No description available.

Nous avons découvert que TRF2 est aussi sur-exprimée au niveau des cellules endothéliales de nombreux types de cancers humains alors qu’elle n’est pas détectable dans les vaisseaux des tissus sains adjacents. Des cellules endothéliales extraites de tumeurs ex-vivo manifestent une expression supérieure de TRF2, une migration et une prolifération accrues et une aptitude à former des tubules élevée, comparées aux endotheliums isolées de tissus sains. La sur-expression de cette protéine in vitro dans des cellules endothéliales primaires et ex-vivo entraine l’augmentation de la prolifération, de la migration et de la capacité de ces dernières à former des tubules. La diminution de l’expression de TRF2 conduit à l’effet inverse. Par ailleurs, la modulation de l’expression de TRF2 n’affecte pas la proportion de cellules apoptotiques. De même, les variations des niveaux d’expression de TRF2 n’induisent aucune réponse aux dommages à l’ADN et les modifications des facultés angiogéniques sont indépendantes d’ATM. Les effets angiogéniques de TRF2 semblent donc distincts des fonctions télomériques. Etant donné que le facteur de transcription WT1 (Wilms’ tumour suppressor 1) est fortement exprimé dans les vaisseaux de tumeurs humaines et régule les propriétés angiogéniques des cellules endothéliales, nous nous sommes penché sur la régulation potentielle de TRF2 par WT1. WT1 se lie en effet sur le promoteur de TRF2 pour activer sa transcription. Enfin, nous avons démontré que l’activité angiogénique de TRF2 réside en partie dans sa capacité à se fixer sur le promoteur du gène codant pour le récepteur angiogénique à activité tyrosine kinase PDGFRβ et à activer sa transcription. / We discovered that TRF2 is expressed in endothelial cells of many human cancer types but not in the vessels of healthy adjacent tissues. Endothelial cells derived from tumours ex vivo exhibited a significantly increased TRF2 expression, and a higher migration, proliferation and tube formation potential as endothelium obtained from healthy tissues. In vitro TRF2 over-expression in primary or ex vivo endothelial cells resulted in an increased proliferation, migration and tube formation, while silencing of TRF2 led to the opposite results. No changes in apoptosis could be observed. Interestingly, modulation of TRF2 in endothelium does not induce DNA damage responses and the observed changes in the angiogenic behaviour are ATM –independent. The angiogenic effects of TRF2 seem therefore to be uncoupled from its telomeric function. Since the transcription factor WT1 (Wilms’ tumour suppressor 1) is highly expressed in human tumour vessels and mediates angiogenic properties of endothelial cells, we investigated whether TRF2 expression could be regulated by WT1. Indeed, WT1 binds the TRF2 promoter and activates its transcription. Finally, we demonstrated that TRF2 promotes angiogenesis by binding to the promoter of the gene encoding for the angiogenic tyrosine kinase receptor PDGFRβ and activating its transcription.

Die Phospholipid-Hydroperoxid Glutathion-Peroxidase (phGPx) ist ein monomeres Selenoprotein, welches innerhalb der Familie der Glutathion-Peroxidasen aufgrund seiner breiten Substratspezifität und der Fähigkeit Proteinthiole zu modifizieren eine Sonderstellung einnimmt. Vom Gen der phGPx werden nach heutigem Kenntnisstand drei verschiedene Protein-Isoformen gebildet. Die mitochondriale Isoform enthält am N-Terminus ein mitochondriales Insertionssignal und wird bevorzugt im Testis exprimiert. Von einem im Leserahmen stromabwärts liegenden Startkodon wird die kürzere, ubiquitär exprimierte zytosolische Isoform synthetisiert. Eine dritte phGPx-Isoform besitzt eine N-terminale nukleäre Lokalisationssequenz (kodiert von einem alternativen Exon 1) und wird vornehmlich in den Kernen post-meiotischer Zellen der Spermatogenese gefunden. Aufgabe dieser Arbeit war es, die molekularen Mechanismen zu untersuchen, die am Zustandekommen des vielfältigen Expressionsmusters der phGPx-Isoformen beteiligt sind. Im ersten Teil der Arbeit wurden transkriptionelle Regulationsmechanismen der phGPx-Expression untersucht. Im proximalen Promotorbereich (-100 bp – +228 bp) des phGPx-Gens wurden unter in vitro (Supershift-Assay) und in vivo (Chromatin-Immunopräzipitation) Bedingungen die Transkriptionsfaktoren Sp1 und NF-Y identifiziert, die an drei GC-reiche Motive beziehungsweise zwei inverse CCAAT-Boxen binden. Darüber hinaus konnten in kompetetiven Gelshift-Assays im proximalen Promotorbereich zwei Bindungssequenzen identifiziert werden, die von Faktoren der Smad-Familie gebunden werden. Funktionelle in vitro Promotorstudien mit mutierten Promotorkonstrukten zeigten, dass die Mutagenesen der Sp1- und NF-Y Bindestellen einen starken Einfluss auf die Reportergenaktivität hatten. Im zweiten Teil der Arbeit wurden durch Untersuchungen von Protein-RNA-Interaktionen post-transkriptionelle Mechanismen der Expressionsregulation studiert. Mit Hilfe des in vivo Ansatzes des Hefe Drei-Hybrid Systems wurde der Guanin-reiche Sequenz bindende Faktor 1 (GRSF1) identifiziert, der in der 5’-untranslatierte Region der mitochondrialen phGPx-mRNA bindet. In RNA Gelshift-Assays wurde die Spezifität dieser Interaktion bestätigt und näher charakterisiert. Schließlich wurden für GRSF1 und die phGPx Expressionsprofile in murinen Gewebe erstellt sowie die zeitabhängige Expression beider Proteine während der Embryogenese verfolgt. Die auffällig ähnlichen Expressionsmuster lassen ähnliche Regulationsmechanismen vermuten. Die in dieser Arbeit identifizierten trans-regulatorischen Proteine Sp1, NF-Y, Smad und GRSF1 sollten an der differentiellen Expression der phGPx-Isoformen beteiligt sein. / The Phospholipid Hydroperoxide Glutathione Peroxidase (phGPx) is a monomeric selenoprotein that is unique in the family of Glutathione Peroxidases due to its low substrate specificity and its ability to oxidise protein thiols. Three different isoforms are known to derive from one common gene. The mitochondrial Isoform contains an N-terminal mitochondrial insertion sequence and is preferentially expressed in postpubertal testis. The shorter, ubiquitously expressed, cytosolic isoform is expressed from an in-frame start codon. A third isoform contains an N-terminal nuclear localization signal coded for by an alternative exon 1 and is preferentially expressed in the nuclei of post-meiotic spermatides. The aim of the present study is to investigate the molecular mechanisms leading to the different isoforms and causing their tissue specific expression pattern. In the first part of this work transcriptional regulatory mechanisms will be analysed. Within the proximal promoter region (-100 to +228 bp) of the phGPx gene the transcription factors SP1 and NF-Y were identified to bind to three GC-boxes and two CCAAT-boxes respectively using in vitro methods (Supershift Assays) and in vivo methods (Chromatine immunoprecipitation). Moreover, performing competitive gel shift assays two binding elements for the smad family of transcription factors could be identified. Functional in vitro reporter gene assays provided evidence that the mutagenesis of the binding sequences for NF-Y and Sp1 has a strong impact on promoter activity. In the second part of this work post-transcriptional events in the expression regulation of the phGPx were analysed on the basis of protein/RNA interactions. Applying the in vivo approach of the yeast three hybrid system the Guanin-riche sequence binding factor 1 (GRSF1) could be identified binding to the 5’-untranslated region of the mitochondrial phGPx messenger. RNA mobility shift assays were performed to further characterize the specificity of this protein/RNA interaction. Eventually, the tissue distribution of GRSF1 and phGPx was studied in murine tissues and their expression kinetics were followed during murine embryogenesis. The obvious parallel expression kinetics for mitochondrial phGPx and GRSF1 suggest common regulatory mechanisms for these two genes. All the identified trans-regulatory elements are very likely to be involved in the differential expression regulation of the phGPx isoforms.

No description available.

No / Resveratrol is a polyphenolic phytoestrogen that has been shown to exhibit potent anti-oxidant, anti-inflammatory, and anti-catabolic properties. Increased osteoclastic and decreased osteoblastic activities result in bone resorption and loss of bone mass. These changes have been implicated in pathological processes in rheumatoid arthritis and osteoporosis. Receptor activator of NF-kappaB ligand (RANKL), a member of the TNF superfamily, is a major mediator of bone loss. In this study, we investigated the effects of resveratrol on RANKL during bone morphogenesis in high density bone cultures in vitro. Untreated bone-derived cell cultures produced well organized bone-like structures with a bone-specific matrix. Treatment with RANKL induced formation of tartrate-resistant acid phosphatase-positive multinucleated cells that exhibited morphological features of osteoclasts. RANKL induced NF-kappaB activation, whereas pretreatment with resveratrol completely inhibited this activation and suppressed the activation of IkappaBalpha kinase and IkappaBalpha phosphorylation and degradation. RANKL up-regulated p300 (a histone acetyltransferase) expression, which, in turn, promoted acetylation of NF-kappaB. Resveratrol inhibited RANKL-induced acetylation and nuclear translocation of NF-kappaB in a time- and concentration-dependent manner. In addition, activation of Sirt-1 (a histone deacetylase) by resveratrol induced Sirt-1-p300 association in bone-derived and preosteoblastic cells, leading to deacetylation of RANKL-induced NF-kappaB, inhibition of NF-kappaB transcriptional activation, and osteoclastogenesis. Co-treatment with resveratrol activated the bone transcription factors Cbfa-1 and Sirt-1 and induced the formation of Sirt-1-Cbfa-1 complexes. Overall, these results demonstrate that resveratrol-activated Sirt-1 plays pivotal roles in regulating the balance between the osteoclastic versus osteoblastic activity result in bone formation in vitro thereby highlighting its therapeutic potential for treating osteoporosis and rheumatoid arthritis-related bone loss.