Department of Medical Microbiology and Immunology, Texas A & M University Health Science Center, College Station, Texas
Correspondence: Vernon L. Tesh, Department of Medical Microbiology and Immunology, Texas A & M University Health Science Center, Rm. 407 Reynolds Medical Building, College Station, TX 77843-1114. E-mail: tesh{at}medicine.tamu.edu
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(TNF-) detectable in urine. Thus, sequelae may
develop following the localized production of proinflammatory cytokines
within the kidneys. A possible source of these cytokines are
macrophages, which respond to the toxins by producing TNF-. We
have shown previously that THP-1 cells produce soluble TNF- in
response to the toxins, whose production requires host-cell
tyrosine-kinase activity and toxin-enzymatic activity. To further
examine signaling pathways involved in TNF- expression, we
determined that JNK1 and -2 and p38, but not ERK1 or -2, were
phosphorylated following toxin exposure. Blockade of p38 activation
reduced TNF- production following Shiga toxin 1 treatment. Finally,
we present a model of the ribotoxic stress response triggered in human
macrophages by Shiga toxins.
Key Words: cytokines • ERK • HUS • ribosome • signal transduction
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A group of structurally and functionally related toxins comprise the
Shiga toxin family, including the prototypical Shiga toxin (Stx) of
Shigella dysenteriae serotype 1 and toxins designated Stx1,
Stx2, Stx2c, Stx2d, Stx2e, and Stx2f produced by E. coli
[4
]. All Shiga toxins are AB5 molecules that
inhibit protein synthesis by catalyzing the depurination of a single
adenine residue from the 28S ribosomal RNA, thereby inhibiting
elongation factor-1-dependent peptide elongation [5
6
7
].
The pentameric ring of B subunits mediates toxin binding to target
cells through interaction with the neutral glycolipid
globotriaosylceramide (Gb3) [8
, 9
].
Experiments using cultured human vascular endothelial cells derived
from a variety of sources demonstrated that the cells were markedly
sensitized to the cytotoxic action of Shiga toxins by stimulation with
the proinflammatory cytokines tumor necrosis factor (TNF-) or
interleukin (IL)-1ß [10
11
12
]. The sensitization
phenomenon was associated with the up-regulated expression of Gb3 on
the cell surface leading to increased toxin binding and internalization
[11
]. Monocytes or macrophages exposed to purified Shiga
toxins in vitro respond by increased expression and secretion of
TNF- and IL-1ß, suggesting that cells of the myeloid lineage found
within the kidneys and CNS may be important sources of the cytokines
that sensitize endothelial cells to the toxins.
Our studies to elucidate the mechanism(s) of Stx-induced
proinflammatory cytokine expression have demonstrated that exposure of
fully differentiated THP-1 cells (a human monocytic cell line) to
purified Stxs in vitro results in the following events: the toxins bind
the cells and are internalized; there is rapid nuclear translocation of
nuclear factor-
B (NF-B), coincident with the degradation of
cytoplasmic IB; there is increased AP-1 binding activity;
transcriptional activation of the TNF- gene is detected by Northern
blot analysis; and increased levels of soluble TNF- and IL-1ß are
detected in culture supernatants [13
]. Cytokine
production requires enzymatically active toxins. Finally, TNF-
transcription and production by THP-1 cells in response to toxin
treatment are decreased by broad-spectrum protein kinase C (PKC) and
protein tyrosine kinase (PTK) inhibitors [14
]. In this
study, we further characterize the intracellular signaling pathways
involved in Stx1-induced TNF- production by THP-1 cells by screening
for activation of the terminal kinases of the major mitogen-activated
protein kinase (MAPK) pathways, the c-jun NH2-terminal
kinase (JNK), extracellular signal-regulated kinase (ERK), and p38
kinase cascades. These three pathways are known to be involved in the
expression of TNF- following treatment of monocytes with a variety
of stimulants, such as bacterial lipopolysaccharides (LPS)
[15
]. The MAPK pathways consist of a series of kinases,
which sequentially phosphorylate downstream kinases to transmit
intracellular signals. These pathways typically involve the activation
of a MAP kinase kinase kinase (MAPKKK), which phosphorylates and
activates a MAP kinase kinase (MAPKK), which in turn phosphorylates a
MAPK. Once activated, the MAPKs phosphorylate downstream cellular
substrates such as transcription factors and other protein kinases
[16
]. All isoforms of the MAPKs studied here are
activated by dual phosphorylation at threonine and tyrosine residues.
Using antibodies specifically recognizing the phosphorylated forms of
the MAPKs, we examined JNK, ERK, and p38 in Stx1-treated THP-1 cells
for possible activation of these pathways, as well as a possible role
for the p38 cascade in the production of TNF-.
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Cell lines
The human myelogenous leukemia cell line THP-1 was purchased
from American Type Culture Collection (ATCC; Manassas, VA) and
maintained in RPMI-1640 (Gibco-BRL, Grand Island, NY) supplemented with
penicillin (50 U/ml), streptomycin (50 µg/ml), and 10% fetal bovine
serum (FBS; Hyclone Laboratories, Logan, UT) at 37°C in humidified
5% CO2. Prior to use in experiments, THP-1 cells were
induced to differentiate into the mature macrophage-like state by
treatment with phorbol myristate acetate (PMA; Sigma Chemical Co., St.
Louis, MO) at 50 ng/ml for 48 h. Differentiated cells were washed
twice with cold Dulbecco’s phosphate-buffered saline (PBS; Sigma
Chemical Co.) and incubated with fresh media lacking PMA with daily
media changes for 72 h. The murine macrophage cell line RAW 264.7
(kindly provided by Dr. Roderick McCallum, Texas A & M University
Health Science Center, College Station, TX) was maintained in
Dulbecco’s modified Eagle’s medium (DMEM; Gibco-BRL) with the above
supplements.
Preparation of cellular extracts
THP-1 cells (5x106) were differentiated with PMA in
60 mm culture dishes in 5.0 ml supplemented RPMI-1640. Eighteen hours
prior to stimulation, cells were washed twice in cold Dulbecco’s PBS,
and media was replaced with RPMI-1640 with penicillin/streptomycin and
0.5% FBS to reduce endogenous MAPK activity. A similar procedure was
used for RAW 264.7 cells, where 5 x 106 cells were
plated in 60 mm tissue-culture dishes and allowed to adhere overnight.
The cells were washed twice with cold PBS and serum starved for 18 h in DMEM containing penicillin/streptomycin and 0.5% FBS prior to
stimulation. PMA-differentiated THP-1 or RAW 264.7 cells were
stimulated with one of the following reagents for 15 min: PMA (Sigma
Chemical Co.) at 200 ng/ml, purified LPS derived from E.
coli O111:B4 (Sigma Chemical Co.) at 10 µg/ml, or anisomycin
(Calbiochem, La Jolla, CA) at 1.0 µg/ml or were stimulated for 1 h with Stx1 at 800 ng/ml, Stx1 and LPS (800 ng/ml and 10 µg/ml,
respectively), or ricin (Sigma Chemical Co.) at 100 pg/ml or 10
µg/ml. The cells were washed twice in cold Dulbecco’s PBS and lysed
in modified RIPA buffer [1.0% Nonidet P-40 (NP-40), 1.0%
Na-deoxycholate, 150 mM NaCl, 10 mM Tris-HCl (pH 7.5), 5.0 mM
Na-pyrophosphate, 1.0 mM NaVO4, 5.0 mM NaF, 1.0 µg/ml
aprotinin, 1.0 µg/ml leupeptin, and 0.1 mM phenylmethylsulfonyl
fluoride (PMSF)] for 15 min at 4°C. Extracts were collected and
passed through 22- and 27-gauge needles to shear DNA. The extracts were
cleared by centrifugation at 15,000 g for 15 min. Cleared
extracts were stored at -80°C until used. For dose-response
analysis, differentiated THP-1 cells were exposed to increasing
concentrations of Stx1 (50 ng/ml–1 µg/ml) for 1 h followed by
extraction. Kinetic analysis of p38 and JNK was performed by incubating
differentiated THP-1 cells with 800 ng/ml Stx1 for increasing amounts
of time followed by extraction and analysis.
Western blot analysis
Protein content in cell extracts prepared from stimulated THP-1
cells was determined using the Micro BCA Protein Assay Kit (Pierce,
Rockford, IL). Equal amounts of proteins (60–80 µg protein per gel
lane) were separated by 12% Tris-glycine sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred
to polyvinylidene difluoride (PVDF) membranes. Membranes were blocked
with TBS [20 mM Tris (pH 7.6) and 137 mM NaCl] containing 0.1% Tween
20 and 3.0% bovine serum albumin (BSA). The following antibodies from
New England Biolabs (Beverly, MA) suspended in TBS-0.1% Tween 20 and
3.0% BSA were used to detect activated kinases: Phosphorylated ERK1
and -2 were detected with p44/42 MAPK (Thr202/Tyr204) monoclonal
antibody (mAb) E10, phosphorylated JNK1 and -2 were detected with
stress-activated protein kinase (SAPK)/JNK (Thr183/Tyr185) mAb G9, and
phosphorylated p38 was detected using polyclonal antiphospho-p38
(Thr180/Tyr182) antibodies. The primary antibodies were bound by a
polyclonal antimouse or antirabbit horseradish peroxidase (HRP)
conjugate (Amersham, Arlington Heights, IL) and detected by enhanced
chemiluminescence (ECL; Amersham). Blots were stripped with stripping
buffer [10 mM ß-mercaptoethanol, 2.0% SDS, and 62.5 mM Tris-HCl (pH
6.7)] and reprobed with anti-ERK1 (sc-93-G), anti-JNK1 (sc-571), or
anti-p38 antibodies (sc-535; Santa Cruz Biotechnology, Santa Cruz, CA),
which bind irrespective of phosphorylation state. These antibodies were
selected to demonstrate equal loading and to show that the levels of
the respective kinases were unchanged as a result of the activity of
the protein-synthesis inhibitors. Blots were analyzed densitometrically
using an IS1000 imaging system (Alpha Innotech Corp., Leandro, CA). The
results are presented as a percentage of basal activity defined as
(phosphorylated form of kinase/unphosphorylated form) x
100%/(phosphorylated form of kinase in unstimulated
cells/unphosphorylated form in unstimulated cells). Data shown
are means SE from at least three independent experiments.
Analysis of TNF-
Concentrations of immunoreactive TNF- in culture supernatants
were determined using a commercial enzyme-linked immunosorbent assay
(ELISA) kit (Quantikine, R&D Systems, Minneapolis, MN), following the
manufacturer’s instructions. The sensitivity of this assay is 4.4
pg/ml.
Inhibitor studies
The p38 inhibitor SB202190 was purchased from Calbiochem and
dissolved in dimethyl sulfoxide (DMSO) at a concentration of 20 mM. For
use in inhibitor studies, a concentration of 3 µM was prepared in
RPMI-1640 medium. Cells were pretreated for 30 min with the inhibitor
or vehicle only. Supernatants were removed and replaced with media
containing Stx1 or LPS with the inhibitor, with inhibitor alone, or
with DMSO alone and incubated for an additional 24 h. Supernatants
were then collected and analyzed for TNF- production by ELISA. Total
organic solvents in all assays were <0.1%. SB202190 was not cytotoxic
at the concentrations used in the studies as assessed by the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)
assay [19
].
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production
in response to Stx1 treatment required toxin-enzymatic activity and
monocyte PTK activity [14
], we hypothesized that Stx1
treatment of THP-1 cells may activate the JNK cascade. Using Western
blot analysis, we found that following the exposure of THP-1 cells to
Stx1 for 1 h, JNK 1 and -2 phosphorylation increased (Fig. 1A
and B). JNK 1 and -2 phosphorylation state was also
increased by treatment with the protein-synthesis inhibitors anisomycin
and ricin. Like Stx1, anisomycin and ricin act on the
peptidyltransferase center of the ribosome to mediate protein-synthesis
inhibition, and anisomycin and ricin have been shown to activate the
JNK cascade in rat fibroblasts [24
25
26
]. There have been
several studies demonstrating the activation of JNK following treatment
of monocytes with high levels of LPS [27
]. Therefore, we
used high concentrations of LPS as a positive control for JNK
activation. Treatment of cells with LPS increased JNK phosphorylation
but to a lesser extent compared with Stx1 treatment. Treatment with a
combination of Stx1 and LPS did not result in JNK activation profiles
markedly different from treatment with Stx1 alone.
 View larger version (48K): [in a new window] |
Figure 1. Stx1-induced phosphorylation of JNK1 and -2 in differentiated THP-1
cells. Cells (5x106) were exposed to PMA (200 ng/ml), LPS
(10 µg/ml), or anisomycin (1 µg/ml) for 15 min or to Stx1 (800
ng/ml), Stx1 and LPS (800 ng/ml and 10 mg/ml, respectively), or ricin
(10 µg/ml) for 1 h followed by extraction, as described in
Materials and Methods. Cell extracts were then analyzed by SDS-PAGE and
Western blotting using a mAb that recognizes the phosphorylated form of
JNK1 and -2. Blots were stripped and reprobed with anti-JNK1, which
binds irrespective of phosphorylation state to verify equal loading.
(A) Graphical representation of densitometric results of three
independent experiments. Percentages were calculated as described in
Materials and Methods. (B) Representative Western blot of
antiphospho-JNK1 and -2 (upper panel) and its corresponding anti-JNK1
blot (lower panel).
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 View larger version (37K): [in a new window] |
Figure 2. Dose-response and kinetic analysis of JNK1 and -2 phosphorylation in
differentiated THP-1 cells. (A) Dose-response analysis. Cells
(5x106) were exposed to increasing doses of Stx1 (50
ng/ml–1 µg/ml, as indicated) for 1 h followed by extraction and
Western blotting as described for Figure 1
. Upper panel, graphical
representation of densitometric results from three independent
experiments; lower panel, representative Western blots corresponding to
antiphospho-JNK1 and -2 (upper blot) and anti–JNK1 and -2 (lower
blot). (B) Kinetic analysis. Cells (5x106) were exposed to
800 ng/ml Stx1 for increasing amounts of time (15 min–6 h, as
indicated), followed by extraction and Western blotting as described in
Figure 1
. Upper panel, graphical representation of densitometric
results from three independent kinetic analyses; lower panel,
representative Western blots corresponding to antiphospho-JNK1 and -2
(upper blot) and anti-JNK1 and -2 (lower blot). Both sets of
experiments included anisomycin (1 µg/ml for 15 min) as a positive
control.
|
 View larger version (44K): [in a new window] |
Figure 3. Stx1 does not induce the phosphorylation of ERK1 and -2 in
differentiated THP-1 cells. Cells (5x106) were exposed to
PMA (200 ng/ml), LPS (10 µg/ml), or anisomycin (1 µg/ml) for 15 min
or to Stx1 (800 ng/ml), Stx1 and LPS (800 ng/ml and 10 µg/ml,
respectively), or ricin (10 µg/ml) for 1 h followed by
extraction, as described in Materials and Methods. Cell extracts were
then analyzed by SDS-PAGE and Western blotting using a mAb that
recognizes the phosphorylated form of ERK1 and -2. Blots were stripped
and reprobed with anti-ERK1, which binds irrespective of
phosphorylation state to verify equal loading. (A) Graphical
representation of densitometric results of phosphorylated ERK1 and -2
from three independent experiments. Percentages were calculated as
described in Materials and Methods. (B) Upper and lower panels,
respectively, representative Western blot of antiphospho-ERK1 and -2
and its corresponding anti-ERK1 blot.
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expression in response to LPS
treatment [37
, 38
]. We hypothesized that
p38 may be phosphorylated in response to treatment with Stx1.
Differentiated THP-1 cells were treated for 1 h with Stx1, LPS,
Stx1 + LPS, or two doses of ricin, or for 15 min with anisomycin.
Cells were then lysed, and p38 activation was determined by Western
blotting (Fig. 4A
and B). All stimulants tested caused increased p38
activation over basal levels. Similar to the dose response for JNK1
activation, p38 phosphorylation was maximal at a toxin dose of 800
ng/ml and decreased at the highest toxin concentration used in the
analysis (Fig. 5 A
). The p38 kinase is phosphorylated rapidly in response to Stx1
treatment, reaching maximal levels at 15 min and decreasing over a
6 h time period (Fig. 5B)
.
 View larger version (38K): [in a new window] |
Figure 4. Stx1 induces the phosphorylation of p38 in differentiated THP-1 cells.
Cells (5x106) were exposed to LPS (10 µg/ml) or
anisomycin (1 µg/ml) for 15 min or to Stx1 (800 ng/ml), Stx1 and LPS
(800 ng/ml and 10 µg/ml, respectively), or ricin (100 pg/ml or 10
µg/ml) for 1 h followed by extraction, as described in Materials
and Methods. Cell extracts were then analyzed by SDS-PAGE and Western
blotting using polyclonal antibodies that recognize the phosphorylated
form of p38. Blots were stripped and reprobed with anti-p38, which
binds irrespective of phosphorylation state to verify equal loading.
(A) Graphical representation of densitometric results of three
independent experiments. Percentages were calculated as described in
Materials and Methods. (B) Representative Western blot of
antiphospho-p38 (upper panel) and its corresponding anti-p38 blot
(lower panel).
|
 View larger version (36K): [in a new window] |
Figure 5. Dose-response and kinetic analysis of p38 phosphorylation in
differentiated THP-1 cells. (A) Dose-response analysis. Cells
(5x106) were exposed to increasing doses of Stx1 (50
ng/ml–1 µg/ml, as indicated) for 1 h followed by extraction and
Western blotting as described for Figure 4
. Upper panel, graphical
representation of densitometric results from three independent
experiments; lower panel, representative antiphospho-p38 blot (upper
blot) and corresponding anti-p38 blot (lower blot). (B) Kinetic
analysis. Cells (5x106) were exposed to 800 ng/ml Stx1 for
increasing amounts of time (15 min–6 h, as indicated), followed by
extraction and Western blotting as described in Figure 4
. Upper panel,
graphical representation of densitometric results from three
independent kinetic analyses; lower panel, representative Western blots
of antiphospho-p38 blot (upper blot) and corresponding anti-p38 blot
(lower blot). Both sets of experiments included anisomycin (1 µg/ml
for 15 min) as a positive control.
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expression expression, we used SB202190, a pyrimidyl
imidazole inhibitor of p38 activity [38
,
39
]. Differentiated THP-1 cells were treated with
SB202190 for 30 min, and the medium was then replaced with media
containing Stx1 or LPS (200 ng/mL) and SB202190. After 24 h, supernatants were harvested (400 ng/mL), and immunoreactive TNF-
levels were determined by ELISA (Table 1
). In the presence of the p38 inhibitor, we detected an
approximately 1.5-fold decrease in TNF- synthesis following
stimulation with Stx1 or LPS. Thus, activation of the p38 MAPK cascade
appears necessary for optimal TNF- expression in response to these
stimuli. |
View this table: [in a new window] |
Table 1. Effect of p38 Inhibition on Stx-1-mediated TNF-
Production
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The findings that toxin enzymatic activity and target-cell PTK activity
were required for cytokine expression suggested that Shiga
toxin-mediated damage to ribosomes may activate a cellular-stress
signaling response, ultimately leading to the expression and secretion
of TNF- and other cytokines. Support for the hypothesis that
ribosomal damage triggers intracellular signaling cascades comes from
the work of Iordanov et al. [26
] in which rat
fibroblasts were treated with the protein-synthesis inhibitors
anisomycin or ricin. Although these protein-synthesis inhibitors are
structurally different from the Shiga toxins, all three inhibitors
share the property of acting on the peptidyltransferase center of the
ribosome. Following treatment of fibroblasts with anisomycin or ricin,
increased JNK1 activity was detected. The authors referred to this
phenomenon as the ribotoxic stress response. We suggest that the Shiga
toxins be added to this category of ribotoxins, because treatment of
THP-1 cells with purified Stx1 activated the SAPK cascades JNK and p38.
Using a TNF- translational construct, Swantek et al.
[15
] showed that JNK was involved in the regulation of
TNF- translation following exposure of murine macrophages to LPS. In
addition, JNK is also believed to be involved in transcriptional
activation, particularly of genes regulated by AP-1 because of the
ability of activated JNK to phosphorylate c-Jun [42
].
Using electrophoretic mobility shift assays, we demonstrated increased
DNA binding by AP-1 in THP-1 nuclear lysates after Stx1 treatment,
suggesting the possibility that JNK may be involved in this
transcriptional activation event [13
]. Kojima et al.
[43
] demonstrated an increase in the phosphorylation of
c-Jun in the Caco-2 cell line after exposure to Stx1. Collectively,
these data suggest that in response to ribotoxic stress following the
exposure of THP-1 cells to Stx1, JNK1 and -2 isoforms may be activated
to regulate TNF- expression at transcriptional and
post-transcriptional stages.
We show that treatment of THP-1 cells with Stx1 results in the
activation of p38, and the p38 inhibitor SB202190 partially reduces the
quantities of secreted TNF- detectable after treatment of cells with
Stx1. The linkage between activation of the p38 cascade and cytokine
expression remains to be fully characterized. Wang et al.
[39
] demonstrated that in LPS-treated human monocytes,
p38 activation was associated with enhanced stability of cytokine mRNA
transcripts; i.e., in the presence of SB202190, the stabilities of
TNF-, IL-6, and macrophage-inflammatory protein-1 (MIP-1)
mRNAs decreased. In addition, p38 activation has been linked to control
of TNF- mRNA translation by regulating the phosphorylation of the
translation initiation factor eIF4E [44
].The precise
biological role of p38 activation in Stx1-treated THP-1 cells is
currently unknown. Work in our laboratory is being conducted to
understand the relationship between p38 activation and TNF-
transcriptional activation, translation initiation, and mRNA stability.
The result of Shiga toxin-induced activation of JNK and p38 cascades may not be limited to induction of proinflammatory cytokines. Thorpe et al. [45 ] demonstrated that human intestinal epithelial cells exposed to Stx1 or Stx2 express the chemokine IL-8, and IL-8 expression was inhibited by SB202190 treatment, linking Shiga toxin-induced chemokine expression with p38 activation. Thus, activation of the JNK and p38 cascades may represent a universal cellular response to protein-synthesis inhibitors acting at a particular site in the ribosome. Whether a cell type will respond to Shiga toxins by JNK and p38 activation may be determined by levels of membrane expression of Gb3, relative ratios of Gb3 isoforms (short-carbon chain vs. long-carbon chain fatty acids; [46 ]), and the appropriate routing of the toxins to the ER. The mechanism(s) regulating the types of genes activated following Shiga toxin-induced JNK and/or p38 activation (e.g., chemokines vs. cytokines) remains to be determined. Figure 6 is a summary of the known intracellular signaling events that have been determined to occur in differentiated THP-1 cells following Stx1 exposure.
 View larger version (113K): [in a new window] |
Figure 6. Summary of intracellular signaling events determined to occur in
differentiated THP-1 cells following Stx1 exposure and leading to the
expression of TNF-. Following binding and internalization of
enzymatically active Stx1, ribosomal damage at the peptidyltransferase
center generates a stress response leading to the activation of the
kinases PKC [14
], JNK1 and -2, and p38. Blockade of PKC
and p38 leads to a reduction in TNF- production, although the
mechanisms by which these kinases regulate the transcription and/or
translation of TNF- are still not clearly understood. In addition,
cytoplasmic IB is degraded with a coincident activation and nuclear
translocation of NF-B and an increase in binding of AP-1
[13
]. These two transcription factors may interact with
the TNF- promoter to up-regulate transcription in response to the
toxin.
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Received July 25, 2001; accepted August 21, 2001.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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gene transcription and nuclear translocation of the transcriptional activators nuclear factor-B and activator protein-1 Blood 92,558-566) translation: glucocorticoids inhibit TNF- translation by blocking JNK/SAPK Mol. Cell. Biol. 17,6274-6282[Abstract]-sarcin/ricin loop in the 28S rRNA Mol. Cell. Biol. 17,3373-3381[Abstract]This article has been cited by other articles:
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