Published online before print November 21, 2005
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Department of Medical Microbiology and Immunology, Texas A & M University System Health Science Center, College Station, Texas
1 Correspondence: Department of Medical Microbiology and Immunology, 407 Reynolds Medical Building, Texas A & M University System Health Science Center, College Station, TX 77843-1114. E-mail: tesh{at}medicine.tamhsc.edu
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Key Words: interleukin-1ß • interleukin-8 • ribotoxic stress response
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(TNF-), interleukin (IL)-1ß, IL-6, and a number of CC and CXC chemokines [12
13
14
15
]. The localized production of TNF- and IL-1ß may contribute to pathogenesis by up-regulating the expression of Gb3 on target endothelial cells, thereby sensitizing the cells to the action of Stxs [16
17
18
]. Chemokines may be essential for the infiltration of monocytes and neutrophils into sites of vascular damage [15
, 19
].
The precise signaling pathways activated by Stxs to elicit cytokine production are not known. However, mitogen-activated protein kinases (MAPKs) are involved in intracellular signaling for cytokine expression as well as cell proliferation and/or apoptosis [20
21
22
]. MAPK cascades are categorized into three major pathways: c-Jun N-terminal kinases (JNK), p38 MAPKs, and extracellular signal-regulated kinases (ERK), and each MAPK contains multiple isoforms. Studies using a rat fibroblast cell line and human monocytic and intestinal epithelial cell lines have shown that anisomycin, ricin, -sarcin, and Stxs, protein synthesis inhibitors that share the property of acting on the 28S rRNA, activate the JNK and p38 MAPK cascades [23
24
25
]. In earlier studies measuring MAPK activation in differentiated, macrophage-like THP-1 cells, we noted that a p38 MAPK inhibitor partially blocked soluble TNF- production induced by Stx1 [24
]. We also noted that in contrast to LPS, which mediated a rapid increase in cytokine expression, Stx1 induced the prolonged expression or release of cytokines, a phenomenon which is in part a result of the stabilization of cytokine and chemokine mRNA transcripts [15
, 26
]. The prolonged activation of MAPK cascades by Stxs may be critical for transcript stabilization and prolonged expression of cytokines. Finally, it has recently been shown that the transient activation of MAPKs promotes signaling for cell survival, and the prolonged activation of MAPKs may be important for triggering apoptosis [27
, 28
]. We showed that after a 12-h exposure to Stx1 and LPS, 
40% of differentiated THP-1 cells were induced to undergo apoptosis [29
], suggesting that MAPK cascades may be activated for prolonged times in toxin-treated cells. Therefore, we have examined the capacity of Stx1 to induce the prolonged activation of all three MAPK cascades and studied their roles in Stx1 and/or LPS-induced cytokine expression.
Stxs may regulate macrophage cytokine expression at transcriptional and post-transcriptional stages. Treatment of THP-1 cells with Stx1 resulted in the activation and nuclear translocation of nuclear factor-
B and activation of activator protein-1 [30
]. Activation of the transcription factors was associated with increased levels of TNF- and IL-1ß mRNA transcripts in toxin-treated cells. Stxs also affect cytokine and chemokine expression at a post-transcriptional stage by altering mRNA decay rates [15
, 26
, 31
]. In eukaryotic cells, however, translation initiation may be the rate-limiting step of protein expression. Initiation involves the modulation of protein-protein and protein-RNA interactions and is under control of diverse signal transduction pathways [32
]. LPS was shown to increase the translation rate of endogenous or transiently transfected TNF mRNA by two- to threefold in the murine monocytic RAW264.7 cell line [33
]. LPS activates translation initiation through protein kinase cascades, which ultimately alter the phosphorylation status of eukaryotic initiation factor 4E (eIF4E) and its binding protein eIF4E-BP or phosphorylated heat- and acid-stable protein (PHAS)-1 [34
, 35
]. Recently, Colpoys et al. [36
] showed that Stxs activate eIF4E in the human intestinal epithelial cell line Hct-8. Therefore, we hypothesized that in addition to stabilizing mRNAs, Stxs may also affect the translation of cytokine mRNAs through modulation of signaling pathways to further increase translation initiation. We examined the capacity of Stx1 to phosphorylate eIF4E in THP-1 cells and the requirement of eIF4E activation in increased protein synthesis activity and the production of cytokines. The requirement for Stx1 enzymatic activity in eIF4E phosphorylation was examined using purified Stx1 B subunits and Stx1 holotoxins containing site-directed mutations in the toxin-active site. Stx1 and anisomycin, protein synthesis inhibitors that activate the ribotoxic stress response, were shown to trigger eIF4E activation in THP-1 cells.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Toxin preparations
Purified Stx1 was prepared from cell lysates obtained from E. coli DH5 harboring plasmid pCKS112, which contains the stx1 operon under control of a thermoinducible promoter [37
]. Stx1 was purified from cell lysates by sequential ion exchange and chromatofocusing chromatography. Purity of toxins was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with silver staining and by Western blot analysis. Prepared toxins contained <0.1 ng endotoxin per ml determined by Limulus amoebocyte lysate assay (Associates of Cape Cod, Inc., East Falmouth, MA). Purified Stx1-B subunits were the kind gift of Dr. Cheleste Thorpe (New England Medical Center-Tufts University, Boston, MA). Purified Stx1 holotoxin containing an A subunit double mutation (E167Q; R170L) lacking N-glycosidase activity [38
] was the kind gift of Dr. Yoshifumi Takeda (Jissen Women’s University, Tokyo, Japan). Purified LPS derived from the enterohemorrhagic E. coli serotype O111 was purchased from Sigma Chemical Co.
Cell lines
The human myelogenous leukemia cell line THP-1 [39
] was obtained from American Type Culture Collection (Manassas, VA) and cultured in RPMI-1640 medium (Gibco-BRL, Grand Island, NY) supplemented with penicillin (100 U/ml), streptomycin (100 ug/ml), and 10% fetal bovine serum (FBS; Hyclone Laboratories, Logan, UT). Cells were maintained at 37°C in 5% CO2 in a humidified incubator. THP-1 cells were differentiated to the macrophage-like state using medium containing phorbol 12-myristate 13-acetate (PMA; 50 ng/ml) for 48 h. The medium was then replaced with medium lacking PMA for 3 days, changing the medium every day. Cells were challenged with Stx1 and/or LPS for the indicated time periods, and cell extracts were prepared using the procedure below.
Preparation of cellular lysates
THP-1 cells (5x106) were washed once in cold Dulbecco’s phosphate-buffered saline and suspended in RPMI with 0.5% FBS prior to stimulation. Cells were serum-starved for 18 h to reduce endogenous kinase activity. Cells were stimulated with Stx1 (400 ng/ml) or LPS (200 ng/ml) or both stimulants in medium containing 0.5% FBS for various time periods as indicated in the figures. Cells were harvested and lysed at 4°C in modified radioimmunoprecipitation assay buffer [1.0% Nonidet P-40, 1.0% Na-deoxycholate, 150 mM NaCl, 50 mM Tris-HCl (pH 7.5), 0.25 mM Na-pyrophosphate, 2 mM each sodium vanadate and sodium fluoride, 10 µg/ml aprotinin, 1.0 µg/ml leupeptin and pepstatin, and 200 mM phenylmethylsulfonyl fluoride]. Extracts were collected and cleared by centrifugation at 15,000 g for 10 min. Cleared extracts were stored at –80°C until further use for Western blot analysis as described below.
Western blot analysis
Cell extracts prepared from stimulated THP-1 cells were used for determination of protein content using the Micro BCA protein assay kit (Pierce, Rockford, IL). Equal amounts of proteins (60–80 µg protein per gel lane) were separated by SDS-PAGE using 8% and 12% acrylamide gels and transferred to nitrocellulose membranes, which were blocked with 5% milk prepared in Tris-buffered saline (TBS)/Tween 20 [200 mM Tris (pH 7.6), 1.38 M NaCl, containing 0.1% Tween 20] and incubated overnight at 4°C with various primary antibodies specific for JNK, p38, and ERK MAPKs or eIF4E in 5% bovine serum albumin made in TBS with 0.1% Tween 20. Membranes were then incubated with the corresponding secondary antibodies (rabbit/mouse immunoglobulin G coupled with horseradish peroxidase) for 2 h at room temperature. Bands were visualized using the Western Lightning chemiluminescence system (NEN-Perkin Elmer, Boston, MA). The intensities of protein bands captured on autoradiography film were quantitated using Bio-Rad Imager quantification software (Bio-Rad, Hercules, CA). Fold induction was calculated, as stimulated protein band intensity values divided by unstimulated control protein band intensity values after normalizing for loading controls. Data shown are from at least three independent experiments.
Measurement of protein synthesis
Protein synthesis was measured by the incorporation of [3H]-leucine into trichloroacetic acid-insoluble material. THP-1 cells were cultured in 24-well plates, incubated in the presence or absence of Stx1 (400 ng/ml), with or without pretreatment with Mnk1 inhibitor CGP57380 (50 µM), or incubated with the ribotoxic stress inducer anisomycin (1.0 µg/ml) for various time-points. Thirty minutes before each time-point, [3H]-leucine (4.0 µCi) was added into the culture medium containing 0.05 g/L leucine (RPMI 1640) and incubated for 30 min at 37°C. Cells were solubilized, and incorporation of [3H]-leucine into nascent polypeptides was stopped by incubation with 0.1 M KOH for 10 min at 37°C. Proteins were precipitated by the addition of 400 µl 20% trichloroacetic acid (TCA), incubation on ice for 15 min, and centrifugation at 6000 g for 5 min. Supernatants were discarded, and the precipitates were washed with 95% ethanol two times and allowed to air dry. Precipitates were solubilized in 250 µl 0.5M KOH and transferred into scintillation vials containing 10 ml scintillant. Radioactivity was measured using a scintillation counter (Beckman LS8000, Beckman Instruments Inc., Fullerton, CA).
Analysis of IL-1ß and IL-8 production
THP-1 cells (2x106 cells/ml) were treated with inhibitors specific for JNK1/2 (SP600125; 50 µM), p38 (SB203580; 20 µM), and ERK1/2 (PD98059; 50 µM) MAPKs alone, in combinations of two inhibitors, or all three together or treated with the Mnk1-specific inhibitor (CGP57380; 50 µM) for 1 h prior to challenge with Stx1 and/or LPS. The cells were incubated at 37°C in a humidified 5% CO2 incubator for 24 h. Cell-free supernatants were collected and used for quantification of cytokine production with human IL-1ß and IL-8 Quantikine sandwich enzyme-linked immunosorbent assay (ELISA) kits from R & D Systems (Minneapolis, MN). Supernatants (200 µL in the case of IL-8 or 50 µl in the case of IL-1ß) were added to duplicate wells on the ELISA plates. Following the manufacturer’s protocol, absorbance at 450 nm (A450) and A570 was measured using an ELISA plate reader (Dynatech MR5000, Dynatech Laboratories, Chantilly, VA.) Each assay was repeated three times, and mean cytokine values ± SEM were represented as pg/ml (IL-1ß) or ng/ml (IL-8).
Statistical analysis
Statistical analyses of kinetics data (fold-induction from three independent experiments) of JNK, p38, and ERK1/2 MAPK and eIF4E activation were performed using two-way ANOVA with the Duncan multiple-range test for post-hoc comparisons (SAS Institute, Cary, NC). For MAPK and Mnk1 inhibitor studies and protein synthesis inhibition assays, the results were compiled from three independent experiments, and t-test was used to calculate the significance of inhibition from the Stx1, LPS, and Stx1 + LPS-treated cells.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Figure 1. Activation of JNK1/2 (p54/p46) after stimulation of THP-1 cells with Stx1, LPS, or Stx1 + LPS. (A) Differentiated THP-1 cells (5x106 cells/well) were treated with purified Stx1 (400 ng/ml), LPS (200 ng/ml), or both for the indicated time periods. Cell lysates were prepared, and protein was quantified according to the procedure in Materials and Methods. Equal quantities of proteins were subjected to SDS-PAGE, and separated proteins were transferred to nitrocellulose membranes. Western blotting was performed using a phospho-JNK1/2-specific antibody (WB: Phospho JNK). Membranes were stripped and reprobed with antibodies recognizing the activated and nonactivated forms of JNK1/2 [WB: JNK (loading control)]. Blots shown are representative of three independent experiments. Depiction of the kinetics of JNK1/2 activation in response to treatment with (B) Stx1, (C) LPS, or (D) Stx1 + LPS. The densities of bands from at least three independent experiments were quantified, and fold-increase ± SEM was compared with control cells represented as line graphs. *, Time-points at which Stx1 + LPS-induced JNK1/2 activation significantly differed from cells treated with Stx1 or LPS alone (P<0.005). **, Time-point at which LPS-induced JNK1/2 activation was significantly different from Stx1-treated cells. C, Zero-hour control values.
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9.5-fold increased above controls, were reached at 6 h and 12 h of LPS and Stx1 stimulation, respectively. Differences between Stx1 and LPS-mediated p38 activation at these peak values were statistically significant (P<0.05). With the exception of the 1-h time-point, treatment of THP-1 cells with Stx1 + LPS compared with treatment with either stimulant alone resulted in significantly higher (P<0.001) p38 MAPK activation. A major difference noted between JNK and p38 activation triggered by Stx1 + LPS was that phospho-JNK levels declined rapidly after 1 h, whereas p38 MAPK levels remained elevated over the course of the experiments.
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Figure 2. Activation of p38 MAPK in THP-1 cells treated with Stx1, LPS, or Stx1 + LPS. (A) Differentiated THP-1 cells were treated with Stx1, LPS, or both for the indicated time periods. Cell lysates were subjected to Western blotting using a phospho-p38 MAPK-specific antibody (WB: P-p38). Blots were stripped and reprobed with antibodies recognizing activated and nonactivated forms of p38 MAPK [WB: p38 (loading control)]. Blots shown are representative of three independent experiments. MW, Molecular weight. (B) Depiction of the kinetics of p38 MAPK activation in response to treatment with Stx1 ( ), LPS ( ), or Stx1 + LPS ( ). The densities of bands from at least three independent experiments were quantified, and fold-increase ± SEM was compared with control cells. *, Time-points at which Stx1 + LPS-induced p38 MAPK activation differed significantly from cells treated with Stx1 or LPS alone (P<0.005). #, Time-points at which LPS-induced p38 MAPK activation differed significantly from Stx1-treated cells. C, Zero-hour control values.
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Figure 3. Activation of ERK1/2 (p44/p42) after stimulation of THP-1 cells with Stx1, LPS, or Stx1 + LPS. (A) Differentiated THP-1 cells were treated with Stx1, LPS, or both for the indicated time periods. Cell lysates were subjected to Western blotting using a phospho-ERK1/2-specific antibody (WB: P-Erk 1/2). Membranes were stripped and reprobed with antibodies recognizing the activated and nonactivated forms of ERK1/2 [WB: Erk 1/2 (loading control)]. Blots shown are representative of three independent experiments. Depiction of the kinetics of ERK1/2 activation in response to treatment with (B) Stx1, (C) LPS, or (D) Stx1 + LPS. The densities of bands from at least three independent experiments were quantified, and fold-increase ± SEM was compared with control cells represented as line graphs. *, Time-points at which Stx1 + LPS-induced ERK1/2 activation significantly differed from cells treated with Stx1 or LPS alone (P<0.005). C, Zero-hour control values.
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transcripts and soluble protein were induced in THP-1 cells treated with Stx1 + LPS [26
, 41
]. In the presence of both stimulants, optimal levels of eIF4E phosphorylation were detected, peaking at an 11-fold increase over control cells at 1 h, and activation was maintained over 4 h of toxin stimulation. These values were significantly different (P<0.01) compared with treatment with Stx1 or LPS alone. After 6 h, eIF4E phosphorylation returned to control values for all stimulants (data not shown). We failed to detect significant differences in eIF4E activation in unstimulated control cells over a 24-h time period (data not shown).
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Figure 4. Phosphorylation of eIF4E in THP-1 cells treated with Stx1, LPS, or Stx1 + LPS. (A) Differentiated THP-1 cells (5x106) were treated with purified Stx1 (400 ng/ml), LPS (200 ng/ml), or Stx1 + LPS for the indicated time periods. Cells were lysed, and soluble proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes as described in Materials and Methods. Membranes were probed with antibodies specific for the phosphorylated form of eIF4E (WB: P-eIF4E). Blots were stripped and reprobed with antibodies recognizing the activated and nonactivated forms of eIF4E [WB: eIF4E (loading control)]. Blots shown are representative of three independent experiments. (B) Graphical representation of the kinetics of eIF4E phosphorylation in cells treated with Stx1 (), LPS (), or both (). Fold increases ± SEM were calculated from densities of phospho-specific bands compared with basal values for each time-point from at least three independent experiments. *, Time-points that significantly differed (P<0.05) between THP-1 cells treated with Stx1 + LPS versus treatment with Stx1 or LPS alone. **, Time-point that significantly differed between treatment with Stx1 versus LPS alone. #, Time-point that significantly differed between THP-1 cells treated with Stx1 versus unstimulated control cells. C, Unstimulated control values.
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, IL-1ß, or IL-8 from differentiated THP-1 cells in vitro [26
, 29
, 41
]. Smith et al. [25
] showed that heat-inactivated Stx1 and a single-site Stx1 A subunit mutant failed to activate p38 and JNK in human intestinal epithelial cells. If MAPK activation were required for eIF4E activation, contributing to the increased expression of cytokines, we hypothesized that Stxl B subunits and Stx1 holotoxin molecules containing a double mutation in the active site of the A subunit (E167Q; R170L) would be unable to induce activation of eIF4E. As shown in Figure 5A
and 5B
, neither Stx1 B subunits alone nor the Stx1A– (E167Q; R170L) double mutant were capable of inducing eIF4E phosphorylation above basal activation levels. Stxs and anisomycin have been shown to elicit the ribotoxic stress response as defined by the rapid activation of JNK and p38 MAPKs [23
24
25
], and we show here (Figs. 4
and 5C)
that both protein synthesis inhibitors activate eIF4E in THP-1 cells. These data suggest that the ribotoxic stress response may activate eIF4E through the stress-activated protein kinase cascades.
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Figure 5. Effect of Stx1 B subunits, enzymatic mutant holotoxin Stx1A– (E167Q; R170L), and the ribotoxic stress inducer anisomycin on eIF4E phosphorylation. Differentiated THP-1 cells (5x106 cells/well) were treated with (A) purified Stx1 B subunits (800 ng/ml), (B) enzymatic mutant holotoxin Stx1A– (E167Q; R170L; 400 ng/ml), or (C) anisomycin (1.0 µg/ml) for the indicated time periods. Cell lysates were prepared, equal concentrations of proteins were separated by SDS-PAGE, and the proteins were transferred to nitrocellulose membranes, which were probed with antibodies recognizing activated eIF4E (WB: P-eIF4E). The same membranes were stripped and probed with eIF4E antibody recognizing activated and nonactivated forms of eIF4E to check for equal protein loading (WB: eIF4E). Data shown are representative blots from at least three independent experiments. C, Zero-hour control.
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Figure 6. Effect of Mnk1 and MAPK inhibitors on eIF4E activation. Differentiated THP-1 cells were treated with (A) the Mnk1 inhibitor CGP57380 (20 and 40 µM) or (B) a combination of the JNK1/2 inhibitor SP600125 (50 µM), p38 MAPK inhibitor SB203580 (20 µM), and ERK1/2 inhibitor PD98059 (50 µM) for 1 h prior to stimulation with Stx1 (400 ng/ml) for 4 or 6 h. Cell lysates were prepared, and soluble proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes, which were probed with antibodies recognizing the activated form of eIF4E (WB: P-eIF4E). Blots were stripped and reprobed with antibodies recognizing the activated and nonactivated forms of eIF4E for equal protein loading (WB: eIF4E). Dose-dependent inhibition of Stx1-induced eIF4E phosphorylation by the Mnk1 inhibitor is shown in A, lanes 4 and 5 (20 µM and 40 µM CGP57380, respectively). Pretreatment of cells with vehicle alone [dimethyl sulfoxide (DMSO)] prior to Stx1 exposure failed to alter eIF4E activation (A, lanes 2 and 3; B, lane 2). Treatment of cells with the inhibitors alone did not alter eIF4E activation (data not shown). (C) Western blotting was performed on cell lysates derived from THP-1 cells pretreated with the Mnk1 inhibitor (40 and 60 µM) for 1 h before exposure to LPS (200 ng/ml) or Stx1 + LPS (400 and 200 ng/ml, respectively) for 1 or 2 h (WB: P-eIF4E). Blots were stripped and reprobed to check for equal protein loading (WB: eIF4E). Dose-dependent inhibition of eIF4E phosphorylation induced by LPS or Stx1 + LPS by the Mnk1 inhibitor is shown in C, lanes 4 and 5 (40 µM and 60 µM CGP57380, respectively). Pretreatment of cells with vehicle alone (DMSO) prior to LPS or Stx1 + LPS treatment failed to alter eIF4E activation (C, lanes 2 and 3). Data shown are representative blots from at least three independent experiments. C, Zero-hour control.
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Figure 7. Effect of Mnk1 inhibition on total protein synthesis. Differentiated THP-1 cells (1.0x106 cells/well) were treated with Stx1 (400 ng/ml), with or without pretreatment with the Mnk1 inhibitor CGP57380 (CGP; 50 µM) for 1 h or were treated with the Mnk1 inhibitor alone or with anisomycin (1.0 µg/ml) for the indicated time periods. [3H]-Leucine incorporation into TCA-insoluble material was measured by the procedure described in Materials and Methods. The data are expressed as percentage [3H]-leucine incorporation in comparison with basal protein synthesis (untreated cells). The data are derived from triplicate values at each time-point from two independent experiments. *, A significant difference (P<0.05) between the values for cells treated with Stx1 alone versus Stx1 + CGP57380-treated cells.
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Table 1. Effect of Pretreatment with JNK (SP600125), p38 (SB203580), and ERK (PD98059) MAPK Inhibitors or Mnk1 Inhibitor (CGP57380) on the Production of Soluble IL-1ß in Stx1 and/or LPS-Treated THP-1 Cells
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Table 2. Effect of Pretreatment with JNK (SP600125), p38 (SB203580), and ERK (PD98059) MAPK Inhibitors or Mnk 1 Inhibitor (CGP57380) on the Production of Soluble IL-8 in Stx1 and/or LPS-Treated THP-1 Cells
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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MAPK interacting kinases (Mnk)1/2 are serine/threonine kinases involved in eIF4E activation [46
, 47
]. Mnk1 has been shown to have low basal activity, which is enhanced by agents that activate the ERK and p38 MAPKs. Mnk2, in contrast, has high basal activity, which is not enhanced by agents that activate MAPKs, and Mnk2 is thought to be primarily responsible for maintenance of basal levels of eIF4E phosphorylation [35
, 44
]. We have used a low molecular weight Mnk1-specific inhibitor (CGP57380 [48
]) to show that Mnk1 mediates Stx1- and LPS-induced eIF4E phosphorylation. At the concentrations of the Mnk1 inhibitor used in this study, we did not detect toxic effects caused by the inhibitor alone (data not shown). Our findings are consistent with the recent report of Colpoys et al. [36
], showing that Stxs induce eIF4E activation in the Hct-8 intestinal epithelial cell line. We also show that the Mnk1 activating kinases ERK1/2 and p38, as well as JNK1/2, are phosphorylated in response to Stx1. ERK1/2 transduces signals important for growth in response to serum factors and mitogens, and NIH3T3 cells overexpressing ERK1/2, show an elevated basal level of eIF4E phosphorylation [49
]. JNK1/2 and p38 MAPKs are activated by cell stressors such as ultraviolet light, DNA damage, and protein synthesis inhibition and regulate cell growth, differentiation, apoptosis, and cytokine expression [20
21
22
]. The kinetic evaluation of MAPK activation showed that Stx1 induced the activation of JNK and p38 MAPKs, but maximal activation required 6 and 12 h of toxin stimulation, respectively. The induction of JNK and p38 MAPK activation by LPS was rapid but not prolonged, returning to near basal levels within the time course of the experiments. Stx1 was not a vigorous inducer of ERK1/2 activation, and LPS mediated prolonged activation of ERK1/2. The simultaneous exposure of THP-1 cells to Stx1 + LPS produced the highest levels of MAPK phosphorylation, although levels of activated JNK and ERK declined over time, and levels of phospho-p38 remained elevated. We speculate that the decline in activated JNK and ERK levels in Stx1 + LPS-treated cells may be a result of apoptosis, as we have shown, using deoxyuridine triphosphate nick-end labeling (TUNEL) staining, that 40% of macrophage-like THP-1 cells are TUNEL-positive 12 h after treatment with Stx1 + LPS [29
]. Yet, in the face of significant apoptosis, p38 activation is maintained in Stx1 + LPS-treated cells.
Stx1 enzymatic activity and the ribotoxic stress response appear to be necessary to trigger eIF4E activation. Stx1 enzymatic mutant holotoxin or purified Stx1 B subunits did not activate eIF4E. These data correlate with earlier studies showing that purified B subunits do not activate MAPK cascades or trigger cytokine expression [24 , 26 , 41 ]. Stx1 and anisomycin are protein synthesis inhibitors acting on the 28S rRNA component of eukaryotic ribosomes to block the peptidyl transferase reaction and activate the ribotoxic stress response. Stx1 and anisomycin activated eIF4E in THP-1 cells. Finally, eIF4E activation appears to be important in mediating a transient increase in protein synthesis in THP-1 cells intoxicated with Stx1, as a Mnk1-specific inhibitor blocked the rapid increase in protein synthesis but did not affect the long-term inhibition of protein synthesis seen with prolonged exposure to Stx1.
The consequences of prolonged activation of MAPKs by Stx1 in THP-1 cells may be correlated with the prolonged activation of cytokine genes and/or the stabilization of cytokine mRNA transcripts, resulting in increased levels of IL-1ß and IL-8 being detected in culture supernatants. In support of this, ricin treatment of murine monocytic cells activated MAPKs, which were required for the expression of several proinflammatory mediators and transcription factors [50 ]. The biological consequences of transient versus prolonged activation of MAPK cascades remain to be fully characterized but may involve such fundamental processes as increased inflammation and cytokine expression and signaling for cell survival or programmed cell death. In light of these observations, we measured soluble IL-1ß and IL-8 levels produced by Stx1, LPS, or Stx1 + LPS-treated THP-1 cells cultured in the presence of single MAPK inhibitors or in the presence of combinations of inhibitors. A partial reduction in IL-1ß and IL-8 production was observed with individual inhibitors (data not shown) and with combinations of the inhibitors. In our hands, the p38 and ERK cascades appeared most important in Stx1-induced signaling, as inhibitors of these MAPKs were most effective at blocking cytokine expression. However, interpretation of the data was confounded by the fact that treatment of THP-1 cells with JNK + p38 inhibitors alone reproducibly increased IL-1ß and IL-8 expression above basal levels expressed by unstimulated cells, suggesting that these MAPK cascades are normally involved in the negative regulation of cytokine expression. Inhibitors of all three major MAPK cascades and the Mnk1-specific inhibitor significantly reduced (73–96%) cytokine expression compared with stimulation with Stx1 and/or LPS without the inhibitors. The data presented here suggest that all three kinase cascades may contribute to increased cytokine expression. Previous work using NIH3T3 cells suggested that more than one kinase family was involved in regulating eIF4E phosphorylation. ERK1/2, activated in the presence of serum, contributed to eIF4E phosphorylation, which was inhibited by the ERK1/2 inhibitor PD98059 but not by the p38 MAPK inhibitor SB203580, whereas eIF4E activation, mediated by p38 MAPK, was inhibited by the p38 inhibitor but not by the ERK inhibitor [51 ]. We show here that use of inhibitors for all three kinase families, as well as the Mnk1-specific inhibitor, reduced eIF4E phosphorylation in response to Stx1 and/or LPS.
The precise role of cytokines in the pathogenesis of HUS requires additional study. Serum TNF- and IL-1ß levels in HUS patients have been reported to range from 44 to 568 pg/ml and 55 to 70 pg/ml, respectively [52
, 53
]. These values are in the range induced by purified Stx1 but less than the values induced by Stx1 + LPS from THP-1 cells in vitro [26
, 30
]. However, detection of elevated serum cytokines is not a consistent finding in patients with HUS. Karpman et al. [54
] detected elevated serum TNF- levels (defined as >25 pg/ml) in only seven of 31 HUS patients, and Murata et al. [53
] reported that only six of 12 HUS patients had elevated serum TNF- levels, and nine of 12 had elevated serum IL-8 levels on the first day of hospitalization. There is evidence that cytokines may be induced in a focal manner in HUS, and sites of tissue damage may correspond with sites of cytokine expression. For example, mice administered purified Stxs developed acute tubular necrosis [37
]. Harel et al. [55
] infused purified Stx1 into TNF- promoter:chloramphenicol acetyltransferase (CAT) transgenic mice and detected CAT activity only in the kidneys. In contrast to HUS, numerous studies have shown that prolonged dysregulation of cytokine expression is a hallmark of endotoxic shock and can lead to a systemic "spillover effect" in which cytokines mediate deleterious effects. Thus, although we have shown differences in ERK activation, in the kinetics and extent of JNK, p38, and eIF4E activation, and in the amounts of soluble IL-1ß and IL-8 produced in macrophage-like THP-1 cells treated with Stx1 versus LPS, both bacterial products appear to signal through MAPKs and eIF4E. Therefore, additional factors may be responsible for the different pathophysiological characteristics of HUS and endotoxic shock.
Received June 13, 2005; revised October 6, 2005; accepted October 10, 2005.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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on human vascular endothelial cells in vitro Infect. Immun. 59,4173-4179-sarcin/ricin loop in the 28S rRNA Mol. Cell. Biol. 17,3373-3381 J. Leukoc. Biol. 71,107-114
radiation: duration of JNK activation may determine cell death and proliferation J. Biol. Chem. 271,31929-31936 gene transcription and nuclear translocation of the transcriptional activators nuclear factor-B and activator protein-1 Blood 92,558-566 by lipopolysaccharide J. Biol. Chem. 273,974-980 expression: requirements for toxin enzymatic activity and monocyte protein kinase C and protein tyrosine kinases Infect. Immun. 68,5183-5189This article has been cited by other articles:
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R. P. Cherla, S.-Y. Lee, R. A. Mulder, M.-S. Lee, and V. L. Tesh Shiga Toxin 1-Induced Proinflammatory Cytokine Production Is Regulated by the Phosphatidylinositol 3-Kinase/Akt/Mammalian Target of Rapamycin Signaling Pathway Infect. Immun., September 1, 2009; 77(9): 3919 - 3931. [Abstract] [Full Text] [PDF]
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K. A. D. Sauter, A. R. Melton-Celsa, K. Larkin, M. L. Troxell, A. D. O'Brien, and B. E. Magun Mouse Model of Hemolytic-Uremic Syndrome Caused by Endotoxin-Free Shiga Toxin 2 (Stx2) and Protection from Lethal Outcome by Anti-Stx2 Antibody Infect. Immun., October 1, 2008; 76(10): 4469 - 4478. [Abstract] [Full Text] [PDF]
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 H. K. Bae and J. J. Pestka Deoxynivalenol Induces p38 Interaction with the Ribosome in Monocytes and Macrophages Toxicol. Sci., September 1, 2008; 105(1): 59 - 66. [Abstract] [Full Text] [PDF]
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 C. Zanchi, C. Zoja, M. Morigi, F. Valsecchi, X. Y. Liu, D. Rottoli, M. Locatelli, S. Buelli, A. Pezzotta, P. Mapelli, et al. Fractalkine and CX3CR1 Mediate Leukocyte Capture by Endothelium in Response to Shiga Toxin J. Immunol., July 15, 2008; 181(2): 1460 - 1469. [Abstract] [Full Text] [PDF]
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M. K. Stone, G. L. Kolling, M. H. Lindner, and T. G. Obrig p38 Mitogen-Activated Protein Kinase Mediates Lipopolysaccharide and Tumor Necrosis Factor Alpha Induction of Shiga Toxin 2 Sensitivity in Human Umbilical Vein Endothelial Cells Infect. Immun., March 1, 2008; 76(3): 1115 - 1121. [Abstract] [Full Text] [PDF]
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M. Brigotti, D. Carnicelli, E. Ravanelli, A. G. Vara, C. Martinelli, R. R. Alfieri, P. G. Petronini, and P. Sestili Molecular Damage and Induction of Proinflammatory Cytokines in Human Endothelial Cells Exposed to Shiga Toxin 1, Shiga Toxin 2, and {alpha}-Sarcin Infect. Immun., May 1, 2007; 75(5): 2201 - 2207. [Abstract] [Full Text] [PDF]
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S.-Y. Lee, R. P. Cherla, and V. L. Tesh Simultaneous Induction of Apoptotic and Survival Signaling Pathways in Macrophage-Like THP-1 Cells by Shiga Toxin 1 Infect. Immun., March 1, 2007; 75(3): 1291 - 1302. [Abstract] [Full Text] [PDF]
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J. Pestka and H.-R. Zhou Toll-Like Receptor Priming Sensitizes Macrophages to Proinflammatory Cytokine Gene Induction by Deoxynivalenol and Other Toxicants Toxicol. Sci., August 1, 2006; 92(2): 445 - 455. [Abstract] [Full Text] [PDF]
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