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First published online 6 May 2003
doi: 10.1242/jcs.00461

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NF-B signalling is inhibited by glucocorticoid receptor and STAT6 via distinct mechanisms

¹ Biosciences Building, School of Biological Sciences, University of Liverpool, Crown Street, Liverpool L69 7ZB, UK
² Department of Medicine, University of Manchester, Stopford Building, Manchester M13 9PT, UK
³ Lead Generation, Molecular Biology, AstraZeneca R&D Charnwood, Bakewell Road, Loughborough LE11 5RH, UK

View larger version (17K): [in a new window]   Fig. 1. Functional activity of EGFP-GR in HeLa cells. (A) HeLa cells transiently transfected with pTAT3-luc and either pEGFP-GR or control pEGFP-N1 expression vectors were treated with dexamethasone for 20 hours prior to harvesting. TAT3-directed luciferase expression was measured and plotted relative to the expression for 0 nM dexamethasone-treated cells for each transfection. n=3, error bars±s.e. (B) HeLa cells were transiently transfected with pNF-B-luc together with various EGFP expression vectors. EGFP expression vectors expressing fusions with p65, GR or both, or a control EGFP were transfected into the cells. Cells were pre-treated for 40 minutes with either medium alone or with 10 nM dexamethasone (in medium) prior to treatment with 10 ng ml-1 TNF for 6 hours. The luciferase activity in cell lysates was determined in pairs of samples either treated with TNF alone or with dexamethasone and TNF. The relative luciferase activity obtained between these values reflects the level of inhibition obtained through prior stimulation with dexamethasone. These ratios are plotted as percentage inhibition for each different transfection condition. All transfections were significantly different to one another as calculated by 5% least significant difference. n=3, error bars±s.e.

View larger version (102K): [in a new window]   Fig. 2. Confocal microscopy of p65-dsRed translocation. Green fluorescence represents either EGFP-GR (A) or EGFP (B); red fluorescence represents p65-dsRed. Times after TNF treatment (minutes) are shown. (A) Merged images of p65-dsRed and EGFP-GR. p65-dsRed nuclear localization is very brief in cells expressing EGFP-GR and treated with 10 nM dexamethasone for 40 minutes prior to TNF (10 ng ml-1) treatment. (B) p65-dsRed and EGFP fluorescence are shown as separate channels for clarification. p65-dsRed (left) shows increased nuclear localization in cells expressing control EGFP (right) and treated with TNF (10 ng ml-1).

View larger version (21K): [in a new window]   Fig. 3. Modulation of p65-dsRed translocation by glucocorticoids. HeLa cells expressing p65-dsRed and either EGFP (A) or EGFP-GR (B) were pretreated for 40 minutes with medium, 10 nM dexamethasone (Dex) or 10 nM RU486, followed by confocal microscopy (data not shown). Cells were then treated with 10 ng ml-1 TNF and monitored every 2 minutes by confocal microscopy. p65-dsRed subcellular fluorescence was analysed and plotted as a ratio of nuclear:cytoplasmic fluorescence relative to the ratio at 0 minutes. (C) The time of 50% maximal nuclear import to 50% nuclear export of p65-dsRed from the data represented in A and B was determined for each treatment and plotted as the half-life of nuclear occupancy. Calculation of the 5% least significance difference between treatments showed all to be significantly different to one another (*) with the exception of EGFP and EGFP-GR transfected cells pretreated with dexamethasone. Each treatment was performed in triplicate with a minimum of 20 cells per experiment. Data plotted represent mean ratio per cell ± s.d.(n–1) for each experiment.

View larger version (13K): [in a new window]   Fig. 4. Inhibition of NF-B-mediated transcription by STAT6. HeLa cells were transiently transfected with pNF-B-luc together with various EGFP expression vectors. EGFP expression vectors expressing fusions with p65, STAT6 or both, or a control EGFP were transfected into the cells. Cells were pretreated for 40 minutes with either medium alone or 10 ng ml-1 IL-4 (in medium) prior to treatment with 10 ng ml-1 TNF for 6 hours. The luciferase activity in cell lysates was determined in pairs of samples treated either with TNF alone or with IL-4 and TNF. The relative luciferase activity obtained between these values reflects the level of inhibition obtained through prior stimulation with IL-4. These ratios are plotted as percentage inhibition for each different transfection condition. IL-4 treatment of EGFP-STAT6-expressing cells significantly inhibited transcription compared with all other transfection conditions (5% least significant difference). n=3, error bars±s.e.

View larger version (24K): [in a new window]   Fig. 5. Repression of p65 activation by IL-4/STAT6. HeLa cells transiently transfected with p65-dsRed and either EGFP or EGFP-STAT6 were pretreated with 10 ng ml-1 IL-4 or medium for 40 minutes. Subsequent stimulation with 10 ng ml-1 TNF was followed by confocal microscopy for 80 minutes. (A) Fluorescence images of cells expressing p65-dsRed (left; red fluorescence) and EGFP-STAT6 (right; green fluorescence) treated with IL-4 and TNF, showing marked inhibition of p65-dsRed translocation compared with cells expressing EGFP treated with TNF (Fig. 2B). Positions of nuclei are highlighted in grey in the first EGFP-STAT6 image. Times after TNF treatment are shown in minutes. (B) Quantification of p65-dsRed translocation inhibition by activation and expression of EGFP-STAT6. p65-dsRed localization was analysed and quantified as described in Fig. 3. Each treatment was performed in triplicate with a minimum of 16 cells per experiment. Data plotted represent mean nuclear:cytoplasmic ratio per cell±s.d.(n–1) for each experiment.

View larger version (37K): [in a new window]   Fig. 6. Modulation of TNF-induced IB degradation. (A) HeLa cells transiently transfected with pIB-EGFP and p65-dsRed were pretreated for 40 minutes with 10 ng ml-1 IL-4, 10 nM dexamethasone or medium prior to TNF (10 ng ml-1) stimulation. Cytoplasmic IB-EGFP fluorescence for individual cells was plotted relative to the fluorescence at 0 minutes. Each treatment was performed in triplicate with a minimum of 14 cells per experiment. Error bars±s.d.(n–1). (B) HeLa cells were pretreated with either 10 ng ml-1 IL-4 or medium for 40 minutes. Cells were harvested in lysis buffer at various time points (as shown) following treatment with 10 ng ml-1 TNF. Samples were blotted with an anti-phospho-IB antibody.