Distinct expression pattern of microtubule-associated protein-2 in human oligodendrogliomas and glial precursor cells.

Microtubule-associated protein 2 (MAP2), a protein linked to the neuronal cytoskeleton in the mature central nervous system (CNS), has recently been identified in glial precursors indicating a potential role during glial development. In the present study, we systematically analyzed the expression of MAP2 in a series of 237 human neuroepithelial tumors including paraffin-embedded specimens and tumor tissue microarrays from oligodendrogliomas, mixed gliomas, astrocytomas, glioblastomas, ependymomas, as well as dysembryoplastic neuroepithelial tumors (DNT), and central neurocytomas. In addition, MAP2-immunoreactive precursor cells were studied in the developing human brain. Three monoclonal antibodies generated against MAP2A-B or MAP2A-D isoforms were used. Variable immunoreactivity for MAP2 could be observed in all gliomas with the exception of ependymomas. Oligodendrogliomas exhibited a consistently strong and distinct pattern of expression characterized by perinuclear cytoplasmic staining without significant process labeling. Tumor cells with immunoreactive bi- or multi-polar processes were mostly encountered in astroglial neoplasms, whereas the small cell component in neurocytomas and DNT was not labeled. These features render MAP2 immunoreactivity a helpful diagnostic tool for the distinction of oligodendrogliomas and other neuroepithelial neoplasms. RT-PCR, Western blot analysis, and in situ hybridization confirmed the expression of MAP2A-C (including the novel MAP2+ 13 transcript) in both oligodendrogliomas and astrocytomas. Double fluorescent laser scanning microscopy showed that GFAP and MAP2 labeled different tumor cell populations. In embryonic human brains, MAP2-immunoreactive glial precursor cells were identified within the subventricular or intermediate zones. These precursors exhibit morphology closely resembling the immunolabeled neoplastic cells observed in glial tumors. Our findings demonstrate MAP2 expression in astrocytic and oligodendroglial neoplasms. The distinct pattern of immunoreactivity in oligodendrogliomas may be useful as a diagnostic tool. Since MAP2 expression occurs transiently in migrating immature glial cells, our findings are in line with an assumed origin of diffuse gliomas from glial precursors.

Neural Wiskott-Aldrich syndrome protein modulates Wnt signaling and is required for hair follicle cycling in mice.

The Rho family GTPases Cdc42 and Rac1 are critical regulators of the actin cytoskeleton and are essential for skin and hair function. Wiskott-Aldrich syndrome family proteins act downstream of these GTPases, controlling actin assembly and cytoskeletal reorganization, but their role in epithelial cells has not been characterized in vivo. Here, we used a conditional knockout approach to assess the role of neural Wiskott-Aldrich syndrome protein (N-WASP), the ubiquitously expressed Wiskott-Aldrich syndrome-like (WASL) protein, in mouse skin. We found that N-WASP deficiency in mouse skin led to severe alopecia, epidermal hyperproliferation, and ulceration, without obvious effects on epidermal differentiation and wound healing. Further analysis revealed that the observed alopecia was likely the result of a progressive and ultimately nearly complete block in hair follicle (HF) cycling by 5 months of age. N-WASP deficiency also led to abnormal proliferation of skin progenitor cells, resulting in their depletion over time. Furthermore, N-WASP deficiency in vitro and in vivo correlated with decreased GSK-3beta phosphorylation, decreased nuclear localization of beta-catenin in follicular keratinocytes, and decreased Wnt-dependent transcription. Our results indicate a critical role for N-WASP in skin function and HF cycling and identify a link between N-WASP and Wnt signaling. We therefore propose that N-WASP acts as a positive regulator of beta-catenin-dependent transcription, modulating differentiation of HF progenitor cells.

Generation and phenotypic analysis of protein S-deficient mice.

Protein S (PS) is an important natural anticoagulant with potentially multiple biologic functions. To investigate further the role of PS in vivo, we generated Pros(+/-) heterozygous mice. In the null (-) allele, the Pros exons 3 to 7 have been excised through conditional gene targeting. Pros(+/-) mice did not present any signs of spontaneous thrombosis and had reduced PS plasma levels and activated protein C cofactor activity in plasma coagulation and thrombin generation assays. Tissue factor pathway inhibitor cofactor activity of PS could not be demonstrated. Heterozygous Pros(+/-) mice exhibited a notable thrombotic phenotype in vivo when challenged in a tissue factor-induced thromboembolism model. No viable Pros(-/-) mice were obtained through mating of Pros(+/-) parents. Most E17.5 Pros(-/-) embryos were found dead with severe intracranial hemorrhages and most likely presented consumptive coagulopathy, as demonstrated by intravascular and interstitial fibrin deposition and an increased number of megakaryocytes in the liver, suggesting peripheral thrombocytopenia. A few E17.5 Pros(-/-) embryos had less severe phenotype, indicating that life-threatening manifestations might occur between E17.5 and the full term. Thus, similar to human phenotypes, mild heterozygous PS deficiency in mice was associated with a thrombotic phenotype, whereas total homozygous deficiency in PS was incompatible with life.

Immunoelectron microscopical identification of the uncoupling protein in brown adipose tissue mitochondria.

The distribution of the uncoupling protein (UCP) in brown adipocyte mitochondria of the hibernant Muscardinus avellanarius was obtained by ultrastructural immunocytochemistry. In both cryosections and sections of Lowicryl-embedded material UCP was localized in the mitochondrial cristae of brown adipocytes, but not in liver mitochondria. It should now be possible to easily identify the morphology of cells committed to BAT differentiation in the tissue as well as in cell culture.

The role of the Estrogen-Responsive B box protein in skin morphogenesis, wound repair and skin disease

SUMMARY: EBBP is a poorly characterized member of the RBCC/TRIM family (RING finger B-box coiled-coilltripartite motif). It is ubiquitously expressed, but particularly high levels are found in keratinocytes. There is evidence that EBBP is involved in inflammatory processes, since it can interact with pro-interleukin-1 ß (prolL-1 ß) in human macrophages and keratinocytes, and its downregulation results in reduced secretion of IL-1 ß. IL-1ß activation and secretion requires the proteolytic cleavage of prolL-1ß by caspase-1, which in turn is actìvated by a protein complex called the inflammasome. As it has been demonstrated that EBBP can bind two different proteins of the inflammasome (NALP-1 and caspase 1), we assumed that EBBP plays a role in the regulation of inflammation and that the inflammasome, which has as yet only been described in ínflammatory cells, may also exist in keratinocytes. Indeed, I could show in my thesis that the inflammasome components are expressed in human keratinócytes at the RNA and protein level and also in vivo in human epidermis. After irradiation with a physiological dose of UVB, keratinocytes activated prolL-1ß and secreted prolL-1 a, IL-1 ß, prolL-18 and inflammasome proteins, although all these proteins lack a classical signal peptide. The secretion was dependent on caspase-1 activity, but not on de novo protein synthesis. Knock-down of NALP1 and -3, caspase-1 and -5, EBBP and Asc strongly reduced the secretion of IL-1 ß, demonstrating that also in keratinocytes inflammasome proteins are directly involved in maturation of this cytokine. These results demonstrate for the first time the presence of an active inflammasome in non-professional immune cells. Moreover, they show that UV irradiation is a stimulus for inflammasome activation in keratinocytes. For the analysis of the ín vivo functions of EBBP, transgenic mice overexpressing EBBP in the epidermis were generated. To examine the influence of EBBP overexpression on inflammatory processes, we subjected the mice to different challenges, which induce inflammation. Wound-healing, UVB irradiation and delayed hypersensitivity were tested, but we did not observe any phenotype in the K14-EBBP mice. Besides, a conditional ebbp knockout mouse has been obtained, which will allow to determine the effects of EBBP gene deletion in different tissues and organs. RESUME: EBBP est un membre encore mal connu de la famille des RBCC/TRIM (RING finger B-box coiled-coil/tripartite motif). Il est exprimé de manière ubiquitaire, et en particulier dans les kératinocytes. EBBP étant capable d'interagir avec la prointerleukine-1 ß (prolL-1 ß) dans les macrophages et les kératinocytes humains et de réguler la sécrétion de l'IL-1 ß, il est très probable que cette protéine est impliquée dans l'inflammation. L'activation et la sécrétion de l'IL-1 ß requièrent le clivage protéolytique de son précurseur prolL-1ß par la caspase-1, qui est elle-même activée par un complexe protéique appelé l'inflammasome. Comme il a été démontré qu'EBBP peut lier deux protéines de l'inflammasome (NALP-1 et caspase-1), nous avons émis l'hypothèse qu'EBBP joue un rôle dans la régulation de l'inflammation et que l'inflammasome, jusqu'ici décrit exclusivement dans des cellules inflammatoires, existe dans les kératinocytes. En effet, j'ai pu montrer dans ma thèse que les composants de l'inflammasome sont exprimés dans les kératinocytes humains ainsi que in vivo dans l'épiderme humain. Après irradiation avec une dose, physiologique d'UVB, les kératinocytes activent la prolL-1 ß et sécrètent la prolL-1a, l'IL-1 ß, la prolL-18 et des protéines de l'inflammasome, bien que toutes ces protéines soient dépourvues de peptide signal. La sécrétion dépend de la caspase-1 mais pas de la synthèse protéique de novo. Le knock-down de NALP-1 et -3, des caspase-1 et -5, d'EBBP et d'Asc réduit de manière marquée la sécrétion d'IL-1 ß, démontrant que dans les kératinocytes également, les protéines de l'inflammasome sont impliquées directement dans la maturation de cette cytokine. Ces résultats démontrent pour la première fois la présence d'un inflammasome actif dans des cellules immunitaires non professionnelles. De plus, ils montrent que l'irradiation aux UV est un stimulus pour l'activation de l'inflammasome dans les kératinocytes. Pour l'analyse des fonctions d'EBBP in vivo, nous avons généré des souris transgéniques qui surexpriment EBBP dans l'épiderme. En vue d'examiner l'influence de la surexpression d'EBBP sur le processus inflammatoire, nous avons soumis ces souris à differents modèles d'inflammation. Nous avons testé cicatrisation, UVB et hypersensibilité retardée, mais n'avons pas observé de phénotype chez les souris transgéniques. En parallèle, nous avons également généré des souris knock-out pour ebbp qui devraient nous permettre de déterminer les effets de la suppression d'EBBP dans différents tissus et organes.

Expression of human estrogen receptor mutants in Xenopus oocytes: correlation between transcriptional activity and ability to form protein-DNA complexes.

The human estrogen receptor (hER) is a trans-acting regulatory protein composed of a series of discrete functional domains. We have microinjected an hER expression vector (HEO) into Xenopus oocyte nuclei and demonstrate, using Western blot assay, that the hER is synthesized. When nuclear extracts from oocytes were prepared and incubated in the presence of a 2.7 kb DNA fragment comprising the 5' end of the vitellogenin gene B2, formation of estrogen-dependent complexes could be visualized by electron microscopy over the estrogen responsive element (ERE). Of crucial importance is the observation that the complex formation is inhibited by the estrogen antagonist tamoxifen, is restored by the addition of the hormone and does not take place with extracts from control oocytes injected with the expression vector lacking the sequences encoding the receptor. The presence of the biologically active hER is confirmed in co-injection experiments, in which HEO is co-introduced with a CAT reporter gene under the control of a vitellogenin promoter containing or lacking the ERE. CAT assays and primer extensions analyses reveal that both the receptor and the ERE are essential for estrogen induced stimulation of transcription. The same approach was used to analyze selective hER mutants. We find that the DNA binding domain (region C) is essential for protein--DNA complex formation at the ERE but is not sufficient by itself to activate transcription from the reporter gene. In addition to region C, both the hormone binding (region E) and amino terminal (region A/B) domains are needed for an efficient transcription activation.(ABSTRACT TRUNCATED AT 250 WORDS)

Tailed Duplex DNA is the preferred substrate for Rad51 protein-mediated homologous pairing.

Purpose of the Study: To elucidate the mechanism of homologous recombination and double-strand break repair mediated by the eukaryotic recombination pin, Rad51.

Identification of MAGI1 as a tumor-suppressor protein induced by cyclooxygenase-2 inhibitors in colorectal cancer cells.

Cyclooxyganase-2 (COX-2), a rate-limiting enzyme in the prostaglandin synthesis pathway, is overexpressed in many cancers and contributes to cancer progression through tumor cell-autonomous and paracrine effects. Regular use of non-steroidal anti-inflammatory drugs or selective COX-2 inhibitors (COXIBs) reduces the risk of cancer development and progression, in particular of the colon. The COXIB celecoxib is approved for adjunct therapy in patients with Familial adenomatous polyposis at high risk for colorectal cancer (CRC) formation. Long-term use of COXIBs, however, is associated with potentially severe cardiovascular complications, which hampers their broader use as preventive anticancer agents. In an effort to better understand the tumor-suppressive mechanisms of COXIBs, we identified MAGUK with Inverted domain structure-1 (MAGI1), a scaffolding protein implicated in the stabilization of adherens junctions, as a gene upregulated by COXIB in CRC cells and acting as tumor suppressor. Overexpression of MAGI1 in CRC cell lines SW480 and HCT116 induced an epithelial-like morphology; stabilized E-cadherin and β-catenin localization at cell-cell junctions; enhanced actin stress fiber and focal adhesion formation; increased cell adhesion to matrix proteins and suppressed Wnt signaling, anchorage-independent growth, migration and invasion in vitro. Conversely, MAGI1 silencing decreased E-cadherin and β-catenin localization at cell-cell junctions; disrupted actin stress fiber and focal adhesion formation; and enhanced Wnt signaling, anchorage-independent growth, migration and invasion in vitro. MAGI1 overexpression suppressed SW480 and HCT116 subcutaneous primary tumor growth, attenuated primary tumor growth and spontaneous lung metastasis in an orthotopic model of CRC, and decreased the number and size of metastatic nodules in an experimental model of lung metastasis. Collectively, these results identify MAG1 as a COXIB-induced inhibitor of the Wnt/β-catenin signaling pathway, with tumor-suppressive and anti-metastatic activity in experimental colon cancer.

Nucleic acid-binding properties of the RRM-containing protein RDM1.

RDM1 (RAD52 Motif 1) is a vertebrate protein involved in the cellular response to the anti-cancer drug cisplatin. In addition to an RNA recognition motif, RDM1 contains a small amino acid motif, named RD motif, which it shares with the recombination and repair protein, RAD52. RDM1 binds to single- and double-stranded DNA, and recognizes DNA distortions induced by cisplatin adducts in vitro. Here, we have performed an in-depth analysis of the nucleic acid-binding properties of RDM1 using gel-shift assays and electron microscopy. We show that RDM1 possesses acidic pH-dependent DNA-binding activity and that it binds RNA as well as DNA, and we present evidence from competition gel-shift experiments that RDM1 may be capable of discrimination between the two nucleic acids. Based on reported studies of RAD52, we have generated an RDM1 variant mutated in its RD motif. We find that the L119GF --> AAA mutation affects the mode of RDM1 binding to single-stranded DNA.

Differential involvement of peroxisome-proliferator-activated receptors alpha and delta in fibrate and fatty-acid-mediated inductions of the gene encoding liver fatty-acid-binding protein in the liver and the small intestine.

Liver fatty-acid-binding protein (L-FABP) is a cytoplasmic polypeptide that binds with strong affinity especially to long-chain fatty acids (LCFAs). It is highly expressed in both the liver and small intestine, where it is thought to have an essential role in the control of the cellular fatty acid (FA) flux. Because expression of the gene encoding L-FABP is increased by both fibrate hypolipidaemic drugs and LCFAs, it seems to be under the control of transcription factors, termed peroxisome-proliferator-activated receptors (PPARs), activated by fibrate or FAs. However, the precise molecular mechanism by which these regulations take place remain to be fully substantiated. Using transfection assays, we found that the different PPAR subtypes (alpha, gamma and delta) are able to mediate the up-regulation by FAs of the gene encoding L-FABP in vitro. Through analysis of LCFA- and fibrate-mediated effects on L-FABP mRNA levels in wild-type and PPARalpha-null mice, we have found that PPARalpha in the intestine does not constitute a dominant regulator of L-FABP gene expression, in contrast with what is known in the liver. Only the PPARdelta/alpha agonist GW2433 is able to up-regulate the gene encoding L-FABP in the intestine of PPARalpha-null mice. These findings demonstrate that PPARdelta can act as a fibrate/FA-activated receptor in tissues in which it is highly expressed and that L-FABP is a PPARdelta target gene in the small intestine. We propose that PPARdelta contributes to metabolic adaptation of the small intestine to changes in the lipid content of the diet.

CHARACTERIZATION OF EPSTEIN-BARR VIRUS-ENCODED LATENT MEMBRANE PROTEIN 1

Le virus d'Epstein-Barr (EBV), un virus de la famille des gammaherpesvirus, infecte plus de 95% de la population adulte mondiale. EBV est associé à plusieurs types de cancers dont le lymphome de Hodgkin, le lymphome de Burkitt et le carcinome nasopharyngé. La protéine membranaire de latence 1 (LMP1), l'oncogène principal d'EBV, est une protéine membranaire intégrale composée d'une petite extrémité N-terminale cytoplasmique, six segments transmembranaires (TMs) lié par de petites boucles et un long domaine C-terminale cytoplasmique. Le gène de LMP1, BNLF-1, est très polymorphe et plusieurs variants de la protéine LMP1 ont été décrits. Parmi les variants de LMP1 la majeure différence décrite est leur capacité à activer le facteur de transcription NF-κB. Nous avons défini des polymorphismes permettant aux variants d'avoir une activation accrue de NF-κB comparé au prototype B95-8 LMP1. Tous les polymorphismes cruciaux identifiés dans notre étude se trouvent dans les TMs 4 et 5 de LMP1. Nous avons étudié l'implication de chaque paire de TMs dans l'association à la membrane, l'auto-agrégation, la liaison aux partenaires cellulaires de LMP1 TRAF3 et β-TrCP, ainsi que pour NF-κB. De plus, nous avons décrit un nouveau rôle pour LMP1 consistant à inhiber l'activation contrôlée par MAVS de ISRE et du promoteur d'IFNβ. En résumé, nous avons observé que les différentes paires de TMs, ainsi que les deux boucles intracellulaires, ne sont pas équivalents. Dans l'ensemble, notre étude a montré que les TMs jouent un rôle clé dans les interactions protéine-protéine et la signalisation et qu'ils peuvent être considérés comme des régulateurs essentiels des activités de LMP1.

Involvement of the RNA-binding protein ARE/poly(U)-binding factor 1 (AUF1) in the cytotoxic effects of proinflammatory cytokines on pancreatic beta cells.

AIMS/HYPOTHESIS: Chronic exposure of pancreatic beta cells to proinflammatory cytokines leads to impaired insulin secretion and apoptosis. ARE/poly(U)-binding factor 1 (AUF1) belongs to a protein family that controls mRNA stability and translation by associating with adenosine- and uridine-rich regions of target messengers. We investigated the involvement of AUF1 in cytokine-induced beta cell dysfunction. METHODS: Production and subcellular distribution of AUF1 isoforms were analysed by western blotting. To test for their role in the control of beta cell functions, each isoform was overproduced individually in insulin-secreting cells. The contribution to cytokine-mediated beta cell dysfunction was evaluated by preventing the production of AUF1 isoforms by RNA interference. The effect of AUF1 on the production of potential targets was assessed by western blotting. RESULTS: MIN6 cells and human pancreatic islets were found to produce four AUF1 isoforms (p42>p45>p37>p40). AUF1 isoforms were mainly localised in the nucleus but were partially translocated to the cytoplasm upon exposure of beta cells to cytokines and activation of the ERK pathway. Overproduction of AUF1 did not affect glucose-induced insulin secretion but promoted apoptosis. This effect was associated with a decrease in the production of the anti-apoptotic proteins, B cell leukaemia/lymphoma 2 (BCL2) and myeloid cell leukaemia sequence 1 (MCL1). Silencing of AUF1 isoforms restored the levels of the anti-apoptotic proteins, attenuated the activation of the nuclear factor-κB (NFκB) pathway, and protected the beta cells from cytokine-induced apoptosis. CONCLUSIONS/INTERPRETATION: Our findings point to a contribution of AUF1 to the deleterious effects of cytokines on beta cell functions and suggest a role for this RNA-binding protein in the early phases of type 1 diabetes.

Capsule permeability via polymer and protein ingress/egress.

Static incubation tests, where microcapsules and beads are contacted with polymer and protein solutions, have been developed for the characterization of permselective materials applied for bioartificial organs and drug delivery. A combination of polymer ingress, detected by size-exclusion chromatography, and protein ingress/ egress, assessed by gel electrophoresis, provides information regarding the diffusion kinetics, molar mass cutoff(MMCO) and permeability. This represents an improvement over existing permeability measurements that are based on the diffusion of a single type of solute. Specifically, the permeability of capsules based on alginate, cellulose sulfate, polymethylene-co-guanidine were characterized as a function of membrane thickness. Solid alginate beads were also evaluated. The MMCO of these capsules was estimated to be between 80 and 90 kDa using polymers, and between 116-150 kDa with proteins. Apparently, the globular shape of the proteins (radius of gyration (Rg) of 4.2-4.6 nm) facilitates their passage through the membrane, comparatively to the polysaccharide coil conformation (Rg of 6.5-8.3 nm). An increase of the capsule membrane thickness reduced these values. The MMCO of the beads, which do not have a membrane limiting their permselective properties, was higher, between 110 and 200 kDa with dextrans, and between 150 and 220 kDa with proteins. Therefore, although the permeability estimated with biologically relevant molecules is generally higher due to their lower radius of gyration, both the MMCO of synthetic and natural watersoluble polymers correlate well, and can be used as in vitro metrics for the immune protection ability of microcapsules and microbeads. This article shows, to the authors' knowledge, the first reported concordance between permeability measures based on model natural and biological macromolecules.