 |
INTRODUCTION |
Septic shock is the detrimental consequence of the host
response to a bacterial infection. Septic shock involves hypotension, disseminated intravascular coagulation, and acute organ dysfunction accompanying the inflammatory and procoagulant responses. Hypotension and the signs of inadequate organ perfusion are the major
manifestations of sepsis. The changes in the vascular reactivity do not
depend on infectious pathogens but on disturbances in the coagulation and fibrinolytic cascade (1-5). The cascade of inflammatory and clotting reactions induces the development of disseminated
intravascular coagulation, and microparticles cause the acute
generation of thrombin (6). It has been shown that the level of the
thrombin and antithrombin complexes is higher in patients with sepsis
than in healthy control subjects (7). In sepsis, the level of
antithrombin III (ATIII),1 an
endogenous coagulation inhibitor, is reduced as a result of complex
formation with multiple activated clotting factors (8, 9). The plasma
ATIII level is low in septic patients as a consequence of ATIII
consumption in severe sepsis (10). It is highly likely that the
activation of prothrombin to thrombin by the coagulation pathway
increases the level of unbound free thrombin in these patients.
Vascular hyporeactivity is attributable to excess nitric oxide (NO)
production, a key gaseous molecule inducing the collapse of the
cardiovascular system, by an inducible form of NOS (11). The inducible
nitric-oxide synthase (iNOS) expression and NO production greatly
affect the inflammatory processes (12, 13). Proinflammatory cytokines
such as the tumor necrosis factor-
induce NO production (14, 15).
Activated protein C, as a natural anticoagulant, regulates the
coagulation system by inhibiting thrombin generation and attenuating
the inflammatory responses induced by lipopolysaccharide (LPS).
Protein C prevents LPS-induced hypotension by inhibiting excess
NO production (16). ATIII inhibits nuclear factor-
B (NF-
B)
activation in monocytes and endothelial cells (17). In addition, it has
been shown that ATIII prevents LPS-induced hypotension by inhibiting
NOS induction in animals (18).
Macrophages, which are effector cells in eliminating microorganisms and
other noxious elements, participate in many complex immunological and
inflammatory processes. They produce the cytokines that recruit other
inflammatory cells, which are responsible for the diverse effects of
inflammation. Septic shock syndrome results from an excessive
triggering of endogenous inflammatory mediators, which are released
primarily by activated macrophages (19).
In view of the imbalance between thrombin and antithrombin in septic
patients as a result of the exhaustion of antithrombin and excess NO as
culminating factors in vascular hyporeactivity, this study investigated
the effect of thrombin on the production of NO in macrophages. In
addition, the signaling pathways responsible for the induction of iNOS
by thrombin were examined. This study reports for the first time that
thrombin induces iNOS through I-
B
phosphorylation and subsequent
NF-
B activation via the protein kinase C (PKC) and c-Jun N-terminal
kinase (JNK) pathways in macrophages. Thrombin exerts mitogenic
proliferation through the receptors coupled with the G
12
and G
13 proteins belonging to the G
12
subfamily (20-22). We determined whether thrombin-induced NF-
B
activation occurred via a pathway involving G
12/13. In view of the uncertainty of the PKC linkage to the G
12
subfamily, we assessed whether PKC was associated with the
G
12 family in the activation of NF-
B by thrombin. The
G
12 protein is linked to the Rho-directed guanine
nucleotide exchange factor, p115, and the GTPase-activating protein,
RasGAP1 (23, 24). The G
12/13 proteins are implicated in
the Rho-dependent cytoskeletal shape change and JNK
activation (20, 25, 26). This study further determined that JNK was
involved in the degradation of the phosphorylated I-
B
downstream
of G
12/13.
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EXPERIMENTAL PROCEDURES |
Reagents--
LB30057 was the kind gift from LG Biotech Inc.
(Daeduk, Korea). [
-32P]ATP (3000 mCi/mmol) was
obtained from Amersham Biosciences. Horseradish peroxidase-conjugated
goat anti-rabbit IgG and 5-bromo-4-chloro-3-indolyl phosphate/nitro
blue tetrazolium were supplied from Invitrogen. Alkaline
phosphatase-conjugated goat anti-mouse IgG was purchased from
Kirkegaard & Perry Laboratories (Gaithersburg, MD). Anti-c-Rel (p65),
anti-p50, and anti-I-
B
antibodies were obtained from Santa Cruz
Biotechnology (Santa Cruz, CA). Anti-phospho-I-
B
antibody was
supplied from New England Biolabs (Beverly, MA). Anti-iNOS antibody was
obtained from Transduction Laboratories (Lexington, KY) or Santa Cruz
Biotechnology. Horseradish peroxidase-conjugated and fluorescein
isothiocyanate-conjugated anti-rabbit IgG were obtained from
Zymed Laboratories Inc. (San Francisco, CA).
PD98059 was obtained from Calbiochem. Thrombin and other reagents
in the molecular studies were supplied from Sigma. The Limulus
Amoebocyte Lysate test (i.e. an endotoxin test using the gel
clot method) showed that the thrombin was endotoxin-free with the
sensitivity limit of 0.06 enzyme units/ml. Activated mutants of
G
12/13 (G
12/13QL), wild types
of G
12/13 (G
12/13W), and JNK1 dominant
negative mutant (KmJNK1) were kindly provided from Dr. N. Dhanasekaran
(Fels Institute for Cancer Research and Molecular Biology, Department
of Biochemistry, Temple University).
Cell Culture--
The Raw264.7 cell line was obtained from
American Type Culture Collection (ATCC, Manassas, VA) and maintained in
Dulbecco's modified Eagle's medium containing 10% fetal bovine
serum, 50 units/ml penicillin, and 50 µg/ml streptomycin at 37 °C
in a humidified atmosphere with 5% CO2. Raw264.7 cells
were plated at a density of 5 × 106/10-cm2 dish and preincubated for 24 h
at 37 °C. For all experiments, cells were grown to 80-90%
confluency and were subjected to no more than 20 cell passages. To
compare NO production, Raw264.7 cells were incubated with 1 µg/ml of
LPS (Escherichia coli 026:B6; Difco).
Assay of NO Production--
NO production was monitored by
measuring the nitrite level in the culture medium. This was performed
by mixing with Griess reagent (1% sulfanilamide, 0.1%
N-1-naphthylenediamine dihydrochloride, and 2.5% phosphoric
acid). Absorbance was measured at 540 nm after incubation for 10 min.
Immunoblot Analysis--
SDS-PAGE and immunoblot analyses were
performed according to the procedures published previously (27). Cells
were lysed in the buffer containing 20 mM Tris·Cl (pH
7.5), 1% Triton X-100, 137 mM sodium chloride, 10%
glycerol, 2 mM EDTA, 1 mM sodium orthovanadate, 25 mM
-glycerophosphate, 2 mM sodium
pyrophosphate, 1 mM phenylmethylsulfonyl fluoride, and 1 µg/ml leupeptin. Cell lysates were centrifuged at 10,000 × g for 10 min to remove debris. The proteins were
fractionated using a 7.5% separating gel to assess the level of iNOS,
whereas I-
B
and its phosphorylated form were determined using a
12% separating gel. Briefly, the fractionated proteins were
electrophoretically transferred to nitrocellulose paper. Cytosolic iNOS
was immunoblotted with monoclonal anti-iNOS antibody, whereas
polyclonal anti-I-
B
and anti-phosphorylated I-
B
antibodies
were used to assess I-
B
and its phosphorylated form,
respectively. Cell lysates were centrifuged at 10,000 × g for 10 min to remove debris. Activated JNK, p38 kinase,
and ERK in cell lysates were immunochemically assessed using the
specific antibodies, which recognized the active phosphorylated forms.
The levels of unphosphorylated JNK, p38 kinase, and ERK1/2 were
measured using the respective antibody directed against each MAP
kinase. The secondary antibodies were horseradish peroxidase- or
alkaline phosphatase-conjugated anti-IgG antibody. Nitrocellulose paper
was developed using 5-bromo-4-chloro-3-indolyl phosphate/4-nitro blue
tetrazolium chloride or developed using ECL chemiluminescence system
(Amersham Biosciences).
Preparation of Nuclear Extracts--
Culture dishes were washed
with ice-cold PBS. The dishes were then scraped and transferred to
microtubes. Cells were allowed to swell by adding 100 µl of lysis
buffer (10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.5% Nonidet P-40, 1 mM
dithiothreitol, and 0.5 mM phenylmethylsulfonyl fluoride).
Tubes were vortexed to disrupt cell membranes. The samples were
incubated for 10 min on ice and centrifuged for 5 min at 4 °C.
Pellets containing crude nuclei were resuspended in 50 µl of the
extraction buffer, containing 20 mM HEPES (pH 7.9), 400 mM NaCl, 1 mM EDTA, 1 mM
dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride, and
then incubated for 30 min on ice. The samples were centrifuged at
15,800 × g for 10 min to obtain the supernatant
containing nuclear extracts. The nuclear extracts were stored at
70 °C until use.
Gel Retardation Assay--
A double-stranded DNA probe for the
consensus sequence of nuclear factor-
B (NF-
B,
5'-AGTTGAGGGGACTTTCCCAGGC-3') was used for gel shift
analysis after end-labeling of the probe with
[
-32P]ATP and T4 polynucleotide kinase.
The reaction mixture contained 2 µl of 5× binding buffer containing
20% glycerol, 5 mM MgCl2, 250 mM
NaCl, 2.5 mM EDTA, 2.5 mM dithiothreitol, 0.25 mg/ml poly(dI-dC), and 50 mM Tris·Cl (pH 7.5), 8 µg of
nuclear extracts, and sterile water in a total volume of 10 µl.
Incubations were initiated by addition of 1-µl probe (106
cpm) and continued for 20 min at room temperature. In some experiments, an aliquot of nuclear extracts (8 µg each) was incubated with 2 µg
of highly specific anti-p65 and/or p50 antibody (NF-
B) at room
temperature for 1 h, according to the method described previously (28). Samples were loaded onto 4% polyacrylamide gels at 100 V. The
gels were removed, fixed, and dried, followed by autoradiography.
Immunocytochemistry of p65--
Cells were grown on Lab-TEK
chamber slides® (Nalge Nunc International Corp.) and incubated in
serum-deprived medium for 24 h. Standard immunocytochemical method
was used as described previously (29). For immunostaining, cells were
fixed in 100% methanol for 30 min and washed three times with PBS.
After blocking in 5% bovine serum albumin in PBS for 1 h at room
temperature or overnight at 4 °C, cells were incubated for 1 h
with polyclonal rabbit anti-p65 antibody (1:100) in PBS containing
0.5% bovine serum albumin. Cells were incubated with fluorescein
isothiocyanate-conjugated goat anti-rabbit IgG (1:100) after serial
washings with PBS. Counter-staining with propidium iodide verified the
location and integrity of nuclei. Stained cells were washed and
examined using a laser scanning confocal microscope (Leica TCS NT,
Leica Microsystems, Wetzlar, Germany).
Transient Transfection--
Cells were plated at a density of
0.5 × 106 cells/well in a 6-well dish and transfected
the following day. Briefly, cells were incubated with
G
12/13W or G
12/13QL plasmid (1 µg each
of the plasmid DNA) and 3 µl of LipofectAMINE® reagent (Invitrogen)
in 1 ml of antibiotic-free MEM for 3 h. Culture medium was changed with serum-free MEM with antibiotics, and cells were further incubated for 12 h to assess iNOS expression and for 1-3 h to monitor
NF-
B activity. The transfection efficiency was ~50%, as
determined by transfection with the lacZ reporter
gene. For phorbol 12-myristate 13-acetate (PMA) experiments, cells were
treated with 1 µM PMA for 18 h and transfected with
G
12/13QL plasmid.
Stable Transfection with JNK1 Dominant Negative Mutant
Plasmid--
Cells were transfected using Transfectam® according to
the manufacturer's instruction (Promega, Madison, WI). Raw264.7 cells were replated 24 h before transfection at a density of 2 × 106 cells in a 10-cm2 plastic dish.
Transfectam® (20 µl) was mixed with 10 µg of a JNK1 dominant
negative mutant (JNK1(
)) plasmid in 2.5 ml of minimal essential
medium (MEM). Cells were transfected by addition of MEM containing each
plasmid and Transfectam® and then incubated at 37 °C in a
humidified atmosphere of 5% CO2 for 6 h. After
addition of 6.25 ml of MEM with 10% fetal bovine serum, cells were
incubated for an additional 48 h, and geneticin was added to
select the resistant colonies. In the present study, a mixture of
stably transfected JNK1(
) clones were used. JNK1(
) cells had no
inducible JNK activity, as monitored by phosphorylation of glutathione
S-transferase-c-Jun (stimulation by 10 µg/ml bovine
collagen(I) for 1 h). The expression of JNK1, but not JNK2, was
decreased 43% by JNK1(
) transfection.
Statistical Methods--
One-way analysis of variance procedures
were used to assess significant differences among treatment groups. For
each significant effect of treatment, the Newman-Keuls test was used
for comparisons of multiple group means. The criterion for statistical
significance was set at p < 0.05 or p < 0.01.
 |
RESULTS |
Induction of iNOS by Thrombin--
The effect of thrombin on NO
production in macrophages was first assessed. Thrombin (10 units/ml,
12 h) increased the NO production by 4-5-fold. NO production was
maintained for at least 48 h (Fig. 1A). Western blot analysis
confirmed iNOS induction by thrombin at the time points examined (Fig.
1A). LPS (1 µg/ml), which was used as a positive control,
induced iNOS with a concomitant increase in NO production (Fig.
1A). The extent of iNOS induction by thrombin was comparable
with that by LPS. iNOS induction by thrombin was potentiated by the
presence of serum (data not shown). In order to characterize the
effects of thrombin on the iNOS expression per se, the
subsequent experiments were conducted with cells starved of serum for
24 h.

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Fig. 1.
Induction of iNOS by thrombin.
A, the time course of iNOS induction and NO production. The
macrophages were incubated with or without thrombin (10 units/ml) or
LPS (1 µg/ml), and the iNOS levels were immunochemically determined.
The amount of nitrite in the medium was monitored, as described under
"Experimental Procedures." B, the effect of varying
concentrations of thrombin on iNOS induction. Both iNOS expression and
NO production were measured in the cells incubated with 1, 10, or 100 unit(s) of thrombin per ml for 18 h. C, the effect of
LB30057 on the induction of iNOS by thrombin. The cells were treated
with 0.1-100 µM of LB30057 for 30 min and further
incubated with thrombin (10 units/ml) for 18 h. Subsequently, the
iNOS expression level was assessed. Each lane was loaded with 30 µg
of the cytosolic proteins. The data represent the mean ± S.E.
with 6 separate experiments.
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The effect of varying thrombin concentrations on the iNOS expression
was next examined. Although 1 unit/ml thrombin (i.e. equivalent to the plasma concentration observed in healthy control animals (30)) affected iNOS expression minimally, 10 units/ml thrombin
or greater notably induced the protein (Fig. 1B). This was
in agreement with the Western blot results. Therefore, thrombin exhibited a threshold effect. The level of thrombin was increased severalfold by sepsis with a reciprocal decrease in the ATIII levels
(7, 10). Hence, the concentration used in this study was considered
appropriate for assessing the role of thrombin in sepsis.
LB30057 is a direct thrombin inhibitor with a Ki of
0.38 nM (31). Treatment of the Raw264.7 cells with 1 µM or greater LB30057 completely blocked the induction of
iNOS by thrombin (18 h), which demonstrated that thrombin induces iNOS
per se presumably through the thrombin receptor (Fig.
1C). Reverse transcriptase-PCR analysis confirmed that the
iNOS induction by thrombin accompanied an increase in the mRNA
level (data not shown).
Activation of NF-
B Transcription Factor--
iNOS expression is
controlled primarily by the transcription factor, NF-
B (32). In
order to determine whether or not the iNOS induction by thrombin was
mediated by NF-
B activation, the nuclear extracts prepared from the
cells treated with thrombin for 0.5-12 h were probed with the
radiolabeled NF-
B consensus oligonucleotide (Fig.
2A). NF-
B was activated by
thrombin with a band intensity of a slow migrating p65/p50 complex
being increased from 30 min to 12 h. The p50/p50 homodimer complex
migrated slightly faster in the cells treated with thrombin. Supershift
analysis was carried out using anti-p65 and anti-p50 antibodies
to confirm whether or not the retarded band consisted of the p65 and
p50 proteins. A 20-fold excess of the NF-
B probe abolished the band retardation (Fig. 2B). Either anti-p65 or the anti-p50
antibodies supershifted the retarded band. The addition of both
anti-p65 and anti-p50 antibodies also caused a supershift with the
reduction in the band intensity of the p65/p50 complex (Fig.
2B). These results suggest that the p65 and p50 proteins
were the components actively binding to the NF-
B-binding site. The
specificity of the thrombin effect on NF-
B activation was verified
by LB30057 (10 µM) (Fig. 2C).

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Fig. 2.
Gel shift analyses of
NF- B transcription complexes. A gel shift
assay was performed using the nuclear extracts prepared from the
Raw264.7 cells cultured with or without thrombin (10 units/ml) for
0.5-12 h. Each lane contained 8 µg of the nuclear extracts and 5 ng
of the labeled NF- B consensus oligonucleotide. A, gel
shift analysis of NF- B binding to DNA. B, supershift of
NF- B bound with DNA. An antibody competition experiment was
conducted by incubating the nuclear extracts prepared from the cells
treated with thrombin (10 units/ml) for 3 h, with the specific
antibodies (2 µg each) directed against the p65 or p50 protein. A
competition study was carried out by adding a 20-fold excess of the
unlabeled NF- B-binding oligonucleotide. The
arrowhead(s) indicate(s) the p65/p50 dimer bound
with DNA (closed arrowhead) and the supershifted NF- B DNA
complex (open arrowhead). C, inhibition of
NF- B binding to the NF- B consensus oligonucleotide by LB30057.
LB30057 (10 µM) inhibited the thrombin-inducible NF- B
binding to the NF- B consensus oligonucleotide (3 h). The results
were confirmed by repeated experiments.
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Because p65 was the major component of the NF-
B activated by
thrombin, this study determined the translocation of p65 into the
nucleus. The Raw264.7 cells were treated with thrombin for 30 min to
3 h, fixed, and permeabilized. Immunocytochemistry showed that the
p65 protein was located mainly in the cytoplasm of the control cells
(Fig. 3A). In contrast, the
p65 protein moved into the nucleus after the thrombin treatment. The
nuclear integrity was confirmed by propidium iodide staining of the
identical cells (Fig. 3A). The proteolytic degradation of
the I-
B
subunit preceded the translocation of NF-
B to the
nucleus. These studies were extended to determine whether or not
NF-
B activation by thrombin resulted from the degradation of
I-
B
(Fig. 3B). I-
B
phosphorylation preceded
I-
B
degradation. Thrombin (10 unit/ml) increased I-
B
phosphorylation at 15 min (Fig. 3B). The I-
B
level was
subsequently decreased between 30 min and 1 h. Therefore, thrombin
activates NF-
B through I-
B
degradation following its
phosphorylation.

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Fig. 3.
Nuclear translocation of p65, phosphorylation
of I- B , and
degradation of I- B by
thrombin. A, immunofluorescence subcellular
localization of the p65 protein. The p65 protein was
immunocytochemically detected using anti-p65 antibodies. Thrombin (10 units/ml) caused the p65 protein to migrate toward the nucleus or to
translocate into the nucleus over a period ranging from 30 min to
3 h. The same fields were counter-stained with propidium iodide to
locate the nuclei. B, phosphorylation of I- B and
I- B degradation. The phosphorylated I- B and I- B
protein levels were both immunochemically determined in the cells
treated with thrombin between 15 min and 3 h. The data represent
the mean ± S.E. with 3 separate experiments (significant as
compared with control, *, p < 0.05; **,
p < 0.01).
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iNOS Induction and NF-
B Activation by
G
12/13--
Previously, it was reported (20) that
thrombin binding to its receptor stimulates the guanine nucleotide
exchange of G
12 subfamily proteins. This study was
interested in whether or not the G
12/13 subunits were
responsible for NF-
B-mediated iNOS induction. G
12QL
or G
13QL expression also notably increased NO production
in the cells (Fig. 4A). In
order to confirm that thrombin induced iNOS in the Raw264.7 cells
through the G
12/13 pathway, the cells were transfected
with the either G
12W, G
13W, G
12QL, or G
13QL (G
12 or
G
13 activated mutant) plasmid and then treated with
thrombin. Thrombin induced iNOS in the cells transfected with the
G
12/13W plasmid, which was comparable with that by
either G
12QL or G
13QL. iNOS was not
inducible in the cells transfected with the G
15QL
(G
15 activated mutant) plasmid, which was used as a
negative control. This study next determined whether or not the
activated mutants of G
12/13 stimulated p65/p50 complex
binding to the NF-
B consensus oligonucleotide. Either G
12QL or G
13QL increased the band
intensity of the p65/p50 DNA complex (Fig. 4B).

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Fig. 4.
NF- B-mediated
induction of iNOS by activated
G 12/13. A,
induction of iNOS by thrombin in the cells transfected with the plasmid
encoding the wild type G 12 (G 12W) or
G 13 (G 13W), or by an activated mutant of
G 12 (G 12QL) or G 13
(G 13QL) (1 µg DNA per well). The control cells were
transfected with the pcDNA plasmid. The effect of
G 15QL (1 µg DNA per well, negative control) on iNOS
expression was compared with that by the tumor necrosis factor-
(TNF- , 5 ng/ml, 18 h). The cells transfected with
the G 12W, G 13W, G 12QL, or
G 13QL plasmid (0.25 µg DNA per well) were exposed to
thrombin (10 units/ml) for 12 h. The cells transfected with the
G 15QL plasmid (0.25 µg of DNA per well) were used as a
negative control. The iNOS protein and nitrite production levels were
monitored 12 h after transfecting the Raw264.7 cells (3 h) with
the plasmid encoding for G 12QL or G 13QL.
The data represent the mean ± S.E. of 4 separate experiments
(significant compared with control, **, p < 0.01).
B, representative gel shift analysis of NF- B binding to
DNA. The NF- B binding activity was assessed 1 or 3 h after the
transient transfection of the cells (3 h) with the
G 12/13QL plasmid. Each lane contained 8 µg of the
nuclear extracts and 5 ng of the labeled NF- B-binding
oligonucleotide.
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PKC-mediated NF-
B Activation by Thrombin Downstream of
G
12/13--
Thrombin induced cell differentiation via
the PKC-dependent pathway (34). The potential role of PKC
in the NF-
B-mediated iNOS induction by thrombin was assessed in the
PKC-depleted cells. The cells were treated with PMA for 18 h for
PKC depletion, which was followed by exposure to thrombin (10 units/ml)
for 24 h. Pretreatment of the cells with PMA completely inhibited
both the iNOS induction and the increase in NO production by thrombin
(Fig. 5A). PKC depletion also
inhibited NF-
B activation, I-
B
phosphorylation, and I-
B
degradation by thrombin (Fig. 5, B and C). These
results show that PKC plays a role in the I-
B
phosphorylation by
thrombin.

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Fig. 5.
The effect of PMA pretreatment on
NF- B-mediated induction of iNOS by
thrombin. A, inhibition of thrombin-inducible iNOS
expression by PKC depletion. The iNOS protein and nitrite production
were monitored 24 h after exposing the PMA-pretreated (18 h) cells
to thrombin (10 units/ml). The data represent the mean ± S.E. of
4 separate experiments (significant compared with the control, **,
p < 0.01; significant compared with thrombin, ##,
p < 0.01). B, gel shift analysis of NF- B
binding to DNA. The NF- B binding activity was assessed 6 h
after exposing the PMA-pretreated (18 h) cells to thrombin.
C, effect of PKC depletion on I- B phosphorylation and
degradation by thrombin. The phosphorylated I- B and I- B
proteins were immunochemically assessed 15-30 min after exposing the
PMA-pretreated (18 h) cells to thrombin. D, inhibition
of G 12QL- or G 13QL-inducible iNOS
expression by PKC depletion. iNOS induction in the cells
transfected with the G 12/13QL plasmid was compared with
that in the G 12/13QL-transfected PKC-depleted cells. The
iNOS level was assessed 18 h after transfecting the cells that had
been pretreated with PMA for 18 h with the plasmid encoding for
G 12/13QL. Results were confirmed by repeated
experiments.
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In the case of the pathways of the G
12 protein-coupled
receptors, the PKC-dependent phosphorylation is linked to
G
12/13 activation (35). As an approach to determine
whether or not PKC controlled iNOS induction downstream of
G
12/13, iNOS expression was monitored in the
G
12/13QL-transfected cells, which had been depleted of
PKC. PKC depletion completely inhibited the iNOS induction by
G
12/13QL (Fig. 5D).
The Role of MAP Kinases in the iNOS Induction by Thrombin--
The
MAP kinases were involved in iNOS expression (36). Subsequently, we
determined whether the MAP kinases including JNK, p38 kinase, and
ERK1/2 controlled iNOS induction by thrombin. Thrombin activated all
three MAP kinases in the Raw264.7 cells (Fig.
6A). JNK and ERK1/2
phosphorylation was distinct at 30 min to 1 h, which gradually
returned toward the control levels (i.e. 3-6 h). p38 kinase
was only weakly phosphorylated by thrombin. Cells stably
transfected with the dominant negative mutants or chemical inhibitors
were used to assess the role of each MAP kinase in the induction of
iNOS. JNK inhibition by the stable transfection with a dominant
negative mutant of JNK1 (JNK1(
)) completely suppressed iNOS induction
by thrombin (Fig. 6B). In contrast, p38 kinase inhibition by
SB203580 (10 µM) enhanced the enzyme expression (Fig.
6C). PD98059 (50 µM) failed to affect the iNOS
expression level. Hence, the induction of iNOS by thrombin was
regulated by the distinct and opposed functions of JNK and p38
kinase.

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Fig. 6.
Activation of MAP kinases by thrombin and the
role of MAP kinases in the induction of iNOS. A,
activation of the MAP kinases by thrombin. The cells were cultured with
or without thrombin (10 units/ml) for 15 min to 6 h, and the
extent of JNK, p38 kinase, and ERK1/2 activation was assessed by
immunoblotting of the respective phosphorylated form of MAP kinase. The
unphosphorylated JNK, p38 kinase, and ERK1/2 were immunoblotted for the
controls. The results were confirmed by repeated experiments.
p-JNK, phosphorylated JNK; p-p38 kinase,
phosphorylated p38 kinase; p-ERK1/2, phosphorylated ERK1/2.
B, the role of JNK in inducing iNOS by thrombin. iNOS
expression and the extent of NO production were measured 18 h
after exposing either the control cells or JNK1( ) cells to thrombin.
The data represent the mean ± S.E. of 6 separate experiments
(significant as compared with control, **, p < 0.01;
significant compared with thrombin, ##, p < 0.01).
C, the effect of MAP kinase inhibition on the induction of
iNOS by thrombin. The cells were treated with either SB203580
(SB, 10 µM) or PD98059 (PD, 50 µM) for 30 min and further incubated with thrombin (10 units/ml) for 18 h. The data represent the mean ± S.E. of 4 separate experiments (significant compared with the control, **,
p < 0.01; significant compared with thrombin, ##,
p < 0.01).
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Coupling of G
12/13 to JNK--
The activation of
the G proteins stimulates the MAP kinases in a variety of cells (26,
37, 38). This study determined whether or not the MAP kinase pathways
were connected with G
12/13 by using either JNK1(
)
cells or chemical inhibitors. First, the iNOS protein was monitored in
the JNK1(
) cells that were transiently transfected with the plasmid
encoding for either G
12QL or G
13QL. The
expression of the active mutant of G
12/13 failed to
induce iNOS in the JNK1(
) cells (Fig.
7). SB203580 slightly enhanced iNOS
induction in the cells expressing G
12/13QL (Fig. 7).
PD98059 did not alter the induction of iNOS by G
12QL or
G
13QL. Therefore, JNK and p38 kinase oppositely function
downstream of G
12/13.

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Fig. 7.
The role of MAP kinases in the induction of
iNOS by G 12/13QL. The iNOS
protein expression level was determined 18 h after transfecting
the control cells or JNK1( ) cells with the plasmid encoding the
activated mutant of either G 12 or G 13.
The transfected cells were also treated with either SB203580
(SB, 10 µM) or PD98059 (PD, 50 µM) for 18 h. The data represent the mean ± S.E. with 3 separate experiments (significant compared with
G 12/13QL alone, *, p < 0.05).
|
|
JNK-dependent Degradation of Phosphorylated
I-
B
--
In order to assess whether or not the JNK pathway
controls the NF-
B activation by thrombin, the nuclear extracts,
which were prepared from the cells treated with thrombin for 3 h,
were probed with the radiolabeled NF-
B consensus oligonucleotide. In
the JNK1(
) cells, thrombin did not increase the NF-
B binding
activity (Fig. 8A,
left). The basal NF-
B binding to DNA was lower in the JNK1(
) cells than in the control cells (Fig. 8A,
right). This is consistent with the observation that the nuclear
translocation of p65 was blocked by the JNK1(
) stable transfection
(Fig. 8B). However, I-
B
was not degraded by the
presence of thrombin in the JNK1(
) cells (Fig. 8C). The
phosphorylated I-
B
level in the JNK1(
) cells treated with
thrombin was comparable with that of the control (Fig.
8C). These results support the notion that the JNK
pathway is responsible for the degradation of phosphorylated I-
B
.

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Fig. 8.
The role of JNK in
NF- B DNA binding,
I- B phosphorylation,
and I- B degradation
by thrombin. A, gel shift analysis of NF- B binding
to the NF- B consensus oligonucleotide. Either the control or
JNK1( ) cells were incubated with or without thrombin (10 units/ml)
for 3 h. Each lane contained 8 µg of the nuclear extracts and 5 ng of the labeled NF- B-binding oligonucleotide. B,
immunocytochemistry of the p65 protein in the control or JNK1( )
cells. The cells were incubated with or without thrombin for 1 h
and subjected to immunocytochemistry. The same fields were
counter-stained with propidium iodide to locate the nuclei.
C, phosphorylation and degradation of I- B by thrombin.
The I- B phosphorylation level was assessed 15 min after treating
the cells with thrombin, whereas I- B degradation was measured 30 min after the treatment. The data represent the mean ± S.E. of 3 separate experiments (significant as compared with control cells or
JNK1( ) cells, *, p < 0.05; **, p < 0.01).
|
|
 |
DISCUSSION |
Overproduction of the proinflammatory cytokines in sepsis
(e.g. tumor necrosis factor-
) is critically involved in
activating the coagulation system, leading to vascular hyporeactivity
and disseminated intravascular coagulation (39, 40). LPS and/or the
cytokines induce iNOS expression in many cell types (41). NO and the
major inflammatory mediators are produced mainly by activated
macrophages. Excessive NO production by iNOS plays a crucial role in
activating the immune system and in the proinflammatory effects during
LPS-induced septic shock (42-44). In particular, NO induces the
collapse of vascular reactivity and the pathologic alterations (13,
45). In clinical situations, there is a discrepancy between the serum
endotoxin level and the mortality of the patients with Gram-negative
sepsis (46). The persistent NO production in septic patients may result
from other unknown mediator(s) that are generated by LPS. The
endogenous coagulation inhibitors reverse the vascular hyporeactivity
induced by LPS (16, 18). Protein C and ATIII have been studied
extensively as the protective modulators of septic shock (47, 48).
Thrombin, a serine protease of the trypsin family, is a key enzyme of
the blood clotting system. It is the key coagulant molecule that is
commonly involved in two independent (e.g. contact and extrinsic systems) activation pathways. The coagulation pathway involves a series of reactions, which culminate in the production of
sufficient thrombin (6). Thrombin converts fibrinogen to fibrin and
participates in regulating numerous physiological and pathological
processes. Thrombomodulin serves as a thrombin receptor. When thrombin
is bound to thrombomodulin, it loses its procoagulant activity, and its
inactivation by ATIII is accelerated with an enhancement of
protein C activation (6). The generation of excessive thrombin
leads to thrombosis, which is the major cause of morbidity and
mortality. Thrombin in close proximity to the active mediators also
plays a role in the diverse cellular responses in the vascular and
avascular tissues (49). It has been reported that thrombin potentiates
both interferon-
and tumor necrosis factor-
-induced NO production
in C6 glioma cells (50). Thrombin stimulates the proliferation of
smooth muscle cells and vascular disturbances through NF-
B
activation (51). This study found for the first time that thrombin
induces iNOS in the macrophages via members of the G
12
family. The inflammatory cytokines exert their biological effects
through the cytokine receptor superfamily. Hence, the regulatory
mechanisms for iNOS induction by thrombin appear to differ from those
by inflammatory cytokines. This study found a threshold effect for
thrombin in iNOS induction. The concentration of 10 units/ml markedly
increased iNOS expression, whereas 1 unit/ml thrombin has a minimal
effect. The threshold effect may reflect the septic pathological
situation, wherein the level of unbound activated thrombin is increased
as a result of the conversion of prothrombin to thrombin and the
reciprocal consumption of ATIII in sepsis.
NF-
B is a pleiotropic regulator of many genes (e.g. iNOS)
involved in the immune and inflammatory responses (32). This study
found that iNOS induction and excess NO production by thrombin are
mediated primarily by NF-
B activation. NF-
B exists in the cytoplasm of unstimulated cells in a quiescent form bound to its inhibitor (52). Thrombin was found to activate the p65/p50 NF-
B DNA
binding complex and to induce the nuclear translocation of the p65 protein.
In this study, the role of the G
12 family members
downstream of thrombin signaling (35) in NF-
B-mediated iNOS
induction was verified by experiments using an activated mutant of
G
12 or G
13. It was shown that thrombin
induces the stress fiber assembly via G
12- or
G
13-coupled receptor activation (20). The
Gq-coupled receptor activates the downstream signals in a
PKC-dependent, fully PKC-independent, or partially
PKC-dependent pathway (54, 55). Thrombin differentiates the
normal lung fibroblasts to a myofibroblast phenotype via its receptor
via a protein kinase C-dependent pathway (34). Macrophage
activation by external stimuli causes the phosphorylation and
degradation of I-
B
. I-
B kinase activation by LPS is dependent
on PKC and ERK (56). This study found that PKC was involved in the
NF-
B-mediated iNOS induction by thrombin via the phosphorylation and
degradation of I-
B
. PKC depletion prevented the induction of iNOS
by the activated mutants of G
12/13, which raised the
notion that the PKC pathway functions downstream of
G
12/13 activation.
Thrombin activated ERK in the endothelial cells (57). While this report
was being revised, it was reported that thrombin activated p38 kinase
in the platelets, which led to NF-
B-dependent leukocyte
recruitment (58). In this study, it was found that all three MAP
kinases JNK, p38 kinase, and ERK1/2, were activated by thrombin. Among
the MAP kinases, the JNK pathway was responsible for the induction of
iNOS by thrombin, which was strongly supported by the lack of iNOS
induction in the thrombin-treated JNK1(
) cells. The p38 kinase
pathway oppositely regulated iNOS induction by thrombin. ERK1/2
activation was not responsible for iNOS induction, as evidenced by the
results from the chemical inhibitor. The lack of iNOS induction by
G
12/13 in the JNK1(
) cells supports the concept that
JNK serves as an essential pathway downstream of the
G
12/13 proteins. Again, JNK and p38 kinase oppositely
control the NF-
B-mediated iNOS induction by the activated mutants of G
12/13. Thus, the induction of iNOS by thrombin was
regulated by the opposed functions of JNK and p38 kinase downstream of
G
12/13.
The gel shift and immunoblot analyses revealed that the pathway
involving JNK controlled NF-
B activation in response to thrombin. The increase in NF-
B DNA binding activity and the nuclear
translocation of p65 were both completely abolished by the JNK1(
)
transfection. The diminished NF-
B DNA binding activity in the
JNK1(
) cells may be due in part to the decrease in the basal NF-
B
activity. This study found for the first time that thrombin failed to
degrade I-
B
in the JNK1(
) cells despite its I-
B
phosphorylation. These results strongly support the belief that the JNK
pathway was responsible for degrading the phosphorylated I-
B
. The
time course in I-
B
phosphorylation and degradation by thrombin
paralleled that in JNK activation.
The ubiquitin-proteasome pathway controls the timed destruction of the
phosphorylated I-
B
in order to activate NF-
B (33, 59).
Recently, it was shown that the activation of the stress-activated protein kinase, JNK, by the forced expression of the constitutively active mutants of JNKK2 and members of the Jun family leads to the
accumulation of
-TrCP, which mediates the ubiquitination of the
phosphorylated I-
B
via the recruitment of a ubiquitin ligase
complex (53). Therefore, it is highly likely that the accumulation of
phosphorylated I-
B
and the failure of I-
B
degradation by
thrombin in the JNK1(
) cells might result from the inhibition of the
ubiquitin-proteasome pathway.
In summary, this study demonstrated that thrombin plays an important
role in the vascular responsibility by inducing iNOS and NO production.
In addition, thrombin activates the pathway coupled with
G
12/13 for enzyme induction. G
12/13
activation then leads to the PKC-dependent phosphorylation
of I-
B
and the JNK-mediated I-
B
degradation. The cellular
signaling pathways, by which thrombin induces iNOS, may serve as the
pharmacological targets for both preventing and treating vascular
hyporeactivity in septic patients.