From the Departments of Internal Medicine and
§ Surgery, University of Texas Southwestern and the Dallas
Veterans Affairs Medical Center, Dallas, Texas 75216 and the
¶ Webb-Waring Institute, Denver, Colorado 80262
Received for publication, October 8, 2002, and in revised form, March 3, 2003
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ABSTRACT |
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Activation of endothelial cell NF- Activation of vascular endothelial NF- Despite articulation of this universal pathway, a number of details
concerning NF- Human endothelial cells express a functional NADPH oxidase that appears
to be essential for transmitting signals initiated by mechanical shear,
human immunodeficiency virus type 1 Tat, tumor necrosis factor, phorbol
ester, and growth factors (17-21). Because of the diverse phenotypic
responses invoked by these various stimuli, we recently suggested that
one basis for signal specificity of reactive oxidants may be through
spatial site direction of the NADPH oxidase to relevant signaling
complexes. As an example, a two-hybrid screen using the full-length
oxidase adapter p47phox yielded the signaling protein TRAF4 as
a functional binding partner (22). In this latter screen, TRAF4 was the
only cDNA recovered, perhaps because of the intramolecular masking
of the core binding domains of p47 in the intact molecule. In the
present study, we used a truncated p47phox lacking a purported
autoinhibitory C terminus as a screening bait and recovered the NF- Plasmid Construction--
All of the PCR amplifications for
subcloning or mutagenesis were performed with either Pfu
Turbo (Stratagene) or Pfx Platinum (Invitrogen). All of the
new constructs were confirmed by sequencing. pGBKT7-p47,
pGBKT7-p47(205-390), pGBKT7-p47(1-205), pGBKT7-p47(1-298), pCINF-p47, pGEX-p47, and p47-GFP were constructed as previously described (18, 22). pGBKT7-p47(153-286), pGBKT7-p47(153-219), pGBKT7-p47(223-286), and pGBKT7-p47(153-219;R193) were constructed by
PCR amplification of the corresponding domains between EcoRI and SalI sites with the addition of stop codons and ligation
into the Gal4-BD shuttle vector pGBKT7 (Clontech).
pGBKT7-p47(4-125) was derived by PCR amplification between
NcoI and EcoRI sites and ligation into pGBKT7.
pGEX-p47(1-298) was made by PCR amplification of p47(1-298) between
EcoRI sites with addition of a stop codon and ligation into
pGEX-2TK. Full-length RelA was amplified between XhoI and
XbaI sites from a HUVEC library and ligated directly into pCI-neo (Promega) to create pCIN-RelA. pNF- Yeast Two-hybrid Screening and Mating Analysis--
A human
endothelial GST Pull-down--
BL21-RP E. coli were transformed
with pGEX, pGEX-p47, or pGEX-p47(1-298) and induced at 37 °C for
3 h with isopropyl- Coimmunoprecipitation--
Phoenix-293 (Fx) cells were
electroporated with pCINF-p47, expressing FLAG-tagged full-length
p47phox. After 24 h, the cells were extracted at 4 °C
for 30 min in lysis buffer (150 mM NaCl, 20 mM
Tris, pH 7.5, 1 mM EDTA, 1 mM EGTA, 1% Triton
X-100, 2.5 mM sodium pyrophosphate, 1 mM
Microscopy--
HUVEC were electroporated with p47-GFP with or
without RelA-PR and then grown on fibronectin-coated slides. Following
IL-1 NF- Electrophoretic Mobility Shift Assay--
HUVEC were infected
with respective adenoviruses for 1 h as above and the following
day were stimulated with IL-1 RelA Translocation and I- RelA Phosphorylation--
After infection with adenoviruses,
HUVEC were serum-starved overnight and then incubated in phosphate-free
Dulbecco's modified Eagle's medium for 30 min.
[32P]Orthophosphate (100 µCi/ml) and 1% fetal calf
serum were then added, and the cells were incubated for another 4 h. After stimulation with IL-1 Oxidant Production--
Oxidant production was assessed as the
oxidation of 2',7'-dichlorofluorescin diacetate (DCF; Molecular Probes)
(18) with modifications. Briefly, HUVEC were cotransfected with
pDsRed2-C1 (Clontech) and either pCIneo, pCIN-RelA
(301-431), or pSh-p67(V204A). After 24 h, the cells were loaded
with 10 µM DCF for 20 min, washed extensively, stimulated
with IL-1 RelA Binds to the Tandem SH3 Domains of p47phox--
To
relieve autoinhibitory folding of the p47phox bait protein, we
deleted the C-terminal 92 residues containing two proposed proline-rich motifs and all of the serines known to be phosphorylated in
p47phox of stimulated phagocytes (25, 26). This truncation
appears to be equivalent to a phosphorylated full-length
p47phox in opening core binding domains (26, 27). Upon
screening the HUVEC GAL4-AD library, three clones were identified that
encoded RelA in frame with the GAL4-AD. All three clones encoded
residues 170-551, encompassing part of the N-terminal Rel homology
domain, a mid-protein region of unknown function, and the C-terminal
transactivation domains. Using a yeast mating technique, binding of
RelA to p47(1-298) was confirmed (Fig.
1a). In contrast,
RelA did not bind to the full-length p47phox protein,
consistent with masking of the RelA binding site by the C terminus of
p47phox. Deletion analysis suggested binding of RelA to the
tandem SH3 domains of p47phox (153-286) but not to the
N-terminal PX domain (4-125) or C-terminal tail containing
proline- and arginine-rich domains. Although each SH3 domain bound RelA
in isolation, SH3a (153-219) bound its target with 2.3-fold greater
avidity compared with SH3b (223-286) as measured by lacZ
expression. The binding of SH3a to RelA appeared to depend on its SH3
surface and not some unrelated colocalized motif, because destruction
of the tryptophan bridge (p47(153-219;R193)) (18, 28, 29) abolished
binding activity. Unexpectedly, SH3a when accompanied by the N-terminal
half of the protein (1-205), and SH3b, when accompanied by the C
terminus (205-390), did not bind RelA. Although it is thought that
full-length p47phox in its native configuration involves
intramolecular interactions between SH3a and the C-terminal tail (28)
as well as SH3b and the N-terminal PX domain (30), it is likely that
binding function in these truncated derivatives is lost because of
intramolecular masking of SH3a by the PX domain and SH3b by the C
terminus. This would suggest that the binding domains of
p47phox are not entirely functionally independent and may adopt
alternate tertiary structures based on the specific truncation.
RelA also bound GST-p47(1-298) in vitro (Fig.
1b), suggesting a direct binding between proteins. The
smaller, ~54-58-kDa protein seen in this figure is consistent and
specifically binds GST-p47(1-298), likely representing a partially
translated or proteolyzed RelA fragment. Lack of RelA binding to the
full-length GST-p47(1-390) is consistent with the yeast mating data.
In whole cells, endogenous RelA specifically coprecipitated with
FLAG-tagged full-length p47phox (Fig. 1c),
suggesting that in mammalian cells the RelA-binding site of
p47phox is at least partially exposed. In support of this
interpretation, endogenous p47phox coprecipitated with
endogenous RelA in unstimulated HUVEC, suggesting a preformed complex
(Fig. 1d). Binding did not quantitatively increase following
IL-1 p47phox Overexpression Increases IL-1
In contrast to activation of RelA, its nuclear translocation and DNA
binding did not appear to be affected by p47phox
overexpression. IL-1 p47phox Binds to a Proline-rich RelA
Mid-region--
Various truncations of RelA were translated in
vitro to identify a general p47-binding domain within RelA. Well
established regions of RelA include the Rel homology domain
(approximately residues 1-300) and the C-terminal transactivation
domains (TA1, 521-551; TA2, 431-520) (32); however, neither of these
regions bound GST-p47(1-298) (Fig.
4a). Instead, the mid-region
between these two domains (301-431) avidly bound GST-p47(1-298).
Because SH3 domains such as those of p47phox are known to
identify proline-rich ligands, it is noteworthy that prolines comprise
32 of the 131 residues (24%) in this RelA mid-region. Specifically,
five motifs within this RelA proline-rich region (RelA-PR) present
candidate left-handed type II polyproline helices, characterized by the
consensus
As further evidence that p47phox binds RelA-PR, we found that
the RelA-PR peptide blocked binding of full-length RelA to
GST-p47(1-298) in vitro (Fig. 4c). In HUVEC,
endogenous RelA also colocalized strongly with p47-GFP in focal
peripheral structures in unstimulated (not shown) and
IL-1
To further investigate the potential functional importance of an
interaction between endogenous p47phox and RelA, we attempted
to decrease IL-1 IL-1 Characteristic of most adapter proteins, p47phox displays
multiple binding surfaces that include an N-terminal PX domain, tandem SH3 domains, a variant proline-rich remnant, a basic region, and a
C-terminal proline-rich motif. A wealth of studies have demonstrated the importance of most of these domains in self-association or in
binding to other oxidase subunits, whereas few studies have documented
binding to non-oxidase moieties. Here, we demonstrate binding of the
tandem SH3 domains of p47phox to RelA. Both yeast mating and
GST pull-down experiments suggested masking of the RelA-binding domain
by the C terminus of p47phox, consistent with present models of
intramolecular folding of this protein (26-28, 36). The precise
mechanism of interaction, however, at this point remains unclear.
Physiologic binding was also demonstrated by coprecipitation and
colocalization studies in intact endothelial cells, suggesting at least
partial unmasking of the RelA binding surface in vivo, even
under unstimulated conditions. Thus, the physical state of
p47phox may differ in resting endothelial cells as compared
with its state in vitro or in resting phagocytes. In the
latter cell type, p47phox is thought to exist in an
unphosphorylated, folded state. In endothelial cells, however, we have
previously noted quantitative association of endogenous p47phox
with the cytoskeleton (18), suggesting a binding function not present
in resting neutrophils, and have further noted phosphorylation of
p47phox in unstimulated
HUVEC.2 In further support of
an open form of p47phox, a recent study demonstrated
coprecipitation of this protein with other NADPH oxidase
components, including p22phox, in unstimulated endothelial
cells (37).
Colocalization of the two proteins in endothelial cells was not diffuse
but rather concentrated at peripheral dorsolateral protrusions.
Importantly, p47phox localizes to the cortical cytoskeleton of
endothelial cells and is highly concentrated in edge ruffles (18, 22).
RelA similarly has been noted to associate with actin structures (38),
and disruption of actin polymerization blocks activation of NF- Overexpression of p47phox in HUVEC augmented the otherwise weak
activation of a The selective effect of p47phox overexpression on RelA
phosphorylation and activation but not translocation is consistent with a recent series of studies suggesting that NF- As further evidence that p47phox participates in endogenous
NF- Interference with the NADPH oxidase through expression of a
trans-dominant interfering mutant of p67phox, chemical
inhibition of the oxidase, or scavenging of O Although the specific targets of oxidants in the IL-1 signaling cascade
were not identified in this study, protein-tyrosine phosphatases are
known to be reversibly inactivated by OB
by interleukin (IL)-1 constitutes an event critical to the progression
of the innate immune response. In this context, oxidants have been
associated with NF-
B activation, although the molecular source and
mechanism of targeting have remained obscure. We found that RelA,
essential for NF-
B activation by IL-1, was associated with the NADPH
oxidase adapter protein p47phox in yeast two-hybrid,
coprecipitation, and in vitro binding studies. RelA and
p47-GFP also colocalized in endothelial cells in focal submembranous
dorsoventral protrusions. Overexpression of p47phox synergized
with IL-1
in the activation of an artificial
B-luciferase reporter and specifically augmented IL-1
-induced RelA
transactivation activity. p47phox overexpression also greatly
increased IL-1
-stimulated RelA phosphorylation, whereas it had no
effect on I-
B degradation or on RelA nuclear translocation or
B
binding. The tandem SH3 domains of p47phox were found to
associate with a proline-rich mid-region of RelA (RelA-PR) located
between the Rel homology and transactivation domains. The RelA-PR
peptide blocked interaction of p47phox and RelA, and ectopic
expression of RelA-PR abrogated IL-1
-induced transactivation of the
NF-
B-dependent E-selectin promoter. Further, suppression
of NADPH oxidase function through the inhibitor diphenylene iodonium, the superoxide dismutase mimetic Mn(III)
tetrakis(4-benzoic acid)porphyrin (MnTBAP), or expression of a dominant
interfering mutant of a separate NADPH oxidase subunit (p67(V204A))
decreased IL-1
-induced E-selectin promoter activation, suggesting
that p47phox facilitates NF-
B activation through linkage
with the NADPH oxidase. IL-1
rapidly increased tyrosine
phosphorylation of IL-1 type I receptor-associated proteins, suggesting
that oxidants may operate through inactivation of local
protein-tyrosine phosphatases in the proximal IL-1
signaling pathway
leading to RelA activation.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B by
IL-1
1 during the
innate immune response drives the production of a number of proteins, such as tumor necrosis factor, intercellular adhesion molecule 1, and E-selectin, necessary for the full expression of acute inflammation. A linear activation sequence following engagement of the
type I IL-1 receptor (IL-1RI) has been intensely studied and involves
binding of the MyD88 and Tollip adapters to the IL-1 receptor chains
(1, 2) with subsequent recruitment of IL-1 receptor-associated kinase
(1, 3). IL-1 receptor-associated kinase appears to leave the receptor
complex to bind TRAF6, which associates with the mitogen-activated
protein 3-kinase TAK1 through the adapter TAB2 (4, 5). TAK1 in turn
activates NF-
B-inducing kinase, which acts upstream of the I-
B
kinase complex (6). This latter complex phosphorylates I-
B
(7-11), marking it for ubiquitination and degradation, with consequent
unmasking of the nuclear localization sequence of Rel family members,
nuclear translocation of the active NF-
B complex, and
transactivation of NF-
B- responsive genes.
B activation remain unresolved. In particular, numerous studies suggest an important role for endogenous oxidants in
the activation of NF-
B by IL-1. IL-1 increases oxidant production, and strategies designed to diminish oxidant production or augment oxidant scavenging cause decreased IL-1-induced NF-
B activity (12-14). However, exogenous H2O2 does not
replicate the time course of cytokine-induced NF-
B translocation
(15), and the involvement of oxidants in IL-1 signaling may be
cell-specific (15, 16). In addition, the mechanism of endogenous
oxidant involvement is unclear, because none of the signaling elements
listed above present obvious redox-responsive switches. Further, the
source of oxidants and the mechanism by which oxidants target the
NF-
B pathway have not been identified.
B
family member p65/RelA. Further evidence suggests the participation of
p47phox in RelA phosphorylation and transactivation but not
I-
B degradation or translocation of RelA into the nucleus.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B-luciferase was
obtained from Clontech and consisted of four
sequential
B consensus sites (GGGAATTTCC) upstream of the
herpes simplex virus-TK TATA-like promoter. pRL-TK and pFR-luc
were obtained from Promega and Stratagene, respectively. A plasmid
expressing Gal4-RelA was the kind gift of Dr. M. Lienhard Schmitz. All
of the RelA truncations were derived by PCR amplification of RelA
segments between XhoI and XbaI sites with
insertion of stop codons, cloning into pCR4-TOPO blunt (Invitrogen),
and then subcloning into pCI-neo. pGEX-RelA-PR was constructed by
amplification of RelA (301-431) between BamHI and
EcoRI sites and ligation into pGEX-2TK. pELAM-luc was
previously described (23). p67phox cDNA was obtained from
the American Type Culture Collection, and a V204A mutation was
introduced with PCR mutagenesis. The mutant was then subcloned into the
XbaI and KpnI sites of pShuttle (Clontech) to create pSh-p67(V204A). Adenoviruses
harboring wild type p47phox and lacZ were previously
described (22), and a similar technique was used to generate
Ad-p47(W193R). Equivalent expression of wild type p47phox and
p47(W193R) was demonstrated by Western blot at an equal multiplicity of
infection (not shown). lacZ transgene was expressed in
>95% of HUVEC.
phage Gal4-AD library was constructed as previously
reported (22) and dropped out in the shuttle vector pAD-GAL4-2.1.
Saccharomyces cerevisiae AH109, which harbor two auxotrophic
reporter genes (ADE2 and HIS3) and
lacZ, were stably transformed with pGBKT7-p47(1-298), which
was found to lack autonomous transactivation activity. Transformants
were selected and secondarily transformed with the endothelial library
using lithium acetate. A high stringency screen was performed,
requiring auxotrophic selection for bait vector (tryptophan), library
vector (leucine), and both interaction reporters (histidine and
adenine), in addition to lacZ expression (filter lift
assay). Positive colonies were restreaked, and single clones were
retested. Candidate library plasmids were passaged through
Escherichia coli, stably transformed into AH109, tested for
autonomous transactivation, and mated to Y187 yeast harboring the bait
vector, and the diploids were retested for auxotrophy and
lacZ expression. Interactions between RelA and different
p47phox domains were tested by mating Gal4-AD-RelA-transformed
AH109 with Y187 expressing Gal4-BD fusions with various p47phox
truncations and assessing quantitative
-galactosidase expression. Briefly, the diploids were selected with leucine- and
tryptophan-deficient media and replated under selection. Overnight
liquid cultures of diploid colonies were diluted and grown to mid-log
phase in selection medium, adjusted to identical
A600 readings, then washed twice, and
resuspended in 0.3 ml of Z buffer (60 mM
Na2HPO4, 40 mM
NaH2PO4, pH 7.0, 10 mM KCl, and 1 mM MgSO4). 0.1 ml of cell suspension was then
freeze-thawed three times using liquid nitrogen and vortexed with glass
beads, and the supernatant was added to 0.7 ml of Z buffer with 0.27%
-mercaptoethanol. Color was developed using 0.16 ml of
o-nitrophenyl
-D-galactopyranoside (4 mg/ml in Z buffer) and stopped with 0.4 ml of 1 M
Na2CO3. The tubes were centrifuged at
14,000 × g, and the A420 was
measured. Negative controls included Gal4-BD or Gal4-AD without fusions.
-D-thiogalactopyranoside. The
cells were lysed, and GST fusions were captured on
glutathione-Sepharose (Amersham Biosciences). Fusion protein
concentrations were estimated by Coomassie Blue PAGE. Full-length or
truncated RelA was transcribed using the T7 promoter of pCI-neo and
translated in vitro in the presence of
[35S]methionine using a kit (TNT quick
coupled; Promega). Translated products were assessed on a gel using
autoradiography and normalized prior to addition to the binding
reaction. Binding, washing, and analysis were performed as described
(22). Similar studies were performed in the presence of RelA-PR peptide
by bacterial expression of GST-RelA (301-431), purification on
GSH-Sepharose, and release of the peptide with thrombin cleavage.
-glycerophosphate, 1 mM Na3VO4,
1 µg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride), sonicated once for 5 s, and centrifuged at 6000 × g for 20 min at 4 °C. The supernatants were precleared
with protein G-agarose (Amersham Biosciences) and then
immunoprecipitated with anti-FLAG (clone M2, Sigma) or isotype control,
collecting conjugates at 1500 × g between washes. The
immunoblots were performed first using anti-RelA (Santa Cruz) and then
anti-p47phox (gift of Dr. Bernard Babior). For coprecipitation
of endogenous proteins, RelA was immunoprecipitated from HUVEC
using mouse anti-RelA (Santa Cruz) followed by immunoblot using rabbit
anti-p47phox and then rabbit anti-RelA. To assess tyrosine
phosphorylation, HUVEC were transfected with pCIneo, pSh-p67(V204A), or
pCIN-RelA-PR, stimulated with 2 ng/ml human IL-1
(Peprotech) for
varying times, and then lysed as above. IL-1RI was immunoprecipitated
(rabbit polyclonal N-20, Santa Cruz) after adjusting for total protein, and an immunoblot for phosphotyrosine (Upstate Biotechnology, Inc.) was performed.
stimulation, cells were washed twice with PBS, fixed with 3%
formaldehyde in PBS, washed twice, and permeabilized in 50 mM NaCl, 3 mM MgCl2, 200 mM sucrose, 10 mM HEPES, pH 7.4, and 0.5%
Triton X-100 for 5 min. The cells were blocked with 3% bovine serum
albumin in PBS and incubated with anti-RelA (Santa Cruz) at 1:200 in
1% bovine serum albumin at room temperature for 1 h. After three
washes in 1% PBS, the cells were incubated with rhodamine-conjugated secondary antibody (1:400) at room temperature for 1 h. After two
washes in 1% bovine serum albumin and two washes in PBS, the cells
were mounted with ProLong antifade (Molecular Probes), and the images
acquired with a Zeiss Axiovert S100TV LSM 410 laser scanning system.
B and RelA Activation--
For assessment of NF-
B
activation, HUVEC were first infected with the appropriate adenovirus
for 1 h at a multiplicity of infection of 1:100, recovered
overnight and then synchronized at the G1-S transition with
thymidine. Six hours after thymidine release, the cells were
electroporated with pNF-
B-luciferase and pRL-TK. 24 h later,
the cells were stimulated with 2 ng/ml IL-1
. The cells were lysed
6 h later, and both firefly and Renilla luciferase were
assessed using a dual luciferase kit (Promega). All of the signals were
normalized using the Renilla transfection control, which did
not significantly change with transgene expression or IL-1
stimulation. Activation of RelA was assessed by cotransfection of HUVEC
with the Gal4-BD-RelA fusion, the Gal4-luciferase reporter pFR-luc, and
pRL-TK. Transactivation of the Gal4 minimal promoter was assessed
6 h after IL-1
addition and was normalized to
Renilla luciferase. NF-
B activity was also assessed by
transactivation of the E-selectin promoter. HUVEC were transfected
after cell cycle synchronization using FuGENE 6 (Roche Applied Science)
with pELAM-luc and pRL-TK. Either pCIN-RelA (301-341), pSh-p67(V204A), or pCIneo were cotransfected. After 24 h, the cells were
stimulated with IL-1
(2 ng/ml) for 6 h, and the luciferase
activity was measured. In some studies, HUVEC were treated with
diphenylene iodonium (DPI; 10 µM) or MnTBAP (100 µM) for 30 min prior to IL-
1 stimulation.
for 30 min prior to lysis in cold
lysis buffer (10 mM HEPES, pH 7.8, 10 mM KCl,
0.1 mM EDTA, 2 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and
10 µg/ml pepstatin A) on ice for 15 min. The cells were disrupted in
0.6% Nonidet P-40 with vortexing, and the nuclei were recovered by
centrifugation and extracted at 4 °C for 30 min 10% glycerol, 20 mM HEPES, pH 7.8, 420 mM NaCl, 5 mM
EDTA, 5 mM dithiothreitol, and 1 mM
phenylmethylsulfonyl fluoride. Equal protein equivalents were incubated
with 32P-labeled oligonucleotides from the
B3 site of
the tumor necrosis factor promoter, as previously described (24). For
supershift assays, the nuclear extracts were incubated with respective
antibodies for 30 min prior to addition of the labeled probe. All of
the supershift antibodies were from Santa Cruz. The samples were
fractionated on a 4% acrylamide gel prior to autoradiography.
B Degradation--
After infection
with adenoviruses, HUVEC were stimulated with IL-1
(2 ng/ml) and
lysed at various time intervals. Equal protein equivalents of lysate
were loaded onto 12% PAGE gels and immunoblotted for I-
B
to
assess degradation. For RelA translocation, HUVEC nuclei were isolated
and extracted as above, and equal protein equivalents were
immunoblotted with anti-RelA (Santa Cruz).
(2 ng/ml) for 10 min, the cells were
lysed on ice for 5 min in lysis buffer containing 0.1% SDS and 0.5%
deoxycholate. The cells were sheared through a 23-gauge needle
10 times and pelleted at 13,000 × g for 15 min. After
preclearing and normalization for protein, RelA was immunoprecipitated
and washed four times in lysis buffer with SDS and deoxycholate, twice
in lysis buffer containing 0.5 M NaCl, and once in 50 mM Tris, pH 7.0. One third of the immunoprecipitate was
immunoblotted for RelA, and the remainder was analyzed by autoradiography.
(2 ng/ml) for 5 min, and analyzed by flow cytometry. The
DCF fluorescence (FL1 channel) of DsRed-expressing cells (FL2 channel)
was assessed. Separation of red and green channels was established
using cells expressing DsRed, cells loaded with DCF, and cells with
both or neither.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (24K):
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Fig. 1.
RelA associates with p47phox.
a, p47phox and various truncations were fused to the
Gal4-BD and transformed into Y187 yeast. Interaction with RelA was
assessed by mating to AH109 yeast harboring Gal4-AD-RelA and
quantification of -galactosidase in solution. p47(1-298) but not
full-length p47phox interacted with RelA. The tandem SH3
domains of p47phox were necessary and sufficient for this
interaction, with the greatest lacZ expression attained with
SH3ab, then SH3a, and then SH3b. SH3a containing a W198R mutation
displayed minimal interaction. The values are the means ± S.E. of
four experiments. b, GST pull-down demonstrating that
[35S]methionine-labeled RelA directly bound
GST-p47(1-298) but not GST-p47(1-390) (full-length) or GST alone. A
more quickly migrating band is likely partially translated or
proteolyzed RelA. c, Fx cells were transfected with empty
vector or FLAG-p47. Endogenous RelA coprecipitated with FLAG-p47 using
anti-FLAG antibodies in transfected but not untransfected cells and did
not coprecipitate using isotype control antibodies in FLAG-p47-transfected cells. d, at base line or
following stimulation with IL-1
, HUVEC lysates were
immunoprecipitated (IP) with either irrelevant or anti-RelA
antibodies and immunoblotted (IB) for p47phox.
Endogenous p47phox coprecipitated with endogenous RelA in both
IL-1
-stimulated and unstimulated HUVEC.
stimulation, suggesting a constitutive nature for this
interaction in endothelial cells.
-induced RelA
Activation--
Transactivation of an NF-
B consensus-luciferase
reporter by IL-1
was consistently low (122% of control) in
Ad-lacZ-infected HUVEC (Fig.
2, a and b). This
did not appear to be due to spurious effects of adenoviral infection,
interference with the normalizing Renilla reporter, passage
number, transfection technique, or low specific activity of IL-1
(data not shown) and may suggest that transactivation of this
artificial promoter construct in HUVEC by IL-1
is fundamentally
inefficient. Indeed, in a recent report, fluorocytometric enrichment of
high responding HUVEC clones stably transfected with an NF-
B
reporter was required to demonstrate response to IL-1
(31). Despite
this low efficiency, overexpression of p47phox, while having
minimal effect alone, increased IL-1
-stimulated NF-
B-luciferase
activity 2-fold above control (Fig. 2b). Overexpression of
the oxidase-inactive mutant p47(R193), which also would not be expected
to bind RelA, had no effect on IL-1
-induced NF-
B-luciferase activity. A similar pattern was observed for the transactivating activity of RelA specifically. The minimal induction of Gal4-RelA by
IL-1
was potentiated by p47phox overexpression (Fig.
2c).
View larger version (27K):
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Fig. 2.
p47phox overexpression potentiates
IL-1 -induced NF-
B
activation. a, whole cell lysate immunoblot
(IB) of HUVEC infected with Ad-lacZ, Ad-p47, or
Ad-p47(R193) with antibodies to p47phox. b, HUVEC
were infected with the indicated viruses and transfected with
NF-
B-luciferase and then exposed to IL-1
(2 ng/ml for 6 h).
IL-1
stimulated only a minimal increase in luciferase activity in
Ad-lacZ-infected cells, which was augmented in
Ad-p47-infected cells (p < 0.05). b, HUVEC
were cotransfected with Gal4-RelA and pFR-luc to assess transactivation
by RelA. Ad-p47 infection potentiated Gal4-RelA activation by IL-1
(p < 0.05). Reporter activity in a and
b were normalized for transfection with Renilla
luciferase. Histograms are the means ± S.E. of four
experiments.
increased NF-
B DNA binding in
Ad-lacZ-infected HUVEC, and overexpression of
p47phox did not alter either basal or IL-1
-stimulated gel
shift patterns (Fig. 3a). The
nucleoproteins in both Ad-lacZ (not shown) and Ad-p47-infected HUVEC stimulated with IL-1
(Fig. 3b)
contained both RelA and p50 (NF-
B1). As expected, immunoblots
confirmed translocation of RelA within 10 min of IL-1
stimulation,
and this translocation was unaffected by p47phox overexpression
(Fig. 3b). Similarly, p47phox overexpression did not
raise basal RelA levels in either cytosolic (not shown) or nuclear
fractions. Further, degradation of I-
B
was demonstrated within
15-30 min after IL-1
stimulation, with partial recovery by 60 min,
and this pattern was again unchanged by p47phox overexpression
(Fig. 3c). However, p47phox greatly affected RelA
phosphorylation (Fig. 3d). In Ad-lacZ-infected HUVEC, IL-1
treatment increased RelA phosphorylation 1.9-fold, whereas in Ad-p47-infected cells, IL-1
increased RelA
phosphorylation 8.7-fold (averages of three experiments).
View larger version (37K):
[in a new window]
Fig. 3.
p47phox overexpression
increases IL-1 -induced RelA phosphorylation
but not nuclear translocation. a, HUVEC were stimulated
with 2 ng/ml IL-1
for 30 min prior to gel shift assay.
B binding
increased equally in Ad-lacZ and Ad-p47-infected cells.
Supershift demonstrates the presence of RelA and p50 in
B complexes.
Ab, antibody. b, HUVEC were stimulated with
IL-1
for the indicated durations and nuclear RelA assessed by
immunoblot. Equivalent translocation of RelA occurred in
Ad-lacZ (upper panel) and Ad-p47-infected
(lower panel) cells. c, HUVEC were stimulated
with IL-1
for the indicated times, and I-
B
degradation was
assessed with immunoblots of whole cell lysate. The IL-1
-induced
degradation of I-
B
was unchanged in Ad-lacZ
(upper panel) compared with Ad-p47-infected (lower
panel) cells. d, RelA was metabolically labeled and
immunoprecipitated from HUVEC. Cells stimulated with IL-1
for 10 min
had greatly increased RelA [32P]phosphorylation when
infected with Ad-p47 compared with Ad-lacZ-infected cells
(upper panel). The lower panel shows an
immunoblot (IB) for RelA of same samples. Each of the panels
is representative of at least two individual experiments.
PX
PX(R) (Fig. 4b).
Although the most N-proximal of these (DPRPPPR) varies from the
consensus by inclusion of an acidic rather than hydrophobic first
residue, this motif is well conserved in mouse (EPRPPTR) and chick
(EPRPPRR) RelA proteins. In contrast, three of the remaining four
polyproline motifs are poorly preserved in other species, with the
remaining motif (APGPPQA) being represented in mouse (TPGPPQS) but not
chick RelA.
View larger version (30K):
[in a new window]
Fig. 4.
p47phox binds RelA (301-431).
a, GST pull-down shows direct binding of full-length RelA,
RelA (301-431), and RelA (170-431) to GST-p47(1-298). RelA did not
bind to GST alone. b, the p47-binding region of RelA (within
residues 301-341) comprises a proline-rich segment containing five
polyproline sites that are candidate type II SH3-binding sequences. The
invariant prolines of the PXXP motif are boxed,
and the residue numbers are shown. c, GST pull-down of
full-length RelA by GST-p47(1-298). Purified RelA-PR decreased direct
binding of RelA to GST-p47(1-298).
-stimulated (Fig. 5,
a-c) cells. Such submembranous structures appeared to
extend dorsally in Z sections (not shown). Ectopic expression of the
RelA-PR truncation greatly diminished physical colocalization of
endogenous RelA within these discreet p47-GFP collections (Fig. 5,
d-f).
View larger version (128K):
[in a new window]
Fig. 5.
Colocalization of RelA and p47-GFP.
HUVEC were transfected with p47-GFP and stimulated with IL-1 (2 ng/ml) 24 h later. RelA was immunostained with
rhodamine-conjugated secondary antibodies. p47-GFP (green
channel, first column) colocalized with RelA (red
channel, second column) in discreet regions
concentrating in focal protrusions (a-c). d-f,
cotransfection of p47-GFP with RelA-PR caused loss of colocalization.
Note persistent nuclear localization of RelA despite RelA-PR
expression.
-induced activation of endogenous NF-
B through
ectopic expression of RelA-PR, which we are not aware of as having
other binding partners. Because IL-1
only weakly activated the
consensus NF-
B-luciferase reporter, we instead used the native
E-selectin promoter, relevant to endothelial cells, to produce a more
robust IL-1
-induced signal. This promoter harbors three NF-
B
sites, at least two of which cooperate functionally and are required
for activity (33). As expected for this native endothelial-specific
promoter, IL-1
increased ELAM-luciferase activity in HUVEC and in
parallel caused a low level of oxidant production (Fig.
6a). Expression
of the RelA-PR region had minimal effects on oxidant production but
completely blocked IL-1
-induced E-selectin promoter activation. To
further implicate p47phox, we utilized a separate subunit of
the NADPH oxidase, p67phox harboring a mutation in the
purported activation domain (p67(V204A)) (34). This latter mutant has
been shown to act as a transdominant inhibitor of the NADPH oxidase
in vitro and in recombinant COS-phox cells (34,
35). As shown in Fig. 6a, coexpression of p67(V204A) also
significantly decreased IL-1
-induced E-selectin promoter activation
as well as oxidant production. As further evidence that p47phox
acts on NF-
B through its participation in the NADPH oxidase, we
found that the oxidase inhibitor DPI and the membrane-permeant superoxide dismutase mimetic MnTBAP also blocked IL-1
-induced E-selectin promoter transactivation (Fig. 6b). DPI also
blocked IL-1
-induced phosphorylation of endogenous RelA, consistent
with the effects of p47phox-dependent oxidants on
RelA phosphorylation (Fig. 6c).
View larger version (21K):
[in a new window]
Fig. 6.
NADPH oxidase participates in
NF- B activation. a, HUVEC were
cotransfected with pELAM-luc and one of the indicated vectors and
stimulated with 2 ng/ml IL-1
for 6 h. Top panel,
IL-1
-induced transactivation of the E-selectin promoter was
decreased by expression of the RelA proline-rich segment (RelA-PR) and
by a dominant negative NADPH oxidase subunit (DN-p67)
(p < 0.05). Bottom panel, IL-1
-induced
oxidant production was decreased by DN-p67 (p < 0.05).
Rel. Act., relative activity. b, IL-1
-induced
transactivation of the E-selectin promoter was decreased by the
superoxide dismutase mimetic MnTBAP (100 µM, 30 min of pretreatment) and by the oxidase inhibitor
DPI (10 µM, 30 min pretreatment) (p < 0.05). The histograms are the means ± S.E. of four individual determinations.
Cont, control. c, endogenous RelA was
metabolically labeled with [32P]orthophosphate. IL-1
(2 ng/ml) increased RelA phosphorylation roughly 2-fold, whereas DPI
blocked IL-1
-dependent RelA phosphorylation.
IB, immunoblot.
Increases Tyrosine Phosphorylation of IL-1RI-associated
Proteins--
Because oxidants are known to increase tyrosine
phosphorylation events, we searched for an increase in such events in
the proximal IL-1 signaling pathway. Several proteins were shown to coprecipitate with the IL-1RI, including a ~98-kDa protein that was
tyrosine phosphorylated within 2 min of IL-1
stimulation (Fig.
7a). In addition, ~139-,
106-, 45-, 42-, and 39-kDa proteins displayed increased tyrosine
phosphorylation 3-5 min after IL-1
stimulation. Expression of
p67(V204A) decreased IL-1
-dependent tyrosine
phosphorylation of these proteins (Fig. 7b), consistent with
involvement of the NADPH oxidase in IL-1
-induced protein tyrosine
phosphorylation. In addition, the RelA-PR peptide also decreased
tyrosine phosphorylation of these proteins (Fig. 7c), suggesting that localization of the oxidase to a RelA complex is
necessary for the targeting of oxidants to IL-1 receptor-linked proteins.
View larger version (43K):
[in a new window]
Fig. 7.
IL-1 increases
tyrosine phosphorylation of IL-1RI-associated proteins. HUVEC were
treated with 2 ng/ml IL-1
for the indicated times, and IL-1RI was
immunoprecipitated. The immunoblot is with anti-phosphotyrosine.
Prominent and rapid tyrosine phosphorylation of multiple proteins
occurred (a). Expression of p67(V204A) (DN-p67,
b) or RelA-PR (c) decreased IL-1
-induced
tyrosine phosphorylation of IL-1R1-associated proteins. The results are
representative of three experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B by
phorbol ester (39). Thus, association of p47phox with RelA in
such focal areas may reflect the ability of the cytoskeleton to
function as a dynamic scaffold for signaling complexes.
B consensus promoter by IL-1
, suggesting a
functional significance for this association. Several observations
suggest that this enhancement of NF-
B activity was not a nonspecific effect of p47phox overexpression. First, the anticipated
nonspecific effect of p47phox binding would be to sequester
RelA or sterically hinder binding of transcription complexes and
therefore decrease, rather than increase, NF-
B activation. Second,
p47-related enhancement of NF-
B was specific for IL-1
and was not
seen in unstimulated cells or cells stimulated with tumor necrosis
factor (not shown). Third, p47phox overexpression specifically
increased IL-1
-induced RelA phosphorylation and activation but not
nuclear importation or I-
B
degradation. Thus, p47phox
binding also did not appear to simply displace I-
B
from RelA, consistent with the physically separate binding regions of RelA for
I-
B
and p47phox.
B activation requires signaling through both the well described pathway leading to I-
B
degradation and translocation of NF-
B elements into the nucleus and,
in addition, a distinct pathway resulting in RelA phosphorylation and
activation. For instance, phosphorylation of RelA on Ser529
of its C-terminal transactivation domain increases its transactivation activity but not its nuclear translocation (40), and genetic ablation
of either TRAF2-associated kinase (T2K) or GSK-3
decreases IL-1-induced NF-
B activation but not p65 translocation or I-
B degradation in vivo (41, 42). Of note, both IL-1
and
H2O2 increase RelA phosphorylation (43, 44).
Activation of RelA appears to involve both phosphatidylinositol
3-kinase and Akt, and interventions that block these two kinases
selectively decrease RelA phosphorylation and activation but not
translocation (45-48). Importantly, Rac1, another component of the
NADPH oxidase, also appears necessary for IL-1
-induced RelA
activation but not translocation (49, 50).
B activation, we decreased IL-1
-induced transactivation of the highly NF-
B-dependent E-selectin promoter through
overexpression of the RelA p47-binding domain. This experiment did not
involve overexpression of p47phox, again suggesting specificity
of the preceding studies. This latter domain (RelA-PR) appears to be a
proline-rich region with no other presently defined function.
Therefore, RelA-PR overexpression would not be expected to antagonize
homo- or heterodimerization through the N-terminal Rel homology domain
nor to interfere with the transactivation function of the C-terminal
transactivation domains. Indeed, this RelA mid-region in isolation does
not possess basal RelA transactivation squelching activity
(32).
B-dependent promoter transactivation by IL-1
,
inferring that p47phox may act through its known function as a
constituent of the NADPH oxidase. This result was unexpected, because
the p47phox SH3a domain, which appears to increase the avidity
of p47phox for RelA, is traditionally thought to act instead as
a ligand for the C-terminal polyproline motif of p22phox, thus
promoting functional oxidase assembly (28, 36). Several models may
explain this apparent discrepancy. First, a recent study provides
convincing evidence that critical assembly of the oxidase involves
non-SH3 regions of p47phox interacting with a cytosolic loop of
p22phox (residues 51-63) remote from its polyproline motif
(51), allowing the possibility for p47-SH3a to interact simultaneously
with another protein. Second, because p47-SH3b also binds RelA, it is
possible that this binding site tethers RelA to the oxidase or that
RelA is "traded" from SH3a to SH3b upon docking with the oxidase.
The functional significance of p47-SH3b has not been clearly
identified, because it appears to be relatively unimportant for
O
pathway. Indeed, protein tyrosine
phosphorylation events are necessary for NF-
B activation but not
nuclear translocation (53). Consistent with this hypothesis, we found
that IL-1
rapidly increased tyrosine phosphorylation of a number of
IL-1RI-associated proteins, and this phosphorylation was decreased by
suppression of NADPH oxidase activity (p67(V204A)) or delocalization of
the oxidase (RelA-PR). IL-1
has previously been shown to increase
tyrosine phosphorylation of the IL-1RI itself in Saos2 cells (54),
although we did not find this effect in endothelial cells. In this
latter study, however, tyrosine phosphorylation was essential for
phosphatidylinositol 3-kinase recruitment and activation by IL-1
,
suggesting a potential link between oxidants and the
phosphatidylinositol 3-kinase/Akt/RelA phosphorylation pathway. Taken
together, our data suggest that the oxidase may be tethered in to a
RelA-containing complex through interactions with p47phox, to
direct oxidant production to susceptible proteins acting upstream
of RelA phosphorylation.
![]() |
ACKNOWLEDGEMENT |
---|
We acknowledge the expert technical assistance of Ginny Poffenberger.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants R01-HL61897 and R01-HL67256 and by the Medical Research Service of the Department of Veterans Affairs and the Robert Wood Johnson Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dallas VAMC, MC
151, 4500 S. Lancaster Rd., Dallas, TX 75216. Tel.:
214-857-0753; Fax: 214-857-0340; E-mail:
lance.terada@med.va.gov.
Published, JBC Papers in Press, March 4, 2003, DOI 10.1074/jbc.M210314200
2 R. F. Wu, Y. Gu, Y. C. Xu, F. E. Nwariaku, and L. S. Terada, manuscript submitted.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: IL, interleukin; IL-1RI, type I IL-1 receptor; HUVEC, human umbilical vein endothelial cell; TK, thymidine kinase; MnTBAP, Mn(III) tetrakis(4-benzoic acid)porphyrin; PX, phox; GST, glutathione S-transferase; GFP, green fluorescent protein; PBS, phosphate-buffered saline; DPI, diphenylene iodonium; DCF, 2',7'-dichlorofluorescin diacetate; Ad, adenovirus.
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