610 Medizin und Gesundheit
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Forkhead box O (FoxO) transcription factors are conserved proteins involved in the regulation of life span and age-related diseases, such as diabetes and cancer. Stress stimuli or growth factor deprivation promotes nuclear localization and activation of FoxO proteins, which—depending on the cellular context—can lead to cell cycle arrest or apoptosis. In endothelial cells (ECs), they further regulate angiogenesis and may promote inflammation and vessel destabilization implicating a role of FoxOs in vascular diseases. In several cancers, FoxOs exert a tumor-suppressive function by regulating proliferation and survival. We and others have previously shown that FoxOs can regulate these processes via two different mechanisms: by direct binding to forkhead-responsive elements at the promoter of target genes or by a poorly understood alternative process that does not require direct DNA binding and regulates key targets in primary human ECs. Here, we performed an interaction study in ECs to identify new nuclear FoxO3 interaction partners that might contribute to FoxO-dependent gene regulation. Mass spectrometry analysis of FoxO3-interacting proteins revealed transformation/transcription domain–associated protein (TRRAP), a member of multiple histone acetyltransferase complexes, as a novel binding partner of FoxO family proteins. We demonstrate that TRRAP is required to support FoxO3 transactivation and FoxO3-dependent G1 arrest and apoptosis in ECs via transcriptional activation of the cyclin-dependent kinase inhibitor p27\(^{kip1}\) and the proapoptotic B-cell lymphoma 2 family member, BIM. Moreover, FoxO–TRRAP interaction could explain FoxO-induced alternative gene regulation via TRRAP-dependent recruitment to target promoters lacking forkhead-responsive element sequences.
NFATc1 plays a critical role in double-negative thymocyte survival and differentiation. However, the signals that regulate Nfatc1 expression are incompletely characterized. Here we show a developmental stage-specific differential expression pattern of Nfatc1 driven by the distal (P1) or proximal (P2) promoters in thymocytes. Whereas, preTCR-negative thymocytes exhibit only P2 promoter-derived Nfatc1β expression, preTCR-positive thymocytes express both Nfatc1β and P1 promoter-derived Nfatc1α transcripts. Inducing NFATc1α activity from P1 promoter in preTCR-negative thymocytes, in addition to the NFATc1β from P2 promoter impairs thymocyte development resulting in severe T-cell lymphopenia. In addition, we show that NFATc1 activity suppresses the B-lineage potential of immature thymocytes, and consolidates their differentiation to T cells. Further, in the pTCR-positive DN3 cells, a threshold level of NFATc1 activity is vital in facilitating T-cell differentiation and to prevent Notch3-induced T-acute lymphoblastic leukaemia. Altogether, our results show NFATc1 activity is crucial in determining the T-cell fate of thymocytes.
Azole resistance of the fungal pathogen Aspergillus fumigatus is an emerging problem. To identify novel mechanisms that could mediate azole resistance in A. fumigatus, we analyzed the transcriptome of a mitochondrial fission/fusion mutant that exhibits increased azole tolerance. Approximately 12% of the annotated genes are differentially regulated in this strain. This comprises upregulation of Cyp51A, the azole target structure, upregulation of ATP-binding cassette (ABC) superfamily and major facilitator superfamily (MFS) transporters and differential regulation of transcription factors. To study their impact on azole tolerance, conditional mutants were constructed of seven ABC transporters and 17 transcription factors. Under repressed conditions, growth rates and azole susceptibility of the mutants were similar to wild type. Under induced conditions, several transcription factor mutants showed growth phenotypes. In addition, four ABC transporter mutants and seven transcription factor mutants exhibited altered azole susceptibility. However, deletion of individual identified ABC transporters and transcription factors did not affect the increased azole tolerance of the fission/fusion mutant. Our results revealed the ability of multiple ABC transporters and transcription factors to modulate the azole susceptibility of A. fumigatus and support a model where mitochondrial dysfunctions trigger a drug resistance network that mediates azole tolerance of this mold.
Out of the myriad of potential DNA binding sites of the glucocorticoid receptor (GR) found in the human genome, only a cell-type specific minority is actually bound, indicating that the presence of a recognition sequence alone is insufficient to specify where GR binds. Cooperative interactions with other transcription factors (TFs) are known to contribute to binding specificity. Here, we reasoned that sequence signals preventing GR recruitment to certain loci provide an alternative means to confer specificity. Motif analyses uncovered candidate Negative Regulatory Sequences (NRSs) that interfere with genomic GR binding. Subsequent functional analyses demonstrated that NRSs indeed prevent GR binding to nearby response elements. We show that NRS activity is conserved across species, found in most tissues and that they also interfere with the genomic binding of other TFs. Interestingly, the effects of NRSs appear not to be a simple consequence of changes in chromatin accessibility. Instead, we find that NRSs interact with proteins found at sub-nuclear structures called paraspeckles and that these proteins might mediate the repressive effects of NRSs. Together, our studies suggest that the joint influence of positive and negative sequence signals partition the genome into regions where GR can bind and those where it cannot.
1,25-dihydroxyvitamin D3 (1,25D3) was reported to induce premature organismal aging in fibroblast growth factor-23 (Fgf23) and klotho deficient mice, which is of main interest as 1,25D3 supplementation of its precursor cholecalciferol is used in basic osteoporosis treatment. We wanted to know if 1,25D3 is able to modulate aging processes on a cellular level in human mesenchymal stem cells (hMSC). Effects of 100 nM 1,25D3 on hMSC were analyzed by cell proliferation and apoptosis assay, beta-galactosidase staining, VDR and surface marker immunocytochemistry, RT-PCR of 1,25D3-responsive, quiescence-and replicative senescence-associated genes. 1,25D3 treatment significantly inhibited hMSC proliferation and apoptosis after 72 h and delayed the development of replicative senescence in long-term cultures according to beta-galactosidase staining and P16 expression. Cell morphology changed from a fibroblast like appearance to broad and rounded shapes. Long term treatment did not induce lineage commitment in terms of osteogenic pathways but maintained their clonogenic capacity, their surface marker characteristics (expression of CD73, CD90, CD105) and their multipotency to develop towards the chondrogenic, adipogenic and osteogenic pathways. In conclusion, 1,25D3 delays replicative senescence in primary hMSC while the pro-aging effects seen in mouse models might mainly be due to elevated systemic phosphate levels, which propagate organismal aging.
Abstract
Sulphur is an essential element that all pathogens have to absorb from their surroundings in order to grow inside their infected host. Despite its importance, the relevance of sulphur assimilation in fungal virulence is largely unexplored. Here we report a role of the bZIP transcription factor MetR in sulphur assimilation and virulence of the human pathogen Aspergillus fumigatus. The MetR regulator is essential for growth on a variety of sulphur sources; remarkably, it is fundamental for assimilation of inorganic S-sources but dispensable for utilization of methionine. Accordingly, it strongly supports expression of genes directly related to inorganic sulphur assimilation but not of genes connected to methionine metabolism. On a broader scale, MetR orchestrates the comprehensive transcriptional adaptation to sulphur-starving conditions as demonstrated by digital gene expression analysis. Surprisingly, A. fumigatus is able to utilize volatile sulphur compounds produced by its methionine catabolism, a process that has not been described before and that is MetR-dependent. The A. fumigatus MetR transcriptional activator is important for virulence in both leukopenic mice and an alternative mini-host model of aspergillosis, as it was essential for the development of pulmonary aspergillosis and supported the systemic dissemination of the fungus. MetR action under sulphur-starving conditions is further required for proper iron regulation, which links regulation of sulphur metabolism to iron homeostasis and demonstrates an unprecedented regulatory crosstalk. Taken together, this study provides evidence that regulation of sulphur assimilation is not only crucial for A. fumigatus virulence but also affects the balance of iron in this prime opportunistic pathogen.
Author Summary
Invasive pulmonary aspergillosis (IPA) is a life-threatening disease that affects primarily immunosuppressed patients. During the last decades the incidence of this disease that is accompanied by high mortality rates has increased. Since opportunistic pathogenic fungi, unlike other pathogens, do not express specific virulence factors, it is becoming more and more clear that the elucidation of fungal metabolism is an essential task to understand fungal pathogenicity and to identify novel antifungal targets. In this work we report genetic inactivation of the sulphur transcription regulator MetR in Aspergillus fumigatus and subsequent study of the resulting phenotypes and transcriptional deregulation of the mutant. Here we show that regulation of sulphur assimilation is an essential process for the manifestation of IPA. Moreover, a regulatory connection between sulphur metabolism and iron homeostasis, a further essential virulence determinant of A. fumigatus, is demonstrated in this study for the first time. A deeper knowledge of sulphur metabolism holds the promise of increasing our understanding of fungal virulence and might lead to improved antifungal therapy.
INTRODUCTION: Recently, we could show that angiotensin II, the reactive peptide of the blood pressure-regulating renin-angiotensin-aldosterone-system, causes the formation of reactive oxygen species and DNA damage in kidneys and hearts of hypertensive mice. To further investigate on the one hand the mechanism of DNA damage caused by angiotensin II, and on the other hand possible intervention strategies against end-organ damage, the effects of substances interfering with the renin-angiotensin-aldosterone-system on angiotensin II-induced genomic damage were studied.
METHODS: In C57BL/6-mice, hypertension was induced by infusion of 600 ng/kg • min angiotensin II. The animals were additionally treated with the angiotensin II type 1 receptor blocker candesartan, the mineralocorticoid receptor blocker eplerenone and the antioxidant tempol. DNA damage and the activation of transcription factors were studied by immunohistochemistry and protein expression analysis.
RESULTS: Administration of angiotensin II led to a significant increase of blood pressure, decreased only by candesartan. In kidneys and hearts of angiotensin II-treated animals, significant oxidative stress could be detected (1.5-fold over control). The redox-sensitive transcription factors Nrf2 and NF-κB were activated in the kidney by angiotensin II-treatment (4- and 3-fold over control, respectively) and reduced by all interventions. In kidneys and hearts an increase of DNA damage (3- and 2-fold over control, respectively) and of DNA repair (3-fold over control) was found. These effects were ameliorated by all interventions in both organs. Consistently, candesartan and tempol were more effective than eplerenone.
CONCLUSION: Angiotensin II-induced DNA damage is caused by angiotensin II type 1 receptor-mediated formation of oxidative stress in vivo. The angiotensin II-mediated physiological increase of aldosterone adds to the DNA-damaging effects. Blocking angiotensin II and mineralocorticoid receptors therefore has beneficial effects on end-organ damage independent of blood pressure normalization.
Depending on the environmental conditions, the pathogenic yeast Candida albicans can undergo different developmental programs, which are controlled by dedicated transcription factors and upstream signaling pathways. C. albicans strains that are homozygous at the mating type locus can switch from the normal yeast form (white) to an elongated cell type (opaque), which is the mating-competent form of this fungus. Both white and opaque cells use the Ste11-Hst7-Cek1/Cek2 MAP kinase signaling pathway to react to the presence of mating pheromone. However, while opaque cells employ the transcription factor Cph1 to induce the mating response, white cells recruit a different downstream transcription factor, Tec1, to promote the formation of a biofilm that facilitates mating of opaque cells in the population. The switch from the white to the opaque cell form is itself induced by environmental signals that result in the upregulation of the transcription factor Wor1, the master regulator of white-opaque switching. To get insight into the upstream signaling pathways controlling the switch, we expressed all C. albicans protein kinases from a tetracycline-inducible promoter in a switching-competent strain. Screening of this library of strains showed that a hyperactive form of Ste11 lacking its N-terminal domain (Ste11ΔN467) efficiently stimulated white cells to switch to the opaque phase, a behavior that did not occur in response to pheromone. Ste11ΔN467-induced switching specifically required the downstream MAP kinase Cek1 and its target transcription factor Cph1, but not Cek2 and Tec1, and forced expression of Cph1 also promoted white-opaque switching in a Wor1-dependent manner. Therefore, depending on the activation mechanism, components of the pheromone-responsive MAP kinase pathway can be reconnected to stimulate an alternative developmental program, switching of white cells to the mating-competent opaque phase.
Die Rolle von NFAT-Transkriptionsfaktoren bei der Regulation der Apoptose peripherer T-Zellen
(2006)
In der vorliegenden Arbeit wurde die Rolle der NFAT-Transkriptionsfaktoren NFATc2 und NFATc3 beim AICD (Activation induced cell death) von peripheren T-Lymphozyten untersucht. Dazu wurde die Auslösbarkeit der Apoptose mittels Anti-CD3-Antikörper bei Wildtyp- bzw. Knock-out-Mäusen mit folgender NFAT-Ausstattung verglichen: NFAT c2+/+c3-/-, c2-/-c3+/+, c2-/-c3-/+, c2-/-c3-/-. Mittels FACS-Analyse von T-Helfer-Zellen aus den Lymphknoten dieser Mäuse zeigte sich, dass die CD3-vermittelte Apoptose - im Gegensatz zur Fas-vermittelten - mit dem Gesamtgehalt der Zellen an NFATc2 und NFAT c3 korreliert und diesbezüglich eine direkte Proportionalität angenommen werden kann.
Interleukin-5 ist ein Th2-Cytokin, das eosinophile Granulozyten aktiviert und B-Zellen zur Produktion von IgE stimuliert. Bei der Entstehung von allergischen (wie z.B. Asthma) und atopischen Reaktionen spielt die erhöhte Ausschüttung von IL-5 eine wichtige Rolle. Der Interleukin-5-Promoter weist unter anderem Bindestellen für NF-AT-Faktoren und GATA-3 auf. NF-ATc ist ein Mitglied der NF-AT („Nuclear Factor of Activated T-cells“)-Transkriptionsfaktoren, die an den verschiedensten immunologischen Funktionen beteiligt sind, vor allem aber an der Steuerung der Cytokingene. GATA-3 ist ein wichtiger Th2-spezifischer Zinkfinger-Transkriptionsfaktor aus der Familie der GATA-Faktoren, die an eine gemeinsame WGATAR-Sequenz der DNA binden. Molkentin et al. zeigten 1998, daß NF-AT3 und GATA-4 in Herzmuskelzellen physikalisch interagieren und daß ihre funktionelle Kooperation bei der Aktivierung verschiedener Promotoren letztendlich zur Entwicklung einer Herzhypertrophie beiträgt. In dieser Arbeit sollte untersucht werden, ob eine ähnliche physikalische Interaktion zwischen NF-ATc und GATA-3 in T-Zellen stattfindet. Zu diesem Zweck wurden im ersten Teil der Arbeit Coimmunopräzipitationen durchgeführt. Dabei konnte in mit NF-ATc und GATA-3 cotransfizierten 293T Zellen eine spezifische in vivo Interaktion der beiden Transkriptionsfaktoren nachgewiesen werden. Im zweiten Teil der Arbeit sollten mittels GST-„pulldown“-Experimenten die für die Interaktion wichtigen Proteindomänen von NF-ATc und GATA-3 bestimmt werden. Im ersten Schritt wurden die dafür benötigten Plasmide konstruiert. Im zweiten Schritt erfolgte die bakterielle Expression und nachfolgende Aufreinigung der GST-Fusionsproteine. Mit GST wurde jeweils eine N- und C-terminale Hälfte von NF-ATc und GATA-3 fusioniert. Mit diesen rekombinanten Proteinen wurden die „pulldown“-Experimente durchgeführt. Dabei konnte eine Interaktion des C-terminalen Anteils (enthält den zweiten Zinkfinger) von GATA-3 mit NF-ATc detektiert werden. Nachfolgende Ergebnisse deuteten auf eine Interaktion des C-terminalen Anteils (enthält die „Rel-Similarity-Domain“) von NF-ATc mit GATA-3 hin. Analog zeigten Molkentin et al., daß die RSD von NF-AT3 mit dem C-terminalen Zinkfinger von GATA-4 in Herzmuskelzellen interagiert. Die Interaktion von NF-ATc und GATA-3 scheint nicht nur physikalisch zu existieren, sondern auch funktionell von Bedeutung zu sein. In Luciferase-Reporteressays, die in unserem Labor durchgeführt wurden, zeigte sich bei Cotransfektion von NF-ATc und GATA-3 im Vergleich zu Einzeltranfektionen eine drastische Aktivitätssteigerung des IL-5 Promoters. Diese Ergebnisse weisen – wiederum analog zu den Vorgänge im Herzen - auf eine funktionelle Kooperation der beiden Transkriptionsfaktoren bei der Steuerung des IL-5 Promoters hin.