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• W2012642617 abstract "Increased synthesis of insulin-like growth factor-1 is induced in murine macrophages by prostaglandin E2 (PGE2) and tumor necrosis factor-α (TNFα). Accordingly, we have investigated mechanisms regulating synthesis of PGE2 that might contribute to autocrine/paracrine effects on insulin-like growth factor-1 production. In response to zymosan, TNFα specifically induced a 5-fold increase in PGE2 synthesis, at the same time decreasing PGD2 production in a reciprocal fashion. Activators of cyclic AMP-dependent protein kinase (PKA), such as PGE2 itself or dibutyryl cyclic AMP, did not modify PGE2 production by themselves but potentiated the TNFα-induced increase in PGE2; this effect required both RNA and protein synthesis. No significant change in arachidonate release or production of other eicosanoids was observed. The inducible form of cyclooxygenase-2 (COX2) but not of the constitutive form COX1 was implicated in the generation of both PGE2 and PGD2 in these cells by use of specific inhibitors and effects of dexamethasone. Neither COX1 nor COX2 protein levels were affected by TNFα or PKA activators used alone, whereas in association, marked up-regulation of COX2 mRNA and protein was observed. Incubations of cells carried out with PGH2demonstrated that PGE2 synthase activity was increased after a TNFα pretreatment. Taken together, our results suggest that TNFα induced a switch from the PGD2 to PGE2synthesis pathway by regulating PGE2 synthase expression and/or activity and that activators of PKA markedly potentiated the TNFα-induced increase in PGE2 through up-regulation of COX2 gene expression. Increased synthesis of insulin-like growth factor-1 is induced in murine macrophages by prostaglandin E2 (PGE2) and tumor necrosis factor-α (TNFα). Accordingly, we have investigated mechanisms regulating synthesis of PGE2 that might contribute to autocrine/paracrine effects on insulin-like growth factor-1 production. In response to zymosan, TNFα specifically induced a 5-fold increase in PGE2 synthesis, at the same time decreasing PGD2 production in a reciprocal fashion. Activators of cyclic AMP-dependent protein kinase (PKA), such as PGE2 itself or dibutyryl cyclic AMP, did not modify PGE2 production by themselves but potentiated the TNFα-induced increase in PGE2; this effect required both RNA and protein synthesis. No significant change in arachidonate release or production of other eicosanoids was observed. The inducible form of cyclooxygenase-2 (COX2) but not of the constitutive form COX1 was implicated in the generation of both PGE2 and PGD2 in these cells by use of specific inhibitors and effects of dexamethasone. Neither COX1 nor COX2 protein levels were affected by TNFα or PKA activators used alone, whereas in association, marked up-regulation of COX2 mRNA and protein was observed. Incubations of cells carried out with PGH2demonstrated that PGE2 synthase activity was increased after a TNFα pretreatment. Taken together, our results suggest that TNFα induced a switch from the PGD2 to PGE2synthesis pathway by regulating PGE2 synthase expression and/or activity and that activators of PKA markedly potentiated the TNFα-induced increase in PGE2 through up-regulation of COX2 gene expression. Macrophages are known to generate prostaglandins (PGs) 1The abbreviations used are: PG, prostaglandin; IGF-1, insulin-like growth factor-1; dbcAMP, dibutyryl cyclic AMP; PKA, cyclic AMP-dependent protein kinase; COX, cyclooxygenase; PLA2, phospholipase A2; AA, arachidonic acid; GAPDH, glyceraldehyde 3-phosphodehydrogenase; CHX, cycloheximide; AD, actinomycin D; TNFα, tumor necrosis factor-α. in response to various stimuli such as endotoxin (1Lee S.H. Soyoola E. Chanmugam P. Hart S. Sun W. Zhong H. Liou S. Simmons D. Hwang D. J. Biol. Chem. 1992; 267: 25934-25938Abstract Full Text PDF PubMed Google Scholar, 2Hempel S.L. Monick M.M. Hunninghake W. J. Clin. Invest. 1994; 93: 391-396Crossref PubMed Scopus (289) Google Scholar), phorbol myristate acetate (3Bonney R.J. Wightman P.D. Dahlgren M.E. Davies P. Kuehl F.A. Humes J.L. Biochim. Biophys. Acta. 1980; 633: 410-421Crossref PubMed Scopus (43) Google Scholar), or phagocytic particles (4Humes J.L. Bonney R.J. Pelus L. Dahlgren M.E. Sadowski S.J. Kuehl F.A. Davies P. Nature. 1977; 269: 149-151Crossref PubMed Scopus (406) Google Scholar). In addition to playing important roles in such biologic processes as cell proliferation (5Elias J.A. Rossman M.D. Zurier R.B. Daniele R.P. Am. Rev. Respir. Dis. 1985; 131: 94-99PubMed Google Scholar), inflammatory and immune responses (6Phipps R.P. Stein S.H. Roper R.L. Immunol. Today. 1991; 12: 349-352Abstract Full Text PDF PubMed Scopus (496) Google Scholar, 7Roper R.L. Conrad D.H. Warner G. Phipps R.P. J. Immunol. 1990; 145: 2644-2651PubMed Google Scholar, 8Betz M. Fox B.S. J. Immunol. 1991; 146: 108-113PubMed Google Scholar), and the production of extracellular matrix proteins (9Diaz A. Munoz E. Johnston R. Korn J.H. Jimenez S.A. J. Biol. Chem. 1993; 268: 10364-10371Abstract Full Text PDF PubMed Google Scholar), prostaglandins may also act in an autocrine/paracrine fashion to modulate the responses of the macrophages themselves (10Chu E. Casey L.C. Harris J.E. Braun D.P. J. Clin. Immunol. 1993; 13: 49-57Crossref PubMed Scopus (9) Google Scholar, 11Russell S.W. Pace J.L. J. Leukocyte Biol. 1984; 35: 291-301Crossref PubMed Scopus (26) Google Scholar, 12Taffet S.M. Russell S.W. J. Immunol. 1981; 126: 424-427PubMed Google Scholar). We have previously shown that in murine macrophages, prostaglandin E2 (PGE2) increased the synthesis of insulin-like growth factor-1 (IGF-1), a growth factor for fibroblasts, by a TNFα-independent signaling pathway. The effect of TNFα on this PGE2-induced process was additive (13Fournier T. Riches D.W.H. Winston B.W. Rose D.M. Young S.K. Noble P.W. Lake F.R. Henson P.M. J. Immunol. 1995; 155: 2123-2133PubMed Google Scholar). Since macrophages are themselves a potent source of PGE2, autocrine up-regulation of IGF-1 was a distinct possibility. Furthermore, TNFα might itself alter the IGF-1 response by, in part, enhancing production of PGE2. Accordingly, in this study, we addressed the effects of TNFα and PGE2 on PGE2 production in murine bone marrow-derived macrophages. Since PGE2 acts by receptor-mediated generation of cyclic AMP and activation of protein kinase A (PKA), the effect of direct addition of dibutyryl cyclic AMP (dbcAMP) was also examined. The first enzymatic step in eicosanoid synthesis is the release of free arachidonic acid (AA) from membrane phospholipid. Depending on the cell type and stimulus, different forms of phospholipase A2(PLA2), including an 85-kDa cytosolic PLA2(cPLA2) (14Schalkwijk C.G. de Vet E. Pfeilschifter J. van den Bosch H. Eur. J. Biochem. 1992; 210: 169-176Crossref PubMed Scopus (70) Google Scholar, 15Lin L.L. Lin A.Y. Knopf J.L. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6147-6151Crossref PubMed Scopus (519) Google Scholar), 14-kDa secreted group II PLA2(sPLA2) (16Fonteh A.N. Bass D.A. Marshall L.A. Seeds M. Samet J.M. Chilton F.H. J. Immunol. 1994; 152: 5438-5446PubMed Google Scholar, 17Barbour S.E. Dennis E.A. J. Biol. Chem. 1993; 268: 21875-21882Abstract Full Text PDF PubMed Google Scholar, 18Murakami M. Kudo I. Inoue K. J. Biol. Chem. 1993; 268: 839-844Abstract Full Text PDF PubMed Google Scholar), or a calcium-independent cytosolic PLA2 (19Ramanadham S. Gross R.W. Han X. Turk J. Biochemistry. 1993; 32: 337-346Crossref PubMed Scopus (123) Google Scholar, 20Lehman J. Brown K.A. Ramanadham S. Turk J. Gross R.W. J. Biol. Chem. 1993; 268: 20713-20716Abstract Full Text PDF PubMed Google Scholar), have each been described to play a role in mediating AA release and subsequent prostaglandin synthesis. Agents that stimulate AA release in macrophages, including zymosan, phorbol 12-myristate 13-acetate, calcium ionophore A23187, and okadaic acid, can activate cPLA2 by enhancing serine phosphorylation of the enzyme (21Qiu Z.-H. de Carvalho M.S. Leslie C.C. J. Biol. Chem. 1993; 268: 24506-24513Abstract Full Text PDF PubMed Google Scholar). More recently, a role for mitogen-activated protein kinase has been described in mediation of agonist-induced activation of cPLA2 (22Lin L.L. Wartmann M. Lin A.Y. Knopf J.L. Seth A. Davis R.J. Cell. 1993; 72: 269-278Abstract Full Text PDF PubMed Scopus (1657) Google Scholar, 23Qiu Z.-H. Leslie C.C. J. Biol. Chem. 1994; 269: 19480-19487Abstract Full Text PDF PubMed Google Scholar). Cytokine-induced changes in gene expression of sPLA2 have been reported in rat mesangial cells (24Konieczkowski M. Sedor J.R. J. Clin. Invest. 1993; 92: 2425-2532Crossref Scopus (36) Google Scholar) and human synovial cells (25Angel J. Berenbaum F. Le Denmat C. Nevalainen T. Masliah J. Fournier C. Eur. J. Biochem. 1994; 226: 125-131Crossref PubMed Scopus (109) Google Scholar), but the activity of these enzymes is presumably also regulated by the extent and location of their secretion. Prostaglandin endoperoxide-synthase or cyclooxygenase (COX) catalyzes the conversion of AA to PGH2, which is then metabolized by one or more terminal synthases to a variety of biologically active prostanoids (26Vane J. Nature. 1994; 367: 215-216Crossref PubMed Scopus (696) Google Scholar). Cyclooxygenase is a key enzyme in prostanoid synthesis and possesses both fatty acid cyclooxygenase activity (producing PGG2 from AA) and PG hydroperoxidase activity (converting PGG2 to PGH2). COX2 is a recently described form of cyclooxygenase that is induced in a number of cells by proinflammatory stimuli, which contrasts to the lack of induction seen with the previously characterized constitutive enzyme, COX1 (27Feng L. Xia Y. Garcia G.E. Hwang D. Wilson C.B. J. Clin. Invest. 1995; 95: 1669-1675Crossref PubMed Scopus (456) Google Scholar). Thus, COX2 is thought to contribute to the generation of prostanoids at sites of inflammation (28Murakami M. Matsumoto R. Austen K.F. Arm J.P. J. Biol. Chem. 1994; 269: 22269-22275Abstract Full Text PDF PubMed Google Scholar), (see Ref. 29Otto J.C. Smith W.L. J. Lipid Mediat. Cell Signal. 1995; 12: 139-156Crossref PubMed Scopus (180) Google Scholar for review). In the current study, the ability of murine macrophages to synthesize PGE2 in response to a phagocytic particle (zymosan) was selectively induced by TNFα. In contrast, a concomitant decrease in PGD2 production was observed after TNFα priming, suggesting a striking switch from production of PGD2 to PGE2 and regulation of PG synthase activity. In addition, we show herein that activators of PKA markedly potentiated the TNFα-induced increase in PGE2 through up-regulation of COX2 gene expression. Our results suggest that (i) PGE2might act as an autocrine mediator to stimulate and to maintain the differentiation of uncommitted macrophages into IGF-1 producing cells and (ii) this differentiated cell type may participate in an exocrine fashion to regulate the inflammatory response by its ability to synthesize and release PGE2. C3H/HeJ mice were bred in the Biological Resource Center at the National Jewish Center. The C3H/HeJ strain is lipopolysaccharide hyporesponsive and was chosen to minimize the effects of trace lipopolysaccharide contamination (30Pace J.L. Russell S.W. Leblanc P.A. Murasko D.M. J. Immunol. 1985; 134: 977-981PubMed Google Scholar). Approximately 8-week-old mice were killed by CO2 narcosis. Dulbecco's modified Eagle's medium was obtained from Bio-Whittaker (Walkersville, MD). Fetal bovine serum (Hybri-Max) as well as all chemical compounds were purchased from Sigma except for valeryl salicylate and NS-398, which were purchased from Cayman Chemical (Ann Arbor, MI). Nonidet P-40 was purchased from Boehringer Mannheim, and nitrocellulose membranes were purchased from Bio-Rad. Recombinant murine TNFα was generously provided by Genentech, Inc. Zileuton (A64077) was obtained from Abbott Laboratories (North Chicago, IL). Zymosan was purchased from Sigma and opsonized using a human serum pool as described (3Bonney R.J. Wightman P.D. Dahlgren M.E. Davies P. Kuehl F.A. Humes J.L. Biochim. Biophys. Acta. 1980; 633: 410-421Crossref PubMed Scopus (43) Google Scholar). The antibodies used included generous gifts of antibody to PGE2 and PGD2 (Dr. J. Maclouf, INSERM, Paris), 6-keto-PGF1α (Dr. K. Allen, Colorado State University, Fort Collins, CO) and TXB2 (Dr. Frank Fitzpatrick, University of Colorado Health Science Center, Denver). Antibodies for LTB4 and LTC4 were purchased from Advanced Magnetics (Boston, MA). RNAzol B was purchased from Tel-Test, Inc. (Friendswood, TX), and BCA protein assay was purchased from Pierce. Murine COX1 cDNA probe was purchased from Cayman Chemicals (Ann Arbor, MI), and murine COX2 mRNA analysis was performed using a 1.8-kilobase pair cDNA insert kindly provided by Dr Voelkel (UCHSC, Denver). Human glyceraldehyde 3-phosphodehydrogenase (GAPDH) cDNA was provided by J. Shannon (National Jewish Center, Denver). Both monoclonal antibodies were kindly provided by Dr. J. Maclouf (INSERM, Paris). Murine bone marrow-derived macrophages were obtained using a technique previously described in detail (31Riches D.W. Henson P.M. Remigio L.K. Catteral J.F. Strunk R.C. J. Immunol. 1988; 141: 180-188PubMed Google Scholar). Dulbecco's modified Eagle's medium containing 2 mm glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin, 0.37% (w/v) NaHCO3, 10% (v/v) heat-inactivated fetal bovine serum, and 10% (v/v) L929 cell-conditioned medium as a source of colony-stimulating factor-1 was used for the isolation, culture, and stimulation of murine bone marrow-derived macrophages. Bone marrow cells were flushed asceptically from the dissected pelvises, femurs, and tibias of C3H/HeJ mice with a jet of complete medium directed through a 25-gauge needle to form a single cell suspension. The bone marrow cells were dispensed at a concentration of 0.45 × 106 cells/well in 24-well tissue culture plates for the eicosanoid study, 11 × 106 cells/100-mm diameter culture dishes for protein immunoblotting, and at a concentration of 1.8 × 106 cells/well in 6-well tissue culture plates for RNA analysis. The cells were matured at 37 °C under a 10% (v/v) CO2 atmosphere for 5 days before use. After incubation of macrophages for the indicated period of time with various stimuli, the cell monolayers were washed twice in Ca2+, Mg2+-free phosphate-buffered saline, pH 7.4; then, cells were incubated for 1 h with the phagocytic stimulus (50 particles of opsonized zymosan per cell) or with 10 μm exogenous AA in 2% BSA-Dulbecco's modified Eagle's medium. The culture medium was centrifuged to remove nonadherent cells and assayed for AA metabolites using enzyme immunoassay methods previously described (32Pradelles P. Grassi J. Maclouf J. Anal. Chem. 1985; 57: 1170-1173Crossref PubMed Scopus (645) Google Scholar, 33Westcott J.Y. Johnston K. Batt A. Wenzel S.E. Voelkel N.F. J. Appl. Physiol. 1990; 68: 2640-2648Crossref PubMed Scopus (57) Google Scholar). LTB4, LTC4, TXB2, and 6-keto-PGF1α (the stable metabolite of PGI2) were assayed directly from fresh supernatants while PGD2and PGE2 were measured as their methyl oximes after derivatization with methoxamine as described by Kelly et al.(34Kelly R.W. Deam S. Cameron M.J. Seamark R.F. Prostaglandins Leukotrienes Med. 1986; 24: 1-14Abstract Full Text PDF PubMed Scopus (122) Google Scholar). Macrophage monolayers were scraped in 500 μl of 0.5N sodium hydroxide, and total protein was determined using a BCA protein assay. Eicosanoid levels were all expressed as picograms per microgram of macrophage protein. Total content of free AA in both cell lysates and supernatants was measured using a quantitative mass spectrometric assay (35Hadley J.S. Fradin A. Murphy R.C. Biochem. Environ. Mass Spectrom. 1988; 15: 175-178Crossref PubMed Scopus (30) Google Scholar). Briefly, macrophage monolayers maintained in culture medium (0.45 × 105 cells/ml) were lysed by adding 1 ml of MeOH directly in the well, and lysates (2 ml) were acidified to pH 3 with 100 μl of 1 N HCl. Free AA was extracted twice with one volume of hexane and dried under vacuum. The pentafluorobenzyl ester was synthesized by adding a 10% solution of pentafluorobenzyl bromide in acetonitrile (50 μl) and an equal volume of a 10% solution of diisopropylamine in acetonitrile and then allowed to stand at room temperature for 10 min. After vacuum evaporation of reagents, the derivative was dissolved in 100 μl of hexane for electron capture ionization mass spectrometric analysis (36Antoine C. Murphy R.C. Henson P.M. Maclouf J. Biochim. Biophys. Acta. 1992; 1128: 139-146Crossref PubMed Scopus (31) Google Scholar). Free arachidonic acid levels were expressed as nanograms per well using a calibration curve. Mouse macrophages were isolated, cultured, and stimulated as described above. Prostaglandin synthase activity was performed according to Kelner and Uglik (37Kelner M.J. Uglik S.F. Biochem. Biophys. Res. Commun. 1994; 198: 298-303Crossref PubMed Scopus (7) Google Scholar). After a 16-h incubation period, cells (2 × 106) were collected by scraping and resuspended in 100 μl of cold buffer (50 mm TRIS, pH 7.5, 0.1 m sodium chloride, 0.1 m EDTA). The cells were lysed by freezing/thawing them twice in a acetone-dry ice bath and centrifuged at 14,000 rpm in a microcentrifuge. Supernatants were warmed to 37 °C in a water bath for 1 min, and reduced glutathione (6 μg in 10 μl) was added for 1 min. Then, 150 ng of PGH2 was added for 1 min. The reaction was terminated by adding 10 μl of a 25 mm FeCl2 solution. Spontaneous conversion was evaluated by incubating PGH2 in buffer alone with no cell. The concentrations of PGE2 and PGD2 at 0 and 1 min were determined by enzyme immunoassay. The expression of COX1, COX2, and GAPDH mRNA transcripts was determined by Northern analysis. Total cellular RNA was extracted from macrophage monolayers with 1 ml of RNAzol B for 1.8 × 106 cells (38Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63189) Google Scholar). 10 μg of total RNA was electrophoresed under denaturing conditions through a 1.2% agarose-formaldehyde gel and then transferred to NYTRAN hybridization filters as described (39Noble P.W. Lake F.R. Henson P.M. Riches D.W.H. J. Clin. Invest. 1993; 91: 2368-2375Crossref PubMed Scopus (309) Google Scholar). Total RNA was covalently linked to the membrane by UV cross-linking (0.12 joules) using a Stratalinker 1800 (Stratagene). The blots were hybridized with 106 dpm/ml of 32P-labeled cDNA probes for 18 h and washed to a final stringency of 0.2 × SSC at 42 °C, and autoradiograms were prepared by exposure to Kodak X-OMAT AR film at −70 °C. To ensure that the differences in transcript expression were specific to COX1 and COX2 mRNA and not due to differential loading of RNA, the blots were stripped in 2% glycerol for 3 min at 100 °C and reprobed with GAPDH. The density of the autoradiographic signals was quantified by scanning densitometry combined with integration using the “Image 1.35” software run on a Macintosh microcomputer. The results were expressed as a ratio of COX to GAPDH expression as indicated. Macrophage monolayers were scraped into ice-cold lysis buffer containing 50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 0.1% SDS, 1% Nonidet P-40, 10 mmEDTA, 1 mm phenylmethylsulfonyl fluoride, 10 mmdithiothreitol, and 1 mm aprotinin. 50 μg of total protein determined using a BCA protein assay were separated in SDS/10% polyacrylamide gels and transferred onto nitrocellulose membranes. The blots were washed in Tris-buffered saline (20 mm Tris, pH 7.6, 137 mm NaCl) with 0.05% (v/v) Tween 20 (TBST), blocked overnight with 5% (w/v) fat-free dry milk in TBST. The same blot was probed with a monoclonal antibody for COX1, stripped, and then reprobed with a monoclonal antibody for COX2. Results are presented as the mean ± S.E. for at least three separate experiments. Comparisons between groups were made using the student's paired t test. Murine bone marrow-derived macrophages were cultured for 5 days in the presence of colony-stimulating factor-1, followed by priming with TNFα, PGE2, or dbcAMP alone or in combination for a period of 16 h. After the culture medium was removed and cell monolayers washed, macrophages were stimulated for 1 h with 50 particles per cell of opsonized zymosan in fresh culture medium to promote production and release of arachidonic acid metabolites. Previous analysis had shown this amount and time of exposure to zymosan to be optimal (data not shown). As shown in Fig.1, 10 ng/ml TNFα led to a 5-fold increase in PGE2 production, whereas no change was observed in response to either 0.1 mm dbcAMP or 1 μmPGE2 treatment alone. However, when cells were incubated with the combination TNFα+dbcAMP or TNFα+PGE2, synergistic responses were observed corresponding to a 4-fold increase and a 2-fold increase compared with TNFα alone for dbcAMP and PGE2, respectively. To determine the point at which the TNFα and PGE2 were exerting their effect, the release of free AA was analyzed to explore the possibility that these effectors may affect the balance of deacylation and reacylation. To block metabolism of free AA released by COX and 5-lipoxygenase (5-LO), cells were incubated in the presence of 5 μm indomethacin to block the COX activity (40Futaki N. Takahishi S. Yokoyama M. Arai I. Higuchi S. Otomo S. Prostaglandins. 1994; 47: 55-59Crossref PubMed Scopus (800) Google Scholar) and 5 μm zileuton to inhibit 5-lipoxygenase (41Wenzel S.E. Trudeau J.B. Kaminsky D.A. Cohn J. Martin R.J. Westcott J.Y. Am. J. Crit. Care Med. 1995; 152: 897-905Crossref Scopus (192) Google Scholar) for the 1-h stimulation period with zymosan. In Fig.2, intracellular plus extracellular AA content is shown, as determined by mass spectrometry and expressed as nanogram/0.45 × 106 cells. Independently of the presence of the inhibitors, no significant variations in AA release were observed in response to any of the stimuli. Differences observed in AA release between the presence and absence of the COX and 5-lipoxygenase inhibitors are likely due to utilization of AA as substrate. Similar results were obtained when AA release was analyzed after the cells had been labeled with [3H]AA (data not shown). These results suggest that the effect of TNFα on PGE2 levels with or without dbcAMP is not due to enhanced phospholipase activity. To determine whether the observed induced increase in PGE2 production was exclusive to PGE2, the effect on other AA metabolites known to be produced by macrophages was examined. The production of two cyclooxygenase metabolites, TXB2 and 6-keto-PGF1α (the stable PGI2 metabolite), as well as two 5-lipoxygenase products (LTC4 and LTB4), were quantified in the supernatant of cells primed for 18 h with the various stimuli and then stimulated with zymosan. Table I shows that untreated cells released high levels of TXB2 (12.95 pg/μg), whereas release of the other eicosanoids was low. In contrast to PGE2 (Fig. 1), TNFα alone or in combination with dbcAMP did not significantly increase the production of any of these four AA metabolites studied. Interestingly, a slight but significant increase in TXB2 and LTB4 release was observed in dbcAMP-treated cells.Table ITXB2, 6-keto-PGF1α, LTC4, and LTB4 production in murine macrophagesTXB26-keto-PGF1αLTC4LTB4Untreated12.95 ± 1.860.46 ± 0.041.29 ± 0.540.33 ± 0.0310 ng/ml TNFα7.91 ± 1.200.88 ± 0.510.717 ± 0.390.29 ± 0.110.1 mM dbcAMP16.93 ± 3.09*1.10 ± 0.801.74 ± 0.980.56 ± 0.05*TNFα + dbcAMP10.57 ± 0.831.95 ± 1.240.78 ± 0.460.71 ± 0.25Release of arachidonic acid metabolites was quantified in the supernatant as described in Fig. 1 and under “Experimental Procedures.” Results are expressed as picograms per microgram protein, and values represent the mean ± S.E. of 5 (TXB2), 4 (LTB4), and 3 (LTC4) different cultures or represent the mean ± variation of two independent experiments (6-keto-PGF1α). *, p < 0.05 compared with values of untreated. Open table in a new tab Release of arachidonic acid metabolites was quantified in the supernatant as described in Fig. 1 and under “Experimental Procedures.” Results are expressed as picograms per microgram protein, and values represent the mean ± S.E. of 5 (TXB2), 4 (LTB4), and 3 (LTC4) different cultures or represent the mean ± variation of two independent experiments (6-keto-PGF1α). *, p < 0.05 compared with values of untreated. In contrast to the effect of the cytokine on thromboxane, PGI2, or leukotrienes, TNFα had a dramatic inhibitory effect on the production of PGD2. Fig.3 A depicts the dose-dependent variations in PGE2 and PGD2 synthesis and release in response to increasing concentration of TNFα. Whereas unprimed macrophages, when stimulated with zymosan, synthesize and release high amounts of PGD2(22.89 pg ± 4.99 pg of PGD2/μg of total protein;n = 6), the release of PGE2 by these same cells remained at a low level (1.75 pg ± 0.34 pg/μg). However, when cells were incubated for a 16-h period with increasing concentration of TNFα (0.01 to 100 ng/ml), a simultaneous decrease in PGD2 release and increase in PGE2 production was observed that dropped to 7.5 ± 0.88 pg/μg and increased to 10.33 ± 0.3 pg/μg, respectively (at 10 ng/ml TNFα). Interestingly, the overall amount of released prostaglandins (PGE2+PGD2) remained unchanged (15.2 pg/μg for no TNFα versus 15.5 with 10 ng/ml TNFα stimulation; Fig. 3 A). The same reciprocal pattern of prostaglandin production was seen when cells were incubated with 10 ng/ml TNFα for 16 h and then stimulated for 1 h with 10 μmexogenous AA instead of zymosan (Fig. 3 B), suggesting that the role of zymosan is to stimulate PLA2, thus generating the AA, and that the effects of TNFα are downstream of this step. All together, the results shown in Fig. 3 suggest that TNFα might act by switching the synthesis of prostaglandins from the PGD2 to the PGE2 pathway. To further investigate the molecular basis involved in the regulation of PGE2 and PGD2 synthesis in murine macrophages, cells were treated with 10 ng/ml TNFα alone or in combination with 0.1 mmdbcAMP in the presence or absence of 5 μg/ml cycloheximide or 0.5 μg/ml actinomycin D. As illustrated in Fig.4, the observed increase in PGE2 production after stimulation of cells with TNFα alone or in combination with dbcAMP was completely abolished by either cycloheximide or actinomycin D, suggesting an absolute requirement for both RNA and protein synthesis. In contrast, neither cycloheximide nor actinomycin D prevented the inhibition of PGD2 production by TNFα. Interestingly, only cycloheximide but not actinomycin D inhibited basal PGD2 levels. These results suggest that, unlike the TNFα+dbcAMP-mediated induction of PGE2synthesis, basal production of PGD2 does not require new RNA synthesis. The observed decrease in PGD2 synthesis following cycloheximide treatment might reflect the turnover of constitutive enzyme(s) involved in the basal production of this prostaglandin. Since protein and RNA synthesis were involved in the enhanced production of PGE2by the combination of TNFα+dbcAMP and because basal production of PGD2 was dependent of protein synthesis, we next examined steady state mRNA levels of the constitutive (COX1) and the inducible form (COX2) of cyclooxygenase. Messenger RNA levels were analyzed by Northern blot after a 12-h stimulation period, and hybridization with a GAPDH cDNA probe was performed to ensure for equal loading of total RNA. As shown in Fig.5, COX1 mRNA was constitutively expressed in control macrophages while COX2 mRNA level remained undetectable. Neither 1 μm PGE2 nor 0.1 mm dbcAMP affected the expression of either COX1 or COX2, whereas 10 ng/ml TNFα inhibited the basal expression of COX1 mRNA and seemed to slightly increase COX2 mRNA levels. When cells were incubated with the combination TNFα+PGE2 or dbcAMP, elevation in COX2 mRNA levels was observed. The TNFα-induced inhibition of COX1 mRNA expression was not significantly altered by the presence of PGE2 or dbcAMP. As seen in Fig.6, Western blot analysis of macrophage whole cell lysates using specific monoclonal antibodies for COX1 and COX2 showed that the COX2 protein levels paralleled those of the transcript. The low content of COX2 protein in untreated cells was not affected by any of the stimuli used alone but was enhanced when cells were incubated with the combination of TNFα+dbcAMP. In contrast, the basal expression of COX1 protein remained unchanged in response to any of the treatments. Although the TNFα-induced down-regulation of COX1 mRNA levels was observed at 12 h, no significant change in the corresponding protein was seen after 18 h. This latter observation suggests that the constitutive form of COX may be relatively stable so that protein levels were not yet affected at 18 h despite the absence of transcript at 12 h.Figure 6Anti-COX2 and anti-COX1 immunoblots of macrophage lysates. Macrophage whole cell lysates after a 16-h stimulation period were probed with monoclonal antibodies for COX1 and COX2. Comparison to a protein ladder and to the migration of pure protein (standard) identify immunoreactive COX1 and COX2. As described by others (58Sirois J. Richards J.S. J. Biol. Chem. 1992; 267: 6382-6388Abstract Full Text PDF PubMed Google Scholar), murine COX2 migrates as two bands, the lower band resulting from inefficient N-glycosylation that has no effects on enzyme activity. This Western blot is representative of three separate experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To further characterize which COX isozyme was involved in the up-regulated synthesis of PGE2, inhibitors of COX1 and/or COX2 activities were used. As shown in Fig.7 A, the C" @default.
• W2012642617 created "2016-06-24" @default.
• W2012642617 creator A5030906578 @default.
• W2012642617 creator A5060331150 @default.
• W2012642617 creator A5089263670 @default.
• W2012642617 date "1997-12-01" @default.
• W2012642617 modified "2023-09-27" @default.
• W2012642617 title "Tumor Necrosis Factor-α Inversely Regulates Prostaglandin D2 and Prostaglandin E2 Production in Murine Macrophages" @default.
• W2012642617 cites W1488753908 @default.
• W2012642617 cites W1501606466 @default.
• W2012642617 cites W1519478770 @default.
• W2012642617 cites W1527859504 @default.
• W2012642617 cites W1537493052 @default.
• W2012642617 cites W1538690208 @default.
• W2012642617 cites W1548653314 @default.
• W2012642617 cites W1554644961 @default.
• W2012642617 cites W1587123510 @default.
• W2012642617 cites W1601845527 @default.
• W2012642617 cites W1641540149 @default.
• W2012642617 cites W1964705779 @default.
• W2012642617 cites W1978111778 @default.
• W2012642617 cites W1980132152 @default.
• W2012642617 cites W1987175676 @default.
• W2012642617 cites W1993667392 @default.
• W2012642617 cites W2011508883 @default.
• W2012642617 cites W2013190602 @default.
• W2012642617 cites W2015871485 @default.
• W2012642617 cites W2019404076 @default.
• W2012642617 cites W2022037486 @default.
• W2012642617 cites W2025325421 @default.
• W2012642617 cites W2030997003 @default.
• W2012642617 cites W2034277711 @default.
• W2012642617 cites W2034679499 @default.
• W2012642617 cites W2037764011 @default.
• W2012642617 cites W2040455391 @default.
• W2012642617 cites W2048152958 @default.
• W2012642617 cites W2060789551 @default.
• W2012642617 cites W2062003642 @default.
• W2012642617 cites W2077532004 @default.
• W2012642617 cites W2081273158 @default.
• W2012642617 cites W2081822067 @default.
• W2012642617 cites W2083918642 @default.
• W2012642617 cites W2093585097 @default.
• W2012642617 cites W2094752831 @default.
• W2012642617 cites W2111144017 @default.
• W2012642617 cites W2120654193 @default.
• W2012642617 cites W2123758031 @default.
• W2012642617 cites W2130964473 @default.
• W2012642617 cites W2146388445 @default.
• W2012642617 cites W2146643436 @default.
• W2012642617 cites W2297797255 @default.
• W2012642617 cites W2330794658 @default.
• W2012642617 cites W2402328530 @default.
• W2012642617 cites W2410510205 @default.
• W2012642617 cites W4249032892 @default.
• W2012642617 cites W4294216491 @default.
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