The nuclear hormone receptor peroxisome proliferator-activated receptor beta/delta potentiates cell chemotactism, polarization, and migration.

After an injury, keratinocytes acquire the plasticity necessary for the reepithelialization of the wound. Here, we identify a novel pathway by which a nuclear hormone receptor, until now better known for its metabolic functions, potentiates cell migration. We show that peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) enhances two phosphatidylinositol 3-kinase-dependent pathways, namely, the Akt and the Rho-GTPase pathways. This PPARbeta/delta activity amplifies the response of keratinocytes to a chemotactic signal, promotes integrin recycling and remodeling of the actin cytoskeleton, and thereby favors cell migration. Using three-dimensional wound reconstructions, we demonstrate that these defects have a strong impact on in vivo skin healing, since PPARbeta/delta-/- mice show an unexpected and rare epithelialization phenotype. Our findings demonstrate that nuclear hormone receptors not only regulate intercellular communication at the organism level but also participate in cell responses to a chemotactic signal. The implications of our findings may be far-reaching, considering that the mechanisms described here are important in many physiological and pathological situations.

Protein-protein interactions between adenovirus DNA polymerase and nuclear factor I mediate formation of the DNA replication preinitiation complex.

The in vitro adenovirus (Ad) DNA replication system provides an assay to study the interaction of viral and host replication proteins with the DNA template in the formation of the preinitiation complex. This initiation system requires in addition to the origin DNA sequences 1) Ad DNA polymerase (Pol), 2) Ad preterminal protein (pTP), the covalent acceptor for protein-primed DNA replication, and 3) nuclear factor I (NFI), a host cell protein identical to the CCAAT box-binding transcription factor. The interactions of these proteins were studied by coimmunoprecipitation and Ad origin DNA binding assays. The Ad Pol can bind to origin sequences only in the presence of another protein which can be either pTP or NFI. While NFI alone can bind to its origin recognition sequence, pTP does not specifically recognize DNA unless Ad Pol is present. Thus, protein-protein interactions are necessary for the targetting of either Ad Pol or pTP to the preinitiation complex. DNA footprinting demonstrated that the Ad DNA site recognized by the pTP.Pol complex was within the first 18 bases at the end of the template which constitutes the minimal origin of replication. Mutagenesis studies have defined the Ad Pol interaction site on NFI between amino acids 68-150, which overlaps the DNA binding and replication activation domain of this factor. A putative zinc finger on the Ad Pol has been mutated to a product that fails to bind the Ad origin sequences but still interacts with pTP. These results indicate that both protein-protein and protein-DNA interactions mediate specific recognition of the replication origin by Ad DNA polymerase.

Scaffold attachment factor B1 directly interacts with nuclear receptors in living cells and represses transcriptional activity.

Transcriptional activity relies on coregulators that modify the chromatin structure and serve as bridging factors between transcription factors and the basal transcription machinery. Using the DE domain of human peroxisome proliferator-activated receptor gamma (PPARgamma) as bait in a yeast two-hybrid screen of a human adipose tissue library, we isolated the scaffold attachment factor B1 (SAFB1/HET/HAP), which was previously shown to be a corepressor of estrogen receptor alpha. We show here that SAFB1 has a very broad tissue expression profile in human and is also expressed all along mouse embryogenesis. SAFB1 interacts in pull-down assays not only with PPARgamma but also with all nuclear receptors tested so far, albeit with different affinities. The association of SAFB1 and PPARgamma in vivo is further demonstrated by fluorescence resonance energy transfer (FRET) experiments in living cells. We finally show that SAFB1 is a rather general corepressor for nuclear receptors. Its change in expression during the early phases of adipocyte and enterocyte differentiation suggests that SAFB1 potentially influences cell proliferation and differentiation decisions.

Retention half times in the skeleton of plutonium and Sr-90 from above-ground nuclear tests: A retrospective study of the Swiss population

Plutonium and Sr-90 are considered to be among the most radiotoxic nuclides produced by the nuclear fission process. In spite of numerous studies on mammals and humans there is still no general agreement on the retention half time of both radionuclides in the skeleton in the general population. Here we determined plutonium and Sr-90 in human vertebrae in individuals deceased between 1960 and 2004 in Switzerland. Plutonium was measured by sensitive SF-ICP-MS techniques and Sr-90 by radiometric methods. We compared our results to the ones obtained for other environmental compartments to reveal the retention half time of NBT fallout Pu-239 and Sr-90 in trabecular bones of the Swiss population. Results show that plutonium has a retention half time of 40 +/- 14 years. In contrast Sr-90 has a shorter retention half time of 13.5 +/- 1.0 years. Moreover Sr-90 retention half time in vertebrae is shown to be linked to the retention half time in food and other environmental compartments. These findings demonstrate that the renewal of the vertebrae through calcium homeostatic control is faster for Sr-90 excretion than for plutonium excretion. The precise determination of the retention half time of plutonium in the skeleton will improve the biokinetic model of plutonium metabolism in humans. (C) 2010 Elsevier Ltd. All rights reserved.

Nuclear triiodothyronine receptor expression is regulated by axon-Schwann cell contact.

Thyroid hormones, which play an important role in the development and regeneration of the nervous system, require the presence of specific nuclear T3 receptors (NT3R). In this study we provide evidence that NT3R expression by Schwann cells was up-regulated in response to a loss of axonal contact in vitro and in vivo. In dorsal root ganglia explant cultures, Schwann cells which accompanied axons (nerve fibres) were devoid of NT3R. When Schwann cells were orphaned from axon contact by axon transection, all the nuclei of these cells displayed NT3R immunoreactivity. Similar results were obtained in situ; in adult rat sciatic nerve, Schwann cells which ensheathed healthy axons never expressed NT3R immunoreactivity. After sciatic nerve transection in vivo the nuclei of Schwann cells deprived of axonal contact displayed a clear NT3R immunoreaction.

Enhanced excitation-coupled Ca(2+) entry induces nuclear translocation of NFAT and contributes to IL-6 release from myotubes from patients with central core disease.

Prolonged depolarization of skeletal muscle cells induces entry of extracellular calcium into muscle cells, an event referred to as excitation-coupled calcium entry. Skeletal muscle excitation-coupled calcium entry relies on the interaction between the 1,4-dihydropyridine receptor on the sarcolemma and the ryanodine receptor on the sarcoplasmic reticulum membrane. In this study, we directly measured excitation-coupled calcium entry by total internal reflection fluorescence microscopy in human skeletal muscle myotubes harbouring mutations in the RYR1 gene linked to malignant hyperthermia (MH) and central core disease (CCD). We found that excitation-coupled calcium entry is strongly enhanced in cells from patients with CCD compared with individuals with MH and controls. Furthermore, excitation-coupled calcium entry induces generation of reactive nitrogen species and enhances nuclear localization of NFATc1, which in turn may be responsible for the increased IL-6 released by myotubes from patients with CCD.

Gene transfer into Xenopus hepatocytes: transcriptional regulation by members of the nuclear receptor superfamily.

A procedure to culture Xenopus laevis hepatocytes that allows the cells in primary culture to be subjected to gene transfer experiments has been developed. The cultured cells continue to present tissue-specific markers such as expression of the albumin gene or estrogen-controlled vitellogenin gene expression, which are both restricted to liver. Two efficient and reproducible gene transfer procedures have been adapted to the Xenopus hepatocytes, namely lipofection and calcium phosphate-mediated precipitation. The transcription of transfected reporter genes controlled by estrogen-, glucocorticoid- or peroxisome proliferator-response elements was stimulated by endogenous or co-transfected receptor in a ligand-dependent manner. Furthermore, the expression of a reporter gene under the control of the entire promoter of the vitellogenin B1 gene mimicked the expression of the chromosomal vitellogenin gene with respect to basal and estrogen-induced activity. Thus, this culture-transfection system will prove very useful to study the regulation of genes expressed in the liver under the control of various hormones or xenobiotics.

Extremity exposure in nuclear medicine therapy with 90Y-labelled substances - Results of the ORAMED project

90Y-labelled radiopharmaceuticals offer promising prospects for radionuclide therapies of tumours, e.g. radioimmunotherapies (RIT), (EANM, 2007), peptide receptor radiotherapies (PRRT), (Otte et al., 1998), and selective internal radiotherapies (SIRT), (Salem and Thurston, 2006). 90Y, an almost pure high-energy beta radiation emitter (Eβ,max = 2.28 MeV), is a favourable radionuclide for therapeutic purposes. However, when preparing and performing these therapies, high activities of 90Y (>1 GBq) are to be manipulated and technicians, physicians and nurses may receive high skin exposures to the hands. If radiation protection standards are low, the exposure of staff can exceed the annual skin dose limit of 500 mSv. Within a particular work package (WP4) of the ORAMED project, comprehensive measurements in nuclear medicine departments of several hospitals in 6 European countries were carried out. The study focussed on 90Y-labelled substances such as Zevalin® and DOTATOC to achieve a representative database on staff exposure. This paper summarises the most important results and conclusions for individual monitoring of skin exposure of staff.

An adeno-associated virus-based intracellular sensor of pathological nuclear factor-κB activation for disease-inducible gene transfer.

Stimulation of resident cells by NF-κB activating cytokines is a central element of inflammatory and degenerative disorders of the central nervous system (CNS). This disease-mediated NF-κB activation could be used to drive transgene expression selectively in affected cells, using adeno-associated virus (AAV)-mediated gene transfer. We have constructed a series of AAV vectors expressing GFP under the control of different promoters including NF-κB -responsive elements. As an initial screen, the vectors were tested in vitro in HEK-293T cells treated with TNF-α. The best profile of GFP induction was obtained with a promoter containing two blocks of four NF-κB -responsive sequences from the human JCV neurotropic polyoma virus promoter, fused to a new tight minimal CMV promoter, optimally distant from each other. A therapeutical gene, glial cell line-derived neurotrophic factor (GDNF) cDNA under the control of serotype 1-encapsidated NF-κB -responsive AAV vector (AAV-NF) was protective in senescent cultures of mouse cortical neurons. AAV-NF was then evaluated in vivo in the kainic acid (KA)-induced status epilepticus rat model for temporal lobe epilepsy, a major neurological disorder with a central pathophysiological role for NF-κB activation. We demonstrate that AAV-NF, injected in the hippocampus, responded to disease induction by mediating GFP expression, preferentially in CA1 and CA3 neurons and astrocytes, specifically in regions where inflammatory markers were also induced. Altogether, these data demonstrate the feasibility to use disease-activated transcription factor-responsive elements in order to drive transgene expression specifically in affected cells in inflammatory CNS disorders using AAV-mediated gene transfer.

Calpastatin-mediated inhibition of calpains in the mouse brain prevents mutant ataxin 3 proteolysis, nuclear localization and aggregation, relieving Machado-Joseph disease.

Machado-Joseph disease is the most frequently found dominantly-inherited cerebellar ataxia. Over-repetition of a CAG trinucleotide in the MJD1 gene translates into a polyglutamine tract within the ataxin 3 protein, which upon proteolysis may trigger Machado-Joseph disease. We investigated the role of calpains in the generation of toxic ataxin 3 fragments and pathogenesis of Machado-Joseph disease. For this purpose, we inhibited calpain activity in mouse models of Machado-Joseph disease by overexpressing the endogenous calpain-inhibitor calpastatin. Calpain blockage reduced the size and number of mutant ataxin 3 inclusions, neuronal dysfunction and neurodegeneration. By reducing fragmentation of ataxin 3, calpastatin overexpression modified the subcellular localization of mutant ataxin 3 restraining the protein in the cytoplasm, reducing aggregation and nuclear toxicity and overcoming calpastatin depletion observed upon mutant ataxin 3 expression. Our findings are the first in vivo proof that mutant ataxin 3 proteolysis by calpains mediates its translocation to the nucleus, aggregation and toxicity and that inhibition of calpains may provide an effective therapy for Machado-Joseph disease.

Apports de la médecine nucléaire en cardiologie préventive: l'exemple du syndrome métabolique [Value of nuclear medicine in preventive cardiology: the metabolic syndrome example]

Metabolic syndrome represents a grouping of risk factors closely linked to cardiovascular diseases and diabetes. At first, nuclear medicine has no direct application in cardiology at the level of primary prevention, but positron emission tomography is a non invasive imaging technique that can assess myocardial perfusion as well as the endothelium-dependent coronary vasomotion--a surrogate marker of cardiovascular event rate--thus finding an application in studying coronary physiopathology. As the prevalence of the metabolic syndrome is still unknown in Switzerland, we will estimate it from data available in the frame of a health promotion program. Based on the deleterious effect on the endothelium already observed with two components, we will estimate the number of persons at risk in Switzerland.

The biology of cancer stem cells : part 1: nuclear translocation of CD44 : part 2: IMP2 in glioblastoma cancer stem cells

SummaryCancer stem cells (CSC) are poorly differentiated, slowly proliferating cells, with high tumorigenic potential. Some of these cells, as it has been shown in leukemia, evade chemo- and radiotherapy and recapitulate the tumor composed of CSC and their highly proliferative progeny. Therefore, understanding the molecular biology of those cells is crucial for improvement of currently used anti-cancer therapies.This work is composed of two CSC-related projects. The first deals with CD44, a frequently used marker of CSC; the second involves Imp2 and its role in CSC bioenergetics. PART 1. CD44 is a multifunctional transmembrane protein involved in migration, homing, adhesion, proliferation and survival. It is overexpressed in many cancers and its levels are correlated with poor prognosis. CD44 is also highly expressed by CSC and in many malignancies it is used for CSC isolation.In the present work full-lenght CD44 nuclear localization was studied, including the mechanism of nuclear translocation and its functional role in the nucleus. Full-length CD44 can be found in nuclei of various cell types, regardless of their tumorigenic potential. For nuclear localization, CD44 needs to be first inserted into the cell membrane, from which it is transported via the endocytic pathway. Upon binding to transportinl it is translocated to the nucleus. The nuclear localization signal recognized by transportinl has been determined as the first 20 amino acids of the membrane proximal intracellular domain. Nuclear export of CD44 is facilitated by exportin Crml. Investigation of the function of nuclear CD44 revealed its implication in de novo RNA synthesis.PART 2. Glioblastoma multiforme is the most aggressive and most frequent brain malignancy. It was one of the first solid tumors from which CSC have been isolated. Based on the similarity between GBM CSC and normal stem cells expression of an oncofetal mRNA binding protein Imp2 has been investigated.Imp2 is absent in normal brain as well as in low grade gliomas, but is expressed in over 75% GBM cases and its expression is higher in CSC compared to their more differentiated counterparts. Analysis of mRNA transcripts bound by Imp2 and its protein interactors revealed that in GBM CSC Imp2 may be implicated in mitochondrial metabolism. Indeed, shRNA mediated silencing of protein expression led to decreased mitochondrial activity, decreased oxygen consumption and decreased activity of respiratory chain protein complex I. Moreover, lack of Imp2 severely affected self-renewal and tumorigenicity of GBM CSC. Experimental evidence suggest that GBM CSC depend on mitochondrial oxidative phosphorylation as an energy producing pathway and that Imp2 is a novel regulator of this pathway.RésuméLes cellules cancéreuses souches sont des cellules peu différentiées, à proliferation lente et hautement tumorigénique. Ces cellules sont radio-chimio résistantes et sont capable reformer la tumeur dans sont intégralité, reproduisant l'hétérogénéité cellulaire présent dans la tumeur d'origine. Pour améliorer les therapies antitumorales actuelles il est crucial de comprendre les mécanismes moléculaires qui caractérisent cette sous-population de cellules hautement malignes.Ce travail de thèse se compose de deux projets s'articulant autour du même axe :Le CD44 est une protéine multifonctionnelle et transmembranaire très souvent utilisée comme marqueur de cellules souches tumorales dans différents cancers. Elle est impliquée dans la migration, l'adhésion, la prolifération et la survie des cellules. Lors de ce travail de recherche, nous nous sommes intéressés à la localisation cellulaire du CD44, ainsi qu'aux mécanismes permettant sa translocation nucléaire. En effet, bien que principalement décrit comme un récepteur de surface transmembranaire, le CD44 sous sa forme entière, non clivée en peptides, peut également être observé à l'intérieur du noyau de diverses cellules, quel que soit leur potentiel tumorigénique. Pour passer ainsi d'un compartiment cellulaire à un autre, le CD44 doit d'abord être inséré dans la membrane plasmique, d'où il est transporté par endocytose jusqu'à l'intérieur du cytoplasme. La transportai permet ensuite la translocation nucléaire du CD44 via une « séquence signal » contenue dans les 20 acides aminés du domaine cytoplasmique qui bordent la membrane. A l'inverse, le CD44 est exporté du noyau grâce à l'exportin Crml. En plus des mécanismes décrits ci-dessus, cette étude a également mis en évidence l'implication du CD44 dans la synthèse des ARN, d'où sa présence dans le noyau.Le glioblastome est la plus maligne et la plus fréquente des tumeurs cérébrales. Dans ce second projet de recherche, le rôle de IMP2 dans les cellules souches tumorales de glioblastomes a été étudié. La présence de cette protéine oncofoetale a d'abord été mise en évidence dans 75% des cas les plus agressifs des gliomes (grade IV, appelés glioblastomes), tandis qu'elle n'est pas exprimée dans les grades I à III de ces tumeurs, ni dans le cerveau sain. De plus, IMP2 est apparue comme étant davantage exprimée dans les cellules souches tumorales que dans les cellules déjà différenciées. La baisse de l'expression de IMP2 au moyen de shRNA a résulté en une diminution de l'activité mitochondriale, en une réduction de la consommation d'oxygène ainsi qu'en une baisse de l'activité du complexe respiratoire I.L'inhibition de IMP2 a également affecté la capacité de renouvellement de la population des cellules souches tumorales ainsi que leur aptitude à former des tumeurs.Lors de ce travail de thèse, une nouvelle fonction d'un marqueur de cellules souches tumorales a été mise en évidence, ainsi qu'un lien important entre la bioénergétique de ces cellules et l'expression d'une protéine oncofoetale.

Additional data for nuclear DNA give new insights into the phylogenetic position of Sorex granarius within the Sorex araneus group.

Many species contain genetic lineages that are phylogenetically intermixed with those of other species. In the Sorex araneus group, previous results based on mtDNA and Y chromosome sequence data showed an incongruent position of Sorex granarius within this group. In this study, we explored the relationship between species within the S. araneus group, aiming to resolve the particular position of S. granarius. In this context, we sequenced a total of 2447 base pairs (bp) of X-linked and nuclear genes from 47 individuals of the S. araneus group. The same taxa were also analyzed within a Bayesian framework with nine autosomal microsatellites. These analyses revealed that all markers apart from mtDNA showed similar patterns, suggesting that the problematic position of S. granarius is best explained by an incongruent behavior by mtDNA. Given their close phylogenetic relationship and their close geographic distribution, the most likely explanation for this pattern is past mtDNA introgression from S. araneus race Carlit to S. granarius.