy control mechanism. We also identified protein inhibitor of activated stat y (PIASy), a SUMO E3 ligase, as a new interacting partner of ERRα which promotes the sumoylation of ERRα and represses its transcriptional activity in a PDSM-dependent manner. Furthermore, by showing that an ERRα phosphoS19-specific antibody, but not a polyclonal ERRα antibody with a minimal affinity for phosphorylated S19, allows detection of endogenous sumoylated ERRα in mouse liver extract, we confirmed that the ERRα phospho-sumoyl switch is functional in vivo. / ERRα is highly expressed in the kidney, but its role in this organ is unknown. Therefore, we used a combination of physiological studies, gene expression and genome-wide location analysis to explore the role of ERRα in the kidney. A defect in sodium and potassium homeostasis was observed in the ERRα null mice, which correlated with the ERRα renal transcriptional program comprising key sodium and potassium channels. Furthermore, telemetry monitoring revealed that the ERRα null mice display a significantly reduced blood pressure at nighttime and this correlated the renal transcriptional program of ERRα comprising genes involved in blood pressure regulation. In addition, we identified the Renin-Angiotensin pathway genes as direct ERRα target genes in the kidney. These results identify a role for ERRα in renal sodium/potassium handling, intra-renal renin-angiotensin pathway, blood pressure regulation and possibly hypertension. / Les récepteurs reliés aux récepteurs de l'estrogène (ERRs) sont ubiquitaires et constitutivement actifs et le rôle joué par les modifications post-traductionnelles sur leur activité transcriptonnelle est peu connu. Nous avons démontré qu'un motif consensus de sumoylation phospho-dependente (PDSM) situé dans le domaine N-terminal diminue l'activité transcriptionnelle des ERRs sur des promoteurs à élements de réponses multiples grâce à un mécanisme de contrôle de la synergie. Nous avons aussi établi que les ERRs intéragissent avec la E3-SUMO-ligase PIASy et que cette dernière promouvoit la sumoylation du ERRα de manière phospho-dépendante. De plus, en montrant que la forme sumoylée endogène de ERRα dans de l'extrait de foie de souris n'était détectable qu'avec un anticorps spécifique dirigé contre la sérine 19 phoshorylée nous avons confirmé la validité de l'interrelation phosphorylation-sumoylation dans un contexte physiologique in vivo. / Même si le haut niveau d'expression de ERRα dans les reins est reconnu, son rôle dans cet organe est inconnu. Nous avons donc utilisé une approche combinatoire d'analyses physiologiques, d'expression génique et d'identification de sites spécifiques de liaison à l'ADN au niveau génomique afin d'explorer plus avant le rôle physiologique de ERRα dans le rein. Nous avons observé que les souris knock-out pour le gène de ERRα présentent un problème au niveau de l'homéostasie sodique et potassique corrèlant directement avec le programme transcriptionnel rénal qui comprends plusieurs canaux sodiques et potassiques importants. De plus, la mesure des paramètres cardiovasculaires par télémétrie a révélé que les souris knock-out pour le gène du ERRα ont une pression sanguine nocturne plus faible qui corrèle avec le programme transcriptionnel comprenant plusieurs gènes influençant la pression sanguine. Nous avons aussi identifié les promoteurs de certains gènes composant le système rénine-angiotensine comme gènes cibles potentiels de ERRα dans les reins. Nos résultats suggèrent une implication du ERRα dans le contrôle de la pression sanguine basale et possiblement dans l'hypertension.
324

The eIF2alpha phosphorylation pathway as a novel component of PI3K signaling with implications in tumor treatment

Mounir, Zineb January 2011 (has links)
Inhibition of protein synthesis by phosphorylation of eIF2alpha on serine 51 is mediated by a family of kinases activated by various forms of stress. The biological outcome of eIF2alpha phosphorylation can either be cytoprotective or pro-apoptotic. Translation is also regulated at the initiation step by modulation of eIF4E downstream of the PI3K/Akt/mTOR signaling pathway. Our research reveals that the PI3K/Akt/mTOR pathway can also regulate translation initiation by modulation of eIF2alpha kinase activity and eIF2alpha phosphorylation. Our findings provide evidence that the tumor suppressor PTEN activates the PKR- eIF2alpha phosphorylation pathway independently of its phosphatase activity but requires an intact PDZ-binding motif. Activation of the PKR- eIF2alpha phosphorylation pathway is essential for the anti-proliferative and pro-apoptotic functions of PTEN. Our work is in line with the pro-apoptotic functions of PKR and shows its requirement for the tumor suppressive functions of PTEN. The second important effector downstream of PI3K signaling, Akt, regulates a wide variety of biological processes. Our research shows that the eIF2alpha kinase PERK is a novel substrate of Akt. Indeed, Akt phosphorylates PERK on threonine 799 which inhibits its catalytic activity and ability to induce eIF2alpha phosphorylation. We further demonstrate that PERK activation and eIF2alpha phosphorylation counteract the biological functions of Akt in response to stress conditions including ER stress and oxidative stress. Our findings show that the cytoprotective PERK- eIF2alpha phosphorylation pathway is activated following treatment with pharmacological inhibitors of PI3K and Akt. We demonstrate that inactivation of the PERK-eIF2alpha phosphorylation pathway increases the susceptibility of tumor cells to death by pharmacological inhibitors of the PI3K/Akt pathway.The last arm of PI3K signaling is the mTORC1 pathway which is mainly involved in translational control. Our research reveals that mTORC1 interacts with PERK leading to its activation and induction of eIF2alpha phosphorylation independently of the kinase activity of mTOR. We further demonstrate that pharmacological inhibition of mTORC1 by rapamycin specifically induces PERK activation and eIF2alpha phosphorylation without initiating an ER stress response. Moreover, our findings reveal that the interaction between mTORC1 and PERK is increased following rapamycin treatment suggesting that a preferential localization of mTORC1 to the ER might be responsible for its ability to regulate PERK activity. Our research reveals a novel link between mTOR signaling and translational control through the eIF2alpha phosphorylation pathway. / La phosphorylation de la sous unité alpha du facteur eIF2 sur le résidu sérine 51, qui permet l'inhibition de la synthèse des protéines, est induite par une famille de kinases activées par différentes formes de stress. Cette phosphorylation peut avoir des effets pro-apoptotiques ou prolifératifs. La traduction menant à la synthèse de protéines est également contrôlée à l'étape de l'initiation par la régulation du facteur d'initiation eIF4E en aval de la voie de signalisation PI3K et de ses effecteurs Akt/PKB et mTOR. Nos recherches démontrent que la voie de signalisation PI3K/Akt/mTOR peut aussi réguler l'initiation de la traduction en modulant l'activité des kinases d'eIF2alpha et la phosphorylation du facteur eIF2alpha. Nos résultats prouvent que le suppresseur de tumeur PTEN active la phosphorylation du facteur eIF2alpha par sa kinase PKR, indépendamment de son activité phosphatase tout en requérant le domaine PDZ de PTEN. L'activation de PKR et l'induction de la phosphorylation du facteur eIF2alpha sont essentiels aux fonctions anti- prolifératives et pro- apoptotiques de PTEN. Nos résultats sont en accord avec les fonctions pro-apoptotiques connues de PKR et démontrent la nécessité de PKR pour les propriétés de suppresseur de tumeurs de PTEN. Akt, le second effecteur important en aval de la voie signalisation PI3K, régule un très grand nombre de processus biologiques. Nos recherches établissent que la kinase du facteur eIF2alpha PERK est un nouveau substrat d'Akt. En effet, Akt phosphoryle le résidu thréonine 799 de PERK ce qui inhibe son activité catalytique ainsi que sa capacité à phosphoryler son substrat eIF2alpha. Ainsi, nos recherches montrent que l'activation de PERK et la phosphorylation du facteur eIF2alpha éliminent les fonctions biologiques d'Akt en réponse aux conditions de stress tel que le stress du réticulum endoplasmique et le stress oxydatif. De plus, nos résultats illustrent que la voie cytoprotective de PERK conduisant à la phosphorylation du facteur eIF2alpha est activée suite aux traitements par les inhibiteurs pharmacologiques de PI3K et d'Akt. Ainsi, nous démontrons que l'inactivation de la voie cytoprotective de PERK menant à la phosphorylation du facteur eIF2alpha augmente la sensibilité des cellules cancéreuses à la mort cellulaire suite aux traitements par les inhibiteurs pharmacologiques de la voie de signalisation PI3K/Akt. Le dernier élément de la voie de signalisation PI3K est la voie de mTORC1, dont la fonction principale est le contrôle de la traduction. Nos recherches établissent une interaction entre mTORC1 et PERK menant à son activation et à la phosphorylation du facteur eIF2alpha indépendamment de l'activité kinase de mTOR. De plus, nous démontrons que l'utilisation de l'inhibiteur pharmacologique de mTORC1, la rapamycine, induit spécifiquement l'activation de PERK et la phosphorylation du facteur eIF2alpha sans toutefois initier un stress du réticulum endoplasmique. Par conséquent, nos recherches mettent en évidence que l'interaction entre mTORC1 et PERK est augmentée suite aux traitements par la rapamycine ce qui suggère l'existence d'une relocalisation de mTORC1 vers le réticulum endoplasmique qui pourrait être responsable de sa capacité à réguler l'activation de PERK. Nos recherches révèlent un nouveau lien entre la voie de signalisation mTOR et le contrôle de la traduction à travers la voie de phosphorylation du facteur eIF2alpha.
325

Characterization of the Tet-On Grb 7 and Tet-On 14-3-3 sigma mouse models

Bergeron, Alison January 2011 (has links)
ErbB2 is overexpressed and amplified in about 30% of all breast tumors and is correlated with poor patient prognosis. ErbB2 knock in mouse models mimic the amplification in chromosome 11 (human chromosome 17q21-25) comprising of ErbB2 and Grb7 and a deletion in chromosome 4 (human chromosome 1p35-36) including 14-3-3σ seen in human breast cancers. Grb7 is an adaptor protein known to regulate cell migration and transcription by interacting with a cell migration protein FAK and a transcription factor protein FHL2. Grb7 overexpression has been linked to an increase in metastasis and poor patient outcome. 14-3-3σ is negative cell cycle protein that is upregulated by p53. Interestingly, 14-3-3σ has a role in down regulating ErbB2 expression by sequestering EGR2 from the nucleus to the cytoplasm. These results suggest that 14-3-3σ deletion and Grb7 overexpression in ErbB2 tumors represents an interesting target to study as its deletion may be a targeted event in the development of ErbB2 tumors.To elucidate the role of Grb7 and 14-3-3σ I generated a mouse model utilizing the Tet-On mouse model system whereby these two proteins are overexpressed in the mouse mammary epithelium. Through this study we confirmed successful localized overexpression of both Grb7 and 14-3-3σ in the mouse mammary gland epithelium. Further, we were able to confirm that individual expression of both Grb7 and 14-3-3σ lead to a ductal outgrowth defect during mammary gland development. These observations confirm that Grb7 and 14-3-3σ both play a role in mouse mammary gland development. / ErbB2 est au-dessus d'exprimer et d'amplifier dans environ 30% de toutes les tumeurs de sein et est corrélé avec le pronostic patient pauvre. Le coup en ErbB2 dans des modèles de souris imitent l'amplification en chromosome 11 (chromosome humain 17q21-25) comportant d'ErbB2 et de Grb7 et une suppression en chromosome 4 (chromosome humain 1p35-36) comprenant le  14-3-3σ vu dans les cancers du sein humains. Grb7 est une protéine d'adapteur connue pour régler la migration et la transcription de cellules par l'interaction avec une protéine FAK de migration de cellules et une protéine FHL2 de facteur de transcription. Grb7 au-dessus d'expression a été lié à une augmentation de métastase et de résultats patients pauvres. Le  14-3-3σ est une protéine négative de cycle de cellules qui upregulated par p53. Intéressant, 14-3-3σ a un rôle en réglant vers le bas l'expression ErbB2 en séquestrant EGR2 du noyau au cytoplasme. Ces résultats suggèrent que la suppression 14-3-3σ et le Grb7 au-dessus de l'expression dans les tumeurs ErbB2 représente une cible intéressante pour étudier pendant que sa suppression peut être un événement visé dans le développement des tumeurs ErbB2. Ces résultats suggèrent que la suppression 14-3-3σ et le Grb7 au-dessus de l'expression dans les tumeurs ErbB2 représente une cible intéressante pour étudier pendant que sa suppression peut être un événement visé dans le développement des tumeurs ErbB2. Pour élucider le rôle de Grb7 et de 14-3-3 j'ai produit d'une utilisation de modèle de souris Tet-Sur le système modèle de souris par lequel ces deux protéines soient plus de exprimées dans l'épithélium mammaire de souris. Par cette étude nous avons confirmé réussi localisé au-dessus de l'expression de Grb7 et de 14-3-3σ dans l'épithélium de glande mammaire de souris. De plus, nous pouvions confirmer que l'expression individuelle des deux Grb7 et le  14-3-3 mènent à un défaut ductal de conséquence pendant le développement de glande mammaire. Ces observations confirment que Grb7 et 14-3-3σ les deux jeu un rôle dans le développement de glande mammaire de souris.
326

The inflammatory role of late gestation lung 1«(Lgl1)»

Bao, Tim January 2011 (has links)
Deficient alveolar maturation in bronchopulmonary dysplasia (BPD) is characterized by distal airspace enlargement, disruption of elastin fibers and goblet cell hyperplasia, which are further exacerbated by supplemental oxygen and/or mechanical ventilation at birth. Glucocorticoids (GCs) are known to stimulate alveolarization and were the preferred treatment for BPD. However, prolonged use of GCs provoke severe side-effects and is no longer recommended. Our lab cloned late-gestation lung 1 (Lgl1), a GC-induced, developmentally regulated, secreted glycoprotein that is speculated to play a role in early lung branching morphogenesis and as a modulator of alveolar formation. We demonstrate that Lgl1 is profoundly suppressed in our OVA-induced asthma model. Combined with our previous findings that associate a deficiency in Lgl1 with inflammation, we propose that Lgl1 is a multifunctional protein involved in early and late lung development in addition to its role in inflammation. / Le problème de maturation alvéolaire dans les poumons des enfants atteints de dysplasie bronchopulmonaire (DBP) est caractérisé par l'élargissement des espaces aériens du poumon distal, par la désintégration des fibres d'élastine et par l'hyperplasie des cellules caliciformes. Ces effets sont exacerbés par l'oxygène et/ou la ventilation mécanique donnés à la naissance aux bébés prématurés. Les glucocorticoïdes (GCs) sont reconnus comme étant des stimulants de l'alvéolarisation et représentaient le traitement favorisé pour les nouveau-nés souffrant de DBP. Cependant, il a été démontré que l'utilisation prolongée des GCs provoque des effets secondaires sévères et leur utilisation n'est plus recommandée de nos jours. Notre laboratoire a cloné le gène late-gestation lung 1 (Lgl1) codant pour une protéine qui est induite par les GCs et dont l'expression est finement régulée durant le développement. Lgl1 est une glycoprotéine secrétée qui pourrait jouer un rôle dans la morphogénèse du poumon, dans le processus de ramification pulmonaire et dans la modulation de la formation des alvéoles. Nous démontrons que l'expression de Lgl1 est grandement diminuée dans un modèle d'asthme induit par l'administration d'ovalbumine (OVA). Nous avons démontré précédemment qu'une déficience du gène Lgl1 est associée à l'inflammation pulmonaire et nous proposons que Lgl1 est une protéine multifonctionnelle qui serait impliquée dans le développement précoce et tardif du poumon, en plus de son rôle dans les processus inflammatoires.
327

Loss of downregulation : a mechanism for oncogenic activation of receptor tyrosine kinase-derived oncoproteins

Mak, Hayley Hoi Lam. January 2006 (has links)
Many human cancers have been associated with the deregulation of receptor tyrosine kinases. This occurs through various molecular mechanisms, including gene amplification, chromosome translocation and point mutations some of which involve loss of negative regulatory signals. Negative regulation of RTKs involves their internalisation and subsequent degradation in the lysosome. RTK oncoproteins activated following chromosomal translocation are no longer transmembrane proteins and would be predicted to escape lysosomal degradation. In this study, we addressed whether the Tpr-Met oncogene, produced via chromosomal translocation, escapes downregulation thereby leading to its oncogenic activity. Unlike the Met receptor, Tpr-Met is localized to the cytoplasm and lacks the binding site for Cbl, E3 ubiquitin ligases. Ubiquitination of RTKs, including the Met receptor, target them for efficient degradation in the lysosome. To address whether the subcellular localization of Tpr-Met, and/or loss of its Cbl binding site, is important for its oncogenic activity, Tpr-Met variants, targeted to the plasma membrane with and without Cbl recruitment were examined. The presence of a Cbl binding site and ubiquitination of cytosolic Tpr-Met oncoproteins does not alter Tpr-Met transforming activity, or protein stability, and these proteins fail to enter the endocytic pathway. However membrane targeting allows Tpr-Met to enter the endocytic pathway and transformation and protein stability are decreased in a Cbl dependent manner. These data demonstrate that the oncogenic activity of Tpr-Met is in part dependent on its ability to escape normal down-regulatory mechanisms and provides a paradigm for many RTK-derived oncoproteins activated following chromosomal translocation.
328

Distinct regulation of CDPCux : by cyclinCdk complexes

Santaguida, Marianne Theresa. January 2005 (has links)
Cyclins are short-lived proteins that "cycle" through the cell cycle and determine the timing of activity of cyclin-dependent kinases (Cdks). Cyclin/Cdks regulate the progression through each phase of the cell cycle, presumably by phosphorylating key proteins. To date, only a few Cdk targets have been identified and it is not clear how Cdk complexes differ from one another. I have discovered that the CDP/Cux protein can bind to, and be phosphorylated by, several cyclin/Cdk complexes. Yet, my evidence demonstrates that each complex regulates CDP/Cux differently. The goal of my thesis is to document the interaction of CDP/Cux with several cyclin/Cdks and to characterize the effects of these interactions on CDP/Cux. / I investigated three separate cyclin/Cdk complexes that interact with CDP/Cux. I determined that CDP/Cux is a target of the cyclin A/Cdk1 and cyclin A/Cdk2 complexes. However each kinase differentially regulated CDP/Cux. Phosphorylation of CDP/Cux by cyclin A/Cdk1 inhibited its DNA binding and transcriptional activities in G2 phase of the cell cycle. Although both cyclin A/Cdk1 and cyclin A/Cdk2 were recruited to CDP/Cux in a similar fashion, cyclin A/Cdk2 did not phosphorylate the same sites in CDP/Cux as cyclin A/Cdk1. Furthermore cyclin A/Cdk2 did not inhibit CDP/Cux DNA binding or transcriptional activities. Cyclin A/Cdk2 allowed CDP/Cux activity in S phase. CDP/Cux is the first physiological target of cyclin A/Cdk identified that is differentially regulated depending on the Cdk present in the complex. Finally, I determined that cyclin D1/Cdk4 negatively regulates the generation of the activated, proteolytically processed, CDP/Cux isoform. This regulation is achieved by phosphorylation and inhibition of proteolytic processing in the G1 phase. The processed CDP/Cux isoform accelerates cell cycle progression, and consequently generates cells that are small in size. Therefore, cyclin D1/Cdk4 may play a role in cell growth (the size of cells) by delaying the timing of CDP/Cux proteolytic processing. / Overall, my work demonstrates that several cyclin/Cdk complexes interact with CDP/Cux and regulates CDP/Cux in a distinct manner. The control of CDP/Cux activity by cyclin/Cdk complexes in G1, S and G2/M phase contributes to a greater understanding the regulation of CDP/Cux and its role in the cell cycle.
329

The role of Gas6-Axl in vascular biology /

Hasanbasic, Ines. January 2006 (has links)
Gas6, the product of a growth arrest specific gene, is a member of the vitamin K-dependent family of gamma-carboxylated proteins and the ligand for the tyrosine kinase receptor Axl. Gas6-Axl interactions are important in many biological functions such as cell survival, mitogensis and regulation of thrombosis. We show that gas6 plays an important role in endothelial cell survival and that its post-translational modification (gamma-carboxylation) is necessary for its biologic activity. Using flow cytometry, we first demonstrate that gas6 can prevent apoptosis induced by serum starvation of primary cultures of human umbilical vein endothelial cells (HUVECs). This effect is mediated through activation of the phosphatidylinositol 3-kinase (PI3 kinase) and Akt pathway, known anti-apoptosic regulators. In addition, in the presence of the PI3 kinase inhibitor, wortmannin, gas6 is unable to induce Akt phosphorylation during serum-stress resulting in endothelial cell apoptosis. Similarly, dominant negative Akt constructs prevent gas6-induced endothelial cell survival, underscoring the importance of Akt activation in gas6-mediated survival. / Several downstream regulators of this survival pathway were also identified in HUVECs, namely, NF-kappaB as well as the antiapoptotic and proapoptotic proteins Bcl-2 and caspase 3, respectively. Luciferase assay experiments indicate that gas6 induces a rapid increase in NF-kappaB transcriptional activity. We also show that NF-kappaB is phosphorylated early after gas6 treatment as evidenced by Western Blot analysis. As well, the level of Bcl-2 protein increased, supporting the notion that the Bcl-2 antiapoptotic pathway is stimulated, and levels of caspase 3 activation, a know proapoptotic regulator, are significantly reduced with gas6 treatment. These initial results indicate that gas6-Axl interactions are an important mediator of endothelial cell survival during serum stress through activation of classical antiapoptotic pathway, namely, Akt phosphorylation, NF-kappaB activation, increased Bcl-2 protein expression, and a reduction in caspase 3 activation. / A most intriguing feature of gas6 as a mediator of cell survival is its unusual posttranslational modification, gamma-carboxylation. Thus, understanding the role of Gla domain of gas6 in gas6-Axl interaction is of fundamental importance since gamma-carboxylation may influence the anti-apoptotic property of gas6-Axl interaction. Here for the first time we show that Gla domain of gas6 is indispensable for its anti-apoptotic function. Initially, we show that carboxylated gas6 binds to phosphatidylserine-containing phospholipid membranes in an analogous manner to other gamma-carboxylated proteins whereas decarboxylated gas6 does not. Further, the gamma-carboxylation inhibitor, warfarin, abrogates gas6-mediated protection of NIH3T3 fibroblasts from serum starvation-induced apoptosis. A similar effect is observed on endothelium where only carboxylated but not decarboxylated gas6 protects endothelial cells from serum starvation-induced apoptosis. In addition, we also demonstrate that proper gamma-carboxylation of gas6 affects activation of its downstream targets, Axl and Akt. These results thus clearly indicate that this post-translational modification imparts a significant role in function of gas6. / Therefore, we propose that gas6 plays important role in endothelial cell survival and that its post-translational modification, gamma-carboxylation, is crucial for this function.
330

Molecular mechanisms and signaling pathways induced by TGF [beta] for transcriptional repression of target genes

Lacerte, Annie. January 2006 (has links)
Signaling by growth factors from the TGFbeta/activin has a profound impact on cancer development. They can either negatively regulate tumor development by their ability to inhibit cell growth, induce apoptosis and limit the number of cell divisions through inhibition of telomerase activity, or positively promote cancer through the induction of angiogenesis, epithelial to mesenchymal transition, and the suppression of the immune response. These ligands bind to serine-threonine kinase receptors that phosphorylate the Smad proteins: Smad2 and Smad3. Once phosphorylated, Smad2/3 cooperate with Smad4 to activate or repress expression of TGFbeta and activin target genes. It is the combined expression/repression of the numerous target genes that determine the physiological outcome of TGFbeta and activin signaling. In this thesis, we have characterized the molecular mechanisms by which TGFbeta and activin repress two target genes that are often overexpressed in human tumors. First, we studied the inhibitory effects of TGFbeta/activin on cell growth and prolactin (PRL) production in pituitary lactotroph tumor cells, as prolactinomas are tumors that secrete large amounts of PRL, causing endocrine and reproductive disorders. We showed that activin represses PRL expression through the Smad pathway and the tumor suppressor menin by downregulating the expression of Pit-1, a pituitary transcription factor essential for PRL expression. Second, we wanted to understand how TGFbeta inhibits hTERT gene expression, the gene encoding for the protein component of telomerase. In 90% of cancers, telomerase is reactivated through the induction of hTERT, allowing cancer cells to become immortal. Using the keratinocyte HaCaT cell line, we showed that TGFbeta rapidly increased E2F-1 expression, through the Smad pathway, Erk and p38 kinases. We identify four binding sites for E2F as critical for hTERT inhibition by TGFbeta. Moreover, interfering with E2F activity also abolishes hTERT inhibition by TGFbeta. These data identify E2F-1 as the intermediate factor required by TGFbeta to repress hTERT expression. Understanding the mechanisms used by TGFbeta and activin to regulate their target genes is important to improve our basic knowledge on how cancers progress. More importantly, it can create new avenues for cancer therapy development.

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