Dynamics of the STAT3 Transcription Factor: Nuclear Import Dependent on Ran and Importin-β1

The signal transducer and activator of transcription-3 (STAT3) induces transcription of genes that control differentiation, inflammation, proliferation, and tumor cell invasion. Cytokines such as interleukin-6 and interferon stimulate the specific tyrosine phosphorylation of STAT3, which confers its ability to bind consensus DNA targets. In addition, unphosphorylated STAT3 has been demonstrated to induce specific gene expression. STAT3 must gain entrance to the nucleus to impact transcription, however access to the nucleus is a tightly regulated process. Because nuclear trafficking is critical to the function of STAT3, we investigated the molecular mechanisms by which STAT3 is imported to the nucleus. Live cell imaging techniques were used with STAT3 tagged with green fluorescence protein (GFP) or photoactivatable GFP to follow the cellular dynamics of both unphosphorylated and tyrosine phosphorylated forms. Cytokine activation did not alter the rate of STAT3 nuclear import or nuclear export. In addition, Förster resonance energy transfer experiments revealed homomeric interaction of unphosphorylated STAT3 dependent on its amino terminus, but this dimerization is not necessary for its nuclear import. Previous work demonstrated the adapter importin-α3 binds to STAT3 and is required for nuclear import. To determine whether STAT3 nuclear import is mediated by the importin-α/importin-β1 heterodimer, the effects of siRNA to importin-β1 were evaluated. Results indicate STAT3 nuclear import is dependent on the function of importin-β1. Since the Ran GTPase is necessary to bind importin-β1 in the nucleus for release of importin-α-cargo, the effect of a GTPase deficient mutant of Ran was tested. Expression of the Ran interfering mutant inhibited STAT3 nuclear import. This study defines importin-α/importin-β1/Ran as the molecular mechanism by which STAT3 traffics to the nucleus

Serielle Analyse der Genexpression während der Rattenleberregeneration durch Ovalstammzellen

Die Leber besitzt die einzigartige Fähigkeit der Selbstregeneration nach Schädigung. Adulte Hepatozyten sind in der Lage, in der normalen Leber, ein perfektes Gleichgewicht zwischen Zellzuwachs und Zelluntergang aufrecht zu erhalten. Bei Schädigung des Lebergewebes können sie durch ihre Fähigkeit zur Proliferation eine Regeneration der Leber herbeiführenUnter bestimmten Umständen wie z.B. Hepatokarzinogenese oder einer durch Drogen, Viren oder Toxine induzierten chronischen Leberschädigung, wenn die proliferative Fähigkeit der Hepatozyten beeinträchtigt ist, kommt es zur Rekrutierung fakultativer hepatischer Stammzellen den so genannten Ovalzellen die in der Lage sind Hepatozyten und Gallengangszellen zu generieren. Es kann daher angenommen werden, dass die Ovalzellen ein zweites Kompartiment sind, dass an der Regeneration der Leber beteiligt ist, wenn diese massiv geschädigt und die Proliferation der Hepatozyten suprimiert ist.Um die Rattenleberregeneration durch Ovalzellen zu untersuchen wurde das Protokoll der 70% igen partiellen Hepatektomie (PH) und einer zusätzlichen Behandlung mit 2-Acetoamidofluoren (2-AAF) verwendet. Die Entwicklung, Proliferation und Differenzierung der Ovalzellen in vivo wurde durch histologische Techniken charakterisiert. Zwischen Tag 1 und 3 nach PH konnte die Induktion von Ovalzellen innerhalb des Portalfeldes beobachtet werden, dabei exprimierten sie den typischen onkofetalen Marker -Fetoprotein (AFP) sowohl auf mRNA wie auch auf Proteinebene. Sie waren in der Lage zu proliferieren und sich zu Albumin-exprimierenden Hepatozyten und zu Zytokeratin 7 (CK 7) Gallengangszellen zu differenzieren.Das Genexpressionsmuster während der frühen Stadien der Ovallzellproliferation und differenzierung wurde mittels einer seriellen Analyse der Genexpression (SAGE) untersucht. Insgesamt wurden 153, 057 tags analysiert: 52,343 tags aus normalen Lebern, 50,502 tags aus den mit 2-AAF behandelten Kontrollen und 50,212 tags aus einem frühen Stadium der Ovallzellproliferation. Durch die vergleichende Analyse der Transkriptome konnten 45 Gene identifiziert werden, die während der Ovallzellregeneration differenziell exprimiert werden. Dabei waren 27 hochreguliert und 18 herunterreguliert. Die temporale Regulation dieser Gene wurde durch Real Time PCR untersucht. Unter den hochregulierten Genen fanden sich Zellzyklusgene wie CDC42 und Cyclin D1; Zytoskelett-assotiierte Proteine wie Stathmin 1 und E-Tropomodulin; Signaltransduktion triggernde Gene wie CDC151 und Lipopolysaccharid bindendes Protein; Transportergene wie Na+/Pi-Kotransporter 4, Phosphatidylcholin Transferprotein und ATPase H+ 34 kDa Lysosomaltransporter; antiapoptotische Gene wie Thioredoxinlike 2. Herunterreguliert sind z. B. Gene des Lipidmetabolismus wie Hydroxysäurereduktase 3 und Steroyl-CoA-Desaturase.Die Regulation der Proteinexpression von CDC42 und Cyclin D1 wurde mittels Western-Blot untersucht. Wir konnten zeigen, dass CDC42 und Cyclin D1 in zeitlicher Übereinstimmung mit AFP koexprimiert werden. Unsere Befunde zeigen, dass CDC42 und Cyclin D1 am Mechanismus der Ovallzellproliferation während der Leberregeneration beteiligt sein könnten

Dengue-2 Virus-Like Particle (VLP) Based Vaccine Elicits the Highest Titers of Neutralizing Antibodies When Produced at Reduced Temperature

A dengue vaccine capable of rapidly eliciting a robust and balanced immunity against the four virus serotypes after only a few immunizations is greatly needed. We describe a new strategy to develop dengue vaccines based on the assembly of virus-like particles (VLPs) utilizing the structural proteins CprME together with a modified complex of the NS2B/NS3 protease, which enhances particle formation and yield. These VLPs are produced in mammalian cells and resemble native dengue virus as demonstrated by negative staining and immunogold labelling electron microscopy (EM). We found that VLPs produced at lower temperature (31 degrees C) were recognized by conformational monoclonal antibodies (MAbs) 4G2, 3H5 and C10 whereas VLPs produced at higher temperature (37 degrees C) were not recognized by these MAbs. To investigate the significance of these conformational discrepancies in vaccine performance, we tested the immunogenicity of VLP vaccines produced at 31 degrees C or 37 degrees C. Mice immunized with the VLP vaccine produced at 31 degrees C (VLP-31 degrees C) elicited the highest titer of neutralizing antibodies when compared to those elicited by equivalent doses of the vaccine produced at 37 degrees C (VLP-37 degrees C), inactivated dengue virus vaccine or to the titer of a human anti-dengue-2 convalescence serum reference. Our results demonstrate that the conformation of the E protein displayed on the VLP vaccine plays a critical role in the induction of highly neutralizing antibodies. These findings will guide development of a tetravalent vaccine capable of eliciting a robust and balanced neutralizing response against the four-dengue serotypes regardless of background immunity

The schistosome enzyme that activates oxamniquine has the characteristics of a sulfotransferase

Available evidence suggests that the antischistosomal drug oxamniquine is converted to a reactive ester by a schistosome enzyme that is missing in drug-resistant parasites. This study presents data supporting the idea that the active ester is a sulfate and the activating enzyme is a sulfotransferase. Evidence comes from the fact that the parasite extract loses its activating capability upon dialysis, implying the requirement of some dialyzable cofactor. The addition of the sulfate donor 3'-phosphoadenosine 5'-phosphosulfate (PAPS) restored activity of the dialyzate, a strong indication that a sulfotransferase is probably involved. Classical sulfotransferase substrates like b-estradiol and quercetin competitively inhibited the activation of oxamniquine. Furthermore, these substrates could be sulfonated in vitro using an extract of sensitive (but not resistant) schistosomes. Gel filtration analysis showed that the activating factor eluted in a fraction corresponding to a molecular mass of about 32 kDa, which is the average size of typical sulfotransferase subunits. Ion exchange and affinity chromatography confirmed the sulfotransferase nature of the enzyme. Putative sulfotransferases present in schistosome databases are being examined for their possible role as oxamniquine activators

IL-6 promotes MYC-induced B cell lymphomagenesis independent of STAT3.

The inflammatory cytokine IL-6 is known to play a causal role in the promotion of cancer, although the underlying mechanisms remain to be completely understood. Interplay between endogenous and environmental cues determines the fate of cancer development. The Eμ-myc transgenic mouse expresses elevated levels of c-Myc in the B cell lineage and develops B cell lymphomas with associated mutations in p53 or other genes linked to apoptosis. We generated Eμ-myc mice that either lacked the IL-6 gene, or lacked the STAT3 gene specifically in B cells to determine the role of the IL-6/JAK/STAT3 pathway in tumor development. Using the Eμ-myc lymphoma mouse model, we demonstrate that IL-6 is a critical tumor promoter during early stages of B cell lymphomagenesis. IL-6 is shown to inhibit the expression of tumor suppressors, notably BIM and PTEN, and this may contribute to advancing MYC-driven B cell tumorigenesis. Several miRNAs known to target BIM and PTEN are upregulated by IL-6 and likely lead to the stable suppression of pro-apoptotic pathways early during the tumorigenic process. STAT3, a classical downstream effector of IL-6, appears dispensable for Eμ-myc driven lymphomagenesis. We conclude that the growth-promoting and anti-apoptotic mechanisms activated by IL-6 are critically involved in Eμ-myc driven tumor initiation and progression, but the B cell intrinsic expression of STAT3 is not required