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B
in NF-B Activation by an Oxidative Stress1
*
Laboratory of Virology, Institute of Pathology, University of Liege, Liege, Belgium;
Oncogenesis, Differentiation, and Signal Transduction Laboratory, Institut de Recherche sur le Cancer-Centre Nationale de la Recherche Scientifique, Villejuif, France;
Institut National de la Santé et de la Recherche Médicale, Unit 99, Hôpital Henri Mondor, Creteil, France; and
§
Unit of Infectious Diseases, AZ Sint-Jan, Brugge, Belgium
 |
Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B involves the
signal-dependent degradation of basally phosphorylated inhibitors such
as IB
. In response to proinflammatory cytokines or mitogens, the
transduction machinery has recently been characterized, but the
activation mechanism upon oxidative stress remains unknown. In the
present work, we provide several lines of evidence that NF-B
activation in a T lymphocytic cell line (EL4) by hydrogen peroxide
(H2O2) did not involve phosphorylation of the
serine residues 32 and 36 in the amino-terminal part of IB.
Indeed, mutation of Ser32 and Ser36 blocked
IL-1ß- or PMA-induced NF-B activation, but had no effect on its
activation by H2O2. Although IB was
phosphorylated upon exposure to H2O2, tyrosine
residue 42 and the C-terminal PEST (proline-glutamic
acid-serine-threonine) domain played an important role. Indeed,
mutation of tyrosine 42 or serine/threonine residues of the PEST domain
abolished NF-B activation by H2O2, while it
had no effect on activation by IL-1ß or PMA-ionomycin. This
H2O2-inducible phosphorylation was not
dependent on IB kinase activation, but could involve casein kinase
II, because an inhibitor of this enzyme
(5,6-dichloro-1-ß-D-ribofuranosyl-benzimidazole) blocks
NF-B activation. H2O2-induced IB
phosphorylation was followed by its degradation by calpain proteases or
through the proteasome. Taken together, our findings suggest that
NF-B activation by H2O2 involves a new
mechanism that is totally distinct from those triggered by
proinflammatory cytokines or mitogens. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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During an inflammatory response, lymphocytes are exposed to ROS (mainly
hydrogen peroxide and hypochlorous acid) derived from activated
macrophages and neutrophils. If not generated in too great an amount,
ROS can be tolerated by lymphocytes and used as a second messenger in
signal transduction processes to regulate gene expression, such as that
activated during immune and inflammatory responses (10).
One of the downstream targets of ROS is NF-B, which is the first
eukaryotic transcription factor shown to respond directly to oxidative
stress (11).
The NF-B transcription factor binds specific DNA sequences as
dimers. These are constituted of proteins belonging to the Rel/NF-B
family. In mammals, this family contains proteins p50, p52, p65 (RelA),
RelB, and c-Rel (Rel) (12, 13). These five proteins harbor
a related, but nonidentical, 300-aa-long Rel homology domain that is
responsible for dimerization, nuclear translocation, and specific DNA
binding. In addition, RelA, RelB, and c-Rel, but not p50 or p52,
contain one or two trans-activating domains. p50 and p52
derive from cytoplasmic precursors named p105 and p100, respectively.
NF-B complexes are sequestered in the cytoplasm of most resting
cells by inhibitory proteins belonging to the IB family
(14, 15, 16, 17), comprised of IB, IBß, IB
, p100,
and p105.
In the case of cells stimulated by proinflammatory cytokines such as
IL-1ß or TNF-, the signal transduction pathway leading to the
phosphorylation and degradation of IB proteins has recently been
clarified in HeLa and L293 cells (18, 19, 20, 21 ; see Refs.
22 and 23 for review). It is included in a
700- to 900-kDa complex called signalsome involving the IB
kinases (IKK- and -ß) and the NF-B essential modulator (NEMO).
Important partners are proteins associated with the TNF- or IL-1
receptors, the NF-B-inducing kinase, and mitogen-activated protein
kinase kinase-1. The IKKs phosphorylate the IB protein on serines
32 and 36, a reaction that targets IB for ubiquitination and
rapid degradation by the 26S proteasome. These reactions are extremely
rapid, with the cellular IB protein being completely degraded
within minutes following cell stimulation before being resynthesized to
replenish the IB cytoplasmic pool. This rapid and transient
degradation of IB contrasts with its slow and sustained
disappearance, observed by several authors, when cells are induced by
an extracellular generation of ROS, such as that which occurs during
inflammation (11, 24, 25, 26). These differences in the
kinetics of IB degradation very likely reflect the existence of
various signaling pathways leading to IB phosphorylation.
In this study, we investigated the NF-B activation mechanism in a
murine T lymphocytic cell line treated with hydrogen peroxide
(H2O2) and showed that
other IB amino acids than serines 32 and 36 are required for its
degradation and subsequent NF-B translocation to the nucleus.
Tyrosine 42 and serine/threonine residues of the PEST (proline glutamic
acid serine threonine) domain of IB play a crucial role in the
activation mechanism by
H2O2 and calpain proteases,
and the proteasome are likely to be involved in the degradation of
IB.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The EL4 mouse lymphoma cell line was obtained from American Type Culture Collection (Manassas, VA). Cells were cultured in RPMI 1640 and ultraglutamine with 10% FCS (BioWhittaker, Petit Rechain, Belgium). They were seeded in fresh medium containing 2% FCS 12–24 h before any experiment.
EL4 cells expressing human IB S32,36A, IB Y42F, or IB
S283A, T291A, T299A mutant proteins were obtained after electroporation
of the parental EL4 cells with 80 µg of a plasmid bearing the
corresponding human IB mutated gene under control of the CMV
promoter together with the gene conferring neomycin resistance or with
an empty plasmid (EL4/2 cells). Cells were selected over a period of 21
days in the presence of 1 mg/ml neomycin before being cloned by flow
cytometry and expanded individually. Expression of the human IB
mutant proteins was assessed by Western blot analysis.
Chemicals
All chemicals were of reagent grade from UCB (Brussels, Belgium) or Sigma (St. Louis, MO). Ionomycin was obtained from Calbiochem (La Jolla, CA). H2O2 was purchased from Merck (Darmstadt, Germany) and was diluted just before being added to cells. H2O2 concentrations were calculated by UV spectrophotometry based on an extinction coefficient value of 80. Lactacystin was a gift from Martin Kroll (Pasteur Institute, Paris, France) or was purchased from Boston Biochemicals (Boston, MA), Z-Asp-Glu-Val-Asp-FMK (Z-DEVD) was obtained from EPS (Livermore, CA), E64-d was obtained from Peptide International (Louisville, KY), and DRB was purchased from Biomol (Boston, MA). A cocktail of phosphatase inhibitors was made of 1 mM Na3VO4, 10 mM ß-glycerophosphate, 5 mM NaF, and 200 nM okadaic acid.
Plasmids
Plasmid bearing the IB S32,36A mutated gene under the
control of the CMV promoter was a gift from P. Baeuerle (Micromet,
Munchen, Germany). This plasmid gave rise to the expression of a
IB S32,36A tagged with an extension 15 aa long at the C-terminal.
The plasmid containing the IB S283A, T291A, T299A gene under the
control of the CMV promoter was a gift from J. Hiscott (Montreal,
Canada), and the plasmid expressing IB Y42F was a gift from J.-F.
Peyron (Nice, France). The pNF-B-Luc reporter construct contains
five B sites from the HIV-1 long terminal repeat cloned upstream of
the luciferase gene (Stratagene, La Jolla, CA).
Generation of oxidative stress
EL4 cells (1 x 106/ml culture
medium) were cultured in 75-cm2 culture flasks
before being treated with
H2O2 (0–500 µM). EL4
cell treatment with H2O2
was performed as described previously (7), and 5 x
106 cells were subsequently harvested at various
times (between 10 and 240 min) to prepare nuclear and cytoplasmic
protein extracts for NF-B EMSAs and Western blot analysis,
respectively. After 48 h, EL4 cells were counted, and cytotoxicity
was estimated using trypan blue exclusion.
Nuclear and cytoplasmic protein extraction
The method used was described previously (27). Briefly, at various times after the oxidative stress, 5 x 106 EL4 cells were washed in 1 ml of cold PBS and centrifuged at 15,000 x g for 15 s, resuspended in 200 µl of cold hypotonic buffer (10 mM HEPES-KOH, 2 mM MgCl2, 0.1 mM EDTA, 10 mM KCl, 1 mM DTT, and 0.5 mM PMSF, pH 7.9), left on ice for 10 min, and then vortex mixed and centrifuged at 15,000 x g for 30 s. Aliquots of supernatant containing the cytoplasmic proteins were quickly frozen in liquid nitrogen and stored at -80°C. The pellets of nuclei were gently resuspended in 15 µl of cold saline buffer (50 mM HEPES-KOH, 50 mM KCl, 300 mM NaCl, 0.1 mM EDTA, 10% (w/v) glycerol, 1 mM DTT, and 0.5 mM PMSF, pH 7.9) and left for 20 min on ice. After centrifugation (15,000 x g for 15 min at 4°C), aliquots of supernatant containing the nuclear proteins were kept in liquid nitrogen and stored at -80°C. Protein concentrations were measured with the Bio-Rad protein assay (Munich, Germany).
Electrophoretic mobility shift assay
The method used was previously described (7), with
slight modifications. Briefly, 5 µg of nuclear proteins were
incubated for 30 min at room temperature with 0.2 ng of
32P-labeled oligonucleotidic probe, 2 µg of
BSA, and 1 µg of poly(dI-dC)-poly(dI-dC) (Pharmacia Biotech Benelux,
Rozendaal, The Netherlands) in 20 mM HEPES-KOH, 75 mM NaCl, 1 mM EDTA,
5% (v/v) glycerol, 0.5 mM MgCl2, and 1 mM DTT,
pH 7.9, buffer (final volume, 10 µl). DNA-protein complexes were then
resolved on a nondenaturing 6% (w/v) polyacrylamide gel run for 4
h at 180 V in x0.25 TBE (2.5 mM Tris, 2.5 mM
H3BO3, and 2 mM EDTA, pH
8.5). The gel was dried and autoradiographed on Fuji x-ray film
(General Electric, Antwerp, Belgium). For supershift experiments,
protein extracts were preincubated with Abs against p50, p65, c-Rel,
p52, and RelB (polyclonal Abs from Santa Cruz Biotechnology, Santa
Cruz, CA) for 15 min on ice before addition of the labeled B probe.
The oligonucleotide probe (5'-GGTTACAAGGGACTTTCCGCTG-3';
Eurogentec, Seraing, Belgium) was labeled by in-filling with the Klenow
DNA polymerase (Roche, Mannheim, Germany) as described previously
(24). Specific activity was always
108 cpm/µg.
Transient transfection assays
EL4 or EL4-S32,36A cells were grown in six-well plates for 2
days in RPMI 1640 and ultraglutamine with 10% FCS (BioWhittaker) and
transfected with 5 µg of B-Luc reporter plasmid. Plasmids were
mixed in OptiMEM (Life Technologies, Little Chalfont, U.K.), added to
Fugene liposomes (9 µl; Roche) for 15 min at room temperature and
loaded on cells in 2 ml of RPMI containing 10% FCS for 24 h. Then
cells were treated with PMA (0.2 µM)-ionomycin (2 µM) or
H2O2 (10, 50, 100, or 300
µM). After treatment, cells were cultivated for 24 h and then
washed twice in PBS, lysed for 15 min, and centrifuged at 15,000
x g for 4 min. Luciferase activities corrected for the
amount of protein (Bio-Rad protein assay) were measured in
supernatants.
Western blotting
IB protein was detected by Western blot analysis using a
specific mAb (MAD10B, a gift from R. Hay, St. Andrews, Scotland) or the
anti-human full-length IB polyclonal Ab (Euromedex, Souffel
Weyersheim, France). The MAD10B Ab recognizes a 20-aa stretch within
the amino-terminal region of IB. Cytoplasmic extracts were
prepared at various times after the
H2O2-mediated treatment by
hypotonic lysis, pelleting the nuclei, and collecting the supernatant
fraction (7). Cytoplasmic proteins were added to a loading
buffer (10 mM Tris-HCl (pH 6.8), 1% SDS, 25% glycerol, 0.1 mM 2-ME,
and 0.03% bromophenol blue), boiled, electrophoresed on a 10 or 12%
polyacrylamide-SDS gel, and electrotransferred to Immobilon-P membranes
(Millipore, Bedford, MA). Filters were incubated in a primary Ab for
120 min at room temperature (1/500 dilution) and in
peroxidase-conjugated rabbit anti-mouse IgG (1/1000 dilution; DAKO,
Copenhagen, Denmark) for 60 min at room temperature and finally
analyzed using Amersham’s enhanced chemiluminescence system (Amersham,
Aylesbury, U.K.) with Fuji x-ray films.
Immunoprecipitation and in vitro IKK assay
Cytoplasmic extracts (500 µg) prepared in hypotonic buffer as
described above supplemented with 3 mM EDTA, 1 mM
Na3VO4, 1 mM NaF, 0.5 mM
ß-glycerophosphate, and protease inhibitors (Complete, Roche) were
incubated in immunoprecipitation buffer (50 mM Tris, 250 mM NaCl, 3 mM
EDTA, 3 mM EGTA, 0.1% Nonidet P-40, 1 mM
Na3VO4, 1 mM NaF, 0.5 mM
ß-glycerophosphate, and Complete) with 5 µl of anti-IKK Ab
(H744, Santa Cruz Biotechnology) or 1 µl of anti-NEMO Ab (from
Dr. A. Israel, Pasteur Institute) together with 40 µl of protein
A-agarose (Pharmacia, Uppsala, Sweden) overnight at 4°C.
Immunoprecipitates were collected by centrifugation and washed three
times in the immunoprecipitation buffer and then twice in kinase buffer
(50 mM Tris, 100 mM NaCl, 2 mM MgCl2, 1 mM DTT,
0.5 mM PMSF, 1 mM Na3VO4, 1
mM NaF, 0.5 mM ß-glycerophosphate, and Complete).
Immunoprecipitates were then resuspended in 40 µl of kinase buffer
supplemented with [
-32P]ATP in the presence
of 500 ng of the wild-type GST-IB1–54 or
mutant GST-IB1–54 (S32,36A; gift from Dr.
R. Gaynor, University of Texas, Dallas, TX) and were incubated at
30°C for 30 min. Reactions were stopped by the addition of SDS
loading buffer and were submitted to SDS-PAGE. The gel was then dried
and autoradiographed. To check loading and confirm the presence of
IKKs, the upper part of the SDS-PAGE was analyzed by Western blotting
using the anti-IKK Ab (H744 Ab, Santa Cruz Biotechnology).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B DNA binding activity induced by
H2O2 is slow and sustained
NF-B activation and subsequent IB degradation kinetics
were first analyzed in control EL4/2 cells following induction by PMA
(0.2 µM) and ionomycin (2 µM), IL-1ß (100 U/ml), or
H2O2 (300 µM). The time
course of NF-B activation by
H2O2 was dramatically
different from that induced by IL-1ß or PMA-ionomycin. As shown in
Fig. 1
(left and
central panels), addition of PMA-ionomycin to EL4/2 cells
gave rise to an important and rapid activation of NF-B, as detected
by EMSA. The complex was already detectable 15 min after induction, and
the band intensity gradually decreased up to 240 min following
induction. Similar results were observed with IL-1ß stimulation, but
the kinetics were more transient, with NF-B almost completely
disappearing from the nucleus after 120 min. Analysis of the fate of
IB in cytoplasmic extracts from these PMA-ionomycin- or
IL-1ß-stimulated cells revealed that IB was rapidly degraded
(after 15 min) before being resynthesized rather rapidly (from
30–240 min).
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B induction were recorded (Fig. 1
, right panel).
Indeed, NF-B was faintly detected in the nucleus of cells 30 min
after treatment, but maximal activation was observed after 60 min, with
NF-B induction remaining elevated up to 240 min after addition of
H2O2. The slow and
sustained NF-B activation by
H2O2 strongly contrasted
with the rapid and transient activation induced by either PMA-ionomycin
or proinflammatory cytokines such as IL-1ß and TNF- (data not
shown). Western blot analysis revealed that IB proteolysis
occurred concomitantly with the nuclear appearance of NF-B, reaching
its maximum at 60 min, and that newly synthesized forms appeared at
later times (Fig. 1). Other IB molecules were also followed; among
them only IBß was also degraded upon
H2O2 induction (data not
shown). These data suggest that the mechanism by which
H2O2 caused NF-B
activation and IB degradation differs from that used by IL-1ß
or PMA-ionomycin.
IL-1ß and PMA-ionomycin are known to cause phosphorylation of
IB at S32,36, leading to degradation through a
proteasome-dependent pathway. We thus verified the implication of these
residues in H2O2-mediated
IB proteolysis.
To characterize the molecular mechanisms involved in
H2O2-mediated NF-B
activation, we generated EL4 mouse lymphoma cell lines expressing
mutated human IB inhibitory proteins. EL4 cells expressing the
human IB gene mutated at S32 and S36 or at position Y42 were
called EL4-S32,36A or EL4-Y42F, respectively; those expressing the
human IB gene mutated at S283, T291, and S299 were called
EL4-PEST.
S32 and S36 of IB are not required for NF-B activation by
H2O2
In the case of EL4-S32,36A cells,
H2O2 stimulation led to
NF-B activation quite similar to that described in control EL4/2
cells, except for the maximum, which was somewhat delayed to 180 min
(Fig. 2, left panel). On the
other hand, the presence of S32,36A mutated IB protein strongly
reduced the NF-B induction mediated by the IL-1ß stimulation (Fig. 2, right panel). The very transient activation of NF-B
could very well be due to the translocation of NF-B bound to the
endogenous wild-type murine IB molecules. Similar behavior was
observed after stimulation with PMA-ionomycin (data not shown). In
addition, three independent EL4-S32,36A cell clones expressing
different amounts of the human mutated protein gave identical results
(data not shown).
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B
activation by H2O2
In the case of EL4-Y42F and EL4-PEST cells, EMSAs revealed that
the presence of these mutated IB molecules almost completely
prevented NF-B activation by
H2O2, even at longer time
points (up to 5 h; Fig. 2, left panel). The weak
NF-B activation probably came from the translocated NF-B bound to
wild-type endogenous IB molecules. EL4 cells bearing the Y42F
IB mutation were still able to be induced by IL-1ß to the same
extent as in control cells (Fig. 2, right panel). Comparable
results were observed with EL4-PEST cells, but NF-B activation by
IL-1ß was prolonged up to 180 min. Because the lack of NF-B
activation by H2O2 in the
EL4-Y42F and -PEST cells was reminiscent of a cytotoxic effect, we
decided to restimulate them with IL-1ß for 30 min following 180
min of treatment with H2O2.
As shown in Fig. 2 (central panel), this subsequent
stimulation led to NF-B activation, demonstrating that the lack
of NF-B activation in EL4-Y42F and -PEST cells was not due to a
cytotoxic effect or to the loss of an important component of the
NF-B signal transduction machinery. For these experiments, two
EL4-Y42F and three EL4-PEST cell clones were tested with identical
results. Moreover, the intracytoplasmic p50 and RelA levels were very
similar in unstimulated EL4/2 and EL4-Y42F and -PEST cells,
demonstrating that the noninducibility of NF-B in the latter two
cell lines was not due to a depletion of RelA and p50 in the
intracytoplasmic pool (data not shown).
Because NF-B induction by
H2O2 was not inhibited by
IB mutated at serines 32 and 36, we decided to analyze its
composition by supershift experiments. As shown in Fig. 3A, addition of Abs directed
against p50 and RelA to nuclear extracts from
H2O2- or
PMA-ionomycin-stimulated EL4-S32,36A cells induced large, but similar,
supershifts. Abs against c-Rel, RelB, and p52 (data not shown) had no
effect, demonstrating that induction by
H2O2 predominantly induced
the classical NF-B heterodimer in EL4-S32,36A clones. A similar
complex (p50/RelA) was detected in the control cell line stimulated
with H2O2 (data not
shown).
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B induction by
H2O2 in EL4-S32,36A cells
was transcriptionally active, transient transfection assays were
conducted. As shown in Fig. 3B, stimulation of the
EL4-S32,36A cells with a B-LUC construct gave rise to
transcriptional activity that increased with the
H2O2 concentration (up to
5-fold) and was similar to that obtained in the control cells (Fig. 3B, right panel). On the other hand, while
induction of the EL4/2 cells with PMA-ionomycin gave rise to
a 12-fold increase in the B-dependent transcriptional activity, this
was sharply reduced in EL4-S32,36A cells (Fig. 3B,
left and right panels). These data demonstrated
that the NF-B complex translocated by
H2O2 in the nucleus of
EL4/2 cells was transcriptionally active and confirmed that the
presence of the S32,36A form of IB strongly impaired the NF-B
transcriptional activity induced by PMA-ionomycin.
H2O2 induces both wild-type and
S32,36A-mutated IB degradation
Western blot analysis made it possible to distinguish the
endogenous murine IB from the mutated human molecule.
Furthermore, electrophoretic separation of the two molecules was
facilitated by the expression of a tagged fusion protein
(S32,36A-IB). Stimulation of EL4-S32,36A cells with PMA-ionomycin
for 30 min revealed that the endogenous murine IB molecule was
rapidly degraded after addition of PMA-ionomycin, whereas the mutated
molecule remained undegraded (Fig. 4A, left
panels). It should be emphasized that this mutated molecule
progressively accumulated after induction with PMA-ionomycin. Identical
results were observed after stimulation with IL-1ß or clones
expressing various amounts of the mutated IB (Fig. 4A,
left panels, and data not shown). These observations helped
us to understand why a weak and transient NF-B induction was
detected in EL4-S32,36A cells treated with PMA-ionomycin. Indeed,
NF-B nuclear translocation was very likely due to degradation of the
wild-type murine IB molecule, responsible for the weak transient
release of NF-B (Fig. 2). In contrast, H2O2 induced
degradation of both the wild-type and mutated forms (Fig. 4A, right panels). The mutated S32,36A IB
molecule was degraded after 60 min and reached a maximum after 120 min,
at which time 60–70% of the mutated S32,36A IB molecules were
degraded. This behavior was repeatedly observed in several independent
clones (Fig. 4A and data not shown). Because the expression
of the mutated gene in EL4-S32,36A cells was variable depending on the
clone, we also followed IB degradation after
H2O2 stimulation in a clone
expressing a low amount of the mutated S32,36A IB molecule. In
that clone, the mutated IB molecule first started to accumulate
before being degraded with similar kinetics as in the other clones
(data not shown). In conclusion,
H2O2 treatment
systematically led to degradation of S32,36A-mutated IB in
contrast to what was observed with PMA-ionomycin or IL-1ß.
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B phosphorylation
Cytokine-inducible phosphorylation of IB at S32,36 was
necessary for its degradation and subsequent NF-B activation.
Because IB degradation was observed following treatment with
H2O2 even when serines 32
and 36 were substituted for alanines, we were interested to know
whether this treatment promoted IB phosphorylation at other
sites. We examined IB phosphorylation after treatment with
H2O2 in the presence of
lactacystin, a proteasome inhibitor (28), and several
phosphatase inhibitors to abolish phosphorylated IB degradation.
Cell extracts were prepared at time points preceding the appearance of
nuclear NF-B. These experimental conditions allowed us to visualize
phosphorylated murine IB protein after 5 min of stimulation with
PMA-ionomycin and 10–20 min after addition of
H2O2 (Fig. 4B).
However, when the mutated IB form was analyzed, no upshifted band
was visualized after treatment with
H2O2.
IKKs are not activated after treatment with H2O2
To confirm our data showing that mutated S32,36A IB
molecules can always be degraded after oxidative stress, we
investigated the effect of
H2O2 treatment on the
induction of IKK activity. These kinases have been shown to
specifically phosphorylate IB at S32 and S36. To examine this
activity, IKKs were immunoprecipitated using either an anti-IKK
Ab or an anti-NEMO Ab. Control experiments with IL-1ß or
PMA-ionomycin stimulations dramatically increased IKK activity 5 min
after stimulation, as shown by phosphorylation of the wild-type
GST-IB fusion protein (Fig. 5,
upper panel). This reaction was specific, because no
phosphorylation of the mutated GST-IB S32,36A was detected under
these conditions (Fig. 5, lower panel). In contrast, no IKK
activity was detectable at any times after
H2O2 stimulation (Fig. 5, upper panel). Again, no phosphorylation of IB was
observed when the substrate was replaced by its mutated counterpart
(Fig. 5, lower panel). These results indicate that IKKs were
not involved in IB phosphorylation after treatment with
H2O2.
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B proteins are not degraded in response
to H2O2 stimulation
IB proteolysis was then analyzed in EL4 -PEST cells. As
shown in Fig. 6, analysis of both
endogenous wild-type and mutated IB forms revealed that after
induction with IL-1ß, both IB molecules were degraded and
resynthesized with kinetics similar to those observed with EL4/2
control cells subjected to identical conditions. Inversely, degradation
of the mutated IB was totally inhibited after treatment with
H2O2, explaining why
NF-B activation was undetectable in these clones (Fig. 2). It should
be noted that the level of mutated gene expression was rather high in
the EL4-PEST cells and very similar in all the clones tested. These
data indicated that serine and threonine residues (S283, T291, and
T299) situated around the PEST sequence were required for IB
degradation and subsequent NF-B activation following treatment with
H2O2.
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B forms, whereas no
degradation of the mutated molecule was observed after
H2O2 induction (Fig. 6).
The separation of human and murine IB forms was rendered
difficult by the quite similar electrophoretic mobility of the two
proteins as well as by overexpression of the mutated molecule. In
conclusion, these data indicated that the Y42 residue of IB
protein was also required for IB degradation and subsequent
NF-B activation following treatment with
H2O2. The various clones
tested exhibited similar behaviors.
H2O2 induces a degradation of IB that
is partially proteasome independent
Since we demonstrated that the IB degradation pathway
following H2O2 very likely
involved different residues from those important in the response to
cytokines, we investigated the role of the 26S proteasome in
H2O2-mediated IB
proteolysis. To ascertain proteasome involvement in IB
proteolysis, NF-B activation was followed in the presence or the
absence of lactacystin. EMSA analysis showed that pretreatment of cells
with lactacystin inhibited NF-B translocation after treatment with
PMA-ionomycin, whereas it had only a weak effect on NF-B
translocation induced by
H2O2 (Fig. 7, left and central
panels). Taken together, these observations indicate that
treatment of EL4/-S32,36A cells with H2O2 led to
inducible IB phosphorylation and that degradation upon
stimulation could be conducted by a proteasome-independent
mechanism.
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B degradation after treatment with
H2O2
Because caspases were recently shown to be capable of cleaving
IB (29), EL4/2 cells were treated with PMA-ionomycin
or H2O2 in the presence of
100 µM Z-DEVD, which inhibits caspase-3, -6, -7, -8, and -10.
Nevertheless, under our experimental conditions, addition of Z-DEVD did
not modify NF-B activation after treatment with either PMA-ionomycin
or H2O2, indicating that
caspases were not involved in IB degradation (data not
shown).
Another protease, calpain, was recently suggested to be involved in
NF-B regulation (30, 31). E64-d has been shown to be a
calpain protease inhibitor (32), although its absolute
specificity in inhibiting calpain has been debated. However, E64-d has
several important characteristics, such as being easily taken up by
cells and inhibiting calpain proteases without inhibiting the
proteasome. EL4/-S32,36A cells were therefore incubated in the presence
of E64-d before being treated with PMA-ionomycin or
H2O2. As shown in Fig. 7
(right panel), E64-d partially inhibited NF-B activation
in EL4/-S32,36A cells treated with 300 µM
H2O2, demonstrating that
calpain was involved in the degradation process.
To ensure that H2O2
addition by itself is not involved in the degradation mechanism,
IB was transcribed and translated in vitro before being subjected
to H2O2 for 30 and 60 min.
Under these conditions, IB was not altered by the treatment,
confirming that oxidative treatment by itself did not lead to IB
degradation (data not shown).
Caseine kinase II is involved in IB phosphorylation by
H2O2
Caseine kinase II has been shown to be involved in constitutive
phosphorylation of several serine and threonine residues of the PEST
region of IB (33). To investigate whether casein
kinase II could be part of the induced IB degradation and NF-B
activation by H2O2, EL4/2
cells were treated with 300 µM
H2O2 for 2 h in the
presence of DRB (0.2 mM), which specifically inhibits casein kinase II
(34) without inducing any cytotoxic effect. As shown in
Fig. 8, EMSA analysis revealed that
NF-B activation was inhibited with 0.2 mM DRB, while no effect on
NF-B induction mediated by IL-1ß was observed with this DRB
concentration. DRB had no effect on NF-B activation when EL4/2 cells
were treated with PMA-ionomycin (data not shown). This indicates that
casein kinase II could well be involved in NF-B activation in EL4/2
cells after H2O2 treatment.
It must be noted that the higher DRB concentrations (0.5 or 1 mM) were
cytotoxic for the cells (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B activation process triggered by
H2O2 that is clearly
different from the well-characterized IB S32,36
phosphorylation-K21,22 ubiquitination-proteasome pathway. Indeed,
NF-B activation by H2O2
in a lymphoma cell line (EL4) is associated with a slow and sustained
activation of NF-B through an atypical mechanism of IB
degradation. It does not require serine 32 and 36 phosphorylation by
IKK, but probably relies on an unmodified tyrosine residue 42 and the
carboxyl-terminal PEST sequence to be phosphorylated by casein kinase
II. The degradation involves the calpain proteases and the proteasome.
The NF-B complex activated by
H2O2 in EL4 cells is the
heterodimer p50/RelA and is transcriptionally active (data not
shown).
The Rel/NF-B activation induced by several extracellular signals,
such as proinflammatory cytokines (IL-1ß and TNF-), requires
proteolysis of the associated IB by the phosphorylation-dependent
ubiquitin-proteasome pathway (reviewed in Refs. 35 and
36). Only four exceptions to this general signaling
pathway have been reported to date: 1) NF-B activation following
exposure to pervanadate or hypoxia-reoxygenation, which induces
IB phosphorylation at tyrosine 42 and dissociation from NF-B
without degradation (37); 2) a continuous nuclear
transport of p50-c-Rel dimer induced by high level basal degradation of
associated IB requiring free calcium (30); 3)
NF-B activation by amino acid analogues through a redox-controlled
IB degradation by the proteasome without apparent IB
phosphorylation (38); and 4) IB phosphorylation by
p90rsk in response to mitogens (39)
or for mucin overexpression in epithelial cells by Pseudomonas
aeruginosa (40). These latter data suggest that
different transduction pathways leading to NF-B activation may
coexist within a cell, with one pathway either being preferentially
triggered by a defined stimulus or one stimulus triggering a different
pathway in another cell type. It has recently been shown that short
wavelength UV (UV-C) activates NF-B independently of phosphorylation
at serine residues 32 and 36 (41, 42). Furthermore,
expression of a catalytically inactive IKK-ß mutant did not prevent
NF-B activation by UV-C (42), indicating that distinct
activation mechanisms can function in parallel within one cell
type.
In the case of oxidative stress mediated by
H2O2, a common observation
is the slow and sustained activation of NF-B correlating with
IB degradation (this work and Refs. 24 and
43 ; see Ref. 44 for review). This same
phenomenon was observed when cells were induced by other agents known
to generate oxidative stress, such as UV light (45),
hypochlorous acid (24), or photosensitization (25, 26). Because these kinetics of activation strongly contrast with
those initiated by proinflammatory cytokines, we postulated that the
respective transduction mechanisms are different. A first striking
observation made in this work is the nonrequirement of intact serine 32
and 36 residues for NF-B activation and IB degradation.
However, the integrity of either tyrosine 42 or the PEST sequence is
needed for IB degradation and NF-B activation by
H2O2. Numerous authors have
reported that the PEST sequence is essential for IB degradation
and stabilization and also for the rapid proteolysis of the
unassociated IB (32, 46, 47, 48, 49, 50). Three residues (S283,
T291, and T299) are important for phosphorylation by casein kinase II
and constitutive phosphorylation of IB in vivo (33).
The PEST sequence has been shown not to be required for IB
signal-induced degradation, but its removal stabilizes free IB in
unstimulated cells (51). This stabilization of IB
mutated in the PEST sequence correlates well with our observation of a
very low nuclear NF-B content in uninduced EL4-PEST cells. However,
we found that mutation of these three residues within the PEST sequence
abolishes IB-inducible degradation by
H2O2. The role of the PEST
sequence in response to
H2O2 contrasts with its
noninvolvement in the responses to IL-1ß and TNF- in EL4 (see
above) or to TNF- in 293 cells (50). Reinforcing our
data, it has recently been shown that a stretch between amino acid
positions 277 and 287 of the carboxyl-terminal part of IB is
important for the early onset of NF-B after UV irradiation of
primary human skin fibroblasts and HeLa cells (41). Thus,
our data together with reports from the literature demonstrate that the
carboxyl-terminal part of IB is important for inducible
phosphorylation. Casein kinase II could very well be involved in this
reaction. It should be pointed out, however, that multiple kinase
activities are likely to be involved, because tyrosine, serine, and
threonine residues have been shown to be important.
Because IKK- and -ß have been shown to selectively phosphorylate
S32,36 residues (22, 23), it is very likely that these two
kinases are not responsible for the
H2O2-induced
phosphorylation of the S283 and T291,299 residues of the PEST sequence.
Casein kinase II, on the other hand, has been found to physically
associate with the PEST sequence of IB, inducing a multisite
(serine/threonine) phosphorylation required for basal and HIV-induced
degradation of IB in vitro (46). It has recently
been shown that purified endogenous casein kinase II effectively
phosphorylated serine 32 of IB and that when associated with
p90rsk, this kinase complex is capable of
phosphorylating both degradation-relevant serines in IB and also
exists as a preassembled kinase-substrate complex containing all known
IB isoforms and most Rel family members (51). These
last data together with many other reports, such as in U937, Chinese
ovary, and rat lymphoid cells treated with bufalin (52),
insulin (53), and prolactin (54), demonstrate
the inducibility of casein kinase II. It is thus clear that casein
kinase II activity may be stimulated by various stimuli, and oxidative
stress is probably one of them. Indeed, A170, which is an oxidative
stress-inducible protein with a zinc finger, two PEST sequences, and
many potential phosphorylation sites for serine/threonine kinases, is
phosphorylated in activated macrophages by two 40- and 44-kDa kinases
similar to and ' subunits of casein kinase II (55).
How phosphorylation of the PEST sequence by casein kinase II in
response to H2O2 targets
IB for degradation is still unknown.
We have also shown that inhibition of calpain proteases partially
prevents NF-B activation in EL4 cells in response to
H2O2. From these
experiments we deduced that phosphorylated IB protein on S/T
residues in the PEST sequence becomes a substrate for digestion by
calpains. This conclusion is reinforced by recent data showing that the
PEST sequence of IB is critical for its calpain-dependent
degradation (56). Furthermore, the IB-PEST domain
binds to the calmodulin-like domain of the large subunit of µ-calpain
and acts as a modulator in promoting the physical association and
subsequent degradation by µ-calpain. Although IB degradation is
attributed primarily to the ubiquitin-proteasome pathway, there have
recently been a number of alternate proteolytic mechanisms described
for IB, including some that specifically implicate isoforms of
calpain as the direct IB protease (30, 31, 56, 57, 58).
Among the known substrates of calpains are peptides with large
aliphatic or aromatic side chains, but there exist at least two other
motifs: the presence of PEST sequences or of calmodulin-binding domains
(see Ref. 59 for review). PEST sequences have frequently
been considered a necessary feature of proteins cleaved by calpain. The
rationale for this proposal is the acidic chain character of the PEST
sequence, which could sequester Ca2+ until its
concentration would become high enough to satisfy the calpain
requirement. Calpains are cysteine proteases activated by
Ca2+ binding to a catalytic subunit of about 80
kDa (59). While most of the mechanism of calpain
activation is still a matter of debate, it is known that a rise in the
Ca2+ concentration, heterodimerization, and
membrane association (60) are important means for in vivo
activation. Regulation of the free intracellular calcium concentration
plays a major role in physiological signal transduction and has been
shown to be important for NF-B activation (61). It has
recently been shown that TNF--inducible IB proteolysis in
HepG2 liver cells is mediated by calpain (31). This
inducible calpain proteolytic activity occurs coincidentally with the
particulate-to-cytosol redistribution of the catalytic µ-calpain
subunit into the IB compartment (31). Exposure to
agents that produce hydroperoxides or the addition of exogenous
hydroperoxides also causes elevation of intracellular
Ca2+ in some cells (60). Because
Ca2+ release after exposure to
H2O2 is well documented
together with calpain activation under oxidative stress conditions
(62, 63), it is clear why IB phosphorylation in the
PEST sequence becomes a substrate for degradation by calpains.
In conclusion, this work supports the important function of tyrosine 42
and the carboxyl-terminal PEST sequence in the phosphorylation and
degradation of IB upon exposure to
H2O2. This sequence of
events raises the question of how casein kinase II is activated by
oxidative stress and why S/T residues within the PEST sequence, which
have been shown to be important for constitutive phosphorylation, also
play a role in inducible IB degradation.
|
Acknowledgments |
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|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Jacques Piette, Laboratory of Virology, Institute of Pathology B23, University of Liege, B-4000 Liege, Belgium.
3 Abbreviations used in this paper: ROS, reactive oxygen species; IKK, IB kinase; E64-d, L-3-trans-ethoxycarbonyloxirane-2-carbonyl]-L-leucine (3-methyl) amide; DRB, 5,6-dichloro-1-ß-D-ribofuranosyl-benzimidazole; NEMO, NF-B essential modulator; PEST, proline-glutamic acid-serine-threonine; Z-DEVD, Z-Asp-Glu-Val-Asp-FMK.
Received for publication November 5, 1999. Accepted for publication February 3, 2000.
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