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• W1988594080 abstract "In this study, we investigated the role of PGE2 in mouse mastocytoma P-815 cell adhesion to extracellular matrix proteins (ECMs) in vitro. We report that PGE2 accelerated ProNectin FTM (a proteolytic fragment of fibronectin)-mediated adhesion, which was abolished by addition of the GRGDS peptide, an inhibitor of the RDG binding site of ProNectin FTM. We show that the cAMP level and cAMP-regulated protein kinase (PKA) activity are critical mediators of this PGE2 effect, because the cell-permeable cAMP analogue 8-Br-cAMP accelerated P-815 cell adhesion to ProNectin FTM and the pharmacological inhibitor of PKA, H-89, blocked PGE2-mediated adhesion. Consistent with mRNA expression of the Gs-coupled EP4- and Gi-coupled EP3-PGE receptor subtypes, P-815 cell adhesion was accelerated by treatment with a selective EP4 agonist, ONO-AE1–329, but not a selective EP1/EP3 agonist, sulprostone. However, simultaneous treatment with ONO-AE1–329 and sulprostone resulted in augmentation of both the cAMP level and cell adhesion. The augmentation of EP3-mediated cAMP synthesis was dose-dependent, without affecting the half-maximal concentration for EP4-mediated Gs-activity, which was inhibited by a Gi inhibitor, pertussis toxin. In conclusion, these findings suggest that PGE2 accelerates RGD-dependent adhesion via cooperative activation between EP3 and EP4 and contributes to the recruitment of mast cells to the ECM during inflammation. In this study, we investigated the role of PGE2 in mouse mastocytoma P-815 cell adhesion to extracellular matrix proteins (ECMs) in vitro. We report that PGE2 accelerated ProNectin FTM (a proteolytic fragment of fibronectin)-mediated adhesion, which was abolished by addition of the GRGDS peptide, an inhibitor of the RDG binding site of ProNectin FTM. We show that the cAMP level and cAMP-regulated protein kinase (PKA) activity are critical mediators of this PGE2 effect, because the cell-permeable cAMP analogue 8-Br-cAMP accelerated P-815 cell adhesion to ProNectin FTM and the pharmacological inhibitor of PKA, H-89, blocked PGE2-mediated adhesion. Consistent with mRNA expression of the Gs-coupled EP4- and Gi-coupled EP3-PGE receptor subtypes, P-815 cell adhesion was accelerated by treatment with a selective EP4 agonist, ONO-AE1–329, but not a selective EP1/EP3 agonist, sulprostone. However, simultaneous treatment with ONO-AE1–329 and sulprostone resulted in augmentation of both the cAMP level and cell adhesion. The augmentation of EP3-mediated cAMP synthesis was dose-dependent, without affecting the half-maximal concentration for EP4-mediated Gs-activity, which was inhibited by a Gi inhibitor, pertussis toxin. In conclusion, these findings suggest that PGE2 accelerates RGD-dependent adhesion via cooperative activation between EP3 and EP4 and contributes to the recruitment of mast cells to the ECM during inflammation. mast cell prostaglandin heterotrimeric GTP-binding protein pertussis toxin phorbol 12-myristate 13-acetate phosphate-buffered saline 4-morpholineethanesulfonic acid phosphatidylinositol 3-kinase cAMP-dependent protein kinase extracellular matrix Differentiated mast cells (MCs),1 which originate from bone marrow stem cells, traffic throughout the circulation and adhere to the extracellular matrix (ECM) in various tissues. MCs are widely distributed in tissues throughout the body, especially in connective tissues, serosal cavities, and on mucosal surfaces under normal physiological conditions. This characteristic distinguishes MCs from other bone marrow-derived hematopoietic cells, such as basophils, neutrophils, and eosinophils. MCs congregate around nerves, blood vessels, and lymphatic vessels. MCs therefore interact with not only the ECM but with other cells as well. As well known for rodent connective tissue-typed MCs and mucosal-typed MCs, the biological activity of MCs vary with their interactions with the ECM and other cells.MCs are widely distributed along basement membranes, indicating that MCs might adhere to laminin. Supporting this fact, mouse bone marrow-derived mast cells (BMMC) have been reported to adhere to laminin, when the cells were activated by phorbol 12-myristate 13-acetate (PMA) (1Thompson H.L. Burbelo P.D. Segui-Real B. Yamada Y. Metcalfe D.D. J. Immunol. 1989; 143: 2323-2327PubMed Google Scholar, 2Thompson H.L. Burbelo P.D. Yamada Y. Kleiman H.K. Metcalfe D.D. J. Immunol. 1989; 143: 4188-4192PubMed Google Scholar, 3Thompson H.L. Burbelo P.D. Yamada Y. Kleiman H.K. Metcalfe D.D. Immunology. 1991; 72: 144-149PubMed Google Scholar) or antigen-stimulated aggregation of FcεRI (4Thompson H.L. Burbelo P.D. Metcalfe D.D. J. Immunol. 1990; 145: 3425-3431PubMed Google Scholar). In addition to laminin, MCs can adhere to other matrix components such as fibronectin (5Dastych J. Costa J.J. Thompson H.L. Metcalfe D.D. Immunology. 1991; 73: 478-484PubMed Google Scholar) and vitronectin (6Bianchine P.J. Burd P.R. Metcalfe D.D. J. Immunol. 1992; 149: 3665-3671PubMed Google Scholar, 7Gurish, M. F., Bell, A. F., Smith, T. J., Ducharme, L. A., Wang, R.-K., and Weis, J. H. (149) J. Immunol. 149, 1964–1972.Google Scholar). As with laminin, the adherence of BMMC to fibronectin has been reported to occur through activation with PMA or after aggregation of FcεRI. These adherence activities required calcium (3Thompson H.L. Burbelo P.D. Yamada Y. Kleiman H.K. Metcalfe D.D. Immunology. 1991; 72: 144-149PubMed Google Scholar). In contrast to BMMC, the mouse PT18 cell line spontaneously adhered to laminin (1Thompson H.L. Burbelo P.D. Segui-Real B. Yamada Y. Metcalfe D.D. J. Immunol. 1989; 143: 2323-2327PubMed Google Scholar), and human skin mast cells also spontaneously adhered to laminin and fibronectin (8Walsh L.J. Kaminer M.S. Lazarus G.S. Lavker R.M. Murphy G.F. Lab. Invest. 1991; 65: 433-439PubMed Google Scholar). These previous findings indicate that the interactions between MCs and matrix components may depend on the cells involved and the kinds of stimuli.PGE2, which is involved in inflammation section (9Ohuchi K. Yoshino S. Kanaoka K. Tsurufuji S. Levine L. Int. Archs. Allergy Appl. Immun. 1982; 68: 326-331Crossref PubMed Scopus (42) Google Scholar), affects both differentiation and growth of MCs in vitro. PGE2 enhances mast cell differentiation from cord blood mononuclear cells (10Saito H. Ebisawa M. Tachimoto H. Shichijo M. Fukagawa K. Matsumoto K. Iikura Y. Awaji T. Tsujimoto G. Yanagida M. Uzumaki H. Takahashi G. Tsuji K. Nakahata T. J. Immunol. 1996; 157: 343-350PubMed Google Scholar) and in the fibroblast co-culture system (11Kameyoshi Y. Morita E. Tanaka T. Hiragun T. Yamamoto S. Arch. Dermatol. Res. 2000; 292: 240-247Crossref PubMed Scopus (17) Google Scholar). Very recently, Dormond et al. (12Dormond O. Foletti A. Paroz C. Ruegg C. Nat. Med. 2001; 7: 1041-1047Crossref PubMed Scopus (269) Google Scholar) reported that a COX-2 inhibitor suppressed αVβ3-dependent HUVEC spreading, migration, and angiogenesis through Rac activation (12Dormond O. Foletti A. Paroz C. Ruegg C. Nat. Med. 2001; 7: 1041-1047Crossref PubMed Scopus (269) Google Scholar), and PGE2 accelerated αVβ3-mediated HUVEC responses in a cAMP-dependent manner (13Dormond O. Bezzi M. Mariotti A. Ruegg C. J. Biol. Chem. 2002; 277: 45838-45846Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). However, no reports have published the effects of PGE2 on MC adhesion to the ECM.The PGE2 receptors (EP) are comprised of four subtypes, EP1, EP2, EP3, and EP4, which are coupled to different G proteins and signal pathways (14Coleman R.A. Smith W.L. Narumiya S. Pharmacol. Rev. 1994; 46: 205-229PubMed Google Scholar, 15Narumiya S. Sugimoto Y. Ushikubi F. Physiol. Rev. 1999; 79: 1193-1226Crossref PubMed Scopus (0) Google Scholar). Among these subtypes, EP2 and EP4 couple to Gs, resulting in increases of intracellular cAMP concentrations (16Katsuyama M. Nishigaki N. Sugimoto Y. Morimoto K. Negishi M. Narumiya S. Ichikawa A. FEBS Lett. 1995; 372: 151-156Crossref PubMed Scopus (161) Google Scholar, 17Honda A. Sugimoto Y. Namba T. Watabe A. Irie A. Negishi M. Narumiya S. Ichikawa A. J. Biol. Chem. 1993; 268: 7759-7762Abstract Full Text PDF PubMed Google Scholar), while EP3 couples to Gi, causing a decrease in cAMP levels (18Sugimoto Y. Namba T. Honda A. Hayashi Y. Negishi M. Ichikawa A. Narumiya S. J. Biol. Chem. 1992; 267: 6463-6466Abstract Full Text PDF PubMed Google Scholar, 19Sugimoto Y. Negishi M. Hayashi Y. Namba T. Honda A. Watabe A. Hirata M. Narumiya S. Ichikawa A. J. Biol. Chem. 1993; 268: 2712-2718Abstract Full Text PDF PubMed Google Scholar, 20Irie A. Sugimoto Y. Namba T. Harazono A. Honda A. Watabe A. Negishi M. Narumiya S. Ichikawa A. Eur. J. Biochem. 1993; 217: 313-318Crossref PubMed Scopus (164) Google Scholar, 21Irie A. Segi E. Sugimoto Y. Ichikawa A. Negishi M. Biochem. Biophys. Res. Commun. 1994; 204: 303-309Crossref PubMed Scopus (57) Google Scholar). Very recently, we and other investigators have reported that activation of the Gi-coupled EP3 receptor was able to augment adenylyl cyclase activity via stimulation of a Gs-coupled receptor (22Hatae N. Yamaoka K. Sugimoto Y. Negishi M. Ichikawa A. Biochem. Biophys. Res. Commun. 2002; 290: 162-168Crossref PubMed Scopus (29) Google Scholar, 23Southall M.D. Vasko M.R. J. Biol. Chem. 2001; 276: 16083-16091Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). We previously reported that the mouse EP3β receptor was able to augment EP2-induced adenylyl cyclase activity in both EP2- and EP3-transfected COS-7 cells (22Hatae N. Yamaoka K. Sugimoto Y. Negishi M. Ichikawa A. Biochem. Biophys. Res. Commun. 2002; 290: 162-168Crossref PubMed Scopus (29) Google Scholar). Southall and Vasko (23Southall M.D. Vasko M.R. J. Biol. Chem. 2001; 276: 16083-16091Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar) showed that the simultaneous depletion of rat EP3C and EP4 was essential to abolish PGE2-stimulated cAMP production and neuropeptide release in rat sensory neurons (23Southall M.D. Vasko M.R. J. Biol. Chem. 2001; 276: 16083-16091Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). These permissive actions between the two subtypes may be involved in the events of a number of physiological actions of PGE2 (15Narumiya S. Sugimoto Y. Ushikubi F. Physiol. Rev. 1999; 79: 1193-1226Crossref PubMed Scopus (0) Google Scholar).Here we extended the analyses of the effects of PGE2on adhesion of mouse mastocytoma P-815 cells to the fibronectin component in a PKA-dependent manner, and we examined the correlation between cAMP levels and adhesion in P-815 cells to see whether EP3 could augment EP4-induced cAMP synthesis.DISCUSSIONPGE2 was previously reported to affect both differentiation and growth of mast cells in vitro. For example, PGE2 is essential for the differentiation of IL-3-dependent mouse mast cells from spleen cells (30Hu Z.Q. Asano K. Seki H. Shimamura T. J. Immunol. 1995; 155: 2134-2142PubMed Google Scholar), PGE2 enhanced human mast cell differentiation from cord blood mononuclear cells by inhibiting the production of macrophage-derived GM-CSF (10Saito H. Ebisawa M. Tachimoto H. Shichijo M. Fukagawa K. Matsumoto K. Iikura Y. Awaji T. Tsujimoto G. Yanagida M. Uzumaki H. Takahashi G. Tsuji K. Nakahata T. J. Immunol. 1996; 157: 343-350PubMed Google Scholar), and PGE2 enhanced the growth of differentiated mast cells in a fibroblast co-culture system (11Kameyoshi Y. Morita E. Tanaka T. Hiragun T. Yamamoto S. Arch. Dermatol. Res. 2000; 292: 240-247Crossref PubMed Scopus (17) Google Scholar). It is therefore possible that PGE2 plays a role in the interaction of mast cells with fibroblastic cells and extracellular matrix components. The interaction between mast cells and extracellular matrix components have profound influences on the targeting of mast cell progenitors to specific locations, the distribution of mast cell subsets, and the biological responsiveness of mast cells in tissues. The ability of mast cells to adhere to fibronectin, which involves the RGD sequence located within the cell-attachment domain of the fibronectin molecule, may play a role in the migration of mast cells in various tissues (5Dastych J. Costa J.J. Thompson H.L. Metcalfe D.D. Immunology. 1991; 73: 478-484PubMed Google Scholar). In the present report, we found that PGE2 is able to stimulate the adhesion of P-815 cells to Pronectin-F through the RGD cell attachment domain of fibronectin, and that this adherent activity is mediated via a cAMP-dependent pathway induced by the activation of the EP4 and/or EP3 receptors. It is possible that the cAMP-protein kinase A pathway may be involved in the induction of these cell attachment molecules, as PGE2-induced cell attachment was inhibited by treatment with H-89 (Fig. 3B) and cycloheximide (data not shown). However, neither PGE2 nor 8Br-cAMP augmented the expression of VLA-5, one of the fibronectin receptors (data not shown). Therefore, cycloheximide may affect the signaling pathway of the fibronectin receptors. Further experiments are required to clarify these points.Prostaglandin E2 acts through binding to its specific receptors, which are comprised of four subtypes, EP1, EP2, EP3, and EP4 (14Coleman R.A. Smith W.L. Narumiya S. Pharmacol. Rev. 1994; 46: 205-229PubMed Google Scholar, 15Narumiya S. Sugimoto Y. Ushikubi F. Physiol. Rev. 1999; 79: 1193-1226Crossref PubMed Scopus (0) Google Scholar). Among them, EP3 and EP4 couple to Gi and Gs, and result in inhibition and stimulation of adenylyl cyclase activity, respectively (17Honda A. Sugimoto Y. Namba T. Watabe A. Irie A. Negishi M. Narumiya S. Ichikawa A. J. Biol. Chem. 1993; 268: 7759-7762Abstract Full Text PDF PubMed Google Scholar, 18Sugimoto Y. Namba T. Honda A. Hayashi Y. Negishi M. Ichikawa A. Narumiya S. J. Biol. Chem. 1992; 267: 6463-6466Abstract Full Text PDF PubMed Google Scholar, 19Sugimoto Y. Negishi M. Hayashi Y. Namba T. Honda A. Watabe A. Hirata M. Narumiya S. Ichikawa A. J. Biol. Chem. 1993; 268: 2712-2718Abstract Full Text PDF PubMed Google Scholar, 20Irie A. Sugimoto Y. Namba T. Harazono A. Honda A. Watabe A. Negishi M. Narumiya S. Ichikawa A. Eur. J. Biochem. 1993; 217: 313-318Crossref PubMed Scopus (164) Google Scholar, 21Irie A. Segi E. Sugimoto Y. Ichikawa A. Negishi M. Biochem. Biophys. Res. Commun. 1994; 204: 303-309Crossref PubMed Scopus (57) Google Scholar). The mouse EP3 receptor is comprised of three isoforms, EP3α, EP3β, and EP3γ, which differ in their C terminus. Among them, EP3β, which was used in this experiment is known to be coupled to the Gi protein (19Sugimoto Y. Negishi M. Hayashi Y. Namba T. Honda A. Watabe A. Hirata M. Narumiya S. Ichikawa A. J. Biol. Chem. 1993; 268: 2712-2718Abstract Full Text PDF PubMed Google Scholar). Very recently, activation of a Gi-coupled receptor has been reported to augment the adenylyl cyclase activity induced by the stimulation of a Gs-coupled receptor in COS-7 cells. For example, activation of Gi-coupled receptors such as α2 adrenoreceptor (29Fereman A.D. Conklin B.R. Schrader K.A. Reed R.R. Bourne H.R. Nature. 1992; 356: 159-161Crossref PubMed Scopus (480) Google Scholar) and bradykinin B2 receptor (32Hanke S. Nurnberg B. Groll D.H. Liebmann C. Mol. Cell. Biol. 2001; 21: 8452-8460Crossref PubMed Scopus (30) Google Scholar) lead to the augmentation of Gs-stimulated adenylyl cyclase in COS-7 cells. This synergistic effect has not been clearly shown in mammalian cells. Southall and Vasko (23Southall M.D. Vasko M.R. J. Biol. Chem. 2001; 276: 16083-16091Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar) showed that the simultaneous depletion of both rat EP3C and EP4 was essential for abolishing PGE2-stimulated cAMP production and neuropeptide release in rat sensory neurons (23Southall M.D. Vasko M.R. J. Biol. Chem. 2001; 276: 16083-16091Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). In the current experiment we showed that the Gi-coupled EP3 agonist sulprostone augmented cAMP formation induced by the EP4 agonist ONO-AE1-329 in P-815 cells. Similarly, we found that sulprostone was able to augment cAMP formation in P-815 cells activated by an IP agonist, carbacyclin, suggesting that the augmentative effects of EP3 can be observed irrespective of Gs activation. The mechanism underlying these phenomena was thought to be via Gβγ-mediated activation of type IV adenylyl cyclase (29Fereman A.D. Conklin B.R. Schrader K.A. Reed R.R. Bourne H.R. Nature. 1992; 356: 159-161Crossref PubMed Scopus (480) Google Scholar), because pretreatment with PT inhibited the augmentation by the activation of the Gi-coupled EP3 receptor, and P-815 cells express type IV adenylyl cyclase (data not shown). Therefore, Gβγ-mediated activation of type IV adenylyl cyclase may be involved in EP3-mediated signaling to augment EP4-stimulated adenylyl cyclase activity in P-815 cells.Although the involvement of phosphatidylinositol 3-kinase (PI3K) in cell adhesion to matrix proteins is shown in a variety of cell types (33Wu C. J. Cell Sci. 1999; 112: 4485-4489Crossref PubMed Google Scholar), the role of PI3K in mast cell adhesion is still unknown. Kinashiet al. (31Kinashi T. Escobedo J.A. Williams L.T. Takatsu K. Springer T.A. Blood. 1995; 86: 2086-2090Crossref PubMed Google Scholar) reported that the adhesion of platelet-derived growth factor receptor-expressed bone marrow-derived mast cells was inhibited by the addition of the PI3K inhibitor wortmannin. To understand whether PI3K is activated by PGE2 during P-815 cell adhesion, we examined the effect of the PI3K inhibitors, wortmannin (100 nm) and LY294002 (10 μm) on EP3/EP4-agonist induced cell adhesion to fibronectin. As a result, these inhibitors showed an additive effect on the augmentation of PGE2-induced cell adhesion (wortmannin: 81.7 ± 0.1% and LY294002: 82.4 ± 0.2%, compared with PGE2stimulation alone: 24.8 ± 0.6%). Furthermore, each inhibitor alone had an effect on cell adhesion (wortmannin: 20.4 ± 0.1% and LY294002: 24.1 ± 0.1%, compared with the absence of these inhibitors (3.3 ± 0.1%)). Therefore, these results indicate that P-815 cell attachment to fibronectin is regulated by two independent signaling pathways involving PKA and PI3K. Further experiments are necessary to clarify the fundamental differences in the signaling involving PKA and PI3K in P-815 cell attachment to fibronectin. In summary, this study clearly demonstrates that two subtypes of the PGE2 receptor, EP3 and EP4, are cooperatively involved in PGE2-evoked and cAMP-mediated functions of P-815 cell adhesion. Differentiated mast cells (MCs),1 which originate from bone marrow stem cells, traffic throughout the circulation and adhere to the extracellular matrix (ECM) in various tissues. MCs are widely distributed in tissues throughout the body, especially in connective tissues, serosal cavities, and on mucosal surfaces under normal physiological conditions. This characteristic distinguishes MCs from other bone marrow-derived hematopoietic cells, such as basophils, neutrophils, and eosinophils. MCs congregate around nerves, blood vessels, and lymphatic vessels. MCs therefore interact with not only the ECM but with other cells as well. As well known for rodent connective tissue-typed MCs and mucosal-typed MCs, the biological activity of MCs vary with their interactions with the ECM and other cells. MCs are widely distributed along basement membranes, indicating that MCs might adhere to laminin. Supporting this fact, mouse bone marrow-derived mast cells (BMMC) have been reported to adhere to laminin, when the cells were activated by phorbol 12-myristate 13-acetate (PMA) (1Thompson H.L. Burbelo P.D. Segui-Real B. Yamada Y. Metcalfe D.D. J. Immunol. 1989; 143: 2323-2327PubMed Google Scholar, 2Thompson H.L. Burbelo P.D. Yamada Y. Kleiman H.K. Metcalfe D.D. J. Immunol. 1989; 143: 4188-4192PubMed Google Scholar, 3Thompson H.L. Burbelo P.D. Yamada Y. Kleiman H.K. Metcalfe D.D. Immunology. 1991; 72: 144-149PubMed Google Scholar) or antigen-stimulated aggregation of FcεRI (4Thompson H.L. Burbelo P.D. Metcalfe D.D. J. Immunol. 1990; 145: 3425-3431PubMed Google Scholar). In addition to laminin, MCs can adhere to other matrix components such as fibronectin (5Dastych J. Costa J.J. Thompson H.L. Metcalfe D.D. Immunology. 1991; 73: 478-484PubMed Google Scholar) and vitronectin (6Bianchine P.J. Burd P.R. Metcalfe D.D. J. Immunol. 1992; 149: 3665-3671PubMed Google Scholar, 7Gurish, M. F., Bell, A. F., Smith, T. J., Ducharme, L. A., Wang, R.-K., and Weis, J. H. (149) J. Immunol. 149, 1964–1972.Google Scholar). As with laminin, the adherence of BMMC to fibronectin has been reported to occur through activation with PMA or after aggregation of FcεRI. These adherence activities required calcium (3Thompson H.L. Burbelo P.D. Yamada Y. Kleiman H.K. Metcalfe D.D. Immunology. 1991; 72: 144-149PubMed Google Scholar). In contrast to BMMC, the mouse PT18 cell line spontaneously adhered to laminin (1Thompson H.L. Burbelo P.D. Segui-Real B. Yamada Y. Metcalfe D.D. J. Immunol. 1989; 143: 2323-2327PubMed Google Scholar), and human skin mast cells also spontaneously adhered to laminin and fibronectin (8Walsh L.J. Kaminer M.S. Lazarus G.S. Lavker R.M. Murphy G.F. Lab. Invest. 1991; 65: 433-439PubMed Google Scholar). These previous findings indicate that the interactions between MCs and matrix components may depend on the cells involved and the kinds of stimuli. PGE2, which is involved in inflammation section (9Ohuchi K. Yoshino S. Kanaoka K. Tsurufuji S. Levine L. Int. Archs. Allergy Appl. Immun. 1982; 68: 326-331Crossref PubMed Scopus (42) Google Scholar), affects both differentiation and growth of MCs in vitro. PGE2 enhances mast cell differentiation from cord blood mononuclear cells (10Saito H. Ebisawa M. Tachimoto H. Shichijo M. Fukagawa K. Matsumoto K. Iikura Y. Awaji T. Tsujimoto G. Yanagida M. Uzumaki H. Takahashi G. Tsuji K. Nakahata T. J. Immunol. 1996; 157: 343-350PubMed Google Scholar) and in the fibroblast co-culture system (11Kameyoshi Y. Morita E. Tanaka T. Hiragun T. Yamamoto S. Arch. Dermatol. Res. 2000; 292: 240-247Crossref PubMed Scopus (17) Google Scholar). Very recently, Dormond et al. (12Dormond O. Foletti A. Paroz C. Ruegg C. Nat. Med. 2001; 7: 1041-1047Crossref PubMed Scopus (269) Google Scholar) reported that a COX-2 inhibitor suppressed αVβ3-dependent HUVEC spreading, migration, and angiogenesis through Rac activation (12Dormond O. Foletti A. Paroz C. Ruegg C. Nat. Med. 2001; 7: 1041-1047Crossref PubMed Scopus (269) Google Scholar), and PGE2 accelerated αVβ3-mediated HUVEC responses in a cAMP-dependent manner (13Dormond O. Bezzi M. Mariotti A. Ruegg C. J. Biol. Chem. 2002; 277: 45838-45846Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). However, no reports have published the effects of PGE2 on MC adhesion to the ECM. The PGE2 receptors (EP) are comprised of four subtypes, EP1, EP2, EP3, and EP4, which are coupled to different G proteins and signal pathways (14Coleman R.A. Smith W.L. Narumiya S. Pharmacol. Rev. 1994; 46: 205-229PubMed Google Scholar, 15Narumiya S. Sugimoto Y. Ushikubi F. Physiol. Rev. 1999; 79: 1193-1226Crossref PubMed Scopus (0) Google Scholar). Among these subtypes, EP2 and EP4 couple to Gs, resulting in increases of intracellular cAMP concentrations (16Katsuyama M. Nishigaki N. Sugimoto Y. Morimoto K. Negishi M. Narumiya S. Ichikawa A. FEBS Lett. 1995; 372: 151-156Crossref PubMed Scopus (161) Google Scholar, 17Honda A. Sugimoto Y. Namba T. Watabe A. Irie A. Negishi M. Narumiya S. Ichikawa A. J. Biol. Chem. 1993; 268: 7759-7762Abstract Full Text PDF PubMed Google Scholar), while EP3 couples to Gi, causing a decrease in cAMP levels (18Sugimoto Y. Namba T. Honda A. Hayashi Y. Negishi M. Ichikawa A. Narumiya S. J. Biol. Chem. 1992; 267: 6463-6466Abstract Full Text PDF PubMed Google Scholar, 19Sugimoto Y. Negishi M. Hayashi Y. Namba T. Honda A. Watabe A. Hirata M. Narumiya S. Ichikawa A. J. Biol. Chem. 1993; 268: 2712-2718Abstract Full Text PDF PubMed Google Scholar, 20Irie A. Sugimoto Y. Namba T. Harazono A. Honda A. Watabe A. Negishi M. Narumiya S. Ichikawa A. Eur. J. Biochem. 1993; 217: 313-318Crossref PubMed Scopus (164) Google Scholar, 21Irie A. Segi E. Sugimoto Y. Ichikawa A. Negishi M. Biochem. Biophys. Res. Commun. 1994; 204: 303-309Crossref PubMed Scopus (57) Google Scholar). Very recently, we and other investigators have reported that activation of the Gi-coupled EP3 receptor was able to augment adenylyl cyclase activity via stimulation of a Gs-coupled receptor (22Hatae N. Yamaoka K. Sugimoto Y. Negishi M. Ichikawa A. Biochem. Biophys. Res. Commun. 2002; 290: 162-168Crossref PubMed Scopus (29) Google Scholar, 23Southall M.D. Vasko M.R. J. Biol. Chem. 2001; 276: 16083-16091Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). We previously reported that the mouse EP3β receptor was able to augment EP2-induced adenylyl cyclase activity in both EP2- and EP3-transfected COS-7 cells (22Hatae N. Yamaoka K. Sugimoto Y. Negishi M. Ichikawa A. Biochem. Biophys. Res. Commun. 2002; 290: 162-168Crossref PubMed Scopus (29) Google Scholar). Southall and Vasko (23Southall M.D. Vasko M.R. J. Biol. Chem. 2001; 276: 16083-16091Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar) showed that the simultaneous depletion of rat EP3C and EP4 was essential to abolish PGE2-stimulated cAMP production and neuropeptide release in rat sensory neurons (23Southall M.D. Vasko M.R. J. Biol. Chem. 2001; 276: 16083-16091Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). These permissive actions between the two subtypes may be involved in the events of a number of physiological actions of PGE2 (15Narumiya S. Sugimoto Y. Ushikubi F. Physiol. Rev. 1999; 79: 1193-1226Crossref PubMed Scopus (0) Google Scholar). Here we extended the analyses of the effects of PGE2on adhesion of mouse mastocytoma P-815 cells to the fibronectin component in a PKA-dependent manner, and we examined the correlation between cAMP levels and adhesion in P-815 cells to see whether EP3 could augment EP4-induced cAMP synthesis. DISCUSSIONPGE2 was previously reported to affect both differentiation and growth of mast cells in vitro. For example, PGE2 is essential for the differentiation of IL-3-dependent mouse mast cells from spleen cells (30Hu Z.Q. Asano K. Seki H. Shimamura T. J. Immunol. 1995; 155: 2134-2142PubMed Google Scholar), PGE2 enhanced human mast cell differentiation from cord blood mononuclear cells by inhibiting the production of macrophage-derived GM-CSF (10Saito H. Ebisawa M. Tachimoto H. Shichijo M. Fukagawa K. Matsumoto K. Iikura Y. Awaji T. Tsujimoto G. Yanagida M. Uzumaki H. Takahashi G. Tsuji K. Nakahata T. J. Immunol. 1996; 157: 343-350PubMed Google Scholar), and PGE2 enhanced the growth of differentiated mast cells in a fibroblast co-culture system (11Kameyoshi Y. Morita E. Tanaka T. Hiragun T. Yamamoto S. Arch. Dermatol. Res. 2000; 292: 240-247Crossref PubMed Scopus (17) Google Scholar). It is therefore possible that PGE2 plays a role in the interaction of mast cells with fibroblastic cells and extracellular matrix components. The interaction between mast cells and extracellular matrix components have profound influences on the targeting of mast cell progenitors to specific locations, the distribution of mast cell subsets, and the biological responsiveness of mast cells in tissues. The ability of mast cells to adhere to fibronectin, which involves the RGD sequence located within the cell-attachment domain of the fibronectin molecule, may play a role in the migration of mast cells in various tissues (5Dastych J. Costa J.J. Thompson H.L. Metcalfe D.D. Immunology. 1991; 73: 478-484PubMed Google Scholar). In the present report, we found that PGE2 is able to stimulate the adhesion of P-815 cells to Pronectin-F through the RGD cell attachment domain of fibronectin, and that this adherent activity is mediated via a cAMP-dependent pathway induced by the activation of the EP4 and/or EP3 receptors. It is possible that the cAMP-protein kinase A pathway may be involved in the induction of these cell attachment molecules, as PGE2-induced cell attachment was inhibited by treatment with H-89 (Fig. 3B) and cycloheximide (data not shown). However, neither PGE2 nor 8Br-cAMP augmented the expression of VLA-5, one of the fibronectin receptors (data not shown). Therefore, cycloheximide may affect the signaling pathway of the fibronectin receptors. Further experiments are required to clarify these points.Prostaglandin E2 acts through binding to its specific receptors, which are comprised of four subtypes, EP1, EP2, EP3, and EP4 (14Coleman R.A. Smith W.L. Narumiya S. Pharmacol. Rev. 1994; 46: 205-229PubMed Google Scholar, 15Narumiya S. Sugimoto Y. Ushikubi F. Physiol. Rev. 1999; 79: 1193-1226Crossref PubMed Scopus (0) Google Scholar). Among them, EP3 and EP4 couple to Gi and Gs, and result in inhibition and stimulation of adenylyl cyclase activity, respectively (17Honda A. Sugimoto Y. Namba T. Watabe A. Irie A. Negishi M. Narumiya S. Ichikawa A. J. Biol. Chem. 1993; 268: 7759-7762Abstract Full Text PDF PubMed Google Scholar, 18Sugimoto Y. Namba T. Honda A. Hayashi Y. Negishi M. Ichikawa A. Narumiya S. J. Biol. Chem. 1992; 267: 6463-6466Abstract Full Text PDF PubMed Google Scholar, 19Sugimoto Y. Negishi M. Hayashi Y. Namba T. Honda A. Watabe A. Hirata M. Narumiya S. Ichikawa A. J. Biol. Chem. 1993; 268: 2712-2718Abstract Full Text PDF PubMed Google Scholar, 20Irie A. Sugimoto Y. Namba T. Harazono A. Honda A. Watabe A. Negishi M. Narumiya S. Ichikawa A. Eur. J. Biochem. 1993; 217: 313-318Crossref PubMed Scopus (164) Google Scholar, 21Irie A. Segi E. Sugimoto Y. Ichikawa A. Negishi M. Biochem. Biophys. Res. Commun. 1994; 204: 303-309Crossref PubMed Scopus (57) Google Scholar). The mouse EP3 receptor is comprised of three isoforms, EP3α, EP3β, and EP3γ, which differ in their C terminus. Among them, EP3β, which was used in this experiment is known to be coupled to the Gi protein (19Sugimoto Y. Negishi M. Hayashi Y. Namba T. Honda A. Watabe A. Hirata M. Narumiya S. Ichikawa A. J. Biol. Chem. 1993; 268: 2712-2718Abstract Full Text PDF PubMed Google Scholar). Very recently, activation of a Gi-coupled receptor has been reported to augment the adenylyl cyclase activity induced by the stimulation of a Gs-coupled receptor in COS-7 cells. For example, activation of Gi-coupled receptors such as α2 adrenoreceptor (29Fereman A.D. Conklin B.R. Schrader K.A. Reed R.R. Bourne H.R. Nature. 1992; 356: 159-161Crossref PubMed Scopus (480) Google Scholar) and bradykinin B2 receptor (32Hanke S. Nurnberg B. Groll D.H. Liebmann C. Mol. Cell. Biol. 2001; 21: 8452-8460Crossref PubMed Scopus (30) Google Scholar) lead to the augmentation of Gs-stimulated adenylyl cyclase in COS-7 cells. This synergistic effect has not been clearly shown in mammalian cells. Southall and Vasko (23Southall M.D. Vasko M.R. J. Biol. Chem. 2001; 276: 16083-16091Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar) showed that the simultaneous depletion of both rat EP3C and EP4 was essential for abolishing PGE2-stimulated cAMP production and neuropeptide release in rat sensory neurons (23Southall M.D. Vasko M.R. J. Biol. Chem. 2001; 276: 16083-16091Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). In the current experiment we showed that the Gi-coupled EP3 agonist sulprostone augmented cAMP formation induced by the EP4 agonist ONO-AE1-329 in P-815 cells. Similarly, we found that sulprostone was able to augment cAMP formation in P-815 cells activated by an IP agonist, carbacyclin, suggesting that the augmentative effects of EP3 can be observed irrespective of Gs activation. The mechanism underlying these phenomena was thought to be via Gβγ-mediated activation of type IV adenylyl cyclase (29Fereman A.D. Conklin B.R. Schrader K.A. Reed R.R. Bourne H.R. Nature. 1992; 356: 159-161Crossref PubMed Scopus (480) Google Scholar), because pretreatment with PT inhibited the augmentation by the activation of the Gi-coupled EP3 receptor, and P-815 cells express type IV adenylyl cyclase (data not shown). Therefore, Gβγ-mediated activation of type IV adenylyl cyclase may be involved in EP3-mediated signaling to augment EP4-stimulated adenylyl cyclase activity in P-815 cells.Although the involvement of phosphatidylinositol 3-kinase (PI3K) in cell adhesion to matrix proteins is shown in a variety of cell types (33Wu C. J. Cell Sci. 1999; 112: 4485-4489Crossref PubMed Google Scholar), the role of PI3K in mast cell adhesion is still unknown. Kinashiet al. (31Kinashi T. Escobedo J.A. Williams L.T. Takatsu K. Springer T.A. Blood. 1995; 86: 2086-2090Crossref PubMed Google Scholar) reported that the adhesion of platelet-derived growth factor receptor-expressed bone marrow-derived mast cells was inhibited by the addition of the PI3K inhibitor wortmannin. To understand whether PI3K is activated by PGE2 during P-815 cell adhesion, we examined the effect of the PI3K inhibitors, wortmannin (100 nm) and LY294002 (10 μm) on EP3/EP4-agonist induced cell adhesion to fibronectin. As a result, these inhibitors showed an additive effect on the augmentation of PGE2-induced cell adhesion (wortmannin: 81.7 ± 0.1% and LY294002: 82.4 ± 0.2%, compared with PGE2stimulation alone: 24.8 ± 0.6%). Furthermore, each inhibitor alone had an effect on cell adhesion (wortmannin: 20.4 ± 0.1% and LY294002: 24.1 ± 0.1%, compared with the absence of these inhibitors (3.3 ± 0.1%)). Therefore, these results indicate that P-815 cell attachment to fibronectin is regulated by two independent signaling pathways involving PKA and PI3K. Further experiments are necessary to clarify the fundamental differences in the signaling involving PKA and PI3K in P-815 cell attachment to fibronectin. In summary, this study clearly demonstrates that two subtypes of the PGE2 receptor, EP3 and EP4, are cooperatively involved in PGE2-evoked and cAMP-mediated functions of P-815 cell adhesion. PGE2 was previously reported to affect both differentiation and growth of mast cells in vitro. For example, PGE2 is essential for the differentiation of IL-3-dependent mouse mast cells from spleen cells (30Hu Z.Q. Asano K. Seki H. Shimamura T. J. Immunol. 1995; 155: 2134-2142PubMed Google Scholar), PGE2 enhanced human mast cell differentiation from cord blood mononuclear cells by inhibiting the production of macrophage-derived GM-CSF (10Saito H. Ebisawa M. Tachimoto H. Shichijo M. Fukagawa K. Matsumoto K. Iikura Y. Awaji T. Tsujimoto G. Yanagida M. Uzumaki H. Takahashi G. Tsuji K. Nakahata T. J. Immunol. 1996; 157: 343-350PubMed Google Scholar), and PGE2 enhanced the growth of differentiated mast cells in a fibroblast co-culture system (11Kameyoshi Y. Morita E. Tanaka T. Hiragun T. Yamamoto S. Arch. Dermatol. Res. 2000; 292: 240-247Crossref PubMed Scopus (17) Google Scholar). It is therefore possible that PGE2 plays a role in the interaction of mast cells with fibroblastic cells and extracellular matrix components. The interaction between mast cells and extracellular matrix components have profound influences on the targeting of mast cell progenitors to specific locations, the distribution of mast cell subsets, and the biological responsiveness of mast cells in tissues. The ability of mast cells to adhere to fibronectin, which involves the RGD sequence located within the cell-attachment domain of the fibronectin molecule, may play a role in the migration of mast cells in various tissues (5Dastych J. Costa J.J. Thompson H.L. Metcalfe D.D. Immunology. 1991; 73: 478-484PubMed Google Scholar). In the present report, we found that PGE2 is able to stimulate the adhesion of P-815 cells to Pronectin-F through the RGD cell attachment domain of fibronectin, and that this adherent activity is mediated via a cAMP-dependent pathway induced by the activation of the EP4 and/or EP3 receptors. It is possible that the cAMP-protein kinase A pathway may be involved in the induction of these cell attachment molecules, as PGE2-induced cell attachment was inhibited by treatment with H-89 (Fig. 3B) and cycloheximide (data not shown). However, neither PGE2 nor 8Br-cAMP augmented the expression of VLA-5, one of the fibronectin receptors (data not shown). Therefore, cycloheximide may affect the signaling pathway of the fibronectin receptors. Further experiments are required to clarify these points. Prostaglandin E2 acts through binding to its specific receptors, which are comprised of four subtypes, EP1, EP2, EP3, and EP4 (14Coleman R.A. Smith W.L. Narumiya S. Pharmacol. Rev. 1994; 46: 205-229PubMed Google Scholar, 15Narumiya S. Sugimoto Y. Ushikubi F. Physiol. Rev. 1999; 79: 1193-1226Crossref PubMed Scopus (0) Google Scholar). Among them, EP3 and EP4 couple to Gi and Gs, and result in inhibition and stimulation of adenylyl cyclase activity, respectively (17Honda A. Sugimoto Y. Namba T. Watabe A. Irie A. Negishi M. Narumiya S. Ichikawa A. J. Biol. Chem. 1993; 268: 7759-7762Abstract Full Text PDF PubMed Google Scholar, 18Sugimoto Y. Namba T. Honda A. Hayashi Y. Negishi M. Ichikawa A. Narumiya S. J. Biol. Chem. 1992; 267: 6463-6466Abstract Full Text PDF PubMed Google Scholar, 19Sugimoto Y. Negishi M. Hayashi Y. Namba T. Honda A. Watabe A. Hirata M. Narumiya S. Ichikawa A. J. Biol. Chem. 1993; 268: 2712-2718Abstract Full Text PDF PubMed Google Scholar, 20Irie A. Sugimoto Y. Namba T. Harazono A. Honda A. Watabe A. Negishi M. Narumiya S. Ichikawa A. Eur. J. Biochem. 1993; 217: 313-318Crossref PubMed Scopus (164) Google Scholar, 21Irie A. Segi E. Sugimoto Y. Ichikawa A. Negishi M. Biochem. Biophys. Res. Commun. 1994; 204: 303-309Crossref PubMed Scopus (57) Google Scholar). The mouse EP3 receptor is comprised of three isoforms, EP3α, EP3β, and EP3γ, which differ in their C terminus. Among them, EP3β, which was used in this experiment is known to be coupled to the Gi protein (19Sugimoto Y. Negishi M. Hayashi Y. Namba T. Honda A. Watabe A. Hirata M. Narumiya S. Ichikawa A. J. Biol. Chem. 1993; 268: 2712-2718Abstract Full Text PDF PubMed Google Scholar). Very recently, activation of a Gi-coupled receptor has been reported to augment the adenylyl cyclase activity induced by the stimulation of a Gs-coupled receptor in COS-7 cells. For example, activation of Gi-coupled receptors such as α2 adrenoreceptor (29Fereman A.D. Conklin B.R. Schrader K.A. Reed R.R. Bourne H.R. Nature. 1992; 356: 159-161Crossref PubMed Scopus (480) Google Scholar) and bradykinin B2 receptor (32Hanke S. Nurnberg B. Groll D.H. Liebmann C. Mol. Cell. Biol. 2001; 21: 8452-8460Crossref PubMed Scopus (30) Google Scholar) lead to the augmentation of Gs-stimulated adenylyl cyclase in COS-7 cells. This synergistic effect has not been clearly shown in mammalian cells. Southall and Vasko (23Southall M.D. Vasko M.R. J. Biol. Chem. 2001; 276: 16083-16091Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar) showed that the simultaneous depletion of both rat EP3C and EP4 was essential for abolishing PGE2-stimulated cAMP production and neuropeptide release in rat sensory neurons (23Southall M.D. Vasko M.R. J. Biol. Chem. 2001; 276: 16083-16091Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). In the current experiment we showed that the Gi-coupled EP3 agonist sulprostone augmented cAMP formation induced by the EP4 agonist ONO-AE1-329 in P-815 cells. Similarly, we found that sulprostone was able to augment cAMP formation in P-815 cells activated by an IP agonist, carbacyclin, suggesting that the augmentative effects of EP3 can be observed irrespective of Gs activation. The mechanism underlying these phenomena was thought to be via Gβγ-mediated activation of type IV adenylyl cyclase (29Fereman A.D. Conklin B.R. Schrader K.A. Reed R.R. Bourne H.R. Nature. 1992; 356: 159-161Crossref PubMed Scopus (480) Google Scholar), because pretreatment with PT inhibited the augmentation by the activation of the Gi-coupled EP3 receptor, and P-815 cells express type IV adenylyl cyclase (data not shown). Therefore, Gβγ-mediated activation of type IV adenylyl cyclase may be involved in EP3-mediated signaling to augment EP4-stimulated adenylyl cyclase activity in P-815 cells. Although the involvement of phosphatidylinositol 3-kinase (PI3K) in cell adhesion to matrix proteins is shown in a variety of cell types (33Wu C. J. Cell Sci. 1999; 112: 4485-4489Crossref PubMed Google Scholar), the role of PI3K in mast cell adhesion is still unknown. Kinashiet al. (31Kinashi T. Escobedo J.A. Williams L.T. Takatsu K. Springer T.A. Blood. 1995; 86: 2086-2090Crossref PubMed Google Scholar) reported that the adhesion of platelet-derived growth factor receptor-expressed bone marrow-derived mast cells was inhibited by the addition of the PI3K inhibitor wortmannin. To understand whether PI3K is activated by PGE2 during P-815 cell adhesion, we examined the effect of the PI3K inhibitors, wortmannin (100 nm) and LY294002 (10 μm) on EP3/EP4-agonist induced cell adhesion to fibronectin. As a result, these inhibitors showed an additive effect on the augmentation of PGE2-induced cell adhesion (wortmannin: 81.7 ± 0.1% and LY294002: 82.4 ± 0.2%, compared with PGE2stimulation alone: 24.8 ± 0.6%). Furthermore, each inhibitor alone had an effect on cell adhesion (wortmannin: 20.4 ± 0.1% and LY294002: 24.1 ± 0.1%, compared with the absence of these inhibitors (3.3 ± 0.1%)). Therefore, these results indicate that P-815 cell attachment to fibronectin is regulated by two independent signaling pathways involving PKA and PI3K. Further experiments are necessary to clarify the fundamental differences in the signaling involving PKA and PI3K in P-815 cell attachment to fibronectin. In summary, this study clearly demonstrates that two subtypes of the PGE2 receptor, EP3 and EP4, are cooperatively involved in PGE2-evoked and cAMP-mediated functions of P-815 cell adhesion. We thank Dr. M. P. L. Caton of Rhone-Poulenc Ltd. for the generous gift of sulprostone. We are grateful to Dr. Manabu Negishi of the Laboratory of Molecular Neurobiology, Graduate School of Biostudies, University of Kyoto for valuable advice on this study." @default.
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• W1988594080 title "Induction of Adherent Activity in Mastocytoma P-815 Cells by the Cooperation of Two Prostaglandin E2 Receptor Subtypes, EP3 and EP4" @default.
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