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First published online August 3, 2005
doi: 10.1242/10.1242/jcs.02460

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Cationic cell-penetrating peptides interfere with TNF signalling by induction of TNF receptor internalization

¹ Department of Molecular Biology, Institute for Cell Biology, University of Tübingen, Auf der Morgenstelle 15, Tübingen, 72076, Germany
² Department of Immunology, Institute for Cell Biology, University of Tübingen, Auf der Morgenstelle 15, Tübingen, 72076, Germany
³ Institute for Cell Biology and Immunology, University of Stuttgart, Allmandring 31, 70569 Stuttgart, Germany

View larger version (49K): [in a new window]   Fig. 1. Effect of the uptake mechanism on the cargo bioactivity. (A) HeLa cells were either incubated with Smac-Antp for 30 minutes at 37°C or electroporated with Smac with increasing concentrations of each peptide, harvested with trypsin/EDTA and analysed by flow cytometry. Error bars represent the standard deviation of the mean of three independent experiments (a.u., absorbance units). (B) HeLa cells were either incubated or electroporated with medium containing Smac-Antp or Smac (each 5 µM) and analysed by laser scanning microscopy. The left panels show fluorescein fluorescence, the right panels are transmission images. Scale bars: 20 µm. (C) HeLa cells were either incubated with increasing concentrations of Smac-Antp or with Rev-Antp for 30 minutes at 37°C or electroporated with Smac or Rev peptides. After removal of peptides cells were stimulated with the TNF-R1-specific mutant TNF (100 ng/ml) and CHX (2 µg/ml) for 6 hours at 37°C, then scraped off the surface, washed and lysed. Caspase-3 activity was determined in cell lysates using a fluorogenic caspase-3 substrate and expressed as fold activation by dividing the caspase-3 activity in treated cells by the activity in untreated cells (no peptide, no TNF/CHX). Error bars represent the standard deviation of the mean of three independent experiments. (D) Cells were either incubated or electroporated with medium containing peptides. After washing, cells were treated, in triplicate, for 24 hours at 37°C with TNF-R1-specific mutant TNF (100 ng/ml) in the presence of CHX (2.0 µg/ml). Finally, crystal violet staining cell was used as a measure of cell viability.

View larger version (11K): [in a new window]   Fig. 2. Inhibition of TNF/CHX-mediated caspase-3 activation by Antp. HeLa cells were incubated with medium containing increasing concentrations of Antp for 30 minutes at 37°C. Then cells were washed and apoptosis was induced by stimulation with TNF-R1-specific mutant TNF (100 ng/ml) and CHX (2 µg/ml). (A) For measurements of caspase-3 activity cells were harvested 6 hours after the induction of apoptosis. The error bars represent the standard deviation of the mean of three independent experiments. (B) Determination of cell viability was performed 24 hours after TNF/CHX treatment using crystal violet staining.

View larger version (10K): [in a new window]   Fig. 3. Inhibition of IL-8 induction by cell-penetrating peptides. (A) HeLa cells were incubated with medium containing the indicated peptides (40 µM) for 30 minutes at 37°C. After washing, cells were stimulated with TNF-R1-specific mutant TNF (100 ng/ml) for 5 hours (black bars) or left untreated (grey bars). IL-8 present in the supernatants as determined by ELISA was normalized on the cell number determined by MTT assay. (B) Cells were incubated with increasing concentrations of Antp or R9 for 30 minutes, stimulated with TNF-R1-specific mutant TNF (100 ng/ml) for 5 hours and IL-8 expression was determined. The error bars represent the standard deviation of the mean of three samples.

View larger version (114K): [in a new window]   Fig. 4. Inhibition of p65 nuclear translocation. (A) HeLa cells were incubated with medium containing the indicated peptide (30 µM) for 30 minutes, washed and then stimulated with TNF-R1-specific mutant TNF (100 ng/ml) for 30 minutes. Cells were then fixed and p65 was detected by immunofluorescence. (B) HeLa cells were incubated with increasing concentrations of Antp peptide for 30 minutes and then stimulated with the TNF-R1-specific mutant TNF (100 ng/ml). To quantify the inhibitory effect of Antp on p65 nuclear translocation, cells were subdivided into three groups and counted manually: cells with a clear nuclear staining (++), cells with equal levels of cytoplasmic and nuclear staining (+) and cells with fluorescence confined to the cytoplasm (–). For each condition more than 200 cells were counted and analysed in three independent experiments. Scale bars: 20 µm (A) and 50 µm (B).

View larger version (96K): [in a new window]   Fig. 5. Influence of bafilomycin A1 on the cellular localization of CPPs and on the nuclear translocation of p65. (A) HeLa cells were treated with Baf A1 (300 nM) for 30 minutes, washed and incubated with Antp or R9 (each 30 µM) for a further 30 minutes at 37°C before being examined by confocal microscopy at RT. (B) In a second experiment, after incubation with Baf A1 and peptide as above, cells were washed with medium and stimulated with the TNF-R1-specific mutant TNF (100 ng/ml) for 30 minutes. After fixation the distribution of p65 was determined by immunofluorescence microscopy. The change of the subcellular distribution of the CPPs upon fixation precluded the parallel detection of CPP localization and NF-B translocation. Scale bars: 20 µm.

View larger version (17K): [in a new window]   Fig. 6. Peptide-mediated downregulation of TNF-R1 but not Fas. (A) HeLa cells were incubated with increasing concentrations of Antp, R9 and Tat for 30 minutes at 37°C. (B) HeLa cells were incubated either with medium alone or with medium containing the TACE inhibitor TAPI-1 (20 µM) for 1 hour at 37°C, then washed and incubated with increasing concentrations of Antp for a further 30 minutes in the presence or absence of inhibitor. (A and B) Cells were harvested with trypsin/EDTA and incubated with the TNF-R1-specific antibody H398 (5 µg/ml) for 1 hour at 4°C. (C) HeLa cells were incubated with increasing concentrations of Antp or R9 for 30 minutes at 37°C, washed, harvested with trypsin/EDTA and incubated with monoclonal anti-CD95 (Fas) antibody (5 µg/ml) for 1 hour at 4°C. (A-C) Cells were washed and incubated with a secondary antibody (Cy5-labelled, goat anti-mouse, 1:500) for 30 minutes at 4°C. For the determination of residual receptor on the cell surface the mean fluorescence in the untreated groups served as a reference (100%). The error bars represent the standard deviation of the mean of three independent experiments.

View larger version (117K): [in a new window]   Fig. 7. Peptide-mediated internalization of TNF-R2. After immunostaining of TNF-R2 with TNF-R2-specific antibody/Zenon Alexa Fluor 647 conjugates, HeLa-TNF-R2 cells were washed and left unstimulated (A), stimulated with TNF-R2-specific mutant TNF (100 ng/ml) for 30 minutes at 37°C (B) or incubated with Antp or R9 (each 5 µM) for 1 hour at 37°C (C,D). Cells were analysed by multichannel confocal microscopy: (A-D) receptor staining; (E,F) distribution of fluorescent peptides; (G,H) superposition of both channels. Scale bar: 20 µm.

View larger version (68K): [in a new window]   Fig. 8. CPP mediate internalization of receptors in a dynamin-dependent but lipid raft-independent manner. (A) HeLadynK44A cells were incubated with or without tetracycline for 48 hours at 37°C, washed and then incubated with medium containing 20 µg/ml Alexa Fluor 633-labelled transferrin for 30 minutes at 37°C. After washing, cells were analysed by confocal microscopy. (B) HeLadynK44A cells were transfected by electroporation with a plasmid coding for human TNF-R2 and incubated for 48 hours at 37°C either with or without tetracycline. TNF-R2 was labelled with a TNF-R2-specific antibody/Zenon Alexa Fluor 647 conjugate. Cells were incubated with Antp (20 µM) for 30 minutes at 37°C, or remained untreated. After washing with medium cells were analysed by multichannel confocal microscopy. (C) EGFR was labelled with Zenon Alexa Fluor 647 conjugate for 30 minutes at 4°C, then cells were washed and incubated with R9 peptide for 30 minutes at 37°C. Scale bar: 20 µm. (D,E) HeLa cells were treated with methyl-ß-cyclodextrin (MßCD; 5 mM) for 30 minutes then incubated with Antp in the presence or absence of the inhibitor for further 30 minutes. Cells were then harvested with trypsin/EDTA, TNF-R1 was immunostained and cells were analysed by flow cytometry. Peptide-associated fluorescence (fluorescein) and Alexa Fluor 647 signal were detected simultaneously.

View larger version (147K): [in a new window]   Fig. 9. Antp-mediated internalization of TNF-R2 occurs without recruitment of TRAF2. HeLa-TNF-R2 cells were transfected by electroporation with a plasmid coding for a TRAF2-GFP fusion protein. After 24 hours TNF-R2 was stained using a TNF-R2-specific antibody/Zenon Alexa Fluor 647 conjugate. After washing, cells were either stimulated with TNF-R2-specific mutant TNF (300 ng/ml, 30 minutes at 37°C) or incubated with unlabelled Antp (20 µM, 30 minutes at 37°C) or left untreated: (A-C) receptor staining; (D-F) TRAF2-GFP; (G-I) superposition of both channels. Scale bar: 20 µm.

View larger version (114K): [in a new window]   Fig. 10. Tat protein induces internalization of TNF-R2. After immunostaining of TNF-R2 with a TNF-R2-specific antibody/Zenon Alexa Fluor 647 conjugate, HeLa-TNF-R2 cells were washed and incubated with fluorescein-labelled Tat protein. Cells were analysed by multichannel confocal microscopy; the first images were taken immediately (2 minutes) after protein addition and further images were taken as indicated. Scale bar: 20 µm.