LKB1 est un gène suppresseur de tumeur qui code une kinase « maitre » dont l’activité contrôle la polarité et la prolifération cellulaire en les coordonnant avec l’état métabolique de la cellule. Ce travail a abouti à l’identification d’une nouvelle isoforme LKB1 appelée ∆N-LKB1 qui est générée par transcription alternative et initiation interne de la traduction de l'ARNm LKB1. La protéine ∆N-LKB1 est délétée de sa partie N-terminale incluant une partie de son domaine kinase. Bien que la protéine N-LKB1 soit catalytiquement inactive, elle potentialise l'effet activateur de la protéine LKB1 sur sa cible principale l’APMK, senseur énergétique de la cellule, via une interaction directe avec le domaine d'auto-inhibition de l’AMPK. En revanche, ∆N-LKB1 interfère négativement avec la capacité de LKB1 à induire la polarité cellulaire. Enfin, en utilisant des approches in vitro et in vivo, nous avons montré que N-LKB1 possède une propriété oncogénique intrinsèque. N-LKB1 est exprimée seule dans la lignée NCI-H460 issue du cancer du poumon. L’inhibition de l’expression de N-LKB1 dans les cellules NCI-H460 induit une diminution de la survie de ces cellules et inhibe leur pouvoir oncogénique quand elles sont greffées dans la souris nude. Nous avons donc identifié une nouvelle isoforme LKB1 qui stimule l’adaptation métabolique LKB1-dépendante, mais qui inhibe la polarité cellulaire contrôlée par LKB1. Le suppresseur de tumeur LKB1 ainsi que l’oncogène N-LKB1 sont codé par le même gène, ce qui peut expliquer certains des effets paradoxaux de LKB1 durant la tumorigenèse. / The LKB1 tumor suppressor gene encodes a master kinase that coordinates the regulation of energetic metabolism, cell growth and cell polarity. We now report the identification of a novel isoform of LKB1 named N-LKB1 that is generated through alternative transcription and internal initiation of translation of the LKB1 mRNA. The N-LKB1 protein lacks the N-terminal region and a portion of the kinase domain. Although N-LKB1 is catalytically inactive, it potentiates the stimulating effect of LKB1 on the AMP-activated protein kinase (AMPK) metabolic sensor through a direct interaction with the regulatory auto-inhibitory domain of AMPK. Contrasting, N-LKB1 negatively interferes with the LKB1 polarizing activity. Finally, combining in vitro and in vivo approaches, we showedthat N-LKB1 has an intrinsic oncogenic property. N-LKB1 is expressed solely in the lung cancer cell line, NCI-H460. Silencing of N-LKB1 decreased survival of NCI-H460 cells and inhibited their tumorigenicity when engrafted in nude mice. In conclusion, we have identified a novel LKB1 isoform that enhances the LKB1-controlled AMPK metabolic activity but inhibits LKB1-induced polarizing activity. Both, the LKB1 tumor suppressor and the oncogene, N-LKB1, are expressed from the same locus and this may account for some of the paradoxical effects of LKB1 during tumorigenesis.

Le VHC infecte les hépatocytes, cellules polarisées du foie. Le cycle de réplication du VHC est dépendant du métabolisme lipidique de la cellule hôte. Mais la relation entre VHC, polarité cellulaire et métabolisme lipidique est mal connue. Nous avons démontré que SHIP2 joue un rȏle important dans l’établissement de la polarité apicobasale des cellules épithéliales. La protéine core du HCV induit la perte de polarité cellulaire et diminue l’expression de la phosphatase SHIP2. La réintroduction de SHIP2 dans les cellules exprimant core restitue la polarité cellulaire et diminue l’expression de core. SHIP2 agit aussi sur l’accumulation et l’organisation des gouttelettes lipidiques qui sont des éléments cellulaires nécessaires à la réplication du VHC. Ces résultats montrent le rôle de SHIP2 dans la polarité cellulaire et le désigne comme une cible intéressante pour des recherches dans la lutte contre les infections du VHC. / HCV infects hepatocytes, polarized cells of the liver. HCV replication cycle is dependent on lipid metabolism of the host cell. But the relationship between HCV cell polarity and lipid metabolism is unknown. We demonstrated that SHIP2 plays an important role in establishment of the apicobasal epithelial cell polarity. The HCV core protein induces loss of cell polarity and decreases the expression of the phosphatase SHIP2. The reintroduction of SHIP2 in cells expressing core restores cell polarity and decreases the expression of core protein. SHIP2 also negatively affect lipid droplets, which are important for HCV replication. These results show the role of SHIP2 in cell polarity and designate it as an attractive target for research in the fight against HCV infection.

No description available.

<p>RhoGTPases are proteins working as molecular switches as they bind and hydrolyze GTP. They are in their active conformation when GTP is bound and are then able to interact with their effector proteins, which relay the downstream signaling. When the GTP is hydrolyzed to GDP, the RhoGTPase is inactivated. RhoGTPases have been shown to be activated by a variety of stimuli and they are implicated in regulation of diverse cellular processes, including cell migration, cell cycle progression, establishment of cell polarity and transformation. </p><p>We identified mammalian Par6 as a novel effector protein for the RhoGTPases Cdc42 and Rac1. The <i>Caenorhabditis elegans</i> homologue of Par6 had previously been shown to be essential for cell polarity development in the worm embryo. We found that endogenous Par6 colocalized with the tight junction protein ZO-1 in MDCKII epithelial cells. Par6 also interacted with mammalian Par3, another member of the <i>par</i> (for partitioning defective) gene family, first identified in <i>C.elegans</i>. Endogenous Par3 also localized to tight junctions in epithelial cells. This suggested that Par6 and Par3 are part of a complex regulating cell polarity also in mammalian cells. The interaction between Par6 and activated Cdc42 and Rac1 suggested a role for these RhoGTPases in the regulation of this complex.</p><p>Co-expression of Par6 together with PKCζ, induced a dramatic change in cell morphology. The cells rounded up and long cellular extensions, resembling neurites, were formed. The ability to induce these changes in cell morphology was found to be dependent on the direct interaction between Par6 and PKCζ, as well as on the kinase activity of PKCζ. We observed that cells co-expressing mPar6C and PKCζ contained bundled microtubules and microtubules that hade been acetylated, indicating that the microtubules were stabilized. </p><p>To investigate the roles of RhoGTPases in PDGF-induced gene expression we performed cDNA microarray analyses on AG01518 human foreskin fibroblasts in which we over-expressed the dominant negative forms of Cdc42, Rac1 and RhoA. We found that the expression of 16 genes, out of the 45 up-regulated by PDGF-BB, were inhibited ≥50% in the presence of dominant negative Cdc42, Rac1 or RhoA. 19 other genes were down-regulated by one or two of the dominant RhoGTPases. Our data implied that the expression of many PDGF-BB induced genes can be affected by RhoGTPase signaling. </p><p>In conclusion, the work presented here has increased the knowledge of the involvement of RhoGTPase signaling in establishment of cell polarity and gene regulation.</p>

RhoGTPases are proteins working as molecular switches as they bind and hydrolyze GTP. They are in their active conformation when GTP is bound and are then able to interact with their effector proteins, which relay the downstream signaling. When the GTP is hydrolyzed to GDP, the RhoGTPase is inactivated. RhoGTPases have been shown to be activated by a variety of stimuli and they are implicated in regulation of diverse cellular processes, including cell migration, cell cycle progression, establishment of cell polarity and transformation. We identified mammalian Par6 as a novel effector protein for the RhoGTPases Cdc42 and Rac1. The Caenorhabditis elegans homologue of Par6 had previously been shown to be essential for cell polarity development in the worm embryo. We found that endogenous Par6 colocalized with the tight junction protein ZO-1 in MDCKII epithelial cells. Par6 also interacted with mammalian Par3, another member of the par (for partitioning defective) gene family, first identified in C.elegans. Endogenous Par3 also localized to tight junctions in epithelial cells. This suggested that Par6 and Par3 are part of a complex regulating cell polarity also in mammalian cells. The interaction between Par6 and activated Cdc42 and Rac1 suggested a role for these RhoGTPases in the regulation of this complex. Co-expression of Par6 together with PKCζ, induced a dramatic change in cell morphology. The cells rounded up and long cellular extensions, resembling neurites, were formed. The ability to induce these changes in cell morphology was found to be dependent on the direct interaction between Par6 and PKCζ, as well as on the kinase activity of PKCζ. We observed that cells co-expressing mPar6C and PKCζ contained bundled microtubules and microtubules that hade been acetylated, indicating that the microtubules were stabilized. To investigate the roles of RhoGTPases in PDGF-induced gene expression we performed cDNA microarray analyses on AG01518 human foreskin fibroblasts in which we over-expressed the dominant negative forms of Cdc42, Rac1 and RhoA. We found that the expression of 16 genes, out of the 45 up-regulated by PDGF-BB, were inhibited ≥50% in the presence of dominant negative Cdc42, Rac1 or RhoA. 19 other genes were down-regulated by one or two of the dominant RhoGTPases. Our data implied that the expression of many PDGF-BB induced genes can be affected by RhoGTPase signaling. In conclusion, the work presented here has increased the knowledge of the involvement of RhoGTPase signaling in establishment of cell polarity and gene regulation.

No description available.

Les anomalies du tube neural (ATN), incluant l'anencéphalie et le spina-bifida, représentent un groupe de malformations congénitales très fréquentes chez l'homme. Ces anomalies sont causées par un défaut partiel ou complet de la fermeture du tube neurale au cours de l'embryogenèse. Les ATN ont une étiologie complexe et multifactorielle impliquant des facteurs environnementaux et génétiques. La voie de signalisation non-canonique du Frizzled (Fz)/Dishevelled (Dvl) contrôle la polarité cellulaire planaire (PCP) et le processus morphogénétique appelé l’extension convergente qui est essentiel pour la gastrulation et la fermeture du tube neural. Très important, des mutations des gènes de cette voie étaient fortement associées aux ATN chez la souris et l’humain. Scribble est un gène de la voie PCP qui cause une sévère ATN chez la souris Circletail. Notre étude vise à analyser le rôle de SCRIBBLE1 dans les ATN humains par des analyses de séquence de son cadre de lecture et ses jonctions exon-introns. Notre étude comporte 396 patients recrutés au Centre Spina Bifida de l’hôpital Gaslini en Gènes, Italie et 83 patients recrutés au Centre Spina Bifida de l’hôpital Sainte Justine. Les patients sont affectés par plusieurs formes d’ATN. Nous avons identifié neuf mutations rares et non synonymes chez 10 patients, p.Asp93Ala (c. 435G>A), p.Gly145Arg (c. 278A>C), p.Gly263Ser (c. 786C>A), p.Gly469Ser (c. 1405G>A), p.Pro649His (c. 1946C>A), p.Gln808His (c. 2424G>T), p.Val1066Met (c. 3196G>A), p.Arg1150Gln (c. 3480G>A) et p.Thr1422Met (c. 4266C>T). Cinque mutations, p.Gly263Ser, p.Pro649His, p.Gln808His, p.Arg1150Gln, p.Thr1422Met, étaient absentes dans les contrôles analysés et prédites d’être pathogéniques in silico. Cette étude montre que des mutations rares dans SCRIB1 pourraient augmenter le risque des ATN dans une fraction des patients. L’identification des gènes prédisposant aux ATN nous aidera à mieux comprendre les mécanismes pathogéniques impliqués dans ces maladies. / Neural tube defects (NTDs), including anencephaly and spina bifida, represent a group of very common birth defects in humans. These anomalies are caused by a partial or complete failure of neural tube closure during embryogenesis. NTDs have a multifactorial etiology involving environmental and genetic factors. The non-canonical signaling pathway Frizzled (Fz) / Dishevelled (Dvl) controls the planar cell polarity (PCP) and the morphogenetic process called convergent extension (CE) which is essential for gastrulation and neural tube closure. Importantly, mutations in genes of this pathway were strongly associated with NTDs in mice and humans. Scribble is a PCP gene that causes a severe NTD mouse Circletail. Scribble binds to another PCP protein, Stbm / Vang, and they cooperate together for the stability of the PCP pathway. Our study aims at investigating the role of SCRIBBLE1 in human NTDs by sequence analyses of its open reading frame and exon-intron junctions. The cohort included in this study consisted of 396 patients recruited at the Spina Bifida Centre of Gaslini Hospital in Genoa, Italy, and 83 patients recruited at the Spina Bifida Center of the Sainte Justine Hospital, Montreal, Canada. Patients were affected by several forms of NTDs. We identified nine non-synonymous and rare mutations in 10 patients: p.Asp93Ala (c. 435G>A), p.Gly145Arg (c. 278A>C), p.Gly263Ser (c. 786C>A), p.Gly469Ser (c. 1405G>A), p.Pro649His (c. 1946C>A), p.Gln808His (c. 2424G>T), p.Val1066Met (c. 3196G>A), p.Arg1150Gln (c. 3480G>A) and p.Thr1422Met (c. 4266C>T). Five of those mutations, p.Gly263Ser, p.Pro649His, p.Gln808His, p.Arg1150Gln, p.Thr1422Met, were absent in all controls analyzed and were predicted to be pathogenic using bioinformatics. Our study demonstrates that rare mutations in SCRIB1 could predispose to NTDs in a fraction of patients. The identification of genes that predispose to ATN will help us better understand the pathogenic mechanisms involved in these diseases.

La voie de la polarité planaire cellulaire (PCP), aussi connue sous le nom de la voie non-canonique du Frizzled/Dishevelled, contrôle le processus morphogénétique de l'extension convergente (CE) qui est essentiel pour la gastrulation et la formation du tube neural pendant l'embryogenèse. La signalisation du PCP a été récemment associée avec des anomalies du tube neural (ATN) dans des modèles animaux et chez l'humain. Prickle1 est une protéine centrale de la voie PCP, exprimée dans la ligne primitive et le mésoderme pendant l'embryogenèse de la souris. La perte ou le gain de fonction de Prickle1 mène à des mouvements de CE fautifs chez le poisson zèbre et la grenouille. PRICKLE1 interagit directement avec deux autres membres de la voie PCP, Dishevelled et Strabismus/Vang. Dans notre étude, nous avons investigué le rôle de PRICKLE1 dans l'étiologie des ATN dans une cohorte de 810 patients par le re-séquençage de son cadre de lecture et des jonctions exon-intron. Le potentiel pathogénique des mutations ainsi identifiées a été évalué par des méthodes bioinformatiques, suivi par une validation fonctionnelle in vivo dans un système poisson zèbre. Nous avons identifié dans notre cohorte un total de 9 nouvelles mutations dont sept: p.Ile69Thr, p.Asn81His, p.Thr275Met, p.Arg682Cys et p.Ser739Phe, p.Val550Met et p.Asp771Asn qui affectent des acides aminés conservés. Ces mutations ont été prédites in silico d’affecter la fonction de la protéine et sont absentes dans une large cohorte de contrôles de même origine ethnique. La co-injection de ces variantes avec le gène prickle1a de type sauvage chez l’embryon de poisson zèbre a démontré qu’une mutation, p.Arg682Cys, modifie dans un sens négatif le phénotype du défaut de la CE produit par pk1 de type sauvage. Notre étude démontre que PK1 peut agir comme facteur prédisposant pour les ATN chez l’humain et élargit encore plus nos connaissances sur le rôle des gènes de la PCP dans la pathogenèse de ces malformations. / The planar cell polarity pathway (PCP) or the non-canonical Frizzled/Dishevelled pathway controls the morphogenetic process of convergent extension (CE) that is essential during embryogenesis for gastrulation and neural tube formation. Recently, PCP signalling was associated with neural tube defects (NTD) in humans and animal models. The core PCP protein, Prickle1, is expressed in the primitive streak and mesoderm during mouse embryogenesis. Both gain and loss of function of Prickle1 cause faulty CE movements in zebrafish and the frog. PRICKLE1 physically interacts with two other core PCP members, Dishevelled and Strabismus/Vang. In the present study we investigated the role of PRICKLE1 in the aetiology of NTDs in a large cohort of 810 patients through resequencing of its open reading frame and exon-intron junctions. The pathogenicity of the identified mutations was assessed through bioinformatics methods followed by a functional validation in a zebrafish system, in vivo. We identified in our cohort a total of nine novel mutations, of which seven affected conserved amino acids: p.Ile69Thr, p.Asn81His, p.Thr275Met, p.Arg682Cys, p.Ser739Phe, p.Val550Met and p.Asp771As. These mutations were predicted to affect the function of the protein in silico and were absent in a large cohort of ethnically-matched controls. Co-injection of these variants with the wild type pk1 in zebrafish oocytes revealed that one mutation, p.Arg682Cys, antagonized the CE phenotype induced by the wild-type zebrafish prickle1a in a dominant fashion. Our study demonstrates that PRICKLE1 can represent a predisposing factor for human NTDs and further expands our knowledge on the role that PCP genes in the pathogenesis of these malformations.

Thesis (Ph. D.) -- University of Texas Southwestern Medical Center at Dallas, 2005. / Vita. Bibliography: 108-115.

No description available.