Die intrazelluläre Weiterleitung von Interferonsignalen von der Zytoplasmamembran zum Zellkern wird vermittelt über den Signaltransduktor und Aktivator der Transkription 1 (STAT1), welcher in seiner tetrameren Form als Transkriptionsfaktor an Immunantworten beteiligt ist. In diesem Projekt wurde der Protomerenaustausch zwischen STAT1-Dimeren unter kinetischen Gesichtspunkten untersucht und dabei dieser Prozess als ein potentiell geschwindigkeitsbestimmender Schritt des Aktivierungs-/Inaktivierungs-Zyklus von STAT1 identifiziert. Die Daten unterstützen einen alternativen Mechanismus für den Wechsel zwischen der parallelen und antiparallelen Konformation von STAT1-Dimeren basierend auf der Dissoziation und nachfolgenden Reassoziation von Protomeren, bei dem reziproke Interaktionen innerhalb des N-terminalen Domänendimers zur Stabilisierung eines intermediären Konformationsübergangs nicht benötigt werden. Durch Bindung an spezifische DNA-Zielbereiche, als Gamma-aktivierte Sequenzen (GAS) bezeichnet, wird die Dynamik des Protomerenaustauschs wesentlich beeinträchtigt. In der Sequenz des für das zytoskelettale Strukturprotein Ezrin kodierenden humanen EZR-Gens wurde mittels in silico Analyse ein doppeltes GAS-Motiv als mögliche STAT1-Zielsequenz identifiziert und die Bindung von STAT1-Dimeren an jedes der beiden Elemente sowie eine moderate Geninduktion bestätigt. Allerdings zeigen Mäuse mit einer N-terminalen Substitutionsmutation von STAT1, welche die kooperative DNA-Bindung beeinträchtigt, sowie auch ein kompletter funktioneller Knockout des Stat1-Gens keine veränderte Expression von Ezrin und Moesin in Knochenmarkszellen verglichen mit Mäusen, die das Wildtyp-Molekül exprimieren. In einem Myokardinfarktmodell durch Ligatur des Ramus interventricularis anterior zeigen männliche Mäuse mit Expression der Interferon-γ-irresponsiven STAT1-Mutante höhere Überlebensraten, während weibliche Tiere vor den nachteiligen Effekten des kardialen Remodellings in der frühen Phase geschützt sind. In entzündlichen myokardialen Infiltraten dieser Tiere wurde ein geringfügig höheres Expressionsniveau an tyrosinphosphoryliertem STAT1 nachgewiesen, während die Gesamtproteinmenge an STAT1 gegenüber dem Wildtyp reduziert war. Zellen aus lymphatischen Organen STAT1-defizienter Tiere mit experimenteller autoimmuner Enzephalomyelitis, die als Modell einer T-Helfer-Zell-vermittelten Autoimmunerkrankung verwendet wurde, zeigten einen hyperproliferativen Phänotyp und sezernierten größere Mengen an IFNγ und IL-17A. Injektion dieser Mäuse mit Lipopolysaccharid während der Induktionsphase der experimentellen autoimmunen Enzephalomyelitis hob den hyperproliferativen Phänotyp vollständig auf. Zusammenfassend demonstrieren die Ergebnisse aus dieser Arbeit die Bedeutung einer kooperativen DNA-Bindung und Tetramerstabilisierung von STAT1 im Zusammenspiel komplexer immunologischer Prozesse auch in Abwesenheit infektiöser Pathogene und unterstreichen zudem die Schlüsselrolle von tyrosinphosphoryliertem STAT1 bei der Verknüpfung zwischen angeborenem und erworbenem Immunsystem.

<p>All aspects of cell life are regulated by signal transduction mechanisms. This thesis describes the regulatory roles of a few key signal transduction molecules involved in three major biological responses. The studied pathways include platelet derived growth factor (PDGF)-BB induced transformation of murine fibroblasts, interferon (IFN)-γ stimulated monocyte activation and all-trans retinoic acid (ATRA) induced myeloid differentiation. </p><p>We found that intact phosphoinositide 3OH-kinase (PI3K) activity is essential in the signaling pathway that leads to the morphological alterations and migration pattern characteristic of PDGF-BB transformed NIH/sis and NIH/COL1A1 fibroblasts. Furthermore, our data indicated that the small Rho-GTPase, Rac1 is the predominant mediator of these signals downstream of PI3K.</p><p>The study of the IFN-γ induced activation of monocytic U-937 cells showed that upregulation of the high affinity receptor for IgG (FcγRI) is dependent on the coordination of several regulatory events: the PKR-mediated serine 727 phosphorylation of Stat1, the expression of the hematopoietic lineage specific transcription factor PU.I, and the activation of the NFκB pathway.</p><p>ATRA-induced differentiation and cell cycle arrest are impaired in U-937 sublines expressing phosphorylation deficient Stat1 (Stat1Y701F and Stat1S727A). The findings in paper III indicated that the expression pattern of the myeloid specific transcription factors Stat2, ICSBP and c/EBPε was altered in the sublines and that intact Stat1 activation is critical for maintaining the balance of the transcriptional network during ATRA induced terminal differentiation.</p><p>Finally, ATRA-induced differentiation and growth arrest were blocked by treatment with the IKKα/β inhibitor BMS345541 or by ectopic expression of the NFκB super repressor IκBα (S32A/S36A). The fact that IκB(AA) sublines differentiated normally in response to vitamin D3, showed that NFκB inhibition specifically affected ATRA induced responses. Notably we suggest that the activity of the NFκB pathway may interfere with the differentiation process via a direct effect on the RAR/RXR mediated transcription.</p>

All aspects of cell life are regulated by signal transduction mechanisms. This thesis describes the regulatory roles of a few key signal transduction molecules involved in three major biological responses. The studied pathways include platelet derived growth factor (PDGF)-BB induced transformation of murine fibroblasts, interferon (IFN)-γ stimulated monocyte activation and all-trans retinoic acid (ATRA) induced myeloid differentiation. We found that intact phosphoinositide 3OH-kinase (PI3K) activity is essential in the signaling pathway that leads to the morphological alterations and migration pattern characteristic of PDGF-BB transformed NIH/sis and NIH/COL1A1 fibroblasts. Furthermore, our data indicated that the small Rho-GTPase, Rac1 is the predominant mediator of these signals downstream of PI3K. The study of the IFN-γ induced activation of monocytic U-937 cells showed that upregulation of the high affinity receptor for IgG (FcγRI) is dependent on the coordination of several regulatory events: the PKR-mediated serine 727 phosphorylation of Stat1, the expression of the hematopoietic lineage specific transcription factor PU.I, and the activation of the NFκB pathway. ATRA-induced differentiation and cell cycle arrest are impaired in U-937 sublines expressing phosphorylation deficient Stat1 (Stat1Y701F and Stat1S727A). The findings in paper III indicated that the expression pattern of the myeloid specific transcription factors Stat2, ICSBP and c/EBPε was altered in the sublines and that intact Stat1 activation is critical for maintaining the balance of the transcriptional network during ATRA induced terminal differentiation. Finally, ATRA-induced differentiation and growth arrest were blocked by treatment with the IKKα/β inhibitor BMS345541 or by ectopic expression of the NFκB super repressor IκBα (S32A/S36A). The fact that IκB(AA) sublines differentiated normally in response to vitamin D3, showed that NFκB inhibition specifically affected ATRA induced responses. Notably we suggest that the activity of the NFκB pathway may interfere with the differentiation process via a direct effect on the RAR/RXR mediated transcription.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2008. / Title from first page of PDF file (viewed June 6, 2008). Includes bibliographical references.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2008. / Title from first page of PDF file (viewed June 6, 2008). Includes bibliographical references.

Indiana University-Purdue University Indianapolis (IUPUI) / Progression of Chlamydia into upper reproductive tract epithelium and the induction of subsequent immune responses to infection are major contributors to Chlamydia-induced pathogenesis of the genital tract. We reported that C. muridarum infection of the oviduct epithelial cells (OEs) secrete IFN-β in a TLR3 dependent manner. However, we showed that the C. muridarum infected TLR3-deficient OEs were still able to secrete minimal amounts of IFN-β into the supernatants, which is suggestive that there are other signaling pathways that contribute to Chlamydia-induced IFN-β synthesis in these cells. Previous studies describing the activation of the JAK/STAT signaling pathway during Chlamydia infection of cervical epithelial cells proposes a putative role for STAT1 in the synthesis of type I IFNs during Chlamydia infection. The present study investigated the role of STAT1 in Chlamydia-induced IFN-β production in OEs. OEs were infected with Chlamydia muridarum and analyzed at 24 hours by RT-PCR and western blot to determine STAT1 expression. STAT (-/-) OEs were infected and IFN-β production measured by ELISA. Quantitative real-time PCR analyses were performed at 6 and 16 hour post-infection to elucidate the mechanisms involved in IFN-β production during infection. Fluorescent microscopy was used to observe changes in Chlamydia replication. STAT1 activation and expression were significantly increased in wild-type (WT) OEs upon infection. TLR3 (-/-) OEs showed diminished STAT1 protein activation and expression. Augmented STAT1 protein expression corresponded to STAT1 mRNA levels. ELISA analyses revealed significantly less IFN-β production in infected STAT1 (-/-) OEs compared to WT OEs. Quantitative real-time PCR data showed that gene expression of IFN-β and of type I IFN signaling components were significantly increased during late stage Chlamydia infection, dependent on STAT1. Temporal regulation and increases in expression of IFN-α subtypes during infection were STAT1-dependent. Our results implicate STAT1-mediated signaling as a contributor to the C. muridarum-induced synthesis of IFN-β and other type I IFNs in OEs. We previously described a major role for TLR3 in the early-stage Chlamydia-induced synthesis of IFN-β in OEs; the results from this study suggest a role for STAT1 in the synthesis of type I IFNs that occurs during early and late stages of infection.

No description available.

No description available.

本實驗室於先前利用莫氏水迷津試驗篩選學習快與學習慢的大白鼠，取出其海馬迴組織並進行聚合酶連鎖反應差異顯示(PCR differential display)，結果顯示學習快與學習慢的大白鼠背側海馬迴之間共有98個cDNA片段有差異表現。把這些cDNA片段進行定序並利用BLAST資料庫比對，其中一個cDNA片段為大白鼠的pias1 [protein inhibitor of activated STAT1 (signal transducer and activator of transcription 1)] 基因。為了瞭解pias1基因的表現是否和空間學習有所關聯，隨機把大白鼠分成兩組，一組為有訓練組別(有空間線索與隱藏式平台)，另一組為無訓練的組別(沒有平台，作為游泳的控制組)同時進行莫氏水迷津學習試驗。試驗完畢，取出海馬迴組織進行即時定量聚合酶連鎖反應與西方墨點法來分析PIAS1的mRNA與蛋白質的表現。結果顯示有水迷津訓練的大白鼠，其PIAS1的mRNA與蛋白質表現皆明顯的高於無訓練的組別。為了更進一步確認PIAS1在空間學習中所扮演的角色，我們利用基因轉染的技術，轉染PIAS1 siRNA至大白鼠海馬迴CA1區域抑制PIAS1的表現。我們發現轉染PIAS1 siRNA至CA1區域會抑制大白鼠在水迷津的行為表現，然而轉染野生型的PIAS1質體基因至CA1區域卻會增進水迷津試驗的學習能力，同時我們也以西方墨點法發現，當轉染PIAS1 siRNA會增加STAT1 Tyr701的磷酸化，而轉染PIAS1 WT則會抑制STAT1 Tyr701的磷酸化。為了探討PIAS1促進記憶形成的分子機制，我們發現當轉染突變型的STAT1 Y701F質體基因至CA1區域，會抑制PIAS1 siRNA所造成記憶的損害。這些實驗結果代表著PIAS1會抑制STAT1 Tyr701的磷酸化，而PIAS1促進記憶的形成可能是藉由抑制STAT1 Tyr701的磷酸化而達成。另外，我們也單獨轉染突變型的STAT1 Y701F質體基因至CA1區域，水迷津實驗結果顯示會促進空間記憶的形成。目前PIAS1在免疫的角色已有許多研究證實，但是本篇研究是第一個提出PIAS1會參與哺乳類動物學習與記憶形成探討。 / Our laboratory has previously identified 98 cDNA fragments by using PCR differential display from rat dorsal hippocampus that are differentially expressed between fast learners and slow learners from the water maze learning task. After sequencing and BLAST analysis, one of these cDNA fragments encodes the rat pias1 [protein inhibitor of activated STAT1 (signal transducer and activator of transcription 1)] gene. In order to determine whether pias1 gene expression is associated with spatial learning, naïve rats were randomly assigned to the trained group (with visual cues and platform been present) and the non-trained group (without the platform as the swimming control). The dorsal hippocampus from these animals was dissected out at the end of the training and was subjected to RNA and protein extraction for real-time PCR and Western blot analysis of PIAS1 expression, respectively. Results revealed that the pias1 mRNA level and protein level was both higher in the hippocampus of trained rats than non-trained rats. To further examine the role of PIAS1 involved in spatial learning and memory, the specific PIAS1 siRNA was used to knockdown the expression of PIAS1 in rat hippocampal CA1 region. We found that transfection of PIAS1 siRNA to the CA1 area impaired water maze performance, whereas transfection of the wild-type PIAS1 DNA plasmid to the CA1 area facilitated water maze performance in rats. Meanwhile, PIAS1 siRNA increased STAT1 phosphorylation at Tyr701 whereas PIAS1 WT decreased STAT1 phosphorylation at this residue. In the examination of molecular mechanism underlying PIAS1-mediated memory facilitation, we have found that transfection of the STAT1 Y701F mutant plasmid antagonized the memory-impairing effect of PIAS1 siRNA, whereas transfection of STAT1 Y701F alone facilitated spatial memory formation. These results together suggest that one of the molecular mechanisms underlying PIAS1-mediated memory facilitation is through decreased STAT1 phosphorylation at Tyr701. All these manipulations did not affect visible platform learning in rats. In addition to the well documented role of PIAS1 in the immune system, here we have been the first to demonstrate a novel role of PIAS1 involved in spatial memory formation in rats.

In type 1 diabetes (T1D) a combination of genetic predisposition and environmental factors triggers islet inflammation (insulitis) leading to a selective and gradual destruction of the pancreatic beta cells. Beta cells mainly die through apoptosis, triggered at least in part by pro-inflammatory cytokines such as IL-1β, TNF-α and IFN-γ. Recent findings suggest that the mitochondrial pathway of cell death is involved in this death cascade. Array analysis indicated that TNF-α+IFN-γ induces transcription factors such as NF-ĸB, STAT1, and AP-1 in beta cells. We presently aimed to examine the pathway(s) of apoptosis triggered by TNF-α+IFN-γ in beta cells. <p>TNF-α+IFN-γ induces beta cell apoptosis through the intrinsic pathway of cell death. This involved activation of the BH3 only proteins DP5, PUMA and Bim. Knockdown (KD) of either DP5 or PUMA or both led to a partial protection of INS-1E cells (12-20%), while silencing Bim led to about 60% protection against cytokine-induced apoptosis. Bim is transcriptionally induced by activated STAT1. TNF-α+IFN-γ also induces downregulation of Bcl-XL, an anti-apoptotic Bcl-2 gene which inhibits Bim. Knocking down Bcl-XL alone led to increase in apoptosis, but this was prevented by the parallel KD of Bim.<p>The ultimate goal of our research is to protect beta cells from the autoimmune assault. Previous data revealed that JunB inhibits ER stress and apoptosis in beta cells treated with IL-β+IFN-γ. Here, TNF-α+IFN-γ up-regulated the expression of JunB which was downstream of activated NF-ĸB. JunB KD exacerbated TNF-α+IFN-γ induced beta cell death in primary rat beta cells and INS-1E cells. The gene networks affected by JunB were studied by microarray analysis. JunB regulates 20-25% of the cytokine-modified beta cell genes, including the transcription factor ATF3 and Bcl-XL. ATF3 expression was increased in cytokine-treated human islets and in vitro silencing of JunB led to >60% reduction in ATF3 overexpression. We confirmed direct JunB regulation of the ATF3 promoter by its binding to an ATF/CRE site. Silencing of ATF3 aggravated TNF-α+IFN-γ induced cell death in beta cells and led to the downregulation of Bcl-XL expression in INS-1E cells. Pharmacological upregulation of JunB using forskolin led to upregulation of ATF3 and consistent protection of these cells against cytokine-induced cell death, while genetic overexpression of JunB in mice increased ATF3 expression in the pancreatic islets and reversed the pro-apoptotic effects of cytokines on beta cells (±40 % protection). <p>As a whole, our findings indicate that TNF-α+IFN-γ triggers beta cell apoptosis by the upregulation of the pro-apoptotic protein Bim and downregulation of the Bcl-XL protein. These deleterious effects are at least in part antagonized by JunB via activation of ATF3. <p><p>Dans le diabète de type 1 (DT1), la combinaison de facteurs génétiques de prédisposition et de l'environnement déclenche l'inflammation des îlots de Langerhans (insulite) conduisant à une destruction sélective et progressive des cellules bêta du pancréas. Les cellules bêta meurent principalement d’apoptose, déclenchée au moins en partie par les cytokines pro-inflammatoires sécrétées par les cellules immunitaires comme l’IL-β, le TNF-α l’IFN-γ. De récentes découvertes suggèrent que la voie mitochondriale de la mort cellulaire jouerait un rôle dans la mort de ces cellules. L'analyse de réseaux de gène utilisant les biopuces d’ADN indique que l’association TNF-α+IFN-γ induit l’activation de facteurs de transcription tels que NF-ĸB, STAT1 et AP-1 dans la cellule bêta. Dans ce contexte, nous avons cherché à examiner les voies de l'apoptose déclenchées par le TNF-α+IFN-γ dans la cellule bêta. <p>En présence de TNF-α+IFN-γ les cellules bêta meurent par apoptose via la voie intrinsèque. L’activation des protéines pro-apoptotiques « BH3-seulement » dont DP5, PUMA et Bim étaient en cause de cette apoptose. Le « knockdown »1 (KD), de DP5 ou de PUMA, ou des deux en même temps conduit à une protection partielle des cellules INS-1E (12-20%), tandis que le KD de Bim conduit à environ 60% de protection contre l’apoptose induite par cette combinaison de cytokines. La transcription de Bim est induite par STAT1 activé. Parallèlement à la régulation positive de Bim, TNF-α+IFN-γ conduit à la régulation négative de la protéine Bcl-XL. Bcl-XL est une protèine anti-apoptotique de la famille de protèines Bcl-2 qui en general inhibe Bim. Réduire l’expression de Bcl-XL seul induit une augmention de l'apoptose, alors que le KD de Bim et Bcl-XL en parallèle empêche l'apoptose.<p>Le but ultime de notre recherche est de protéger les cellules bêta des agressions autoimmunitaires. Les données antérieures ont révélé que JunB inhibe le stress du réticulum endoplasmique et l'apoptose dans les cellules bêta traitées avec IL-β+IFN-γ. Nous avons observé que TNF-α+IFN-γ induit l'expression de JunB qui se produit en aval de NF-ĸB activé. Il est important de noter que l’inactivation de JunB par des agents interférants de l’ARN (siRNA) exacerbe la mort des cellules primaires bêta de rat et de cellules INS-1E induite par les cytokines. Les réseaux de gènes touchés par JunB ont été étudiés grâce a l'analyse en microréseaux. JunB règule 20-25% des gènes modifiés par des cytokines dans les cellules bêta, y compris le facteur de transcription ATF3 et Bcl-XL. L’expression d’ATF3 est augmenté dans les îlots humains traités avec les cytokines et la répression in vitro de JunB conduit à une réduction de >60% de l’expression d’ATF3. Nous avons confirmé la régulation d’ATF3 par JunB en montrant que JunB est directement lié au promoteur d’ATF3 via le site ATF/CRE. La diminution d’expression d’ATF3 en presence de TNF-α+IFN-γ a aggravé la mort cellulaire induite dans les cellules bêta et a conduit à la régulation négative de l'expression de Bcl-XL dans les cellules INS-1E. L’augmentation pharmacologique de JunB dans les cellules INS-1E par l’utilisation de forskolin a conduit à la régulation positive en aval d’ATF3 et par conséquente à la protection de cellules bêta vis-a-vis de effets indésirables des cytokines. Dans cette optique, la surexpression génétique de JunB dans le modèle Ubi-JunB de souris transgénique a conduit à une surexpression d’ATF3 dans les îlots pancréatiques et a permir d’inverser les effets pro-apoptotiques de cytokines sur la cellule bêta (protection ± 40%).<p>Globalement, ces résultats indiquent que TNF-α+IFN-γ déclenche l'apoptose des cellules bêta par la régulation positive du gène pro-apoptotique Bim et la régulation négative du gène anti-apoptotique Bcl-XL. Ces effets indésirables sont inhibé en partie par JunB via l’activation de ATF3.<p><p>1Pas d’équivalent en français. Signifie la réduction de l’expression d’un gène via utilisation d’un siRNA (agent interférant de l’ARN).<p> / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished