Regulation of Delayed Prostaglandin Production in Activated P388D1 Macrophages by Group IV Cytosolic and Group V Secretory Phospholipase A2s
1999; Elsevier BV; Volume: 274; Issue: 18 Linguagem: Inglês
10.1074/jbc.274.18.12263
ISSN1083-351X
AutoresHiroyuki Shinohara, Marı́a A. Balboa, Chris A. Johnson, Jesús Balsinde, Edward A. Dennis,
Tópico(s)Drug Transport and Resistance Mechanisms
ResumoGroup V secretory phospholipase A2 (sPLA2) rather than Group IIA sPLA2 is involved in short term, immediate arachidonic acid mobilization and prostaglandin E2 (PGE2) production in the macrophage-like cell line P388D1. When a new clone of these cells, P388D1/MAB, selected on the basis of high responsivity to lipopolysaccharide plus platelet-activating factor, was studied, delayed PGE2 production (6–24 h) in response to lipopolysaccharide alone occurred in parallel with the induction of Group V sPLA2 and cyclooxygenase-2 (COX-2). No changes in the level of cytosolic phospholipase A2(cPLA2) or COX-1 were observed, and Group IIA sPLA2 was not detectable. Use of a potent and selective sPLA2 inhibitor, 3-(3-acetamide 1-benzyl-2-ethylindolyl-5-oxy)propanesulfonic acid (LY311727), and an antisense oligonucleotide specific for Group V sPLA2revealed that delayed PGE2 was largely dependent on the induction of Group V sPLA2. Also, COX-2, not COX-1, was found to mediate delayed PGE2 production because the response was completely blocked by the specific COX-2 inhibitor NS-398. Delayed PGE2 production and Group V sPLA2expression were also found to be blunted by the inhibitor methylarachidonyl fluorophosphonate. Because inhibition of Ca2+-independent PLA2 by an antisense technique did not have any effect on the arachidonic acid release, the data using methylarachidonyl fluorophosphonate suggest a key role for the cPLA2 in the response as well. Collectively, the results suggest a model whereby cPLA2 activation regulates Group V sPLA2 expression, which in turn is responsible for delayed PGE2 production via COX-2. Group V secretory phospholipase A2 (sPLA2) rather than Group IIA sPLA2 is involved in short term, immediate arachidonic acid mobilization and prostaglandin E2 (PGE2) production in the macrophage-like cell line P388D1. When a new clone of these cells, P388D1/MAB, selected on the basis of high responsivity to lipopolysaccharide plus platelet-activating factor, was studied, delayed PGE2 production (6–24 h) in response to lipopolysaccharide alone occurred in parallel with the induction of Group V sPLA2 and cyclooxygenase-2 (COX-2). No changes in the level of cytosolic phospholipase A2(cPLA2) or COX-1 were observed, and Group IIA sPLA2 was not detectable. Use of a potent and selective sPLA2 inhibitor, 3-(3-acetamide 1-benzyl-2-ethylindolyl-5-oxy)propanesulfonic acid (LY311727), and an antisense oligonucleotide specific for Group V sPLA2revealed that delayed PGE2 was largely dependent on the induction of Group V sPLA2. Also, COX-2, not COX-1, was found to mediate delayed PGE2 production because the response was completely blocked by the specific COX-2 inhibitor NS-398. Delayed PGE2 production and Group V sPLA2expression were also found to be blunted by the inhibitor methylarachidonyl fluorophosphonate. Because inhibition of Ca2+-independent PLA2 by an antisense technique did not have any effect on the arachidonic acid release, the data using methylarachidonyl fluorophosphonate suggest a key role for the cPLA2 in the response as well. Collectively, the results suggest a model whereby cPLA2 activation regulates Group V sPLA2 expression, which in turn is responsible for delayed PGE2 production via COX-2. Arachidonic acid (AA) 1The abbreviations used are: AA, arachidonic acid; PAF, platelet-activating factor; LPS, bacterial lipopolysaccharide; cPLA2, Group IV cytosolic phospholipase A2; sPLA2, secretory phospholipase A2; COX, cyclooxygenase (prostaglandin H2synthase); MAFP, methylarachidonyl fluorophosphonate; PGE2, prostaglandin E2; iPLA2, Ca2+-independent phospholipase A21The abbreviations used are: AA, arachidonic acid; PAF, platelet-activating factor; LPS, bacterial lipopolysaccharide; cPLA2, Group IV cytosolic phospholipase A2; sPLA2, secretory phospholipase A2; COX, cyclooxygenase (prostaglandin H2synthase); MAFP, methylarachidonyl fluorophosphonate; PGE2, prostaglandin E2; iPLA2, Ca2+-independent phospholipase A2mobilization and the generation of prostaglandins by major immunoinflammatory cells such as macrophages and mast cells usually occur in two phases. The immediate phase, which takes minutes and is elicited by Ca2+-mobilizing agonists such as platelet-activating factor (PAF), is characterized by a burst of AA liberation. In some cells such as P388D1 macrophages (1Balsinde J. Dennis E.A. J. Biol. Chem. 1996; 271: 6758-6765Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar, 2Balsinde J. Balboa M.A. Dennis E.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7951-7956Crossref PubMed Scopus (171) Google Scholar) and MMC-34 mast cells (3Reddy S.T. Herschman H.R. J. Biol. Chem. 1997; 272: 3231-3237Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar), this burst is mainly produced by a secretory phospholipase A2 (sPLA2) but is strikingly regulated by the cytosolic Group IV phospholipase A2(cPLA2).The delayed phase of prostaglandin production is accompanied by the continuous supply of AA over long incubation periods spanning several hours. There is some discrepancy about the identity of the PLA2 isoform(s) involved in the delayed phase. Despite this phase being independent of a Ca2+ increase, the cPLA2 has often been suggested to be critically involved (3Reddy S.T. Herschman H.R. J. Biol. Chem. 1997; 272: 3231-3237Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 4Murakami M. Nakatani Y. Atsumi G. Inoue K. Kudo I. Crit. Rev. Immunol. 1997; 17: 225-284Crossref PubMed Google Scholar, 5Murakami M. Kuwata H. Amakasu Y. Shimbara S. Nakatani Y. Atsumi G. Kudo I. J. Biol. Chem. 1997; 272: 19891-19897Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). However, other studies have suggested the quantitatively more important role of the sPLA2, an enzyme that is dramatically induced during long term incubation of the cells with a variety of stimuli (4Murakami M. Nakatani Y. Atsumi G. Inoue K. Kudo I. Crit. Rev. Immunol. 1997; 17: 225-284Crossref PubMed Google Scholar, 5Murakami M. Kuwata H. Amakasu Y. Shimbara S. Nakatani Y. Atsumi G. Kudo I. J. Biol. Chem. 1997; 272: 19891-19897Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 6Bingham III, C.O. Murakami M. Fujishima H. Hunt J.E. Austen K.F. Arm J.P. J. Biol. Chem. 1996; 271: 25936-25944Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). There is, however, agreement that COX-2, another enzyme whose expression is augmented dramatically after long term stimulation, is absolutely required for long term PGE2 production, irrespective of the constitutive presence of COX-1 (7Herschman H.R. Biochim. Biophys. Acta. 1996; 1299: 125-140Crossref PubMed Scopus (1160) Google Scholar, 8Murakami M. Matsumoto R. Urade Y. Austen K.F. Arm J.P. J. Biol. Chem. 1995; 270: 3239-3246Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 9Langenbach R. Morham S.G. Tiano H.F. Loftin C.D. Ghanayem B.I. Chulada P.C. Mahler J.F. Lee C.A. Goulding E.H. Kluckman K.D. Kim H.S. Smithies O. Cell. 1995; 83: 483-492Abstract Full Text PDF PubMed Scopus (1035) Google Scholar).Using a new clone of the P388D1 macrophage-like cells termed P388D1/MAB, we provide herein evidence for the involvement of Group V sPLA2 in delayed PGE2production. Furthermore, our results suggest that Group V sPLA2 expression is dependent upon the activation of Group IV cPLA2.DISCUSSIONA striking hallmark of the immunoinflammatory response is the generation of oxygenated derivatives of AA such as the prostaglandins. The response of major prostaglandin-secreting cells such as macrophages and mast cells to proinflammatory stimuli is generally biphasic (4Murakami M. Nakatani Y. Atsumi G. Inoue K. Kudo I. Crit. Rev. Immunol. 1997; 17: 225-284Crossref PubMed Google Scholar). The first phase is completed within minutes after the addition of the stimulus, whereas the second phase usually takes several hours (4Murakami M. Nakatani Y. Atsumi G. Inoue K. Kudo I. Crit. Rev. Immunol. 1997; 17: 225-284Crossref PubMed Google Scholar). Using the murine macrophage-like cell line P388D1, we have been studying the molecular mechanisms responsible for AA mobilization and prostaglandin production in response to LPS/PAF. When primed by LPS, these cells will respond to Ca2+-mobilizing stimuli such as PAF by generating a rapid burst of free AA, part of which is converted to prostaglandins such as PGE2. Strikingly, this process is completed within a few minutes after the addition of PAF (19Balsinde J. Barbour S.E. Bianco I.D. Dennis E.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11060-11064Crossref PubMed Scopus (127) Google Scholar). No free AA or prostaglandins are produced after the immediate phase is completed, not even after several hours of cell exposure to LPS/PAF (13Glaser K.B. Asmis R. Dennis E.A. J. Biol. Chem. 1990; 265: 8658-8664Abstract Full Text PDF PubMed Google Scholar). Such a behavior, which is abnormal for a macrophage cell, has prevented us from studying the molecular mechanisms responsible for delayed prostaglandin production in macrophages. In an attempt to overcome this problem, we subcloned the P388D1 cells by limit dilution, and selecting on the basis of high responsivity to LPS/PAF, we obtained a clone termed MAB, which shows enhanced sensitivity to LPS/PAF in the immediate phase (min) and exhibits a delayed response (h) to LPS alone.Using the MAB clone, we have characterized the LPS-induced delayed prostaglandin production in terms of the role played by distinct PLA2 enzymes and their coupling with downstream COX enzymes during LPS signaling. Our previous work on the immediate response of the cells to LPS/PAF highlighted the very important role played by the novel Group V sPLA2 as the provider of most of the free AA directed to PGE2 biosynthesis (11Balboa M.A. Balsinde J. Winstead M.V. Tischfield J.A. Dennis E.A. J. Biol. Chem. 1996; 271: 32381-32384Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar). Herein, several lines of evidence suggest that Group V sPLA2 also behaves as a major provider of AA for the delayed phase of PGE2production in LPS-treated cells. First, delayed [3H]AA release and PGE2 production correspond with the induction of Group V sPLA2 mRNA and enhanced secretion of a sPLA2-like activity to the supernatants, with no change in the constitutive levels of cPLA2 and no detectable induction of Group IIA sPLA2. Second, delayed PGE2 production is strongly blunted by LY311727, a selective sPLA2 inhibitor. Third, an antisense oligonucleotide specific for Group V sPLA2 (11Balboa M.A. Balsinde J. Winstead M.V. Tischfield J.A. Dennis E.A. J. Biol. Chem. 1996; 271: 32381-32384Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar) suppresses Group V sPLA2 activity and inhibits delayed PGE2 production. Our conclusions in this regard fully agree with recent works by Kudo and co-workers (20Murakami M. Shimbara S. Kambe T. Kuwata H. Winstead M.V. Tischfield J.A. Kudo I. J. Biol. Chem. 1998; 273: 14411-14423Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar, 21Murakami M. Kambe T. Shimbara S. Kudo I. J. Biol. Chem. 1999; 274: 3103-3115Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar) that were published while this manuscript was under review. By using transfection techniques, Kudo and co-workers (20Murakami M. Shimbara S. Kambe T. Kuwata H. Winstead M.V. Tischfield J.A. Kudo I. J. Biol. Chem. 1998; 273: 14411-14423Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar, 21Murakami M. Kambe T. Shimbara S. Kudo I. J. Biol. Chem. 1999; 274: 3103-3115Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar) have also documented the importance of Group V sPLA2 in delayed AA release and PGE2 production.Our data have also implicated the cPLA2 as an important step in LPS signaling by enabling the subsequent action of the sPLA2. Thus the cPLA2 inhibitor MAFP (1Balsinde J. Dennis E.A. J. Biol. Chem. 1996; 271: 6758-6765Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar) markedly blocked both long term [3H]AA release and Group V sPLA2 mRNA induction. Collectively, these results suggest an intriguing cross-talk between the cPLA2 and the Group V sPLA2 for the delayed phase of prostaglandin production in macrophages. This is a very interesting concept because cross-talk appears to exist as well between these two enzymes during the immediate phase of prostaglandin production (1Balsinde J. Dennis E.A. J. Biol. Chem. 1996; 271: 6758-6765Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar, 2Balsinde J. Balboa M.A. Dennis E.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7951-7956Crossref PubMed Scopus (171) Google Scholar). In the immediate phase, cPLA2 activation generates a rapid and early burst of free AA inside the cell that enables sPLA2activation by an as yet unidentified mechanism (1Balsinde J. Dennis E.A. J. Biol. Chem. 1996; 271: 6758-6765Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar, 2Balsinde J. Balboa M.A. Dennis E.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7951-7956Crossref PubMed Scopus (171) Google Scholar). In the delayed phase, cPLA2 activation influences sPLA2apparently by regulating sPLA2 mRNA levels.Cross-talk between cPLA2 and sPLA2 in the immediate phase of prostaglandin production was also found to take place in mast cells (3Reddy S.T. Herschman H.R. J. Biol. Chem. 1997; 272: 3231-3237Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar) when the same protocol originally used in macrophages (1Balsinde J. Dennis E.A. J. Biol. Chem. 1996; 271: 6758-6765Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar) was employed. Furthermore, a recent study by Kuwataet al. (22Kuwata H. Nakatani Y. Murakami M. Kudo I. J. Biol. Chem. 1998; 273: 1733-1740Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar) about fibroblasts suggests that cross-talk between cPLA2 and sPLA2 in the delayed phase could also constitute a general mechanism of activation. Using a different cPLA2 inhibitor, arachidonyl trifluoromethyl ketone, Kuwata et al. (22Kuwata H. Nakatani Y. Murakami M. Kudo I. J. Biol. Chem. 1998; 273: 1733-1740Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar) found that cPLA2inhibition blocked sPLA2 expression in fibroblasts, leading to reduced PGE2 generation. The study by Kuwata et al. (22Kuwata H. Nakatani Y. Murakami M. Kudo I. J. Biol. Chem. 1998; 273: 1733-1740Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar) is interesting not only because it supports the possible universality of cross-talk between cPLA2 and sPLA2 but because the sPLA2 expressed by rat fibroblasts is a Group IIA enzyme, not Group V. This lends further support to the emerging notion that Group IIA and Group V sPLA2 may be functionally redundant (23Tischfield J.A. J. Biol. Chem. 1997; 272: 17247-17250Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar). In addition, Kuwata et al. (22Kuwata H. Nakatani Y. Murakami M. Kudo I. J. Biol. Chem. 1998; 273: 1733-1740Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar) were able to show that overcoming cPLA2 inhibition by exogenous AA partially restored the Group IIA sPLA2 expression. These results suggest that the AA mobilized by cPLA2 is responsible for cross-talk between cPLA2 and sPLA2 (22Kuwata H. Nakatani Y. Murakami M. Kudo I. J. Biol. Chem. 1998; 273: 1733-1740Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). This is again reminiscent of what happens in the immediate phase of activation, wherein the cPLA2-derived AA is also responsible for cross-talk between cPLA2 and sPLA2, albeit by different mechanisms (1Balsinde J. Dennis E.A. J. Biol. Chem. 1996; 271: 6758-6765Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar, 2Balsinde J. Balboa M.A. Dennis E.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7951-7956Crossref PubMed Scopus (171) Google Scholar). Unfortunately, inhibition by MAFP of Group V sPLA2expression and activity could not be reversed in our macrophage studies with LPS alone by supplementing the medium with exogenous AA (up to 100 μm). This was not unexpected because P388D1cells manifest an extraordinarily high capacity to import free AA from exogenous sources and incorporate it into membranes (19Balsinde J. Barbour S.E. Bianco I.D. Dennis E.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11060-11064Crossref PubMed Scopus (127) Google Scholar, 24Balsinde J. Bianco I.D. Ackermann E.J. Conde-Frieboes K. Dennis E.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8527-8531Crossref PubMed Scopus (255) Google Scholar, 25Balsinde J. Dennis E.A. Eur. J. Biochem. 1996; 235: 480-485Crossref PubMed Scopus (29) Google Scholar), which is much higher than that of most other cells (26Surette M.E. Chilton F.H. Biochem. J. 1998; 330: 915-921Crossref PubMed Scopus (29) Google Scholar). Thus, the half-life of the free AA in the cell would be too short to adequately mimic the low but continued production of AA-derived cPLA2 upon long term LPS exposure.A model has recently emerged suggesting differential actions of COX-1 and COX-2 by virtue of differential coupling to distinct PLA2s (2Balsinde J. Balboa M.A. Dennis E.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7951-7956Crossref PubMed Scopus (171) Google Scholar, 3Reddy S.T. Herschman H.R. J. Biol. Chem. 1997; 272: 3231-3237Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 6Bingham III, C.O. Murakami M. Fujishima H. Hunt J.E. Austen K.F. Arm J.P. J. Biol. Chem. 1996; 271: 25936-25944Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 8Murakami M. Matsumoto R. Urade Y. Austen K.F. Arm J.P. J. Biol. Chem. 1995; 270: 3239-3246Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 20Murakami M. Shimbara S. Kambe T. Kuwata H. Winstead M.V. Tischfield J.A. Kudo I. J. Biol. Chem. 1998; 273: 14411-14423Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar, 21Murakami M. Kambe T. Shimbara S. Kudo I. J. Biol. Chem. 1999; 274: 3103-3115Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar, 27Murakami M. Nakatani Y. Kudo I. J. Biol. Chem. 1996; 271: 30041-30051Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). Thus, depending on whether cPLA2 or sPLA2 is the provider of free AA, either COX-1 or COX-2 would be responsible for PGE2release. However, which PLA2 form couples to which COX isoform appears to depend strongly on cell type. We have recently demonstrated that the immediate, PAF receptor-mediated phase of PGE2 production in LPS-primed macrophages involves sPLA2 coupling to COX-2 (2Balsinde J. Balboa M.A. Dennis E.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7951-7956Crossref PubMed Scopus (171) Google Scholar). The current results support a similar kind of coupling for the delayed PGE2 production in LPS-treated cells. Identical coupling has been suggested by Arm and co-workers (6Bingham III, C.O. Murakami M. Fujishima H. Hunt J.E. Austen K.F. Arm J.P. J. Biol. Chem. 1996; 271: 25936-25944Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar) for the delayed phase of PGE2 generation in mast cells. These results raise another interesting concept regarding the regulation of PGE2 during both phases of activation. As is the case for AA release (Fig. 2 A), we have observed that the amount of PGE2 generated during the Ca2+-dependent short term stimulation is comparable to the amount produced in the late phase. It follows from this comparison that although the effector enzymes involved in the response are the same (i.e. cPLA2, sPLA2, COX-2), the regulatory mechanisms differ. Thus, in the short phase at low levels of COX-2, it appears that the dramatic burst in AA release is what determines the amount of PGE2produced. In contrast, in the delayed phase at comparably lower AA availability, it appears that both the induction of large amounts of COX-2 protein and of the AA provider, Group V sPLA2, determine the amount of PGE2 produced.It is important to note, however, that our results have not excluded that a minor fraction of the long term PGE2 produced in response to LPS could arise from the AA generated by the cPLA2. Should this be the case, some cPLA2/COX-2 coupling may exist as well, similar to what has been suggested by Reddy and Herschman (3Reddy S.T. Herschman H.R. J. Biol. Chem. 1997; 272: 3231-3237Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar) for delayed PGD2production in mast cells and by Murakami et al. (5Murakami M. Kuwata H. Amakasu Y. Shimbara S. Nakatani Y. Atsumi G. Kudo I. J. Biol. Chem. 1997; 272: 19891-19897Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar) in cells derived from Group IIA-deficient mice. The striking feature of the current work is that although COX-1 is present in active form in the P388D1 cells (2Balsinde J. Balboa M.A. Dennis E.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7951-7956Crossref PubMed Scopus (171) Google Scholar), it appears to be spared from the process of long term PGE2 production. This finding remains unexplained but has recently been recognized in other cell types as well (6Bingham III, C.O. Murakami M. Fujishima H. Hunt J.E. Austen K.F. Arm J.P. J. Biol. Chem. 1996; 271: 25936-25944Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 8Murakami M. Matsumoto R. Urade Y. Austen K.F. Arm J.P. J. Biol. Chem. 1995; 270: 3239-3246Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 22Kuwata H. Nakatani Y. Murakami M. Kudo I. J. Biol. Chem. 1998; 273: 1733-1740Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). Recent work by Spencer et al. (16Spencer A.G. Woods J.W. Arakawa T. Singer I.I. Smith W.L. J. Biol. Chem. 1998; 273: 9886-9893Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar) showed no differences in the distribution of COX-1 versus COX-2 among subcellular fractions in a variety of cells. Thus subcellular compartmentalization may not be the cause for COX-1 not being utilized during LPS signaling. Other putative explanations may include the existence of COX-selective regulatory components, selective coupling to terminal PG synthases, or kinetic differences in AA utilization by the two isoforms.In summary, we have established a subclone of P388D1 cells, MAB, that displays long term responsiveness to LPS in terms of PGE2 generation. We have confirmed (11Balboa M.A. Balsinde J. Winstead M.V. Tischfield J.A. Dennis E.A. J. Biol. Chem. 1996; 271: 32381-32384Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar) that these cells express Group V sPLA2, not Group IIA sPLA2, and found that (i) Group V sPLA2 is a key enzyme in long term AA mobilization as well and (ii) Group V sPLA2 is functionally coupled to COX-2. Furthermore, our results have suggested that cPLA2 plays a key role in long term AA mobilization, at least partly by regulating the expression of Group V sPLA2. Arachidonic acid (AA) 1The abbreviations used are: AA, arachidonic acid; PAF, platelet-activating factor; LPS, bacterial lipopolysaccharide; cPLA2, Group IV cytosolic phospholipase A2; sPLA2, secretory phospholipase A2; COX, cyclooxygenase (prostaglandin H2synthase); MAFP, methylarachidonyl fluorophosphonate; PGE2, prostaglandin E2; iPLA2, Ca2+-independent phospholipase A21The abbreviations used are: AA, arachidonic acid; PAF, platelet-activating factor; LPS, bacterial lipopolysaccharide; cPLA2, Group IV cytosolic phospholipase A2; sPLA2, secretory phospholipase A2; COX, cyclooxygenase (prostaglandin H2synthase); MAFP, methylarachidonyl fluorophosphonate; PGE2, prostaglandin E2; iPLA2, Ca2+-independent phospholipase A2mobilization and the generation of prostaglandins by major immunoinflammatory cells such as macrophages and mast cells usually occur in two phases. The immediate phase, which takes minutes and is elicited by Ca2+-mobilizing agonists such as platelet-activating factor (PAF), is characterized by a burst of AA liberation. In some cells such as P388D1 macrophages (1Balsinde J. Dennis E.A. J. Biol. Chem. 1996; 271: 6758-6765Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar, 2Balsinde J. Balboa M.A. Dennis E.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7951-7956Crossref PubMed Scopus (171) Google Scholar) and MMC-34 mast cells (3Reddy S.T. Herschman H.R. J. Biol. Chem. 1997; 272: 3231-3237Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar), this burst is mainly produced by a secretory phospholipase A2 (sPLA2) but is strikingly regulated by the cytosolic Group IV phospholipase A2(cPLA2). The delayed phase of prostaglandin production is accompanied by the continuous supply of AA over long incubation periods spanning several hours. There is some discrepancy about the identity of the PLA2 isoform(s) involved in the delayed phase. Despite this phase being independent of a Ca2+ increase, the cPLA2 has often been suggested to be critically involved (3Reddy S.T. Herschman H.R. J. Biol. Chem. 1997; 272: 3231-3237Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 4Murakami M. Nakatani Y. Atsumi G. Inoue K. Kudo I. Crit. Rev. Immunol. 1997; 17: 225-284Crossref PubMed Google Scholar, 5Murakami M. Kuwata H. Amakasu Y. Shimbara S. Nakatani Y. Atsumi G. Kudo I. J. Biol. Chem. 1997; 272: 19891-19897Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). However, other studies have suggested the quantitatively more important role of the sPLA2, an enzyme that is dramatically induced during long term incubation of the cells with a variety of stimuli (4Murakami M. Nakatani Y. Atsumi G. Inoue K. Kudo I. Crit. Rev. Immunol. 1997; 17: 225-284Crossref PubMed Google Scholar, 5Murakami M. Kuwata H. Amakasu Y. Shimbara S. Nakatani Y. Atsumi G. Kudo I. J. Biol. Chem. 1997; 272: 19891-19897Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 6Bingham III, C.O. Murakami M. Fujishima H. Hunt J.E. Austen K.F. Arm J.P. J. Biol. Chem. 1996; 271: 25936-25944Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). There is, however, agreement that COX-2, another enzyme whose expression is augmented dramatically after long term stimulation, is absolutely required for long term PGE2 production, irrespective of the constitutive presence of COX-1 (7Herschman H.R. Biochim. Biophys. Acta. 1996; 1299: 125-140Crossref PubMed Scopus (1160) Google Scholar, 8Murakami M. Matsumoto R. Urade Y. Austen K.F. Arm J.P. J. Biol. Chem. 1995; 270: 3239-3246Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 9Langenbach R. Morham S.G. Tiano H.F. Loftin C.D. Ghanayem B.I. Chulada P.C. Mahler J.F. Lee C.A. Goulding E.H. Kluckman K.D. Kim H.S. Smithies O. Cell. 1995; 83: 483-492Abstract Full Text PDF PubMed Scopus (1035) Google Scholar). Using a new clone of the P388D1 macrophage-like cells termed P388D1/MAB, we provide herein evidence for the involvement of Group V sPLA2 in delayed PGE2production. Furthermore, our results suggest that Group V sPLA2 expression is dependent upon the activation of Group IV cPLA2. DISCUSSIONA striking hallmark of the immunoinflammatory response is the generation of oxygenated derivatives of AA such as the prostaglandins. The response of major prostaglandin-secreting cells such as macrophages and mast cells to proinflammatory stimuli is generally biphasic (4Murakami M. Nakatani Y. Atsumi G. Inoue K. Kudo I. Crit. Rev. Immunol. 1997; 17: 225-284Crossref PubMed Google Scholar). The first phase is completed within minutes after the addition of the stimulus, whereas the second phase usually takes several hours (4Murakami M. Nakatani Y. Atsumi G. Inoue K. Kudo I. Crit. Rev. Immunol. 1997; 17: 225-284Crossref PubMed Google Scholar). Using the murine macrophage-like cell line P388D1, we have been studying the molecular mechanisms responsible for AA mobilization and prostaglandin production in response to LPS/PAF. When primed by LPS, these cells will respond to Ca2+-mobilizing stimuli such as PAF by generating a rapid burst of free AA, part of which is converted to prostaglandins such as PGE2. Strikingly, this process is completed within a few minutes after the addition of PAF (19Balsinde J. Barbour S.E. Bianco I.D. Dennis E.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11060-11064Crossref PubMed Scopus (127) Google Scholar). No free AA or prostaglandins are produced after the immediate phase is completed, not even after several hours of cell exposure to LPS/PAF (13Glaser K.B. Asmis R. Dennis E.A. J. Biol. Chem. 1990; 265: 8658-8664Abstract Full Text PDF PubMed Google Scholar). Such a behavior, which is abnormal for a macrophage cell, has prevented us from studying the molecular mechanisms responsible for delayed prostaglandin production in macrophages. In an attempt to overcome this problem, we subcloned the P388D1 cells by limit dilution, and selecting on the basis of high responsivity to LPS/PAF, we obtained a clone termed MAB, which shows enhanced sensitivity to LPS/PAF in the immediate phase (min) and exhibits a delayed response (h) to LPS alone.Using the MAB clone, we have characterized the LPS-induced delayed prostaglandin production in terms of the role played by distinct PLA2 enzymes and their coupling with downstream COX enzymes during LPS signaling. Our previous work on the immediate response of the cells to LPS/PAF highlighted the very important role played by the novel Group V sPLA2 as the provider of most of the free AA directed to PGE2 biosynthesis (11Balboa M.A. Balsinde J. Winstead M.V. Tischfield J.A. Dennis E.A. J. Biol. Chem. 1996; 271: 32381-32384Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar). Herein, several lines of evidence suggest that Group V sPLA2 also behaves as a major provider of AA for the delayed phase of PGE2production in LPS-treated cells. First, delayed
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