The MAP kinase-activated protein kinase 2 (MK2) contributes to the Shiga toxin-induced inflammatory response

Abstract

Infection with Shiga toxin (STx)-producing bacteria can progress to a toxemic, extraintestinal injury cascade known as haemolytic uremic syndrome (HUS), the leading cause of acute renal failure in children. Mounting evidence suggests that STx activates stress response pathways in susceptible cells and has implicated the p38 mitogen-activated protein kinase (MAPK) pathway. More importantly, some of the pathology associated with HUS is believed to be a result of a STx-induced inflammatory response. From a siRNA screen of the human kinome adapted to a high-throughput format, we found that knock-down of the MAPK-activated protein kinase 2 (MK2), a downstream target of the p38 MAPK, protected against Shiga toxicity. Further characterization of the in vitro role of MK2 revealed that STx activates the p38-MK2 stress response pathway in both p38- and MK2-dependent manners in two distinct cell lines. MK2 activation was specific to damage to the ribosome by an enzymatically active toxin and did not result from translational inhibition per se. Genetic and chemical inhibition of MK2 significantly decreased the inflammatory response to STx. These findings suggest that MK2 inhibition might play a valuable role in decreasing the immuopathological component of STx-mediated disease.

Fig. 1

Knock-down of MK2 protects against Shiga toxicity. A siRNA screen of 646 human kinase and kinase-associated genes was adapted to a 96-well format (see Experimental procedures). Shown are two representative plates, run in duplicate (circles and squares), demonstrating light levels of 80 different kinase siRNAs after a 24-h treatment with STx1 (1 ng/mL). Well number represents each well of HeLa-Fluc cells transfected with 50 nM siRNA targeting a specific kinase, and corresponding light levels are shown on the y-axis. Control wells have been excluded (see Supplementary Fig. 2). A hit was considered any knock-down that maintained luminescence at least 3 standard deviations above STx1-treated controls (dotted line). Knock-down of MK2 (siRNA sequence provided in Experimental Procedures) conferred the highest protection against Shiga toxicity. Mean and standard deviations for each set of duplicate plates were determined by GraphPad Prism. MK2, mitogen-activated protein kinase-activated protein kinase 2.

Fig. 2

STx1 specifically induces activation of the p38-MK2 pathway in both HeLa and HMVEC in time- and dose-dependent manners. A. HeLa cells (left) or HMVEC (right) were treated with no toxin or STx1 (10 ng/mL) for the indicated times, and lysates were probed with the indicated antibodies. Activation of the p38-MK2 pathway is observed by 60 min following exposure to STx1, while stimulation of this pathway in HMVEC can be seen as early as 30 min. B. HeLa cells (left) or HMVEC (right) were treated with either no toxin or increasing STx1 concentrations for 2 h. Lysates were probed as in (A). For both cell lines, as little as 0.1 ng/mL STx1 was able to activate the p38-MK2 pathway. For (A) and (B), actin staining served as a loading control. C. HeLa cells were pretreated with DMSO (0.5% v/v) or the p38 inhibitor SB202190 (10 μM) for 1 h prior to a 30-min exposure to STx1 (100 ng/mL). Equal fractions of lysates were probed with the indicated antibodies, with MK2 serving as a loading control. Inhibition of p38 eliminates STx1-induced MK2 activation. In addition, treatment with 100 ng/mL heat-inactivated STx1 (HI-STx1) or the STxB subunit (100 ng/mL) did not activate MK2. Heat inactivation of STx1 involved incubating the toxin for 12 h at 95°C.

Fig. 2

STx1 specifically induces activation of the p38-MK2 pathway in both HeLa and HMVEC in time- and dose-dependent manners. A. HeLa cells (left) or HMVEC (right) were treated with no toxin or STx1 (10 ng/mL) for the indicated times, and lysates were probed with the indicated antibodies. Activation of the p38-MK2 pathway is observed by 60 min following exposure to STx1, while stimulation of this pathway in HMVEC can be seen as early as 30 min. B. HeLa cells (left) or HMVEC (right) were treated with either no toxin or increasing STx1 concentrations for 2 h. Lysates were probed as in (A). For both cell lines, as little as 0.1 ng/mL STx1 was able to activate the p38-MK2 pathway. For (A) and (B), actin staining served as a loading control. C. HeLa cells were pretreated with DMSO (0.5% v/v) or the p38 inhibitor SB202190 (10 μM) for 1 h prior to a 30-min exposure to STx1 (100 ng/mL). Equal fractions of lysates were probed with the indicated antibodies, with MK2 serving as a loading control. Inhibition of p38 eliminates STx1-induced MK2 activation. In addition, treatment with 100 ng/mL heat-inactivated STx1 (HI-STx1) or the STxB subunit (100 ng/mL) did not activate MK2. Heat inactivation of STx1 involved incubating the toxin for 12 h at 95°C.

Fig. 2

STx1 specifically induces activation of the p38-MK2 pathway in both HeLa and HMVEC in time- and dose-dependent manners. A. HeLa cells (left) or HMVEC (right) were treated with no toxin or STx1 (10 ng/mL) for the indicated times, and lysates were probed with the indicated antibodies. Activation of the p38-MK2 pathway is observed by 60 min following exposure to STx1, while stimulation of this pathway in HMVEC can be seen as early as 30 min. B. HeLa cells (left) or HMVEC (right) were treated with either no toxin or increasing STx1 concentrations for 2 h. Lysates were probed as in (A). For both cell lines, as little as 0.1 ng/mL STx1 was able to activate the p38-MK2 pathway. For (A) and (B), actin staining served as a loading control. C. HeLa cells were pretreated with DMSO (0.5% v/v) or the p38 inhibitor SB202190 (10 μM) for 1 h prior to a 30-min exposure to STx1 (100 ng/mL). Equal fractions of lysates were probed with the indicated antibodies, with MK2 serving as a loading control. Inhibition of p38 eliminates STx1-induced MK2 activation. In addition, treatment with 100 ng/mL heat-inactivated STx1 (HI-STx1) or the STxB subunit (100 ng/mL) did not activate MK2. Heat inactivation of STx1 involved incubating the toxin for 12 h at 95°C.

Fig. 3

STx-induced MK2 activation is p38-specific. HeLa cells (left) or HMVEC (right) were pretreated with the indicated compounds 30 min prior to a 2-h exposure to STx2 (10 ng/mL). Lysates were probed with the indicated antibodies. Two distinct p38 inhibitors, SB203580 (2.65 μM) and the p38 inhibitor III (0.5 μM), prevented STx2-mediated Hsp27 phosphorylation, while the JNK inhibitor SP600125 (25 μM) showed no effect, similar to STx2-treated cells treated lacking compound treatment (“STx2 alone”). “No toxin” refers to cells lacking compound and toxin treatment. β-actin staining served as a loading control.

Fig. 4

STx activation of the p38-MK2 pathway is dependent on MK2 activity. A. Overexpression of catalytically inactive MK2 (MK2-DN) eliminates STx1-induced Hsp27 phosphorylation. HeLa cells were transduced with adenoviral constructs expressing luciferase (pAd-Luc), wild-type MK2 (MK2-WT), or catalytically inactive MK2 (MK2-DN; see Methods). Cells were then exposed to STx1 (10 ng/mL) for 6 h, and equal fractions of lysates were probed with the indicated antibodies. B. HMVEC were pretreated with DMSO (0.5% v/v; top) or the MK2 inhibitor PHA-781089 (20 μM; bottom) for 1 h prior to exposure to STx2 (1 ng/mL) for the indicated times. Lysates were probed with the indicated antibodies. Chemical inhibition of MK2 prevents STx-induced Hsp27 phosphorylation. HI-STx2, heat-inactivated STx2 (1 ng/mL). For (A) and (B), actin staining served as a loading control.

Fig. 4

STx activation of the p38-MK2 pathway is dependent on MK2 activity. A. Overexpression of catalytically inactive MK2 (MK2-DN) eliminates STx1-induced Hsp27 phosphorylation. HeLa cells were transduced with adenoviral constructs expressing luciferase (pAd-Luc), wild-type MK2 (MK2-WT), or catalytically inactive MK2 (MK2-DN; see Methods). Cells were then exposed to STx1 (10 ng/mL) for 6 h, and equal fractions of lysates were probed with the indicated antibodies. B. HMVEC were pretreated with DMSO (0.5% v/v; top) or the MK2 inhibitor PHA-781089 (20 μM; bottom) for 1 h prior to exposure to STx2 (1 ng/mL) for the indicated times. Lysates were probed with the indicated antibodies. Chemical inhibition of MK2 prevents STx-induced Hsp27 phosphorylation. HI-STx2, heat-inactivated STx2 (1 ng/mL). For (A) and (B), actin staining served as a loading control.

Fig. 5

Activation of the p38-MK2 pathway by STx depends on toxin adherence and intracellular trafficking and appears to be part of a ribotoxic stress response. A. HMVEC were pretreated with DMSO (0.5% v/v), GCA (10 μM), or P1 glycoprotein (1 μg/mL) for 30 min prior to exposure to STx1 (10 ng/mL) or media alone (“No Toxin”). Cells were allowed to internalize toxin for 2 h at 37°C prior to lysis and probing with the indicated antibodies. Pretreatment with the P1 glycoprotein inhibited STx1-mediated phosphorylation of p38 and Hsp27, and GCA treatment similarly blocked activation of the p38-MK2 pathway compared to DMSO-treated cells (“Toxin alone”). Pretreatment with GCA, however, had no effect on activation of this pathway following a 2-h exposure to anisomycin (10 ng/mL). B. HMVEC were exposed to various translational inhibitors for 2 h, and the phosphorylation status of p38 and Hsp27 was assessed by Western blotting. Only inhibitors that are known to cause direct damage to the ribosome (STx1, STx2, and ricin) induced p38 and Hsp27 phosphorylation, while translational inhibitors acting through different mechanisms (DT, PE, Puro, CHX) showed little to no activation. “None” refers to cells lacking compound and toxin treatment. STx1, Shiga toxin 1 (10 ng/mL); STx2, Shiga toxin 2 (10 ng/mL); GCA, Golgicide A; DT, diphtheria toxin (1 μg/mL); PE, Pseudomonas exotoxin A (1 μg/mL); Puro, puromycin (10 μg/mL); CHX, cycloheximide (100 μg/mL). For both (A) and (B), actin staining served as a loading control.

Fig. 5

Activation of the p38-MK2 pathway by STx depends on toxin adherence and intracellular trafficking and appears to be part of a ribotoxic stress response. A. HMVEC were pretreated with DMSO (0.5% v/v), GCA (10 μM), or P1 glycoprotein (1 μg/mL) for 30 min prior to exposure to STx1 (10 ng/mL) or media alone (“No Toxin”). Cells were allowed to internalize toxin for 2 h at 37°C prior to lysis and probing with the indicated antibodies. Pretreatment with the P1 glycoprotein inhibited STx1-mediated phosphorylation of p38 and Hsp27, and GCA treatment similarly blocked activation of the p38-MK2 pathway compared to DMSO-treated cells (“Toxin alone”). Pretreatment with GCA, however, had no effect on activation of this pathway following a 2-h exposure to anisomycin (10 ng/mL). B. HMVEC were exposed to various translational inhibitors for 2 h, and the phosphorylation status of p38 and Hsp27 was assessed by Western blotting. Only inhibitors that are known to cause direct damage to the ribosome (STx1, STx2, and ricin) induced p38 and Hsp27 phosphorylation, while translational inhibitors acting through different mechanisms (DT, PE, Puro, CHX) showed little to no activation. “None” refers to cells lacking compound and toxin treatment. STx1, Shiga toxin 1 (10 ng/mL); STx2, Shiga toxin 2 (10 ng/mL); GCA, Golgicide A; DT, diphtheria toxin (1 μg/mL); PE, Pseudomonas exotoxin A (1 μg/mL); Puro, puromycin (10 μg/mL); CHX, cycloheximide (100 μg/mL). For both (A) and (B), actin staining served as a loading control.

Fig. 6

MK2 inhibition reduces the STx1-induced inflammatory response. A. HeLa cells were transduced with a control adenoviral construct (pAd-Luc; black bars), a construct expressing wild-type MK2 (MK2-WT; gray bars), or a construct expressing a catalytically inactive MK2 (MK2-DN; white bars) for 24 h prior to treating with STx1 (10 ng/mL) for 6 h. The levels of IL-6 and TNFα mRNA were determined by qPCR and are expressed as a fold change above mRNA levels in transduced cells lacking STx1 (see Experimental Procedures). Data points represent duplicate data from three independent experiments (mean ±S.D.). Sample means were compared using a two-tailed Student's t-test for independent samples. B. HeLa cells were pretreated with DMSO (0.5% v/v; black bars), the MK2 inhibitor PHA-781089 (20 μM; gray bars), or the p38 inhibitor SB202190 (10 μM; white bars) for 1 h prior to a 6-h exposure to STx1 (100 ng/mL). The levels of IL-6 and TNFα mRNA were determined by qPCR and are expressed as a fold change above mRNA levels in compound-treated cells in the absence of STx1. Data points represent duplicate data from three independent experiments (mean ±S.D.). Sample means were compared as in (A). n.s. denotes no significant change, * significant change (p<0.05), ** highly significant change (p<0.01).

Fig. 6

MK2 inhibition reduces the STx1-induced inflammatory response. A. HeLa cells were transduced with a control adenoviral construct (pAd-Luc; black bars), a construct expressing wild-type MK2 (MK2-WT; gray bars), or a construct expressing a catalytically inactive MK2 (MK2-DN; white bars) for 24 h prior to treating with STx1 (10 ng/mL) for 6 h. The levels of IL-6 and TNFα mRNA were determined by qPCR and are expressed as a fold change above mRNA levels in transduced cells lacking STx1 (see Experimental Procedures). Data points represent duplicate data from three independent experiments (mean ±S.D.). Sample means were compared using a two-tailed Student's t-test for independent samples. B. HeLa cells were pretreated with DMSO (0.5% v/v; black bars), the MK2 inhibitor PHA-781089 (20 μM; gray bars), or the p38 inhibitor SB202190 (10 μM; white bars) for 1 h prior to a 6-h exposure to STx1 (100 ng/mL). The levels of IL-6 and TNFα mRNA were determined by qPCR and are expressed as a fold change above mRNA levels in compound-treated cells in the absence of STx1. Data points represent duplicate data from three independent experiments (mean ±S.D.). Sample means were compared as in (A). n.s. denotes no significant change, * significant change (p<0.05), ** highly significant change (p<0.01).