Am J Physiol Cell Physiol 295: C1427-C1433, 2008.
First published September 24, 2008; doi:10.1152/ajpcell.00218.2008
0363-6143/08 $8.00
RECEPTORS AND SIGNAL TRANSDUCTION
Essential role of EP3 subtype in prostaglandin E2-induced adhesion of mouse cultured and peritoneal mast cells to the Arg-Gly-Asp-enriched matrix
Mariko Sakanaka,1
Satoshi Tanaka,1,2
Yukihiko Sugimoto,3 and
Atsushi Ichikawa2
1Department of Immunobiology, School of Pharmacy and Pharmaceutical Sciences, and 2Institute for Biosciences, Mukogawa Women's University, Koshien, Nishinomiya, Hyogo; and 3Department of Physiological Chemistry, Graduate School of Pharmaceutical Sciences, Kyoto University, Yoshida, Sakyo-ku, Kyoto, Japan
Submitted 22 April 2008
; accepted in final form 18 September 2008
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ABSTRACT
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Accumulating evidence has indicated that mast cells can modulate a wide variety of immune responses. Migration and adhesion play a critical role in regulation of tissue mast cell function, in particular, under inflammatory conditions. We previously demonstrated that prostaglandin (PG) E2 stimulates adhesion of a mouse mastocytoma cell line, P-815, to the Arg-Gly-Asp (RGD)-enriched matrix through cooperation between two PGE2 receptor subtypes: EP3 and EP4 (Hatae N, Kita A, Tanaka S, Sugimoto Y, Ichikawa A. J Biol Chem 278: 17977–17981, 2003). We here investigated PGE2-induced adhesion of IL-3-dependent bone marrow-derived cultured mast cells (BMMCs). In contrast to the elevated cAMP-dependent adhesion of P-815 cells, EP3-mediated Ca2+ mobilization plays a pivotal role in PGE2-induced adhesion of BMMCs. Adhesion and Ca2+ mobilization induced by PGE2 were abolished in the Ptger3–/– BMMCs and were significantly suppressed by treatment with pertussis toxin, a phospholipase C inhibitor, U-73122, and a store-operated Ca2+ channel inhibitor, SKF 36965, indicating the involvement of Gi-mediated Ca2+ influx. We then investigated PGE2-induced adhesion of peritoneal mast cells to the RGD-enriched matrix. EP3 subtype was found to be the dominant PGE receptor that expresses in mouse peritoneal mast cells. PGE2 induced adhesion of the peritoneal mast cells of the Ptger3+/+ mice, but not that of the Ptger3–/– mice. In rat peritoneal mast cells, PGE2 or an EP3 agonist stimulated both Ca2+ mobilization and adhesion to the RGD-enriched matrix. These results suggested that the EP3 subtype plays a pivotal role in PGE2-induced adhesion of murine mast cells to the RGD-enriched matrix through Ca2+ mobilization.
Gi; Ca2+ mobilization; store-operated Ca2+ channel; bone marrow-derived cultured mast cells
MAST CELL IS GENERALLY REGARDED as a key effector cell in parasite infection and anaphylactic response. However, accumulating evidence suggests that mast cell plays critical roles in much broader ranges of immune responses, such as peripheral tolerance and bacterial infection (19, 20). Migration and adhesion play critical roles in regulation of mast cell functions in tissues, in particular under emergency conditions. Previous studies have indicated that mast cells are often accumulated in inflamed tissues, such as in the airways in atopic asthma and synovial tissues in rheumatoid arthritis.
Prostaglandin E2 (PGE2) is one of the major eicosanoids produced during inflammatory responses. Although mast cells are not the major source of PGE2, a wide variety of cells that can generate PGE2 are found in close proximity of mast cells, such as smooth muscle cells, respiratory epithelial cell, fibroblasts, and macrophages (2, 4, 9, 13). Based on these findings, we hypothesized that PGE2 can affect migration and adhesion of tissue mast cells by acting on the specific receptors. Using a mouse mastocytoma, P-815, our laboratory previously demonstrated that PGE2 induces the adhesion to the Arg-Gly-Asp (RGD)-enriched matrix through elevation of cAMP levels, which results from cooperation between two PGE2 receptor subtypes: EP3 and EP4 (7). EP3 subtype is known to be coupled to multiple G proteins, such as Gi, Gs, and G12 (17, 23). In P-815 cells, augmentation of the EP4-mediated cAMP generation by the EP3 subtype was suppressed by treatment with pertussis toxin (PTX), indicating that the EP3 subtype is coupled to Gi and liberation of β
-subunit may be involved in augmented cAMP generation. Very recently, Weller et al. demonstrated that PGE2 can function as a chemotactic factor of mast cells by acting on the EP3 subtype (28). Although the EP4 subtype is expressed in bone marrow-derived cultured mast cells (BMMCs), PGE2-induced chemotaxis is mediated solely by the EP3 subtype; PGE2-induced chemotactic responses were suppressed by treatment with PTX and mimicked by an EP1/EP3 agonist. Since accumulating evidence suggests that the dominant receptor that is responsible for PGE2-induced responses in mast cells depends on the species and tissue localization (3), it is possible that the EP4-independent pathway is involved in PGE2-induced adhesion of BMMCs. We here characterized the PGE2-induced adhesion of BMMCs and peritoneal mast cells to the RGD-enriched matrix and found that the EP3 subtype plays a dominant role in adhesion of BMMCs and peritoneal cells.
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MATERIALS AND METHODS
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Animals.
Specific-pathogen-free, 6-wk-old female C57BL/6 mice were obtained from Japan SLC (Hamamatsu, Japan). The Ptger3–/– mice (25) were back-crossed for at least eight generations to the C57BL/6 background. This study was approved by the Committee on Animal Experiments of Mukogawa Women's University and conformed with the Guiding Principles in the Care and Use of Animals of the American Physiological Society.
Materials.
Sulprostone was a generous gift from Dr. M. P. L. Caton of Rhone-Poulenc. ONO-AE-248 and ONO-AE1-437 were generously supplied by ONO Pharmaceuticals (Osaka, Japan). ProNectin F (ProF) and ProNectin L (ProL) were generously supplied by Sanyo Chemical Industries (Kyoto, Japan). The following materials were obtained from the sources indicated: GRGDS peptide from PEPTIDE Institute (Osaka, Japan); Cellmatrix (type I-P collagen) from Nitta Gelatin (Osaka, Japan); Histodenz, bovine serum albumin (BSA), thapsigargin, and an anti-dinitrophenyl IgE (clone SPE-7) from Sigma-Aldrich (St. Louis, MO); PTX from Seikagaku (Tokyo, Japan); PGE2 from Cayman Chemical (Ann Arbor, MI); 12-O-tetradecanoyl-phorbol 13-acetate (TPA) and Gö6976 from Calbiochem (San Diego, CA); SKF 96365 from TOCRIS (Bristol, UK); thrombin from bovine plasma and fura-2 AM from NACALAI TESQUE, (Kyoto, Japan); U-73343 and U-73122 from BIOMOL (Exeter, UK); RNeasy Mini Kit from QIAGEN (Valencia, CA); Moloney murine leukemia virus (MMLV) reverse transcriptase from Invitrogen (Carlsbad, CA); and Taq DNA polymerase from TOYOBO (Osaka, Japan). All other chemicals were commercial products of reagent grade.
Preparation of BMMCs.
BMMCs were prepared as described previously with a minor modification (24). Bone marrow cells were cultured in RPMI-1640 containing 10% heat-inactivated FBS, 50 µM β-mercaptoethanol, and 0.1 mM nonessential amino acids at 37°C in a fully humidified 5% CO2 atmosphere for 4–5 wk in the presence of 10 ng/ml IL-3 instead of WEHI-3-conditioned medium. Greater than 95% of the viable cells were confirmed to be immature mast cells, as assessed by staining with acidic toluidine blue (pH 2.5).
Measurement of cell adhesion.
Twelve-well culture plates were coated with 10 µg/ml ProF, 10 µg/ml ProL, 0.3 mg/ml Cellmatrix, or 3% BSA. BMMCs were seeded on the well at a density of 1 x 106 cells·ml–1·well–1 and incubated in the medium containing 10 µM indomethacin. The adherent cells were recovered by treatment with trypsin. Cell number of the adherent and nonadherent cells was counted using the COULTER Z2 cell counter (Beckman Coulter, Fullerton, CA). Percentages of the adherent cells were calculated according to the following formula: cell adhesion (%) = the number of adherent cells x 100/(the number of adherent cells + the number of nonadherent cells).
Measurement of cytosolic Ca2+ concentrations.
Cytosolic Ca2+ concentration was measured with fura-2 AM, as previously described (24). Fluorescent intensities were measured, at an excitation wavelength of 340 or 380 nm and an emission wavelength of 510 nm, with a fluorescence spectrometer (CAF-110, Jasco, Tokyo, Japan).
Purification of peritoneal mast cells.
Mouse peritoneal cells were recovered with Tyrode-HEPES buffer (130 mM NaCl, 5 mM KCl, 1.4 mM CaCl2, 1 mM MgCl2, 5.6 mM glucose, 10 mM HEPES-NaOH, pH 7.3) containing 0.1% gelatin (THG buffer) and centrifuged for 5 min at 800 g. The peritoneal cells were suspended in THG buffer, layered onto gradient solution containing 25% (wt/vol) Histodenz, and centrifuged for 15 min at 500 g. The resultant cell pellet was twice washed with PBS to obtain purified peritoneal mast cells (>90% purity, confirmed by Alcian blue/Safranin-O staining). In the case of purification of rat peritoneal mast cells, 0.05% gelatin and 22.5% (wt/vol) Histodenz were alternatively used, and density gradient separation was performed at 45 g.
RT-PCR.
Total RNAs were isolated from BMMCs or the purified peritoneal mast cells using RNeasy Mini Kit, according to the manufacturer's instructions. Total RNAs were reverse transcribed using MMLV reverse transcriptase and amplified by PCR using a primer offset for each PGE receptor subtypes: EP1 (1,014 bp), 5'-ACC CTG CAT CCT GAG CAG CAC TGG CCC TCT-3' (forward) and 5'-CGA TGG CCA ACA CCA CCA ACA CCA GCA GGG-3' (reverse); EP2 (604 bp), 5'-TTC ATA TTC AAG AAA CCA GAC CCT GGT GGC-3' (forward) and 5'-AGG GAA GAG GTT TCA TCC ATG TAG GCA AAG-3' (reverse); EP3 (608 bp), 5'-ATC CTC GTG TAC CTG TCA CAG CGA CGC TGG-3' (forward) and 5' TGC TCA ACC GAC ATC TGA TTG AAG ATC ATT-3' (reverse); EP4 (601 bp), 5'-GAC TGG ACC ACC AAC GTA ACG GCC TAC GCC-3' (forward) and 5'-ATG TCC TCC GAC TCT CTG AGC AGT GCT GGG-3' (reverse).
Measurement of peritoneal mast cell adhesion.
Eight-well chamber slides (AGC Techno glass, Funahashi, Japan) were coated with 10 µg/ml ProF or 3% BSA. Peritoneal cells were collected in Tyrode-HEPES buffer containing 0.1% BSA and resuspended in RPMI-1640 medium containing 10% heat-inactivated FBS. Peritoneal cells were seeded on the chamber in the presence of 10 µM indomethacin at a density of 1 x 105 cells·0.5 ml–1·well–1 (mouse peritoneal cells) or of 1 x 104 cells·0.5 ml–1·well–1 (rat peritoneal mast cells). The cells were incubated for 30 min at 37°C before stimulation. The adherent cells were fixed by 100 mM sodium phosphate, pH 7.4, containing 3% sucrose, 2% paraformaldehyde, and 0.1% glutaraldehyde, and stained with the acidic toluidine blue. Percentages of mast cells in the total peritoneal cells were determined based on the count of the cells stained with 2% Giemsa solution.
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RESULTS
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PGE2-induced adhesion of BMMCs to ProF.
Our laboratory previously reported the PGE2-induced adhesion of P-815 cells to an RGD-enriched matrix, ProF (7). In the present study, we investigated the effects of PGE2 on adhesion activity of BMMCs. PGE2 significantly augmented the adhesion of BMMCs to ProF or ProL, but not to type I collagen or BSA (Fig. 1A). The stimulated adhesion to ProF was suppressed in the presence of the GRGDS peptide (Fig. 1B), indicating that BMMCs adhere to ProF via the RGD sequence as well as P-815 cells did. The PGE2-induced adhesion to ProF was a saturable and concentration-dependent response (Fig. 1C).
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Fig. 1. Prostaglandin E2 (PGE2)-induced adhesion of bone marrow-derived cultured mast cells (BMMCs). A: BMMCs were incubated with (solid bars) or without (open bars) 0.1 µM PGE2 for 1 h in the wells coated with ProNectin F (ProF), ProNectin L (ProL), type I collagen (collagen I), or bovine serum albumin (BSA). The value of **P < 0.01 is regarded as significant by Student's t-test (vs. without PGE2). B: BMMCs were stimulated with (solid bars) or without (open bars) 0.1 µM PGE2 for 1 h in the wells coated with ProF in the presence (GRGDS) or absence (none) of 200 µM GRGDS peptide. The value of **P < 0.01 is regarded as significant by Student's t-test (vs. none). C: BMMCs were incubated for 1 h in the wells coated with ProF in the presence of the indicated concentrations of PGE2. Values are presented as means ± SE; n = 3 (A), n = 4 (B), n = 3 (C).
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EP3 subtype is essential for PGE2-induced adhesion and Ca2+ mobilization.
We then investigated the time course of adhesion of BMMCs to ProF. PGE2-induced adhesion was transient, and the adhesion level returned to the base level 6 h after the stimulation (Fig. 2A). PGE2-induced adhesion was well mimicked by sulprostone (EP1 and EP3 agonist) and ONO-AE-248 (EP3 agonist), but not by ONO-AE-1-437 (EP4 agonist), indicating the involvement of the EP3 subtype. We, therefore, investigated the adhesion using BMMCs derived from the Ptger3–/– mice. PGE2-induced adhesion was abolished in the Ptger3–/– BMMCs (Fig. 2B). No significant difference between the Ptger3+/+ and Ptger3–/– BMMCs was found in adhesion to ProF stimulated with TPA or monomeric IgE (16), indicating that the machinery required for the adhesion to ProF is not impaired in the Ptger3–/– BMMCs (Fig. 2, C and D). BMMCs from the Ptger3+/+ mice were found to express both the EP3 and EP4 subtype mRNAs, as previously reported (Fig. 2E).
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Fig. 2. EP3 subtype is essential for PGE2-induced adhesion of BMMCs. A and B: time courses of the adhesion response of the Ptger3+/+ (A) or Ptger3–/– (B) BMMCs are presented. BMMCs were incubated in the wells coated with ProF for the periods indicated in the presence of vehicle alone (EtOH), 0.1 µM PGE2 (PGE2), 0.1 µM sulprostone (Sulp), 1 µM ONO-AE-248 (EP3-A), or 1 µM ONO-AE-1-437 (EP4-A). C and D: the Ptger3+/+ or Ptger3–/– BMMCs were incubated with (solid bars) or without (open bars) 0.1 µM 12-O-tetra-decanoyl-phorbol 13-acetate (TPA) (C), or 1 µg/ml an anti-dinitrophenyl IgE (SPE-7) (D). Values are presented as means ± SE (n = 3). E: total RNAs were isolated from Ptger3+/+ and Ptger3–/– BMMC (+/+ and –/–) and subjected to RT-PCR analyses. The amplified products for the EP1, EP2, EP3, and EP4 subtype were detected. Total RNAs from various tissues (EP1: kidney; EP2: uterus; EP3: brain; EP4: intestine) were used as the positive controls (P). Each reaction was performed in the absence of RNA as the negative control (N).
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Gi-mediated Ca2+ mobilization is one of the signaling events downstream of the EP3 subtype (10). PGE2 induced a drastic Ca2+ mobilization in the Ptger3+/+ BMMCs, followed by a sustained phase with a moderate cytosolic Ca2+ level (Fig. 3). Similar responses were observed in the Ptger3+/+ BMMCs stimulated with sulprostone and ONO-AE-248, but not with ONO-AE1-437. In the Ptger3–/– BMMCs, PGE2-induced Ca2+ mobilization was drastically attenuated, and no EP agonists tested could induce Ca2+ mobilization (Fig. 3 and data not shown). Treatment with thrombin (Fig. 3) or monomeric IgE (data not shown) induced similar levels of Ca2+ mobilization responses in the Ptger3+/+ and Ptger3–/– BMMCs. In contrast to the drastic elevation of cAMP levels in P-815 cells stimulated with PGE2, PGE2 failed to induce cAMP elevation in BMMCs (data not shown).
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Fig. 3. Ca2+ mobilization in BMMCs induced by various PGE receptor agonists. The Ptger3+/+ (left) and Ptger3–/– (right) BMMCs were stimulated with 0.1 µM PGE2, 0.1 µM Sulp, 1 µM ONO-AE-248 (EP3-A), 1 µM ONO-AE-1-437 (EP4-A), or 1 U/ml thrombin, and the cytosolic Ca2+ levels were measured using fura-2 AM. The time points of stimulation are indicated by the arrows. Bars = 1 min. This is a representative figure of three independent experiments showing similar results.
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Characterization of EP3-mediated adhesion and Ca2+ mobilization in BMMCs.
Since the EP3 subtype is known to be coupled to multiple trimeric G proteins, we then investigated the signaling pathway downstream of the EP3 subtype. PGE2-induced adhesion and Ca2+ mobilization in BMMCs were strikingly suppressed by pretreatment with PTX, indicating the dominant role of Gi/Go (Fig. 4, A and B). Pretreatment of BMMCs with a specific inhibitor of phospholipase C, which is known to be activated upon Gβ liberation from trimeric Gi complex, abolished the adhesion responses and Ca2+ mobilization induced by PGE2 (Fig. 4, C and D). It remains unknown which kinds of Ca2+ channels are involved in the Ca2+ influx induced by the EP3 subtype. We found that SKF 96365, a specific inhibitor of store-operated Ca2+ channels, has a potential to block the PGE2-induced Ca2+ mobilization and adhesion (Fig. 4, E and F). These results collectively indicate that the EP3 subtype is coupled to Gi and induces Ca2+ mobilization, which is maintained by persistent activation of the store-operated Ca2+ channels.
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Fig. 4. Characterization of EP3-mediated adhesion and Ca2+ mobilization. A: BMMCs were pretreated with pertussis toxin (PTX) or without PTX (control) (0.1 mg/ml) for 1 h and then incubated with (solid bars) or without (open bars) 0.1 µM PGE2 for 1 h. The values are presented as means ± SE (n = 3). The value **P < 0.01 is regarded as significant by the Student's t-test (vs. none). B: BMMCs were pretreated as described above and then stimulated with 0.1 µM PGE2 (indicated by the arrows). The cytosolic Ca2+ levels were measured. Bars = 1 min. This is a representative figure of three independent experiments showing similar results. C: BMMCs were pretreated with (U-73343 and U-73122) or without (control) an inhibitor for phospholipase C (4 µM) for 7 min and then incubated with (solid bars) or without (open bars) 0.1 µM PGE2 for 1 h. The values are presented as means ± SE (n = 3). The value **P < 0.01 is regarded as significant by Student's t-test (vs. none). D: BMMCs were pretreated as described above and then stimulated with 0.1 µM PGE2 (indicated by the arrows). The cytosolic Ca2+ levels were measured. Bars = 1 min. This is a representative figure of three independent experiments showing similar results. E: BMMCs were stimulated with (solid bars) or without (open bars) 0.1 µM PGE2 for 1 h in the presence (SKF 96365) or absence (control) of 50 µM SKF 96365. The values are presented as means ± SE (n = 3). The value **P < 0.01 is regarded as significant by Student's t-test (vs. none). F: BMMCs were pretreated with or without 50 µM SKF 96365 for 1 min and then stimulated with 0.1 µM PGE2 (indicated by the arrows). The cytosolic Ca2+ levels were measured. Bars = 1 min. This is a representative figure of three independent experiments showing similar results.
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Deficiency in PGE2-induced adhesion in peritoneal mast cells of the Ptger3–/– mice.
Several functions specific to mature mast cells, such as cationic secretagogue-induced degranulation, are not reproduced in BMMCs, even though BMMCs can well mimic the functions of immature mast cells. It is of great interest to determine whether mature mast cells utilize the EP3 or EP4 subtype for adhesion. We then investigated the PGE2-induced adhesion to ProF in peritoneal mast cells, which are regarded as typical connective tissue-type mast cells. Purified peritoneal mast cells were found to exclusively express the EP3 subtype by RT-PCR analyses (Fig. 5A). PGE2-induced adhesion of peritoneal mast cells to ProF was concentration dependent, and the EC50 value of this response was similar to that in BMMCs (Figs. 5B and 1C). In the Ptger3–/– peritoneal cells, PGE2-induced adhesion of mast cells was completely abolished, whereas a similar level of adhesion was observed in the presence of TPA compared with the Ptger3+/+ peritoneal cells (Fig. 5, C–E). No significant difference was found in the number of peritoneal mast cells between the Ptger3+/+ and Ptger3–/– mice (percentages of mast cells in total peritoneal cells, Ptger3+/+ 2.43 ± 0.380%, Ptger3–/– 2.44 ± 0.516%). PGE2-induced adhesion of the peritoneal mast cells was significantly suppressed by SKF 36965, indicating that the adhesion should require Ca2+ influx (Fig. 5F). In addition, the GRGDS peptide blocked the PGE2-induced adhesion of the peritoneal mast cells (Fig. 5G). These results indicate that the EP3 subtype mediates the PGE2-induced adhesion of mouse peritoneal mast cells in a similar manner to that of BMMCs.
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Fig. 5. Detection of PGE2 receptor subtype mRNAs and PGE2-induced adhesion of mouse peritoneal mast cells to ProF. A: total RNA from mouse purified peritoneal mast cells (pMC) was isolated and subjected to RT-PCR analyses, and the amplified PCR products for the EP1, EP2, EP3, and EP4 subtype were detected. Total RNAs from various tissues were used as positive controls (P): kidney for the EP1, uterus for the EP2, brain for the EP3, and intestine for the EP4 subtype. PCR was performed without RNA as a negative control (N). B: the peritoneal cells were incubated for 1 h in wells coated with ProF in the presence of the indicated concentrations of PGE2, and the number of adhered mast cells was counted. The values are presented as means ± SE (n = 6). C and D: peritoneal cells form the Ptger3+/+ (C) or Ptger3–/– (D) mice were incubated with or without 0.1 µM PGE2 (EtOH and PGE2) or 10 nM TPA (DMSO and TPA) in the wells coated with ProF (solid bars) or BSA (open bars) for 1 h. The number of adhered mast cells was counted. The values are presented as means ± SE (n = 6–7). The values *P < 0.05 and **P < 0.01 are regarded as significant by one-way ANOVA (Dunnett's post hoc test, vs. EtOH). E: mouse peritoneal cells from the Ptger3+/+ (a, b, and c) or Ptger3–/– (d, e, and f) mice were incubated without (a and d) or with 0.1 µM PGE2 (b and e) or 10 nM TPA (c and f) for 1 h and stained by the acidic toluidine blue. The cells that exhibit metachromasy (mast cells) are indicated by the arrows. Bar = 10 µm. F and G: the peritoneal cells were stimulated with or without 0.1 µM PGE2 (EtOH and PGE2) or 10 nM TPA (DMSO and TPA) in the wells coated with ProF in the presence (solid bars) or absence (open bars) of 50 µM SKF 96365 (F) or 200 µM GRGDS (G). The values are presented as means ± SE (B: n = 7, C: n = 6). The values **P < 0.01 are regarded as significant by one-way ANOVA (Dunnett's post hoc test, vs. without SKF 96365 or GRGDS).
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EP3-mediated Ca2+ mobilization and adhesion of rat peritoneal mast cells.
Although the inhibitory effects of SKF 36965 on the adhesion of peritoneal mast cells imply the requirement of PGE2-induced Ca2+ influx, it was found to be quite difficult to determine the cytosolic Ca2+ levels using purified mouse peritoneal mast cells in a reproducible manner. We then used rat peritoneal mast cells to investigate PGE2-induced signal transduction, since rat peritoneal mast cells can be readily purified and have been used as the good model for connective tissue-type mast cells. The adhesion of the peritoneal mast cells to ProF was significantly augmented by PGE2 or the EP3 agonist, but not by the EP4 agonist (Fig. 6A). The PGE2- or EP3 agonist-induced adhesion was abolished in the presence of the GRGDS peptide or SKF 36965, as well as in the mouse peritoneal mast cells (Fig. 6A). PGE2 and the EP3 agonist were found to induce Ca2+ mobilization in the rat peritoneal mast cells, which was completely suppressed by SKF 36965 (Fig. 6B). These results suggest that PGE2 can evoke Ca2+ mobilization and stimulate the adhesion to ProF in the rat peritoneal mast cells, which can be mimicked by the EP3 agonist.
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Fig. 6. PGE2-induced Ca2+ mobilization and adhesion of rat peritoneal mast cells. A: rat peritoneal mast cells were stimulated with or without 1 µM PGE2 [solid bars; control (EtOH), dotted bars], 1 µM ONO-AE-248 (stripped bars), 1 µM ONO-AE1-437 (shaded bars), or 30 nM TPA [right panel, solid bars; control (DMSO), open bars] for 30 min in the presence (GRGDS) or absence (none) of 200 µM GRGDS or 50 µM SKF 96365 in the wells coated with ProF. Basal adhesion levels are presented (left panel, open bars). The values are presented as means ± SE (n = 3–4). The values *P < 0.05 are regarded as significant by one-way ANOVA (Dunnett's post hoc test vs. EtOH). B: rat peritoneal mast cells were pretreated with (SKF 96365) or without (none) 50 µM SKF 96365 for 45 min and then stimulated with 1 µM PGE2, 1 µM ONO-AE-248, or 1 U/ml thrombin. Cytosolic Ca2+ levels were measured using fura-2 AM. The time points of stimulation are indicated by the arrows. Bars = 1 min. This is a representative figure of three independent experiments showing similar results.
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DISCUSSION
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In contrast to the adhesion through cAMP elevation by cooperation between the EP3 and EP4 subtypes in P-815 cells (7), the EP3 subtype was found to play an essential role in PGE2-induced adhesion of BMMCs and peritoneal mast cells through Ca2+ mobilization. The signaling pathways utilized in mast cells stimulated by PGE2 can be classified into two types: one is mediated through cAMP elevation by the Gs-coupled subtype, such as EP2 and EP4; and the other is through Ca2+ mobilization by the Gi-coupled subtype, EP3. PGE2-induced cAMP elevation suppressed the histamine release upon antigen stimulation in rat peritoneal and human lung mast cells (12, 22) and resulted in release of vascular endothelial growth factor in human cord blood-derived cultured mast cells through activation of the EP2 subtype (1). In these kinds of mast cells, cAMP elevation plays a key role in the PGE2-induced signal transduction. On the other hand, PGE2 or an EP1/EP3 agonist, sulprostone, was reported to stimulate IL-6 release and potentiate degranulation upon antigen stimulation in a mouse IL-3-dependent mast cell line, MC9 (6). Nguyen et al. (21) demonstrated PGE2-induced Ca2+ mobilization and PGE1-mediated augmentation of antigen-induced degranulation in BMMCs, both of which are abolished in BMMCs derived from the EP3-deficient mice. They also showed that PGE2 failed to enhance cAMP formation in BMMCs, although the expression of the EP4 subtype was detected. These results indicate that the action of PGE2 is mediated exclusively by the EP3 subtype in BMMCs and MC9 cells. Considering the characteristics of mast cells involved in these PGE2-induced responses together, degrees of maturation, species difference, and/or malignant conversion may determine the available EP subtypes and their roles in the signal transduction. BMMCs were found to express both the EP3 and EP4 subtype (21, 28, and this study), which is similar in the expression pattern to P-815 cells. Further studies are required to clarify how the signaling pathway downstream of the EP3 subtype is differentially regulated, since the EP3 subtype augments EP4-mediated cAMP production in P-815 cells but induces Ca2+ mobilization in BMMCs and rat peritoneal mast cells.
Our laboratory characterized in detail the EP3-mediated Ca2+ mobilization in the present study. We found that PGE2-induced Ca2+ increase consists mainly of Ca2+ influx, which is suppressed by a specific inhibitor for store-operated Ca2+ channels, SKF 96365. Since the adhesion of BMMCs and rat peritoneal mast cells was completely suppressed in the presence of the inhibitors that suppressed Ca2+ mobilization induced by PGE2, it is likely that Gi-mediated Ca2+ influx plays a pivotal role downstream of the EP3 subtype, at least in the adhesion response. PTX-sensitive adhesion of BMMCs to fibronectin was also reported with the other mediators, such as thrombin and 5-hydroxytryptamine (15, 26). Thrombin was reported to induce the adhesion of BMMCs by acting on protease-activated receptor-1, which was accompanied by degranulation and IL-6 production (26). Although the author did not measure the cytosolic Ca2+ levels, these responses should be mediated by Ca2+ mobilization, as well as in the present study.
It remained unknown which subtypes of the EP receptors are expressed in mature tissue mast cells. We demonstrated that the EP3 subtype is the only detectable EP subtype in mouse peritoneal mast cells and that PGE2-induced adhesion is abolished in the Ptger3–/– mast cells. Kunikata et al. (14) demonstrated that antigen-induced histamine release is significantly suppressed by PGE2 or ONO-AE-248 (EP3 agonist) in lung tissues, and such suppressive effect was abolished in the Ptger3–/– lung tissues. Since it remains controversial whether the EP3 subtype suppresses or potentiates degranulation upon antigen stimulation (5, 6, 14, 21), much attention should be paid to the heterogeneity of tissue mast cells. In regard to mouse peritoneal mast cells, we observed no detectable degranulation in the presence of PGE2, whereas high concentrations of TPA induced significant levels of degranulation (data not shown).
Recent studies demonstrated that mast cells can undergo chemotaxis in response to various chemoattractants, such as histamine, 5-hydroxytryptamine, PGE2, leukotriene B4, and sphingosine-1-phosphate (8, 11, 15, 27, 28). These results indicate that mast cells have a tendency to accumulate at the inflamed site that is characterized by production of these chemoattractants. We noticed that the adhesion of BMMCs in response to PGE2 is rapid and transient. Process of mast cell activation can be generally classified into two phases: mast cells release histamine and neutral proteases through degranulation and generate a series of lipid mediators in the immediate phase, whereas they produce chemokines and cytokines in the late phase (18). Considering this time course, it is likely that PGE2-induced transient adhesion to fibronectin restricts chemotaxis of mast cells during the immediate phase. PGE2 may regulate the localization of tissue mast cells under inflammatory conditions and potentiate the impact of proinflammatory mediators generated by mast cells.
We conclude that the EP3 subtype is essential for PGE2-induced adhesion of mouse BMMCs and rat peritoneal mast cells through Ca2+ mobilization. It is likely that EP3-mediated Ca2+ mobilization is mediated by the Gi-phospholipase C axis. This pathway may play a critical role in regulation of mast cell adhesion under inflammatory conditions.
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GRANTS
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This study was supported, in part, by a grant from Naito Foundation, Sankyo Foundation of Life Science, the Fugaku Trust for Medical Research, and by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Science, Sports and Technology of Japan.
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FOOTNOTES
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Address for reprint requests and other correspondence: S. Tanaka, Dept. of Immunobiology, School of Pharmacy and Pharmaceutical Sciences, Mukogawa Women's Univ., Koshien, Nishinomiya, Hyogo 663-8179, Japan (e-mail: s_tanaka{at}mukogawa-u.ac.jp)
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES
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1. Abdel-Majid RM, Marshall JS. Prostaglandin E2 induces degranulation-independent production of vascular endothelial growth factor by human mast cells. J Immunol 172: 1227–1236, 2004.[Abstract/Free Full Text]2. Bonazzi A, Bolla M, Buccellati C, Hernandez A, Zarini S, Vigano T, Fumagalli F, Viappiani S, Ravasi S, Zannini P, Chiesa G, Folco G, Sala A. Effect of endogenous and exogenous prostaglandin E2 on interleukin-1β-induced cyclooxigenase-2 expression in human smooth-muscle cells. Am J Respir Crit Care Med 162: 2272–2277, 2000.[Abstract/Free Full Text]
3. Boyce JA. Mast cells and eicosanoid mediators: a system of reciprocal paracrine and autocrine regulation. Immunol Rev 217: 168–185, 2007.[CrossRef][Web of Science][Medline]
4. Faour WH, He Y, He QW, de Ladurantaye M, Quintero M, Mancini A, Di Battista JA. Prostaglandin E2 regulates the level and stability of cyclooxygenase-2 mRNA through activation of p38 mitogen-activated protein kinase in interleukin-1β-treated human synovial fibroblasts. J Biol Chem 276: 31720–31731, 2001.[Abstract/Free Full Text]
5. Feng C, Beller EM, Bagga S, Boyce JA. Human mast cells express multiple EP receptors for prostaglandin E2 that differentially modulate activation responses. Blood 107: 3243–3250, 2006.[Abstract/Free Full Text]
6. Gomi K, Zhu FG, Marshall JS. Prostaglandin E2 selectively enhances the IgE-mediated production of IL-6 and granulocyte-macrophage colony-stimulating factor by mast cells through an EP1/EP3-dependent mechanism. J Immunol 165: 6545–6552, 2000.[Abstract/Free Full Text]
7. Hatae N, Kita A, Tanaka S, Sugimoto Y, Ichikawa A. Induction of adherent activity in mastocytoma P-815 cells by the cooperation of two prostaglandin E2 receptor subtypes, EP3 and EP4. J Biol Chem 278: 17977–17981, 2003.[Abstract/Free Full Text]
8. Hofstra CL, Desai PJ, Thurmond RL, Fung-Leung WP. Histamine H4 receptor mediates chemotaxis and calcium mobilization of mast cells. J Pharmacol Exp Ther 305: 1212–1221, 2003.[Abstract/Free Full Text]
9. Humes JL, Bonney RJ, Pelus L, Dahlgren ME, Sadowski SJ, Kuehl FA Jr, Davies P. Macrophages synthesis and release prostaglandins in response to inflammatory stimuli. Nature 269: 149–151, 1977.[CrossRef][Web of Science][Medline]
10. Irie A, Segi E, Sugimoto Y, Ichikawa A, Negishi M. Mouse prostaglandin E receptor EP3 subtype mediates calcium signals via Gi in cDNA-transfected Chinese hamster ovary cells. Biochem Biophys Res Commun 204: 303–309, 1994.[CrossRef][Web of Science][Medline]
11. Jolly PS, Bektas M, Olivera A, Gonzalez-Espinosa C, Proia RL, Rivera J, Milstien S, Spiegel S. Transactivation of sphingosine-1-phosphate receptors by FceRI triggering is required for normal mast cell degranulation and chemotaxis. J Exp Med 199: 959–970, 2004.[Abstract/Free Full Text]
12. Kaliner M, Austen KF. Cyclic AMP, ATP, and reversed anaphylactic histamine release from rat mast cells. J Immunol 112: 664–674, 1974.[Abstract/Free Full Text]
13. Kowalski ML, Pawliczak R, Wozniak J, Siuda K, Poniatowska M, Iwaszkiewicz J, Kornatowski T, Kaliner MA. Differential metabolism of arachidonic acid in nasal polyp epithelial cells cultured from aspirin-sensitive and aspirin-tolerant patients. Am J Respir Crit Care Med 161: 391–398, 2000.[Abstract/Free Full Text]
14. Kunikata T, Yamane H, Segi E, Matsuoka T, Sugimoto Y, Tanaka S, Tanaka H, Nagai H, Ichikawa A, Narumiya S. Suppression of allergic inflammation by the prostaglandin E receptor subtype EP3. Nat Immunol 6: 524–531, 2005.[CrossRef][Web of Science][Medline]
15. Kushnir-Sukhov NM, Gilfillan AM, Coleman JW, Brown JM, Bruening S, Toth M, Metcalfe. 5-Hydroxytryptamine induces mast cell adhesion and migration. J Immunol 177: 6422–6432, 2006.[Abstract/Free Full Text]
16. Lam V, Kalesnikoff J, Lee CW, Hernandez-Hansen V, Wilson BS, Oliver JM, Krystal G. IgE alone stimulates mast cell adhesion to fibronectin via pathways similar to those used by IgE + antigen but distinct from those used by Steel factor. Blood 102: 1405–1413, 2003.[Abstract/Free Full Text]
17. Macias-Perez IM, Zent R, Carmosino M, Breyer MD, Breyer RM, Pozzi A. Mouse EP3
, β and receptor variants reduce tumor cell proliferation and tumorigenesis in vivo. J Biol Chem 283: 12538–12545, 2008.[Abstract/Free Full Text]
18. Metcalfe DD, Baram D, Mekori YA. Mast cells. Physiol Rev 77: 1033–1079, 1997.[Abstract/Free Full Text]
19. Metz M, Grimbaldeston MA, Nakae S, Piliponsky AM, Tsai M, Galli SJ. Mast cells in the promotion and limitation of chronic inflammation. Immunol Rev 217: 304–328, 2007.[CrossRef][Web of Science][Medline]
20. Metz M, Maurer M. Mast cell-key effector cells in immune responses. Trends Immunol 28: 304–328, 2007.
21. Nguyen M, Solle M, Audoly LP, Tilley SL, Stock JL, McNeish JD, Coffman TM, Dombrowicz D, Koller BH. Receptors and signaling mechanisms required for prostaglandin E2-mediated regulation of mast cell degranulation and IL-6 production. J Immunol 169: 4586–4593, 2002.[Abstract/Free Full Text]
22. Peachell PT, MacGlashan DW Jr, Lichtenstein LM, Schleimer RP. Regulation of human basophil and lung mast cell function by cyclic adenosine monophosphate. J Immunol 140: 571–579, 1988.[Abstract]
23. Sugimoto Y, Narumiya S. Prostaglandin E receptors. J Biol Chem 282: 11613–11617, 2007.[Abstract/Free Full Text]
24. Tanaka S, Takasu Y, Mikura S, Satoh N, Ichikawa A. Antigen-independent induction of histamine synthesis by immunoglobulin E in mouse bone marrow derived mast cells. J Exp Med 196: 229–235, 2002.[Abstract/Free Full Text]
25. Ushikubi F, Segi E, Sugimoto Y, Murata T, Matsuoka T, Kobayashi T, Hizaki H, Tuboi K, Katsuyama M, Ichikawa A, Tanaka T, Yoshida N, Narumiya S. Impaired febrile response in mice lacking the prostaglandin E receptor subtype EP3. Nature 395: 281–284, 1998.[CrossRef][Web of Science][Medline]
26. Vliagoftis H. Thrombin induces mast cell adhesion to fibronectin: evidence for involvement of protease-activated receptor-1. J Immunol 169: 4551–4558, 2002.[Abstract/Free Full Text]
27. Weller CL, Collington SJ, Brown JK, Miller HR, Al-Kashi A, Clark P, Jose PJ, Hartnell A, Williams TJ. Leukotriene B4, an activation product of mast cells, is a chemoattractant for their progenitors. J Exp Med 201: 1961–1971, 2005.[Abstract/Free Full Text]
28. Weller CL, Collington SJ, Hartnell A, Conroy DM, Kaise T, Barker JE, Wilson MS, Taylor GW, Jose PJ, Williams TJ. Chemotactic action of prostaglandin E2 on mouse mast cells acting via the PGE2 receptor 3. Proc Natl Acad Sci USA 104: 11712–11717, 2007.[Abstract/Free Full Text]
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