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INTRODUCTION |
Human monocytes can be induced to express a variety of genes
involved in immune and inflammatory responses and cell adhesion. Following stimulation with microbial products like lipopolysaccharide (LPS),1 monocytes secrete
and/or express several inflammatory factors such as TNF
, IL-12,
IL-1, IL-6, nitric oxide, granulocyte-macrophage colony-stimulating
factor, chemokines such as IL-8, RANTES (regulated on activation normal
T cell expressed), macrophage chemotactic protein-1 and macrophage
inflammatory protein-1/
, the adhesion molecule ICAM-1, and the
stimulatory complex for T cells, i.e. B7 and MHC class II molecules.
The pleiotropic transcription factor NF-
B plays an important role in
the transcriptional regulation of all these genes (reviewed in Ref. 1).
NF-B occurs in both homo- and heterodimeric forms. The most common
transcriptionally active form is a p50/p65 heterodimer (reviewed in
Ref. 2). In unstimulated cells, NF-B is localized in the cytosol
bound to inhibitor proteins, collectively termed IB. Stimulation
results in IB phosphorylation, ubiquitination, and proteosomal
degradation, followed by the rapid translocation of NF-B to the
nucleus where it binds to specific B elements within promoters (1,
2).
Several studies have shown that the transactivating activity of NF-B
requires DNA binding and interaction with coactivators that bridge
various transcriptional activators and components of the basal
transcriptional machinery. The CREB-binding protein (CBP) is a
ubiquitously expressed nuclear coactivator present in limiting amounts
(reviewed in Ref. 3). A diverse and increasing number of transcription
factors and some elements of the basal transcriptional machinery are
able to form stable physical complexes with and respond to CBP
(reviewed in Refs. 4 and 5). CBP functions as an integrator linking
various transcription factors to the basal transcriptional apparatus,
by binding to the basal transcription factor TFIIB, which in turn
contacts the TATA box-binding protein (TBP) of the TFIID complex in the
basal apparatus (6, 7). The interaction of p65 with CBP is essential
for NF-B transcriptional activity (8, 9), and this interaction can be strengthen by p65 phosphorylation (6, 10), or impeded by competition
from other CBP-binding factors such as CREB, c-Jun, c-Fos, p53, steroid
receptors, c-Myb, and Myo-D (7, 11-13).
Vasoactive intestinal peptide (VIP) and the structurally related
pituitary adenylate cyclase-activating polypeptide (PACAP), two
neuropeptides present in the lymphoid microenvironment, elicit a broad
spectrum of biological functions, including the modulation of innate
and adaptive immunity (reviewed in Refs. 14-17). VIP and PACAP
down-regulate the innate response by inhibiting inducible nitric-oxide
synthase expression and secretion of pro-inflammatory cytokines in
stimulated macrophages (18-23). VIP and PACAP affect the adaptive T
cell response indirectly, by down-regulating B7.1/B7.2 expression and
the subsequent costimulatory function of activated macrophages (24) and
directly by inhibiting IL-2 production and T cell proliferation
(reviewed in Ref. 15). Many of the proinflammatory cytokines and
costimulatory proteins affected by VIP and PACAP are known to be
regulated by NF-B (1). In fact, we have previously demonstrated that
VIP and PACAP inhibit NF-B nuclear translocation and DNA binding to
several promoters in both murine macrophages and T cells (18, 25-27).
However, the effect of VIP and PACAP on NF-B activation in human
monocytes has not been investigated to date. In addition, although we
showed that VIP and PACAP inhibit NF-B DNA binding, a direct effect on NF-B-dependent transcriptional activity has not yet
been addressed. Furthermore, we also asked whether VIP and PACAP could
regulate NF-B transcriptional activity through the regulation of
coactivators. Our data show that VIP and PACAP decrease
NF-B-dependent transcriptional activity in the
LPS-stimulated human monocytic cell line THP-1. This effect is exerted
at multiple levels. The neuropeptides inhibit NF-B nuclear
translocation and DNA binding by inhibiting the IB kinase
(IKK)-mediated IB phosphorylation/degradation. In addition, VIP and
PACAP selectively inhibit the interaction of p65 with CBP, while
increasing interactions between CBP and CREB. Furthermore, by
inhibiting the LPS-induced MEKK1/MEK3/MEK6/p38 MAPK pathway, the two
neuropeptides inhibit TBP activation and its subsequent DNA binding and
interaction with p65. The differential involvement of specific
VIP/PACAP receptors and intracellular pathways was also addressed. The
specific receptor VPAC1 mediates the effects of VIP/PACAP on p65
nuclear translocation, formation of p65·CBP complexes and TBP
activation and formation of p65·TBP complexes. However, whereas a
cAMP-independent pathway is primarily responsible for the effects on
p65 translocation, the cAMP/PKA pathway mediates the effects on the
availability and/or activation of the coactivators CBP and TBP.
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EXPERIMENTAL PROCEDURES |
Reagents--
Synthetic VIP, PACAP-(38), and SB 203580 were
purchased from Calbiochem. The PAC1/VPAC2-antagonist PACAP-(6-38) was
obtained from Peninsula Laboratories (Belmont, CA). The
VPAC1-antagonist [Ac-His1,D-Phe2,Lys15,Arg16,Leu27]VIP-(3-7)-GRF-(8-27)
was kindly donated by Dr. Patrick Robberecht (Universite Libre de
Bruxelles, Belgium). Human recombinant TNF and capture and
biotinylated antibodies against human TNF were purchased from
PharMingen (San Diego, CA). LPS (from Escherichia coli
055:B5), DEAE-dextran, protease inhibitors, and forskolin were
purchased from Sigma, and
N-[2-(p-bromocinnamyl-amino)ethyl]-5-iso-quinolinesulfonamide (H89) was from ICN Pharmaceuticals Inc. (Costa Mesa, CA). Recombinant IB-(1-317) and TFIID (TBP)-tagged fusion proteins and antibodies against p65, p50, IB, IB-kinase (IKK), CREB, p38 MAPK,
MEKK1, MEK3, MEK6, TBP, NF-Y (CBF-A), HMG-I(Y), phosphorylated
IB, and NF-ATp were purchased from Santa Cruz Biotechnology
(Santa Cruz, CA). Antibodies against phosphorylated-p38 MAPK,
phosphorylated-MEK3/6, and phosphorylated-CREB were purchased from New
England Biolabs (Beverly, MA).
Cells--
THP-1, a human leukemic monocytic cell line, was
obtained from American Type Culture Collection (Manassas, VA). THP-1
cells were stimulated with LPS (0.5 µg/ml) or TNF (20 ng/ml) in
the presence or absence of VIP or PACAP-(38)
(10
8 M unless mentioned otherwise).
Plasmids, Transfections, and Luciferase
Assay--
NF-B-dependent gene expression was evaluated
using a luciferase reporter gene driven by four tandem copies of the
enhancer (B4) in a pUC vector
(CLONTECH, Palo Alto, CA).
CRE-dependent gene expression was evaluated using a
luciferase reporter gene (CLONTECH). The plasmid
pRc/RSV-p65 containing the entire cDNA of p65 was kindly provided
Drs. G. J. Nabel and J. Stein through the National Institutes of
Health AIDS Research and Reference Reagent Program. The expression
plasmid, pRc/RSV-mCBP.HA.RK containing the full-length mouse CBP
cDNA with a hemagglutinin (HA) tag (6) was a generous gift of Dr.
R. Goodman. IB degradation was assayed by transiently transfecting
an enhanced green fluorescent protein (EGFP)-tagged IB signaling
probe (CLONTECH) followed by flow cytometry
analysis following the manufacturer's recommendations. Empty vectors
pRc/RSV and pUC-18 (Invitrogen, Carlsbad, CA) were used to maintain a
constant total DNA concentration in each experiment. To assess
variations in transfection efficiencies, the cells were transfected
with 2 µg of the control plasmid pCH110 (Amersham Pharmacia Biotech)
that expresses the lacZ gene. Levels of -galactosidase were determined using the Galacto-Light assay system (Tropix Inc., Bedford, MA) and exhibited <15% variation between samples.
THP-1 cells were transiently transfected with a total of 10-30 µg of
plasmid DNA using DEAE-dextran. Forty eight hours later, the cells were
stimulated with LPS (500 ng/ml) in the absence or presence of VIP or
PACAP, and 6 h later luciferase assays were carried out as
recommended by the manufacturer (Promega). Luciferase activity,
expressed in arbitrary light units, was corrected for protein
concentration or normalized to coexpressed -galactosidase levels.
RNA Extraction and Northern Blot Analysis--
Northern blot
analysis was performed according to standard methods. The probe for
human TNF was generated by RT-PCR as described previously using the
following primers: TNF, 5'-GTTCCTCAGCCTCTTCTCCT-3' and
5'-ATCTATCTGGGAGGGGTCTT-3' (28). Signal quantitation was performed in a
PhosphorImager SI (Molecular Dynamics, Sunnyvale, CA).
Electrophoretic Mobility Shift Assay (EMSA)--
Nuclear
extracts were prepared by the mini-extraction procedure of Schreiber
et al. (29). Double-stranded oligonucleotides (50 ng)
corresponding to the human consensus NF-B site
(5'-AGTTGAGGGGATTTTCCCAGGC-3', Promega), TFIID (TBP)
(5'-GCAGAGCATATAAAA TGAGGTAGGA-3', Santa Cruz Biotechnology), and the
consensus CRE motif (5'-AGAGATT GCCTGACGTCAGAGAGCTAG-3', Santa Cruz
Biotechnology) were end-labeled with [
-32P[ATP. For
EMSAs with THP-1 nuclear extracts, 20,000-50,000 cpm of
double-stranded oligonucleotides, corresponding to ~0.5 ng, were
used. The reaction mixtures (15 µl) were set up containing 0.5-1 ng
of DNA probe, 5 µg of nuclear extract, 2 µg of
poly(dI-dC)·poly(dI-dC), and binding buffer (50 mM
NaCl, 0.2 mM EDTA, 0.5 mM dithiothreitol, 5%
glycerol and 10 mM Tris-HCl, pH 7.5). The mixtures were
incubated on ice for 15 min before adding the probe, followed by
another 20 min at room temperature. Samples were subjected to
electrophoresis in 4% nondenaturing polyacrylamide gels. In
competition and antibody supershift experiments, the nuclear extracts
were incubated for 15 min at room temperature with specific antibodies
(1 µg) or competing cold oligonucleotides (50-fold excess) before the
addition of the labeled probe.
Immunoprecipitation Experiments and Western Blotting--
For
Western blot analysis, whole cell lysates, cytoplasmic fraction, or
nuclear extract (see above) containing 20-30 µg of protein were
subjected to reducing SDS-PAGE (12.5%). After electrophoresis and
electroblotting the membranes were developed with the enhanced chemiluminescence detection system (ECL, Amersham Pharmacia Biotech).
Phosphorylated MEK3 and MEK6 were detected by immunoprecipitation with
anti-MEK3 or anti-MEK6 Abs, followed by SDS-PAGE and immunoblotting
with an Ab against phosphorylated MEK3/6.
Interaction of CBP or TBP with p65 and/or CREB was assessed by
immunoprecipitation of cell extracts (200 µg) with 1-2 µg of anti-p65 or anti-CREB antibodies, followed by treatment with 25 µl of
protein A/G-Sepharose beads (Sigma). After extensive washing and
boiling in 1× SDS sample buffer, the complexes were subjected to
Western blotting with anti-CBP or anti-TBP antibodies. For transient
transfections with the CBP vector, we used an anti-hemagglutinin Ab
(anti-HA; 12CA5 murine monoclonal antibody against influenza HA
peptide; Roche Molecular Biochemicals) to detect transfected HA-tagged
CBP. After detection with an appropriate secondary antibody-conjugated peroxidase, proteins were visualized by enhanced chemiluminescence.
In Vitro Kinase Assays--
In vitro kinase assays
were performed as described previously with some modifications (30).
Endogenous IKK and p38 MAPK were immunoprecipitated from cell
lysates (150-250 µg/sample) by incubation with 0.5 µg of
anti-IKK or anti-p38 MAPK antibodies, for 2 h at 4 °C. The
immune complexes were harvested with protein A/G-Sepharose for 45 min
at 4 °C. The beads were extensively washed and resuspended in 30 µl of kinase buffer with 15 µM ATP, 10 µCi of
[-32P]ATP (3000 Ci/mmol), containing 5 µg of
recombinant IB (for IKK) or TBP (for p38 MAPK). The kinase
reaction was performed at 30 °C for 30 min and stopped by the
addition of 15 µl of 2× SDS sample buffer. Following boiling for 5 min, the samples were subjected to SDS-PAGE (9%), electroblotting, and autoradiography.
Immunoblotting of Proteins Bound to the Proximal Region of the
TNF Promoter--
A double-stranded oligonucleotide spanning the
proximal region of the human TNF promoter (
661 to 1), generated
from THP-1 genomic DNA by PCR (primers used, 5'-TCAGAATGAAA
GAAGAGGGCC-3' and 5'-GGCTGGGTGTGCCAACAACT-3') and biotinylated in our
molecular biology facility, was coupled to Dynabeads M-280 streptavidin (Dynal, Lake Success, NY) according to the manufacturer's
recommendations. The TNF promoter-coupled matrix (250 µg) was
incubated with 25 µl of THP-1 nuclear extract in binding buffer (50 mM NaCl, 5 mM MgCl2, 10 mM Tris, pH 7.5, 1 mM dithiothreitol, 1 mM EDTA, 0.25 µg/ml poly(dI)·poly(dC), and 5%
glycerol) for 20 min at room temperature while mixing every 5 min to
keep the Dynabeads in suspension. The flow-through fractions were
collected; the Dynabeads were washed four times with binding buffer
containing 0.5 µg/ml poly(dI)·poly(dC), and the bound proteins were
solubilized in sample buffer, boiled, and subjected to SDS-PAGE.
Membranes were probed with antibodies against p65, p50, CREB, c-Jun,
TBP, HMG-I(Y), CBP, or NF-Y at dilutions ranging from 1:2,500 to
1:10,000 followed by chemiluminescent detection.
In Vivo Phosphorylation of p65 and TFIID (TBP)--
Confluent
monolayers of THP-1 cells in 10-cm tissue culture dishes were labeled
with 200 mCi of 32PO4/ml (400-800 mCi/ml;
Perkin-Elmer Life Sciences) in phosphate-free RPMI medium with 10%
fetal calf serum for 3 h at 37 °C. Cells were stimulated with
LPS in the absence or presence of VIP or PACAP for 1 h, washed,
and resuspended in lysis buffer (1% Nonidet P-40, 1% sodium
deoxycholate, 0.1% SDS, 0.15 M NaCl, 10 mM
Na3PO4 (pH 7.2), 2 mM EDTA, 50 mM NaF, 0.2 mM Na3VO4,
1 µM okadaic acid, 100 µg/ml phenylmethylsulfonyl
fluoride, 50 µg aprotinin, 10 µg/ml leupeptin, 50 µg/ml
pepstatin), and sonicated. The p65 or TFIID (TBP) were
immunoprecipitated with rabbit anti-p65 or anti-TFIID (TBP) antibodies
(Santa Cruz Biotechnology) and bound to Gammabind (Amersham Pharmacia
Biotech) for 2-12 h at 4 °C. The pellets were washed twice with
high salt, followed by electrophoresis on 8-10% SDS-PAGE gels and autoradiography.
ELISA--
TNF levels in culture supernatants were determined
using a human-specific sandwich ELISA (PharMingen) following the
manufacturer's instructions.
RT-PCR for the Detection of VPAC1, VPAC2, and PAC1 mRNA
Expression--
THP-1 cells were cultured at a concentration of 2 × 106 cells/ml in 100-mm tissue culture dishes and
stimulated with LPS (0.5 µg/ml) for up to 12 h. Two µg of
total RNA was reverse-transcribed, and cDNA was amplified with
specific primers. Glyceraldehyde-3-phosphate dehydrogenase primers
(Stratagene) were used as control. The primers for VPAC1, VPAC2, and
PAC1 receptors have been described before (25).
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RESULTS |
VIP and PACAP Inhibit LPS-induced TNF Production in Human
Monocytes--
We have recently demonstrated that VIP and PACAP
inhibit TNF production in murine peritoneal macrophages and the
macrophage cell line Raw 264.7 (19, 25). To investigate the effects of VIP/PACAP on human monocytes, THP-1 cells were stimulated with LPS in
the presence or absence of VIP or PACAP, and the amounts of secreted
TNF were determined by ELISA. VIP and PACAP inhibit TNF
production in a dose-dependent manner, with a maximum
effect in the concentration range of
108-106
M (Fig. 1A). Both
neuropeptides inhibit in a time-dependent manner TNF
steady-state mRNA levels (Fig. 1B). MG132, a newly
described NF-B inhibitor, shows a similar inhibitory effect on
TNF mRNA (Fig. 1B).
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Fig. 1.
VIP and PACAP inhibit TNF
production in human monocytes. THP-1 cells were treated with
LPS with or without VIP or PACAP (108
M for B) or the NF-B inhibitor MG132 (100 µM). A, supernatant TNF levels were
measured by ELISA (8 h after stimulation). Each result is the mean ± S.D. of four separate experiments performed in duplicate.
B, expression of TNF and -actin mRNA was
determined by Northern blot (2 h after stimulation). One representative
experiment of three is shown. C, Northern blots performed at
different time points after stimulation. Results are expressed in
arbitrary densitometric units normalized for the expression of
-actin and represent the mean ± S.D. of three independent
experiments performed in duplicate.
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VIP and PACAP Inhibit LPS- and TNF-induced
NF-B-dependent Transcription in Monocytes--
Since
LPS up-regulates TNF transcription through an
NF-B-dependent mechanism (31, 32), we determined whether
VIP and PACAP inhibit NF-B transcriptional activity. THP-1 cells
were transiently transfected with the (B)4-luciferase
reporter plasmid, containing four copies of the NF-B consensus site.
Forty eight hours later, the cells were stimulated with LPS or TNF
in either the presence or absence of VIP or PACAP and were assayed for
NF-B-dependent transcription 5 h later. Both LPS
and TNF led to an ~18-fold increase in NF-B transcriptional
activity (Fig. 2A). Treatment with VIP or PACAP strongly inhibits LPS- or TNF-induced NF-B activity (Fig. 2A). The inhibitory effect is
dose-dependent (Fig. 2B).
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Fig. 2.
VIP and PACAP inhibit LPS- and
TNF-induced NF-B
transcriptional activity. THP-1 cells were transfected with the
(B)4-Luc construct (10 µg) and treated 48 h later
with LPS or TNF (10 ng/ml), with or without VIP or PACAP
(108 M for A) for
6 h. Cytosolic extracts (100 µg) were used in luciferase assays.
Fold induction is relative to luciferase activity in unstimulated
cells. Data are expressed as the mean ± S.D. of three independent
experiments performed in duplicate.
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VIP and PACAP Inhibit NF-B Nuclear Translocation by Preventing
LPS-induced Phosphorylation/Degradation of IB--
To assess
whether VIP and PACAP inhibit NF-B DNA binding, EMSAs were
performed. Stimulation of THP-1 cells with LPS led to strong NF-B
binding showing a maximum increase at 2 h after stimulation, and
VIP and PACAP inhibit binding at all time points (Fig.
3A). The binding specificity
was ascertained by the displacement with 50-fold excess of unlabeled
homologous (NF-B) but not nonhomologous oligonucleotide (CRE) (Fig.
3A). Antibody supershift experiments indicate the presence
of both p50 and p65, with no supershift for an irrelevant Ab
(anti-CREB) (Fig. 3A).
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Fig. 3.
VIP and PACAP prevent LPS-induced
IB degradation and the subsequent
NF-B nuclear translocation. A,
VIP and PACAP inhibit NF-B DNA binding. Nuclear extracts from cells
incubated for 2 h with LPS with or without VIP or PACAP
(108 M) were used in EMSA.
Probe, consensus NF-B. Supershift, nuclear
extracts were incubated with polyclonal antibodies against p65, p50, or
CREB for 20 min before the addition of the B probe. One
representative experiment of three is presented. Right
panel, NF-B binding at various time points expressed as
arbitrary densitometric units. Data represent the mean ± S.D. of
three independent assays. B, VIP and PACAP inhibit p65
translocation. Cells were treated with LPS with or without VIP or PACAP
(108 M) for 1 h. Cytosolic
and nuclear proteins were extracted, and Western blot analysis was
performed for p50 and p65. One representative experiment of three is
shown. C, VIP and PACAP prevent IB phosphorylation and
degradation. Left panels, THP-1 cells were stimulated with
LPS with or without VIP or PACAP (108
M). The cytosolic amounts of IB and phosphorylated
IB at different time points were determined by Western blot. One
representative experiment of three is shown. Right panels,
THP-1 cells were transiently transfected with a fluorescent IB-EGFP
signaling probe and treated with LPS with or without
108 M VIP 24 h later. The
percentage of IB+ cells was determined by flow cytometry
at different time points. Similar results were obtained in three
independent experiments. D, VIP and PACAP inhibit IKK
activity. THP-1 cells were stimulated with LPS with or without VIP or
PACAP (108 M) (10 min in
upper panel). IKK activity was assayed in an in
vitro kinase assay. Lower panel, IKK activity is
expressed as arbitrary densitometric units. Data represent the
mean ± S.D. of three independent assays. IKK protein amounts
were determined by immunoblotting (upper panel; control).
E, overexpression of p65 partially reverses the inhibitory
effect of VIP and PACAP. THP-1 cells were transiently cotransfected
with the (B)4-Luc construct (10 µg) and increasing
concentrations (0, 2.5, 5, 10, or 15 µg) of pRSV-p65 vector (p65).
The cells were stimulated 48 h later with LPS with or without
108 M VIP or PACAP and incubated
for 6 h before determining luciferase activity. Fold induction is
relative to luciferase activity in unstimulated cells. Results
represent the mean ± S.D. of three independent experiments
performed in duplicate.
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The primary level of control for NF-B is mediated through its
interaction with the inhibitor IB. VIP and PACAP could inhibit NF-B activity by blocking LPS-induced IB degradation and
subsequent NF-B nuclear translocation. We measured the levels of p65
in cytoplasm and nucleus. As expected, p65 was predominantly localized in the cytoplasm of unstimulated cells, and LPS induced a decrease in
the level of cytoplasmic p65, and an increase in nuclear p65 levels
(Fig. 3B). VIP and PACAP abolished the LPS-induced change in
p65 levels (Fig. 3B), which indicates an inhibition of p65 nuclear translocation. Similar levels of p50 indicate equal protein loading. To determine whether VIP/PACAP interfere with the LPS-induced degradation of IB, we examined cytoplasmic IB levels. As
expected, we observed a time-dependent IB
degradation, paralleled by an increase in IB phosphorylation in
LPS-stimulated cells (Fig. 3C). VIP and PACAP block the
phosphorylation and subsequent degradation of IB (Fig.
3C). In addition, we determined the effect of VIP and PACAP
on IB degradation by transiently transfecting an EGFP-tagged IB
construct in THP-1 cells and assaying the percentage of fluorescent IB+ cells at different time points by flow cytometry.
LPS led to a rapid decrease in IB+ cells, whereas VIP
significantly increased IB half-life in LPS-treated cells (Fig.
3C, right panel).
Since the LPS activation of NF-B requires IKK-mediated
phosphorylation of IB (1, 2), we determined if VIP and PACAP inhibit IKK activity by using an in vitro kinase assay.
Stimulation of THP-1 cells with LPS resulted in a
time-dependent increase in IKK activity, which was
inhibited by VIP and PACAP (Fig. 3D). No differences in
IKK expression were observed (Fig. 3D).
These results demonstrate that VIP and PACAP inhibit NF-B nuclear
translocation and subsequent DNA binding in LPS-stimulated cells by
blocking the IKK-mediated IB phosphorylation/degradation. If the
inhibitory effect of VIP and PACAP on NF-B transcriptional activity
is mediated entirely through the inhibition of NF-B nuclear
translocation, overexpression of p65 should reverse this effect.
Therefore, THP-1 cells were transiently cotransfected with the
(B)4-luciferase reporter plasmid and increasing
concentrations of a vector expressing p65. Increasing concentrations of
p65 only partially reversed the inhibitory effect of VIP and PACAP,
suggesting that the neuropeptides affect more than NF-B nuclear translocation.
Neither VIP nor PACAP Affect LPS-induced p65
Phosphorylation--
Several studies have demonstrated that p65 is
phosphorylated during in vivo NF-B activation, leading to
increased transcriptional activity (10, 33). We examined the effects of
VIP and PACAP on the phosphorylation of transiently transfected p65.
VIP and PACAP had no effect on LPS-induced p65 phosphorylation (Fig.
4, upper panel). Similar
levels of p65 were detected by Western blotting in THP-1 cells
transfected with p65 in the presence or absence of LPS, VIP, or PACAP
(Fig. 4, lower panel).
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Fig. 4.
VIP and PACAP do not affect LPS-induced p65
phosphorylation. THP-1 cells were transiently transfected with the
vector pRSV-p65 (5 µg), labeled 48 h later with
32Pi in phosphate-free media for 3 h, and
stimulated with LPS with or without VIP or PACAP
(108 M) for 1 h. Cell
lysates were subjected to immunoprecipitation with anti-p65, SDS-PAGE,
and autoradiography. Data are representative of four experiments. The
amounts of transfected p65 were determined by immunoblotting of
unlabeled samples with anti-p65 Ab (lower blot).
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VIP and PACAP Promote CREB/CBP Versus p65/CBP Interactions by
Increasing CREB Phosphorylation/Activation--
In addition to DNA
binding, the interaction of p65 with CBP is essential for optimal
NF-B transcriptional activity (9). In addition to p65, CBP interacts
with other factors including CREB (6). Changes in p65 phosphorylation
or competition with other factors for the limiting quantities of
nuclear CBP lead to changes in p65/CBP interactions. THP-1 cells were
stimulated with LPS in the absence or presence of VIP or PACAP, and
total cell lysates were immunoprecipitated with antibodies to p65 or CREB and probed for the presence of CBP. LPS stimulation results in the
appearance of p65·CBP complexes (Fig.
5A). No p65·CBP complexes are detected in unstimulated cells. VIP and PACAP decrease the levels
of p65/CBP and increase the levels of CREB·CBP complexes (Fig.
5A). Moreover, VIP and PACAP induce CREB·CBP instead of p65·CBP complexes even in the presence of overexpressed CBP (Fig. 5A).
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Fig. 5.
VIP and PACAP promote CREB/CBP
versus p65/CBP interactions by increasing CREB
phosphorylation. A, VIP and PACAP promote CBP/CREB and
inhibit CBP·p65 complex formation. Upper panels, THP-1
cells were treated with LPS with or without VIP or PACAP
(108 M) for 1 h. Cell
extracts were subjected to immunoprecipitation (IP) with
anti-CREB or anti-p65 antibodies and analyzed by Western blot with
anti-CBP. Data are representative of four experiments. Lower
panels, THP-1 cells were transiently transfected with the pRSV-CBP
vector containing CBP cDNA coupled to HA tag. After 24 h, the
cells were treated with LPS with or without VIP or PACAP
(108 M) for 1 h. Cell
extracts were subjected to immunoprecipitation (IP) with
anti-CREB or anti-p65 antibodies and analyzed by Western blot with
anti-HA. Data are representative of three experiments. B,
effects of CBP and CBP plus p65 overexpression. Upper panel,
THP-1 cells were cotransfected with the (B)4-Luc
construct (10 µg) and increasing concentrations (0 µg for
1st and 2nd lanes, and 2.5, 5, 10, and 15 µg
for 3rd to 6th lanes, and 5 µg for 7th to
10th lanes) of pRSV-CBP vector (CBP) and increasing
concentrations (0 µg for 1st to 6th
lanes and 2.5, 5, 10, or 15 µg for 7th to
10th lanes) of pRSV-p65. After 48 h, cells
were stimulated with LPS in the absence (1st
lane) or presence of 108
M VIP (2nd to 10th lanes) and
incubated for an additional 5 h before determining luciferase
activity. Fold induction is relative to luciferase activity in
unstimulated cells. Results represent the mean ± S.D. of three
independent experiments performed in duplicate. Lower
panels, THP-1 cells were cotransfected with increasing
concentrations (2.5, 5, 10 and 15 µg for 1st to
4th lanes, 5 µg for 5th to 8th
lanes) of pRSV-p65 vector (p65) and increasing concentrations (0 µg for 1st to 4th lanes and 2.5, 5, 10 and 15 µg for 5th to 8th lanes) of pRSV-CBP
(CBP). After 48 h, cells were stimulated with LPS in the absence
(9th lane) or presence of
108 M VIP (1st to
8th lanes) for 1 h. Cell extracts were subjected to
immunoprecipitation(IP) with anti-p65 and immunoblotted with
anti-HA. Data are representative of four experiments. C, VIP
and PACAP stimulate CREB phosphorylation and nuclear translocation.
THP-1 cells were treated with LPS with or without VIP or PACAP
(108 M) for 1 h. Upper
panels, cell extracts were analyzed by Western blot using
anti-phosphorylated CREB or anti-CREB. Data are representative of four
experiments. Lower panels, cytosolic and nuclear proteins
were extracted and subjected to Western blot with anti-CREB. One
representative experiment of three is shown. D, VIP and
PACAP increase CREB binding activity and subsequent CREB
transactivation. Left panels, nuclear extracts from cells
incubated for 2 h with LPS with or without VIP or PACAP
(108 M) were assayed for DNA
binding by EMSA. Probe, the consensus CRE site.
Supershift, nuclear extracts were incubated with polyclonal
antibodies against p65 or CREB for 20 min before the addition of the
CRE probe. Similar results were observed in other three
experiments. Right panel, THP-1 cells were transfected with
the CRE-Luc construct (10 µg). After 48 h cells were treated
with LPS with or without VIP or PACAP (108
M) for 5 h. Cytosolic extracts (100 µg) were used in
the luciferase assay. Data are expressed as the mean ± S.D. of
relative luciferase units (RLU) from three independent
experiments performed in duplicate.
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To confirm that the VIP/PACAP inhibition of NF-B transcriptional
activity is related to the reduction in p65·CBP complexes, THP-1
cells were cotransfected with the (B)4-luciferase
reporter system and increasing concentrations of p65 and/or CBP.
Expression of increasing concentrations of CBP led a partial reversal
of the inhibitory effect of VIP/PACAP on NF-B activation (Fig.
5B, upper panel). A similar conclusion was reached earlier
regarding p65 (Fig. 3E). However, the coexpression of CBP
(fixed concentration) and p65 (increasing concentrations) completely
reversed the VIP/PACAP effect (Fig. 5B, upper panel). This
correlates with the fact that coexpression of p65 and CBP restored the
p65·CBP complexes to levels observed in the LPS-treated cells (Fig.
5B, lower panel).
The fact that, even in the presence of excess p65, the levels of
p65·CBP complexes and the NF-B transcriptional activity are not
completely restored (Figs. 5B and 3F) indicates
that VIP and PACAP affect the formation of p65·CBP complexes through
both a reduction in nuclear p65 and a direct effect on CBP.
Since VIP receptors are mostly linked to the cAMP/PKA pathway, it is
highly possible that VIP and PACAP activate CREB which then recruits
CBP. Therefore, we analyzed the effects of VIP and PACAP on CREB
phosphorylation. LPS increases CREB phosphorylation slightly as
compared with unstimulated controls (Fig. 5C, upper panels).
In contrast, VIP and PACAP strongly augment the levels of
phosphorylated CREB (Fig. 5C, upper panels). Total CREB
levels were not affected by either treatment. In addition, CREB levels in cytoplasmic, and nuclear extracts were assayed by Western blotting. LPS stimulation results in a slight increase in nuclear CREB, and
treatment with VIP or PACAP leads to high levels of nuclear CREB (Fig.
5C, lower panels).
To determine whether VIP/PACAP-induced CREB phosphorylation correlates
with increased DNA binding and CREB-dependent
transcription, we performed EMSAs using a consensus CRE site and
transient transfections with a CRE-luciferase reporter plasmid. LPS
leads to a slight increase in CRE DNA binding, and VIP and PACAP
strongly augment this binding (Fig. 5D). The binding
specificity was confirmed by using homologous (CRE) and nonhomologous
(NF-B) oligonucleotides as competitors (Fig. 5D). The CRE
complexes are supershifted by an anti-CREB Ab but not by an anti-p65 Ab
(Fig. 5D). In cells transfected with CRE-luciferase
constructs, VIP and PACAP significantly increase the
CRE-dependent transcriptional activity, as compared with
cells treated with LPS alone (Fig. 5D). These results
indicate that VIP and PACAP increase the phosphorylation/activation of CREB which then competes with p65 for limiting amounts of CBP, resulting in increased CREB·CBP and decreased p65·CBP complexes.
VIP and PACAP Reduce LPS-induced TBP DNA Binding Activity and Its
Interaction with p65 by Inhibiting the MEKK1-MEK3/6-p38 MAPK
Pathway--
Since NF-B-driven transcription also depends on the
activation of basal transcription factors, such as TFIIB and TFIID
(TBP), we investigated if VIP and PACAP regulate the basal
transcriptional factors. We determined first the effect of VIP and
PACAP on TBP binding to the TATA box. LPS increases TBP binding, and
VIP and PACAP reduce it to control levels. The specificity of TBP
binding is indicated by competition of 50-fold excess of unlabeled
homologous (TBP), but not nonhomologous, oligonucleotide (NF-AT). The
TBP complexes are supershifted by an anti-TBP Ab but not by an
irrelevant Ab (anti-CREB) (Fig.
6A).
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Fig. 6.
VIP and PACAP reduce TBP nuclear binding and
its interaction with p65 by inhibiting the MEKK1/MEK3/MEK6/p38 MAPK
pathway. A, VIP and PACAP inhibit TBP binding activity.
Nuclear extracts prepared from cells incubated for 2 h with LPS
with or without VIP or PACAP (108
M) were assayed for TBP binding by EMSA. Probe,
consensus TBP site. Supershift, nuclear extracts were
incubated with polyclonal antibodies against TBP or CREB for 20 min
before the addition of the TBP probe. Similar results were observed in
other three experiments. B, VIP and PACAP prevent TBP-p65
interaction. Upper panel, THP-1 cells were treated with LPS
with or without VIP or PACAP (108
M) for 1 h. Cell extracts were subjected to
immunoprecipitation (IP) with anti-p65 and immunoblotted
with anti-TBP. Data are representative of three experiments.
Lower panel, THP-1 cells were transfected with increasing
concentrations (0 µg for 1st and 6th lanes and
2.5, 5, 10, and 15 µg for 2nd to 5th lanes) of
pRSV-p65. After 48 h, cells were stimulated with LPS in the
absence (6th lane) or presence of
108 M VIP (1st to
5th lanes) for 1 h. Cell extracts were
subjected to immunoprecipitation (IP) with anti-p65 and
immunoblotting with anti-TBP. Data are representative of four
experiments. C, VIP and PACAP inhibit in vivo TBP
phosphorylation. THP-1 cells were labeled with
32Pi in phosphate-free media for 3 h.
Cells were treated with medium or LPS with or without VIP
(108 M), PACAP
(108 M), or SB203580 (SB, 0.5 µM) for 1 h. Cell lysates were subjected to
immunoprecipitation with anti-TBP and were electrophoresed. Data are
representative of four experiments. D, VIP and PACAP reduce
p38 MAPK activity by inhibiting the MEKK1/MEK3/6 activation. THP-1
cells were treated with LPS with or without VIP or PACAP
(108 M). Left panels,
at different time points (15 min for blots), p38 MAPK activity was
assayed in an in vitro kinase assay, with TBP as substrate.
p38 MAPK activity is expressed as arbitrary densitometric units. Data
represent the mean ± S.D. of three independent assays. As
control, the amounts of TBP were determined by immunoblotting.
Right panels, the levels of phosphorylated MEK3,
phosphorylated MEK6, and phosphorylated p38 MAPK were determined by
Western blotting (15 min). The amounts of p38 MAPK, MEK3, MEK6, and
MEKK1 were determined by immunoblotting. One representative experiment
of three is shown.
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Since the association of the carboxyl terminus of p65 with TBIIB and
TBP is known to be important for the transcriptional regulation of
NF-B (34, 35), we determined whether VIP and PACAP regulate p65/TBP
interactions by immunoprecipitating cell lysates with anti-p65 Abs and
immunoblotting for TBP. LPS increases this interaction, and VIP and
PACAP inhibit the LPS-induced p65/TBP interaction (Fig. 6B, upper
panel). To determine whether the effect of VIP/PACAP is due to the
previously described inhibition of p65 nuclear translocation, THP-1
cells were transiently transfected with increasing concentrations of
p65. Overexpression of p65 partially reversed the effect of VIP (Fig.
6B, lower panel, compare 2nd to 5th lanes
to the 6th lane). However, the incomplete reversal suggests that
VIP and PACAP might directly regulate TBP activation.
Since TBP is activated following phosphorylation by the p38 MAP kinase,
we evaluated the effect of VIP and PACAP on TBP phosphorylation. Whereas no TBP phosphorylation is observed in unstimulated cells, LPS
induces high levels of phosphorylated TBP (Fig. 6C). VIP and PACAP inhibit TBP phosphorylation, similar to the p38 MAP kinase inhibitor SB 203580 (Fig. 6C). We examined the effect of VIP
and PACAP on p38 MAPK activity with TBP as substrate. Treatment with LPS results in a time-dependent increase in p38 MAPK
activity (Fig. 6D). VIP and PACAP inhibit the LPS-induced
p38 MAPK-mediated phosphorylation of TBP, without affecting TBP and p38
MAPK protein levels (Fig. 6D).
The activation of p38 MAPK in response to LPS or proinflammatory
signals involves a kinase cascade with the upstream activator MEKK1
phosphorylating and activating both MAPK kinases MEK3 and MEK6, which
in turn activate p38 MAPK by phosphorylation at both threonine and
tyrosine residues (36-38). Therefore, we investigated the effect of
VIP and PACAP on phosphorylation of MEK3, MEK6, and p38. LPS leads to
the strong phosphorylation of p38, MEK3 and MEK6, and VIP and PACAP
significantly reduce the phosphorylation of all these kinases (Fig.
6D). None of these treatments affected the expression of any
of the kinases assayed (Fig. 6D). We conclude that the
VIP/PACAP inhibition of TBP phosphorylation is mediated through the
inhibition of the MEKK1/MEK3/MEK6/p38 MAPK cascade.
VIP and PACAP Change the LPS-induced Composition of Nuclear Factors
Bound to TNF Promoter in Human Monocytic Cells--
We next
investigated the effects of VIP and PACAP on the LPS-induced
transcriptional activators binding the proximal regulatory region in
the human TNF promoter, which contains two essential transactivating
binding sites, i.e. the NF-B and CRE elements (31, 32).
Depending on the activation state, the CRE site may bind either CREB or
c-Jun, with CREB preferentially bound in unstimulated cells and c-Jun
in LPS-stimulated cells (25, 32). To determine whether the activators
present in nuclear extracts would bind to the TNF promoter, a
biotinylated affinity matrix spanning the proximal regulatory region of
human TNF promoter was generated and coupled to streptavidin-coated
magnetic beads. This biotinylated probe was incubated with nuclear
extracts from unstimulated or LPS-stimulated THP-1 cells treated with
or without VIP or PACAP. Transcription factor complexes were released
from the magnetic beads by boiling in SDS sample buffer and detected by immunoblotting.
p65 is present in LPS-treated samples, but not in unstimulated or
stimulated cells treated with VIP or PACAP (Fig.
7, p65, input). The p65
present in the LPS-treated cells binds to the TNF promoter region
(Fig. 7, p65, bound). In contrast, p50 is constitutively
expressed in THP-1 cells and binds partially to the TNF promoter
(Fig. 7, p50, input, bound, and flow-thru). The
p50 binding is not affected by LPS, VIP, or PACAP (Fig. 7, p50). CREB is slightly induced by LPS and highly induced by
VIP and PACAP (Fig. 7, CREB, input). All induced CREB binds
to the TNF promoter region (Fig. 7, CREB, bound, and
flow-thru). Both TBP and CBP are constitutively present in
the nucleus, and neither LPS not VIP/PACAP affect their levels (Fig. 7,
CBP and TBP, input). However, whereas CBP from
unstimulated, LPS-stimulated, and VIP/PACAP-treated cells is all bound
to the TNF promoter region, binding of TBP is induced by LPS and
inhibited by VIP and PACAP (Fig. 7, CBP and TBP,
bound). Similar to TBP, c-Jun is present in the nucleus and binds
to the TNF promoter region when the cells are stimulated with LPS,
and this binding is inhibited by VIP and PACAP (Fig. 7,
c-Jun). As a control, we used the nuclear factor-Y
(NF-Y), a transcription factor present in the nucleus which
binds constitutively to various promoters, including TNF. None of
the treatments affected NF-Y binding (Fig. 7, NF-Y).
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Fig. 7.
VIP and PACAP change the composition of
nuclear factors bound to the TNF
promoter. Nuclear extracts prepared from THP-1 cells
incubated for 2 h with LPS with or without VIP or PACAP
(108 M) were incubated with a
biotinylated oligonucleotide spanning the proximal region of the human
TNF promoter. Bound proteins were identified by Western blotting
using the indicated antibodies. One representative experiment of three
is shown.
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Finally, this experimental design allowed us to investigate an
additional possible regulatory element of the NF-B transactivation, i.e. the nonhistone chromosomal proteins of the high
mobility group (HMG)-I(Y) family, two chromatin architectural proteins that play a role in the transcriptional regulation of certain mammalian
genes (39, 40). HMG-I(Y) was shown to enhance the DNA binding of
several transcription factors, including NF-B (41, 42). HMG-I(Y) was
present in the nucleus from unstimulated, LPS-stimulated, and
VIP/PACAP-treated THP-1 cells, and none of the treatments affected its
binding to the TNF promoter, suggesting that HMG-I(Y) is an unlikely
element in the regulation of NF-B activation by VIP and PACAP.
Involvement of VPAC1 and cAMP/PKA in the Effects of VIP and PACAP
on NF-B-mediated Gene Activation--
Although THP-1 cells were
previously shown to express VIP/PACAP-binding sites, primarily coupled
to cAMP production (43), the nature of these binding sites was not
elucidated. We investigated the expression of VPAC1, VPAC2, and PAC1 by
RT-PCR in unstimulated and LPS-stimulated THP-1 cells. Both VPAC1- and
PAC1-specific fragments were amplified from unstimulated and stimulated
monocytes, whereas VPAC2 fragments were only detected in stimulated
cells (Fig. 8A).
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Fig. 8.
Involvement of VPAC1 and cAMP/PKA in
NF-B gene activation and
IKK activity. A, expression of
VPAC1, VPAC2, and PAC1 mRNA in THP-1 cells. Total RNA extracted
from unstimulated and LPS-stimulated (12 h) THP-1 cells was subjected
to RT-PCR with specific primers for VPAC1, VPAC2, and PAC1. One
representative experiment of two is shown. B and
C, THP-1 cells were activated with LPS in the absence
(1st lanes) or presence of VIP
(108 M, 2nd
lanes), or forskolin (106
M, 5th lanes). VPAC1 antagonist
(107 M, 3rd
lanes) or H89 (100 ng/ml, 4th lanes)
were added simultaneously with VIP (108
M). B, NF-B gene activation was analyzed as
described in Fig. 2. Results represent the mean ± S.D. of three
independent experiments performed in duplicate. C,
upper panels, NF-B binding was analyzed 1 h after
stimulation as described in Fig. 3. Lower panels, IKK
activity (20 min) was analyzed with IB as substrate by using an
in vitro kinase assay. For blots, one representative
experiment of three is shown. Dotted lines in graphs
B and C are control values from samples treated
with LPS alone. Results shown in graphs are the mean ± S.D. of
three independent experiments performed in duplicate.
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Our previous studies identified VPAC1 and cAMP as the major mediators
of VIP/PACAP effects on macrophage-derived cytokines (18-21, 22-27).
However, VIP and PACAP inhibit NF-B nuclear translocation in murine
macrophages through a cAMP-independent pathway (18, 25-27). To
determine the receptor and the transduction pathways involved, we used
specific VPAC antagonists and a specific protein kinase A inhibitor
(H89). The VIP inhibition of NF-B transcriptional activity is
completely reversed by the VPAC1 antagonist and only slightly reversed
by increasing concentrations of H89. In addition, forskolin (a
cAMP-inducing agent) mimics only partially the effect of VIP (Fig.
8B). These findings suggest that whereas the effect of VIP
on NF-B activation is entirely VPAC1-dependent, it is mediated by both a cAMP-dependent and a cAMP-independent
pathway. When NF-B DNA binding and IB phosphorylation were
analyzed, we found again that the VPAC1 antagonist completely reversed
the inhibitory effect of VIP and that H89, even at the highest
concentrations used, only minimally reversed this effect. This
correlates with the fact that forskolin inhibits only weakly the
NF-B DNA binding and IB phosphorylation (Fig. 8C).
Therefore, the major pathway for the inhibition of NF-B nuclear
translocation by VIP is non-cAMP mediated.
In contrast, the VPAC1 antagonist and the PKA inhibitor completely
reversed the effect of VIP on CREB phosphorylation, and forskolin
entirely mimicked the effect of VIP (Fig.
9A). A similar conclusion was
reached for the effect of VIP on the preferential induction of
CREB·CBP versus p65·CBP complexes (Fig. 9A).
Finally, the VPAC1 antagonist and H89 also reversed the effects of VIP on the phosphorylation of TBP and the activation of the MEKK1/MEK3/p38 MAPK pathway, with forskolin mimicking the effects of VIP (Fig. 9B). These results suggest that the regulatory activities of
VIP on CBP and TBP are mediated entirely through the cAMP/PKA
pathway.
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Fig. 9.
Involvement of VPAC1 and cAMP/PKA in CREB and
TBP phosphorylation/activation. THP-1 cells were activated with
LPS in the absence (1st lanes) or presence of VIP
(108 M, 2nd
lanes), or forskolin (106
M, 5th lanes). VPAC1 antagonist
(107 M, 3rd
lanes) or H89 (100 ng/ml, 4th lanes)
were added simultaneously with VIP (108
M). A, left panels, phosphorylated
CREB levels and CREB·CBP complexes (1 h after stimulation) were
determined as described in Fig. 5. One representative experiment of
three is shown. Right panel, expression of phosphorylated
CREB levels was analyzed by Western blot, and signal was
densitometrically quantitated. Data are the mean ± S.D. of three
independent experiments performed in duplicate. B, in
vivo phosphorylation of TBP and kinase assays for p38 MAPK and
MEK3 phosphorylation were assessed as described in Fig. 6. One
representative experiment of four is shown. Results of modulation of
p38MAPK activity by different concentrations of H89 are the mean ± S.D. of four independent experiments performed in duplicate.
Dotted lines represent control values of samples treated
with LPS alone.
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DISCUSSION |
VIP and PACAP control inflammatory processes by suppressing the
monocyte/macrophage production of several proinflammatory factors known
to be transcriptionally controlled by NF-B (21-32). Although VIP
and PACAP have been found to inhibit the translocation of NF-B and
its DNA binding in mouse macrophages (18, 25-27), the effects of these
neuropeptides on NF-B-dependent transcriptional activity
and the detailed molecular mechanisms governing this process have still
to be elucidated. The present study shows that VIP and PACAP
specifically inhibit the LPS-induced NF-B transcriptional activity
in the human monocytic cell line THP-1.
VIP and PACAP operate at three different levels to inhibit the NF-B
transcriptional activity. First, VIP and PACAP inhibit p65 nuclear
translocation and subsequent DNA binding. This process is mediated
through the VPAC1 receptor and a non-cAMP transduction pathway. Second,
VIP and PACAP induce CREB phosphorylation, and the phosphorylated CREB
competes with p65 for the coactivator CBP. Third, VIP and PACAP inhibit
the MEKK1/MEK3/6/p38 pathway ultimately affecting the phosphorylation
of TBP and its binding to both p65 and the TNF promoter. The effects
on CBP and TBP are both mediated through VPAC1 and the cAMP/PKA
transduction pathway.
The inhibition of p65 translocation by VIP/PACAP is mediated through
the stabilization of IB. IB, a member of the IB family,
is the major player in the response to inflammatory stimuli. Upon
phosphorylation at specific serine residues by the kinases IKK and
-, IB is ubiquitinated and degraded by the 26 S proteosome (reviewed in Ref. 2). As previously demonstrated for murine macrophages
and T cells (27),2 VIP and
PACAP inhibit IB phosphorylation and its subsequent degradation.
This is accomplished through an inhibitory effect on IKK. Similar
results were obtained in this study. The inhibitory effect of VIP and
PACAP on p65 nuclear translocation and NF-B DNA binding has
functional consequences, since overexpression of p65 partially reverses
this inhibitory effect. The fact that the reversal is incomplete
suggests additional mechanisms for the inhibition of NF-B
transcriptional activity. To investigate this hypothesis several
possibilities were considered.
It has been reported that the catalytic subunit of PKA associates with
the cytosolic NF-B·IB complex phosphorylating p65 and that
p65-mediated transcription is strongly dependent on its phosphorylation
(10). However, our results clearly demonstrate that neither VIP nor
PACAP affect the in vivo LPS-induced phosphorylation of p65.
An additional regulatory element in the NF-B transcriptional
activity is the coactivator CBP. CBP performs an important role in the
integration of diverse signaling pathways by linking p65 with
components of the basal transcriptional machinery, such as TFIIB, TBP,
and histone acetyltransferases (44). The present report demonstrates
that VIP and PACAP indeed inhibit the formation of p65·CBP complexes
and that this event is directly related to the inhibition of NF-B
transcriptional activity. The fact that overexpression of p65 did not
completely reverse the VIP/PACAP inhibition of p65·CBP complex
formation suggests that VIP/PACAP might directly affect CBP. Since CBP
is in limiting amounts in the nucleus and is capable to interact with
several transcriptional factors (3, 6-13), competition for CBP
provides another mechanism for transcriptional regulation (8, 45, 46).
CBP binds to phosphorylated CREB, and formation of CREB·CBP complexes
reduces the CBP available for complexing with p65 (8, 9). VIP/PACAP were shown to induce CREB DNA binding in activated murine macrophages (25, 26). This study shows that VIP and PACAP increase CBP binding to
CREB, replacing p65·CBP with CREB·CBP complexes in LPS-stimulated
THP-1 cells. This is due to VIP/PACAP-induced increases in CREB
phosphorylation/activation. The fact that cotransfections with p65 and
CBP completely reverse the inhibitory effect of VIP/PACAP on NF-B
transcriptional activity, whereas p65 and CBP separately result in only
a partial reversal, suggests that VIP/PACAP operate by inhibiting both
p65 nuclear translocation and CBP availability.
The observation that VIP and PACAP induce high levels of nuclear CREB
was unexpected. In most systems, CREB is localized in the nucleus in
both stimulated and unstimulated cells and becomes transcriptionally
active upon phosphorylation (reviewed in Ref. 47). However, in our
system, the unstimulated THP-1 cells express mostly cytoplasmic CREB,
and VIP/PACAP induce significant levels of nuclear CREB. This might be
a characteristic of this particular cell line, with VIP/PACAP
contributing to the retention of phosphorylated CREB in the nucleus.
In addition, p65 was shown to interact with TBP and TFIIB of the basal
transcriptional complex, and these interactions appear essential for
optimal NF-B-driven transcription (48). The interaction of p65 with
TBP and TFIIB is presumably mediated through CBP (reviewed in Ref. 4).
Our study indicates that VIP and PACAP inhibit LPS-induced TBP binding
to the TATA box and p65/TBP interaction. Similar to CBP, TBP is found
in limiting amounts in the nucleus but is activated following LPS
stimulation by the MEKK1/MEK3/MEK6/p38 MAPK pathway (36-38, 50).
Several studies demonstrated the involvement of p38 MAPK in the
activation of NF-B (50-52). VIP and PACAP inhibit the p38 MAPK
pathway and the subsequent TBP phosphorylation/activation.
Finally, we investigated another possible regulatory element in this
system. HMG-I and HMG-Y are two architectural proteins that facilitate
the assembly of functional nucleoprotein complexes by modifying DNA
conformation and recruiting nuclear proteins to the promoter. HMG-I(Y)
also enhance the DNA binding of NF-B (41). VIP and PACAP did not
affect either the nuclear expression or the DNA binding of HMG-I(Y) in
LPS-stimulated THP-1 cells.
VIP and PACAP act through three specific receptors, i.e.
VPAC1, VPAC2, and PAC1 (53). Human monocytes, including THP-1 cells, have been shown to express VIP/PACAP-binding sites (43), and our data
demonstrate that, similar to mouse macrophages (21, 25), THP-1 cells
express VPAC1 and PAC1 constitutively, and VPAC2 following LPS
stimulation. Although stimulated cells express all three receptors, our
studies using specific antagonists show that VPAC1 is the major
receptor involved in the VIP/PACAP regulation of all the previously
discussed aspects of NF-B-driven transcription.
Intracellular cAMP is the major secondary mediator induced by VPAC1
(53). However, in mos
B-dependent Gene Activation at Multiple Levels in
the Human Monocytic Cell Line THP-1*
§ and
TOP
ABSTRACT
INTRODUCTION
