Originally published In Press as doi:10.1074/jbc.M001051200 on May 4, 2000
J. Biol. Chem., Vol. 275, Issue 32, 24907-24914, August 11, 2000
Activation of NF-
B by Bradykinin through a G
q-
and G
-dependent Pathway That Involves
Phosphoinositide 3-Kinase and Akt*
Ping
Xie
,
Darren D.
Browning§,
Nissim
Hay¶,
Nigel
Mackman
, and
Richard D.
Ye**
From the Departments of Pharmacology and
¶ Molecular Genetics, College of Medicine, University of Illinois,
Chicago, Illinois 60612 and the Department of Immunology, The
Scripps Research Institute, La Jolla, California 92037
Received for publication, February 8, 2000, and in revised form, April 24, 2000
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ABSTRACT |
Recent work has suggested a role for the
serine/threonine kinase Akt and I
B kinases (IKKs) in nuclear factor
(NF)-B activation. In this study, the involvement of these
components in NF-B activation through a G protein-coupled pathway
was examined using transfected HeLa cells that express the B2-type
bradykinin (BK) receptor. The function of IKK2, and to a lesser extent,
IKK1, was suggested by BK-induced activation of their kinase activities
and by the ability of their dominant negative mutants to inhibit
BK-induced NF-B activation. BK-induced NF-B activation and IKK2
activity were markedly inhibited by RGS3T, a regulator of G protein
signaling that inhibits G
q, and by two G
scavengers. Co-expression of Gq potentiated BK-induced
NF-B activation, whereas co-expression of either an activated
Gq(Q209L) or G12 induced
IKK2 activity and NF-B activation without BK stimulation. BK-induced
NF-B activation was partially blocked by LY294002 and by a dominant negative mutant of phosphoinositide 3-kinase (PI3K), suggesting that
PI3K is a downstream effector of Gq and
G12 for NF-B activation. Furthermore,
BK could activate the PI3K downstream kinase Akt, whereas a
catalytically inactive mutant of Akt inhibited BK-induced NF-B
activation. Taken together, these findings suggest that BK utilizes a
signaling pathway that involves Gq,
G12, PI3K, Akt, and IKK for NF-B activation.
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INTRODUCTION |
G protein-coupled receptors
(GPCRs),1 characterized by
their heptahelical structure, constitute a large family of cell surface receptors (1). These receptors are activated by a diverse array of
external stimuli including hormones, neurotransmitters, sensory stimuli, chemoattractants and growth factors. GPCRs transduce signals
through coupling to a collection of heterotrimeric G proteins, thus
generating a broad spectrum of physiological responses (2). Increasing
evidence indicates that GPCRs actively regulate transcription and gene
expression events (3). The signaling pathways connecting GPCRs to
several kinase cascades, including the mitogen-activated protein
kinases ERK1 and ERK2, p38, stress-activated protein kinase, and the
c-Jun kinase, have recently been elucidated. Activation of these
kinases leads to the expression of c-fos and
c-jun, components of the transcription factor activated
protein-1 (AP-1), as well as other transcription factors such as MEF
and ATF2 (4). An increasing number of GPCRs also have been shown to
activate nuclear factor (NF)-B, thereby regulating the expression of
a wide array of inducible genes. GPCRs that have been identified for
their NF-B-activating functions respond to a variety of agonists,
including leukocyte chemoattractants (5-9), thrombin (10-12),
substance P (13), endothelin (14), 5-hydroxytryptamine (15),
lysophosphatidic acid (16), and bradykinin (17). These findings suggest
the presence of multiple signaling pathways for NF-B activation by GPCRs.
NF-B is a dimeric, ubiquitously expressed transcription factor that
plays a critical role in regulating inducible gene expression in immune
and inflammatory responses (18, 19). The target genes that are
regulated by NF-B include cytokines, chemokines, cell adhesion
molecules, growth factors, and immunoreceptors (20). In most cells,
NF-B proteins exist in the cytoplasm in an inactive complex bound to
the IB family of inhibitory proteins. Various stimuli can induce
rapid phosphorylation, ubiquitinylation, and degradation of IB,
resulting in nuclear translocation of NF-B proteins and
transcription activation. A key regulatory step in this pathway is the
activation of a high molecular weight IB kinase (IKK) complex, in
which catalysis is believed to be carried out by multiple kinases
including IKK1 (IKK) and IKK2 (IKK) (19). Much effort has been
made in understanding the signal transduction pathways that regulate
NF-B activation in response to proinflammatory cytokines such as
tumor necrosis factor (TNF) and interleukin-1 (IL-1).
NF-B activation by these inflammatory cytokines is initiated by the
intracellular signaling molecules TNF receptor-associated factors
(TRAF2 and TRAF6), and may involve transforming growth factor
-activated kinase 1 (TAK1), NF-B-inducing kinase (NIK), and IKKs
(19, 20). More recent studies suggest an additional NF-B activation
pathway consisting of the phosphoinositide 3-kinases (PI3K) and its
downstream kinase Akt (also termed protein kinase B, or PKB) (21-23).
These findings demonstrate the presence of parallel signaling pathways
that converge at the point of IKK activation.
The GPCRs known to activate NF-B couple to different G proteins,
including Gq, G13, Gi, and
G16. However, a detailed mechanism underlying
GPCR-induced NF-B activation, including the relative
contribution of G and G proteins and their downstream effectors, has not been delineated. The B2-type bradykinin receptor (B2BKR) is known to couple to multiple G proteins (24, 25) and has
been reported to activate NF-B (17). This study employs B2BKR in a
transient transfection model to investigate the specific involvement of
G and G proteins in NF-B activation. In addition, as a
preliminary effort to delineate the signaling pathways downstream of
heterotrimeric G proteins, we demonstrate that stimulation of NF-B
by this receptor is primarily mediated by IKK2 in a process that
involves PI3K and Akt.
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EXPERIMENTAL PROCEDURES |
Materials--
Bradykinin was purchased from Sigma (St. Louis,
MO). Pertussis toxin (PTX) was from List Laboratories (Campbell, CA).
LY294002 was obtained from CalBiochem (San Diego, CA). Reagents for
luciferase assays and the plasmid pCMV were purchased from Promega
(Madison, WI). A luciferase reporter plasmid was constructed by
ligation of three B binding sequences (5'-AGGGGACTTTCCCA-3') in
tandem into the pGL2-Luc vector (Promega) upstream from the firefly
luciferase cDNA. The green fluorescent protein (GFP) expression
vector EGFP-N1 was from CLONTECH (Palo Alto, CA).
Plasmids containing cDNA inserts for wild-type and constitutively
activated G proteins were gifts from Drs. Cindy Knall and Gary
Johnson (National Jewish Center, Denver, CO), and their
characterization was detailed previously (26). The G1
and G2 constructs were gifts from Dr. Tatyana Voyno-Yasenetskaya (University of Illinois, Chicago). Expression plasmids for RGS3 and RGS3T were kindly provided by Dr. Nickolai Dulin
(University of Illinois, Chicago) and were described previously (27).
The expression vectors of G scavengers, bovine transducin, and
T8ARK-myc with a ARK carboxyl-terminal fragment were kindly provided by Dr. Heidi Hamm (Northwestern University, Chicago) and Dr.
Sivio Gutkind (National Institutes of Health, Bethesda, MD),
respectively. The expression vector of the dominant negative mutant of
p85 (
p85) was prepared as described previously (28). The constructs
of wild-type, constitutively activated, and kinase-deficient mutants of
Akt/PKB were described elsewhere (29). The expression vector of a
dominant negative IB (IBm) was a gift from Dr. Inder Verma
(The Salk Institute, La Jolla, CA). Preparation of the wild-type and
dominant negative mutants of IKK1 and IKK2 in an expression vector were
described previously (30, 31). The human B2BKR expression construct was
prepared by polymerase chain reaction cloning of the entire coding
sequence into the pRK5 expression vector (PharMingen, San Diego, CA).
Cell Culture, Transfection, and Luciferase Assay--
HeLa cells
were maintained in Dulbecco's modified Eagle's medium containing 10%
heat-inactivated fetal bovine serum, 2 mM L-glutamine, 100 international units/ml penicillin, and 50 µg/ml streptomycin. One day prior to transfection, the cells were
seeded in 6-well cell culture plates to give a final density of
40-60% confluency (~5 × 105 cells/well). Cells
were transfected with the B-luciferase reporter, EGFP-N1, B2BKR
expression vector, and other expression constructs as indicated in the
text and figure legends. Transient transfection was performed using
LipofectAMINE Plus reagent (Life Technologies, Inc.), with 0.2 µg of
each DNA construct except as indicated in the figure legends. When
necessary, additional DNA (pCMV) was added to make total DNA of 1 µg/well. 24 h after transfection, cells were starved in
serum-free medium for 16 h and then stimulated with control
solvent, 10 nM BK, 50 ng/ml TNF, or 40 ng/ml IL-1 for
5 h. The B-directed expression of firefly luciferase was determined using luciferase assay reagents from Promega, and the resultant luciferase activities were measured with a Femtomaster FB12
luminometer (Berthold Detection Systems, Pforzheim, Germany). Relative
transfection efficiency and protein expression levels in each sample
were determined by measurement of fluorescent intensity of the
co-transfected GFP in a spectrofluorometer (Photon Technologies International, Monmouth Junction, NJ), with excitation and emission wavelengths of 488 and 507 nm, respectively. Luciferase activities were
then normalized against the level of GFP expression to minimize differences in transfection and protein expression efficiency among
samples. Cell surface expression of B2BKR was detected by flow
cytometry (Becton Dickinson, San Jose, CA) using fluorescein isothiocyanate-labeled BK (10 min at 4 °C; NEN Life Science
Products) and by immunoblotting (see below). B2BKR expression levels
were not altered by co-transfection of the various constructs used in
this study. Unless otherwise indicated in the figure legends, all data
were collected from three independent experiments, each in duplicate.
Normalized data were plotted using Prism software (Version 2.0;
GraphPad, San Diego, CA).
Protein Extraction and Immunoprecipitation--
HeLa cells were
grown in 100-mm cell culture dishes. Transient transfection, culture,
serum starvation, and agonist stimulation were done as described above.
Cells were then rinsed once with ice-cold phosphate-buffered saline and
incubated for 20 min at 4 °C in 1 ml of lysis buffer containing 1%
Nonidet P-40, 50 mM HEPES (pH 7.6), 100 mM
NaCl, 10% glycerol, 1 mM EDTA, 20 mM
-glycerophosphate, 20 mM
p-nitrophenylphosphate, 1 mM sodium
orthovanadate, 1 mM NaF, and 1× Protease Inhibitor Mixture
Set I (CalBiochem). Cellular debris was removed by centrifugation at
14,000 × g for 20 min. Cell lysates were incubated for
3 h at 4 °C with 30 µl of anti-HA-agarose beads (Santa Cruz
Biotechnology, Santa Cruz, CA) or anti-FLAG-agarose beads (Sigma), then
rinsed 3 times with 0.5 ml of lysis buffer, and eluted with 30 µl of
HA peptide (Santa Cruz) or FLAG peptide (Sigma). Aliquots of the
eluates were used for immunoblot and in vitro
phosphorylation assays.
Immunoblot Analysis and Kinase Activity Assays--
Immunoblot
analysis was performed with anti-HA monoclonal antibody (Santa Cruz),
anti-FLAG monoclonal antibody (Sigma), or anti-phospho-Akt polyclonal
antibody (Ser473; New England Biolabs, Beverly, MA). The antibodies
were visualized with horseradish peroxidase-conjugated goat anti-mouse
or anti-rabbit Ig (CalBiochem) using enhanced chemiluminescence
(Pierce). Quantitative measurement of phosphorylated Akt was conducted
using ImageQuant software (Molecular Dynamics, Mountain View, CA). For
the detection of protein expression of the transfected B2BKR and
IBm, total cell lysates were immunoblotted with an anti-B2BKR
monoclonal antibody (Transduction Laboratories, Lexington, KY) and an
anti-IB antibody (New England Biolabs), respectively.
For IB kinase assays, HeLa cells in 100-mm dish were transiently
transfected with 4 µg of B2BKR and 1 µg of HA-tagged IKK1 or
FLAG-tagged IKK2. Cell culture and serum starvation conditions were the
same as in the luciferase assays. Cells were then stimulated with BK or
TNF and lysed. IKKs were immunoprecipitated with anti-HA- or
anti-FLAG-agarose beads and then eluted with HA or FLAG peptide, respectively. The IKK activity assay was performed with 3 µl of eluates and 2 µg of GST-IB (1-54) in 15 µl of kinase buffer containing 20 mM Tris-HCl (pH 7.6), 20 mM
MgCl2, 1 mM EDTA, 20 mM
-glycerophosphate, 20 mM
p-nitrophenylphosphate, 1 mM sodium orthovanadate, 0.4 mM phenylmethylsulfonyl fluoride, 1 mM ATP, 20 mM creatine phosphate, 1× Protease
Inhibitor Mixture Set I (CalBiochem), and 5 µCi of
[-32P]ATP (10 mCi/ml, 6,000 Ci/mmol; Amersham
Pharmacia Biotech), at 37 °C for 30 min. Samples were subsequently
analyzed by 10% SDS-polyacrylamide gel electrophoresis and
autoradiography. The expression vector for GST-IB (1-54) was
constructed, and the proteins were expressed in Escherichia
coli as described previously (32).
For the Akt kinase assay, HeLa cells in 100-mm dishes were transfected
with 4 µg of B2BKR and 1 µg of HA-tagged Akt, cultured and
serum-starved as described above, and stimulated with 10 nM BK or 50 ng/ml TNF for indicated time periods. Cells were lysed, and
Akt was immunoprecipitated with anti-HA-agarose beads and then eluted
with HA peptide. Akt kinase activity assay was performed with 15 µl
of eluates and 0.4 µg of Akt-specific substrate peptide (RPRAATF,
Upstate Biotechnology, Lake Placid, NY) in 15 µl of 2× kinase buffer
at 37 °C for 20 min. Samples were subsequently transferred to P81
phosphocellulose squares (Upstate Biotechnology), rinsed 10 times with
50 ml of 0.75% phosphoric acid, dehydrated in acetone, and dissolved
in 5 ml of scintillation fluid. The resultant radioactivity was
determined with a Beckman LS 3801 scintillation counter.
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RESULTS |
The Function of IKK1 and IKK2 in BK-induced NF-B
Activation--
HeLa cells were transiently transfected with a
B-directed luciferase reporter and an expression vector containing
the human B2BKR cDNA. Mock (vector)-transfected cells did not
respond to BK (up to 100 nM) in luciferase reporter assay
(Fig. 1A) because the
untransfected HeLa cells do not express B2BKR (Fig. 1C and data not shown). In B2BKR-transfected cells, BK induced B-directed luciferase activities in a dose-dependent manner from 0.1 to 500 nM (Fig. 1A). To determine whether
BK-induced NF-B activation requires IB phosphorylation, a
dominant negative mouse IB (IBm) devoid of inducible
phosphorylation (33) was co-expressed in the HeLa cells. IBm
abolished BK-induced NF-B activation as well as that induced by
TNF and IL-1 (Fig. 1B). Expression of B2BKR was not
affected by the co-transfection of IBm, as evidenced by Western
blot analysis (Fig. 1C) and flow cytometry (data not shown).
These results indicate that phosphorylation of IB at
Ser32 and Ser36 is critical for BK-induced
NF-B activation.
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Fig. 1.
Induction of
B-directed luciferase expression by BK in
transfected HeLa cells. HeLa cells were transiently transfected
with constructs encoding a B-driven luciferase reporter and B2BKR.
24 h after transfection, cells were starved in serum-free medium
for 16 h and stimulated with BK for 5 h. A,
induction of B-directed luciferase activities by BK at various
concentrations (conc.). No induction was seen in mock
(vector without B2BKR)-transfected cells. B, inhibition of
the induced luciferase activity by IBm, a mouse IB protein
devoid of inducible phosphorylation. The cells were transfected without
(CTL) or with IBm (0.2 µg) and stimulated with BK
(10 nM), TNF (50 ng/ml), or IL-1 (40 ng/ml) for
5 h prior to luciferase assay. Relative luciferase activities
(RLA) are shown as fold induction. Data shown are the means
and S.D. from two experiments, each with duplicate measurements.
C, expression of B2BKR and IBm in the transfected
cells. Equal amounts of cell lysates were immunoblotted with antibodies
against B2BKR (top) and IB (bottom). The
bands of transfected B2BKR, endogenous IB, and transfected
IBm (migrating slightly faster on SDS-polyacrylamide gel
electrophoresis) are indicated.
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Receptors for several cytokines and lipopolysaccharide preferentially
utilize IKK2 for NF-B activation (30, 31). We examined whether this
is also the case for BK-induced NF-B activation. The K44M mutants of
IKK1 and IKK2, which had been shown to block cytokine-induced NF-B
activation, were co-expressed in the transfected HeLa cells. As shown
in Fig. 2A, expression of
IKK1.DN reduced BK-stimulated NF-B activation by approximately 25%.
A more potent inhibition (~60%) was obtained when IKK2.DN was
co-transfected into the HeLa cells. When used together, these two
dominant negative constructs produced further inhibition of BK-induced
NF-B activation. The inhibition of BK-induced NF-B activation was
not due to the reduced expression of B2BKR, because co-transfection of
IKK1.DN or IKK2.DN did not affect the expression of B2BKR (Fig.
2B). Similar levels of inhibition were observed in the
control cells that were stimulated with TNF, suggesting that BK,
like TNF, preferentially activates IKK2. This notion was further
supported by IKK assays that employed GST-IB (1-54) as a
substrate. Results shown in Fig. 2, C and D,
indicate that, although BK activated both IKK1 and IKK2 with peak
activation at 15 min, the IKK1 activity was transient, whereas the IKK2
activity was more sustained. These results combined suggest an
important role of IKK2 in BK-induced NF-B activation. IKK2 assay was
used in subsequent experiments as an additional measure for G
protein-mediated functions.
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Fig. 2.
Involvement of IKKs in BK-induced
NF-B activation. A, inhibition
of NF-B activation by dominant negative (DN) IKKs. The
K44M mutants of IKK1 and IKK2 (0.2 µg each) were co-transfected into
the HeLa cells, which were then stimulated with BK (10 nM)
or TNF (50 ng/ml). The relative luciferase activities
(RLA) in control cells (without DN IKKs) were set as 100%.
B, expression of B2BKR, IKK1.DN (HA-tagged), and IKK2.DN
(FLAG-tagged), as detected by immunoblotting. Experimental conditions
were the same as in Fig. 1C and are described under
"Experimental Procedures." C and
D, activation of IKK1 (C) and IKK2 (D)
by BK measured in IKK assays using a GST-IB-(1-54) construct as
substrate. IKKs were immunoprecipitated with anti-HA (C)- or
anti-FLAG (D)-agarose beads and then eluted with HA or FLAG
peptide, respectively. Aliquots of the eluates were used for
immunoblotting and kinase activity assays. The 32P-labeled
(top) and Coomassie Blue-stained (middle)
GST-IB-(1-54) substrates as well as immunoblot of IKK1.WT or
IKK2.WT (bottom) are shown. WT, wild type.
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Gq and G Mediate BK-induced Activation of IKK2
and NF-B--
B2BKR couples to heterotrimeric G proteins of the
Gi, Gq/11, and G13 classes
(24, 25). In addition, BK may also couple to Gs under
certain circumstances (34). To determine which G protein(s) is
responsible for BK-induced NF-B activation, the transfected HeLa
cells were first treated with PTX. Our results showed that PTX, at
concentrations sufficient to block fMet-Leu-Phe induced calcium
mobilization and phosphatidylinositol 4,5-bisphosphate hydrolysis (data
not shown), did not alter NF-B activation by BK, TNF, or IL-1
(Fig. 3A). Consistent with
this finding, PTX did not affect IKK2 activation induced by either BK
or TNF (Fig. 3B). We hypothesized that PTX-insensitive
G proteins are responsible for BK-induced NF-B activation. To
evaluate this hypothesis, the wild-type Gq,
G13, and Gi2 were separately co-expressed in HeLa cells. In the absence of BK stimulation, these G proteins produced small increases in B-directed luciferase activity (Fig. 4A). Following BK stimulation,
the cells expressing Gq exhibited an ~75% increase
over the level of luciferase activity induced by BK alone, whereas a
smaller (~20%) increase was observed in the cells expressing
G13. In contrast, Gi2 reduced the level of BK-stimulated luciferase activity by ~20%.
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Fig. 3.
Effect of PTX on BK-induced
NF-B activation. HeLa cells were
transiently transfected as described in Fig. 1. 24 h after
transfection, cells were treated with control solvent (CTL)
or with 100 ng/ml PTX in serum-free medium for 16 h. The cells
were then stimulated with control solvent ( ), 10 nM BK,
50 ng/ml TNF, or 40 ng/ml IL-1 for 5 h prior to luciferase
assay (A). PTX was present in the medium during stimulation.
The relative luciferase activities (RLA) were measured as
fold induction. B, IKK2 assay. Cells were transfected and
treated as above, except that stimulation was carried out for 15 min
(with 10 nM BK) and 10 min (with 50 ng/ml TNF). The
expressed IKK2 was immunoprecipitated from cell lysate, eluted with
FLAG peptide, and used for immunoblotting and kinase activity assays.
The 32P-labeled (top) and Coomassie Blue-stained
(middle) substrates as well as the immunoblot of IKK2.WT
(bottom) are shown. WT, wild type.
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Fig. 4.
A role of
Gq in BK-induced
NF-B activation. A,
Gq potentiates BK-induced NF-B activation in
luciferase assays. HeLa cells were transiently transfected with 0.2 µg each of the expression plasmids for Gq,
G13, and Gi2. After culture and serum
starvation, the cells were stimulated with control solvent
(CTL) or 5 nM BK for 5 h prior to
luciferase assay. Relative luciferase activities (RLA)
presented are the means and S.D. from two independent experiments, each
with duplicate measurements. B, HeLa cells were transfected
as above, without (open bars) or with (solid
bars) a RGS3T expression construct (0.2 µg). The effects of
RGS3T on the luciferase activities induced by BK (10 nM),
TNF (50 ng/ml), and IL-1 (40 ng/ml) were measured and expressed
as fold induction. C, the effect of RGS3T on IKK2 kinase
activity in cells stimulated with BK (10 nM, 15 min) or
TNF (50 ng/ml, 10 min). Various concentrations of RGS3T expression
plasmid were used as indicated. GST-IB-(1-54) was used as a
substrate in the kinase activity assay. The 32P-labeled
(top) and Coomassie Blue-stained (middle)
substrates as well as the immunoblot of wild-type IKK2
(IKK2.WT, bottom) are shown.
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Previous studies have shown that G12 and
G13 activate serum responsive factor through the
downstream effector RhoA (35-37). G13 presumably can
also activate NF-B through this mechanism, since GTPases of the Rho
family have been implicated in NF-B activation (38, 39). In this
study we focused on Gq because it had not been
previously shown to stimulate NF-B activation and because expression
of Gq produced a potent enhancement of the BK-induced
NF-B activation (Fig. 3A). To further examine the role of
Gq, HeLa cells were co-transfected with expression vectors encoding RGS3, a regulator of G protein signaling, and its
shortened form, RGS3T (27, 40). RGS proteins can function as
GTPase-activating proteins (GAP), which facilitate the conversion of
G·GTP to G·GDP and thereby negatively regulate heterotrimeric G protein activation (41, 42). RGS3T has been shown to be a more potent
inhibitor of Gq than RGS3 (40). Our results demonstrated that co-expression of RGS3T markedly reduced BK-stimulated luciferase activity (Fig. 4B). In contrast, the luciferase activities
induced by TNF and IL-1 were only slightly affected by RGS3T
(Fig. 4B). Although less potent, RGS3 also inhibited
BK-induced luciferase activity by ~60% (data not shown). Consistent
with these findings, the BK-induced IKK2 activity was inhibited by the
co-expressed RGS3T in a dose-dependent manner (Fig.
4C). The lack of inhibition of TNF-induced IKK2 activity
indicates the specificity of RGS3T in G protein-mediated NF-B
activation (Fig. 4C).
Inhibition of Gq by RGS3T favors accumulation of
Gq·GDP, an inactivated form of Gq that complexes
with G proteins and limits their functions (41). Thus the
observed inhibitory effect of RGS3T and the potentiation effect of
Gq in the transfected HeLa cells may be a function of
Gq, G, or both. That G proteins play a
direct role in NF-B activation was suggested by the co-expression of
transducin or a carboxyl-terminal fragment of ARK (T8ARK) (43) in
the transfected cells. These scavengers reduced BK-stimulated luciferase activity by approximately 65%, while having little effect
on NF-B activation by TNF and IL-1 (Fig.
5, A and B). In
agreement with the luciferase reporter data, transducin (Fig. 5C) and T8ARK (not shown) also reduced BK-stimulated IKK2
activity without affecting TNF-induced IKK2 activity.
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Fig. 5.
Inhibition of BK-induced
NF-B activation by
G scavengers. HeLa cells
were transfected as described in Fig. 1, without (CTL) or
with expression plasmids encoding bovine transducin (A) or
T8ARK-myc (B). After 16 h of serum starvation, the
cells were stimulated with control solvent (), BK (10 nM), TNF (50 ng/ml), or IL-1 (40 ng/ml) for 5 h
prior to luciferase assay. The B-directed relative luciferase
activities (RLA) are shown as fold induction. C,
inhibition of BK-induced IKK2 activity by transducin. HeLa cells in
100-mm dishes were transfected as described in the legend of Fig. 2. In
some samples, an expression plasmid for transducin was included at
various DNA concentrations as indicated. IKK2 activity was assayed
following stimulation with BK (10 nM, 15 min) or TNF (50 ng/ml, 10 min). The 32P-labeled (top) and
Coomassie Blue-stained (middle) kinase substrates as well as
the immunoblot of wild-type IKK2 (IKK2.WT,
bottom) are shown.
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The direct involvement of Gq and G in NF-B
activation was investigated by expression of
G12 and a Gq mutant
(Q209L) devoid of GTPase activity in the HeLa cells. Expression of
Gq(Q209L) alone resulted in a dramatic increase in
B-directed luciferase activity without BK stimulation (Fig.
6A). Similarly, an ~17-fold increase in luciferase activity was observed when
G12 was co-expressed in the HeLa cells.
Stimulation of NF-B activation by Gq(Q209L) and
G12 proteins was reflected at the level
of IKK activation, as both proteins stimulated the kinase activity of
IKK2 (Fig. 6B). These findings suggest that both
Gq and G12 have a direct function in BK-induced activation of IKK2 and NF-B.
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Fig. 6.
Activation of NF-B
by Gq(Q209L) and
G12.
A, induction of B-directed luciferase activity in cells
transfected with an activated Gq(Q209L), and with
G12. After culture and serum starvation,
luciferase reporter assay was conducted without BK stimulation. In some
samples, the dominant negative IKK2 (IKK2.DN) construct was
co-transfected. B, activation of IKK2 by
Gq(Q209L) and G12. HeLa
cells were transiently transfected with 2 µg of B2BKR or 1 µg of
FLAG-tagged wild-tupe IKK2 (IKK2.WT) in the absence () or
presence (+) of 2 µg of Gq(Q209L) or
G12. IKK2 assay was conducted following
BK stimulation (5 nM for 15 min). The
32P-labeled (top) and Coomassie Blue-stained
(middle) substrates as well as immunoblot of IKK2.WT
(bottom) are shown.
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PI3K and Akt Are Involved in BK-induced NF-B Activation
Downstream of Gq and G--
PI3K is known to be
an effector of heterotrimeric G proteins (44, 45). Recent studies
suggest that PI3K and its downstream serine/threonine kinase Akt play
an important role in NF-B activation by TNF and platelet-derived
growth factor (23, 46). We examined activated PI3K and Akt constructs
for their functions in NF-B activation in the transfected HeLa
cells. Expression of a myristoylated p110 protein (p110.myr) induced an
~20-fold increase of B-directed luciferase activity (Fig.
7A). A much smaller increase
was seen in cells expressing a myristoylated Akt (Akt.myr), but Akt.myr augmented the effect of p110.myr. A partial inhibition of the induced
B activity was obtained by co-expression of IKK2.DN. Taken together,
these findings suggest that in HeLa cells, PI3K and Akt can positively
regulate NF-B activation.
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Fig. 7.
A role of PI3K in BK-induced
NF-B activation. A, PI3K and
Akt are involved in NF-B activation in HeLa cells. The cells were
transfected as described in Fig. 1, with additional expression plasmids
encoding a myristoylated p110 (p110.myr), a myristoylated
Akt (Akt.myr), or a dominant negative IKK2
(IKK2.DN). The B-directed relative luciferase activities
(RLA) were measured without BK stimulation and expressed as
fold induction. B, inhibition of BK-induced NF-B
activation by LY294002. The transfected HeLa cells were treated with 50 µM LY294002 for 30 min and then stimulated with control
solvent (), BK (10 nM), or TNF (50 ng/ml) for 5 h
prior to luciferase assay. LY294002 was present during agonist
stimulation. C, inhibition of NF-B activation by p85,
a dominant negative p85 protein of PI3K. HeLa cells were transiently
transfected without (CTL) or with p85 and with other
plasmids for the luciferase assays as described above. The cells were
stimulated with control solvent (), BK (10 nM), TNF
(50 ng/ml), or IL-1 (40 ng/ml) for 5 h prior to luciferase
assay. D, inhibition of Gq(Q209L)- or
G12-induced NF-B activation by p85. The
experiment was done similarly to that described in Fig. 7A,
except for the inclusion of Gq(Q209L) and
G12 (0.2 µg each) in the transfection
procedure. The induced luciferase activities were measured in the
absence of BK. Maximal response (100%) was measured in the absence of
p85. Data for all experiments are presented as the means and S.D.
from at least two experiments, each with duplicate measurements.
|
|
To investigate the requirement of PI3K in BK-induced NF-B activation
in transfected HeLa cells, we examined the effect of LY294002, a PI3K
inhibitor (47). Our results demonstrated that both the BK-induced and
TNF-induced NF-B activation were dramatically inhibited by
treatment with 50 µM LY294002 prior to agonist
stimulation (Fig. 7B). Co-expression of a dominant negative
mutant of p85 (p85) (28), a regulatory subunit of PI3K, partially
inhibited NF-B activation induced by BK, TNF, or IL-1 (Fig.
7C). Similarly, the B-directed luciferase activities
induced by expression of Gq(Q209L) and
G12 were susceptible to inhibition by
p85 (Fig. 7D). These data confirm a previous finding that
PI3K is involved in BK-induced NF-B activation (48).
To determine whether Akt is a downstream effector of PI3K in the
transfected HeLa cells, we first measured changes in the phosphorylation level of Akt and its kinase activity in response to BK.
As shown in Fig. 8A, BK
stimulated a rapid Akt phosphorylation that peaked at 15 min. Using an
Akt substrate peptide as substrate, we found that BK also induced the
kinase activity of Akt in a time-dependent manner, with
peak kinase activity observed at 15 min (Fig. 8B). The
kinetics of Akt activation paralleled that of IKK2 activation (Fig. 2).
In both experiments, TNF activated Akt to a lesser extent than did
BK.
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|
Fig. 8.
Involvement of Akt in BK-induced
NF-B activation. In A and
B, HeLa cells transfected with HA-tagged Akt were stimulated
with 10 nM BK or 50 ng/ml TNF for the indicated time
periods. Cells were lysed, and Akt was immunoprecipitated with
anti-HA-agarose beads and then eluted with HA peptide. Aliquots of the
eluates were used in assays for immunoblotting (A, bottom
panels) and quantitation (A, top, bar graph) and for
kinase activity measurement (B). An anti-Phospho-Akt
antibody (Ser473) and an anti-HA antibody were used to
detect the phosphorylated Akt (Akt-P) and total Akt,
respectively (A). C, a kinase-deficient Akt
(Akt.KD) inhibits NF-B activation by BK. HeLa cells were
transfected with the relevant plasmids. 16 h after transfection,
serum was removed from culture medium, and the cells were stimulated
with BK (10 nM). Expression of the transfected B2BKR and
Akt.KD (tagged with HA) were detected by immunoblotting analysis.
CTL, control; conc., concentration. In
D, the cells were transfected with Gq(Q209L)
or G12 (0.2 µg each) with or without
Akt.KD (2 µg). Inhibition by Akt.KD on the induced relative
luciferase activities (RLA) was determined as described
above. Data presented are from three independent experiments, each with
duplicate measurements.
|
|
A kinase-deficient Akt mutant (Akt.KD) has been shown to block the
effect of Akt in the inhibition of cell death (49). We examined whether
this Akt mutant (K179M) also affected the BK-stimulated B activity.
Data shown in Fig. 8C indicate that Akt.KD partially inhibited BK-induced NF-B activation in a dose-dependent
manner, while having no obvious effect on the expression level of
B2BKR. Furthermore, Akt.KD also inhibited the luciferase activities
induced by Gq(Q209L) and by
G12 (Fig. 8D). These findings
combined indicate that Akt is a component of the BK signaling pathway
that leads to NF-B activation. However, Akt.KD was not able to
completely abolish the induced luciferase activity by either BK or the
G proteins, suggesting the presence of other parallel signaling pathways for BK-induced NF-B activation.
| |
DISCUSSION |
The transcription factor NF-B plays a critical role in
regulating inducible gene expression in immune and inflammatory
responses as well as in growth and development (18-20). Substantial
progress has been made in understanding the signal transduction
pathways regulating activation of NF-B in response to
proinflammatory cytokines such as TNF and IL-1. Increasing
evidence indicates that ligands for GPCRs can also activate NF-B;
however, the signaling components involved in this process remain to be
characterized. BK, a pluripotent peptide, has been shown to activate
NF-B, which contributes to its proinflammatory functions in tissue
injury and allergy (17). The present study sought to identify the
signaling components involved in BK-induced NF-B activation in
transfected HeLa cells, which have been widely used for delineation of
the signaling pathways for TNF- and IL-1-mediated NF-B
activation (30, 50, 51).
We first determined the potential involvement of the IB kinases IKK1
and IKK2 in BK-induced NF-B activation. IKKs have been established
as a point of convergence for most proinflammatory cytokines that
activate NF-B (20). We found that both IKK1 and IKK2 activities were
induced by BK in a time-dependent manner. In addition,
co-expression of the dominant negative mutants of IKK1 and IKK2
inhibited BK-induced NF-B activation. Our results, together with a
recently published study (52), provide direct evidence for the
involvement of IKKs in GPCR-mediated NF-B activation. Notably, IKK1
activation by BK was transient, while IKK2 activation was sustained. In
addition, IKK2.DN exhibited a more pronounced inhibitory effects on
BK-induced NF-B activation than IKK1.DN. Our observations are
consistent with data obtained from gene targeting studies that suggest
IKK2, but not IKK1, plays a major role in the induction of NF-B
activity in response to inflammatory stimuli (53-56).
A large number of GPCRs, including B2BKR, couple to multiple G
proteins. To determine which G protein(s) is responsible for BK-induced
NF-B activation, we performed co-transfection experiments with three
individual G proteins. Expression of Gq potently enhanced BK-induced NF-B activation. Furthermore, expression of an
activated Gq stimulated IKK2 activity as well as NF-B activation in the absence of agonist. These results, combined with the
lack of inhibition by pertussis toxin, suggest Gq as a
primary G protein that couples B2BKR to NF-B activation.
Gq may directly activate one of its downstream effectors
for this function; alternatively, the G proteins released from
Gq following BK stimulation may also activate NF-B.
This latter possibility was supported by our data showing that
expression of G12 strongly stimulated
IKK2 activity and NF-B activation. Because agonist-stimulated activation of GPCRs is accompanied by release of free G proteins, this function of G may be a widely used mechanism for
GPCR-stimulated NF-B activation. However, many GPCRs that generate
free G12 proteins upon activation do not
mediate NF-B activation. Other receptors, such as the fMet-Leu-Phe
receptor and C5a receptor, activate NF-B only in certain cell types
(7, 8). Thus, the G proteins released during the activation of
these receptors are not sufficient to activate NF-B, and additional
signaling components may be required. Alternatively, there may be a
negative regulatory mechanism that prevents NF-B activation by some
GPCRs. Our finding that RGS3T inhibits BK-induced NF-B activation
provides a first line evidence that the RGS proteins can negatively
regulate GPCR-mediated transcription activation.
In addition to Gq and G12,
we have shown that co-expression of G13 slightly
enhanced BK-stimulated NF-B activation. This finding is compatible
with the function of G13 in activating the small GTPase
RhoA (57-60), which has been demonstrated to stimulate NF-B
activation (38, 39). A previous study using A549 lung epithelial cells
demonstrated partial inhibition of BK-induced NF-B activation by a
dominant negative RhoA construct and by the C3 exoenzyme from
Clostridium botulinum (61), suggesting a function for RhoA
in BK-induced NF-B activation. However, the function of
G13 in BK-induced NF-B activation was not
investigated, and a subsequent work demonstrated inhibition of NF-B
activation by pertussis toxin in A549 cells, suggesting the involvement
of Gi (48). Results obtained from the current work do
not support a positive role of Gi in BK-induced NF-B
activation in HeLa cells, although we cannot exclude the possibility
that the G12 proteins released from
Gi mediate NF-B activation in A549 cells.
Our data suggest that PI3K is a downstream effector of
Gq and 12 and that it is
partially responsible for BK-induced NF-B activation in the
transfected HeLa cells. PI3K is activated by many GPCRs, and its
function in NF-B activation has been suggested in recent studies (9,
23, 46, 48). PI3K may stimulate NF-B activation through a downstream
serine/threonine kinase Akt, as shown recently in TNF- and
platelet-derived growth factor-induced NF-B activation (23, 46). Our
results demonstrated that BK could induce Akt phosphorylation and
stimulate its kinase activity. The time course of the induced Akt
activation is compatible with that of NF-B activation in BK
stimulated cells. A kinase-deficient mutant of Akt partially blocked
BK-induced NF-B activation in a dose-dependent manner.
Furthermore, Akt.KD also inhibited NF-B activation by
G12 and by an activated
Gq. Together, these results suggest that Akt is a
downstream effector of PI3K that plays a role in BK-induced NF-B
activation in HeLa cells. Activation of Akt by PI3K may require
additional intermediate kinases, such as
phosphoinositide-dependent kinase 1 (PDK1) (62). Whether these kinases are necessary for BK-induced NF-B activation remains to be investigated.
In summary, the current study provides direct evidence that
GPCR-mediated NF-B activation shares part of the signaling
mechanisms with that for TNF, including the preferential activation
of IKK2 and a recently identified pathway involving PI3K and Akt. This study also demonstrated that both Gq and
G12 proteins actively participate in
BK-induced NF-B activation and that GPCR-mediated NF-B activation
can be regulated by RGS proteins. These signaling mechanisms are not
found in TNF-induced NF-B activation pathway and are therefore
unique to receptors that couple to heterotrimeric G proteins. Taken
together, our results suggest that the BK signaling pathway leading to
NF-B activation involves B2BKR, Gq,
G12, PI3K, Akt, and IKK2. However,
because inhibition of PI3K and Akt only partially blocked, but did not
completely abolish, NF-B activation by BK, Gq, and
G12, the current study does not exclude the presence of other parallel signaling pathways. Identification of
these other signaling components and pathways in future studies will be
necessary for a more complete understanding of how GPCRs activate
NF-B.
| |
ACKNOWLEDGEMENTS |
We are grateful to Cindy Knall, Gary Johnson,
Sivio Gutkind, Dianqing Wu, Inder Verma, Heidi Hamm, Tatyana
Voyno-Yasenetskaya, Nickolai Dulin, David Donner, and Rong He for
kindly providing the DNA constructs used in this study and for helpful
discussions. We also thank Laura Viise and Marisa McShane for technical assistance.
| |
FOOTNOTES |
*
This work was supported by grants from the National
Institutes of Health (AI40176), the Arthritis Foundation, and the
American Heart Association.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.
§
Recipient of a fellowship from the Arthritis Foundation.
**
An Established Investigator of the American Heart Association. To
whom correspondence should be addressed: Dept. of Pharmacology, MC868,
College of Medicine, University of Illinois, 835 So. Wolcott Ave.,
Chicago, IL 60612. E-mail: yer@uic.edu.
Published, JBC Papers in Press, May 4, 2000, DOI 10.1074/jbc.M001051200
| |
ABBREVIATIONS |
The abbreviations used are:
GPCR, G
protein-coupled receptors;
BK, bradykinin;
B2BKR, type 2 receptor for
bradykinin;
G proteins, guanine nucleotide-binding regulatory proteins;
NF-B, nuclear factor-B;
PTX, pertussis toxin;
RGS, regulators of
G protein signaling;
PI3K, phosphoinositide 3-kinases;
IKKs, IB
kinases;
ERK, extracellular signal-regulated kinase;
TNF, tumor
necrosis factor-;
IL-1, interleukin-1;
GFP, green fluorescence
protein;
myr, myristoylated.
| |
REFERENCES |
| 1.
|
Lefkowitz, R. J.
(1996)
Nat. Biotechnol.
14,
283-286
|
| 2.
|
Bourne, H. R.
(1997)
Curr. Opin. Cell Biol.
9,
134-142
|
| 3.
|
Gutkind, J. S.
(1998)
Oncogene
17,
1331-1142
|
| 4.
|
Gutkind, J. S.
(1998)
J. Biol. Chem.
273,
1839-1842
|
| 5.
|
Brach, M. A.,
de Vos, S.,
Arnold, C.,
Gruss, H. J.,
Mertelsmann, R.,
and Herrmann, F.
(1992)
Eur. J. Immunol.
22,
2705-2711
|
| 6.
|
Kravchenko, V. V.,
Pan, Z.,
Han, J.,
Herbert, J. M.,
Ulevitch, R. J.,
and Ye, R. D.
(1995)
J. Biol. Chem.
270,
14928-14934
|
| 7.
|
Browning, D. D.,
Pan, Z. K.,
Prossnitz, E. R.,
and Ye, R. D.
(1997)
J. Biol. Chem.
272,
7995-8001
|
| 8.
|
Hsu, M. H.,
Wang, M.,
Browning, D. D.,
Mukaida, N.,
and Ye, R. D.
(1999)
Blood
93,
3241-3249
|
| 9.
|
Ye, R. D.,
Pan, Z.,
Kravchenko, V.,
Browning, D. D.,
and Prossnitz, E. R.
(1996)
Gene Expr
5,
205-215
|
| 10.
|
Mari, B.,
Imbert, V.,
Belhacene, N.,
Far, D. F.,
Peyron, J. F.,
Pouyssegur, J.,
Van Obberghen-Schilling, E.,
Rossi, B.,
and Auberger, P.
(1994)
J. Biol. Chem.
269,
8517-8523
|
| 11.
|
Maruyama, I.,
Shigeta, K.,
Miyahara, H.,
Nakajima, T.,
Shin, H.,
Ide, S.,
and Kitajima, I.
(1997)
Ann. N. Y. Acad. Sci.
811,
429-436
|
| 12.
|
Rahman, A.,
Anwar, K. N.,
True, A. L.,
and Malik, A. B.
(1999)
J. Immunol.
162,
5466-5476
|
| 13.
|
Lieb, K.,
Fiebich, B. L.,
Berger, M.,
Bauer, J.,
and Schulze-Osthoff, K.
(1997)
J. Immunol.
159,
4952-4958
|
| 14.
|
Gallois, C.,
Habib, A.,
Tao, J.,
Moulin, S.,
Maclouf, J.,
Mallat, A.,
and Lotersztajn, S.
(1998)
J. Biol. Chem.
273,
23183-23190
|
| 15.
|
Cowen, D. S.,
Molinoff, P. B.,
and Manning, D. R.
(1997)
Mol. Pharmacol.
52,
221-226
|
| 16.
|
Shahrestanifar, M.,
Fan, X.,
and Manning, D. R.
(1999)
J. Biol. Chem.
274,
3828-3833 |
TOP
ABSTRACT
INTRODUCTION
