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B Activation by TNF Via the Activation of p38 Mitogen-Activated Protein Kinase1
Department of Biochemistry, Trinity College, Dublin, Ireland
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B is a central mediator of altered
gene expression during inflammation, and is implicated in a number of
pathologies, including cancer, atherosclerosis, and viral infection. We
report in this study that vitamin C inhibits the activation of NF-B
by multiple stimuli, including IL-1 and TNF in the endothelial cell
line ECV304 and in primary HUVECs. The induction of a NF-B-dependent
gene, IL-8, by TNF was also inhibited. The effect requires millimolar
concentrations of vitamin C, which occur intracellularly in vivo,
particularly during inflammation. Vitamin C was not toxic to cells, did
not inhibit another inducible transcription factor, STAT1, and had no
effect on the DNA binding of NF-B. Inhibition by vitamin C was not
simply an antioxidant effect, because redox-insensitive pathways to
NF-B were also blocked. Vitamin C was shown to block IL-1- and
TNF-mediated degradation and phosphorylation of I-B
(inhibitory
protein that dissociates from NF-B), due to inhibition of I-B
kinase (IKK) activation. Inhibition of TNF-driven IKK activation was
mediated by p38 mitogen-activated protein kinase, because treatment of
cells with vitamin C led to a rapid and sustained activation of p38,
and the specific p38 inhibitor SB203580 reversed the inhibitory effect
of vitamin C on IKK activity, I-B phosphorylation, and NF-B
activation. The results identify p38 as an intracellular target for
high dose vitamin C. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The inducible, higher eukaryotic transcription factor NF-B regulates
the expression of a number of cellular genes involved in immune and
inflammatory responses (10), has antiapoptotic effects
(11, 12, 13), is involved in the replication of several
viruses (10), most notably HIV-1 (14), and
has been implicated in the initiation and development of
atherosclerosis (15, 16). It is activated by diverse
pathogenic signals, including the proinflammatory cytokines IL-1 and
TNF (10). NF-B exists in a latent form in the cytoplasm
of unstimulated cells, comprising a transcriptionally active dimer
bound to an inhibitor protein,
I-B3 (inhibitory
protein that dissociates from NF-B). The currently known subunit
members of the NF-B family in mammals are p50, p65 (RelA), c-Rel,
p52, and RelB, while multiple mammalian forms of I-B also exist,
namely I-B, ß, 
(p105), 
(p100), and 
, and Bcl-3
(17). Most work has focused on the p50/p65 dimer, the
predominant form of NF-B activated in many cells, and its
association with I-B. Upon stimulation with many NF-B
inducers, I-B is rapidly phosphorylated on
Ser32 and Ser36, which
targets the inhibitor protein for ubiquitination and subsequent
degradation by the 26S proteasome (18). A specific E3
ligase that recognizes doubly phosphorylated I-B has now been
identified (19). The released NF-B dimer can then
translocate to the nucleus and activate target genes by binding with
high affinity to B elements in their promoters. The phosphorylation
and degradation of I-B are tightly coupled events, and recently
two I-B kinases (IKKs), termed IKK and IKKß, have been
identified (reviewed in Ref. 20). IKK and ß have been
shown to be activated by IL-1 and TNF, to specifically phosphorylate
Ser32 and Ser36 of
I-B, and to be crucial for NF-B activation by these cytokines
(20, 21, 22). The IKKs are part of a larger multiprotein
complex called the IKK signalsome, which contains a core of IKK and
ß together with IKK (or NF-B essential modulator, NEMO) which
is essential for NF-B activation (23, 24).
Upstream of IKK activation, binding of TNF to TNFR1 aggregates
three TNFR1s together, inducing the association of TNFR-associated
death domain protein with the death domains in the cytosolic region of
TNFR1s (reviewed in Ref. 25). TNFR1-associated death
domain protein then recruits TNFR-associated factor 2 (TRAF2) and
receptor-interacting protein, both of which are essential for NF-B
induction. TRAF2 can then interact with and activate NF-B-inducing
kinase (NIK), which can activate the IKKs (25). In
contrast, binding of IL-1 to its type 1 receptor (IL-1R1) recruits the
IL-1R accessory protein, which leads to recruitment of IL-1R-associated
kinase (25), via the adaptor molecule MyD88
(26). IL-1R-associated kinase then activates TRAF6, which
can associate with the mitogen-activated protein kinase (MAPK) kinase
kinase TGF-ß-activating kinase-1, leading to activation of NIK
(27), and subsequently the IKKs. MAPK/extracellular
signal-regulated kinase kinase kinase-1 has also been implicated in
activation of IKKs by IL-1 and TNF (28), although the
physiological relevance of both NIK and MAPK/extracellular
signal-regulated kinase kinase kinase-1 in IKK activation remains to be
determined (20).
We decided to examine the effect of vitamin C on NF-B, because
although there has been much interest in the effect of redox-modulating
compounds on pathways to NF-B, there are little data on the direct
effect of this antioxidant vitamin on NF-B activation. Furthermore,
NF-B has been implicated in pathologies for which high dose vitamin
C has been suggested as a therapy. In this study, we show for the first
time that millimolar doses of vitamin C inhibit multiple pathways to
NF-B, including IL-1 and TNF, in endothelial cells. Cytokine
stimulation of NF-B was inhibited before phosphorylation of
I-B and activation of IKK. This inhibition was not simply due to
an antioxidant effect. Rather, for TNF, inhibition was mediated by
vitamin C-induced activation of the stress-activated protein kinase
p38 MAPK.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human rIL-1 was a gift from the National Cancer Institute
(Frederick, WA), while human rTNF- was a gift from Dr. Steve Foster
(Zeneca Pharmaceuticals, Macclesfield, U.K.). The NF-B consensus
oligonucleotide was from Promega (Madison, WI). The STAT1 consensus and
mutant oligonucleotides, and the rabbit polyclonal Ab to human
I-B were from Santa Cruz Biotechnology (Santa Cruz, CA).
Anti-phospho-I-B (Ser32), anti-p38
MAPK, and anti-phospho-p38 MAPK
(Thr180/Tyr182) polyclonal
Abs were from New England Biolabs (Beverly, MA). Anti-NEMO antiserum
and plasmids encoding GST/I-B fusion proteins GST/I-B
1–72(1–72) and mutant GST/I-B 1–72(1–72)S32A/S36A were a gift from Dr.
Alain Israel and Dr. Shoji Yamaoka (Institut Pasteur, Paris, France).
GST/I-B fusion proteins were overexpressed and purified by
GST-affinity chromatography using standard techniques. The p38 MAPK
inhibitor SB203580 was purchased from Calbiochem (San Diego, CA) and
dissolved in DMSO. Vitamin C (sodium salt), vitamin E analogues,
PMA, and hydrogen peroxide
(H2O2) were from Sigma
(Poole, U.K.). Vitamin C was dissolved in PBS, pH 7.4, PMA in DMSO,
Trolox in ethanol, -tocopherol acetate in 50% (v/v) ethanol, and
-tocopherol phosphate in 25 mM Tris-HCl, pH 7.4.
Cell culture
The human cell line ECV304 and HUVECs were grown and passaged as described previously (29). All experiments were conducted in complete medium at 37°C. Cell integrity was assessed by measuring lactate dehydrogenase (LDH) release. After treatment of cells, a 100-µl aliquot of medium was removed from each experimental well and assayed for LDH by monitoring spectrophotometrically the decrease in absorbance at 340 nm in the presence of 75 mM Tris-HCl, pH 7.2, containing 150 mM KCl, 0.2 mM NADH, and 4.8 mM sodium pyruvate.
Cell fractionation and preparation of nuclear and cytosolic extracts
Nuclear extracts were prepared as described previously (29). Cytosolic extracts were prepared by removing the supernatant from pelleted nuclei, further centrifugation of this supernatant (13,000 x g, 10 min, 4°C), and removal of the ensuing supernatant into 100 µl storage buffer (10 mM HEPES, pH 7.9, 50 mM KCl, 0.2 mM EDTA, 20% v/v glycerol, 0.5 mM PMSF, 0.5 mM DTT). Protein concentrations were determined using the method of Bradford (30).
Electrophoretic mobility shift assay
Nuclear extracts (2 µg protein) were assayed for NF-B
DNA-binding activity in the EMSA, as described previously
(29). For STAT1 EMSA, 4 µg protein and 50,000 cpm of
radiolabeled STAT1 consensus oligonucleotide were used. For competition
analysis, unlabeled consensus or mutant STAT1 oligonucleotide was
incubated with nuclear extract protein in binding buffer for 20 min at
room temperature, before addition of radiolabeled oligonucleotide.
IL-8 ELISA
HUVECs were seeded in 96-well plates (5 x 104 cells in 200 µl) and 36 h later treated with 20 ng/ml TNF for 6 h with or without a 1-h pretreatment with vitamin C. Cell supernatants were harvested and assayed for IL-8 by ELISA using the DuoSet ELISA Development System for human IL-8 (R&D Systems, Minneapolis, MN), according to the manufacturer’s instructions.
Detection of IL-8 mRNA by RT-PCR
HUVECs were seeded in 10-cm dishes (1 x106 cells in 6 ml) and 24 h later treated with 20 ng/ml TNF for 1 h with or without a 1-h pretreatment with vitamin C. Total RNA was isolated using Tri reagent (Sigma), according to the manufacturer’s recommendations. RT-PCR was then performed using the enhanced avian RT-PCR kit (Sigma) with 2.5 µg total RNA as template. The PCR was performed with an annealing temperature of 55°C for 35 cycles, using primers to IL-8 (human IL-8 PCR Primer Pair; R&D Systems) or to the housekeeping gene aldolase as control.
Anti-I-B immunoblot analysis
Confluent ECV304 cells in six-well plates (3-ml vol) were
treated as described in figure legends. Treatment was terminated by
washing monolayers twice with ice-cold PBS. Total I-B was then
detected, as described previously (29). For detection of
phospho-I-B, 100 µl SDS sample buffer (62.5 mM Tris-HCl (pH
6.8), 2% w/v SDS, 10% glycerol, 50 mM DTT, 0.1% w/v bromophenol
blue) was added directly to cells, which were then sonicated. Lysates
were then resolved by SDS-PAGE, transferred to polyvinylidine
difluoride membranes, and probed with anti-phospho-I-B Ab
according to manufacturer’s instructions.
p38 MAPK assay
Cells were harvested as above, and p38 activation was determined by the appearance of a band on immunoblots specifically recognized by an Ab to phosphorylated p38, because phosphorylation of p38 correlates with kinase activity (31, 32). In each experiment, extracts were also probed using anti-p38 Ab to demonstrate equal protein loading (not shown).
Determination of thiobarbituric acid-reactive substances (TBARS)
Lipid peroxidation was assessed by the TBARS assay, as described previously (29).
IKK assay
Confluent ECV304 cells in 100-mm dishes were treated as
described in figure legends, and monolayers then washed twice with
ice-cold PBS. Cells were lysed in 400 µl lysis buffer (20 mM HEPES,
pH 7.5, 0.1 mM EDTA, 0.1 mM EGTA, 10 mM KCl, 2 mM DTT, 10 mM
MgCl2, 50 mM NaCl, 100 µM sodium orthovanadate,
20 mM ß-glycerol phosphate, 1% Nonidet P-40). Clarified lysates were
precipitated with 1.5 µl anti-NEMO for 1 h at 4°C, and
then 35 µl of 50% protein G-Sepharose was added for 1 h at
4°C. Immune complexes were washed twice with lysis buffer, and then
three times with kinase buffer (20 mM HEPES, pH 7.5, 2 mM DTT, 10 mM
MgCl2, 50 mM NaCl, 100 µM sodium orthovanadate,
20 mM ß-glycerol phosphate). Kinase assays were then performed for 30
min at 30°C in kinase buffer containing 20 µM ATP, 5 µCi
[32-P]ATP, and 1 µg of bacterially
expressed GST/I-B 1–72(1–72) or mutant GST/I-B
1–72(1–72)S32A/S36A. The reaction mixtures were resolved by SDS-PAGE,
followed by autoradiography.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B activation by vitamin C
in endothelial cells
We investigated the effect of vitamin C on a number of stimulants
of NF-B in ECV304 cells. The response of NF-B to all those
stimulants used below have been previously extensively characterized in
both these cells and HUVECs (29). First, cells were
treated with vitamin C for 1 h before stimulation with TNF. Fig. 1
A shows that vitamin C
inhibited TNF-mediated NF-B activation in a dose-dependent manner,
with the effect being apparent at 5 mM (compare lanes 2 and
3), and potent at 10–20 mM (compare lanes 4 and
5 with lane 2). Activation of NF-B by TNF was
completely abolished upon exposing cells to 5 mM vitamin C for 16
h (Fig. 1A, lanes 6–8). Fig. 1B shows
that IL-1, PMA, or H2O2,
all of which activate NF-B in these cells (29), were
also sensitive to a 1-h pretreament with vitamin C: 10–20 mM blocked
the IL-1 response (compare lanes 4 and 5 with
lane 2), 5–10 mM was active against PMA (compare
lanes 7 and 8 with lane 6), while 20
mM inhibited H2O2 (compare
lanes 9 and 10). Fig. 1C shows that
inhibition of NF-B by vitamin C was not a cell line-specific
phenomenon, because primary HUVECs were also sensitive. Activation by
IL-1, TNF, and PMA was all inhibited by 5–20 mM vitamin C. Other cell
types tested that were also sensitive include EL4.NOB1 T lymphoma
cells, and human 132INI astrocytoma cells (not shown).
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B-dependent gene, in HUVECs. Fig. 1
D shows
that TNF treatment of HUVECs led to an increase in both IL-8 mRNA
(measured 1 h post-TNF stimulation by RT-PCR) and secreted IL-8
protein (measured 6 h post-TNF stimulation by ELISA of cell
supernatants). In both cases, pretreament with 5–20 mM vitamin C for
1 h before TNF stimulation led to a dose-dependent inhibition,
while there was no inhibitory effect on constitutive levels of IL-8
release, demonstrating the relevance of vitamin C-mediated NF-B
inhibition to downstream gene induction in endothelial cells.
Specificity of the effect of vitamin C on NF-B activation
We next tested the specificity of the effect of vitamin C on
NF-B. Because vitamin C inhibited all agents tested, the effect may
simply have been due to a general toxic effect on cells. However, this
was found not to be the case, based on a number of indices of toxicity.
Morphologically, cell monolayers of ECV304s and HUVECs appeared normal,
with no detachment of the cells observable after exposure to the doses
of vitamin C used (not shown). Furthermore, as mentioned above, 5–20
mM vitamin C did not inhibit basal levels of IL-8 release during a 7-h
exposure period in which inhibition of an increase in TNF-mediated IL-8
release was observed (Fig. 1D). Fig. 2A shows that membrane
integrity was not affected because there was no increase in LDH release
in response to up to 100 mM vitamin C in ECV304 cells over the 2-h time
course of the experiments shown in Fig. 1, A and
B. Additionally, vitamin C did not induce necrosis or
apoptosis of cells over the course of the experiment shown in Fig. 1A, lanes 6–8, because no DNA smearing or
laddering was detected under these conditions (not shown). A general
effect on inducible transcription factors was also unlikely. Fig. 2B shows that the induction of STAT1 DNA binding by IFN-
was not inhibited by 5–40 mM vitamin C, under identical conditions to
Fig. 1 in which strong inhibition of NF-B was observed.
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B have been shown to act by directly modifying
NF-B and so interfere with its binding to DNA (33, 34).
To explore this possibility for vitamin C, we incubated nuclear
extracts from TNF-stimulated cells with vitamin C for 2 h at
37°C. Fig. 2C shows that this treatment had no inhibitory
effect on NF-B DNA binding in the EMSA at the highest dose of 40 mM
(lane 5), while concentrations of 5–20 mM slightly
potentiated DNA binding (lanes 2–4). This strongly
suggested that vitamin C does not inhibit NF-B by modifying the
complex directly or by interfering with DNA binding. Another
possibility investigated was whether vitamin C chemically interfered
with the release of NF-B from I-B. Fig. 2D shows that
crude cytosolic extracts from control cells had a basal level of
NF-B activity (lane 1). Treatment of these
extracts with a sodium
deoxycholate/3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate
detergent mix led to increased detection of NF-B activity in the
EMSA due to an in vitro release of NF-B from I-B
(lane 2). The profile of NF-B detected in crude
cytosolic extracts, and in the presence of detergent differed somewhat
from that detected in nuclear extracts, in that an extra lower band
seemed to be revealed (lane 1). Importantly,
detergent treatment of extracts from cells treated with 5–40 mM
vitamin C led to a release of similar levels of NF-B to control
cells (compare lanes 4, 6, and 8 with
lane 2). This demonstrated that vitamin C was not altering
the NF-B/I-B complex in a manner that would render it insensitive
to stimulant-induced dissociation, as has been shown for some NF-B
inhibitors (33).
Inhibition of NF-B by vitamin C is not primarily due to its
antioxidant properties
Because vitamin C was inhibiting some pathways to NF-B in
ECV304s and HUVECs which we have previously shown to be insensitive to
antioxidants (particularly IL-1) (29), we suspected that
the effect of vitamin C was not dependent on its antioxidant
properties. To address this, we first determined whether millimolar
vitamin C could act as an antioxidant in our system, by assessing its
ability to block lipid peroxidation. Lipid peroxidation was assessed by
the TBARS assay, a sensitive index of lipid peroxidation that detects
mainly malondialdehyde, an end product of the peroxidation of
polyunsaturated fatty acids and related esters (35). Table I shows that, as previously reported
(29), stimulation of ECV304 cells with TNF for 30 min led
to a small but significant increase in levels of TBARS, here of
123 ± 8% (p < 0.05) above control
levels. Pretreatment of cells with 10 mM vitamin C decreased basal
levels of TBARS to 68 ± 4% of control values, and also
completely blocked the TNF-mediated increase. Hence, vitamin C was
capable of acting as an antioxidant in our experimental system.
However, Table II shows that other
antioxidants failed to mimic the general inhibitory effect of vitamin C
on NF-B, in that they either only inhibited the TNF, and not the
IL-1 pathway, or else were ineffective against both cytokines. As
reported previously (29), butylated hydroxyanisole, a
chain-breaking antioxidant that inhibits lipid peroxidation at the
concentrations tested in this study ( (29), and not
shown), inhibited TNF, but not IL-1.
N-acetyl-L-cysteine, a radical
scavenger and glutathione precursor, inhibited neither IL-1 nor TNF.
Furthermore, the water-soluble vitamin E analogues Trolox,
-tocopherol phosphate, and -tocopherol acetate were also
ineffective against both cytokines (Table II). At the concentrations
and contact times used in this study, -tocopherol acetate has been
shown previously to be active against NF-B in other systems
(36). Additionally, the vitamin E analogues failed to
further potentiate vitamin C-mediated inhibition of IL-1 or TNF (not
shown), even though vitamin C and E have a cooperative antioxidant
effect (4). Together, these results suggested that the
inhibition of NF-B by vitamin C was not dependent on its antioxidant
properties.
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B by inhibiting IKK
activity
Because degradation of I-B is a common key event in the
activation of NF-B by diverse stimuli, the effect of vitamin C on
this stimulant-induced degradation was next determined. Fig. 3A shows that treatment of
ECV304 cells with IL-1 or TNF for 1 h led to degradation of
I-B, as measured by the disappearance of a band specifically
detected by a polyclonal Ab to I-B (compare lanes 2
and 4 with lane 1). Pretreatment of both IL-1-
and TNF-stimulated cells with 20 mM vitamin C for 1 h inhibited
this degradation (lanes 3 and 5). Because
a critical step in many pathways to I-B degradation (including
IL-1 and TNF) is the phosphorylation of Ser32 and
Ser36 on I-B, we tested whether vitamin C
could block this phosphorylation. Fig. 3B shows that
treatment of cells with IL-1 or TNF led to a rapid and transient
increase in phosphorylated I-B, as measured by the appearance of
a band detected by an Ab that specifically cross-reacts with I-B
phosphorylated on Ser32, but does not react with
unphosphorylated I-B. The increase in phospho-I-B in
response to IL-1 was both more rapid and potent than that seen for TNF
(compare lanes 8–10 with lanes 2–4). In both
cases, the presence of 20 mM vitamin C for 1 h before cytokine
treatment of cells inhibited the appearance of phospho-I-B. For
TNF, this was seen as a complete absence of phospho-I-B in the
presence of vitamin C (lanes 5–7), while for IL-1
the appearance of phospho-I-B was both reduced (compare
lane 11 with 9) and delayed (compare lane
12 with 10).
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B activation and
I-B degradation by interfering with I-B phosphorylation.
This strongly suggested that vitamin C would have an effect on the
activity of the IKK complex, given that it contains the specific I-B
kinases IKK and IKKß that can phosphorylate I-B on
Ser32 and Ser36
(20). To investigate this possibility, endogenous IKK
kinase activity was measured by an in vitro anti-NEMO immunocomplex
kinase assay using bacterially expressed GST/I-B 1–72(1–72) as a
substrate. NEMO (or IKK) forms a core IKK complex together with
IKK and IKKß (20). ECV304 cells were treated with
IL-1 or TNF for different times, and then lysates were
immunoprecipitated using anti-NEMO and assayed for IKK activity.
Fig. 4A shows that both IL-1
and TNF treatment induced a rapid increase in phosphorylation of the
I-B substrate. The response to IL-1 was more sustained, and was
still increasing after 30 min (lane 7), while TNF was
more transient, peaking at 15 min (lanes 3) and
decreasing again by 30 min (lane 4). When a mutant
substrate, GST/I-B 1–72(1–72)S32A/S36A, in which the critical Ser
residues phosphorylated by IKKs were replaced with Ala, was used in the
assay, no phosphorylated band was detected in response to IL-1 or TNF
(lanes 9 and 10), confirming the
specificity of the cytokine-induced IKK activity. When cells were
pretreated with 20 mM vitamin C for 1 h, the effect of a
subsequent 15-min stimulation with IL-1 or TNF on IKK activity was
potently inhibited (Fig. 4B, compare lane 2 with
3 and 4 with 5). Conversely, when 20
mM vitamin C was incubated with immunocomplexes from IL-1- and
TNF-stimulated cells directly for 1 h at 37°C just before kinase
assay, there was only a marginal inhibitory effect on IKK activity
(Fig. 4B, lanes 7–10). Hence, vitamin C was inhibiting IKK
activity by a mechanism unlikely to involve a direct effect on
components of the IKK complex.
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Although vitamin C was not directly affecting general kinase
activity, because it didn’t inhibit the IKK kinase assay in vitro,
there was still a possibility that exposing cells to vitamin C led to
an in vivo nondirect effect that could inhibit kinases, such as
depletion of intracellular ATP. Therefore, to confirm the specificity
of the inhibitory effect of vitamin C on IKK, we examined the effect of
vitamin C on the activity of another TNF-responsive kinase, p38 MAPK.
Because phosphorylation of p38 tightly correlates with kinase
activity (31, 32), p38 activation was determined by the
appearance of a band specifically recognized by Ab to phosphorylated
p38. Fig. 5A shows an
experiment in which both NF-B and p38 activity were measured in
response to TNF, both in the presence and absence of vitamin C. Under
the same experimental conditions, vitamin C markedly inhibited
TNF-stimulated NF-B activity (top panel), while
having no inhibitory effect on TNF-mediated p38 activation
(bottom panel). This confirmed that vitamin C was not
inhibiting kinases nonspecifically either in vivo or in vitro.
Surprisingly, when cells were pretreated with vitamin C before addition
of TNF, p38 activity was increased substantially over cells stimulated
with TNF alone (Fig. 5A, bottom panel, compare
lanes 3 and 4 with lane 2).
Furthermore, Fig. 5B shows that vitamin C alone could
activate p38. Treatment of cells with as low as 2 mM vitamin C led to a
modest activation of p38 (compare lane 2 with lane
1), which increased dose dependently with increasing
concentrations of up to 20 mM vitamin C (lanes 3–5).
Pretreatment of cells with the specific p38 inhibitor SB203580
(37, 38) at a concentration of 3 µM for 1 h before
addition of 20 mM vitamin C completely blocked p38 phosphorylation
(compare lane 6 with lane 5). Although SB203580
has been characterized as an inhibitor of p38 kinase activity, it also
inhibits phosphorylation (i.e., activation) of p38, as has been
previously reported and discussed (39). The basis for this
effect is still unexplained. A comparison of the time course of p38
activation by TNF and vitamin C shows the potency of the effect of
vitamin C on p38. Compared with TNF, induction of p38 activity by
vitamin C was more rapid (Fig. 5C, compare lanes
6 and 2), more intense (compare lanes 14 and
11), and more sustained (compare lanes 16 and
13).
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It has been previously shown that rapid activation of p38 MAPK can
be inhibitory toward TNF signaling even though TNF itself activates p38
(40, 41). Because vitamin C potently and rapidly activated
p38 MAPK at concentrations that inhibited IKK activity, we examined
whether IKK inhibition was mediated by p38 MAPK activation. Given that
pretreatment of cells with 3 µM SB203580 effectively blocked
activation of p38 by 20 mM vitamin C (Fig. 5B, lane
6), we tested the effect of this pretreatment on the ability of
vitamin C to inhibit the NF-B system. When cells were incubated with
SB203580 before addition of 20 mM vitamin C, the inhibitory effect of
vitamin C on TNF-mediated NF-B activation was relieved (Fig. 6, top panel). Densitometric
analysis of the bands detected in the EMSA shows that vitamin C reduced
the relative intensity of TNF-induced bands from 4.2 (lane
2) to 3 (lane 3). The presence of SB203580
prevented this decrease, and slightly enhanced the relative intensity
to 5.6 (lane 4). In the absence of vitamin C,
SB203580 had no effect on either constitutive or TNF-stimulated NF-B
EMSA activity (not shown). Furthermore, this relief of vitamin C
inhibition by SB203580 was also apparent when TNF-stimulated I-B
phosphorylation was measured (middle panel). SB203580
prevented the ability of vitamin C to block the appearance of
phospho-I-B induced by TNF (compare lane 4 with
lane 3). Importantly, the effect of SB203580 was shown to be
at the level of IKK activation, because it also relieved vitamin C
inhibition of TNF-stimulated IKK activity (bottom panel). As
in the case of the EMSA, SB203580 had no effect alone on IKK activity
(not shown). Thus, p38 MAPK, activated by vitamin C, can negatively
regulate TNF-mediated NF-B activation by preventing IKK
activation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B activation by diverse
stimuli in endothelial cells. Inhibition was found to be independent of
cell type and activating stimulus, because pathways encompassing IL-1,
TNF, PMA, and H2O2, in
transformed and primary endothelial cells (Fig. 1), and other cell
types (a T cell line and an astrocytoma cell, not shown) proved
sensitive to vitamin C. Another group has reported that in Jurkat T
cells, vitamin C actually enhanced TNF-mediated NF-B activation
(42). However, only one dose of vitamin C was used, 0.2
mM, which is 10–100-fold lower than the concentrations tested in this
study. Lack of inhibition by vitamin C on NF-B in another T cell
line has also been reported (43), but again the dose was
lower; in this study, 1.5 mM. Therefore, it is possible that inhibition
by millimolar vitamin C is a more general phenomenon, although it is
also conceivable that T cells are less sensitive than endothelial cells
to inhibition by vitamin C.
The stimulus-independent inhibition by vitamin C is in contrast to the
more stimulus-specific effects of the commonly used antioxidant
pyrrolidine dithiocarbamate (29), and the general lack of
effect of N-acetyl-L-cysteine on most
of the pathways tested in this study (29, 44, 45).
Moreover, we found that although vitamin C was redox active in ECV304s,
being capable of suppressing lipid peroxidation, the antioxidant
properties of vitamin C were unlikely to be critical to its inhibitory
effect, because it inhibited redox-insensitive pathways to NF-B, and
because other antioxidants failed to mimic or potentiate vitamin C
inhibition. This has important implications for the purported role of
oxidative stress in NF-B activation (46, 47), and in
fact we and others find no compelling evidence for a central role for
oxidative stress in diverse pathways to NF-B (48, 49),
and we have consistently found many pathways to NF-B to be
insensitive to antioxidants (29, 44, 45). Additionally,
where we do see an inhibitory effect with an antioxidant on a pathway
to NF-B, often multiple or unexpected targets are involved,
including direct chemical modification of the NF-B complex itself
(29, 34, 50). Although the kinases that phosphorylate
I-B are now well characterized, there is a notable silence in
relation to the predicted effect of redox modulation on them.
Vitamin C inhibited the increased phosphorylation and degradation of
I-B, and also inhibited the activation of the I-B kinase
complex. To assay IKK activity, antiserum to NEMO (IKK) was used to
immunoprecipitate the IKK complex, because IKK, ß, and most
likely form a core complex that is responsive to IL-1 and TNF, and
critical for I-B phosphorylation (20, 23). IKK and
IKKß are responsible for specifically phosphorylating
Ser32 and Ser36 in response to IL-1 and TNF
(21, 22). Importantly, vitamin C almost completely blocked
this IL-1- and TNF-mediated IKK phosphorylation of I-B. Because
the IKKs also specifically phosphorylate I-Bß, leading to its
degradation (21, 22), vitamin C would probably inhibit
these events as well. Inhibition by vitamin C was not a direct effect
on the IKK complex, because the NEMO-precipitated complex was not
sensitive to direct treatment with vitamin C in vitro. However, the
possibility still exists that a cellular metabolite of vitamin C was
directly targeting the complex.
Surprisingly, vitamin C was shown to be a potent activator of p38 MAPK.
When compared with TNF, a known physiological activator of p38, vitamin
C was a more potent, rapid, and sustained activator. Thus, p38 MAPK is
a novel intracellular target of vitamin C action. This revealed an
important clue as to how vitamin C might inhibit TNF-mediated IKK
activity and NF-B activation, because other groups have recently
shown that rapid activation of p38 by sodium salicylate, sorbitol, or
H2O2 could inhibit
TNF-stimulated I-B and ß degradation, and I-B
phosphorylation (40, 41, 51). Interestingly, p38 has a
well-defined positive regulatory role in the induction of
NF-B-dependent genes regulated by proinflammatory cytokines such as
TNF, but this is downstream of NF-B activation via IKK, at the
levels of transactivation and transcription. For example, it has been
demonstrated that p38 can regulate activation of the RNA polymerase II
general transcription factor TFIID during NF-B-dependent gene
transcription (52) and also can stabilize proinflammatory
cytokine-induced mRNA (53). However, it seems that the
kinetics of p38 activation are critical, in that a rapid and sustained
activation of p38 can lead to inhibition of NF-B, upstream of
transactivation and gene induction. Thus, p38 has a dual role,
depending on the kinetics of activation. The fact that vitamin C
inhibited TNF-induced IL-8 gene expression demonstrates that initial
activation of p38 can override the potential positive effect of p38
downstream of NF-B activation.
In this study, blocking vitamin C-stimulated p38 activation by using
the specific pharmacological p38 inhibitor SB203580 relieved the
inhibition of TNF-mediated NF-B activation, I-B
phosphorylation, and IKK activation. Furthermore, the kinetics of
activation of p38 by vitamin C were consistent with an inhibitory
effect because both p38 activation and the appearance of phospho-I-B
were only strongly apparent after 7-min treatment with TNF, while p38
activation in response to vitamin C was apparent after just 1 min.
Hence, our results confirm the ability of p38 to exert a negative
regulatory effect on TNF-mediated NF-B activation, and also
implicate this effect in the mechanism of vitamin C inhibition. The
inability of TNF to activate IKK in vitamin C-treated cells provides a
rationale for the reported inhibition of TNF-stimulated I-B and
ß degradation, and I-B phosphorylation by p38 (40, 41, 51). Although p38 seemed to be acting at a point between TNF
engaging its receptor and IKK activation, the specific target for p38
in this system is not known.
How vitamin C activates p38 remains to be determined, but it is
unlikely to be due to a general stress effect on cells, such as
hyperosmolarity or low pH, because activation was easily detectable at
2 mM vitamin C (Fig. 5B), while activation by sorbitol is
typically seen at about 300 mM (41), and the vitamin C
solution was buffered. It also remains to be determined as to which
isoforms of p38 are activated in response to vitamin C and involved in
NF-B inhibition. There are four known isoforms of p38, namely ,
ß, , and (54). Generally, p38 is the most
abundant form (55). However, endothelial cells were found
to have higher levels of p38ß, compared with other inflammatory cell
lineages (55), and both p38 and p38ß are inhibited by
SB203580, while the other two isoforms are not (54), so
either or both of p38 and p38ß could be involved here.
Although the concentration of vitamin C used in this study was in the millimolar range, our observations are likely to have relevance during inflammation in vivo. Cells in culture are deficient in vitamin C, unlike in vivo, in which intracellular concentrations are typically 2–5 mM, due to active accumulation into cells against circulating levels of the order of 0.1 mM (56). Some immunologically relevant cells such as neutrophils can accumulate vitamin C up to concentrations of 14 mM when activated during inflammation (57). Vitamin C (5 mM) was inhibitory in our study when added to cells overnight, while shorter incubations required 10–20 mM, which would allow acute accumulation.
Inhibition of NF-B by millimolar vitamin C may also be relevant to
the purported beneficial effects of megadose therapy, whereby exogenous
administration of high dose vitamin C might have an immunomodulatory
effect by subtly suppressing NF-B at sites of inflammation, due to a
greater availability of vitamin C to the plasma during times of acute
intracellular accumulation. High intakes of vitamin C have been
associated with the treatment of viral infections, most notably the
common cold and HIV-1. Rhinovirus is reliant on NF-B-dependent
processes both for cell invasion and for the pathogenesis of infection
(58, 59). As regards HIV-1, clinical improvement was
claimed in AIDS patients who ingested high doses of vitamin C
(9), and it has been shown to suppress HIV-1 replication
in chronically and acutely infected cells (60, 61).
NF-B has a central role in the activation and replication of HIV-1
(10, 14). Our data would also explain a report
demonstrating inhibition of HIV long terminal repeat-directed
transcription by 3–5 mM vitamin C in 293 cells (62).
However, it must be noted that others have also shown that vitamin C
can also suppress HIV expression in an NF-B-independent manner
(43). Other NF-B-dependent pathologies in which
beneficial effects for vitamin C have been claimed include
cardiovascular disease (7, 16) and cancer (6, 10, 11, 12, 13).
In conclusion, we have demonstrated for the first time that millimolar
doses of vitamin C inhibit multiple pathways to NF-B. This
inhibition is mediated by the prevention of IKK activation, which in
the case of TNF is dependent on activation of p38 MAPK. p38 MAPK is
thus a novel intracellular target of vitamin C action. The results may
contribute toward providing a rationale for the purported benefits of
megadose therapy, and suggest that the relationship between vitamin C,
p38 MAPK, and NF-B in vivo warrants further investigation.
|
Acknowledgments |
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B
constructs and anti-NEMO antiserum, and Dr. Dana Philpott (Institut
Pasteur) for advice on the IKK assay. |
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Andrew G. Bowie, Department of Biochemistry, Trinity College, Dublin 2, Ireland.
3 Abbreviations used in this paper: I-B, inhibitory protein that dissociates from NF-B; IKK, I-B kinase; LDH, lactate dehydrogenase; MAPK, mitogen-activated protein kinase; NEMO, NF-B essential modulator; NIK, NF-B-inducing kinase; TBARS, thiobarbituric acid-reactive substances; TRAF, TNFR-associated factor.
Received for publication February 18, 2000. Accepted for publication September 11, 2000.
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