From the
The capability of platelet-activating factor (PAF) to induce
transcription factor activation was examined. In stably transfected
Chinese hamster ovary cells expressing the PAF receptor (CHO-PAFR), PAF
stimulation resulted in the nuclear expression of a DNA binding
activity with specificity to the
Platelet-activating factor (PAF;
1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine)
A
PAF receptor cDNA has been cloned from guinea pig (5) and human
(6-8) cells. The receptor has a seven-transmembrane helical
structure typical for a G protein-coupled receptor. Recent studies have
provided evidence that interaction of PAF to its receptor activates
several signaling pathways(9) . In many types of cells, PAF
stimulates phospholipid turnover via phospholipase C, A
This report describes results
from our study of NF-
Oligonucleotides and their complementary strands for EMSA were from
Promega (Madison, WI) and Santa Cruz Biotechnology. The sequences are:
murine intronic
Results presented in this report demonstrate that PAF is
capable of inducing
The
signaling pathways for PAFR-mediated cellular functions, especially
those related to PAF-induced NF-
Our results indicate that PAF-induced
NF-
The
finding that PAF-induced NF-
We thank Dr. Warner Greene for the gift of the
I
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
B sequence. The p50 and p65
proteins, constituents of the prototypic nuclear factor
B
(NF-
B), were identified as components of the DNA
protein
complexes by antipeptide antibodies in gel supershift as well as UV
cross-linking experiments. PAF induced an initial decrease and
subsequent increase of cytoplasmic I
B
levels, accompanied by
up-regulation of the I
B
messenger RNA, a feature of NF-
B
activation. PAF-induced
B binding activity was detected within 15
min after agonist stimulation, peaked at 30-40 min, and remained
detectable by 2.5 h. SR 27417, a PAF receptor antagonist, blocked
PAF-induced
B binding activity but not that induced by tumor
necrosis factor-
(TNF
). Cholera toxin treatment markedly
reduced PAF-induced
B binding activity, whereas pertussis toxin
had no significant inhibitory effect. Neither of the two toxins
affected the
B binding activity induced by TNF
in the same
cells. In addition to the CHO-PAFR cells, PAF stimulated
B binding
activity in the murine P388D
macrophage and the human ASK.0
B cell lines that express endogenous PAF receptors. These results imply
a potential role of PAF in the regulation of gene expression through a
G protein-coupled transcription factor activation pathway.
(
)is one of the most potent phospholipid agonists known to
date. It is produced by many types of cells, including macrophages,
platelets, basophils, neutrophils, eosinophils, and endothelial
cells(1, 2) . PAF has been shown to play an important
role in inflammatory and immune responses, as well as in heart and
vascular functions(3) . These actions of PAF are mediated mainly
through specific cell surface receptors, although accumulation of
intracellular PAF may also influence cell functions(4) .
,
and D pathways, activates GTPase activity and induces tyrosine
phosphorylation of several proteins. Furthermore, PAF stimulates the
mobilization of intracellular Ca
. Binding of PAF to
its specific receptor has been demonstrated to induce c-fos gene expression in epidermoid carcinoma A-431 (10) and
lymphoblastoid cells (11) and to stimulate transcription of
c-fos, c-jun, and the type 1 collagenase in rabbit
corneal epithelial cells(12) . Recently it has been found that
PAF activates the type 1 human immunodeficiency virus (HIV-1) long
terminal repeat (LTR) in transiently transfected MOLT-4 T-lymphocytic
cells, an effect that could be completely blocked by the PAF receptor
antagonist BN 52021(13) . Another PAF receptor antagonist,
RP55778, has been shown to inhibit HIV-1 expression induced by tumor
necrosis factor-
(TNF
), granulocyte-macrophage colony
stimulating factor, and interleukin-6 (14). These findings suggest a
function of PAF in the regulation of gene expression, although the
nature of the transcription factors involved in PAF-induced gene
expression has yet to be identified.
B as a transcription factor activated by PAF.
NF-
B is a multiprotein factor originally found to bind a decameric
enhancer sequence in the gene for the immunoglobulin
light
chain(15) . It has since been found that NF-
B is a
ubiquitous transcription factor whose DNA binding activity in cells not
expressing the
light chain is suppressed by association with a
cytoplasmic inhibitory protein, I
B(16, 17) .
Various stimuli can cause dissociation of the NF-
B-I
B complex
and the subsequent nuclear translocation of NF-
B, through
signaling pathways that are subjects of extensive
studies(18, 19, 20, 21, 22, 23, 24) .
NF-
B activation has been studied in inflammatory and
immunoregulatory cells (25); however, little is know about its
activation by G protein-coupled receptors. Here we demonstrate that PAF
induces
B binding activity in Chinese hamster ovary cells
expressing the PAF receptor and in murine P388D
macrophages
and human ASK.0 B cells.
Reagents
Platelet-activating factor (C-16) was
obtained from Calbiochem. The PAF antagonist SR 27417 was prepared in
Sanofi Recherche, Toulouse Cedex, France, as
described(26, 27) . [H]PAF was
purchased from DuPont NEN with a specific activity of 60 Ci/mmol.
Cholera and pertussis toxins were from List Laboratory (Campbell, CA).
Lipopolysaccharide (LPS) was isolated from lyophilized Salmonella
minnesota Re595 bacteria as described(28) .
[
-
P]ATP (>5000 Ci/mmol) was from
Amersham Corp. The plasmid pmTNF
(
)was used
for preparation of recombinant murine TNF
from Escherichia
coli. The specific activity of TNF
purified by ion-exchange
chromatography was 7
10
units/mg protein. Rabbit
polyclonal antibodies against the subunits of NF-
B/Rel were
purchased from Santa Cruz Biotechnology (Santa Cruz, CA). They were
raised against 1) a peptide corresponding to the basic NLS sequence and
the NH
-terminal adjacent 11 amino acids of the p105
precursor of the human p50 (anti-p50); 2) a peptide corresponding to
amino acids 3-19 of the human p65 (anti-p65); 3) a peptide
corresponding to carboxyl-terminal 19 amino acids of the human p52
(anti-p52); 4) a peptide corresponding to amino acids 152-176 of
the murine c-Rel protein (anti-c-Rel[N]); and 5) a peptide
corresponding to amino acids 531-550 mapping within the
carboxyl-terminal region of human p65 (anti-p65C). An antibody against
a COOH-terminal peptide (residues 289-317) of I
B
was a
gift from Dr. Warner C. Greene (University of California, San
Francisco, CA) (19). CHO-K1 and P338D
were from ATCC
(Rockville, MD); ASK.0 cells were kindly provided by Dr. William T.
Shearer (Baylor College of Medicine, Houston, TX). The mouse I
B
cDNA and the I
B-CAT constructs (23) were kindly provided by
Dr. Inder M. Verma (The Salk Institute, La Jolla, CA).
chain
B site (underlined),
5`-AGTTGAGGGGACTTTCCCAGGC-3`(NF-
B) (15) and a mutant
B
site with the G
C substitution (underlined) in the NF-
B DNA
binding motif, 5`-AGTTGAGGCGACTTTCCCAGGC-3`. The double-stranded
oligonucleotide (5 pmol) was
P-labeled with T4
polynucleotide kinase.
Cell Culture and Transfection
CHO-K1,
P388D, and ASK.0 cells were maintained in RPMI 1640
containing 10 mM HEPES, 2 mML-glutamine,
penicillin (100 IU/ml), streptomycin (50 µg/ml), and 10% fetal
bovine serum at 37 °C in a humidified 5% CO
environment. The cell line CHO-PAFR was generated by calcium
phosphate co-precipitation of 10 µg of linearized PAF cDNA
construct in the expression vector SFFV.neo (29) for 6 h with
70% confluent CHO-K1 cells. Stably transfected cells were selected
by G418 (500 µg/ml active drug), and 60-70 colonies were
pooled for assays. CHO-K1 cells transfected with human CD14 cDNA gene
(CHO-hCD14 cells) or with empty vector (CHO-RSV cells) were prepared
and have characteristics as described previously(30) . Cells
were serum-deprived in RPMI 1640 without G418 but supplemented with 10
mM HEPES and 2 mML-glutamine for
10-12 h prior to use.
PAF Binding Assays
Membranes were prepared from
CHO-PAFR cells as described(31) . Direct binding with
[H]PAF was carried out in duplicates with 100
µg of membrane proteins, at room temperature for 40 min,
essentially as described by Hwang et al.(32) . For
competition with the PAF receptor antagonist SR 27417, 2 nM [
H]PAF was used with the antagonist at
various concentrations. The binding data were processed with the
SigmaPlot (Jandel Scientific Software) and the LIGAND computer
programs(33) .
Preparation of Nuclear Extracts
Nuclear extracts
were prepared by a modified method of Dignam et
al.(34) . CHO-PAFR, CHO-RSV, and CHO-hCD14 cells were
separately plated at a density of 2 10
cells in
T-25 flasks, and after stimulation, cells were washed three times with
ice-cold phosphate-buffered saline, harvested, and resuspended in 0.4
ml of buffer A (10 mM HEPES, pH 7.9, 10 mM KCl, 0.1
mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol,
0.5 mM phenylmethylsulfonyl fluoride). After 10 min, 23 µl
of 10% Nonidet P-40 was added and mixed for 2 s. Nuclei were separated
from cytosol by centrifugation at 13,000
g for 10 s
and were resuspended in 50 µl of buffer B (20 mM HEPES, pH
7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 0.1
mM phenylmethylsulfonyl fluoride). After 30 min at 4 °C,
lysates were separated by centrifugation (13,000
g, 30
s), and supernatant containing nuclear proteins was transferred to new
vials. The protein concentration of extracts was measured using a
protein dye reagent (Bio-Rad) with bovine serum albumin as standard and
samples were diluted to equal concentration in buffer B for use
directly or storage at -80 °C.
EMSA
Electrophoretic mobility shift assays were
performed by incubating 2.5 µg of the nuclear extract in 12 µl
of binding buffer (5 mM HEPES, pH 7.8, 5 mM MgCl, 50 mM KCl, 0.5 mM dithiothreitol, 0.4 mg/ml poly(dI-dC) (Pharmacia Biotech Inc.),
0.1 mg/ml sonicated double-stranded salmon sperm DNA, and 10% glycerol)
for 10 min at room temperature. Then approximately 20-50 fmol of
P-labeled oligonucleotide probe (30,000-50,000 cpm)
was added, and the reaction mixture was incubated for 10 min at room
temperature. For reactions involving competitor oligonucleotides, the
unlabeled competitor and the labeled probes were premixed before
addition to the reaction mixture. For supershift assays, the reaction
mixture minus the probe was incubated with 2 µl of specific
antibodies for 20 min at room temperature. The
P-labeled
oligonucleotide was then added and incubation continued for 15 min. The
samples were analyzed on 5 or 6% acrylamide gels, which were made in 50
mM Tris borate buffer containing 1 mM EDTA (TBE) or
50 mM Tris, 380 mM glycine, 2 mM EDTA (TGE
buffer) and were pre-electrophoresed for 2 h at 12 V/cm.
Electrophoresis was carried out at the same voltage for 2-2.5 h.
Gel contents were transferred to Whatman DE-81 paper, dried, and
exposed for 3-5 h at -80 °C with an intensifying
screen. Using this method, a nonspecific DNA-protein complex of unknown
origin is sometimes seen in the autoradiograph.
Chloramphenicol Acetyltransferase (CAT) Assay
Five
micrograms each of the IB
promoter-CAT plasmids p0.2kb(WT)CAT
and p0.2kb(M)CAT (23) and the pSVLCAT plasmid (purchased form
Pharmacia and used as a positive control) were separately transfected
into CHO-PAFR cells, together with 1 µg of pCMV
plasmid
(Clonetech), by using the cationic lipid DOTAP (Boehringer Mannheim) as
recommended by the manufacturer. Seven hours after transfection, cells
were washed in serum-free medium, stimulated with PAF (10 nM)
or TNF (100 ng/ml) for 2 h, and collected for CAT assay. Some
transfected cells received no treatment. CAT activities were measured
in crude cellular extracts using
[
C]chloramphenicol (Amersham Corp.) as
substrate, followed by thin layer chromatography to separate the native
from acetylated forms as described(35) . The relative
transfection efficiency was determined by measurement of
-galactosidase activities in cell extracts from the co-transfected
pCMV
.
Ultraviolet (UV) Cross-linking
Analysis
Double-stranded P-radiolabeled
photoreactive oligonucleotide probe containing the
B site was
prepared as described(36, 37, 38) . Briefly, a
22-nucleotide single strand DNA spanning the murine intronic
chain
B site (sequence cited above) was annealed to a
complementary 16-mer oligonucleotide (5`-GCCTGGGAAAGTCCCC-3`), followed
by primer extension with DNA polymerase I (Klenow's fragment,
Exo(-), from Promega) in the presence of 1 mM 5`-bromo-2`-deoxyuridine 5`-triphosphate (BrdUrd), dCTP, and 100
µCi of [
-
P]dATP. UV cross-linking was
performed in solution by irradiation (300 nm, 7000 milliwatts/cm
illuminator, Fotodyne) of the respective binding reaction with 10
µg nuclear extract and [
P]BrdUrd
B-probe for 30 min. After UV cross-linking, the
oligonucleotide-protein adducts were boiled for 2 min in 0.5% SDS,
diluted 5-fold with TSN buffer (10 mM Tris-HCl, pH 8.0, 150
mM NaCl, 0.1% Nonidet P-40), and immunoprecipitated by
incubation at room temperature for 30 min with 5 µg of normal
rabbit IgG or anti-p50, anti-p65 (used as a mixture of the anti-p65 and
anti-p65C, 5 µg of each), and anti-c-Rel antibodies (the latter was
raised against a recombinant protein corresponding to the
amino-terminal 300 residues of the human c-Rel p75, with expected
cross-reactivity to p65; Santa Cruz Biotechnology). Finally, the
P-labeled products were directly analyzed by SDS-PAGE (8%
discontinuous gel) under reducing conditions. In parallel, a
[
P]BrdUrd probe-binding protein complexes were
UV cross-linked in native EMSA gel, excised, and analyzed by SDS-PAGE
as described(37, 39) .
Immunoblotting
Approximately 10 µg of
cytoplasmic extracts, collected after the Nonidet P-40 lysis and
centrifugation steps (see ``Preparation of Nuclear Extracts''
above), were mixed with loading dye, boiled, electrophoresed on a 10%
SDS gel, and transferred to Hybond-ECL nitrocellulose (Amersham).
Filter strips were incubated with primary antibody against the
IB
carboxyl terminus (1:2, 500 dilution) for 30 min at room
temperature, followed by addition of peroxidase-conjugated goat
anti-rabbit IgG at 1:10,000 for 30 min and analysis with enhanced
chemiluminescence reagents (DuPont NEN).
Functional Expression of the Human PAFR in CHO-K1
Cells
The full-length cDNA for the human PAFR (6) was
stably expressed in the CHO-K1 cells. No detectable PAF binding was
found in the untransfected cell line by direct binding with
[H]PAF (not shown). Membranes prepared from the
transfected cells (CHO-PAFR) displayed specific binding to
[
H]PAF with a dissociation constant of 3.96
nM (Fig. 1A); the binding was reduced in a
dose-dependent manner by treatment with the PAFR antagonist SR 27417 (27) (Fig. 1B). Similar binding data was obtained
by using intact cells (not shown). Furthermore, PAF stimulation induced
a calcium mobilization in CHO-PAFR with an EC
of 0.9
nM (data not shown). Thus, PAFR expressed in the transfected
CHO-PAFR cells appeared to have predicted binding and signaling
properties.
Figure 1:
Binding of [H]PAF
with membranes from CHO-PAFR and CHO-K1 cell. A, saturating
binding with membranes prepared from CHO-PAFR. Membrane binding assays
were performed as described under ``Experimental
Procedures.'' Data shown represent average of two experiments,
each measured in duplicate. Inset, Scatchard plot of the
specific binding data with the CHO-PAFR membranes. B,
competitive inhibition of [
H]PAF binding by the
PAFR antagonist SR 27417. The concentration of
[
H]PAF used in these assays was 2 nM,
and unlabeled PAF or SR 27417 was added for competition of binding.
Data shown represent the average from two separate measurements, each
in triplicate.
PAF-induced Nuclear Expression of a
NF-B DNA Binding
Activity
B has been shown to play an important role in
the transcription of genes for many inflammatory factors(25) .
We examined whether PAF, a proinflammatory mediator, is capable of
activating this transcription factor. Nuclear extracts were prepared
from CHO-PAFR and vector-transfected CHO cells after stimulation with
PAF or the control agonist TNF
, and the
B DNA binding
activity was examined by EMSA. Exposure of both CHO-PAFR and
vector-transfected cells to TNF
resulted in a marked increase in
the
B binding activity. In comparison, PAF significantly induced a
B binding activity in CHO-PAFR cells, but only weakly in
vector-transfected cells (Fig. 2A). One nanomolar PAF
was sufficient to produced a near full response, and further increase
in PAF concentration had no additional effect on the activation of
NF-
B (Fig. 2B). This concentration of PAF matched
that for calcium mobilization in CHO-PAFR cells (EC
= 0.9 nM). SR 27417, a PAF receptor
antagonist(27) , blocked PAF-induced
B binding activity but
had no effect on the same activity induced by TNF
(Fig. 2C). These results indicate that PAF-induced
B binding activity is mediated by the specific PAF receptor.
Figure 2:
B DNA binding activity induced by PAF
and TNF
. A,
B DNA binding activity in CHO-K1 cells
transfected with vector only (CHO-RSV, lanes 1-3) or
with the PAFR cDNA (CHO-PAFR, lanes 4-6). The cells were
stimulated with PAF (100 nM) or TNF
(40 ng/ml), as
described under ``Experimental Procedures.'' Nuclear extracts
prepared after 40 min of treatment were analyzed by EMSA, and the
autoradiograph is shown. B, dose response of PAF-induced
B DNA binding activity. The cells were stimulated for 40 min with
PAF at various concentrations before preparation of nucleoprotein
samples. C, the effect of SR 27417 (SR) on PAF- and
TNF
-induced
B binding activity. Following a 30-min incubation
with SR 27417 (10 nM), the cells were treated with 10 nM PAF or with 40 ng/ml TNF
for 30 min and the
B binding
activity was measured by EMSA. The
B DNA
protein complexes
are marked by brackets.
To
test the specificity of the observed DNA-protein interaction, excess
amount of unlabeled B oligonucleotide was included in the assay.
Unlabeled probe at 10- and 100-fold molar excess successfully competed
with the labeled probe (Fig. 3A, lane 2 and 3),
whereas a 100-fold molar excess of a point mutated oligonucleotide had
no effect (Fig. 3A, lane 4).
Figure 3:
Specificity of PAF-induced B binding
activity. A, nuclear extracts prepared from unstimulated (lane 5) or PAF-stimulated cells (50 nM for 40 min; lane 1-4) were used for analysis of the
B binding
activity by EMSA in the absence (lane 1) or presence (lanes 2-4) of competitive unlabeled oligonucleotides. Lanes 2 and 3, competition with an oligonucleotide
containing the
B consensus sequence, at 0.26 and 2.6 pmol,
respectively. Lane 4, competition with an oligonucleotide (2.6
pmol) containing a mutated, nonfunctional
B sequence. The
B
DNA
protein complex is marked by a bracket. B,
PAF and TNF
induced I
B
promoter-directed CAT gene
expression in transiently transfected CHO-PAFR cells. CHO-PAFR cells
were transfected with 5 µg of I
B
promoter-CAT reporter
plasmids (specified at the top) or 5 µg of pSVLCAT
plasmid, together with 1 µg of pCMV
plasmid (for monitoring
transfection efficiency). CAT activity was determined in these cells
after stimulation of PAF or TNF
as described under
``Experimental Procedures.'' The results shown here are
representative of three CAT assays. N.D., not
detectable.
It has been reported
that a B-like site (GGAAATTCCC) is present in the murine
I
B
promoter, and the p65 subunit of NF-
B stimulates
transcription from this promoter in transfected cells(23) . We
co-transfected the CHO-PAFR cells with two CAT constructs driven by the
B site of the I
B
promoter to determine whether PAF could
induce the I
B
promoter-directed CAT gene expression in
CHO-PAFR cells. As illustrated in Fig. 3B, both PAF and
TNF
(as a control) greatly increased the amount of CAT activity
produced in transiently transfected CHO-PAFR cells by the wild-type
I
B
promoter-CAT construct, but not in transfectants
containing the construct with a mutant
B site (CGAAATTAAT). These
data strong suggest that PAF induces a DNA binding activity with
characteristics of the NF-
B.
Presence of the p50 and p65 Subunits in PAF-induced
NF-
The prototypic NF-B DNA
protein Complexes
B is
a heterodimer with the 50 kDa (p50, NFKB1) and 65 kDa (p65, RelA)
protein subunits. Additional proteins have recently been identified
that belongs to the NF-
B/Rel family (reviewed in Ref. 40). We
performed gel supershift assays to determine whether the PAF-induced
DNA
protein complexes contain p50 and p65. With nuclear extracts
from PAF-stimulated cells, the anti-p50 and anti-p65 antibodies induced
a shift of the DNA
protein complexes (Fig. 4A, lanes 2 and 3). Similar results were obtained from cells
stimulated by TNF
(not shown). A quantitative analysis of the
samples revealed that only a small fraction of the DNA
protein
complexes was shifted by the addition of antibodies, possibly due to
the incapability of the antipeptide antibodies to recognize the
complexed proteins as reported in other cell
systems(41, 42) . To further examine the involvement of
p50 and p65, complexes formed with a photoreactive
B probe were UV
cross-linked in native EMSA gel, eluted, and analyzed by SDS-PAGE. This
resulted in three adducts, with relative molecular masses of
approximately 80, 60, and 45 kDa, respectively (Fig. 4B,
lanes 1 and 6). To determine whether p50 and p65 are
present in the DNA
protein complexes, UV cross-linked samples were
immunoprecipitated with antibodies against p50, p65, and c-Rel.
SDS-PAGE analysis of the samples (Fig. 4B, lanes
3-5) indicated recognition of the 60-kDa adduct by the
anti-p50 Ab, whereas the anti-p65 Ab immunoprecipitated a major species
of 80 kDa and a minor species of 45 kDa, respectively. The sizes of
these adducts are similar to those reported with cells from other
species(36, 43) . The 45-kDa minor species is likely to
be the proteolytic product of p65(37) . Similar patterns of
immunoprecipitation were observed in samples stimulated with TNF
(Fig. 4B, lanes 8-10). Thus, these results suggest
that PAF induces nuclear expression of NF-
B containing p50 and
p65.
Figure 4:
Identification of p50 and p65 in the
DNAprotein complexes induced by PAF. A, nuclear extracts
from cells stimulated with PAF (50 nM for 40 min) were
incubated in the presence of specific antibodies (2 µg/sample)
against members of the NF-
B/Rel protein family as noted below: lane 1, control; lanes 2-6, antibodies against
p50 (lane 2), p65 (lane 3), p52 (lane 4),
c-Rel NH
terminus (lane 5), and c-Rel COOH
terminus (lane 6). Samples were analyzed by EMSA (5% gel; TGE
buffer) using an oligonucleotide probe containing the consensus
B
sequence. Control, normal rabbit serum. The anti-p50 and anti-p65
antibodies induced shifts of 14.4 and 6.1% of the complexes,
respectively. B, UV cross-linking of nuclear proteins to
B-specific sequence.
P-Labeled probe containing
BrdUrd was incubated with nuclear extracts from PAF- and TNF-stimulated
CHO-PAFR cells (lanes 1 and 6, respectively).
DNA
protein complexes in EMSA gel were UV cross-linked, excised,
eluted, and resolved by SDS-PAGE. In separate experiments, UV
cross-linked samples were immunoprecipitated with nonspecific (Ctrl, shown in lanes 2 and 7) or specific
antibodies against three members of the NF
B/Rel family of proteins
as noted at the top of each lane and analyzed by SDS-PAGE. The
posi-tions of protein size markers are shown on the left side of the gel autoradiographs.
Kinetics of PAF-induced NF-
PAF
induced a rapid and sustained NF-B Activation
B activation, observed within 15
min and lasted for at least 2.5 h (Fig. 5, upper panel).
The appearance of the DNA
protein complex coincided with the
disappearance of I
B
, as detected by immunoblotting with an
anti-I
B
antibody (Fig. 5, lower panel). This
loss of I
B
was followed by its synthesis at 60 min and by 150
min I
B
reached the prestimulation level. Recently it was
reported that transcription of I
B
was stimulated by NF-
B
activation, and newly synthesized I
B
in turn inhibits
NF-
B
activity(18, 19, 20, 21, 22, 23, 24) .
Results from our experiments indicate that the messenger RNA for
I
B
was up-regulated by nearly 2.5-fold following PAF
stimulation for 60 min (data not shown), consistent with findings by
others using different ligand and cell
systems(18, 19, 21, 23) .
Figure 5:
Time-dependent stimulation of B
binding activity and degradation of I
B
. A,
time-dependent increase in
B binding activity in CHO-PAFR cells.
Nuclear extracts were prepared from cells stimulated with 10 nM PAF at the time points indicated and used for analysis of
B
binding activity by EMSA (5% gel; TBE buffer) and autoradiography. B, degradation of I
B
protein following PAF
stimulation. Cytoplasmic extracts (10 µg) were prepared from
CHO-PAFR cells stimulated with 10 nM PAF at the indicated time
points, resolved by SDS-PAGE, transferred to nitrocellulose membrane,
and detected with a rabbit antiserum specific for a carboxyl-terminal
peptide from I
B
using an enhanced chemiluminescence
assay.
Effects of Bacterial Toxins on PAF-induced NF-
PAFR has been shown to couple to G proteins that are
substrates for ADP-ribosylation catalyzed by pertussis toxin
(PT)(44) . To identify possible G protein-coupled pathways for
PAF-induced NF-B
Activity
B activation, we treated CHO-PAFR cells with
pertussis and cholera toxins (CT) separately. CT, at concentrations
from 0.2 µg/ml to 20 µg/ml, markedly reduced PAF-stimulated
activation of NF-
B (Fig. 6, lanes 6-8),
whereas PT treatment produced only a marginal inhibitory effect at a
relatively high concentration of 2 µg/ml (lanes
3-5). None of the toxins inhibited TNF
-induced
NF-
B activation in the same cells (lanes 9-11).
Figure 6:
Effect
of bacterial toxins on PAF-induced B binding activity. The
CHO-PAFR cells were pretreated with either cholera toxin (CT)
or pertussis toxin (PT) at indicated concentrations for 4 h in
the culture medium. The cells were then stimulated with ligands for 40
min, and nuclear extracts were analyzed by EMSA. The concentrations of
the toxins used with TNF
were 2 µg/ml (PT) and 20 µg/ml
(CT). The ligand concentrations were 10 nM (PAF) and
40 ng/ml (TNF
). Control, without ligand stimulation and
toxin treatment. The bracket marks the
B DNA
protein
complexes.
PAF Induced NF-
Taking into account the capability of PAF to
participate in inflammatory and immune responses, we examined the
possibility of PAF having an effect on NF-B Activation in Cells Expressing the
Endogenous PAFR
B activation in
macrophages and B lymphocytes. The murine P388D
macrophage
and the human ASK.0 B cell lines were chosen for both having been shown
to express high levels of functional PAF
receptors(45, 46) . The results, shown in Fig. 7,
demonstrated that stimulation of P388D
(Fig. 7A) and ASK.0 cells (Fig. 7B)
with PAF induced
B binding activity. It was notable that in both
cases the PAF-stimulated
B binding activity was significantly
reduced by pretreatment of the cells with PAF receptor antagonist SR
27417. In contrast, PAF failed to induce
B binding activity in the
murine RAW264.7 macrophages (not shown), which had no detectable
binding for [
H]PAF(45) . Thus, PAF-induced
NF-
B activation is not restricted to the CHO-PAFR cells and the
ability of PAF to induce
B binding activity appears to require the
cell surface expression of the PAF receptor.
Figure 7:
PAF-induced B binding activity in the
murine macrophage cell line P388D
and human B cell line
ASK.0. P388D
(A) and ASK.0 (B) cells were
stimulated with LPS (100 ng/ml), TNF
(40 ng/ml), or PMA (0.1
µM) for 40 min. In experiments in which the PAF receptor
antagonist SR 27417 (SR) were used, cells were preincubated
for 30 min with SR 27417 (10 nM) followed by stimulation with
10 nM of PAF for 40 min. Nuclear extracts from
agonist-stimulated cells were analyzed by EMSA. To reduce the
constitutive
B binding activity in B cell line, ASK.0 cells were
serum-deprived in RPMI 1640 medium supplemented with 10 mM HEPES and 2 mML-glutamine for 24 h prior to
use. The NF-
B DNA
protein complexes are marked by brackets.
LPS Does Not Induced NF-
LPS is a potent inducer of NF-B Activation in CHO-PAFR
Cells
B activation. In
inflammatory cells such as macrophages, LPS can up-regulate TNF
gene transcription through NF-
B activation(47) . It has
been reported that LPS can stimulate calcium mobilization via PAFR in
transfected CHO-K1 cells(48) . We therefore examined whether LPS
could induce NF-
B activation by binding to the PAFR in the
transfected cells. Under the conditions where PAF- and TNF
-induced
NF-
B could be readily detected, LPS did not induce any detectable
B binding activity at concentrations up to 50 µg/ml (Fig. 8). Thus, LPS did not seem to stimulate NF-
B
activation through binding of the expressed PAF receptor in transfected
CHO-K1 cells.
Figure 8:
Effect of LPS on NF-B activation in
CHO-RSV and CHO-PAFR cells. The cells were stimulated with LPS at
indicated concentrations, PAF (100 nM), or TNF
(40
ng/ml), as described under ``Experiential Procedures.''
Nuclear extracts prepared after 40 min of stimulation were analyzed by
EMSA (6% gel; TGE buffer) using an oligonucleotide probe containing the
decameric
B site. The
B DNA
protein complexes are marked
by a bracket.
B binding activity in CHO-PAFR cells with a
spectrum of characteristics observed during NF-
B activation in
various cell
systems(18, 19, 20, 21, 22, 23, 24) .
This function of PAF was previously unidentified, although PAF has been
shown to stimulate the transcription of the gene for the p150 subunit
of NF-
B(49) . NF-
B activation was elicited by PAF at
nanomolar concentrations and blocked by a specific PAFR antagonist.
Consistent with the cloning data that revealed PAFR as a G
protein-coupled receptor, cholera toxin inhibited PAF-induced NF-
B
activation. The totality of our findings suggest that PAF-induced
NF-
B activation is mediated by a specific cell surface receptor
through a G protein-coupled signaling pathway. PAF stimulation has been
shown to affect gene expression in a number of cases. Recently it was
reported that PAF activates HIV promoter in transfected cells
containing the HIV-LTR(13) . This action of PAF may also be
mediated by the cell surface PAF receptor through NF-
B activation,
because only exogenously applied PAF appeared to activate the HIV
promoter (13) which contains
B sites(50) . PAF could
also induce the expression of collagenase gene in corneal epithelial
cells(12) . This latter finding is in agreement with our
preliminary observation that PAF stimulates the activation of AP-1 in
CHO-PAFR.
(
)The existence of multiple mechanisms
for PAF stimulated gene transcription provides a partial explanation
for the diverse functions of this potent lipid mediator.
B activation, remain to be
investigated. PAFR is functionally coupled to a number of effector
systems, including phospholipase A
and phospholipase C,
through G proteins(51) . In platelets, PAFR couples to a G
protein whose activation reduces the adenylyl cyclase activity,
and PAF stimulation leads to ADP-ribosylation of the G
subunit (44, 52). PT inhibits PAF-induced arachidonic acid
release, PGE
formation, and inositol trisphosphate
production; but it does not affect the calcium mobilization in the
cells(53) . Honda et al. (54) recently reported
activation of MAP kinase in a CHO cells expressing the cloned PAFR.
They showed that MAP kinase activation was inhibited by 60% in
PT-treated cells. These results suggest a correlation between G
activation and PAFR-mediated signal transduction. Our finding
that CT, but not PT, inhibited PAF-induced NF-
B activation
provides evidence that PAFR may couple to more than one G protein for
its various functions. A study has been initiated to determine whether
PAF-stimulated NF-
B activation is mediated by CT-sensitive G
protein in other cells.
B activation in the CHO-PAFR cells was mediated by the specific
PAF receptor. The same receptor does not appear to mediate LPS-induced
NF-
B activation at concentrations up to 50 µg/ml. In a
separate study, we and others found that LPS at nanogram per milliliter
concentrations stimulated NF-
B activation in transfected CHO-K1
cells expressing CD14 (55).
(
)Thus, the CHO-K1
cell line appears to be responsive to LPS through interaction with a
site different from the PAFR. Several groups have shown that LPS could
potentiate certain functions of PAF by up-regulating the cell surface
PAF receptor expression(56, 57, 58) . Whether
this up-regulation results in an additive or synergistic effect on the
activation of NF-
B is currently under investigation.
B activation may have profound
implications. PAFR is expressed in a variety of cells and is believed
to mediate the diverse cellular actions of PAF by coupling to different
signaling pathways(9) . By activating NF-
B, PAF may
participate in the regulation of target gene expression under
physiological as well as pathological conditions. A more intriguing
possibility is that G protein-coupled receptors, to which PAFR belongs,
may play important roles in the regulation of gene expression in
addition to their classical cell activation functions. Further
characterization of these receptors as well as their target genes are
expected to extend our understanding of the molecular actions of PAF
and other relevant agonists for G protein-coupled receptors.
B, nuclear
factor
B; G proteins, guanine nucleotide-binding regulatory
proteins; TNF
, mouse tumor necrosis factor-
; HIV, human
immunodeficiency virus; LTR, long terminal repeat; LPS,
lipopolysaccharide; PT, pertussis toxin; CT, cholera toxin; EMSA,
electrophoretic mobility shift assay; CAT, chloramphenicol
acetyltransferase; BrdUrd, 5`-bromo-2`-deoxyuridine 5`-triphosphate;
CHO, Chinese hamster ovary; PAGE, polyacrylamide gel electrophoresis.
B
antibody, Dr. William Shearer for the ASK.0 cell line,
Drs. Inder Verma and Paul Chiao for the mouse I
B
cDNA probe
and I
B
promoter-CAT plasmids, Dr. Nigel Mackman for
encouragement and discussions, and members of his laboratory for
sharing their methodologies for EMSA and UV cross-linking. We also
thank Dr. William Sha for helpful suggestions and Dr. Eric Prossnitz
for assistance in quantitative analysis of gel shifting data.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.