Subtilase cytotoxin activates PERK, IRE1 and ATF6 endoplasmic reticulum stress-signalling pathways - PubMed (original) (raw)

Subtilase cytotoxin activates PERK, IRE1 and ATF6 endoplasmic reticulum stress-signalling pathways

Jennifer J Wolfson et al. Cell Microbiol. 2008 Sep.

Abstract

Subtilase cytotoxin (SubAB) is the prototype of a new family of AB(5) cytotoxins produced by Shiga toxigenic Escherichia coli. Its cytotoxic activity is due to its capacity to enter cells and specifically cleave the essential endoplasmic reticulum (ER) chaperone BiP (GRP78). In the present study, we have examined its capacity to trigger the three ER stress-signalling pathways in Vero cells. Activation of PKR-like ER kinase was demonstrated by phosphorylation of eIF2alpha, which occurred within 30 min of toxin treatment, and correlated with inhibition of global protein synthesis. Activation of inositol-requiring enzyme 1 was demonstrated by splicing of X-box-binding protein 1 mRNA, while activating transcription factor 6 activation was demonstrated by depletion of the 90 kDa uncleaved form, and appearance of the 50 kDa cleaved form. The rapidity with which ER stress-signalling responses are triggered by exposure of cells to SubAB is consistent with the hypothesis that cleavage by the toxin causes BiP to dissociate from the signalling molecules.

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Figures

Fig. 1

Time-course of eIF2α phosphorylation. Vero cell monolayers were treated with 1 μg ml−1 SubAB, or SubAA272B, for the indicated times. Proteins in cell extracts were separated by SDS-PAGE, electroblotted, probed with anti-phospho-eIF2α, and labelled species were detected using a HRP conjugate and ECL, as described in Experimental procedures. Filters were then stripped and re-probed using anti-eIF2α, to label total eIF2α. An untreated cell extract (cont) was also included. The visible band in the molecular marker (MW) track is 30 kDa.

Fig. 2

Effect of SubAB on eIF2α phosphorylation and protein synthesis. Vero cell monolayers were incubated with the indicated dose of SubAB, or 1 μg ml−1 SubAA272B for 0.5, 1, 2 or 4 h. Levels of eIF2α-P and total eIF2α were then determined by Western blotting, as for Fig. 1. Figures below each lane are fold induction of eIF2α-P following SubAB treatment, relative to treatment with SubAA272B (normalized to loading controls), determined by densitometry using ImageJ 1.38× software. Effects on global protein synthesis were also measured by assessment of [3H]-leucine incorporation into TCA-precipitable material during a 60 min pulse, as described in Experimental procedures. Data are the mean ± SEM from three independent experiments, expressed as a percentage of the untreated control for each time point. NS, not significant; *, P < 0.0001; **, P < 0.002 (two-tailed _t_-test).

Fig. 3

Effect of SubAB on BiP, eIF2α phosphorylation and protein synthesis in PERK −/− (KO) and PERK wt MEFs. MEF monolayers were incubated with 1 μg ml−1 SubAB for various times, as indicated. A. Cleavage of BiP was assessed by Western blotting using anti-BiP C-20 (Santa Cruz), as described previously for Vero cells (Paton et al., 2006). The lanes marked + and − are Vero cells treated for 2 h with or without SubAB respectively. B. Levels of eIF2α-P and total eIF2α were determined by Western blotting, as for Fig. 1; lanes marked + and − denote PERK wt and PERK KO cells respectively. C. Global protein synthesis in PERK wt and PERK KO MEFs were measured as previously described in Fig. 2. Data are the mean ± SD 3H-Leu incorporation, expressed as a percentage of control cells, from a representative experiment conducted in triplicate.

Fig. 4

RT-PCR analysis. RNA extracts from Vero cells treated for 6 h with or without 100 ng ml−1 SubAB, 100 ng ml−1 SubAA272B or 0.2 μg ml−1 thapsigargin were analysed by RT-PCR using primers (Table 1) specific for XBP1, BiP, CHOP, GRP94, ATF4 and GAPDH (internal control), as described in Experimental procedures. The expected mobilities of spliced and unspliced XBP1 amplicons are indicated.

Fig. 5

Time-course. RNA extracts from Vero cells treated with 100 ng ml−1 SubAB for 0–30 h were analysed by RT-PCR using primers (Table 1) specific for the indicated transcripts.

Fig. 6

Quantitative real-time PCR analysis. RNA extracts from Vero cells treated with 100 ng ml−1 SubAB for 0–30 h were reverse-transcribed into cDNA and real-time PCR analysis was then performed using gene-specific primers (Table 2), as described in Experimental procedures. For each time point, the relative amounts of each transcript in SubAB-treated cells were compared with that in untreated cells, using the comparative cycle threshold (2ΔΔCt) method. Data represent the mean ± SD from two independent experiments, expressed as fold change in mRNA compared with the untreated control for each time point (*, P < 0.05; **, P < 0.01; ***, P < 0.001; Student’s unpaired two-tailed _t_-test).

Fig. 7

Time-course of GRP94, CHOP and BiP induction. Vero cells were treated with 100 ng ml−1 SubAB for 0–30 h, and lysates were analysed for GRP94, CHOP and BiP by Western blotting, as described in Experimental procedures. β-actin was used as an internal loading control. For BiP, separate panels showing intact (72 kDa) and cleaved (28 kDa) forms are presented.

Fig. 8

ATF6 activation. Vero cells transfected with p3×FLAG-CMV-7.1-ATF6 were treated with or without 100 ng ml−1 SubAB, 100 ng ml−1 SubAA272B or 0.2 μg ml−1 thapsigargin. At various times thereafter, lysates were prepared from these cells, as well as from untransfected Vero cells, and levels of (A) uncleaved tagged ATF6 (p90ATF6-3×FLAG), or (B) cleaved tagged ATF6 (p50ATF6-3×FLAG), were examined by Western blotting using anti-FLAG. After detection using alkaline phosphatase-conjugate and chromogenic substrate (A) or HRP and chemiluminescence (B), filters were stripped and re-probed with anti-β-actin as a loading control.

Fig. 9

DNA fragmentation assay. Vero cells were treated with 100 ng ml−1 SubAB for the indicated times, or with SubAA272B for 30 h. DNA was then extracted and subjected to agarose gel electrophoresis, as described in Experimental procedures. M denotes molecular size markers.

References

1. Boyce M, Yuan J. Cellular response to endoplasmic reticulum stress: a matter of life or death. Cell Death Differ. 2006;13:363–373. - PubMed
1. Chan SW, Egan PA. Hepatitis C virus envelope proteins regulate CHOP via induction of the unfolded protein response. FASEB J. 2005;19:1510–1512. - PubMed
1. Chong DC, Paton JC, Thorpe CM, Paton AW. Clathrin-dependent trafficking of subtilase cytotoxin, a novel AB5 toxin that targets the ER chaperone BiP. Cell Microbiol. 2007;10:795–806. - PubMed
1. DuRose JB, Tam AB, Niwa M. Intrinsic capacities of molecular sensors of the unfolded protein response to sense alternate forms of endoplasmic reticulum stress. Mol Biol Cell. 2006;17:3095–3107. - PMC - PubMed
1. Eriksson KK, Vago R, Calanca V, Galli C, Paganetti P, Molinari M. EDEM contributes to maintenance of protein folding efficiency and secretory capacity. J Biol Chem. 2004;279:44 600–44 605. - PubMed

Subtilase cytotoxin activates PERK, IRE1 and ATF6 endoplasmic reticulum stress-signalling pathways - PubMed (original) (raw)