Molecular characterization of Unc5CL/ZUD

Résumé : Au cours de l'évolution, les organismes multicellulaires ont développé le système immunitaire afin de pouvoir se défendre contre les pathogènes tel que les bactéries, les virus, et les parasites. La réponse immunitaire doit être finement régulée par différentes voies de signalisation moléculaire, afin d'assurer une efficacité optimale, et d'éviter des dommages tissulaires indésirables. Les résultats expérimentaux décrits dans ce manuscrit, mettent en évidence que la protéine Unc5CL, qui contient un death domain (DD), est impliquée dans la régulation de la réponse immunitaire des muqueuses. Il a été démontré que cette protéine contient aussi un domaine transmembranaire de type III dans sa partie N-terminale, permettant ainsi de l'ancrer et d'exposer sa partie C-terminale dans le cytosol, un prérequis pour la signalisation dans ce compartiment cellulaire. De plus, cette protéine a la capacité d'activer le facteur de transcription NFxB, qui joue un rôle important dans le système immunitaire, ainsi que dans d'autres processus cellulaires essentiels. Le profil transcriptionnel révèle que l'activation de NF-κB induite par Unc5CL conduit principalement à une réponse inflammatoire, qui se caractérise par la production de diverses chimiokines (e.g. CXCL-1, IL-8 et CCL20). Il a également été démontré que Unc5CL requiert les mêmes molécules qui sont utilisées dans la voie de signalisation des récepteurs de la famille toll et de l'interleukine-1. De manière similaire à leur protéine adaptatrice MyD88, Unc5CL a la capacité de recruter, via une interaction homotypique DD-DD, les kinases IRAK1 et IRAK4 qui contiennent elles aussi un DD, permettant ainsi au signal d'être transmis. La production d'un anticorps polyclonal contre le DD de Unc5CL a permis d'identifier des lignées cellulaires et des tissus exprimant cette protéine, ainsi que de déterminer sa localisation sub-cellulaire. Unc5CL a été détecté dans les cellules de la muqueuse utérine et intestinale, ainsi que dans une lignée cellulaire issue d'un adénocarcinome colorectal humain, les CaCo-2. Dans chacun de ces cas, Unc5CL a été principalement détectée au niveau apical des cellules épithéliales polarisées. De manière similaire à PIDD, une protéine impliquée dans la réponse aux dommages à l'ADN, et au constituant des pores nucléaires Nup98, Unc5CL est constitutivement clivé de manière autoprotéolytique, au niveau d'un site HFS. Il est intéressant d'observer que les deux fragments ainsi générés restent fortement associés l'un à l'autre après clivage. Finalement, un criblage protéomique pour identifier un partenaire d'interaction, a mis en évidence l'ubiquitin ligase E3 ITCH, qui régule de manière négative Unc5CL en augmentant sa dégradation. Summary : Multicellular organisms have evolved the immune system in order to defend themselves against pathogens such as bacteria, viruses and eukaryotic parasites. Immune responses have to be tightly orchestrated by signaling mechanisms to achieve optimal effectiveness and minimal tissue damage. The experimental results in this thesis manuscript provide evidence that the death domain (DD)-containing protein Unc5CL might be involved in the regulation of mucosal immune responses. It could be shown that the protein contains an N-terminal type-III transmembrane domain that anchors the protein with its C-terminus exposed to the cytosol, a prerequisite for signaling events in this compartment. Furthermore, the protein has the capacity to activate the transcription factor NF-κB, which plays an important role in the immune system as well as in other essential cellular processes. Transcriptional profiling revealed that Unc5CL-mediated activation of NF-κB mainly leads to an inflammatory response, characterized by the production of chemokines (e.g. CXCL-l, IL-8 and CCL20). Furthermore, it could be shown that Unc5CL requires the same downstream signaling molecules as the evolutionarily ancient tolUinterleukin-1 receptor family. Similar to their adapter protein MyD88, Unc5CL has the capacity to recruit the DD-containing kinases IRAKI and IRAK4 for signaling and can interact with these proteins via homotypic DD-DD interactions. Generation of polyclonal antibodies raised against the DD of Unc5CL allowed the identification of cell lines and tissues that express the endogenous protein as well as to confine its subcellular localization. Unc5CL was detected in primary mucosal uterine and intestinal epithelial cells as well as in the human colorectal adenocarcinoma cell line CaCo-2. In all cases, the protein was mainly localized to the apical face of these polarized epithelial cells. Similar to PIDD, a protein critically involved in responses to DNA damage, and the nuclear pore component Nup98, Unc5CL is constitutively autoproteolytically processed at an HFS site. Interestingly, the two generated cleavage fragments remain tightly associated after processing. Finally, a proteomics screen for interaction partners identified the E3 ubiquitin ligase ITCH as a negative regulator of Unc5CL by targeting the protein for degradation.

Dynamic regulation of notch 1 and notch 2 surface expression during T cell development and activation revealed by novel monoclonal antibodies.

It is well established that Notch signaling plays a critical role at multiple stages of T cell development and activation. However, detailed analysis of the cellular and molecular events associated with Notch signaling in T cells is hampered by the lack of reagents that can unambiguously measure cell surface Notch receptor expression. Using novel rat mAbs directed against the extracellular domains of Notch1 and Notch2, we find that Notch1 is already highly expressed on common lymphoid precursors in the bone marrow and remains at high levels during intrathymic maturation of CD4(-)CD8(-) thymocytes. Notch1 is progressively down-regulated at the CD4(+)CD8(+) and mature CD4(+) or CD8(+) thymic stages and is expressed at low levels on peripheral T cells. Immunofluorescence staining of thymus cryosections further revealed a localization of Notch1(+)CD25(-) cells adjacent to the thymus capsule. Notch1 was up-regulated on peripheral T cells following activation in vitro with anti-CD3 mAbs or infection in vivo with lymphocytic chorio-meningitis virus or Leishmania major. In contrast to Notch1, Notch2 was expressed at intermediate levels on common lymphoid precursors and CD117(+) early intrathymic subsets, but disappeared completely at subsequent stages of T cell development. However, transient up-regulation of Notch2 was also observed on peripheral T cells following anti-CD3 stimulation. Collectively our novel mAbs reveal a dynamic regulation of Notch1 and Notch2 surface expression during T cell development and activation. Furthermore they provide an important resource for future analysis of Notch receptors in various tissues including the hematopoietic system.

Altered growth in male peroxisome proliferator-activated receptor gamma (PPARgamma) heterozygous mice: involvement of PPARgamma in a negative feedback regulation of growth hormone action.

The peroxisome proliferator-activated receptor gamma (PPARgamma) plays a major role in fat tissue development and physiology. Mutations in the gene encoding this receptor have been associated to disorders in lipid metabolism. A thorough investigation of mice in which one PPARgamma allele has been mutated reveals that male PPARgamma heterozygous (PPARgamma +/-) mice exhibit a reduced body size associated with decreased body weight, reflecting lean mass reduction. This phenotype is reproduced when treating the mice with a PPARgamma- specific antagonist. Monosodium glutamate treatment, which induces weight gain and alters body growth in wild-type mice, further aggravates the growth defect of PPARgamma +/- mice. The levels of circulating GH and that of its downstream effector, IGF-I, are not altered in mutant mice. However, the IGF-I mRNA level is decreased in white adipose tissue (WAT) of PPARgamma +/- mice and is not changed by acute administration of recombinant human GH, suggesting an altered GH action in the mutant animals. Importantly, expression of the gene encoding the suppressor of cytokine signaling-2, which is an essential negative regulator of GH signaling, is strongly increased in the WAT of PPARgamma +/- mice. Although the relationship between the altered GH signaling in WAT and reduced body size remains unclear, our results suggest a novel role of PPARgamma in GH signaling, which might contribute to the metabolic disorder affecting insulin signaling in PPARgamma mutant mice.

Cell-free reconstitution of vacuole membrane fragmentation reveals regulation of vacuole size and number by TORC1.

Size and copy number of organelles are influenced by an equilibrium of membrane fusion and fission. We studied this equilibrium on vacuoles-the lysosomes of yeast. Vacuole fusion can readily be reconstituted and quantified in vitro, but it had not been possible to study fission of the organelle in a similar way. Here we present a cell-free system that reconstitutes fragmentation of purified yeast vacuoles (lysosomes) into smaller vesicles. Fragmentation in vitro reproduces physiological aspects. It requires the dynamin-like GTPase Vps1p, V-ATPase pump activity, cytosolic proteins, and ATP and GTP hydrolysis. We used the in vitro system to show that the vacuole-associated TOR complex 1 (TORC1) stimulates vacuole fragmentation but not the opposing reaction of vacuole fusion. Under nutrient restriction, TORC1 is inactivated, and the continuing fusion activity then dominates the fusion/fission equilibrium, decreasing the copy number and increasing the volume of the vacuolar compartment. This result can explain why nutrient restriction not only induces autophagy and a massive buildup of vacuolar/lysosomal hydrolases, but also leads to a concomitant increase in volume of the vacuolar compartment by coalescence of the organelles into a single large compartment.

Molecular changes underlying mammalian phenotypic innovation

Mammals are characterized by specific phenotypic traits that include lactation, hair, and relatively large brains with unique structures. Individual mammalian lineages have, in turn, evolved characteristic traits that distinguish them from others. These include obvious anatom¬ical differences but also differences related to reproduction, life span, cognitive abilities, be¬havior. and disease susceptibility. However, the molecular basis of the diverse mammalian phenotypes and the selective pressures that shaped their evolution remain largely unknown. In the first part of my thesis, I analyzed the genetic factors associated with the origin of a unique mammalian phenotype lactation and I studied the selective pressures that forged the transition from oviparity to viviparity. Using a comparative genomics approach and evolutionary simulations, I showed that the emergence of lactation, as well as the appear¬ance of the casein gene family, significantly reduced selective pressure on the major egg-yolk proteins (the vitellogenin family). This led to a progressive loss of vitellogenins, which - in oviparous species - act as storage proteins for lipids, amino acids, phosphorous and calcium in the isolated egg. The passage to internal fertilization and placentation in therian mam¬mals rendered vitellogenins completely dispensable, which ended in the loss of the whole gene family in this lineage. As illustrated by the vitellogenin study, changes in gene content are one possible underlying factor for the evolution of mammalian-specific phenotypes. However, more subtle genomic changes, such as mutations in protein-coding sequences, can also greatly affect the phenotypes. In particular, it was proposed that changes at the level of gene reg¬ulation could underlie many (or even most) phenotypic differences between species. In the second part of my thesis, I participated in a major comparative study of mammalian tissue transcriptomes, with the goal of understanding how evolutionary forces affected expression patterns in the past 200 million years of mammalian evolution. I showed that, while com¬parisons of gene expressions are in agreement with the known species phylogeny, the rate of expression evolution varies greatly among lineages. Species with low effective population size, such as monotremes and hominoids, showed significantly accelerated rates of gene expression evolution. The most likely explanation for the high rate of gene expression evolution in these lineages is the accumulation of mildly deleterious mutations in regulatory regions, due to the low efficiency of purifying selection. Thus, our observations are in agreement with the nearly neutral theory of molecular evolution. I also describe substantial differences in evolutionary rates between tissues, with brain being the most constrained (especially in primates) and testis significantly accelerated. The rate of gene expression evolution also varies significantly between chromosomes. In particular, I observed an acceleration of gene expression changes on the X chromosome, probably as a result of adaptive processes associated with the origin of therian sex chromosomes. Lastly, I identified several individual genes as well as co-regulated expression modules that have undergone lineage specific expression changes and likely under¬lie various phenotypic innovations in mammals. The methods developed during my thesis, as well as the comprehensive gene content analyses and transcriptomics datasets made available by our group, will likely prove to be useful for further exploratory analyses of the diverse mammalian phenotypes.

Cellular and molecular mechanisms underlying the effects of sleep loss on hippocampal synaptic plasticity

SUMMARY : The function of sleep for the organism is one of the most persistent and perplexing questions in biology. Current findings lead to the conclusion that sleep is primarily for the brain. In particular, a role for sleep in cognitive aspects of brain function is supported by behavioral evidence both in humans and animals. However, in spite of remarkable advancement in the understanding of the mechanisms underlying sleep generation and regulation, it has been proven difficult to determine the neurobiological mechanisms underlying the beneficial effect of sleep, and the detrimental impact of sleep loss, on learning and memory processes. In my thesis, I present results that lead to several critical steps forward in the link between sleep and cognitive function. My major result is the molecular identification and physiological analysis of a protein, the NR2A subunit of NMDA receptor (NMDAR), that confers sensitivity to sleep loss to the hippocampus, a brain structure classically involved in mnemonic processes. Specifically, I used a novel behavioral approach to achieve sleep deprivation in adult C57BL6/J mice, yet minimizing the impact of secondary factors associated with the procedure,.such as stress. By using in vitro electrophysiological analysis, I show, for the first time, that sleep loss dramatically affects bidirectional plasticity at CA3 to CA1 synapses in the hippocampus, a well established cellular model of learning and memory. 4-6 hours of sleep loss elevate the modification threshold for bidirectional synaptic plasticity (MT), thereby promoting long-term depression of CA3 to CA 1 synaptic strength after stimulation in the theta frequency range (5 Hz), and rendering long-term potentiation induction.more difficult. Remarkably, 3 hours of recovery sleep, after the deprivation, reset the MT at control values, thus re-establishing the normal proneness of synapses to undergo long-term plastic changes. At the molecular level, these functional changes are paralleled by a change in the NMDAR subunit composition. In particular, the expression of the NR2A subunit protein of NMDAR at CA3 to CA1 synapses is selectively and rapidly increased by sleep deprivation, whereas recovery sleep reset NR2A synaptic content to control levels. By using an array of genetic, pharmacological and computational approaches, I demonstrate here an obligatory role for NR2A-containing NMDARs in conveying the effect of sleep loss on CA3 to CAl MT. Moreover, I show that a genetic deletion of the NR2A subunit fully preserves hippocampal plasticity from the impact of sleep loss, whereas it does not alter sleepwake behavior and homeostatic response to sleep deprivation. As to the mechanism underlying the effects of the NR2A subunit on hippocampal synaptic plasticity, I show that the increased NR2A expression after sleep loss distinctly affects the contribution of synaptic and more slowly recruited NMDAR pools activated during plasticity-induction protocols. This study represents a major step forward in understanding the mechanistic basis underlying sleep's role for the brain. By showing that sleep and sleep loss affect neuronal plasticity by regulating the expression and function of a synaptic neurotransmitter receptor, I propose that an important aspect of sleep function could consist in maintaining and regulating protein redistribution and ion channel trafficking at central synapses. These findings provide a novel starting point for investigations into the connections between sleep and learning, and they may open novel ways for pharmacological control over hippocampal .function during periods of sleep restriction. RÉSUMÉ DU PROJET La fonction du sommeil pour l'organisme est une des questions les plus persistantes et difficiles dans la biologie. Les découvertes actuelles mènent à la conclusion que le sommeil est essentiel pour le cerveau. En particulier, le rôle du sommeil dans les aspects cognitifs est soutenu par des études comportementales tant chez les humains que chez les animaux. Cependant, malgré l'avancement remarquable dans la compréhension des mécanismes sous-tendant la génération et la régulation du sommeil, les mécanismes neurobiologiques qui pourraient expliquer l'effet favorable du sommeil sur l'apprentissage et la mémoire ne sont pas encore clairs. Dans ma thèse, je présente des résultats qui aident à clarifier le lien entre le sommeil et la fonction cognitive. Mon résultat le plus significatif est l'identification moléculaire et l'analyse physiologique d'une protéine, la sous-unité NR2A du récepteur NMDA, qui rend l'hippocampe sensible à la perte de sommeil. Dans cette étude, nous avons utilisé une nouvelle approche expérimentale qui nous a permis d'induire une privation de sommeil chez les souris C57BL6/J adultes, en minimisant l'impact de facteurs confondants comme, par exemple, le stress. En utilisant les techniques de l'électrophysiologie in vitro, j'ai démontré, pour la première fois, que la perte de sommeil est responsable d'affecter radicalement la plasticité bidirectionnelle au niveau des synapses CA3-CA1 de l'hippocampe. Cela correspond à un mécanisme cellulaire de l'apprentissage et de la mémoire bien établi. En particulier, 4-6 heures de privation de sommeil élèvent le seuil de modification pour la plasticité synaptique bidirectionnelle (SM). Comme conséquence, la dépression à long terme de la transmission synaptique est induite par la stimulation des fibres afférentes dans la bande de fréquences thêta (5 Hz), alors que la potentialisation à long terme devient plus difficile. D'autre part, 3 heures de sommeil de récupération sont suffisant pour rétablir le SM aux valeurs contrôles. Au niveau moléculaire, les changements de la plasticité synaptiques sont associés à une altération de la composition du récepteur NMDA. En particulier, l'expression synaptique de la protéine NR2A du récepteur NMDA est rapidement augmentée de manière sélective par la privation de sommeil, alors que le sommeil de récupération rétablit l'expression de la protéine au niveau contrôle. En utilisant des approches génétiques, pharmacologiques et computationnelles, j'ai démontré que les récepteurs NMDA qui expriment la sous-unité NR2A sont responsables de l'effet de la privation de sommeil sur le SM. De plus, nous avons prouvé qu'une délétion génétique de la sous-unité NR2A préserve complètement la plasticité synaptique hippocampale de l'impact de la perte de sommeil, alors que cette manipulation ne change pas les mécanismes de régulation homéostatique du sommeil. En ce qui concerne les mécanismes, j'ai .découvert que l'augmentation de l'expression de la sous-unité NR2A au niveau synaptique modifie les propriétés de la réponse du récepteur NMDA aux protocoles de stimulations utilisés pour induire la plasticité. Cette étude représente un pas en avant important dans la compréhension de la base mécaniste sous-tendant le rôle du sommeil pour le cerveau. En montrant que le sommeil et la perte de sommeil affectent la plasticité neuronale en régulant l'expression et la fonction d'un récepteur de la neurotransmission, je propose qu'un aspect important de la fonction du sommeil puisse être finalisé au règlement de la redistribution des protéines et du tracking des récepteurs aux synapses centraux. Ces découvertes fournissent un point de départ pour mieux comprendre les liens entre le sommeil et l'apprentissage, et d'ailleurs, ils peuvent ouvrir des voies pour des traitements pharmacologiques dans le .but de préserver la fonction hippocampale pendant les périodes de restriction de sommeil.

A novel interferon-gamma regulated human melanoma-associated antigen, gp33-38, defined by monoclonal antibody Me14-D12. II. Molecular cloning of a genomic probe.

The human Me14-D12 antigen is a cell surface glycoprotein regulated by interferon-gamma (IFN-gamma) on tumor cell lines of neuroectodermal origin. It consists of two non-convalently linked subunits with apparent mol. wt sizes of 33,000 and 38,000. Here we describe the molecular cloning of a genomic probe for the Me14-D12 gene using the gene transfer approach. Mouse Ltk- cells were stably cotransfected with human genomic DNA and the Herpes Simplex virus thymidine kinase (TK) gene. Primary and secondary transfectants expressing the Me14-D12 antigen were isolated after selection in HAT medium by repeated sorting on a fluorescence activated cell sorter (FACS). A recombinant phage harboring a 14.3 kb insert of human DNA was isolated from a genomic library made from a positive secondary transfectant cell line. A specific probe derived from the phage DNA insert allowed the identification of two mRNAs of 3.5 kb and 2.2 kb in primary and secondary L cell transfectants, as well as in human melanoma cell lines expressing the Me14-D12 antigen. The regulation of Me14-D12 antigen by INF-gamma was retained in the L cell transfectants and could be detected both at the level of protein and mRNA expression.

Regulation of the immune response by macrophage migration inhibitory factor: biological and structural features.

The classical T cell cytokine macrophage migration inhibitory factor (MIF) has reemerged recently as a critical mediator of the host immune and stress response. MIF has been found to be a mediator of several diseases including gram-negative septic shock and delayed-type hypersensitivity reactions. Its immunological functions include the modulation of the host macrophage and T and B cell response. In contrast to other known cytokines, MIF production is induced rather than suppressed by glucocorticoids, and MIF has been found to override the immunosuppressive effects of glucocorticoids. Recently, elucidation of the three-dimensional structure of MIF revealed that MIF has a novel, unique cytokine structure. Here the biological role of MIF is reviewed in view of its distinct immunological and structural properties.

Transcriptional regulation of metabolism.

Our understanding of metabolism is undergoing a dramatic shift. Indeed, the efforts made towards elucidating the mechanisms controlling the major regulatory pathways are now being rewarded. At the molecular level, the crucial role of transcription factors is particularly well-illustrated by the link between alterations of their functions and the occurrence of major metabolic diseases. In addition, the possibility of manipulating the ligand-dependent activity of some of these transcription factors makes them attractive as therapeutic targets. The aim of this review is to summarize recent knowledge on the transcriptional control of metabolic homeostasis. We first review data on the transcriptional regulation of the intermediary metabolism, i.e., glucose, amino acid, lipid, and cholesterol metabolism. Then, we analyze how transcription factors integrate signals from various pathways to ensure homeostasis. One example of this coordination is the daily adaptation to the circadian fasting and feeding rhythm. This section also discusses the dysregulations causing the metabolic syndrome, which reveals the intricate nature of glucose and lipid metabolism and the role of the transcription factor PPARgamma in orchestrating this association. Finally, we discuss the molecular mechanisms underlying metabolic regulations, which provide new opportunities for treating complex metabolic disorders.

Positive regulation of the peroxisomal beta-oxidation pathway by fatty acids through activation of peroxisome proliferator-activated receptors (PPAR).

Peroxisome proliferators regulate the transcription of genes by activating ligand-dependent transcription factors, which, due to their structure and function, can be assigned to the superfamily of nuclear hormone receptors. Three such peroxisome proliferator-activated receptors (PPAR alpha, beta, and gamma) have been cloned in Xenopus laevis. Their mRNAs are expressed differentially; xPPAR alpha and beta but not xPPAR gamma are expressed in oocytes and embryos. In the adult, expression of xPPAR alpha and beta appears to be ubiquitous, and xPPAR gamma is mainly observed in adipose tissue and kidney. Immunocytochemical analysis revealed that PPARs are nuclear proteins, and that their cytoplasmic-nuclear translocation is independent of exogenous activators. A target gene of PPARs is the gene encoding acyl-CoA oxidase (ACO), which catalyzes the rate-limiting step in the peroxisomal beta-oxidation of fatty acids. A peroxisome proliferator response element (PPRE), to which PPARs bind, has been identified within the promoter of the ACO gene. Besides the known xenobiotic activators of PPARs, such as hypolipidemic drugs, natural activators have been identified. Polyunsaturated fatty acids at physiological concentrations are efficient activators of PPARs, and 5,8,11,14-eicosatetraynoic acid (ETYA), which is the alkyne homolog of arachidonic acid, is the most potent activator of xPPAR alpha described to date. Taken together, our data suggest that PPARs have an important role in lipid metabolism.

Regulation of phosphate starvation responses in plants: signaling players and cross-talks.

Phosphate (Pi) availability is a major factor limiting growth, development, and productivity of plants. In both ecological and agricultural contexts, plants often grow in soils with low soluble phosphate content. Plants respond to this situation by a series of developmental and metabolic adaptations that are aimed at increasing the acquisition of this vital nutrient from the soil, as well as to sustain plant growth and survival. The development of a comprehensive understanding of how plants sense phosphate deficiency and coordinate the responses via signaling pathways has become of major interest, and a number of signaling players and networks have begun to surface for the regulation of the phosphate-deficiency response. In practice, application of such knowledge to improve plant Pi nutrition is hindered by complex cross-talks, which are emerging in the face of new data, such as the coordination of the phosphate-deficiency signaling networks with those involved with hormones, photo-assimilates (sugar), as well as with the homeostasis of other ions, such as iron. In this review, we focus on these cross-talks and on recent progress in discovering new signaling players involved in the Pi-starvation responses, such as proteins having SPX domains.

Regulation of the expression of Fit insect toxin locus genes in the root-associated biocontrol pseudomonad CHA0

The root-colonizing Pseudomonas fluorescens strain CHA0 is a biocontrol agent of soil-borne plant diseases caused by fungal and oomycete pathogens. Remarkably, this plant-beneficial pseudomonad is also endowed with potent insecticidal activity that depends on the production of a large protein toxin termed Fit (for P. fluorescens insecticidal toxin). In our present work, the genomic locus encoding the P. fluorescens insect toxin is subjected to a detailed molecular analysis. The Fit toxin gene fitD is flanked upstream by the fitABC genes and downstream by the fitE gene that encode the ABC transporter, membrane fusion, and outer membrane efflux components of a type I protein secretion system predicted to function in toxin export. The fitF, fitG, and fitH genes located downstream of fitE code for regulatory proteins having domain structures typical of signal transduction histidine kinases, LysR-type transcriptional regulators, and response regulators, respectively. The role of these insect toxin locus-associated control elements is being investigated with mutants defective for the regulatory genes and with GFP-based reporter fusions to putative promoter regions upstream of the transporter genes fitA and fitE, the toxin gene fitD, and the regulatory genes fitF and fitH. Our preliminary findings suggest that the three regulators interact with known global regulators of biocontrol factor expression to control Fit toxin expression and secretion.

Regulation of endothelial cell integrin function and angiogenesis by COX-2, cAMP and Protein Kinase A.

Angiogenesis, the development of new blood vessels from preexisting vessels, is a key step in tumor growth, invasion and metastasis formation. Inhibition of tumor angiogenesis is considered as an attractive approach to suppress cancer progression and spreading. Adhesion receptors of the integrin family promote tumor angiogenesis by mediating cell migration, proliferation and survival of angiogenic endothelial cells. Integrins up regulated and highly expressed on neovascular endothelial cells, such as alphaVbeta3 and alpha5beta1, have been considered as relevant targets for anti-angiogenic therapies. Small molecular integrin antagonists or blocking antibodies suppress angiogenesis and tumor progression in many animal models, and some of them are currently being tested in cancer clinical trials as anti-angiogenic agents. COX-2 inhibitors exert anti-cancer effects, at least in part, by inhibiting tumor angiogenesis. We have recently shown that COX-2 inhibitors suppress endothelial cell migration and angiogenesis by preventing alphaVbeta3-mediated and cAMP/PKA-dependent activation of the small GTPases Rac and Cdc42. Here we will review the evidence for the involvement of vascular integrins in mediating angiogenesis and the role of COX-2 metabolites in modulating the cAMP/Protein Kinase A pathway and alphaVbeta3-dependent Rac activation in endothelial cells.

A link between FXYD3 (Mat-8)-mediated Na,K-ATPase regulation and differentiation of Caco-2 intestinal epithelial cells.

FXYD3 (Mat-8) proteins are regulators of Na,K-ATPase. In normal tissue, FXYD3 is mainly expressed in stomach and colon, but it is also overexpressed in cancer cells, suggesting a role in tumorogenesis. We show that FXYD3 silencing has no effect on cell proliferation but promotes cell apoptosis and prevents cell differentiation of human colon adenocarcinoma cells (Caco-2), which is reflected by a reduction in alkaline phosphatase and villin expression, a change in several other differentiation markers, and a decrease in transepithelial resistance. Inhibition of cell differentiation in FXYD3-deficient cells is accompanied by an increase in the apparent Na+ and K+ affinities of Na,K-ATPase, reflecting the absence of Na,K-pump regulation by FXYD3. In addition, we observe a decrease in the maximal Na,K-ATPase activity due to a decrease in its turnover number, which correlates with a change in Na,K-ATPase isozyme expression that is characteristic of cancer cells. Overall, our results suggest an important role of FXYD3 in cell differentiation of Caco-2 cells. One possibility is that FXYD3 silencing prevents proper regulation of Na,K-ATPase, which leads to perturbation of cellular Na+ and K+ homeostasis and changes in the expression of Na,K-ATPase isozymes, whose functional properties are incompatible with Caco-2 cell differentiation.