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The Journal of Immunology, 2002, 169: 6474-6481.
Copyright © 2002 by The American Association of Immunologists

Proinflammatory Gene Induction by Platelet-Activating Factor Mediated Via Its Cognate Nuclear Receptor1

A. Marilise Marrache*,{ddagger}, Fernand Gobeil, Jr.{ddagger}, Sylvie G. Bernier{ddagger}, Jana Stankova§, Marek Rola-Pleszczynski§, Sanaa Choufani§, Ghassan Bkaily§, Annie Bourdeau, Martin G. Sirois, Alejandro Vazquez-Tello{ddagger}, Li Fan{ddagger}, Jean-Sébastien Joyal{ddagger}, Janos G. Filep||, Daya R. Varma*, Alfredo Ribeiro-da-Silva*,{dagger} and Sylvain Chemtob2,*,{ddagger}

Departments of * Pharmacology and Therapeutics and {dagger} Anatomy and Cell Biology, McGill University, Montreal, Quebec, Canada; {ddagger} Departments of Pediatrics, Ophthalmology, and Pharmacology, Research Center of Sainte-Justine Hospital, Montreal, Quebec, Canada; § Departments of Pediatrics and Cell Biology, Université de Sherbrooke, Sherbrooke, Quebec, Canada; Institut de Cardiologie de Montréal, Montreal, Quebec, Canada; and || Research Center of Maisonneuve-Rosemont Hospital, Montreal, Quebec, Canada.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
It has been postulated that intracellular binding sites for platelet-activating factor (PAF) contribute to proinflammatory responses to PAF. Isolated nuclei from porcine cerebral microvascular endothelial cells (PCECs) produced PAF-molecular species in response to H2O2. Using FACS analysis, we demonstrated the expression of PAF receptors on cell and nuclear surfaces of PCECs. Confocal microscopy studies performed on PCECs, Chinese hamster ovary cells stably overexpressing PAF receptors, and isolated nuclei from PCECs also showed a robust nuclear distribution of PAF receptors. Presence of PAF receptors at the cell nucleus was further revealed in brain endothelial cells by radioligand binding experiments, immunoblotting, and in situ in brain by immunoelectron microscopy. Stimulation of nuclei with methylcarbamate-PAF evoked a decrease in cAMP production and a pertussis toxin-sensitive rise in nuclear calcium, unlike observations in plasma membrane, which exhibited a pertussis toxin-insensitive elevation in inositol phosphates. Moreover, on isolated nuclei methylcarbamate-PAF evoked the expression of proinflammatory genes inducible nitric oxide synthase and cyclooxygenase-2 (COX-2) and was associated with augmented extracellular signal-regulated kinase 1/2 phosphorylation and NF-{kappa}B binding to the DNA consensus sequence. COX-2 expression was prevented by mitogen-activated protein kinase kinase/extracellular signal-regulated kinase 1/2 and NF-{kappa}B inhibitors. This study describes for the first time the nucleus as a putative organelle capable of generating PAF and expresses its receptor, which upon stimulation induces the expression of the proinflammatory gene COX-2.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Platelet-activating factor (PAF)3 is a potent proinflammatory lipid mediator that in the vascular system cooperates in the activation and recruitment of leukocytes by promoting their adhesion to endothelium. The effects of PAF are mediated through stimulation of a specific receptor. To date, higher mammals and humans have been found to express a single PAF receptor, which belongs to the G protein-coupled receptor (GPCR) superfamily. PAF receptors are distributed on platelets and numerous cells, notably leukocytes, smooth muscle, and endothelium (1). Depending on the cell type, PAF receptor may couple with Gi, Gs, or Gq proteins (2, 3, 4).

Although many responses induced by PAF can result from its interaction with cell surface GPCR, intracrine effects of PAF have also been proposed based largely on pharmacological binding studies (5). But the mechanisms by which PAF can elicit gene expression are unknown, as is the case with most ligands of GPCRs. Some mechanisms recently uncovered to explain GPCR-mediated gene induction implicate endocytosis-associated {beta}-arrestin-c-Src interaction leading to downstream activation of Ras and mitogen-activated protein kinases (MAPKs) and possibly metalloprotease-dependent transactivation of receptor tyrosine kinases involving de novo release of their ligand (6). Alternatively, PAF may exert intracellular actions by generating its own formation (7), inferring possible active intracellular binding sites (8, 9).

There is circumstantial evidence for an intracrine mode of action of PAF. Most newly generated PAF remains cell associated and retained within its producing cells as shown for endothelium and leukocytes (10, 11). Enzymes involved in the biosynthesis of PAF, namely cytosolic phospholipase A2 (cPLA2) and acetyltransferase, have been detected on the nuclear membrane (12, 13). Separate functions for the intracellular and cell surface receptors have been suggested using agents that putatively distinguish these receptors (8, 9). Specifically, immediate effects have been proposed to be mediated by cell surface receptors, whereas regulation of expression of specific genes may be dependent upon intracellular receptors, consistent with the presence of signaling effectors in nuclei, including G proteins (14, 15), ionic channels (16, 17), phospholipases (12, 18), adenylate cyclase (19), kinases (20), and NF-{kappa}B (21). However, the specific identification of intracellular PAF receptors as well as their physiological functions, especially on organelles, which may explain their presumed involvement in gene regulation (8, 22) in particular nuclei, has never been explicitly demonstrated. We hypothesized that PAF receptors may also exist at the cell nucleus where they would contribute in serving particular functions, specifically induction of major proinflammatory genes cyclooxygenase-2 (COX-2) and inducible NO synthase (iNOS). We focused on endothelium and confirmed observations in Chinese hamster ovary (CHO) cells. Our findings unveil 1) the generation of PAF-molecular species and presence of functional PAF receptors at the cell nucleus in situ (on freshly isolated tissue), 2) that stimulation of these nuclear receptors induces expression of major inflammatory immediate-early genes COX-2 and iNOS, and 3) that this induction is MAPK kinase (MEK)- and NF-{kappa}B-dependent. This study establishes an important new mechanism by which PAF regulates gene expression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture and PAF receptor transfection

Primary cultures of porcine cerebral microvascular endothelial cells (PCECs) were grown as described (23). CHO cells were maintained in 1:1 DMEM/Ham’s F-12 nutrient mixture containing 10% FBS, and cell transfection was performed (24). Briefly, human PAF receptor cDNA tagged with c-myc at the 5' end was subcloned into pcDNA3 (Invitrogen, San Diego, CA) expression vector resistant to geneticin. CHO cells (~2.5 x 105 cells/dish) were transiently transfected with the constructs using 2 µg of DNA and 2 µl of FuGENE6 (Roche Diagnostic Systems, Somerville, NJ). Cells were seeded 24 h after transfection and stable CHO cell transfectants (termed CDL8) were isolated by geneticin selection (1 mg/ml). Transfection efficiency was 60–70%.

Preparation of isolated nuclei

Nuclei were isolated from PCECs at 4°C, and the morphological integrity and purity (>98%) were assessed by electron microscopy (Fig. 1GoA) and by near absence of plasma membrane marker 5' nucleotidase (<7%; Sigma-Aldrich, St. Louis, MO) (25). The protein content was measured using a Bio-Rad assay system (Hercules, CA).



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  		FIGURE 1. PAF receptor expression in cerebrovascular endothelial cells. A, Electron micrograph of PCEC nuclear isolation. Bar = 5 µm. B, Cells were stained for surface or intracellular expression of PAF receptor and analyzed by FACS. For nuclear expression, nuclei were first purified and subsequently analyzed as above. Staining with anti-PAF receptor Ab (thick line tracing) and control IgG Ab (background; thin line tracing) is shown. RCN, Relative cell number. Graphs are representative tracings of three separate experiments.

 
PAF production

Isolated nuclei (100 µg) were stimulated with H2O2; the reaction was stopped by adding 4 vol of ethanol and the mixture was placed at -20°C for 1 h. PAF extraction was conducted (26) and PAF-molecular species were quantified by commercial kit (Amersham Pharmacia Biotech, Piscataway, NJ).

Flow cytometry

PAF receptor expression was determined by staining PCECs or nuclei (5 x 105) with a polyclonal goat anti-human PAF receptor Ab (1:25; Santa Cruz Biotechnology, Santa Cruz, CA) or with control normal goat IgG (1:50; Santa Cruz Biotechnology) for 30 min at 4°C in PBS containing 2% FBS. Staining with FITC-conjugated donkey anti-goat IgG (1:50; Cedarlane Laboratories, Hornby, Ontario, Canada) followed this procedure. For intracellular staining, cells were fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 for 15 min at 4°C before staining. The cognate blocking peptide (5-fold excess) was used to validate specificity of immunoreactivity. Cells were processed on a FACSCalibur (BD Biosciences, San Jose, CA) and results were analyzed by CellQuest software (BD Biosciences).

Radioligand binding

Radioligand binding was conducted as previously reported (5). Briefly, 1-O-octadecyl,[octadecyl-9,10-[3H](N)]-2-acetyl-sn-glyceryl-3-phosphorylcholine (specific activity, 160 Ci/mmol; NEN Life Science Products, Boston, MA) was used as labeled ligand, and 10 µM unlabeled methylcarbamate-PAF (C-PAF) (Biomol, Plymouth Meeting, PA), the nonmetabolizable form (27), was used for assessing nonspecific binding. Association-dissociation curve was achieved by incubating isolated nuclei with 1 nM [3H]PAF at different time intervals up to 20 min and was displaced with 1 µM C-PAF at 20 min. Saturation binding was initiated by adding resuspended nuclei (50 µg) to 100 µl of incubation mixture for 30 min at 25°C. Reactions were stopped with cold 10 mM Tris-HCl buffer and samples were filtered through GF/B glass microfiber filters (Whatman, Clifton, NJ). Binding specificity of [3H]PAF (5 nM) on nuclei was assessed by incubating nuclear suspension with closely related lipids: Lyso-PAF; CV 3988 (Biomol), a competitive PAF receptor antagonist; lysophosphatidic acid; and lysophosphatidylcholine (1 µM; Avanti Polar Lipids, Alabaster, AL). PCECs subject to binding identified cell surface PAF binding sites. Values for receptor density (Bmax) and affinity constant (KD) were determined (PRISM software, GraphPad, San Diego, CA).

Immunoblot analyses

Nuclei (100 µg) were treated with C-PAF (1 µM) for various time points for phospho-extracellular signal-regulated kinase (phospho-ERK1/2) immunoblot analyses (25). PAF receptor immunoblot (28) was conducted on nuclear and plasma membrane protein (50 µg) probed with rabbit anti-human PAF receptor (N terminus) Ab (1:10,000; Cayman Chemicals, Ann Arbor, MI). Detection was accomplished with a goat anti-rabbit IgG Ab conjugated with HRP (Pierce, Rockford, IL) and enhanced by chemiluminescence (ECL kit; PerkinElmer, Wellesley, MA).

PAF receptor localization by immunofluorescence

As previously described (29), PCECs were fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 for 15 min. A rabbit anti-human PAF receptor Ab (1:1000) incubation was followed by a goat anti-rabbit FITC-conjugated IgG (1:200) incubation. Nuclear staining was achieved with propidium iodide (PI) dye (3 µg/ml; Molecular Probes, Eugene, OR), and samples were mounted on glass slides with FluoroGuard (Bio-Rad). CDL8 cells were incubated with a mouse anti-Myc Ab (1:2) followed by a goat anti-mouse FITC-conjugated IgG incubation (1:200; Santa Cruz Biotechnology). Isolated nuclei were placed on glass slides previously coated with 3% 3'-aminopropyltriethoxysilane (Sigma-Aldrich) in acetone and treated as above. Samples were examined with an epifluorescence microscope (Axioskop II; Zeiss, Oberkochen, Germany) and a Multi Probe 2001 confocal argon laser scanning system (Molecular Dynamics, Sunnyvale, CA). The primary Ab was omitted for negative controls.

Electron microscopy of PAF receptors

For immunogold labeling, piglets were anesthetized with halothane (2%) and killed with pentobarbital (120 mg/kg, intracardiac) in accordance with regulations of the Canadian Council of Animal Care Committee and with approval of the Sainte-Justine Hospital Animal Care Committee. Animals were perfused via the right ventricle (dorsal aorta clamped) with 4% paraformaldehyde/0.1% glutaraldehyde/15% picric acid in 0.1 M phosphate buffer. This was followed with 4% paraformaldehyde/15% picric acid, and finally 10% sucrose. Brains were removed and immersed in 30% sucrose overnight at 4°C. Tissue sectioning and immunolabeling was done as described elsewhere (30). Rabbit anti-human PAF receptor (1:50) was incubated overnight at 4°C followed by another overnight incubation with goat anti-rabbit gold (1 nm)-conjugated IgG (1:50; British Biocell International, Cardiff, U.K.). After osmication and Epon flat-embedding and re-embedding of the selected areas, ultrathin sections were collected onto formvar-coated grids and contrast stained with uranyl acetate and lead nitrate. The ultrathin sections were observed using a transmission electron microscope (410 LS; Philips, Eindhoven, The Netherlands). In control sections, the primary Ab was preabsorbed with its cognate peptide (500 µg/ml; Cayman Chemicals).

cAMP and inositol phosphate production assays

Decreases in cAMP formation were tested on plasma membrane and nuclei initially stimulated with forskolin (1 µM; Alomone Labs, Jerasulem, Israel). Effects of C-PAF on cAMP generation were determined over 5–10 min at 37°C as described (31), and production was measured by radioimmunoassay kit (Diagnostic Products, Los Angeles, CA).

For inositol phosphate generation, membranes and isolated nuclei were incubated with 2 µCi/ml myo-[2-3H]inositol (Amersham Pharmacia Biotech) for 24–36 h. Thereafter, preparations were treated with C-PAF (100 nM), in the presence or absence of neomycin sulfate (65 µM; Calbiochem, La Jolla, CA) or pertussis toxin (PTX) (25 ng/ml) for 30 min at 37°C in DMEM without phenol red containing 20 mM LiCl/0.05% BSA for PCECs and in buffer (10 mM Tris-HCl, 0.05% BSA, 10 mM LiCl, 25 µM ATP, 90 mM KCl, 0.1 µM CaCl2, 10 mM NaCl, 3 mM MgCl2, 1 mM benzamidine, pH 7.4) for nuclei. Inositol phosphates were extracted on AG1-X8 columns (Bio-Rad).

Calcium signaling

Nuclear calcium mobilization was measured by a spectrofluorometer (PerkinElmer LS 50) and calcium concentration was calculated (32). Nuclei from PCECs, CHO, and CDL8 cells were resuspended in cytosolic-comparable buffer (25 mM HEPES, 125 mM KCl, 2 mM K2HPO4, 200 nM CaCl2, 4 mM MgCl2, pH 7.0). Nuclei were then loaded with 4 µM fura 2 and 0.05% pluronic F-127 (Calbiochem) for 45 min at 4°C. Nuclear suspension (~2 x 105 nuclei/ml) was incubated at 37°C and stimulated thereafter with C-PAF in the absence or presence (15 min before C-PAF) of preactivated PTX (20 µg/ml; Calbiochem) or PAF receptor blockers CV 3988 and PCA 4248 (Biomol) (33).

Stimulation of COX-2 and iNOS gene expression

PCEC nuclei (100–175 µg) were stimulated at 37°C with C-PAF in the absence or presence of CV 3988, PD98059 (1 µM; Calbiochem), and pyrrolidine dithiocarbamate (PDTC) (100 µM; Sigma-Aldrich) at different time points, and total nuclear RNA (nRNA) extraction followed. PCECs starved overnight with DMEM were preincubated with AACOCF3 (20 µM) or cytidine-5'-diphosphocholine (1 µM) (Calbiochem) in HEPES-Tyrode’s stimulation buffer (34) containing 0.1% BSA for 30 min followed by C-PAF (100 nM) incubation. Reactions were stopped by removing buffer and total RNA extracted by RNeasy Protect Mini Kit (Qiagen, Valencia, CA). For inhibition of receptor translocation treatments, C-PAF stimulation was done under potassium-depleted conditions (DMEM:water, 1:1). Total cellular RNA and nRNA (1 µg) were subject to RT-PCR for COX-2 (209 bp) and iNOS (417 bp) genes as described previously (35). Each PCR cycle was 94°C, 30 s; 58°C, 45 s; 72°C, 1 min and was repeated 40 times. 18S rRNA primer (324 bp; Ambion, Austin, TX) was used as internal standard; {beta}-actin (369 bp) was also used for some COX-2 experiments. Primer sequences were 5'-ATGATGTACGCCACAATCTGG-3' and 5'-GTTGAAAAGCAGCTCTGGGTC-3' for COX-2 and 5'-GAGGAGGATCCACMTCTAYCAGGARGARATG-3' and 5'-GGGGAATTCRTARTCYTCRACYTGYTCYTC-3' for iNOS (Sheldon Biotechnology Center, Montreal, Quebec, Canada). Quantification of RNA expression was analyzed by Bioanalyzer-Bio Sizing 2100 (Agilent Technologies, Palo Alto, CA).

Binding of NF-{kappa}B to DNA consensus sequence

PCEC nuclei were stimulated with C-PAF (0.1 µM) for 1 h. Nuclei were then lysed in high-salt buffer (400 mM NaCl) and centrifuged for 30 min at 21,000 x g. Nuclear extracts were dialyzed against buffer containing 20 mM HEPES, 20% glycerol, 100 mM KCl, 0.2 mM EDTA, 1 mM PMSF, 0.5 DTT, pH 7.8. Binding reactions were conducted in a mixture containing 5 mM HEPES, 80 mM KCl, 8% glycerol, 2 mM DTT, pH 7.8, 20 µg of nuclear extract, 2.5 µg of double-stranded poly(dI-dC), and 30 pmol of double-stranded [32P]-labeled oligonucleotide probe with the consensus NF-{kappa}B recognition sequence (5'-AGTTGAGGGGACTTTCCCAGGC-3'). Competition binding was done by incubating a 20- to 50-fold molar excess of nonradioactive double-stranded oligonucleotides, either wild type or mutated (5'-AGTTGAGCTCACTTTCCCAGGC-3'). After incubation (20 min at 30°C), the complexes were resolved by PAGE on 4% gel in 0.5x TBE buffer. Quantification was done by PhosphorImager (Molecular Dynamics).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Nuclear PAF synthesis and intracellular PAF receptor expression detected by flow cytometry

Because PLA2 contributes significantly in the synthesis of PAF (36), is activated by peroxides (37), and is found in nuclei (12), isolated nuclear preparations from PCECs were stimulated with H2O2. H2O2 evoked in nuclei net PAF production in a dose-dependent manner: 1 µM, 4.8 ± 1.5 pg/min/mg protein; 10 µM, 12.5 ± 2.9 pg/min/mg protein.

PCECs expressed PAF receptors at the plasma membrane (mean fluorescence intensity (MFI) = 5; 2.5-fold above background). Permeabilization of cells allowed detection of both cell surface and intracellular PAF receptors (MFI = 25; 6.25-fold above background). Purified nuclei also expressed PAF receptors (MFI = 12; 3-fold above background) (Fig. 1GoB)

[3H]PAF binding to subcellular fractions and immunoblot of PAF receptor

Maximal binding on plasma membrane fraction was higher than on nuclear fractions, whereas affinity constants for PAF in both preparations were similar (Fig. 2GoA). Nuclear binding sites for [3H]PAF appear to be specific for PAF surrogates (C-PAF, Lyso-PAF, and CV 3988), because neither lysophosphatidic acid nor lysophosphatidylcholine was able to displace the radioactive tracer (data not shown). Specific and saturable binding of [3H]PAF were detected on both plasma membrane and nuclear fractions. This binding reached equilibrium in 20 min at 25°C and was reversible (Fig. 2GoB), disclosing similar binding kinetics for PAF on nuclear and plasma membrane fractions (38). In addition, immunoblotting revealed a single PAF receptor-immunoreactive band at 48 kDa on both plasma membrane and nuclear preparations (Fig. 2GoC), consistent with its glycosylation form (39) and suggestive of the same intact protein at the nuclear level.



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  		FIGURE 2. [3H]PAF binding and PAF receptor immunoblot in nuclei of PCECs. A, Maximal specific binding (Bmax) and affinity constant (KD) of [3H]PAF binding on plasma membrane and nuclear fractions. Values are mean ± SEM of three experiments, each performed in duplicate. B, Saturation and dissociation of [3H]PAF binding on nuclear fraction. C, Immunoblot of PAF receptor on plasma membrane (PM) and nuclei (N) fractions migrating at consistent 48 kDa.

 
Nuclear localization of PAF receptors by immunofluorescence

The intracellular PAF receptor distribution in PCECs was identified by immunofluorescence (Fig. 3Go). PAF receptors in PCECs localized to the plasma membrane and to the nucleus (Fig. 3GoIA); PAF receptor immunodetection was confirmed in isolated nuclei of PCECs (Fig. 3GoIB). Merging of confocal micrographs (Fig. 3Go, IB and IC; PAF receptor Ab and PI) supported the presence of nuclear PAF receptors (Fig. 3GoID).



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  		FIGURE 3. I, Immunofluorescent staining of PAF receptor on permeabilized cerebrovascular endothelial cells. PAF receptors were stained with FITC (green) and nuclei were stained with PI (red) and visualized by epifluorescence (IA) or confocal microscopy (IB–ID). IA, Endothelial cells show robust PAF receptor immunoreactivity in nucleus. IB, PAF receptor labeling on isolated nuclei from endothelial cells. IC, Nuclear stain of IB. ID, Merged image of IB and IC showing nuclear localization of PAF receptor (depicted by a yellow-orange staining). Bar = 5 µm. II, Immunofluorescent staining of PAF receptor on permeabilized transfected CHO cells expressing the c-myc-tagged human PAF receptor (CDL8 cells). PAF receptors (detected with Myc Ab) were stained with FITC (green), and nuclei were stained with PI (red) and presented as three-dimensional reconstruction. IIA, PAF receptor labeling on plasma membrane and nuclear region (note nuclear distribution). IIB, Nuclear stain of IIA. IIC, Merged image of IIA and IIB. IID, Virtual staining absence in nontransfected CHO cells. IIE, PAF receptor labeling on nuclei of CDL8 cells. IIF, Nuclear stain of IIE. IIG, Merged image of IIE and IIF revealing colocalization (yellow-orange color). IIH, Absence of staining in nuclei of nontransfected CHO cells. Bar = 10 µm.

 
CHO cells transfected with c-myc-tagged PAF receptor, termed CDL8 cells, displayed similar results. PAF receptor immunostaining (detected with Myc Ab) was found on plasma membrane and nuclei (Fig. 3Go, IIA and IIC) of whole cells as well as on isolated nuclei (Fig. 3Go, IIE and IIG). Nontransfected CHO cells and nuclei (Fig. 3Go, IID and IIH) revealed virtually no staining; immunofluorescence was not detected in the absence of primary Abs (data not shown).

Perinuclear and intranuclear detection of PAF receptors by electron microscopy

Intracellular localization of PAF receptors was ascertained by immunogold electron microscopy on brain tissue from piglets. PAF receptor immunogold staining was identified on the plasma membrane (Fig. 4GoA) and nuclear envelope (Fig. 4GoB) of brain endothelial cells. Incidental immunostaining possibly associated with euchromatin was observed (Fig. 4GoB, inset). Neurons also exhibited consistently nuclear immunostaining for the PAF receptor (data not shown), whereas the abundant glia did not display PAF receptor immunoreactivity. Occasional cytoplasmic detection of PAF receptor was observed, which at higher magnification revealed predominant localization on vesicles as reported (28). No specific immunostaining was noted when the primary Ab was preabsorbed with its cognate peptide (Fig. 4GoC).



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  		FIGURE 4. In situ ultrastructural detection of PAF receptors on endothelial cells of porcine brain tissue sections by electron microscopy. PAF receptor immunoreactivity on plasma membrane (A; arrows) and on nuclear membrane (B; arrowheads), as well as on plasma membrane. C, Absence of immunogold labeling when the anti-PAF receptor Ab is incubated with its cognate peptide. Bar = 0.5 µm. L, Lumen of blood vessel; N, nucleus.

 
Signal transduction of PAF receptors in nuclei

PAF receptor may couple to various G proteins (Gi, Gs, or Gq) (2, 3, 4) affecting different second messengers. We measured cAMP and inositol phosphates in response to stimulation of cell surface and nuclear receptors with C-PAF. On plasma membrane, but not on nuclei, C-PAF significantly augmented inositol phosphate production in a PTX-insensitive manner. This effect was inhibited by an inhibitor of phospholipase C, neomycin sulfate (Fig. 5GoA), suggesting a Gq protein-coupling for the plasma membrane receptor. C-PAF did not cause cAMP generation on both plasma membrane and nuclei (Fig. 5GoA) of PCECs, but rather attenuated forskolin-induced cAMP only in nuclei, suggestive of Gi coupling.



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  		FIGURE 5. Signaling mediators of nuclear PAF receptors. Isolated nuclei were placed in cytosolic-comparable buffer (200 nM CaCl2) as described in Materials and Methods. A, cAMP and inositol phosphate production in plasma membrane and isolated nuclei of PCECs in absence and presence of PAF stimulation. B, Peak calcium transients in isolated nuclei of PCECs as a function of C-PAF concentration. C, Effects of C-PAF receptor antagonists, CV 3988 and PCA 4248, and of PTX on C-PAF-induced peak nuclear calcium transients. Calcium chelator EGTA (0.1 mM) caused similar prevention of C-PAF-induced peak calcium transients. D, Effects of C-PAF on nuclei of PAF receptor-overexpressing CHO cells (CDL8) and nontransfected CHO cells. Data are mean ± SEM of three experiments. *, p < 0.05 compared with bars without asterisks, by one-way ANOVA (factoring for concentration or agents, respectively A–C) or by two-way ANOVA (factoring for cell type and C-PAF concentration, for D) followed by Tukey-Kramer test.

 
We further elucidated factors coupled to the nuclear PAF receptor, and thus studied the effects of PAF on calcium transients. Various calcium pumps and channels have been identified on the nuclear envelope, including IP3, IP4, ryanodine receptors, and Ca2+-ATPase pump (16, 40). C-PAF caused a dose-dependent increase in peak calcium transients in isolated nuclei of PCECs (Fig. 5GoB). This latter effect was abolished by the membrane-impermeable calcium chelator EGTA (0.1 mM) and, more specifically, was diminished by distinct PAF receptor antagonists CV 3988 and PCA 4248 as well as by PTX (Fig. 5GoC), consistent with Gi/o coupling of the receptor (Fig. 5GoA). To ascertain that effects of C-PAF are dependent upon the PAF receptor expression as per receptor occupancy theory, experiments were also conducted on nuclei of CHO and CDL8 cells. C-PAF caused a modest increase in calcium transients in isolated nuclei of CHO cells, which was significantly augmented in nuclei of PAF receptor-overexpressing CDL8 cells (Fig. 5GoD).

Gene transcription by PAF in isolated nuclei

Because calcium plays an instrumental role in DNA replication and gene transcription (20), the functional significance of the PAF receptor at the nucleus also capable of generating its ligand locally was assessed by testing whether stimulation of isolated nuclei with PAF could modulate the inflammatory immediate-early genes COX-2 and iNOS. Stimulation of isolated nuclei of PCECs with C-PAF induced COX-2 and iNOS gene transcription. These effects were blocked by selective PAF receptor antagonist CV 3988 (Fig. 6Go, A and B). Control 18S rRNA expression was unaffected.



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  		FIGURE 6. Effects of C-PAF on iNOS and COX-2 gene transcription: role of ERK and NF-{kappa}B. Isolated nuclei from PCECs were stimulated with C-PAF in absence or presence of PAF receptor antagonist CV 3988, and total nRNA expression of iNOS (A) and COX-2 (B) was measured by RT-PCR. Results are expressed as ratio of gene:18S rRNA concentration. Internal control 18S rRNA as well as {beta}-actin RNA (used only for COX-2) expression did not change. Values are mean ± SEM of three experiments. *, p < 0.05 compared with time 0 value (two-way ANOVA, factoring for time and CV 3988 treatment). C, Western blot of phospho-ERK1/2 in response to C-PAF stimulation of isolated PCEC nuclei. D, Effect of MEK and NF-{kappa}B inhibitors PD98059 and PDTC on C-PAF-elicited COX-2 nRNA in isolated PCEC nuclei. Values are mean ± SEM of three experiments. *, p < 0.05 compared with control. E, Representative NF-{kappa}B-DNA binding after exposure of PCEC nuclei to C-PAF for 1 h (n = 3). F, Effects of internalization inhibitors, brefeldin A and hypotonic potassium-depleted solution, and of cPLA2 inhibitors, AACOCF3 and cytidine-5'-diphosphocholine, on C-PAF-induced COX-2 mRNA in PCECs. COX-2 mRNA was measured by RT-PCR; 18S rRNA was used as internal control and was unaffected by treatments. Values are mean ± SEM of three experiments. *, p < 0.05 compared with control (unstimulated); {dagger}, p < 0.05 compared with C-PAF alone.

 
The mechanisms for this C-PAF-induced COX-2 expression in isolated nuclei were further investigated. Kinases have been found to localize at the cell nucleus (20), are involved in immediate-early gene induction (41), and PAF has been reported to induce MAPK activation (42). C-PAF evoked a time-dependent ERK1/2 phosphorylation in isolated nuclei; total ERK remained unchanged (Fig. 6GoC). The MEK inhibitor PD98059 prevented COX-2 induction by C-PAF in isolated nuclei (Fig. 6GoD). Because MAPK can activate NF-{kappa}B (43), complexes of the latter can be found in the nucleus (21), and COX-2 gene promoter contains NF-{kappa}B binding sequences (44), we tested whether COX-2 expression induced by PAF stimulation is indeed NF-{kappa}B dependent. Stimulation of nuclei with C-PAF (1 h) augmented specific binding of NF-{kappa}B to its DNA consensus sequence (Fig. 6GoE), and NF-{kappa}B inhibitor PDTC prevented C-PAF-evoked rise in COX-2 nRNA in isolated nuclei (Fig. 6GoD). Both ERK1/2 phosphorylation and NF-{kappa}B binding to DNA preceded PAF-evoked COX-2 transcription, concordant with effects of inhibition of these pathways on COX-2 expression.

Because extracellular PAF evokes COX-2 expression (8) and the latter can also be induced by perinuclear stimulation, we attempted to determine the relative contribution of PAF receptor and ligand internalization and autoinduced PAF generation. Inhibitors of clathrin-dependent GPCR internalization, hypotonic potassium-depleted solutions, and brefeldin A (45) did not affect C-PAF-induced COX-2 expression in PCECs (Fig. 6GoF), whereas inhibition of the critical PAF-generating cPLA2 (36) using AACOCF3 and cytidine-5'-diphosphocholine diminished C-PAF-evoked COX-2 expression (Fig. 6GoF), suggesting a role for autostimulated PAF generation (7) in gene induction.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present data uncover the first clear evidence for intracellular, specifically nuclear functional PAF receptors, using a variety of techniques including binding, flow cytometry, immunoblotting, immunofluorescence of native receptors in primary endothelial cells as well as of tagged receptor expressed in recipient CHO cells and, more convincingly, in situ on endothelial cells and neurons of fixed brain. In addition, involvement of these nuclear receptors in stimulating PAF-evoked major proinflammatory genes COX-2 and iNOS is revealed. Finally, a number of signaling mediators of nuclear PAF receptor stimulation have been unveiled; second messengers coupled to nuclear and plasma membrane PAF receptors of the same cell type seem to differ.

Although the presence of some other GPCRs at the nucleus has been reported (29, 46, 47, 48, 49), functionality has only been documented for PGE2 and angiotensin II receptors (29, 46, 50); however, the latter peptide is not produced locally but possibly translocates to the nucleus as shown for parathyroid hormone and somatostatin (51, 52). In contrast, PAF could be generated locally and act on its adjacent receptor in the nucleus ( Figs. 1–4GoGoGoGo). The mechanisms for nuclear localization of PAF receptors are not known, but may involve nuclear localization signal(s) as reported for other GPCRs (53). Indeed a putative nuclear localization signal exists (KKFRKH298–303) at the C-terminal tail of PAF receptors (54). Endoplasmic reticulum retention sequences (55) present on PAF receptor C terminus (NSLK338–341) may also participate in PAF receptor nuclear localization. These sequences may also be implicated in vesicular trafficking (55) and translocation of receptors to the nucleus, as suggested for angiotensin II (56). However, deletion of these C-terminal segments does not alter PAF receptor localization (data not shown). Alternatively, PAF receptors can also undergo agonist-induced receptor internalization via clathrin-coated pits (57). But using classical experimental procedures that interfere with agonist-induced receptor internalization, PAF-induced COX-2 expression was not affected (Fig. 6GoF).

A noteworthy observation relates to signaling mechanisms that seem to differ for the cell surface and nuclear PAF receptors. Stimulation of the former was associated with a rise in inositol phosphate production suggestive of coupling to PTX-insensitve Gq protein, and activation of the latter was associated with a reduction in cAMP formation consistent with coupling to a PTX-sensitive Gi protein (Fig. 5GoA). These findings may imply distinct functions for the PAF receptor, depending on its localization as previously proposed (8, 9). In addition, C-PAF-induced COX-2 transcription in isolated nuclei seems to be MEK/ERK1/2- and NF-{kappa}B dependent (Fig. 6Go, C–E). MEK-1-dependent pathways have recently been reported in endothelial cells (58), and this protein kinase has been found localized at the nucleus (59), consistent with ERK1/2 phosphorylation (Fig. 6GoC). MAPK can in turn activate NF-{kappa}B (43), as seen with PAF (60, 61) and NF-{kappa}B is an important regulator of COX-2 expression (44). It may seem surprising that nuclear stimulation can lead to NF-{kappa}B activation since NF-{kappa}B is mostly complexed to its inhibitory unit I{kappa}B in cytoplasm, and upon stimulus-induced phosphorylation of I{kappa}B, NF-{kappa}B is released to enter the nucleus (62). But like NF-{kappa}B, (nondegraded) free I{kappa}B can also enter the nucleus (62, 63) and possibly reassociate with NF-{kappa}B. I{kappa}B and NF-{kappa}B contain nuclear localization sequences that may explain shuttling of these subunits between the nucleus and cytoplasm, thus having activatable NF-{kappa}B complexes within the nucleus (Fig. 6Go) (21), and in turn regulation of COX-2 transcription.

Different mechanisms have recently been reported to explain MAPK activation and gene induction by extracellular ligand stimulation of GPCRs. It has been shown that upon endothelin and muscarinic stimulation, an unknown matrix metalloprotease is activated to cleave a receptor tyrosine kinase agonist, which in turn leads to ERK phosphorylation (6). Another mechanism for GPCR-mediated MAPK activation documented for {beta}2-adrenergic receptors includes the formation of {beta}-arrestin-c-Src complexes, which during internalization can recruit other signaling molecules to phosphorylate ERK (6). However, our findings do not support these processes in COX-2 induction upon extracellular PAF stimulation (Fig. 6GoF). Metalloprotease inhibitor phenanthroline (data not shown) and blockers of internalization (Fig. 6GoF) did not affect C-PAF-induced COX-2 expression; notably, incorporation of choline-containing phospholipids such as PAF by scramblases is not per se involved in internalization (64). In contrast, contributions of autoinduced generation of PAF (7) are suggested in extracellular PAF-evoked COX-2 expression (Fig. 6GoF). Hence, because PAF formation is to a large extent cPLA2-dependent (36) and cPLA2 activation is associated with its nuclear translocation (12), it is conceivable to suggest based on data presented that PAF can be generated in the vicinity of its nuclear envelope receptor to elicit MAPK activation and COX-2 induction. The present study not only describes the nucleus (envelope) as a putative organelle for PAF generation and site expressing its functional target receptor, but it also provides a novel mechanism for MAPK and (proinflammatory) gene induction.


   Acknowledgments
 
We thank H. Fernandez for technical assistance, M. St-Louis and J. Ouellette for their guidance in electron microscopy sample preparation, S. Pinsonnault for indirect immunofluorescence analysis, and N. Tessier and E. Massicotte for flow cytometry. We would also like to acknowledge Les Fermes Ménard, Inc. (L’Ange Gardien, Quebec, Canada) for their supply of piglets.


   Footnotes
 
1 This work was supported by the Canadian Institutes of Health Research. A.M.M. and F.G. are recipients of studentship and fellowship awards, respectively, from that organization. S.C. is the recipient of a Canada Research Chair. Back

2 Address correspondence and reprint requests to Dr. Sylvain Chemtob, Departments of Pediatrics, Ophthalmology, and Pharmacology, Research Center of Sainte-Justine Hospital, 3175 Côte Sainte-Catherine, Montreal, Quebec, Canada H3T 1C5. E-mail address: sylvain.chemtob{at}umontreal.ca Back

3 Abbreviations used in this paper: PAF, platelet-activating factor; GPCR, G protein-coupled receptor; MAPK, mitogen-activated protein kinase; cPLA2, cytosolic phospholipase A2; COX-2, cyclooxygenase-2; iNOS, inducible NO synthase; CHO, Chinese hamster ovary; MEK, MAPK kinase; PCEC, porcine cerebral microvascular endothelial cell; C-PAF, methylcarbamate-PAF; ERK, extracellular signal-regulated kinase; PI, propidium iodide; PTX, pertussis toxin; PDTC, pyrrolidine dithiocarbamate; n, nuclear; MFI, mean fluorescence intensity. Back

Received for publication May 6, 2002. Accepted for publication September 23, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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