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INTRODUCTION |
Reactive oxygen species
(ROS)1 are normal metabolic
byproducts and intermediates found in many physiological processes.
Three major sources of intracellular ROS include the xanthine/xanthine oxidase system, receptor-coupled NADPH oxidase at the cellular membrane, and the mitochondrial electron transport system (1, 2). ROS
have been increasingly recognized as critical components in disease and
stress-induced cellular injuries such as ischemia/reperfusion (I/R), UV
irradiation, and inflammation. These ROS can lead to direct cellular
damage and can also act as intracellular second messengers to modulate
signal transduction pathways. One such redox-regulated transcription
factor is NF
B (3).
NF
B family members include p50, p52, p65, and c-RelB, which form
homodimeric and heterodimeric transcriptional complexes (4). The
activation of NF
B is controlled by a family of I
B repressor
proteins (I
B
, I
B
, and I
B
) that sequester NF
B in
the cytoplasm (4). Phosphorylation-dependent inactivation of I
B proteins leads to the mobilization of NF
B to the nucleus where it can act as a transcription factor. These phosphorylation pathways have been most extensively studied for I
B
and include two distinct mechanisms involving either serine or tyrosine
phosphorylation of I
B
. The most comprehensively studied pathway
regulating I
B
includes phosphorylation on two serine (32 and 36)
residues by the I
B kinase complex (IKK) (5). This phosphorylation
leads to ubiquitination of I
B
at nearby lysine residues and
degradation by the proteasome. An alternative, less characterized
pathway of NF
B activation acts through tyrosine phosphorylation of
I
B
at residue 42 (6). In contrast to IKK-mediated serine
phosphorylation of I
B
, tyrosine phosphorylation of I
B
is
capable of activating NF
B in the absence of
ubiquitin-dependent degradation of I
B
. However, it is
presently unclear if IKK and/or the I
B
protein-tyrosine kinase
(PTK) interactions with I
B
are functionally modulated by prior
tyrosine or serine phosphorylation of I
B
, respectively. Experimental evidence appears to suggest that prior tyrosine
phosphorylation of I
B
on Tyr-42 may prevent interactions with the
IKK complex and inhibit serine phosphorylation on Ser-32/Ser-36 (7).
Hence, the existence of reciprocal interactions between IKK- and
PTK-mediated phosphorylation of I
B
and the net effect on NF
B
transcriptional activation remains an open question. Although the exact
identity of the I
B tyrosine kinase has not yet been demonstrated
using in vitro reconstitution assays, both PI 3-kinase and
c-Src have been demonstrated to associate with tyrosine phosphorylated
I
B
in T-cells following pervanadate treatment (8) and bone marrow macrophages (BMMs) following TNF
stimulus (9). In addition to
pervanadate, H/R has also been shown to induce tyrosine phosphorylation of I
B
in T-cells in vitro (6) and following I/R injury
to the liver in vivo (10).
The tyrosine kinase p56lck is required for I
B
tyrosine phosphorylation and NF
B activation in T-lymphocytes
following pervanadate treatment (6). Loss of tyrosine kinases
p56lck and ZAP-70 in two Jurkat mutants abolished NF
B
activation and partially suppressed and delayed phosphorylation of
Tyr-42 on I
B
in response to pervanadate treatment (11). However,
this study in T-cells also demonstrated that tyrosine phosphorylation of I
B
was not sufficient to activate NF
B and suggests that both tyrosine and serine kinases act at multiple levels to dissociate the I
B
/NF
B complex. Furthermore, tyrosine phosphorylation of I
B
is observed in BMMs following TNF
treatment, and this
phosphorylation requires c-Src activity (9). Given the historical
dependence of TNF
-mediated activation of NF
B on the IKK complex
and serine phosphorylation of I
B
, the functional involvement of
I
B
tyrosine phosphorylation in response to TNF
appears to be
quite unique to BMMs. Furthermore, the vast majority of studies
evaluating the importance of I
B
tyrosine phosphorylation to date
have been performed in hematopoetically derived T-cells or BMMs. Thus,
the functional relevance of these systems to epithelial models of ischemia/reperfusion remains an open question. Since c-Src can be
directly activated by H2O2 (12), pervanadate
(13), hypoxia (14), or hypoxia/reoxygenation (15), its central
involvement in ROS-mediated IKK and PTK activation of NF
B appears
reasonable. It is also recognized that H2O2 is
capable of activating both IKK- and PTK-dependent pathways
of I
B
phosphorylation and NF
B activation in T-cells (11).
In the present study, we sought to investigate the involvement of c-Src
in the redox-mediated activation of NF
B activation following H/R or
pervanadate treatments in an epithelial cell line (HeLa cells). Since
both IKK-dependent and independent pathways of NF
B
activation have been associated with c-Src activation, we used a number
of adenoviral vectors expressing dominant mutants of IKK
, IKK
,
and I
B
to selectively test for serine or tyrosine I
B
phosphorylation-dependent transcriptional activation of
NF
B. In contrast to previous studies, we have utilized an
NF
B-responsive luciferase reporter gene to directly assess changes
in the transcriptional activation of NF
B. Since the association of
tyrosine-phosphorylated I
B
with PI 3-kinase has been suggested in
proposed models to alter the transcriptional properties of NF
B
dimers (8, 11), direct functional assessment of activation may be more
informative than assessing DNA binding. Triple knockout cell lines
(c-Src
/
, Fyn
/
, Yes
/
) with and without c-Src were also used
to confirm the dependence of I
B
tyrosine phosphorylation on
c-Src. Furthermore, recombinant adenoviral vectors expressing various
ROS scavengers were used to test whether activation of these pathways
contained redox-sensitive components. Results from these studies
indicate that tyrosine phosphorylation of I
B
and NF
B
activation is mediated through redox activation of c-Src.
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EXPERIMENTAL PROCEDURES |
Adenoviral Vectors--
Several E1-deleted recombinant
adenoviral vectors were used to modulate the NF
B pathway and assay
for transcriptional induction of NF
B. Previously described vectors
included the dominant negative mutants Ad.IKK
(KM) (16),
Ad.IKK
(KA) (16), Ad.I
B
(S32A/S36A) (17), and the
NF
B-responsive luciferase reporter vector Ad.NF
Bluc (16).
Ad.BglII was used as an empty vector control (16).
Ad.I
B
(Y42F), which expresses the Y42F mutant form of I
B
,
was generated by cloning the previously described I
B
Y42F cDNA
(6) into pAd.CMVlink (18). All adenoviral vectors, except for
Ad.NF
Bluc, used the CMV enhancer/promoter to express the transgene.
Recombinant adenovirus was purified by two rounds of CsCl
centrifugation and desalted prior to use as described (19).
Cell Culture, Adenoviral Transduction, and Treatments--
HeLa,
SYF, and SYF+c-Src cells were cultured in Dulbecco's Modified Eagle
Medium (DMEM) (Invitrogen) supplemented with 10% fetal bovine serum
(FBS) and 100 µg/ml penicillin and streptomycin. For the tyrosine
phosphorylation assays, HeLa cells were transduced with Ad.IKK
(KM),
Ad.IKK
(KA), Ad.I
B
(S32A/S36A), Ad.I
B
(Y42F), or
Ad.BglII at a multiplicity of infection (MOI) equal to 1000 particles/cell. For the NF
B luciferase reporter assay, HeLa cells were co-infected with Ad.NF
BLuc at an MOI = 500 particles/cell and Ad.IKK
(KM), Ad.IKK
(KA), Ad.I
B
(S32A/S36A), or
Ad.I
B
(Y42F) at an MOI = 1000 particles/cell. Luciferase
reporter assays in SYF and SYF+c-Src cells were performed following
infection with Ad.NF
BLuc alone at an MOI = 500 particles/cell.
Adenoviral infections were performed for 2 h in DMEM without FBS
followed by the addition of an equal volume of 20% FBS, DMEM and
continued incubation for 22 h. Virus-containing media was replaced
at 24 h post-infection with 10% FBS/DMEM. Typically, experiments
were initiated at 24 h post-transduction. Experimental methods
used to induce NF
B were performed according to the following protocols.
Pervanadate Treatment--
Sodium orthovanadate was prepared
fresh in water at a concentration of 500 mM. 40 µl of
sodium orthovanadate and 5 µl of 30% (w/w)
H2O2 was then added to 455 µl
phosphate-buffered saline. This mixture was incubated for 5 min at room
temperature prior to the addition of catalase (200 µg/ml) to remove
the excess H2O2. The pervanadate solution
(final concentration 40 mM) was further incubated for 5 min
at room temperature, immediately diluted in DMEM and applied to cells.
Cells were harvested at 6 h post-pervanadate treatments for NF
B
activation using luciferase assays or as indicated. Control cells were
fed with identical fresh medium that was devoid of pervanadate.
Hypoxia/Reoxygenation--
DMEM (devoid of glucose or
FBS) (Invitrogen) equilibrated in 95% N2, 5%
CO2 or 95% O2, 5% CO2 was used as
hypoxia and reoxygenation medium, respectively. Cells were covered with
minimal hypoxia medium and incubated at 37 °C for 5 h in an
airtight chamber equilibrated with 5% CO2 and 95%
N2. The medium was then replaced with a minimal amount of
reoxygenation medium and incubated further at 37 °C in a chamber
flushed with 5% CO2 and 95% O2. Cells were
harvested 6 h after reoxygenation for NF
B activation luciferase
assays. Control cells were fed with fresh medium at identical times as the hypoxia/reoxygenation samples, but were exposed to 5%
CO2 in atmospheric oxygen.
TNF
Treatment--
Mouse recombinant TNF
(R&D systems,
Minneapolis, MN) was diluted in fresh DMEM medium (10 ng/ml final
concentration) and applied to cells at the time of treatment. Cells
remained exposed to TNF
until they were harvested at 6 h
post-stimulation for NF
B activation luciferase assays. Control cells
were fed at the time of treatment with fresh DMEM medium without
TNF
.
Western Blotting and I
B
Phosphorylation Assays--
Cells
were lysed in RIPA buffer (0.15 M NaCl, 50 mM
Tris pH 7.2, 1% deoxycholate, 1% Triton X-100, 0.1% SDS), and the
protein concentration was determined using a Bio-Rad protein assay
(Bio-Rad, Hercules, CA). 5 µg of cell lysate was resolved on a 10%
SDS-PAGE and then transferred to nitrocellulose membrane using
previously described protocols (18). I
B
protein levels were
determined by Western blot analysis using an anti-I
B
monoclonal
antibody (Santa Cruz Biotechnology, Santa Cruz, CA). To evaluate
I
B
tyrosine phosphorylation, 200 µg of cell lysate was
immunoprecipitated using 2 µg of I
B
antibody (Santa Cruz
Biotechnology) followed by Western blot analysis using
antiphosphotyrosine antibody (Santa Cruz Biotechnology) and standard
protocols (10). Phosphorylated forms of c-Src or total c-Src were
detected using anti-c-SrcPY416, anti-c-SrcPY139, and anti-c-Src
antibodies (Santa Cruz Biotechnology).
NF
B Activation Assays--
NF
B transcriptional activity
was evaluated using an Ad.NF
BLuc reporter vector as previously
described (16). Briefly, cells were infected with Ad.NF
BLuc at an
MOI of 500 particles/cell 24 h prior to TNF-
, pervanadate, or
H/R treatment. 5 µg of total protein from each sample was assayed for
luciferase activity using manufacturer's protocols (Promega, Madison,
WI) in a luminometer as previously reported (16). Luciferase activity
was assessed as relative light units and used as an indicator for the
transcription induction of NF
B. To assess potential global changes
in transcription induced by each type of environmental stimuli, that
were not dependent on NF
B, several experiments were performed
normalizing changes in Ad.NF
BLuc expression to that seen with a
control Ad.CMVLacZ vector (20). In these studies both Ad.NF
BLuc and
Ad.CMVLacZ were co-infected into cells for each of the conditions
examined (MOI = 500 particles/cell for each vector) 24 h
prior to TNF-
, pervanadate, or H/R treatment. Luciferase activity
was then assessed using 5 µg of lysate as described above, and
-galactosidase activity was quantified with 5 µg of lysate using a
previously described protocol (21). Luciferase activity was then
normalized for
-galactosidase expression in reference to the
Ad.BglII infected (no injury) control. Electrophoretic
mobility shift assays for NF
B DNA binding were performed as
previously described using a 32P-labeled NF
B
oligonucleotide probe (18).
In Vitro Kinase Assays--
Two types of in vitro
kinase assays (radioactive and non-radioactive) were used to evaluate
the ability of immunoprecipitated c-Src or IKK
to phosphorylate
GST-I
B
in vitro following different environmental
stimuli. For radioactive in vitro kinase assays, HeLa, SYF,
or SYF+c-Src cells were washed in ice-cold PBS and lysed in 1 ml of
ice-cold RIPA buffer (0.15 M NaCl, 50 mM Tris, pH 7.2, 1% deoxycholate, 1% Triton X-100, 0.1% SDS) followed
by centrifugation at 10,000 rpm for 10 min at 4 °C. The protein
concentration was then determined using a Bio-Rad protein assay
(Bio-Rad, Hercules, CA). 500 µg of protein was immunoprecipitated
with anti-c-Src or anti-IKK
antibodies (Santa Cruz Biotechnology)
and protein A-agarose beads. 1 µg of GST-IkB
protein (Santa
Cruz Biotechnology) was then added to washed protein A pellets in the
presence of 10 µl of kinase buffer (40 mM Hepes, 1 mM
-glycerophosphate, 1 mM
nitrophenolphosphate, 1 mM Na3VO4,
10 mM MgCl2, 2 mM dithiothreitol, 0.3 mM cold ATP, and 10 µCi of
[
-32P]ATP) and incubated at 30 °C for 30 min. The
reaction was terminated by the addition of protein-loading buffer (with
SDS) and boiled at 98 °C for 5 min. Samples were then centrifuged to
remove the agarose beads, and the supernatant was loaded onto a 10%
SDS-PAGE gel. After electrophoresis, proteins were transferred to
nitrocellulose membrane (which reduces the background of free
[
-32P]ATP) and exposed to x-ray film. Non-radioactive
in vitro kinase assays were performed to directly evaluate
the extent of tyrosine phosphorylation of GST-I
B
by
immunoprecipitated c-Src or IKK
. These in vitro kinase
assays were performed identical to the protocol described above except
for the omission of [
-32P]ATP. In vitro
labeled GST-I
B
samples were then evaluated by Western blotting
for the extent of tyrosine phosphorylation using antiphosphotyrosine
antibody (Santa Cruz Biotechnology).
 |
RESULTS |
Transcriptional Activation of NF
B Following Pervanadate or
H/R Treatment Requires I
B
Tyr-42 Phosphorylation and
Is Independent of the IKK Complex and I
B
Serine
Phosphorylation--
NF
B activation can occur through at least two
mechanisms that control I
B
phosphorylation on either tyrosine 42 or serine 32/36. Proinflammatory stimuli such as TNF
are well suited
to activate NF
B through the I
B kinase complex (IKK) that mediates serine phosphorylation of I
B
and ubiquitin-dependent
degradation of I
B
. In contrast, NF
B activation in the liver
following ischemia/reperfusion (I/R) injury (10), and in T-cells
following H/R (6), occurs in the absence of I
B
degradation and is
associated with an increase in tyrosine phosphorylation of I
B
. To
better define the mechanisms involved in NF
B activation following
I/R injury, we developed an in vitro epithelial cell line
model system capable of modulating NF
B activity through tyrosine or
serine phosphorylation of I
B
following H/R, pervanadate, or
TNF
treatments.
To establish that NF
B activation following H/R occurs through a
selective pathway involving tyrosine phosphorylation of I
B
that
is independent of the IKK complex, we utilized several dominant negative mutants to modulate IKK activation and I
B
phosphorylation. NF
B transcriptional activity was evaluated using a
recombinant adenoviral reporter vector (Ad.NF
BLuc) expressing the
NF
B-inducible luciferase gene. As expected and previously reported
in epithelial cell lines, the transcriptional induction of NF
B
following TNF
treatment was significantly inhibited
(p < 0.001) by expression of Ad.IKK
(KA),
Ad.IKK
(KM), or Ad.I
B
(S32A/S36A) in comparison to
Ad.BglII (empty vector control)-transduced cells (Fig.
1A). No inhibition in
TNF
-induced NF
B activation was seen following expression of
Ad.I
B
Y42F. These results confirm the functionality of our vectors
to inhibit IKK-mediated TNF
activation of NF
B and demonstrate a
lack of functional involvement of I
B
Y42 phosphorylation under these conditions. In contrast to findings with TNF
,
I
B
(Y42F) expression significantly inhibited NF
B
transcriptional activation following pervanadate (p < 0.001) or H/R (p < 0.001) treatments (Fig. 1,
A and B). No significant alterations in
pervanadate or H/R-mediated activation of NF
B was seen following
infection with Ad.IKK
(KA), Ad.IKK
(KM), or
Ad.I
B
(S32A/S36A) mutant vectors. Furthermore, when the
induction of NF
B-mediated luciferase expression was normalized
to changes in expression of an irrelevant internal control LacZ
transgene under the control of the CMV promoter, the patterns and
changes for each of the environmental stimuli and dominant mutants
tested were not significantly altered (Fig. 1, A-C). These
data demonstrate that global changes in the overall transcriptional
state of cells cannot account for the specific alterations induced by
the various dominant mutants for a given stimulus.

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Fig. 1.
NF B activation
following pervanadate or H/R treatment is inhibited by
I B (Y42F) but not
I B (S32A/S36A) or
IKK-dominant mutants. HeLa cells were co-infected with
Ad.I B (Y42F), Ad.I B (S32A/S36A), Ad.IKK (KM),
Ad.IKK (KA), or Ad.BglII (MOI of 1000 particles/cell)
together with Ad.NF BLuc (MOI of 500 particles/cell) 24 h before
experimental analysis. Cells were treated with TNF- (10 ng/ml)
(A), pervanadate (100 µM) (B), or
H/R (5 h of hypoxia, 6 h of reoxygenation) (C). Whole
cell extracts were harvested 6 h following treatments, normalized for total protein
content, and evaluated for NF B transcriptional activity using a
luciferase assay. NF B transcriptional activity was determined by the
mean relative luciferase activity (RLA) (± S.E.,
n = 6). D, electrophoretic mobility shift
assays for NF B DNA binding in nuclear extracts harvested from HeLa
cells following TNF (10 ng/ml, 1 h), pervanadate (50 µM, 1 h), or H/R (5 h of hypoxia, 3 h of
reoxygenation) treatments. Exposure times are not equivalent for each
treatment condition. E, HeLa cells were treated with TNF
(10 ng/ml) for 30 min, pervanadate (100 µM) for 30 min,
or H/R (5 h of hypoxia, 30 min of reoxygenation). IKK was
immunoprecipitated with anti-IKK antibody from 500 µg of protein
lysate. The ability of immunoprecipitated IKK to directly
phosphorylate GST-I B fusion protein was evaluated in the presence
of [ 32 -P]ATP in vitro.
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|
To confirm that changes in transcriptional activation of NF
B
mirrored those seen in DNA binding, electrophoretic mobility shift
assays were performed for each of the various stimuli. These results
shown in Fig. 1D confirm that NF
B transcriptional
activation is accompanied by increased DNA binding in nuclear extracts.
Cumulatively, our results evaluating IKK and I
B
mutants suggest
that IKK-mediated serine 32/36 phosphorylation of I
B
does not
play a significant role in regulating NF
B following pervanadate or
H/R stimuli in our HeLa cell line model. To directly evaluate whether
TNF-
imparts selective activation of the IKK complex not observed
following H/R or pervanadate treatments, we performed in
vitro kinase assays with immunoprecipitated IKK
to directly
evaluate IKK activation and ability to phosphorylate GST-I
B
following each of these stimuli. Results from this analysis are shown
in Fig. 1E and demonstrate that TNF-
treatment stimulates
higher levels of IKK activity as compared with H/R and pervanadate
treatments. However, activation of IKK was also observed at lower
levels following both H/R and pervanadate treatments, suggesting that
some overlap in signaling may exist. This apparent overlap may be due
to pervanadate and H/R activation of cytokines, which restimulate cells
through the IKK pathway. These findings substantiate the small
non-significant, but observed, partial inhibition of NF
B
transcriptional activation by IKK mutants seen following H/R and
pervanadate treatments.
Src Inhibitor pp2 Blocks NF
B Activation and I
B
Tyrosine
Phosphorylation Following Pervanadate or H/R
Treatment--
Our results in the HeLa cell model have established
that NF
B activation following H/R or pervanadate treatment is
independent of IKK and serine phosphorylation of I
B
. We next
sought to evaluate candidate upstream factors capable of mediating
tyrosine phosphorylation of I
B
and subsequent NF
B activation.
Src family kinases are widely recognized for their importance in
regulating stress response genes in response to redox-regulated stimuli
such as H/R (15, 22). Furthermore, it has been reported that c-Src
activity was necessary for TNF
-induced tyrosine phosphorylation of
I
B
in BMMs (9). Given the lack of a functional requirement for
I
B
tyrosine phosphorylation in the transcriptional induction of
NF
B following TNF
in our epithelial cell line model, we
investigated whether c-Src might also play a role in NF
B activation
following H/R or pervanadate treatment.
Consistent with the activation of c-Src following H/R or pervanadate
treatment, we observed an increase in both Tyr-416- and Tyr-139-phosphorylated forms of activated c-Src (Fig.
2). H/R treatment demonstrated a greater
increase in both phosphorylated forms while pervanadate treatment more
selectively increased the Tyr-416-phosphorylated form of c-Src. These
findings suggest that indeed c-Src is activated by both pervanadate or
H/R treatment and is consistent with the previously reported
redox-mediated involvement in the activation of c-Src (15). To assign
functional importance to c-Src in the tyrosine phosphorylation of
I
B
and subsequent activation of NF
B, we next evaluated the
effect of the pp2 c-Src inhibitor. Pretreatment of HeLa cells with pp2
significantly inhibited both pervanadate- and H/R-induced NF
B
activation (p < 0.001) (Fig.
3A). Furthermore, the level of
tyrosine phosphorylation of I
B
following pervanadate or H/R
treatment was also significantly reduced in the presence of pp2. These
results are consistent with the hypothesis that c-Src is functionally
required for both I
B
tyrosine phosphorylation and subsequent
activation of NF
B.

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Fig. 2.
Activation of c-Src following pervanadate or
H/R treatment. HeLa cells were treated with pervanadate (100 µM) (A) for 15, 30, and 45 min or hypoxic
media (B) (95% N2, 5% CO2) for
5 h followed by reoxygenation media (95% O2, 5%
CO2) for 15, 30, and 45 min. Both untreated and treated
samples were harvested at the indicated time points into lysis buffer,
and 5 µg of total protein was separated on a 10% SDS-PAGE. Western
blots were evaluated for c-Src tyrosine phosphorylation using two
phosphospecific antibodies that recognize phospho-Tyr-416 and
phospho-Tyr-139 of activated c-Src. The extent of c-Src phosphorylation
is referenced to the total level of c-Src in the sample using an
anti-c-Src antibody that recognized both phosphorylated and
unphosphorylated forms of c-Src.
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Fig. 3.
Inhibition of c-Src activation blocks
NF B transcriptional activation and
I B tyrosine
phosphorylation following pervanadate or H/R treatment.
A, HeLa cells were transduced with Ad.NF BLuc (MOI of 500 particles/cell) 24 h prior to the initiation of experimental
treatment. Cells were then exposed to fresh media with and without pp2
(10 µM) for 30 min, followed by treatment with
pervanadate (100 µM) for 6 h or H/R (hypoxic media;
5% N2, 5% CO2) for 5 h followed by
reoxygenation media (95% O2, 5% CO2) for
6 h. pp2 inhibitor was continually present during pervanadate and
H/R treatments. Whole cell extracts were harvested into lysis buffer,
normalized for total protein content, and evaluated for NF B
activation using a luciferase assay. Results depict the mean (± S.E.,
n = 6) relative luciferase activity (RLA).
HeLa cells were treated with pervanadate (100 µM)
(B) for 30 min or hypoxic media (95% N2, 5%
CO2) (C) for 5 h followed by reoxygenation
media (95% O2, 5% CO2) for 30 min. One group
was pretreated with pp2 (10 µM) for 30 min prior to
pervanadate or H/R. Inhibitor (pp2) was continually present during the
treatments. Cell lysates were harvested and 200 µg of total protein
was immunoprecipitated with anti-I B antibody followed by Western
blotting with an antiphosphotyrosine antibody or anti-I B
antibody.
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|
Tyrosine Phosphorylation of I
B
and NF
B Activation Is
Significantly Reduced in a c-Src, Fyn, and Yes Triple Knockout Cell
Line Following H/R or Pervanadate Treatment--
The
importance of c-Src in mediating tyrosine phosphorylation of I
B
and NF
B activation was further investigated using a knockout cell
line deficient for Src family kinases Src, Fyn, and Yes. These kinases
have similar redundant functions and thus, the knockout of a single
gene will not completely abolish their activity. In SYF cells, Src,
Yes, and Fyn have been all knocked out to establish null Src mutant
activity (23). In SYF+src cell lines, the c-Src activity was
reintroduced into the SYF background. Thus, by comparing these two cell
lines, one can elucidate c-Src function.
Results evaluating the SYF cell line demonstrated a complete loss of
pervanadate- and H/R-induced NF
B transcriptional activation in
comparison to SYF+src cells (p < 0.001) (Fig.
4A). In contrast, there was no
significant difference in TNF
-mediated induction of NF
B in either
of these two cell lines. These results suggest that c-Src activity is
required for NF
B pathways involving tyrosine, but not
serine-mediated phosphorylation of I
B
. To conclusively address
the requirement for c-Src activity to mediate tyrosine phosphorylation
of I
B
, we next evaluated the extent of I
B
tyrosine
phosphorylation in both SYF and SYF+src cells following pervanadate or
H/R treatment. These studies demonstrated that I
B
tyrosine
phosphorylation was significantly reduced in SYF following pervanadate
and completely blocked following H/R as compared with SYF+src cells
(Fig. 4, B and C). Given the previous demonstration of p56Lck function in the activation of
I
B
tyrosine phosphorylation following pervanadate treatment (11),
the residual phosphorylation seen in our c-Src, Fyn, and Yes knockout
cell lines may be due to redundant Lck function. However, our studies
evaluating NF
B activation following pervanadate treatment suggest
that this residual phosphorylation may not be functionally active. In
contrast, our studies evaluating H/R demonstrate for the first time
that I
B
tyrosine phosphorylation and NF
B activation can be
completely blocked in c-Src, Fyn, and Yes knockout cells and fully
restored by c-Src activity alone. These findings suggest that other Src
family kinases (i.e. Lck, Lyn, etc.) play a minor role in
mediating the activation of this pathway in epithelial cells following
H/R.

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Fig. 4.
Tyrosine phosphorylation of
I B and
transcriptional activation of NF B are
significantly reduced in c-Src knockout cell lines. A,
SYF cells or SYF+src cells were transduced with Ad.NF BLuc (MOI of
500 particles/cell) 24 h prior to initiated experiments. Cells
were treated with pervanadate (50 µM) for 6 h or H/R
(5 h of hypoxia, 6 h of reoxygenation), harvested into lysis
buffer, normalized for total protein content, and subjected to
luciferase assays. NF B transcriptional activation was evaluated as
the relative luciferase activity. Results depict the mean (± S.E.,
n = 6) relative luciferase activity (RLA).
SYF or SYF+src cells were treated with pervanadate (50 µM) (B) for 15, 30, and 45 min or hypoxic
media (95% N2, 5% CO2) (C) for
5 h followed by reoxygenation media (95% O2, 5%
CO2) for 15, 30, and 45 min. Both untreated and treated
cell lysates were harvested at the indicated time points, and 200 µg
of total protein was immunoprecipitated with anti-I B antibody,
followed by Western blot analysis with an antiphosphotyrosine antibody
or anti-I B antibody.
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c-Src Phosphorylates IkB
in Vitro--
Having demonstrated that
c-Src activity is required for tyrosine phosphorylation and NF
B
activation, we tested whether c-Src could be the tyrosine kinase that
is directly responsible for tyrosine phosphorylation of I
B
. We
used an in vitro kinase assay to evaluate c-Src tyrosine
kinase activity in SYF cells or SYF+c-src cells following 30 min of
pervanadate treatment. Our results presented in Fig.
5A demonstrate that
immunoprecipitated c-Src from untreated SYF+c-src cells has the ability
to phosphorylate a GST-I
B
fusion protein. Furthermore, as
anticipated, the extent of GST-I
B
phosphorylation is
significantly increased following PV treatment. Similar assays using
SYF cell lysates demonstrated no significant GST-I
B
phosphorylation at baseline, or following pervanadate treatment, and
serve as negative controls for the specificity of c-Src
immunoprecipitation and kinase function. To conclusively demonstrate
that c-Src tyrosine phosphorylates GST-I
B
, we performed cold
in vitro kinase assays and evaluated the phosphorylated
GST-I
B
substrate by Western blotting with antiphosphotyrosine
antibody. These results demonstrated that both H/R and pervanadate
treatments of HeLa cells activates the ability of immunoprecipitated
c-Src to tyrosine phosphorylate I
B
(Fig. 5B).
Furthermore, when similar assays were performed using
immunoprecipitated IKK
, no increase in tyrosine phosphorylation of
I
B
was observed over baseline untreated controls. Cumulatively, these results suggest that c-Src activation following H/R and pervanadate treatment is required for tyrosine phosphorylation of
IkB
. They also suggest that c-Src is likely the direct tyrosine kinase responsible for this phosphorylation event. However, we cannot
rule out the possibility that other tyrosine kinases associated with
c-Src are not also involved in tyrosine phosphorylation of IkB
.

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Fig. 5.
c-Src phosphorylates
I B in
vitro. A, SYF cells or SYF+src cells were
treated with pervanadate (50 µM) for 30 min and then
harvested in RIPA buffer. c-Src was immunoprecipitated with anti-c-Src
antibody from 500 µg of protein lysate. The ability of
immunoprecipitated c-Src to directly phosphorylate GST-I B fusion
protein was then evaluated in the presence of
[ -32P]ATP in vitro. Labeled GST-I B
fusion protein was detected by SDS-PAGE and autoradiography.
B, HeLa cells were treated with pervanadate (100 µM, 30 min), or H/R (5 h of hypoxia, 30 min of
reoxygenation) and evaluated for c-Src activity using a cold in
vitro kinase assay. c-Src or IKK was immunoprecipitated with
anti-c-Src or anti-IKK antibody from 500 µg of protein lysate. The
ability of immunoprecipitated c-Src or IKK to directly
tyrosine-phosphorylate GST-I B fusion protein was evaluated by
Western blotting with antiphosphotyrosine antibody. Immunoreactivity
was detected by ECL and autoradiography.
|
|
Overexpression of Gpx-1 or Catalase, but Not Mn-SOD or Cu,Zn-SOD,
Inhibits Tyrosine Phosphorylation of I
B
and NF
B Activation
Following Pervanadate or H/R--
Activation of NF
B is
widely recognized to be dependent on the redox environment within the
cell. In the context of IKK-mediated activation of NF
B, ROS have
been demonstrated to be a critical component in the activation of both
IKK
(16) and IKK
(24) subunits of the IKK complex following
environmental stimuli. Moreover, H2O2 has been
shown to activate tyrosine phosphorylation of I
B
in T-cells in a
manner similar to pervanadate (11). Given the fact that
H2O2 has been shown to activate c-Src (25) and
the observed dependence of I
B
tyrosine phosphorylation and NF
B transcriptional activation on c-Src activity in our H/R models, we next
sought to investigate whether ROS were a signal component of I
B
tyrosine phosphorylation following H/R.
To investigate the redox-dependence of NF
B transcriptional
activation following pervanadate or H/R treatment, we manipulated the
intracellular redox environment using a set of recombinant adenoviruses
that encoded various ROS-scavenging enzymes. These included Ad.Catalase
or Ad.GPx-1 vectors that degrade H2O2, and Ad.Mn-SOD or Ad.Cu,Zn-SOD vectors that dismutate superoxide anion (O
) into H2O2. Using our
in vitro model, we analyzed the role of these antioxidant
enzymes in regulating I
B
tyrosine phosphorylation and NF
B
transcriptional activation. Results from these studies demonstrated a
significant inhibition (p < 0.001) in both pervanadate
(Fig. 6A) and H/R (Fig.
6C) induction of NF
B transcriptional activity following
expression of GPx-1 or catalase. Consistent with this NF
B activation
data, tyrosine phosphorylation of I
B
following pervanadate or H/R
treatments was also significantly inhibited by GPx-1 or catalase
overexpression (Fig. 6, B and D). In contrast,
overexpression of either Cu,Zn-SOD or Mn-SOD failed to alter I
B
tyrosine phosphorylation or NF
B activation. These findings suggest
that H2O2 is an important redox component in
NF
B activation mediated through I
B-
tyrosine
phosphorylation.

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Fig. 6.
Overexpression of Gpx-1 or
catalase, but not Mn-SOD or Cu,Zn-SOD, inhibits
NF B activation and tyrosine phosphorylation of
I B following
pervanadate or H/R treatment. HeLa cells were co-infected with
Ad.BglII, Ad.Gpx-1, Ad.Catalase, Ad.Cu,Zn-SOD, or Ad.Mn-SOD
(MOI of 1000 particles/cell) together with Ad.NF BLuc (MOI of 500 particles/cell) for 24 h prior to initiating experimental
treatments described below. Cells were treated with per- vanadate (100 µM) (A) for 6 h or
H/R (5 h of hypoxia/6 h of reoxygenation) (C). Whole cell
extracts were normalized for total protein content and subjected to
luciferase assays. NF B transcriptional activity was assessed as the
mean relative luciferase activity (RLA) (± S.E.,
n = 6). For evaluation of I B phosphorylation,
cells were treated with pervanadate (100 µM)
(B) for 30 min or H/R (6 h of hypoxia/30 min of
reoxygenation) (D). Both untreated and treated samples were
harvested into lysis buffer, and 200 µg of total protein was
immunoprecipitated with anti-I B antibody followed by Western blot
analysis with an antiphosphotyrosine antibody or anti-I B
antibody.
|
|
GPx-1 Overexpression Reduces c-Src Kinase Activity--
Having
demonstrated that Gpx-1 overexpression is able to reduce NF
B
activity as well as tyrosine phosphorylation of I
B
, we next
investigated whether GPx-1 expression acts to directly inhibit
activation of c-Src using an in vitro kinase assay. Results from these experiments in HeLa cells demonstrated a significant inhibition in the ability of c-Src to phosphorylate GST-I
B
following pervanadate treatment in the presence of Ad.GPx-1 infection
as compared with Ad.BglII-infected control (Fig.
7). Furthermore, in this assay, transient
treatment with 1 mM H2O2 for 30 min
also significantly activated c-Src kinase function as previously
demonstrated. In summary, our data demonstrate that intracellular
hydrogen peroxide (or hydroxyl radical products) mediates NF
B
activation through regulation of c-Src-dependent I
B
tyrosine phosphorylation. Overexpression of
H2O2 scavengers is able to efficiently reduce
c-Src kinase activity, I
B
tyrosine phosphorylation, and NF
B
activation following H/R or pervanadate injury.

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Fig. 7.
Overexpression of GPx-1 reduces c-Src kinase
activity following pervanadate treatment. HeLa cells were infected
with Ad.BglII or Ad.Gpx-1 (MOI of 1000 particles/cell) for
24 h prior to initiating experimental treatments. Cells were then
treated with pervanadate (100 µM) or hydrogen peroxide (1 mM) for 30 min and then lysed in RIPA buffer. c-Src was
immunoprecipitated with anti-c-Src antibody from 500 µg of protein
lysate. The ability of immunoprecipitated c-Src to directly
phosphorylate GST-I B fusion protein was then evaluated in the
presence of [ -32P]ATP in vitro.
|
|
 |
DISCUSSION |
The physiologic significance of I
B
tyrosine phosphorylation
in mediating NF
B transcriptional activation has remained one of the
poorly understood aspects of this well-studied transcription factor. To
date, all studies evaluating the functional regulation of I
B
tyrosine phosphorylation and NF
B activation have been performed in
T-cells or BMMs. Although the tyrosine phosphatase inhibitor
pervanadate has been shown to be a significant activator of this
pathway, natural physiologic stimuli that induce I
B
tyrosine
phosphorylation have remained elusive. However, TNF
treatment of
BMMs has recently been shown to activate NF
B recruitment to the
nucleus in an I
B
tyrosine phosphorylation-dependent
manner (9). This TNF
-induced pathway of NF
B activation in BMMs
appears to differ significantly from the classical
IKK-dependent pathway involving serine phosphorylation of
I
B
that is active in other epithelial-derived cell types. Other
non-hematopoetic systems have increasingly demonstrated components of
I
B
tyrosine phosphorylation in models of in vitro and
in vivo injury (10, 26, 27). The unique fingerprint of this
pathway appears to be the ability of an I
B
PTK to activate NF
B
in the absence of I
B
proteolytic degradation. In the present
study, we have sought to clarify several issues regarding the
involvement of this I
B
PTK pathway in mediating NF
B
transcriptional activation in epithelial-derived cells following H/R.
Using comparative cell line models to evaluate both I
B
serine and
tyrosine phosphorylation-dependent pathways of NF
B
transcriptional activation, our studies have further characterized an
IKK-independent pathway that regulates NF
B through c-Src activation.
Studies using I
B
phosphorylation mutants and SYF knockout cells
demonstrated that c-Src-mediated transcriptional activation of NF
B
functionally requires tyrosine, but not serine, phosphorylation of
I
B
. Using this HeLa cell model system, we found no functional
requirement for I
B
tyrosine phosphorylation in the
transcriptional activation of NF
B following TNF
stimulus and no
evidence for tyrosine phosphorylation of I
B
following TNF
treatment. Furthermore, our studies evaluating IKK-dominant mutants
demonstrate, for the first time, the lack of IKK involvement in
PTK-mediated pathways of NF
B transcriptional activation following
pervanadate or H/R treatments, while confirming the selective
activation of NF
B by TNF
as mediated through serine phosphorylation of I
B
. These functional studies evaluating the transcriptional activation of NF
B following three independent stimuli suggest that little overlap, if any, exists in IKK and PTK
pathways controlling the I
B
·NF
B complex.
Several similarities and differences between our present studies and
those in BMMs are worth noting. First, pervanadate appears to be a
universal activator of NF
B-requiring I
B
tyrosine
phosphorylation, with consistent results observed in HeLa cells,
T-cells, and BMMs. Second, unlike HeLa cells, treatment of BMMs with
TNF
results in significant activation of NF
B in a manner
dependent on I
B
phosphorylation of tyrosine 42 (9). This
difference underscores the importance of cell type-specific dependences
in the activation of NF
B and I
B
protein tyrosine kinases.
Third, our current studies are the first to directly evaluate the
transcriptional activation of NF
B using an NF
B-responsive
reporter gene. To date, all assays for NF
B activation following
stimulation of I
B
tyrosine phosphorylation had been performed
using NF
B DNA binding.
Intracellular production of ROS has been implicated in the regulation
of numerous signal transduction cascades and in the activation of
NF
B following ischemia/reperfusion injury (1, 28). Furthermore,
c-Src can be directly activated by hydrogen peroxide treatment (12,
25), and stimuli such as angiotensin II can induce c-Src activity,
which is inhibited by antioxidants (29). The association of c-Src
activation following H/R (15) has also suggested that c-Src may act as
a redox sensor in the activation of NF
B. However, other pathways of
NF
B activation involving pro-inflammatory stimuli such as TNF
and
LPS also have redox-sensitive activation components, which are
associated with the IKK complex (16, 24). These studies have suggested
that superoxide formation may be the primary initiating ROS involved in
activation of the IKK complex. Hence, the pathways for redox activation
of NF
B are quite diverse and likely regulated by the spatial
relationship of both specific ROS and the signaling components involved. Our findings in the present study have shed additional light
on the redox diversity of NF
B activation involving tyrosine phosphorylation of I
B
. The demonstration that both Gpx-1 and catalase, but not Mn-SOD or Cu,Zn-SOD, are capable of inhibiting H/R or
pervanadate-induced I
B
tyrosine phosphorylation and NF
B activation, suggests a preference for H2O2
and/or hydroxyl radicals (as a product of H2O2)
in the activation of the I
B
tyrosine kinase. These findings are
consistent with previous reports demonstrating the direct activation of
I
B
tyrosine phosphorylation by H2O2 in
T-cells (11).
Recent evidence has suggested that the p85 subunit of PI3-kinase
associates with tyrosine 42 phosphorylated I
B
but not with unphosphorylated I
B
(8). The catalytic p110 subunit also appears
to be critical in the activation of NF
B. The function of p110 may
involve phosphorylation of NF
B and/or dissociation of the I
B
:
NF
B complex. Since both PI 3-kinase and c-Src have been shown to
associate with one another (30) and both maintain redox-sensitive
components in their activation (31-33), it is plausible that c-Src may
act on this PI 3-kinase complex to mediate NF
B activation. It has
been previously demonstrated that c-Src is at least partially
required for I
B
tyrosine phosphorylation in BMMs following TNF
treatment (9). However, these studies performed in c-Src (
/
) BMMs
demonstrated a delay only in the induction of p50/p65 heterodimers in
the nucleus following the TNF
treatment. In comparison to our
present studies using SYF cells with a c-Src, Fyn, and Yes, knockout
background, we find a more complete block in both I
B
tyrosine
phosphorylation and NF
B transcriptional activation following
both H/R and pervanadate treatment than previously described.
Furthermore this block was completely reversed by reconstitution of
only c-Src activity. These findings highlight the functional redundancy
of Src family kinases and conclusively demonstrate that c-Src is
fully capable of mediating NF
B activation through I
B
tyrosine phosphorylation. Our studies, for the first time, have also
successfully reconstituted I
B
tyrosine phosphorylation with
immunoprecipitated c-Src in an in vitro kinase assay.
Furthermore, the activity of c-Src I
B
tyrosine kinase activity
was modulated in response to H/R and pervanadate treatments in a
redox-dependent fashion. These findings provide the most
conclusive evidence to date that c-Src is able to directly tyrosine
phosphorylate I
B
and that this phosphorylation event is required
for NF
B activation following H/R or pervanadate treatments. The
physiologic relevance of redundant Src family kinases in the activation
of NF
B still remains unclear. However, the redox-regulated
mechanisms that control activation of NF
B by Src family kinases may
be a particularly relevant therapeutic target for organ damage
following I/R injury.