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Ulbricht T.,Leibniz Institute for Age Research | Alzrigat M.,Leibniz Institute for Age Research | Horch A.,Carl Zeiss GmbH | Reuter N.,Friedrich - Alexander - University, Erlangen - Nuremberg | And 7 more authors.
Journal of Cell Biology | Year: 2012

Promyelocytic leukemia (PML) nuclear bodies selectively associate with transcriptionally active genomic regions, including the gene-rich major histocompatibility (MHC) locus. In this paper, we have explored potential links between PML and interferon (IFN)-γ-induced MHC class II expression. IFN-γ induced a substantial increase in the spatial proximity between PML bodies and the MHC class II gene cluster in different human cell types. Knockdown experiments show that PML is required for efficient IFN-γ-induced MHC II gene transcription through regulation of the class II transactivator (CIITA). PML mediates this function through protection of CIITA from proteasomal degradation. We also show that PML isoform II specifically forms a stable complex with CIITA at PML bodies. These observations establish PML as a coregulator of IFN-γ-induced MHC class II expression. © 2012 Ulbricht et al.

Klaus V.,TU Berlin | Hartmann T.,Heinrich Heine University Dusseldorf | Gambini J.,University of Valencia | Graf P.,Heinrich Heine University Dusseldorf | And 4 more authors.
Archives of Biochemistry and Biophysics | Year: 2010

Selected biological effects of 1,4-naphthoquinone, menadione (2-methyl-1,4-naphthoquinone) and structurally related quinones from natural sources - the 5-hydroxy-naphthoquinones juglone, plumbagin and the 2-hydroxy-naphthoquinones lawsone and lapachol - were studied in human keratinocytes (HaCaT). 1,4-naphthoquinone and menadione as well as juglone and plumbagin were highly cytotoxic, strongly induced reactive oxygen species (ROS) formation and depleted cellular glutathione. Moreover, they induced oxidative DNA base damage and accumulation of DNA strand breaks, as demonstrated in an alkaline DNA unwinding assay. Neither lawsone nor lapachol (up to 100μM) were active in any of these assays. Cytotoxic and oxidative action was paralleled by stimulation of stress signaling: all tested quinones except lawsone and lapachol strongly induced phosphorylation of the epidermal growth factor receptor (EGFR) and the related ErbB2 receptor tyrosine kinase. EGFR activation by plumbagin, juglone and menadione was attenuated by a superoxide dismutase mimetic, indicating that ROS-related mechanisms contribute to EGFR activation by these naphthoquinones. © 2010.

Fernau N.S.,Leibniz Institute For Umweltmedizinische Forschung | Fugmann D.,Leibniz Institute For Umweltmedizinische Forschung | Leyendecker M.,Leibniz Institute For Umweltmedizinische Forschung | Reimann K.,Leibniz Institute For Umweltmedizinische Forschung | And 6 more authors.
Journal of Biological Chemistry | Year: 2010

COX-2 (cyclooxygenase-2) is a pivotal player in inflammatory processes, and ultraviolet radiation is a known stimulus for COX-2 expression in skin cells. Here, an induction of COX-2 expression in HaCaT human keratinocytes was observed only upon exposure of cells to UVB (280-320 nm) but not to UVA radiation (320-400 nm), as demonstrated by reverse transcription-PCR and Western blotting. Prostaglandin E2 levels were elevated in cell culture supernatants of HaCaT cells exposed to UVB. COX-2 mRNA stability was dramatically increased by UV Birradiation. Both the stabilization of COX-2 mRNA and the enhancement of COX-2 steady-state mRNA and protein levels caused by UVB were prevented both by inhibition and small interfering RNA-induced depletion of p38MAPK, a kinase strongly activated upon exposure to UVB, suggesting p38 MAPK-dependent mRNA stabilization as a mechanism of UVB-induced COX-2 expression. A dramatic decrease in COX-2 expression induced by UVB was elicited by small interfering RNA-based depletion of a stress-responsive mRNA stabilizing protein regulated by p38MAPK, i.e. HuR; UVB-induced elevation of COX-2 mRNA and protein levels coincided with an accumulation of HuR in the cytoplasm and was attenuated in cells depleted of HuR. Moreover, UVB-induced generation of prostaglandin E2 by HaCaT cells was blunted by HuR depletion, suggesting that stress kinases (such as p38MAPK) as well as HuR are excellent targets for approaches aiming at interfering with induction of COX-2 expression by UVB. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.

Eckers A.,Leibniz Institute For Umweltmedizinische Forschung | Sauerbier E.,Leibniz Institute For Umweltmedizinische Forschung | Sauerbier E.,University of Alberta | Anwar-Mohamed A.,University of Alberta | And 5 more authors.
Archives of Biochemistry and Biophysics | Year: 2011

FHRE-Luc is a promoter reporter construct that is widely used to assess the activity of FoxO (forkhead box, class O) transcription factors. We here demonstrate that this promoter construct responds to exposure of HepG2 human hepatoma cells to known agonists of the aryl hydrocarbon receptor (AhR), 3-methylcholanthrene, benzo(a)pyrene, and 6-formylindolo[3,2-b]carbazole. However, FHRE-Luc activation did not coincide with FoxO DNA binding or changes in Akt-induced FoxO phosphorylation after treatment with AhR agonists. Testing FHRE-Luc deletion constructs and using AhR-deficient cells, we found that FHRE-Luc activation by AhR agonists is due to a functional xenobiotic-response element (XRE) spanning the backbone/insert border of the reporter plasmid. In conclusion, care must be taken when using FHRE-Luc to assess FoxO activity in response to stimuli that potentially interfere with xenobiotic signaling.

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