c-Jun NH2-terminal Kinase-mediated
Redox-dependent Degradation of I
B
ROLE OF THIOREDOXIN IN NF-
B ACTIVATION*
Kumuda C.
Das
From the Department of Molecular Biology, University of Texas
Health Center, Tyler, Texas 75708
Received for publication, July 13, 2000, and in revised form, October 26, 2000
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ABSTRACT |
NF-
B is a redox-sensitive transcription factor
known to be activated by oxidative stress as well as chemical and
biological reductants. Its DNA binding activity requires reduced
cysteines present in the p65 subunit of the dimer. Thioredoxin (Trx) is an endogenous disulfide oxidoreductase known to modulate several redox-dependent functions in the cell. NF-
B was
activated by addition of Escherichia coli thioredoxin in a
redox-dependent manner in A549 cells. Such activation was
accompanied by degradation of I
B in the cytosol. In addition, only
the reduced form of thioredoxin activated NF-
B, whereas the oxidized
form was without any effect. Overexpression of human thioredoxin also
caused activation of NF-
B and degradation of I
B. On the contrary,
dominant-negative redox-inactive mutant thioredoxin expression did not
activate NF-
B, further confirming the redox-dependent
activation of NF-
B. We also investigated the mechanism of activation
of NF-
B by thioredoxin. We demonstrate that thioredoxin activates
c-Jun NH2-terminal kinase (JNK)-signaling cascade, and
dominant-negative expression of mitogen-activated protein kinase kinase
kinase 1 (MEKK1), JNK kinase, or JNK inhibits NF-
B activation by
thioredoxin. In contrast, wild-type MEKK1 or JNK kinase induced NF-
B
activation alone or in combination with thioredoxin expression plasmid.
These findings were also confirmed by NF-
B-dependent
luciferase reporter gene transcription.
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INTRODUCTION |
Nuclear transcription factor
B
(NF-
B)1 is a multi-subunit
factor that can rapidly activate the expression of genes involved in
inflammatory, immune, and acute phase responses (1). Although multiple
forms can exist, the principal active form appears to be a heterodimer
consisting of 50- and 65-kDa subunits (2). The heterodimer remains
bound to an inhibitor protein I
B in the cytoplasm. In response to a
variety of stimuli, I
B is phosphorylated and ubiquitinated, followed
by degradation by the 26 S proteosome (3). This process exposes the
nuclear localization signal, allowing the heterodimeric complex to
interact with the nuclear transport machinery and to translocate to the
nucleus (2). A characteristic of NF-
B is that many different agents
can induce its DNA binding activity (2). Oxidants (4) as well as
reductants (5-7) are known to activate NF-
B. Although
redox-dependent activation of NF-
B is widely recognized,
little is known about how cellular redox status could modulate the
signaling events that are associated with the activation of
NF-
B.
Thioredoxin (Trx) is a potent protein disulfide reductase that
catalyzes protein reduction using reducing equivalents from NADPH in
conjunction with thioredoxin reductase (TR). Remarkably low
concentrations of thioredoxin are effective in reducing disulfides in
insulin, fibrinogen, human chorionic gonadotropin, nitric-oxide synthase, ribonucleotide reductase, glucocorticoid receptors, and other
proteins (8-11). The rate of reduction of insulin disulfide by
thioredoxin was found to be 10,000 times higher than that by dithiothreitol (8). Thus, reduced thioredoxin is an extremely potent
protein disulfide reductase. Intracellularly, most of this ubiquitous
low molecular mass (12 kDa) protein remains reduced (12-13).
Thioredoxin has two critical cysteine residues at the active site,
which in the oxidized protein, form a disulfide bridge located in a
protrusion from the three-dimensional structure of the protein (8). The
flavoprotein thioredoxin reductase catalyzes the
NADPH-dependent reduction of this disulfide (8). Small increases in thioredoxin can cause profound changes in
sulfhydryl-disulfide redox status in protein (8). Additionally,
thioredoxin was shown to restore DNA binding activity of NF-
B in a
cell-free system (14). In the same report, it was shown that redox
regulation of NF-
B activity appeared to be exerted after
dissociation of I
B from the NF-
B complex. Moreover, thioredoxin
was shown to form a complex with p50 subunit of NF-
B (15). In
addition, other reports have shown that critical cysteine 62 in NF-
B
is required to be reduced by thioredoxin for its activation (16). We
reported earlier that reducing thiols can activate NF-
B in intact
cells (7). We also showed that sulfhydryl oxidation or alkylation can
inhibit tumor necrosis factor-
- or interleukin-1-induced NF-
B
activation (17).
Mitogen-activated protein (MAP) kinases are serine/threonine kinases
activated by dual phosphorylation on both a tyrosine and a threonine
(18). These enzymes are important components of signaling pathways that
transduce extracellular stimuli into intracellular responses. There are
three major forms of MAP kinases; extracellular signal-regulating
kinase, c-Jun-NH2-terminal Kinase (JNK) or stress-activated
protein kinase), and p38 MAP kinase. Extracellular signal-regulating
kinase pathway is activated by growth factors and phorbol esters (18).
JNK/stress-activated protein kinase pathway is activated in response to
cellular stresses such as heat shock, UV irradiation, or inflammatory
cytokines (19). Inflammatory cytokines as well as environmental
stresses such as osmotic shock activate p38 MAP kinase (20).
The JNK-signaling cascade functions through the activation of an
initiating kinase such as MAP kinase kinase kinase (MEKK1), which in
turn phosphorylates the MAP kinase kinase (MKK4/SEK1), and MKK4 finally
activates the JNK by phosphorylating the serine and threonine residues
on it (reviewed in Ref. 19). Although the signal transduction cascade
leading to the activation of JNK is relatively well defined, the steps
leading to the phosphorylation of I
B
are poorly understood.
Recent studies demonstrate that I
B
can be phosphorylated by
MEKK1, an upstream kinase of the JNK pathway (21). Moreover, many of
the stimuli that induce NF-
B activation, such as tumor necrosis
factor-
, UV radiation, and lipopolysaccharide, also activate the JNK
signaling cascade. Since phosphorylation of, I
B
is required for
its degradation and Trx can activate NF-
B in intact cells, we
hypothesized a potential role of JNK in Trx-mediated I
B degradation
and activation of NF-
B.
In this report, we demonstrate that thioredoxin activates NF-
B and
causes degradation of I
B. Additionally, we have shown that MEKK1 is
the initiating kinase of the JNK pathway that mediates the NF-
B
activation by thioredoxin. Moreover, we also demonstrate that JNK
subgroup of MAP kinases is activated by redox-active thioredoxin.
Furthermore, we have also shown that thioredoxin can induce
NF-
B-dependent reporter gene expression, and such transcription can be abrogated by inhibition of JNK-signaling intermediates using dominant-negative constructs.
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EXPERIMENTAL PROCEDURES |
Materials--
Escherichia coli thioredoxin was
obtained from Promega Corp. (Madison, WI). Thioredoxin reductase and
human thioredoxin were obtained from American Diagnostica, Inc.
(Greenwich, CT). Anti-p50, anti-p65, pJNK, JNK, and anti-I
B
antibodies were obtained from Santa Cruz Biotechnology (Sant Cruz, CA).
All other materials were obtained in the highest available grade. A549
cells (adenocarcinoma cells) were obtained from ATCC. Lactacystin was
obtained from Sigma.
Cell Culture and Transfections--
A549 cells were cultured in
Kaigan's modified F-12K medium (Life Technologies, Inc.) supplemented
with 10% fetal bovine serum and 100 units of penicillin/streptomycin.
Confluent monolayers were treated with various concentrations of
thioredoxin for different time periods as indicated in the figure
legends. Cell viability was determined by the trypan blue exclusion
method. Human pulmonary artery endothelial cells were obtained from
Clonetics Corp. and propagated in endothelial growth medium (Clonetics,
CA). MEKK1 (pcDNA3-MEKK1) and the kinase-dead dnMEKK1 were generous
gifts of Dr. Tom Maniatis (Harvard University, Boston) and have been described (21). The dominant-negative JNKK expression plasmid (pSR
-dnJNKK) and dominant-negative JNK (pSR
-AFPJNK) expression plasmid were generous gifts of Dr. Gary L. Johnson (National Jewish Medical Center, Denver, CO) and have been described (22). Transfection of various expression plasmids into A549 cells was carried out using
Transfectam reagent (Promega) or Geneporter reagent (Gene Therapy Systems Inc. San Diego, CA) as per manufacturer's protocol.
Site-directed Mutagenesis--
Site-directed mutagenesis of the
redox-active Cys-32 and Cys-35 to serine was performed by a synthesized
oligonucleotide
(5'-TCATTTTGGAAGGCCCAGACCACGTGGC-3') and quick-change mutagenesis kit (Stratagene, La Jolla, CA) as per
manufacturer's protocol. Briefly, double-stranded mutagenic oligonucleotide was synthesized (Genosys) and purified by
polyacrylamide gel electrophoresis. Mutagenic oligonucleotide was added
to the double-stranded plasmid pcDNA3-Trx. The plasmid was
denatured, and the mutagenic-oligonucleotide was annealed by
temperature cycling using Pfu Turbo DNA polymerase. After
temperature cycling, the methylated nonmutated parental template DNA
was digested with DpnI. XL1-Blue supercompetent cells
(Stratagene) were transformed with mutated plasmid. Base substitution
in the mutagenic thioredoxin open reading frame was verified by
sequencing (University of Texas Medical Branch, Galveston, TX). The
mutagenic plasmid (pcDNA3-dnTrx) was amplified for transfection experiments.
Nuclear Extract Preparation--
Nuclear extracts were prepared
as described previously (23). Briefly, 107 cells were
washed in 10 ml of phosphate-buffered saline and centrifuged (1,500 × g for 5 min). The pellet was resuspended in
phosphate-buffered saline (1 ml), transferred into an Eppendorf tube,
and centrifuged again (16,000 × g; 15 s).
Phosphate-buffered saline was removed, and the cell pellet was
resuspended in 400 µl of buffer A (10 mM HEPES, pH 7.8, 10 mM KCl, 0.1 mM EDTA, 2 mM
dithiotheitol, 1 mM phenylmethylsulfonyl fluoride,
leupeptin (0.5 mg/ml), antipain (0.3 mg/ml)) by gentle pipetting. The
cells were allowed to swell on ice for 15 min, after which 25 µl of
10% Nonidet-P40 (Sigma) was added, and the tube was vortexed
vigorously for 10 s. The homogenate was centrifuged for 30 s
in a microcentrifuge. The nuclear pellet was resuspended in buffer C
(20 mM HEPES, pH 7.8, 0.42 M NaCl, 5 mM EDTA, 5 mM dithiotheitol, 1 mM
phenylmethylsulfonyl fluoride, 10% (v/v) glycerol), and the tube was
rocked gently at 4 °C for 30 min on a shaking platform. The nuclear
extract was centrifuged for 10 min in a microcentrifuge at 4 °C, and
the supernatant was frozen at
70 °C in aliquots until the
electrophoretic mobility shift assay (EMSA) was performed. Protein was
quantified by Bradford protein assay (Bio-Rad; Ref. 24).
Electrophoretic Mobility Shift Assay--
For the EMSA, the
NF
B specific oligonucleotide was obtained from Promega Corp.
Oligonucleotide was end-labeled using T4 polynucleotide kinase (Promega) and [
-32P]ATP (NEN) in 10× kinase
buffer (0.5 M Tris-HCl, pH 7.5, 0.1 M
MgCl2, 50 mM dithiotheitol, 1 mM
spermidine, and 1 mM EDTA). For competition studies, 3.5 pmol of unlabeled oligonucleotide was used. Nuclear extract without
labeled oligonucleotide was preincubated for 15 min at 4 °C followed
by a 20-min incubation at room temperature after the addition of
labeled oligonucleotide. The binding reaction contained 10 µg of
sample protein, 5 µl of 5× incubation buffer (20% glycerol, 5 mM MgCl2, 5 mM EDTA, 5 mM dithiotheitol, 500 mM NaCl, 50 mM Tris-HCl, pH 7.5, 0.4 mg/ml calf thymus DNA). In some of
the binding reactions poly(dI-dC) (Amersham Pharmacia Biotech) was
added to a final concentration of 2 µg. The nuclear
protein-32P-oligonucleotide complex was separated from free
32P-labeled oligonucleotide by electrophoresis through a
6% native polyacrylamide gel in a running buffer of 0.25× TBE (5×
TBE = 500 mM Tris, pH 8.0, 450 mM borate,
5 mM EDTA).
Supershift Assay--
For the supershift assay, some of the
binding reactions contained 200 ng of anti-p50 or anti-p65 antibody
(Santa Cruz) along with 2 µg of poly(dI-dC) (Amersham Pharmacia Biotech).
Western Blotting of I
B--
Post-nuclear supernatant was
treated as the cytosolic extract and quantified with Bradford assay
(Bio-Rad, Ref. 24). Equal amounts of protein were resolved on a 10% or
12% SDS-polyacrylamide gel electrophoresis. After electorphoresis,
protein was transferred to a nitrocellulose membrane (Hybond-ECL,
Amersham Pharmacia Biotech) or polyvinylidene difluoride membrane
(Bio-Rad), immunoblotted with anti-I
B (Santa Cruz), and visualized
by the ECL system (Amersham Pharmacia Biotech) using anti-rabbit-HRP
IgG (Santa Cruz).
JNK Activity Assay--
The activity of JNK was assayed by a
nonradioactive assay kit as per the manufacturer's protocol (New
England Biolabs, Beverly, MA). Briefly, stress-activated protein
kinase/JNK was precipitated from the cell lysate by c-Jun fusion
protein bound to glutathione-Sepharose beads. c-Jun contains a high
affinity binding site for stress-activated protein kinase/JNK,
NH2-terminal to the two phosphorylation sites, Ser-63 and
Ser-73. After selectively pulling down JNK using c-Jun fusion protein
beads, the beads were extensively washed, and the kinase reaction was
carried out in the presence of cold ATP in a final volume of 25 µl.
The reaction was stopped with 25 µl of 2× SDS sample buffer and
loaded onto a 10% polyacrylamide gel. Protein was transferred to
nitrocellulose by electroblotting, and c-Jun phosphorylation was
selectively measured using phospho-c-Jun antibody. This antibody
specifically measures JNK-induced phosphorylation of c-Jun at Ser-63, a
site important for the c-Jun-dependent transcriptional activity (20).
NF-
B-luciferase Reporter Assay--
p-TAL-NF-
B-luciferase
reporter vector was obtained from CLONTECH Inc.,
Palo Alto, CA. A549 cells were transiently transfected with various
expression plasmids by Transfectam (Promega) as per manufacturer's protocol. Cells were lysed 48 h post-transfection using reporter lysis buffer (Promega). After lysis, the lysates were
centrifuged for 2 min at 21,000 × g in a
microcentrifuge, and the supernatant was kept frozen at
80 °C.
Luciferase activity was assayed with an automatic microplate
luminometer using a luciferase assay kit (Promega) as per
manufacturer's protocol (Promega). Data was normalized to protein
concentration, and transfection efficiency was normalized with
-gal
expression.
-Galactosidase expression was determined by a
luminescence assay (Tropix) as per manufacturer's protocol and
measured in an automatic microplate luminometer. Data were presented as
fold induction.
 |
RESULTS |
Redox-dependent Activation of NF-
B by E. coli
Thioredoxin in A549 Cells--
A549 cells were incubated with various
amounts of E. coli thioredoxin as shown in Fig.
1A. After incubation, nuclear
extract was prepared, and EMSA was performed using a consensus
oligonucleotide for NF-
B. As demonstrated in Fig. 1A, the
oxidized form of E. coli thioredoxin did not activate
NF-
B. On the contrary, incubation of cells with a
thioredoxin-reducing system (thioredoxin, TR, and NADPH) activated
NF-
B in a time-dependent manner. Maximal activation of
NF-
B occurred at about 2 h in A549 cells (Fig. 1A).
In the dose-response study, oxidized thioredoxin at a concentration of
1-5 µM did not activate NF-
B. On the other hand, a
thioredoxin-reducing system activated NF-
B in a
dose-dependent manner (Fig. 1B). Thioredoxin reductase or NADPH individually or in combination did not activate NF-
B, suggesting the activation of NF-
B by reduced Trx.

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Fig. 1.
Effect of E. coli thioredoxin on
NF- B activation in A549 cells. A,
time course. Confluent A549 cells were incubated with 2 µM oxidized E. coli thioredoxin or the
thioredoxin-reducing system (2 µM E. coli Trx + 0.5 µM TR + 2 mM NADPH) for 1 to 4 h.
After incubation, cells were harvested, and the nuclear extract was
prepared as described under "Experimental Procedures." EMSA was
performed using a NF- B-specific oligonucleotide sequence as
described under "Experimental Procedures." First lane,
unstimulated A549 cells; second-fourth lanes, cells treated
with 2 µM oxidized E. coli Trx for 1-4 h;
fifth through eighth lanes, cells treated with 2 µM E. coli thioredoxin-reducing system for
1-4 h. B, dose response. A549 cells were incubated with the
indicated concentration of Trx-S2 or Trx-(SH)2,
and nuclear extracts and EMSA were performed as described under
"Experimental Procedures." 1-5th lanes, cells treated
with oxidized E. coli thioredoxin for 2 h (1-5
µM); 6-10th lanes, cells treated with 1-5
µM E. coli thioredoxin-reducing system; 11th
lane, cells treated with only NADPH (1 mM); 12th
lane, cells treated with only TR, 0.05 µM;
13th lane, cells treated with TR + NADPH.
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NF-
B Activated by Thioredoxin Is a p50/p65
Heterodimer--
NF-
B complex is formed by p65/p50 heterodimer,
cRel/p50 heterodimer, or p50 homodimer. Thus, to determine the type of
NF-
B complex that is formed by thioredoxin, we performed
gel-supershift assay using anti-p50 or anti-p65 antibodies. As
demonstrated in Fig. 2, the use of
anti-p50 supershifted the NF-
B band, confirming a p50 subunit in the
complex. The NF-
B band was abolished by the use of p65 antibody,
indicating that the DNA contact is exclusively a function of p65
subunit. Use of both antibodies lessened the intensity of the band,
suggesting inhibition of DNA binding.

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Fig. 2.
NF- B activated by
thioredoxin is a p50/65 heterodimer. A549 cells were incubated
with 5 µM Trx-SH (the reducing system as described for
Fig. 1). After incubation, nuclear extracts were prepared, and the
supershift assay was performed as described under "Experimental
Procedures." First lane, untreated A549 cells; second
lane, cells exposed to 5 µM Trx-SH; third
lane, nuclear extracts incubated with 200 ng of anti-p65;
fourth lane, nuclear extracts incubated with 200 ng of
anti-p50; fifth lane, nuclear extracts incubated with 200 ng
of anti-p65 plus 200 ng of anti-p50; sixth lane, competition
reaction with cold NF- B consensus nucleotide.
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Redox-active E. coli Thioredoxin Activates NF-
B in Human
Pulmonary Artery Endothelial Cells (HPAEC)--
A549 cells are
derived from pulmonary epithelial cells and are highly
dedifferentiated. Hence, to demonstrate the activation of NF-
B by
thioredoxin in primary cell cultures, we used pulmonary artery
endothelial cells (HPAEC). HPAEC were cultured as described under
"Experimental Procedures" and were incubated with oxidized or
reduced thioredoxin in a similar manner as that described for the A549
cells. As demonstrated in Fig. 3,
oxidized thioredoxin did not activate NF-
B in HPAEC. On the
contrary, the reduced thioredoxin system induced NF-
B activation in
a time-dependent manner. Maximal activation occurred at
3 h. Therefore, thioredoxin activated NF-
B in primary cells as
well as in transformed cell lines.

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Fig. 3.
Thioredoxin activates
NF- B in HPAEC. HPAEC were maintained as
described under "Experimental Procedures." HPAEC were exposed to
E. coli thioredoxin, both oxidized and the reduced system
(with NADPH and TR as described in Fig. 1) for 1 to 4 h. First
lane, unstimulated HPAEC; second through fifth
lanes, cells treated with 2 µM E. coli Trx-S2 and incubated for 1-4 h; sixth through
ninth lanes, cells treated with E. coli reducing
system (2 µM) for 1-4 h.
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Redox-dependent Degradation of I
B by
Thioredoxin--
Thioredoxin has been shown to activate NF-
B in a
cell-free system (14). Activation of NF-
B by thioredoxin was also
shown to occur after the dissociation of I
B complex form NF-
B.
Recent studies have also demonstrated the activation of
NF-
B-dependent reporter in MCF-7 cells stably expressing
thioredoxin (25). However, the mechanism of such activation has not
been elucidated. In this study, we have shown that externally added
E. coli thioredoxin could activate NF-
B in intact cells.
Thus, to demonstrate whether NF-
B is activated as a consequence of
degradation of I
B by thioredoxin redox, we assayed for I
B in the
cytoplasmic extracts. As shown in the Fig.
4A, I
B was degraded in the
cytoplasm at 2 h when the A549 cells were incubated with a
thioredoxin-reducing system (lane 6). Additionally,
thioredoxin treatment caused I
B degradation in HPAEC after 3 h
(Fig. 4B, lane 7). These time points correlate to
the EMSA studies, demonstrating that degradation of I
B and NF-
B
DNA binding occurring simultaneously.

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Fig. 4.
Degradation of I B by thioredoxin
in A549 cells. A, A549 cells were incubated with 2 µM E. coli oxidized thioredoxin or 2 µM thioredoxin-reducing system for various time periods.
Post-nuclear supernatants were subjected to Western blot analysis using
anti-I B antibodies, as described under "Experimental
Procedures." First lane, untreated A549 cells; second
through fourth lanes, cells incubated with oxidized
thioredoxin for 1, 2, or 4 h; fifth through seventh
lanes, cells incubated with thioredoxin reducing system for
1,2, or 4 h. B, degradation of I B by thioredoxin in
HPAEC. HPAEC were exposed to oxidized or reduced thioredoxin as
mentioned in Fig. 1. Post-nuclear supernatants were subjected to
Western blot analysis using anti-I B antibodies, as described under
"Experimental Procedures." First through fourth lanes,
Trx-S2, 2 µM; fifth through eighth
lanes, Trx-(SH)2, 2 µM. As seen in
the seventh and eighth lanes, I B was degraded by reduced E. coli thioredoxin.
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Redox-active Human Thioredoxin Activates NF-
B--
Human
thioredoxin is similar to E. coli thioredoxin in its redox
function (8). However, the human thioredoxin contains two catalytic
cysteines at positions 32 and 35 and three other structural cysteines
(8). In addition, human thioredoxin is unable to enter the cell (26)
unlike the bacterial thioredoxin. Thus, to investigate whether human
thioredoxin is similar to E. coli thioredoxin in causing the
activation of NF-
B, we overexpressed human thioredoxin by
transfecting an expression vector containing Trx open reading frame
(pcDNA3-Trx) as described under "Experimental Procedures."
Overexpression of thioredoxin induced the activation of NF-
B, as
demonstrated in Fig. 5A
(lane 2). I
B was also degraded in response to Trx
overexpression (Fig. 5B, third lane). To
further verify the role of redox-active cysteines of thioredoxin in
NF-
B activation, we mutated the Cys-32 and Cys-35 by site-directed mutagenesis as described under "Experimental Procedures." The redox-inactive thioredoxin was produced in a dominant-negative manner
when such mutagenic cDNA was cloned to an overexpression vector
(dnTrx) and transfected to A549 cells. Transfection of A549 cells with
dnTrx did not activate NF-
B (Fig. 5A, third
lane) or degrade I
B (Fig. 5B, second
lane), confirming that the redox activity of Trx is required
for the activation of NF-
B (Fig. 5B) in intact cells.

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Fig. 5.
Activation of NF- B by overexpression of
human thioredoxin. A, A549 cells were transfected with
either pcDNA3, pcDNA3-Trx, or pcDNA3-dnTrx as described
under "Experimental Procedures." Nuclear extracts were prepared,
and EMSA was performed as described under "Experimental
Procedures." First lane, pcDNA3 only; second
lane, 4 µg of pcDNA3-Trx; third lane, 4 µg of pcDNA3-dnTrx. B, degradation of I B by
redox-active human thioredoxin. Post-nuclear supernatant of cells
treated with various constructs (as described for Fig. 5A)
was immunoblotted for I B as described under "Experimental
Procedures." First lane, cells transfected with pcDNA3
only; second lane, cells transfected with pcDNA3-dnTrx;
third lane, cells transfected with pcDNA3-Trx.
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Protein Kinase C (PKC) Does Not Mediate I
B Degradation by
Thioredoxin--
Thioredoxin has been shown to activate PKC (27).
Recent reports also indicate that PKC can mediate NF-
B activation in
a variety of cell types (28). Therefore, we hypothesized that PKC may
mediate the NF-
B activation by thioredoxin. We incubated cells with
specific inhibitors of PKC, calphostin C (29) or GF109203X (30), and
then stimulated the cells with E. coli thioredoxin-reducing system. In this experiment, we specifically sought to determine the
degradation of I
B by thioredoxin and the inhibition of
thioredoxin-mediated I
B degradation by PKC inhibitors. As
demonstrated in Fig. 6 (lane 2), thioredoxin-reducing system degraded I
B. However, specific PKC inhibitors calphostin C or GF109203X did not prevent the I
B degradation by thioredoxin, indicating that the PKC pathway is not
involved in the thioredoxin-mediated degradation of I
B and, hence,
the activation of NF-
B.

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Fig. 6.
Protein kinase C does not mediate
I B degradation by thioredoxin. A549 cells
were pre-incubated with GF109203X (15 µM) or calphostin C
(1 µM) for 1 h. After incubation,
Trx-(SH)2 (E. coli system) was added, and cells
were incubated for another period of 2 h, after which nuclear
extract was prepared. Post-nuclear extract was treated as cytosolic
extract. The cytosolic extract was subjected to Western blotting as
described under "Experimental Procedures." First lane,
untreated A549 cells; second lane, 5 µM
Trx-SH2; third lane, calphostin C, 1 mM; fourth lane, calphostin C + Trx-SH2; fifth lane, GF109203X, 15 µM; sixth lane, GF109203X + Trx-SH2, 5 µM.
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Phosphorylation of JNK by E. coli Thioredoxin--
Since JNK
pathway is activated by cytokines or UV radiation, which also activates
NF-
B, we hypothesized that the external addition of E. coli thioredoxin may activate JNK, and such activation may mediate
NF-
B activation. The cytosolic extracts of E. coli thioredoxin-treated cells were subjected to phospho-JNK detection using
phospho-specific antibodies from Santa Cruz. As demonstrated in the
Fig. 7A (lanes
2-4), oxidized E. coli thioredoxin did not activate
the JNK; however, the thioredoxin-reducing system activated JNK after
2 h of incubation (lane 6), a time point similar to the
activation of NF-
B (Fig. 1A). Although we detected
phospho-JNK in thioredoxin-treated cells, there is reason to believe
that total JNK protein may increase due to thioredoxin treatment, and the phospho-specific antibody may loose its specificity at higher JNK
levels. To investigate these possibilities, we detected total JNK level
by using a polyclonal antibody to JNK (Santa Cruz). As demonstrated in
Fig. 7B, there was no change in the total JNK levels,
confirming the specific JNK phosphorylation in response to thioredoxin
treatment. To determine the specificity of JNK activation by
thioredoxin, we also determined the activation of extracellular
signal-regulating kinase or p38 MAP kinases by thioredoxin using
phospho-specific antibodies. We did not detect phospho-extracellular signal-regulating kinase or phospho-p38 in the thioredoxin-treated cells (data not shown). These results suggest that thioredoxin specifically activates JNK-signaling cascade.

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Fig. 7.
Activation of JNK by reduced E. coli
thioredoxin in A549 cells. A, A549 cells were treated
with oxidized or reduced E. coli thioredoxin (2 µM) as described in Fig. 1. Cytosolic extracts were
subjected to Western blotting for phospho-JNK. Immunoreactive bands
were visualized by ECL detection. First lane, unstimulated
A549 cells; second through fourth lanes, cells treated with
E. coli thioredoxin and incubated for 1, 2, or 3 h;
fifth through seventh lanes, cells treated with 2 µM reducing E. coli system and incubated for
1, 2, or 3 h. B, total cellular JNK protein level does
not change in response to thioredoxin treatment. Cell lysates from
E. coli thioredoxin-treated cells (as described in Fig. 1)
were probed with anti-JNK antibody in a Western blotting experiment.
First lane, unstimulated A549 cells; second through fourth
lanes, cells treated with E. coli thioredoxin and
incubated for 1, 2, or 3 h; fifth though seventh lanes,
cells treated with 2 µM reducing E. coli
system and incubated for 1,2 or 3 h. C, activation of
JNK by overexpression of redox-active human thioredoxin in A549 cells.
A549 cells in 60-mm2 dishes were transfected with
pcDNA3, pcDNA3-Trx, or pcDNA3-dnTrx as described under
"Experimental Procedures." Stress-activated protein kinase/JNK was
precipitated from the cell lysates by c-Jun fusion protein bound to
glutathione-Sepharose beads. After selectively pulling down JNK using
c-Jun fusion protein beads, the beads were extensively washed, and the
kinase reaction was carried out in the presence of cold ATP in a final
volume of 25 µl. The reaction was stopped with 25 µl of 2× SDS
sample buffer and loaded onto a 10% polyacrylamide gel. First
lane, cells transfected with pcDNA3 only; second
lane cells transfected with 2 µg of pcDNA3-Trx; third
lane, cells transfected with 4 µg of pcDNA3-Trx;
fourth lane cells transfected with 4 µg of
pcDNA3-dnTrx.
|
|
Redox-active Thioredoxin Increases JNK Activity--
Since reduced
E. coli thioredoxin activated JNK phosphorylation, we sought
to determine whether the overexpression of human thioredoxin could also
activate JNK. Moreover, since human thioredoxin is not permeable into
the cells, overexpression of thioredoxin in the cell is an appropriate
method to study the effect of human thioredoxin on JNK activity. We
have demonstrated that only the redox-active thioredoxin is able to
activate NF-
B. Hence, we also sought to determine the effect of
redox-inactive thioredoxin on JNK activity. A549 cells were transfected
with pcDNA3-Trx or pcDNA3-dnTrx for 24 h followed by cell
lysis, and JNK activity assay was performed as described under
"Experimental Procedures." Redox-active thioredoxin potently
activated JNK, as determined by its ability to phosphorylate c-Jun
(Fig. 7C, lane 3). On the other hand,
redox-inactive mutant thioredoxin was unable to activate JNK (Fig.
7C, lane 4). Thus, the data suggest a
redox-control of JNK activation by thioredoxin.
dnJNKK or dnJNK Inhibit Thioredoxin-induced NF-
B
Activation--
If JNK activation is responsible for NF-
B
activation by thioredoxin, then blocking the JNK activation by
dominant-negative expression of JNK or dominant-negative expression of
JNK kinase should inhibit NF-
B activation by thioredoxin. Therefore,
to delineate the role of the JNK-signaling pathway in NF-
B
activation by thioredoxin, we cotransfected pcDNA3-Trx and dnJNKK
(MKK4/SEK1) or dnJNK expression plasmids into A549 cells. As
demonstrated in Fig. 8A, both
dnJNKK and dnJNK inhibited NF-
B activation by thioredoxin.

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Fig. 8.
dnMEKK1, dnJNK, or dnJNKK inhibit
thioredoxin-induced NF- B activation. A, A549 cells were
transfected with dnMEKK1, dnJNK, or dnJNKK along with pcDNA3-Trx.
Nuclear extracts were prepared 48 h post-transfection, and EMSA
was performed as described under "Experimental Procedures." First
lane, A549 cells transfected with pcDNA3 vector; second
lane, cells transfected with pcDNA3-Trx; third
lane, cells transfected with pcDNA3-Trx and dnMEKK1;
fourth lane, cells transfected with pcDNA3-Trx and
dnJNKK; fifth lane, cells transfected with pcDNA3-Trx
and dnJNK. B, degradation of I B by thioredoxin was
prevented by dnMEKK1, dnJNKK, or dnJNK. A549 cells were transfected
with various expression plasmids as described under "Experimental
Procedures." Nuclear and cytosolic extracts were prepared 24 h
post-transfection. I B bands were detected by Western blotting as
described under "Experimental Procedures." First lane,
A549 cells transfected with pcDNA3 empty vector; second
lane, A549 cells transfected with pcDNA3-Trx; third
lane, cells transfected with pcDNA3-dnMEKK1; fourth
lane, cells transfected with dnJNKK plasmid; fifth
lane, cells transfected with dnJNK plasmid.
|
|
Dominant-negative Kinase-dead MEKK1 Inhibits NF-
B Activation by
Thioredoxin--
MEKK1 is an initiating kinase that activates the JNK
cascade through phosphorylation of MKK4 or the p38 MAP kinase cascade by phosphorylating MKK3/MKK6 (19). Additionally, MEKK1 can directly phosphorylate I
B
kinase, which can cause the phosphorylation of
I
B
and the activation of NF-
B (21). Therefore, activation of
MEKK1 can directly cause NF-
B activation by phosphorylation of
I
B
kinase without involving the JNKK or JNK intermediate signaling steps. Since the JNK pathway is activated by thioredoxin, we
sought to determine the effect of inhibition of JNK cascade by
dominant-negative MEKK1. When pcDNA3-Trx and pcDNA3-dnMEKK1 were cotransfected, we observed inhibition of NF-
B binding (Fig. 8A).
Thioredoxin-mediated I
B Degradation Is Inhibited by dnMEKK1,
dnJNKK, or dnJNK--
We have shown that overexpression of human
thioredoxin causes degradation of I
B. Hence, if dnMEKK1 inhibits
NF-
B activation by thioredoxin, we expect to observe inhibition of
I
B degradation by cotransfection of pcDNA3-Trx and
pcDNA3-dnMEKK1. Additionally, if dnJNK or dnJNKK also inhibits
NF-
B activation by thioredoxin, we would observe degradation of
I
B in cells cotransfected with pcDNA3-Trx and dnJNKK or dnJNK.
Thus, to demonstrate the effect of dnMEKK1, dnJNKK, or dnJNK on I
B
degradation, we cotransfected the plasmids as demonstrated in the Fig.
8B and assayed I
B by Western blotting. As demonstrated in
Fig. 8B, overexpression of Trx degraded I
B. However, co
transfection of dnMEKK1 inhibited I
B degradation by thioredoxin.
Additionally, co-transfection of dnJNKK or dnJNK also prevented I
B
degradation by thioredoxin, suggesting a potential role of the
JNK-signaling cascade in the degradation of I
B by thioredoxin.
Thioredoxin-mediated Activation of NF-
B-luciferase Reporter
Vector Is Inhibited by dnMEKK, dnJNKK, or dnJNK--
To further
confirm the role of the JNK pathway in thioredoxin-mediated NF-
B
activation, we sought to determine the NF-
B-dependent gene transcription using a NF-
B-luciferase reporter construct (CLONTECH). As demonstrated in Fig. 9
(lane 2), there was a 2.5-fold increase in the
NF-
B-dependent luciferase activity by thioredoxin, indicating that thioredoxin in fact induces
NF-
B-dependent gene expression. In addition,
dominant-negative redox-inactive thioredoxin failed to induce
NF-
B-dependent gene expression, demonstrating that the
redox-active cysteines are required for NF-
B-mediated gene
transcription (Fig. 9, lane 9). Furthermore,
NF-
B-mediated luciferase reporter gene transcription was inhibited
when cells were cotransfected with dnMEKK1, dnJNKK, or dnJNK,
demonstrating that the activation of JNK cascade is required in
thioredoxin-induced, NF-
B-mediated gene transcription.

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Fig. 9.
Thioredoxin-mediated
NF- B-luciferase reporter expression is
inhibited by dnMEKK1, dnJNKK, or dnJNK. A549 cells were
transfected with pcDNA-Trx, pcDNA3-dnTrx, dnMEKK1, dnJNKK, or
dnJNK expression plasmids along with pTAL-NF- B-Luc and
pcDNA3.1- -galactosidase expression plasmids, as described under
"Experimental Procedures." Cells were harvested 48 h
post-transfection and lysed by reporter lysis buffer (Promega). The
experiment was done in triplicate and normalized to protein
concentrations. Transfection efficiency was normalized with
-galactosidase expression, determined by a luminescence
-galactosidase assay (Tropix). Lane 1, cells transfected
with pcDNA3 vector; lane 2, cells transfected with
pcDNA3-Trx; lane 3, cells transfected with pcDNA3Trx
and pcDNA3-dnMEKK1; lane 4, cells transfected with
pcDNA3-Trx and dnJNKK; lane 5, cells transfected with
pcDNA3-Trx and dnJNK; lane 6, cells transfected with
only dnMEKK1; lane 7, cells transfected with only dnJNKK;
lane 8, cells transfected with only dnJNK; lane
9, cells transfected with pcDNA3-dnTrx.
|
|
Wild-type MKK4 or MEKK1 Expression Increases Thioredoxin-mediated
NF-
B Activation and NF-
B-dependent Luciferase
Reporter Gene Expression--
To further confirm the role of the JNK
pathway in thioredoxin-mediated NF-
B activation, we cotransfected
pcDNA3-Trx and wild-type MKK4 or MEKK1 expression plasmid into A549
cells and assayed for NF-
B activation by EMSA. Since dnMKK4 or
dnMEKK1 inhibited NF-
B activation and NF-
B-dependent
luciferase expression, we expected an increase in NF-
B activation
with wild-type MKK4 or MEKK1 cotransfection with thioredoxin expression
plasmid. Indeed, there was a significant increase in
NF-
B-dependent luciferase expression (Fig.
10A). In addition,
transfection of wild-type MKK4 or MEKK1 alone or in combination with
trx expression plasmid induced NF-
B activation (Fig.
10B).

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Fig. 10.
Wild-type MKK4 or MEKK1 increase
thioredoxin-mediated NF- B-dependent luciferase reporter expression.
A, A549 cells were transfected with pcDNA-Trx, pcDNA3-MKK4,
or pcDNA3-MEKK1 expression plasmids along with pTAL-NF- B-Luc and
pcDNA3.1- -galactosidase expression plasmids, as described under
"Experimental Procedures." Cells were harvested 48 h
post-transfection and lysed with reporter lysis buffer (Promega). The
experiment was done in triplicate and normalized to protein
concentrations. Transfection efficiency was normalized with
-galactosidase expression, determined by a luminescence
-galactosidase assay (Tropix). First lane, cells
transfected with pcDNA3 vector; second lane, cells
transfected with pcDNA3-Trx; third lane, cells transfected
with pcDNA3-MKK4; fourth lane, cells transfected with
pcDNA3-MEKK1; fifth lane, cells transfected with pcDNA3-Trx
and pcDNA3-MKK4; sixth lane, cells transfected with
pcDNA3-Trx and pcDNA3-MEKK1. B, wild-type MKK4 or MEKK1
increase thioredoxin-mediated DNA binding. A549 cells were transfected
with pcDNA-Trx, pcDNA3-MKK4, or pcDNA3-MEKK1 expression plasmids. Cells
were harvested 48 h-post transfection, and nuclear extracts were
prepared as described under "Experimental Procedures." 10 µg of
nuclear extract was analyzed by EMSA using NF- B-specific
oligonucleotide probe as described under "Experimental Procedures."
First lane, cells transfected with pcDNA3 vector; second
lane, cells transfected with pcDNA3-Trx; third
lane, cells transfected with pcDNA3-MKK4; fourth
lane, cells transfected with pcDNA3-MEKK1; fifth
lane, cells transfected with pcDNA3-Trx and pcDNA3-MKK4;
sixth lane, cells transfected with pcDNA3-Trx and
pcDNA3-MEKK1.
|
|
Lactacystin Inhibited I
B Degradation and p65
Translocation--
We further explored the role of I
B degradation
by thioredoxin in NF-
B activation. Lactacystin inhibits NF-
B by
preventing degradation of I
B due to inhibition of the 26 S
proteosome (31). Hence, if Trx activates NF-
B by degradation of
I
B, inhibiting the 26 S proteosome by lactacystin should inhibit
NF-
B activation and I
B degradation by thioredoxin. Therefore, we
incubated Trx-transfected cells with lactacystin (4 h after
transfection) and determined NF-
B activation by EMSA after 24 h
of treatment. As demonstrated, lactacystin inhibited NF-
B activation
(Fig. 11A, lane
3) and I
B degradation (Fig. 11B, lane 2)
in Trx-transfected cells, further supporting the involvement of I
B
degradation in Trx-mediated NF-
B activation. Additionally, we also
evaluated the effect of Trx expression on p65 translocation. We probed
the nuclear extracts of cells transfected with Trx (as described for
Fig. 11A) with anti-p65 antibody (Santa Cruz) by Western
blotting. As demonstrated in Fig. 11B, Trx-induced p65
translocation to the nucleus, and such translocation was inhibited by
lactacystin.

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Fig. 11.
Lactacystin inhibits thioredoxin-mediated
NF- B activation. A, A549 cells were transfected with
pcDNA3 or pcDNA3-Trx as described under "Experimental
Procedures." 20 µM lactacystin was added to the culture
medium 4 h after transfection, and cells were incubated for
24 h. After incubation, nuclear and cytosolic extracts were
prepared. 10 µg of nuclear extract was analyzed by EMSA using
NF- B-specific oligonucleotide. First lane, cells
transfected with pcDNA3 vector only and treated with lactacystin;
second lane, cells transfected with pcDNA3-Trx; third
lane, cells transfected with pcDNA3-Trx and treated with
20 µM lactacystin for 20 h. B,
lactacystin prevents I B degradation and p65 translocation to the
nucleus induced by thioredoxin. Cytosolic extracts, as described in
A, were subjected to I B (upper panel), and
nuclear extracts were subjected to p65 (lower panel) Western
blotting as described under "Experimental Procedures." First
lane, cells transfected with pcDNA3 and treated with
lactacystin; second lane, cells transfected with
pcDNA3-Trx and treated with lactacystin; third lane,
cells transfected with pcDNA3-Trx.
|
|
 |
DISCUSSION |
In the present report, we have demonstrated that the addition of
E. coli thioredoxin to the culture medium activates NF-
B. We have further provided evidence that oxidized E. coli
thioredoxin was unable to activate NF-
B, whereas the
thioredoxin-reducing system (Trx + NADPH + TR) could activate NF-
B
and degrade I
B in A549 cells. Furthermore, we have shown that
overexpression of human thioredoxin activates NF-
B. In contrast,
dominant-negative redox-inactive human thioredoxin failed to activate
NF-
B. Additionally, we also demonstrate that the JNK-signaling
cascade is activated by thioredoxin. Cotransfection of cells with
pcDNA3-Trx and dominant-negative kinase-dead MEKK1 also inhibited
NF-
B activation by thioredoxin, demonstrating a potential role of
MEKK1 in the activation of NF-
B by thioredoxin. In addition,
cotransfection of cells with pcDNA3-Trx and dnJNKK or dnJNK
inhibited Trx-induced NF-
B activation. Thus, in this report we have
demonstrated, employing two different systems (the external addition of
E. coli thioredoxin and overexpression of human thioredoxin
or its mutant form) that redox-active thioredoxin activates NF-
B,
which is mediated by the initiating kinase of the JNK pathway, MEKK1.
It is possible that thioredoxin that enters the cell could be reduced
by endogenous thioredoxin reductase and NADPH. However, E. coli thioredoxin is not as effective a substrate as the mammalian thioredoxin for mammalian thioredoxin reductase (8), and because of the
oxidizing environment of cell culture medium, it was considered possible that the effect of thioredoxin could be mediated by its oxidized species. Moreover, we have earlier demonstrated that most of
the oxidized E. coli thioredoxin that enters the cell remains in the oxidized form (32). Additionally, for the mammalian TR,
the Km of E. coli thioredoxin is 35 µM, ~14-fold higher than the Km for
mammalian thioredoxin (8). Therefore, using a 2-5 µM
concentration of E. coli thioredoxin, we effectively
prevented the reduction of E. coli thioredoxin by cellular
thioredoxin reductase. In a recent study, we demonstrate that by
using an enzymatic reducing system for reduction of thioredoxin, 85%
of thioredoxin that entered the cell was in fully reduced form (32).
Thus, the use of E. coli thioredoxin was an excellent experimental paradigm in differentiating the redox effect of E. coli thioredoxin from that of endogenous thioredoxin. We further demonstrated that E. coli thioredoxin can also induce
NF-
B activation in primary cultures of pulmonary artery endothelial
cells. Since primary cultures are considered to represent normal
in vivo conditions, our results indicate that activation of
NF-
B by E. coli thioredoxin is not limited to transformed
cell lines.
Mammalian thioredoxin does not enter cultured cells (27). Therefore, to
determine the effect of human thioredoxin on NF-
B activation, we
transiently overexpressed thioredoxin in A549 cells. Thioredoxin
overexpression activated NF-
B 24 h post-transfection (Fig.
5A). However, in the reducing environment of the cell, most of the overexpressed thioredoxin is expected to be in the reduced state, and therefore, activation of NF-
B by overexpressed
thioredoxin could be attributed to its reduced form. To determine the
effect of thioredoxin redox status on NF-
B activation, we mutated
the catalytic Cys-32 and Cys-35 by site-directed mutagenesis. When inserted into a pCMV-directed expression vector, this mutated thioredoxin was expressed in a dominant-negative manner. Overexpression of dominant-negative thioredoxin did not induce NF-
B activation in
A549 cells, indicating the requirement of redox-active cysteines in the
activation of NF-
B.
Previous reports have shown that thioredoxin activates NF-
B after
I
B dissociates from the NF-
B complex, indicating only a reducing
function of thioredoxin (14). Therefore, to understand the mechanism of
activation of NF-
B by thioredoxin, we immunoblotted I
B in the
cytosolic extract. We have shown that the I
B inhibitory protein is
degraded as a result of treatment of cells with 2 µM E. coli thioredoxin-reducing system. There was no
degradation of I
B when cells were incubated with 2 µM
oxidized E. coli thioredoxin. Similar observations were made
with respect to primary cultures of endothelial cells (Fig. 3).
Overexpression of human thioredoxin also degraded I
B (Fig.
5B) and induced translocation of p65 to the nucleus (Fig.
11B). Lactacystin, a 26 S proteosome inhibitor, inhibited
thioredoxin-mediated NF-
B activation and degradation of I
B,
further supporting the role of I
B degradation in NF-
B activation
(Fig. 11). However, expression of dominant-negative redox-inactive
thioredoxin did not degrade I
B. Therefore, it is likely that
thioredoxin redox status modulates an upstream signaling event,
resulting in NF-
B activation.
Since the JNK-signaling cascade is activated by many stimuli that also
activates NF-
B, we hypothesized that JNK or its upstream kinase JNKK
may mediate NF-
B activation by thioredoxin. If this is true, then
dominant-negative JNKK or JNK should inhibit NF-
B activation by
thioredoxin. Indeed, in cotransfection experiments, dnJNKK and dnJNK
inhibited NF-
B activation and prevented degradation of I
B,
indicating a potential role of the JNK pathway in NF-
B activation.
MEKK1 is an initiating kinase of the JNK pathway and activates
MKK4/SEK1, which activates the JNK. Since MEKK1 can directly
phosphorylate I
B
, causing its ubiquitination and subsequent degradation, we reasoned a likely role of MEKK1 in the activation of
NF-
B by thioredoxin. MEKK1 can directly phosphorylate I
B
kinase, causing its activation and subsequent degradation of I
B, or
it can activate MKK4, which in turn can activate JNK. When cells were
cotransfected with pcDNA3-Trx and pcDNA3-dnMEKK1, there was
inhibition of NF-
B activation. However, cotransfection of pcDNA3-Trx and dnJNKK or dnJNK also inhibit NF-
B activation and prevent degradation of I
B. Therefore direct phosphorylation of I
B
kinase by MEKK1 in thioredoxin-stimulated cells is not a likely possibility. Together, these results demonstrate that
thioredoxin activates NF-
B by degradation of I
B, which is
mediated by the JNK-signaling pathway.
Both NF-
B and MAP kinases, particularly JNKs and p38, are activated
by similar agents. A connection between NF-
B and JNK activation has
recently been suggested based on the observation that overexpression of
MEKK1-stimulated NF-
B activation (33-34). Coimmunoprecipitation
assays further demonstrated an interaction between JNK and the NF-
B
subunit c-Rel that could be confirmed by two-hybrid assays (34).
Additionally, studies have demonstrated that dominant-negative
kinase-dead MEKK1 expression was able to inhibit tumor necrosis
factor-
-induced activation of NF-
B (21). However, the exact
mechanism of activation of NF-
B by MEKK1 or the JNK has not been
elucidated. Clearly, further studies are required to delineate the
mechanisms of NF-
B activation by the JNK pathway. Our present data
demonstrate that redox-active thioredoxin was able to activate JNK
pathway, which is initiated at the MEKK1 level. Redox control of MEKK1
activation is rather a novel finding. However, the mechanism of
activation of MEKK1 by thioredoxin awaits further investigation.
Recently, thioredoxin has been shown to migrate to the nucleus upon
stimulation of cells with oxidative stress or other stresses (35).
Since oxidative stress is known to induce and translocate thioredoxin
to the nucleus (35), it is likely that translocated thioredoxin may
modulate the transcription factor by facilitating its DNA binding due
to reduction of sulfhydryls on the DNA binding domain. However, since
I
B
kinase must be phosphorylated to allow the ubiquitin-mediated
degradation, thioredoxin-mediated MEKK1-dependent phosphorylation of I
B may precede the thiol-reducing function of
thioredoxin. Therefore, thioredoxin-mediated NF-
B activation is
likely to be initiated at MEKK1 level, involving the redox-active function of thioredoxin. Additionally, thioredoxin may facilitate the
NF-
B DNA binding in the nucleus by its protein-reducing function after the translocation of the p50/p65 heterodimer.
 |
ACKNOWLEDGEMENTS |
We thank Drs. Roger Davis, Tom Maniatis, and
Gary Johnson for gifts of plasmids.
 |
FOOTNOTES |
*
This work was supported by a Scientist Development Award
from the American Heart Association, National Center, and a research project grant from the American Cancer Society.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: University of Texas
Health Center, 11937 U. S. Hwy 271, Tyler, TX 75708. Tel.: 903-877-7418; Fax: 903-877-7675; E-mail-kumuda@uthct.edu.
Published, JBC Papers in Press, November 3, 2000, DOI 10.1074/jbc.M006206200
 |
ABBREVIATIONS |
The abbreviations used are:
NF-
B, nuclear
transcription factor
B;
Trx, thioredoxin;
TR, thioredoxin reductase;
MAP, mitogen-activated protein;
JNK, c-Jun NH2-terminal
kinase;
JNKK, JNK kinase;
MEKKK, MAP kinase kinase kinase;
dn, dominant-negative;
EMSA, electrophoretic mobility shift assay;
HPAEC, human pulmonary artery endothelial cells;
PKC, protein kinase
C.
 |
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