1 Molecular Biology Graduate Program, University of Iowa College of Medicine,
Iowa City, Iowa, 52242 USA
2 The Center for Gene Therapy, University of Iowa College of Medicine, Iowa
City, Iowa, 52242 USA
3 Department of Anatomy and Cell Biology, University of Iowa College of
Medicine, Iowa City, Iowa, 52242 USA
* Author for correspondence (e-mail: john-engelhardt{at}uiowa.edu)
Accepted 4 September 2002
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Summary |
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Key words: Antisense inhibition, NFB activation, Signal transduction, Apoptosis
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Introduction |
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The most commonly studied pathway of NFB activation involves the
phosphorylation of I
B
on serine residues 32 and 36 by a recently
identified I
B kinase (IKK) complex, which is composed of three
subunits,
, ß and
(Zandi et al., 1997
).
IKK
and IKKß are the two catalytic subunits in the complex.
Phosphorylation of I
B by the IKK complex leads to ubiquitination and
degradation of I
B, which unmasks a nuclear targeting sequence on the
NF
B molecule. This promotes the translocation of NF
B from the
cytoplasm to the nucleus where it becomes an active transcription factor
(Scherer et al., 1995
).
Regulators of IKK activity include NF
B-inducing kinase (NIK), MEKK1 and
NF
B-activating kinase (NAK). These regulators impart signal-specific
activation of NF
B through the IKK complex. TNF-
and IL-1
induction of NF
B is mediated by NIK phosphorylation of IKK
(Hirano et al., 1996
). By
contrast, MEKK1, which is part of the Jun N-terminal kinase/stress-activated
protein kinase pathway, can induce the activation of both IKK
and
IKKß (Lee et al., 1997
).
Most recently, NAK has been shown to directly phosphorylate IKKß
(Tojima et al., 2000
).
An alternative, but less studied mechanism for NFB activation,
involves tyrosine phosphorylation of I
B
leading to dissociation
of NF
B from I
B
without proteolytic degradation
(Fan et al., 1999
). It was
discovered that stimulation of Jurkat T-cells with either pervanadate (a
protein phosphatase inhibitor) or hypoxia/reoxygenation leads to
phosphorylation of I
B
at tyrosine residue 42 and subsequent
NF
B nuclear translocation without I
B proteolysis
(Imbert et al., 1996
).
Similarly, in a neuron cell model, nerve growth factor treatment leads to
NF
B activation through I
B
tyrosine phosphorylation
independent of proteolytic degradation (Bui
et al., 2001
). In support of this mechanism, our group recently
demonstrated that NF
B is activated without concomitant degradation of
I
B in a murine model of liver ischemia/reperfusion (I/R)
(Zwacka et al., 1998
).
Furthermore, tyrosine phosphorylation of I
B
was increased
following I/R injury in the liver, suggesting that this
degradation-independent pathway of NF
B activation is important in I/R
injury (Zwacka et al., 1998
).
Although these previous studies conclusively demonstrate that Y42 of
I
B
is phosphorylated following hypoxia/reoxygenation, the
functional importance of this phosphorylation event in regulating NF
B
transcriptional activation has yet to be proven using a dominant-negative
I
B
Y42 phosphorylation mutant. In the case of both I/R and
hypoxia, these findings have implicated a potentially unique tyrosine kinase
in the phosphorylation of I
B
. Furthermore, this kinase appears
to be distinct from those activated by TNF-
, IL-1, LPS or PMA. The
mechanism that leads to I
B
tyrosine phosphorylation and
NF
B activation remains unclear; nonetheless, several key players have
been identified. C-src, an osteoclast regulatory protein, has been shown to
associate with I
B
and phosphorylate I
B
on tyrosine
42 in murine bone marrow macrophages (BMMs) in a cytokine-specific manner
(Abu-Amer et al., 1998
). Also,
p85, the regulatory subunit of PI 3-kinase, specifically associates with
tyrosine-phosphorylated I
B
through its Src homology domains both
in vitro and in vivo after stimulation of T cells with pervanadate
(Beraud et al., 1999
). These
studies suggest that PI 3-kinase might be a candidate for regulating tyrosine
phosphorylation of I
B
.
As a pro-inflammatory transcription factor, NFB activation in the
initial phase of I/R injury may trigger upregulation of cytokines, including
TNF-
and IL-1, and adhesion molecules, such as ICAM-1, which can
mediate the ensuing subacute inflammatory response
(Fan et al., 1999
). However, in
addition to its pro-inflammatory action, NF
B also plays a protective
role in acute cellular stress responses that inhibit apoptosis following
TNF-
treatment (Liu et al.,
1996
) or ionizing irradiation
(Wang et al., 1996
).
Furthermore, it has been shown in a two-thirds hepatectomy model that
inhibition of NF
B by overexpression of a dominant-negative mutant form
of I
B
increases apoptosis and liver dysfunction
(Iimuro et al., 1998
). It is
currently unclear how the detrimental proinflammatory and the beneficial
anti-apoptotic effects of NF
B activation are regulated in the setting
of various types of injury.
In an effort to design therapeutic approaches to enhance NFB
activation and reduce apoptosis following cellular injuries such as
ischemia/reperfusion, we have generated and characterized a novel adenoviral
vector that expresses an antisense mRNA of I
B
and enhances
NF
B activation. Since two distinct pathways of regulating NF
B
through I
B
serine or tyrosine phosphorylation have been
identified, we have compared how altering the temporal expression profile of
NF
B influences apoptosis following four types of injury that utilize
either serine or tyrosine I
B
kinase activation pathways. Three
recombinant adenoviral vectors expressing a serine mutant
(Ad.I
B
S32/36A), tyrosine mutant (Ad.I
B
Y42F) or
antisense mRNA (Ad.I
B
AS) of I
B
were used to
modulate NF
B activation prior to injury, and the apoptotic outcomes
were compared. Results from these studies suggest that the timing of
NF
B activation during the acute phases of injury can significantly
influence apoptotic outcomes in an injury-stimuli-dependent manner.
Interestingly, activating NF
B prior to injury was anti-apoptotic only
following stimuli dependent on tyrosine kinase activation of I
B
but not IKK-dependent serine phosphorylation of I
B
. These
findings suggest that the temporal regulation of NF
B following injury
is an important feature that influences apoptotic cell fate in a manner which
is also dependent on the pathway of NF
B activation.
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Materials and Methods |
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Cell culture, adenoviral infection and environmental injury
HeLa cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM)
supplemented with 10% fetal bovine serum (FBS) and 100 µg/ml penicillin and
streptomycin. HeLa cells were infected with various recombinant adenoviral
constructs at a multiplicity of infection (moi) ranging from 1000 to 1500
particles/cell. Adenoviral infections were performed for 2 hours in DMEM
without FBS, followed by the addition of an equal volume of DMEM with 20% FBS.
Fresh media was applied to cells when virus was removed at 24 hours
post-infection. Experiments were typically performed at 24-72 hours
post-infection. Experimental stimuli used to induce NFB were performed
by the protocols listed below.
UV irradiation
HeLa cells were washed with PBS three times and irradiated with UV-C at 50
J/m2 in the absence of surface liquid. Fresh medium was quickly
applied to the plates following irradiation. Cells were harvested 2 hours
post-irradiation for EMSA and 6 hours post-irradiation for NFB
activation luciferase assays. Control, mock-irradiated cells were treated in a
similar fashion but were not exposed to UV.
TNF- treatment
Human recombinant TNF- (R&D systems, Minneapolis, MN) was
diluted to 10 ng/ml in fresh medium just prior to use. TNF-
-containing
medium was applied to HeLa cells at the time of stimulation and cells remained
exposed to TNF-
until they were harvested for analysis. Cells were
harvested at 1 hour post-TNF-
treatment for EMSA and 6 hours
post-TNF-
treatment for NF
B activation luciferase assays.
Control cells were fed with fresh medium without TNF-
.
Pervanadate(PV) treatment
500 mM sodium orthovanadate was prepared fresh in water. 40 µl of sodium
orthovanadate was then added to a mixture of 450 µl PBS and 10 µl 30%
(w/w) H2O2. This solution was incubated for 5 minutes at
room temperature prior to the addition of catalase (200 µg/ml final
concentration). The final pervanadate solution (40 mM) was incubated for 5
minutes at room temperature to remove excess H2O2 and
then immediately diluted in DMEM for application to cells. Cells were
continuously exposed to pervanadate containing medium until cells were
harvested for analysis. Harvesting took place at 2 hours post-pervanadate
treatment for EMSA and at 6 hours post-pervanadate treatment for NFB
activation luciferase assays. Control cells were fed fresh medium without
pervanadate.
Hypoxia/reoxygenation (H/R) experiments
DMEM (no glucose, 0%FBS, 1%P/S) equilibrated in 5% CO2/95%
N2 or 5% CO2/95% O2 was used as hypoxia
medium and reoxygenation medium, respectively. HeLa cells were incubated with
hypoxia medium in an airtight chamber equilibrated with 5% CO2/95%
N2 at 37°C for 5 hours. After hypoxia was performed, the medium
was replaced with reoxygenation medium and cells were incubated in a chamber
flushed with 5% CO2/95% O2 at 37°C. Cells were
harvested 3 hours after reoxygenation for EMSA and 6 hours after reoxygenation
for NFB activation luciferase assays. Control cells were fed with fresh
medium at the appropriate times but were exposed to 5% CO2 in
atmospheric oxygen.
Western blotting and electrophoretic mobility shift assays
(EMSA)
Cytoplasmic and nuclear extracts were prepared by standard protocols
(Andrews and Faller, 1991) and
used for western blotting and EMSA, respectively. Briefly,
2x106 HeLa cells were collected into 1.5 ml centrifuge tubes
by blunt scraping with 1 ml PBS. Cells were pelleted for 1 minute and were
then resuspended in 400 µl of cold buffer A (10 mM HEPES pH 7.9, 1.5 mM
MgCl2, 10 mM KCl, 0.5 mM dithiothreitol, 0.2 mM
phenylmethylsulfonyl fluoride). Samples were incubated on ice for 10 minutes,
then vortexed for 10 seconds and finally pelleted by brief centrifugation (1
minute). The supernatant was saved as the cytoplasmic extract for western
blotting, and the pellet was resuspended in 100 µl of storage buffer (20 mM
HEPES, pH 7.9, 25% Glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.21 mM
EDTA, 0.5 mM dithiothreitol and 0.2 mM phenylmethylsulfonyl fluoride). Samples
were then incubated on ice for another 20 minutes followed by centrifugation.
Supernatants were collected for use as nuclear extracts. Protein
concentrations were determined using a Bio-Rad protein assay (Bio-Rad,
Hercules, CA). 5 µg of cytoplasmic extracts were resolved on a 10% SDS-PAGE
and then transferred to nitrocellulose membranes. Following standard protocols
(Zwacka et al., 1998
),
I
B
protein levels were determined by western blotting using
monoclonal anti-I
B
, anti-I
Bß and anti-actin
antibodies (Santa Cruz Biotech, Santa Cruz, California). EMSA analysis and
supershift assays were performed using an NF
B-specific oligonucleotide
(Promega, Madison, WI). The sequence was as follows:
5'-AGTTGAGGGGACTTTCCCAGGC-3'. The double-stranded nucleotides were
end-labeled with
32P-ATP using T4 polynucleotide kinase. 5
µg of nuclear extract was used in each assay for NF
B DNA binding
using standard protocols (Zwacka et al.,
1998
). NF
B antibodies used for supershift EMSA (anti-p50,
anti-p52, anti-c-Rel, anti-RelB and anti-p65) were purchased from Santa Cruz
Biotech.
Apoptosis assays
HeLa cells were infected with either Ad.BglII or Ad.IB
AS for
24 hours at an moi of 1000 particles/cell. Both groups were treated with UV,
TNF-
, pervanadate and hypoxia/reoxygenation. In the TNF-
treated
group, HeLa cells were pretreated with proteasome inhibitor LLnL (40 µM
final concentration) (Calbiochem, La Jolla, California) for 30 minutes prior
to treatment to enhance apoptosis. In all experimental groups, cells were
trypsinized 18 hours following treatment and stained with annexin VFITC
and propidium iodide (PI) for apoptotic analysis. Annexin-VFITC binding
was performed using a kit from Pharmingen (Palo Alto, CA) according to the
manufacturer's protocol. Briefly, 1x106 cells were washed
twice with cold PBS and then resuspended in 1 ml 1xbinding buffer (10 mM
Hepes, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2). 5 µl of
Annexin-VFITC (100 µg/ml) and 10 µl of propidium iodide (50
µg/ml) were then added to 100 µl cell suspensions. Cells were incubated
for 15 minutes in darkness at room temperature. This was followed by the
addition of 400 µl of 1xbinding buffer. Samples were then analyzed by
FACS. Apoptotic cells were identified as an annexin-V-positive population. The
percentage of apoptotic cells was calculated as the fraction of
annexin-V-positive/PI-positive cells.
Luciferase indicator assays for NFB transcriptional
activation
NFB transcriptional activity was assessed using an Ad.NF
BLuc
reporter vector. This construct contains the luciferase gene driven by four
tandem copies of the NF
B consensus sequence fused to a TATA-like
promoter from the Herpes simplex virus thymidine kinase gene
(Sanlioglu et al., 2001
). HeLa
cells were infected with Ad.NF
BLuc 24 hours prior to treatment by UV,
TNF-
, pervanadate or H/R. 5 µg of total protein from each sample was
used to perform luciferase assays. Luciferase activity was measured using a
kit from Promega (Catalog No. E4030, Madison, WI).
IB
phosphorylation assays
Tyrosine phosphorylation of IB
was evaluated by
immunoprecipitation from 200 µg cytoplasmic extracts in 300 µl RIPA
buffer using 2 µg I
B
antibody (Santa Cruz Biotech, Santa
Cruz, CA) overnight at 4°C. 30 µl of Protein-A agarose was then added
and incubated for 4 hours at 4°C. The agarose beads were then washed four
times with ice-cold RIPA buffer before they were resuspended in SDS-PAGE
loading buffer and loaded onto a 10% SDS-PAGE for western blot analysis. The
membrane was probed with an antibody that recognizes phospho-tyrosine residues
(Santa Cruz) at a dilution of 1:1000 following the western protocol outlined
above. Serine phosphorylation of I
B
was detected by standard
western blotting of 5 µg cell lysate using a phosphospecific antibody
(Santa Cruz Biotech, Santa Cruz, California), which recognized phospho-S32/S36
of I
B
.
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Results |
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To experimentally establish that HeLa cells were capable of modulating
NFB through these two independent serine or tyrosine I
B
kinase pathways, we evaluated the state of I
B
serine and
tyrosine phosphorylation following the four types of environmental injury.
Using a phosphospecific antibody that specifically recognizes the
serine-phosphorylated form of I
B
(S32/36), western blot analysis
demonstrated that both UV and TNF-
treatments induced substantial
levels of I
B
serine phosphorylation by 15 to 30 minutes
(Fig. 1A). By contrast, no
significant change in the baseline levels of serine phosphorylation was
observed following pervanadate or H/R treatment. Using immunoprecipitation of
I
B
followed by western blotting with an anti-phosphotyrosine
antibody, we addressed the tyrosine-phosphorylated state of I
B
.
Results from these studies demonstrated that I
B
tyrosine
phosphorylation significantly increased at 15-30 minutes following pervanadate
or H/R treatment (Fig. 1B). By
contrast, no increase in tyrosine phosphorylation of I
B
was
detected following UV or TNF-
treatment. In summary, this data
demonstrates that HeLa cells can be used as a cell model to study NF
B
activation mediated by either serine or tyrosine phosphorylation of
I
B
.
|
IB
(S32/36A) and I
B
(Y42F) mutants
selectively inhibit NF
B activation in an injury-stimulus-dependent
fashion
Although Y42 phosphorylation of IB
has been associated with
NF
B activation following pervanadate and H/R treatments, functional
evidence for the importance of this tyrosine phosphorylation event in the
transcriptional activation of NF
B using dominant-negative mutants of
I
B
is still lacking. To this end, we generated a recombinant
adenovirus encoding the I
B
(Y42F) mutant and evaluated its
ability to inhibit transcriptional activation of an NF
B-responsive
luciferase reporter (also delivered via a recombinant adenovirus vector)
following each of the various types of injury. As an important control for
IKK-mediated pathways of NF
B activation, a similar adenovirus vector
encoding a dominant-negative I
B
(S32/36A) mutant
(Iimuro et al., 1998
) was also
evaluated. Consistent with the finding that I
B
serine
phosphorylation was most significantly induced by TNF-
and UV,
I
B
(S32/36A) expression significantly inhibited NF
B
transcriptional activation following TNF-
(98.7±5.21%,
P<0.001) and UV (74.2±9.1%, P<0.001) treatments
(Fig. 2). By contrast,
I
B
(S32/36A) expression did not significantly inhibit NF
B
activation following pervanadate (P=0.104) or H/R treatment
(P=0.464) (Fig. 2).
These data confirm that the predominant pathway controlling both TNF-
and UV induction of NF
B in HeLa cells occurs through serine
phosphorylation of I
B
. Furthermore, they also demonstrate that
serine phosphorylation of I
B
plays only a minor role in
regulating NF
B transcriptional activity following pervanadate and H/R
stimuli.
|
Using our newly constructed recombinant adenoviral vector expressing the
Y42F mutant of IB
, we next sought to confirm that tyrosine
phosphorylation of I
B
was predominantly responsible for the
transcriptional activation of NF
B following pervanadate or H/R
treatment. Consistent with the tyrosine phosphorylation data,
I
B
(Y42F) expression significantly inhibited NF
B
transcriptional activation following pervanadate (71.6±6.3%,
P<0.01) and H/R (73.7±10.7%, P<0.01) treatments
(Fig. 3). By contrast, both
TNF-
and UV induction of NF
B transcriptional activity was not
significantly altered by expression of I
B
(Y42F)
(P=0.359 and P=0.257, respectively). In summary, these data
demonstrate that the I
B
(Y42F) mutant can selectively inhibit
NF
B transcriptional activation following pervanadate or H/R treatment.
They also confirm that tyrosine phosphorylation of I
B
is the
predominant mechanism of NF
B transcriptional activation by these
stimuli.
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Inhibition of NFB activation through either I
B
tyrosine or serine phosphorylation pathways stimulates apoptosis following
environmental injuries
Although it is widely accepted that activation of NFB following
cellular injury is anti-apoptotic, this phenomena has not been established for
injuries that activate NF
B via tyrosine phosphorylation of
I
B
. The lack of information on this topic is probably due to the
lack of efficient vectors capable of expressing high levels of a
I
B
(Y42F) mutant in the majority of cells. However, since
recombinant adenoviral vectors are capable of expressing high levels of
I
B
mutants, we sought to conclusively address this issue by
comparing the ability of either I
B
(S32/36A) or
I
B
(Y42F) mutant to selectively enhance apoptosis following each
of the previously described types of environmental injury
(Fig. 4). Results from these
experiments demonstrated that expression of the I
B
(S32/36A)
mutant preferentially enhanced apoptosis following TNF-
or UV treatment
to a far greater extent than that seen following expression of the
I
B
(Y42F) mutant (P<0.02). Thus inhibiting NF
B
activation with Ad.I
B
(S32/36A) has a stronger influence on
apoptosis following UV or TNF-
treatment when compared to
Ad.I
B
(Y42F). By contrast, expression of the
I
B
(Y42F) mutant preferentially enhanced apoptosis following H/R
or pervanadate treatment to a greater extent than that seen following
expression of the I
B
(S32/36A) mutant (P<0.03). The
inhibition of NF
B activation with Ad.I
B
(Y42F) had a
stronger influence on apoptosis following pervanadate or H/R treatment
compared with Ad.I
B
(S32/36A). Although this differential effect
of tyrosine and serine mutants of I
B
was not absolute, these
results do functionally substantiate findings for two independent pathways of
NF
B activation. More importantly, they also demonstrate that inhibition
of NF
B activation stimulates apoptosis regardless of the injury pathway
responsible for NF
B activation.
|
Antisense inhibition of IB
protein enhances NF
B
baseline activation
The anti-apoptotic nature of NFB suggests that augmenting NF
B
activation prior to injury might enhance the protective effects of this
molecule. Indeed, such therapeutic strategies appear to be the basis for
protective effects following ischemic preconditioning in the heart
(Morgan et al., 1999
). To this
end, we sought to evaluate whether inhibition of I
B
protein
synthesis using an antisense mRNA approach could reduce apoptosis and improve
cell survival following the various types of environmental injuries. A
recombinant adenovirus (Ad.I
B
AS) encoding I
B
cDNA
in the reversed orientation was tested for its ability to inhibit
I
B
protein expression and enhance NF
B activation. As seen
in Fig. 5A, infection of HeLa
cells with Ad.I
B
AS significantly reduced steady-state levels of
I
B
protein. Interestingly the steady-state level of
I
Bß was increased in the presence of antisense I
B
mRNA. This probably reflects a compensatory mechanism induced by activation of
NF
B. These findings also demonstrated specificity of the antisense
approach to inhibit I
B
expression. Despite the observed changes
in both I
B
and I
Bß levels, no changes in the
steady-state level of the actin internal control were observed.
Ad.I
B
AS infection consequently led to a time-dependent increase
in nuclear NF
B DNA binding that was not observed in Ad.GFP-infected
controls (Fig. 5B). EMSA
supershift assay using antibodies to various NF
B subunits (p50, p52,
c-Rel, RelB, and p65) demonstrated that I
B
AS expression induced
NF
B complexes composed of p50/p65 heterodimers
(Fig. 5C). These results
demonstrate that inhibition of I
B
protein expression using an
antisense mRNA approach significantly elevates baseline levels of NF
B
in the nucleus.
|
Having demonstrated that infection with Ad.IB
AS enhances
baseline NF
B DNA-binding activity in the nucleus by inhibiting
I
B
protein expression, we next sought to confirm that expression
of I
B
AS mRNA enhanced NF
B transcriptional activation
following UV, TNF-
, pervanadate or H/R treatment. In these studies,
HeLa cells were infected with either a control adenovirus, Ad.BglII, or
Ad.I
B
AS at an moi of 1000 particles/cell 24 hours prior to
exposure to UV, TNF-
, pervanadate or H/R. As previously reported, each
of these treatments induced NF
B DNA binding
(Fig. 6); however, the time
course of NF
B activation varied. In uninfected cells, NF
B DNA
binding peaked 2 hours following UV irradiation or pervanadate treatment, 1
hour following TNF-
treatment and 3 hours following H/R treatment (data
not shown). Hence, for comparative studies of Ad.BglII- and
Ad.I
B
AS-infected groups, samples were harvested at the peak time
points of NF
B DNA binding following a given stimulus. Results from this
analysis clearly demonstrated that infection with Ad.I
B
AS
increased nuclear NF
B DNA binding following UV
(Fig. 6A), TNF-
(Fig. 6B), pervanadate
(Fig. 6C) or H/R
(Fig. 6D) treatment. In
summary, these results demonstrate that inhibiting I
B
protein
expression enhances activation of NF
B DNA binding in the nucleus
following exposure to all types of stimuli tested.
|
Since DNA binding is only one indicator of NFB activation, we also
sought to directly evaluate the transcriptional activity of NF
B using
the recombinant adenoviral reporter vector (Ad.NF
BLuc) harboring a
NF
B-inducible luciferase gene. In these studies, HeLa cells were
co-infected with Ad.NF
BLuc and Ad.I
B
AS or Ad.BglII 24
hours prior to treatments. Whole cell extracts were harvested 6 hours
following treatment and luciferase activity was measured as an indicator of
NF
B transcription activation. Consistent with our EMSA result on
NF
B DNA binding, NF
B transcriptional activity was also
significantly enhanced (P<0.01) in the
Ad.I
B
AS-infected groups, as compared to the groups infected by
Ad.BglII, following each of the environmental injuries
(Fig. 7).
|
Activation of NFB prior to injury is pro-apoptotic following
UV or TNF-
treatment but anti-apoptotic following pervanadate or H/R
treatment
On the basis of our findings that dominant-negative phosphorylation mutants
of IB
preferentially enhanced apoptosis following each of the
environmental injuries tested, we hypothesized that elevating the level of
NF
B activation with I
B
AS prior to injury would have the
reverse effect. Interestingly, results from these experiments demonstrated two
distinct functional consequences of I
B
AS expression depending on
the type of environmental injury evaluated
(Fig. 8). Following UV
treatment, I
B
AS expression significantly (P<0.001)
increased apoptosis to 40.6%±2.2 as compared to the Ad.BglII-infected
control group (15.5%±0.8). Similar observations were seen in
TNF-
treated cells, which had a two-fold increase (P<0.01)
in apoptosis in Ad.I
B
AS-infected cells (8.9%±0.5) as
compared to the Ad.BglII control group (4.6%±0.3). In stark contrast,
following PV treatment, I
B
AS expression significantly decreased
(P<0.01) the percentage of apoptotic cells to 16.0%±1.5
compared with the Ad.BglII-infected control group (30.4%±2.0).
Similarly, following H/R, the Ad.I
BAS-infected group demonstrated a
significantly lower (P<0.01) apoptotic rate (4.0%±0.5) as
compared with the control group (6.0%±0.7). These findings demonstrate
that increased NF
B activation during the acute phase of injury can have
either pro-apoptotic or anti-apoptotic effects depending on the type of injury
stimulus. Interestingly, this differential apoptotic effect of enhancing
NF
B activation prior to injury correlated with either serine or
tyrosine I
B
phosphorylation pathway responsible for the
activation of NF
B following injury. Enhancing NF
B activity prior
to injury was pro-apoptotic for stimuli that promote IKK-mediated serine
phosphorylation of I
B
(TNF-
and UV), although it was
anti-apoptotic for stimuli that promote tyrosine phosphorylation of
I
B
(H/R and pervanadate). Although these findings suggest that
enhancing NF
B activity may be a viable strategy for reducing apoptosis
following I/R injury, they also suggest that the temporal regulation of
NF
B is an important component in determining its apoptotic influence
for other types of injuries.
|
![]() |
Discussion |
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Studies establishing this model system confirmed that both UV and
TNF- treatment leads to serine phosphorylation of I
B
. By
contrast, hypoxia/reoxygenation and pervanadate (a tyrosine phosphatase
inhibitor) treatment led to tyrosine phosphorylation of I
B
.
Although these studies are confirmatory in nature, they do establish that this
model system specifically modulates I
B
serine or tyrosine
phosphorylation in an injury-specific manner within the same cell type. The
functional specificity of either I
B
S32/36 or Y42
phosphorylation in promoting NF
B activation in response to specific
injury stimuli was also confirmed using transcriptional reporter assays and
dominant-negative Y42F or S32/36A mutants of I
B
. These studies
for the first time demonstrate that Y42, but not S32/36, phosphorylation of
I
B
is the predominant pathway for transcriptional activation of
NF
B following H/R in an epithelial cell line model.
Although the present study has tried to distinguish between serine and
tyrosine phosphorylation of IB
following each environmental
injury, it is recognized that overlap in these two activation pathways may
exist. For example, it is still debated whether UV irradiation activates
NF
B through IKK-mediated I
B
serine phosphorylation.
Several reports have demonstrated that the dominant mutants IKK
(KM) or
I
B
(S32/36A) can inhibit UV-induced NF
B activation
(Kulms et al., 2000
;
Li et al., 2001
). However,
others have reported that NF
B activation following UV-C irradiation
does not involve the IKK complex or I
B
serine phosphorylation
(Bender et al., 1998
;
Li and Karin, 1998
).
Differences in these results may be cell line dependent. Although most recent
results demonstrate that the majority of UV-induced NF
B activation can
be inhibited by I
B
(S32/36A), the extent of inhibition (74%) was
much less than that seen for TNF-
(99%). Furthermore, 10% of UV-induced
NF
B transcriptional activity was inhibited by the
I
B
(Y42F) mutant, and although this inhibition was not
statistically significant, it does suggest that some overlap in these two
pathways may exist. Alternatively, additional non-I
B
-associated
mechanisms controlling NF
B activation, such as Akt phosphorylation of
p65 (Madrid et al., 2000
), may
also play some minor role in these studies.
Controversies regarding the involvement of NFB in both
anti-apoptotic and pro-apoptotic pathways have been difficult to reconcile
(Lipton, 1997
). In part, the
complexity and diversity of pathways that can activate NF
B has fed this
controversy. Importantly, determinants of cell survival and apoptosis are,
themselves, complex and not solely regulated by a single factor such as
NF
B. Hence, independent pathways of NF
B activation may impart
unique functional consequences to NF
B activity by altering the
composition of signaling intermediates that may act in concert to facilitate
or prevent apoptosis under a given stimulus. The functional consequences of
inhibiting NF
B following pro-inflammatory stimuli such as TNF-
have been well documented to enhance apoptosis in the presence of
I
B
(S32/36A) expression. However, similar information is lacking
for H/R injury using a I
B
(Y42F) mutant. Important to designing
therapeutic strategies for preventing apoptosis following I/R by modulating
NF
B activity was to first establish whether its inhibition of
NF
B was pro- or anti-apoptotic in the setting of H/R. Studies using
either I
B
(S32/36A) or I
B
(Y42F) mutants confirmed
that inhibiting NF
B under all environmental stimuli was proapoptotic.
Although each of these mutants preferentially enhanced apoptosis following
injuries specific for their respective pathways of I
B
phosphorylation, some overlap was seen. For example, although
I
B
(Y42F) expression more significantly increased apoptosis
following H/R than I
B
(S32/36A), the serine mutant also
significantly enhanced apoptosis in comparison with Ad.BglII-infected
controls. This overlap was much higher than that seen in studies evaluating
the specificity of these mutants to transcriptionally activate NF
B
following each of the independent stimuli. We reason that the difference in
the specificity of I
B
mutants to modulate NF
B
transcriptional activation or apoptosis is due to the longer time course
required to achieve apoptosis in the setting of injury. Hence, although the
acute stages of NF
B activation may be selective for serine or tyrosine
I
B
phosphorylation, during the later stages of injury greater
overlap in these two pathways may exist owing to activation of
NF
B-responsive cytokines that restimulate cells through alternative
NF
B pathways. Nonetheless, these studies conclusively demonstrate for
the first time that inhibition of NF
B activation using an
I
B
(Y42F) mutant enhances apoptosis following H/R.
Having demonstrated that activation of NFB is anti-apoptotic
following H/R, we hypothesized that enhancing NF
B activation prior to
injury might provide a protective advantage to cells. In support of this
hypothesis, infection with recombinant adenovirus expressing antisense
I
B
mRNA significantly enhanced NF
B transcriptional
activation and reduced apoptosis following either H/R or PV treatment.
Surprisingly, similar studies in TNF-
- or UV-treated cells gave rise to
the opposite result. Although expression of antisense I
B
mRNA
similarly enhanced NF
B transcriptional activation following either
TNF-
or UV treatment, a significant increase in apoptosis was observed
with these stimuli in antisense I
B
-mRNA-expressing cells. Hence,
elevation of NF
B transcriptional activity prior to injury promoted
apoptosis following TNF-
or UV treatment, although it inhibited
apoptosis following pervanadate or H/R treatment. These findings suggest that
the temporal regulation of NF
B influences apoptosis in a
stimuli-dependent fashion.
One interesting feature of the differential effects of enhancing NFB
on apoptosis following these various stimuli, is the fact that they fell into
two distinct groups associated with a particular pathway for NF
B
activation. For example, both UV- and TNF-
-treated groups, which
demonstrated enhanced apoptosis in the setting of elevated NF
B
activation, utilize S32/36 phosphorylation of I
B
as the pathway
for NF
B activation. By contrast, both H/R- and pervanadate-treated
groups, which demonstrated reduced apoptosis in the setting of elevated
NF
B activation, utilize Y42 phosphorylation of I
B
as the
pathway for NF
B activation. These findings demonstrate that, depending
on the type of environmental injury and the pathway for NF
B activation,
the consequences of enhancing NF
B activation prior to injury can
produce dramatically different phenotypes in relation to programmed cell
death. We hypothesis that this previously unidentified relationship between
IKK or protein tyrosine kinase (PTK) pathways of NF
B activation, and
the ultimate apoptotic consequences of this activation, is probably due to
additional stimuli-specific factors induced under each of the given
conditions. NF
B most probably works in concert with such factors to
determine apoptotic cell fates. This current hypothesis would propose that
activated NF
B following IKK or PTK stimuli is biochemically identical
and that stimuli-specific factors linked to either IKK or PTK pathways of
NF
B activation uniquely alter the subset of injury response genes
transcriptionally activated by NF
B complexes and/or the consequences of
that activation. However, it is interesting that dominant-negative
phosphorylation mutants of I
B
enhanced apoptosis following all
of the environmental stimuli tested. These findings suggest that NF
B
can indeed play an anti-apoptotic role during the later phases of injury.
Taken together, results from experiments using antisense and dominant-negative
mutants of I
B
suggest that the temporal regulation of NF
B
at the time of injury is an important component in determining cell fate.
Evidence for such diversity in NFB involvement in apoptosis is
supported by findings in the literature
(Baichwal and Baeuerle, 1997
;
Jobin and Sartor, 2000
;
Yamamoto and Gaynor, 2001
).
For example, in neuronal cells, NF
B activation mediates
glutamate-induced toxicity and subsequent cell death and blocking NF
B
activation with aspirin or salicylate protects cells from the neurotoxic
effects of glutamate (Grilli et al.,
1996
). Similarly, in a study of serum-starvation-induced
apoptosis, a dominant-negative mutant of the NF
B p65 subunit has been
shown to suppress transcription activity of NF
B and partially inhibit
apoptosis in a human embryonic kidney cell line (293 cells)
(Grimm et al., 1996
). In
contrast to these studies, inhibition of NF
B activation by expression
of a recombinant, dominant-negative serine mutant of I
B
(S32/36A) leads to massive apoptosis in the liver following partial
hepatectomy (Iimuro et al.,
1998
). Similar findings in IKK knockout mice also suggest that
NF
B plays an important role as an anti-apoptotic factor during liver
development (Rudolph et al.,
2000
; Tanaka et al.,
1999
). Since ß-catenin has also been shown to be a substrate
of the IKK complex (Lamberti et al.,
2001
), we also evaluated whether expression of our various
I
B
constructs could affect ß-catenin levels by substrate
competition with the IKK complex and in turn affect the apoptotic outcome.
However, our result demonstrated no significant change in ß-catenin
levels following infection with Ad.I
B
AS, Ad.I
B
(S32/36A) or Ad.I
B
(Y42F) (data not shown).
From a therapeutic standpoint, the present studies suggest that strategies
aimed at enhancing NFB activation to increase cell survival may be
effective for only certain classes of injury stimuli (i.e. I/R) that activate
NF
B through I
B
tyrosine kinase pathways. Such findings
demonstrate that the pathway of NF
B activation and the temporal
regulation of its activation significantly influences apoptotic outcomes. In
support of this concept, a recent study by Lawrence et al. reported that
NF
B activation during the immediate early and late phases of
inflammation plays independent pro-inflammatory and anti-inflammatory roles in
gene expression, respectively (Lawrence et
al., 2001
). Our studies demonstrating that the apoptotic outcomes
of two independent pathways of NF
B activation are differentially
influenced by temporal alteration in the NF
B activity support the
notion of context-dependent roles for NF
B during acute and late phase
responses.
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Acknowledgments |
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References |
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