Activation of the Akt-NF-kappaB pathway by subtilase cytotoxin through the ATF6 branch of the unfolded protein response - PubMed (original) (raw)

. 2009 Jul 15;183(2):1480-7.

doi: 10.4049/jimmunol.0900017. Epub 2009 Jun 26.

Nobuhiko Hiramatsu, Kunihiro Hayakawa, Yasuhiro Tagawa, Maro Okamura, Ryouji Ogata, Tao Huang, Shotaro Nakajima, Jian Yao, Adrienne W Paton, James C Paton, Masanori Kitamura

Affiliations

Activation of the Akt-NF-kappaB pathway by subtilase cytotoxin through the ATF6 branch of the unfolded protein response

Hiroaki Yamazaki et al. J Immunol. 2009.

Abstract

Shiga toxin has the potential to induce expression of inflammation-associated genes, although the underlying mechanisms are not well understood. We examined the effects of subtilase cytotoxin (SubAB), an AB(5) toxin produced by some Shiga toxigenic Escherichia coli, on the activation of NF-kappaB. SubAB is known to be a protease which selectively degrades GRP78/Bip. Treatment of NRK-52E cells with SubAB caused rapid cleavage of GRP78. Following the degradation of GRP78, transient activation of NF-kappaB was observed with a peak at 6-12 h; the activation subsided within 24 h despite the continuous absence of intact GRP78. The activation of NF-kappaB was preceded by transient phosphorylation of Akt. Treatment of the cells with a selective inhibitor of Akt1/2 or an inhibitor of PI3K attenuated SubAB-induced NF-kappaB activation, suggesting that activation of Akt is an event upstream of NF-kappaB. Degradation of GRP78 caused the unfolded protein response (UPR), and inducers of the UPR mimicked the stimulatory effects of SubAB on Akt and NF-kappaB. SubAB triggered the three major branches of the UPR including the IRE1-XBP1, PERK, and ATF6 pathways. Dominant-negative inhibition of IRE1alpha, XBP1, or PERK did not attenuate activation of NF-kappaB by SubAB. In contrast, genetic and pharmacological inhibition of ATF6 significantly suppressed SubAB-triggered Akt phosphorylation and NF-kappaB activation. These results suggested that loss of GRP78 by SubAB leads to transient phosphorylation of Akt and consequent activation of NF-kappaB through the ATF6 branch of the UPR.

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Figures

FIGURE 1

FIGURE 1

Kinetics of cleavage of GRP78 by SubAB. NRK-52E cells were treated with 100 ng/ml (A) or 10 ng/ml (B) of SubAB for up to 24 (A) or 72 h (B) and subjected to Western blot analysis of C-terminal region of GRP78. The level of _β_-actin is shown at the bottom as a loading control.

FIGURE 2

FIGURE 2

Transient activation of NF-κ_B and induction of NF-κ_B-dependent gene expression by SubAB. A, NRK-52E cells were stably transfected with pNF_κ_B-SEAP, and NRK/NF_κ_B-SEAP reporter cells were established. The cells were stimulated with 1 ng/ml IL-1_β or 10 ng/ml TNF-α and subjected to Northern blot analysis of SEAP and MCP-1. Expression of GAPDH is shown at the bottom as a loading control. B and C, NRK/NF_κ_B-SEAP cells were stimulated with serial dilutions of SubAB for 6 h (B) or 10 ng/ml SubAB for indicated time periods (C) and subjected to Northern blot analysis. D, NRK-52E cells were stably transfected with pNF_κ_B-Luc, and NRK/NF_κ_B-Luc cells were established. The cells were stimulated with IL-1_β, TNF-α, or 1 μ_g/ml LPS and subjected to chemiluminescent assay to evaluate luciferase activity. RLU, relative light unit. E and F, NRK/NF_κ_B-Luc cells were treated with 10 ng/ml (E) or 100 ng/ml (F) of SubAB for up to 24 h and subjected to luciferase assay. G, Cells were treated with SubAB or its inactive mutant SubAA272B for up to 9 h and subjected to Western blot analysis of GRP78. H, NRK/NF_κ_B-Luc cells were stimulated with endotoxin-free SubABET– (10 ng/ml) or SubAA272B (10 ng/ml) for up to 24 h and subjected to chemiluminescent assay. I, NRK-52E cells were treated with 100 ng/ml SubAB for up to 6 h and subjected to Western blot analysis of I_κ_B_α and I_κ_B_β_. J, SM/NF_κ_B-SEAP cells were treated with 100 ng/ml SubAB for 9 h and subjected to Northern blot analysis of SEAP and MCP-1. In D–F and H, assays were performed in quadruplicate, and data are expressed as means ± SE. Asterisks indicate statistically significant differences (p < 0.05).

FIGURE 3

FIGURE 3

Transient activation of Akt upstream of NF-_κ_B following exposure to SubAB. A, NRK-52E cells were treated with SubAB for indicated time periods and subjected to Western blot analysis of phosphorylated Akt (P-Akt). The protein level of total Akt is shown at the bottom as a loading control. B–D, NRK/NF_κ_B-Luc cells were treated with SubAB in the absence (−) or presence (+) of 10 _μ_M Akti-1/2 (B and C) or 100 nM wortmannin (D) and subjected to chemiluminescent assay (B and D) or Northern blot analysis of MCP-1 (C). Data are expressed as means ± SE, and asterisks indicate statistically significant differences (p < 0.05).

FIGURE 4

FIGURE 4

Activation of the Akt-NF-_κ_B pathway through the UPR triggered by SubAB. A–C, NRK-52E cells were treated with SubAB and SubAA272B (A), or tunicamycin (1 _μ_g/ml) and thapsigargin (500 nM) (B and C) for indicated time periods and subjected to Northern blot analysis of GRP78 (A and B) or Western blot analysis of phosphorylated Akt (C). D, NRK/NF_κ_B-Luc cells were treated with tunicamycin or thapsigargin for up to 24 h and subjected to chemiluminescent assay. E, Met5A cells were transfected with pNF_κ_B-Luc, treated with tunicamycin (Tun; 10 _μ_g/ml) or thapsigargin (Thap; 1 _μ_M) for 12 h and subjected to luciferase assay. Data are expressed as means ± SE, and asterisks indicate statistically significant differences (p < 0.05).

FIGURE 5

FIGURE 5

Activation of individual branches of the UPR by SubAB. A, NRK-52E cells were treated with SubAB for 1–12 h (top) or 9–24 h (bottom) and subjected to RT-PCR analysis of XBP1 mRNA. XBP1(U), the unspliced form of XBP1; XBP1(S), the spliced form of XBP1. RT(-), without reverse transcriptase. B, NRK/UPRE-Luc cells were treated with SubAB for indicated time periods and subjected to luciferase assay to evaluate activation of UPRE. Data are expressed as means ± SE, and asterisks indicate statistically significant differences (p < 0.05). C, NRK-52E cells were treated with SubAB, and expression of ATF4 was examined by Northern blot analysis. D, NRK-52E cells were transiently transfected with FLAG-ATF6, treated with SubAB, and subjected to Western blot analysis of p90ATF6 using an anti-FLAG Ab.

FIGURE 6

FIGURE 6

Roles of individual branches of the UPR in SubAB-triggered activation of Akt and NF-κ_B. A–C, and E, NRK-52E cells were transiently cotransfected with pNF_κ_B-Luc and pcDNA3.1 (Vector), pCAG-hIRE1_α_K699A (IRE1_α_-DN) (A), pcDNA3.1-dnXBP (XBP1-DN) (B), pcDNA3-hPERK.K621M (PERK-DN) (C), or pcDNA3.1-ATF6_α(171–373)_Δ_AD (ATF6-DN) (E), stimulated with or without SubAB for 12 h and subjected to chemiluminescent assay to evaluate luciferase activity. D, NRK/NF_κ_B-Luc cells were treated with 10–50 _μ_M salubrinal for 12 h and subjected to luciferase assay. F, Mock-transfected NRK/Neo, NRK/ATF6-DN4, and NRK/ATF6-DN9 cells were transiently transfected with pNF_κ_B-Luc, stimulated with SubAB, and subjected to luciferase assay. G, NRK/NF_κ_B-Luc cells were treated with SubAB in the absence or presence of 300 μ_M AEBSF and subjected to chemiluminescent assay. In A–G, assays were performed in quadruplicate. Data are expressed as means ± SE, and asterisks indicate statistically significant differences (p < 0.05). NS, not significant. H and I, NRK/Neo and NRK/ATF6-DN cells (H) or wild-type MEF (WT) and iATF6_α MEF (I) were stimulated by SubAB, and phosphorylation of Akt was evaluated by Western blot analysis.

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