©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Platelet-activating Factor Induces NF-B Activation through a G Protein-coupled Pathway (*)

Vladimir V. Kravchenko , Zhixing Pan , Jiahuai Han , Jean-Marc Herbert (1), Richard J. Ulevitch , Richard D. Ye (§)

From the (1)Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037 and Haemobiology Research Department Sanofi Recherche, Toulouse Cedex, France

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 B sequence. The p50 and p65 proteins, constituents of the prototypic nuclear factor B (NF-B), were identified as components of the DNAprotein complexes by antipeptide antibodies in gel supershift as well as UV cross-linking experiments. PAF induced an initial decrease and subsequent increase of cytoplasmic IB levels, accompanied by up-regulation of the IB 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.


INTRODUCTION

Platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine)()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) .

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, 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.

This report describes results from our study of NF-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, IB(16, 17) . Various stimuli can cause dissociation of the NF-B-IB 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.


EXPERIMENTAL PROCEDURES

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 IB 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 IB cDNA and the IB-CAT constructs (23) were kindly provided by Dr. Inder M. Verma (The Salk Institute, La Jolla, CA).

Oligonucleotides and their complementary strands for EMSA were from Promega (Madison, WI) and Santa Cruz Biotechnology. The sequences are: murine intronic chain B site (underlined), 5`-AGTTGAGGGGACTTTCCCAGGC-3`(NF-B) (15) and a mutant B site with the GC 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).


RESULTS

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 B DNA Binding Activity

NF-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 DNAprotein 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 DNAprotein complex is marked by a bracket. B, PAF and TNF induced IB promoter-directed CAT gene expression in transiently transfected CHO-PAFR cells. CHO-PAFR cells were transfected with 5 µg of IB 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 IB 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 IB promoter to determine whether PAF could induce the IB 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 IB 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-B DNAprotein Complexes

The prototypic NF-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 DNAprotein complexes contain p50 and p65. With nuclear extracts from PAF-stimulated cells, the anti-p50 and anti-p65 antibodies induced a shift of the DNAprotein 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 DNAprotein 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 DNAprotein 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). DNAprotein 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 NFB/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-B Activation

PAF induced a rapid and sustained NF-B activation, observed within 15 min and lasted for at least 2.5 h (Fig. 5, upper panel). The appearance of the DNAprotein complex coincided with the disappearance of IB, as detected by immunoblotting with an anti-IB antibody (Fig. 5, lower panel). This loss of IB was followed by its synthesis at 60 min and by 150 min IB reached the prestimulation level. Recently it was reported that transcription of IB was stimulated by NF-B activation, and newly synthesized IB in turn inhibits NF-B activity(18, 19, 20, 21, 22, 23, 24) . Results from our experiments indicate that the messenger RNA for IB 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 IB. 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 IB 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 IB using an enhanced chemiluminescence assay.



Effects of Bacterial Toxins on PAF-induced NF-B Activity

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 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 DNAprotein complexes.



PAF Induced NF-B Activation in Cells Expressing the Endogenous PAFR

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 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 DNAprotein complexes are marked by brackets.



LPS Does Not Induced NF-B Activation in CHO-PAFR Cells

LPS is a potent inducer of NF-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 DNAprotein complexes are marked by a bracket.




DISCUSSION

Results presented in this report demonstrate that PAF is capable of inducing 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.

The signaling pathways for PAFR-mediated cellular functions, especially those related to PAF-induced NF-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.

Our results indicate that PAF-induced NF-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.

The finding that PAF-induced NF-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.


FOOTNOTES

*
This work was supported by United States Public Health Service Grants AI15136 and GM37696 (to R. J. U.) and AI33503 and GM46572 (to R. D. Y.) and by American Heart Association Grant-in-aid 92-978 (to R. D. Y.). This is publication 8774-IMM from the Scripps Research Institute. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed: Dept. of Immunology, IMM-25, The Scripps Research Institute, La Jolla, CA 92037. Tel.: 619-554-8583; Fax: 619-554-8483.

The abbreviations used are: PAF, platelet-activating factor; PAFR, PAF receptor; NF-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.

V. V. Kravchenko and S. Nedospasov, manuscript in preparation.

V. V. Kravchenko, Z. Pan, J. Han, J.-M. Herbert, R. J. Ulevitch, and R. D. Ye, unpublished data.

V. V. Kravchenko, S. Steineman, L. Kline, and R. J. Ulevitch, submitted for publication.


ACKNOWLEDGEMENTS

We thank Dr. Warner Greene for the gift of the IB antibody, Dr. William Shearer for the ASK.0 cell line, Drs. Inder Verma and Paul Chiao for the mouse IB cDNA probe and IB 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.


REFERENCES
  1. Hanahan, D. J.(1986) Annu. Rev. Biochem.55, 483-509 [CrossRef][Medline] [Order article via Infotrieve]
  2. Snyder, F.(1990) Am. J. Physiol. Cell Physiol.259, C697-C708
  3. Prescott, S. M., Zimmerman, G. A., and McIntyre, T. M.(1990) J. Biol. Chem.265, 17381-17384 [Free Full Text]
  4. Hwang, S. B.(1990) J. Lipid Mediators2, 123-158 [Medline] [Order article via Infotrieve]
  5. Honda, Z., Nakamura, M., Miki, I., Minami, M., Watanabi, T., Seyama, Y., Okado, H., Toh, H., Ito, K., Miyamoto, T., and Shimizu, T.(1991) Nature349, 342-345 [CrossRef][Medline] [Order article via Infotrieve]
  6. Ye, R. D., Prossnitz, E. R., Zou, A., and Cochrane, C. G.(1991) Biochem. Biophys. Res. Commun.180, 105-111 [Medline] [Order article via Infotrieve]
  7. Nakamura, M., Honda, Z., Izumi, T., Sakanaka, C., Mutoh, H., Minami, M., Bito, H., Seyama, Y., Matsumoto, T., Noma, M., and Shimizu, T. (1991) J. Biol. Chem.266, 20400-20405 [Abstract/Free Full Text]
  8. Kunz, D., Gerard, N. P., and Gerard, C.(1992) J. Biol. Chem.267, 9101-9106 [Abstract/Free Full Text]
  9. Chao, W., and Olson, M. S.(1993) Biochem. J.292, 617-629 [Medline] [Order article via Infotrieve]
  10. Tripathi, Y. B., Lim, R. W., Fernandez-Gallardo, S., Kandala, J. C., Guntaka, R. V., and Shukla, S. D.(1992) Biochem. J.286, 527-533 [Medline] [Order article via Infotrieve]
  11. Schulam, G., Kuruvilla, A., Putcha, G., Mangus, L., Franklin-Johnson, J., and Shearer, W. T.(1991) J. Immunol.146, 1642-1648 [Abstract/Free Full Text]
  12. Bazan, H. E. P., Tao, Y., and Bazan, N. G.(1993) Proc. Natl. Acad. Sci. U. S. A.90, 8678-8682 [Abstract/Free Full Text]
  13. Squinto, S. P., Braquet, P., Block, A. L., and Bazan, N. G.(1990) J. Mol. Neurosci.2, 79-84 [Medline] [Order article via Infotrieve]
  14. Weissman, D., Poli, G., Bousseau, A., and Fauci, A. S.(1993) Proc. Natl. Acad. Sci. U. S. A.90, 2537-2541 [Abstract]
  15. Sen, R., and Baltimore, D.(1986) Cell46, 705-716 [Medline] [Order article via Infotrieve]
  16. Baeuerle, P. A., and Baltimore, D.(1988) Science242, 540-546 [Medline] [Order article via Infotrieve]
  17. Haskill, S., Beg, A. A., Tompkins, S. M., Morris, J. S., Yurochko, A. D., Sampson-Johannes, A., Mondal, K., Ralph, P., and Baldwin, A. S., Jr.(1991) Cell65, 1281-1289 [Medline] [Order article via Infotrieve]
  18. Brown, K., Park, S., Kanno, T., Franzoso, G., and Siebenlist, U.(1993) Proc. Natl. Acad. Sci. U. S. A.90, 2532-2536 [Abstract]
  19. Sun, S. C., Ganchi, P. A., Ballard, D. W., and Greene, W. C.(1993) Science259, 1912-1915 [Medline] [Order article via Infotrieve]
  20. Henkel, T., Machleidt, T., Alkalay, I., Krönke, M., Ben-Neriah, Y., and Baeuerle, P. A.(1993) Nature365, 182-185 [CrossRef][Medline] [Order article via Infotrieve]
  21. Scott, M. L., Fujita, T., Liou, H.-C., Nolan, G. P., and Baltimore, D. (1993) Genes & Dev.7, 1266-1276
  22. Rice, N. R., and Ernst, M. K.(1993) EMBO J.12, 4685-4695 [Abstract]
  23. Chiao, P. J., Miyamoto, S., and Verma, I. M.(1994) Proc. Natl. Acad. Sci. U. S. A.91, 28-32 [Abstract]
  24. Cheng, Q., Cant, C. A., Moll, T., Hofer-Warbinek, R., Wagner, E., Birnstiel, M. L., Bach, F. H., and de Martin, R.(1994) J. Biol. Chem.269, 13551-13557 [Abstract/Free Full Text]
  25. Baeuerle, P. A., and Henkel, T.(1994) Annu. Rev. Immunol.32, 141-179 [CrossRef]
  26. Herbert, J. M., Bernat, A., Valette, G., Gigo, V., Lale, A., Laplace, M. C., Lespy, L., Savi, P., Maffrand, J. P., and Le Fur, G.(1991) J. Pharmacol. Exp. Ther.259, 44-51 [Abstract]
  27. Herbert, J.-M.(1992) Biochem. J.284, 201-206 [Medline] [Order article via Infotrieve]
  28. Mathison, J. C., Wolfson, E., Tobias, P. S., and Ulevitch, R. J.(1993) J. Clin. Invest.92, 2053-2058 [Medline] [Order article via Infotrieve]
  29. Fuhlbrigge, R. C., Fine, S. M., Unanue, E. R., and Chaplin, D. D. (1988) Proc. Natl. Acad. Sci. U. S. A.85, 5649-5653 [Abstract]
  30. Kirkland, T. N., Finley, F., Leturcq, D., Moriarty, A., Lee, J. D., Ulevitch, R. J., and Tobias, P. S.(1993) J. Biol. Chem.268, 24818-24823 [Abstract/Free Full Text]
  31. Quehenberger, O., Prossnitz, E. R., Cavanagh, S. L., Cochrane, C. G., and Ye, R. D.(1993) J. Biol. Chem.268, 18167-18175 [Abstract/Free Full Text]
  32. Hwang, S. B., Lee, C. S., Cheah, M. J., and Shen, T. Y.(1983) Biochemistry22, 4756-4763 [Medline] [Order article via Infotrieve]
  33. Munson, P. J., and Rodbard, D.(1980) Anal. Biochem.107, 220-239 [Medline] [Order article via Infotrieve]
  34. Dignam, J. D., Lebovitz, R. M., and Roeder, R. G.(1983) Nucleic Acids Res.11, 1475-1489 [Abstract]
  35. Gorman, C., Moffat, L. F., and Howard, B. H.(1982) Mol. Cell. Biol.2, 1044-1051 [Medline] [Order article via Infotrieve]
  36. Kaufman, P. A., Weinberg, J. B., and Greene, W. C.(1992) J. Clin. Invest.90, 121-129 [Medline] [Order article via Infotrieve]
  37. Oeth, P. A., Parry, G. C., Kunsch, C., Nantermet, P., Rosen, C. A., and Mackman, N.(1994) Mol. Cell. Biol.14, 3772-3781 [Abstract]
  38. Beg, A. A., and Baldwin, A. S., Jr.(1994) Oncogene9, 1487-1492 [Medline] [Order article via Infotrieve]
  39. Fraser, J. K., Tran, S., Nimer, S. D., and Gasson, J. C.(1994) Blood84, 2523-2530 [Abstract/Free Full Text]
  40. Liou, H. C., and Baltimore, D.(1993) Curr. Biol.5, 477-487
  41. Tan, T.-H., Huang, G. P., Sica, A., Ghosh, P., Young, H. A., Longo, D. L., and Rice, N. R.(1992) Mol. Cell. Biol.12, 4067-4075 [Abstract]
  42. Fujita, T., Nolan, G. P., Ghosh, S., and Baltimore, D.(1992) Genes & Dev.6, 775-787
  43. Molitor, J. A., Walker, W. H., Doerre, S., Ballard, D. W., and Greene, W. C.(1990) Proc. Natl. Acad. Sci. U. S. A.87, 10028-10032 [Abstract]
  44. Lad, P. M., Olson, C. V., and Grewal, I. S.(1985) Biochem. Biophys. Res. Commun.129, 632-638 [Medline] [Order article via Infotrieve]
  45. Valone, F. H.(1988) J. Immunol.140, 2389-2394 [Abstract/Free Full Text]
  46. Kuruvilla, A., Putcha, G., and Shearer, W. T.(1991) Biochem. Biophys. Res. Commun.180, 1318-1324 [Medline] [Order article via Infotrieve]
  47. Muller, J. M., Ziegler-Heitbrock, H. W., and Baeuerle, P. A. (1993) Immunobiology187, 233-256 [Medline] [Order article via Infotrieve]
  48. Nakamura, M., Honda, Z., Waga, I., Matsumoto, T., Noma, M., and Shimizu, T.(1992) FEBS Lett.314, 125-129 [CrossRef][Medline] [Order article via Infotrieve]
  49. Tan, X., Sun, X., Gonzalez-Crussi, F. X., Gonzalez-Crussi, F., and Hsueh, W.(1993) FASEB J.7, A216-A216 (abstr.)
  50. Nabel, G., and Baltimore, D.(1987) Nature326, 711-713 [CrossRef][Medline] [Order article via Infotrieve]
  51. Shukla, S. D.(1992) FASEB J.6, 2296-2301 [Abstract/Free Full Text]
  52. Avodonin, P. V., Svitina-Ulitina, I. V., and Kulikov, V. I.(1985) Biochem. Biophys. Res. Commun.131, 307-313 [Medline] [Order article via Infotrieve]
  53. Schlondorff, D., Singhal, P., Hassid, A., Satriano, J. A., and DeCandido, S.(1989) Am. J. Physiol.256, F171-F178
  54. Honda, Z., Takano, T., Gotoh, Y., Nishida, E., Ito, K., and Shimizu, T. (1994) J. Biol. Chem.269, 2307-2315 [Abstract/Free Full Text]
  55. Delude, R. L., Fenton, M. J., Savedra, R., Jr., Perera, P.-Y., Vogel, S. N., Thieringer, R., and Golenbock, D. T.(1994) J. Biol. Chem.269, 22253-22260 [Abstract/Free Full Text]
  56. Glaser, K. B., Asmis, R., and Dennis, E. A.(1990) J. Biol. Chem.265, 8658-8664 [Abstract/Free Full Text]
  57. Liu, H., Chao, W., and Olson, M. S.(1992) J. Biol. Chem.267, 20811-20819 [Abstract/Free Full Text]
  58. Aepfelbacher, M., Ziegler-Heitbrock, H. W., Lux, I., and Weber, P. C. (1992) J. Immunol.148, 2186-2193 [Abstract/Free Full Text]

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