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• W1991453413 abstract "COX-2-dependent prostaglandin (PG) E2 synthesis regulates macrophage MMP expression, which is thought to destabilize atherosclerotic plaques. However, the administration of selective COX-2 inhibitors paradoxically increases the frequency of adverse cardiovascular events potentially through the loss of anti-inflammatory prostanoids and/or disturbance in the balance of pro- and anti-thrombotic prostanoids. To avoid these collateral effects of COX-2 inhibition, a strategy to identify and block specific prostanoid-receptor interactions may be required. We previously reported that macrophage engagement of vascular extracellular matrix (ECM) triggers proteinase expression through a MAPKerk1/2-dependent increase in COX-2 expression and PGE2 synthesis. Here we demonstrate that elicited macrophages express the PGE2 receptors EP1–4. When plated on ECM, their expression of EP2 and EP4, receptors linked to PGE2-induced activation of adenylyl cyclase, is strongly stimulated. Forskolin and dibutryl cyclic-AMP stimulate macrophage matrix metalloproteinase (MMP)-9 expression in a dose-dependent manner. However, an EP2 agonist (butaprost) has no effect on MMP-9 expression, and macrophages from EP2 null mice exhibited enhanced COX-2 and MMP-9 expression when plated on ECM. In contrast, the EP4 agonist (PGE1-OH) stimulated macrophage MMP-9 expression, which was inhibited by the EP4 antagonist ONO-AE3-208. When compared with COX-2 silencing by small interfering RNA or inhibition by celecoxib, the EP4 antagonist was as effective in inhibiting ECM-induced proteinase expression. In addition, ECM-induced MMP-9 expression was blocked in macrophages in which EP4 was silenced by small interfering RNA. Thus, COX-2-dependent ECM-induced proteinase expression is effectively blocked by selective inhibition of EP4, a member of the PGE2 family of receptors. COX-2-dependent prostaglandin (PG) E2 synthesis regulates macrophage MMP expression, which is thought to destabilize atherosclerotic plaques. However, the administration of selective COX-2 inhibitors paradoxically increases the frequency of adverse cardiovascular events potentially through the loss of anti-inflammatory prostanoids and/or disturbance in the balance of pro- and anti-thrombotic prostanoids. To avoid these collateral effects of COX-2 inhibition, a strategy to identify and block specific prostanoid-receptor interactions may be required. We previously reported that macrophage engagement of vascular extracellular matrix (ECM) triggers proteinase expression through a MAPKerk1/2-dependent increase in COX-2 expression and PGE2 synthesis. Here we demonstrate that elicited macrophages express the PGE2 receptors EP1–4. When plated on ECM, their expression of EP2 and EP4, receptors linked to PGE2-induced activation of adenylyl cyclase, is strongly stimulated. Forskolin and dibutryl cyclic-AMP stimulate macrophage matrix metalloproteinase (MMP)-9 expression in a dose-dependent manner. However, an EP2 agonist (butaprost) has no effect on MMP-9 expression, and macrophages from EP2 null mice exhibited enhanced COX-2 and MMP-9 expression when plated on ECM. In contrast, the EP4 agonist (PGE1-OH) stimulated macrophage MMP-9 expression, which was inhibited by the EP4 antagonist ONO-AE3-208. When compared with COX-2 silencing by small interfering RNA or inhibition by celecoxib, the EP4 antagonist was as effective in inhibiting ECM-induced proteinase expression. In addition, ECM-induced MMP-9 expression was blocked in macrophages in which EP4 was silenced by small interfering RNA. Thus, COX-2-dependent ECM-induced proteinase expression is effectively blocked by selective inhibition of EP4, a member of the PGE2 family of receptors. Atherosclerosis is a chronic inflammatory disease characterized by lipid accumulation, macrophage recruitment, smooth muscle proliferation, and fibrosis (1Robbie L. Libby P. Ann. N. Y. Acad. Sci. 2001; 947: 167-179Crossref PubMed Scopus (87) Google Scholar, 2Hansson G.K. N. Engl. J. Med. 2005; 352: 1685-1695Crossref PubMed Scopus (6953) Google Scholar). Macrophage proteinase expression compromises the structural integrity of atherosclerotic lesions by degrading components of the extracellular matrix (ECM), 2The abbreviations used are: ECM, extracellular matrix; COX, cyclooxygenase; PG, prostaglandin; MMP, matrix metalloproteinase; SMC, smooth muscle cell; MAPK, mitogen-activated protein kinase; Erk, extracellular signal-regulated kinase; PBS, phosphate-buffered saline; DPBS, Dulbecco's PBS; DMEM, Dulbecco's modification of Eagle's medium; FBS, fetal bovine serum; SMC, smooth muscle cell; RT, reverse transcription; HRP, horseradish peroxidase; LPS, lipopolysaccharide; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; TNF, tumor necrosis factor; siRNA, small interfering RNA; LE-BSA, low endotoxin bovine serum albumin. 2The abbreviations used are: ECM, extracellular matrix; COX, cyclooxygenase; PG, prostaglandin; MMP, matrix metalloproteinase; SMC, smooth muscle cell; MAPK, mitogen-activated protein kinase; Erk, extracellular signal-regulated kinase; PBS, phosphate-buffered saline; DPBS, Dulbecco's PBS; DMEM, Dulbecco's modification of Eagle's medium; FBS, fetal bovine serum; SMC, smooth muscle cell; RT, reverse transcription; HRP, horseradish peroxidase; LPS, lipopolysaccharide; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; TNF, tumor necrosis factor; siRNA, small interfering RNA; LE-BSA, low endotoxin bovine serum albumin. which contributes to lesion ulceration or rupture and subsequent sequelae of thrombosis, myocardial infarction, and stroke (3Moreno P.R. Falk E. Palacios I.F. Newell J.B. Fuster V. Fallon J.T. Circulation. 1994; 90: 775-778Crossref PubMed Scopus (1050) Google Scholar, 4Shah P.K. Falk E. Badimon J.J. Fernandez-Ortiz A. Mailhac A. Villareal-Levy G. Fallon J.T. Regnstrom J. Fuster V. Circulation. 1995; 92: 1565-1569PubMed Google Scholar, 5Galis Z.S. Sukhova G.K. Lark M.W. Libby P. J. Clin. Investig. 1994; 94: 2493-2503Crossref PubMed Scopus (2192) Google Scholar, 6Galis Z.S. Sukhova G.K. Kranzhöfer R. Clark S. Libby P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 402-406Crossref PubMed Scopus (515) Google Scholar). A substantial body of evidence has identified cyclooxygenase (COX)-2 as a targetable component of the signaling pathway responsible for increased proteinase expression by macrophages in atherosclerotic lesions. COX metabolizes arachidonic acid to an unstable endoperoxide, which is then converted to the principal prostaglandins (PG) by specific synthases (7Hla T. Bishop-Bailey D. Liu C.H. Schaefers H.L. Trifan O.C. Int. J. Biochem. Cell Biol. 1999; 31: 551-557Crossref PubMed Scopus (190) Google Scholar, 8Tanabe T. Tohnai N. Prostaglandins Other Lipid Mediat. 2002; 68–9: 95-114Crossref Scopus (369) Google Scholar). COX-2 expression is elevated in atherosclerotic lesions (9McGeer P.L. McGeer E.G. Yasojima K. Exp. Gerontol. 2002; 37: 925-929Crossref PubMed Scopus (28) Google Scholar, 10Stemme V. Swedenborg J. Claesson H. Hansson G.K. Eur. J. Vasc. Endovasc. Surg. 2000; 20: 146-152Abstract Full Text PDF PubMed Scopus (89) Google Scholar, 11Schonbeck U. Sukhova G.K. Graber P. Coulter S. Libby P. Am. J. Pathol. 1999; 155: 1281-1291Abstract Full Text Full Text PDF PubMed Scopus (383) Google Scholar, 12Baker C.S. Hall R.J. Evans T.J. Pomerance A. Maclouf J. Creminon C. Yacoub M.H. Polak J.M. Arterioscler. Thromb. Vasc. Biol. 1999; 19: 646-655Crossref PubMed Scopus (290) Google Scholar, 13Cipollone F. Prontera C. Pini B. Marini M. Fazia M. De Cesare D. Iezzi A. Ucchino S. Boccoli G. Saba V. Chiarelli F. Cuccurullo F. Mezzetti A. Circulation. 2001; 104: 921-927Crossref PubMed Scopus (357) Google Scholar, 14Belton O. Byrne D. Kearney D. Leahy A. Fitzgerald D.J. Circulation. 2000; 102: 840-845Crossref PubMed Scopus (375) Google Scholar). PGE2, an important mediator of the inflammatory response, stimulates proteinase expression by a variety of cells including macrophages (15Corcoran M.L. Kibbey M.C. Kleinman H.K. Wahl L.M. J. Biol. Chem. 1995; 270: 10365-10368Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 16Shankavaram U.T. DeWitt D.L. Funk S.E. Sage E.H. Wahl L.M. J. Cell. Physiol. 1997; 173: 327-334Crossref PubMed Scopus (98) Google Scholar, 17Ottino P. Bazan H.E.P. Curr. Eye Res. 2001; 23: 77-85Crossref PubMed Scopus (51) Google Scholar, 18Dohadwala M. Batra R.K. Luo J. Lin Y. Kostyantyn K. Pold M. Sharma S. Dubinett S.M. J. Biol. Chem. 2002; 277: 50828-50833Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar). Both PGE synthase and matrix metalloproteinase (MMP) activities are elevated in regions of symptomatic plaques rich in macrophages and susceptible to rupture (13Cipollone F. Prontera C. Pini B. Marini M. Fazia M. De Cesare D. Iezzi A. Ucchino S. Boccoli G. Saba V. Chiarelli F. Cuccurullo F. Mezzetti A. Circulation. 2001; 104: 921-927Crossref PubMed Scopus (357) Google Scholar, 19Cipollone F. Fazia M. Iezzi A. Ciabattoni G. Pini B. Cuccurullo C. Ucchino S. Spigonardo F. De Luca M. Prontera C. Chiarelli F. Cuccurullo F. Mezzetti A. Arterioscler. Thromb. Vasc. Biol. 2004; 24: 1259-1265Crossref PubMed Scopus (109) Google Scholar). Moreover, treatment of low density lipoprotein receptor-deficient mice with a specific COX-2 inhibitor resulted in reduced aortic atherosclerosis (20Burleigh M.E. Babaev V.R. Oates J.A. Harris R.C. Gautam S. Riendeau D. Marnett L.J. Morrow J.D. Fazio S. Linton M.F. Circulation. 2002; 105: 1816-1823Crossref PubMed Scopus (281) Google Scholar), and statin-dependent plaque stabilization was associated with decreased COX-2, PGE synthase, and MMP activities (21Cipollone F. Fazia M. Iezzi A. Zucchelli M. Pini B. De Cesare D. Ucchino S. Spigonardo F. Bajocchi G. Bei R. Muraro R. Artese L. Pitattelli A. Chiarelli F. Cuccurullo F. Mezzetti A. Circulation. 2003; 107: 1479-1485Crossref PubMed Scopus (142) Google Scholar, 22Hernandez-Presa M.A. Martin-Ventura J.L. Ortego M. Gomez-Hernandez A. Tunon J. Hernandez-Vargas P. Blanco-Colio L.M. Mas S. Aparicio C. Ortega L. Vivanco F. Gerique J.G. Diaz C. Hernandez G. Egido J. Atherosclerosis. 2002; 160: 49-58Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 23Ganne F. Vasse M. Beaudeux J.L. Peynet J. Francois A. Mishal Z. Chartier A. Tobelem G. Vannier J.P. Soria J. Soria C. Thromb. Haemostasis. 2000; 84: 680-688Crossref PubMed Scopus (80) Google Scholar, 24Bellosta S. Via D. Canavesi M. Pfister P. Fumagalli R. Paoletti R. Bernini F. Arterioscler. Thromb. Vasc. Biol. 1998; 18: 1671-1678Crossref PubMed Scopus (522) Google Scholar). Finally, a naturally occurring polymorphism in the COX-2 promoter that is associated with reduced expression appears to protect against myocardial infarction and stroke (25Cipollone F. Toniato E. Martinotti S. Fazia M. Iezzi A. Cuccurullo C. Pini B. Ursi S. Vitullo G. Averna M. Arca M. Montali A. Campagna F. Ucchino S. Spigonardo F. Taddei S. Virdis A. Ciabattoni G. Notarbartolo A. Cuccurullo F. Mezzetti A. J. Am. Med. Assoc. 2004; 291: 2221-2228Crossref PubMed Scopus (240) Google Scholar). Taken together, these data suggest that selective inhibition of COX-2 would lead to reduced PGE2 and MMP expression, resulting in greater plaque stability. However, recent data indicate that administration of COX-2-selective inhibitors increases adverse cardiovascular events (26Ray W.A. Stein M. Daugherty J.R. Hall K. Arbogast P.G. Griffin M.R. Lancet. 2002; 360: 1071-1073Abstract Full Text Full Text PDF PubMed Scopus (492) Google Scholar, 27Mukherjee D.M. Nissen S.E. Topol E.J. J. Am. Med. Assoc. 2001; 286: 954-959Crossref PubMed Scopus (1673) Google Scholar). The explanation for this effect is not fully understood; however, inhibition of COX-2 results in the loss of all downstream PGs, some of which have anti-inflammatory functions (28De Gaetano G. Donati M.B. Cerletti C. Trends Pharmacol. Sci. 2003; 24: 245-252Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). Likewise, it has been suggested that selective COX-2 inhibitors block PGI2 production by vascular endothelium, without inhibiting COX-1-dependent platelet thromboxane A2 expression, thereby supporting a pro-thrombotic state (29McAdam B.F. Catella-Lawson F. Mardini I.A. Kapoor S. Lawson J.A. FitzGerald G.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 272-277Crossref PubMed Scopus (1189) Google Scholar, 30Cheng Y. Austin S.C. Rocca B. Koller B.H. Coffman T.M. Grosser T. Lawson J.A. FitzGerald G.A. Science. 2002; 296: 539-541Crossref PubMed Scopus (724) Google Scholar). Finally, several cyclooxygenase-independent effects of the COX-2 inhibitors have been described (31Hwang D.H. Fung V. Dannenberg A.J. Neoplasia. 2002; 4: 91-97Crossref PubMed Google Scholar), which may contribute to adverse cardiovascular events. Despite the adverse cardiovascular events associated with selective COX-2 inhibition, the COX-2-PGE2-MMP-9 axis remains an attractive target to block macrophage proteinase expression at sites of chronic inflammation. In this regard, we previously reported that macrophage engagement of vascular smooth muscle cell (SMC)-derived ECM triggers proteinase expression through a protein kinase C-dependent activation of MAPKerk1/2, which leads to increased COX-2 expression and PGE2 synthesis (32Khan K.M. Howe L.R. Falcone D.J. J. Biol. Chem. 2004; 279: 22039-22046Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Selective inhibition of macrophage COX-2 activity blocked ECM-induced proteinase expression and ECM-induced PGE2 production, and MMP-9 expression was markedly reduced in COX-2–/– macrophages compared with wild type macrophages (32Khan K.M. Howe L.R. Falcone D.J. J. Biol. Chem. 2004; 279: 22039-22046Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). In studies reported here, we have determined whether selective inhibition of the PGE2 receptor family (EP1–4) can attenuate ECM-induced MMP-9 expression by macrophages. We demonstrate that thioglycollate-elicited macrophages express EP1–4. When plated on ECM, their expression of EP2 and EP4, receptors linked to PGE2-induced activation of adenylyl cyclase (33Regan J.W. Life Sci. 2003; 74: 143-153Crossref PubMed Scopus (382) Google Scholar), is strongly stimulated. Although forskolin and dibutryl cAMP stimulate macrophage MMP-9 expression in a dose-dependent manner, the selective EP2 agonist (butaprost) has no effect on macrophage MMP-9 expression, and macrophages from EP2 null mice exhibited enhanced COX-2 and MMP-9 expression when plated on ECM. In contrast, a selective EP4 agonist (PGE1-OH) stimulated macrophage MMP-9 expression, which was inhibited by the EP4 antagonist ONO-AE3-208. Finally, preincubation of macrophages with the EP4 antagonist or inhibition of their EP4 expression by siRNA was as effective in inhibiting ECM-induced MMP-9 expression as treatment with the selective COX-2 inhibitor celecoxib or inhibition of COX-2 expression by siRNA. Thus, COX-2-dependent ECM-induced proteinase expression is markedly attenuated by selective inhibition of EP4, a member of the PGE2 family of receptors. Isolation of Peritoneal Macrophages—Thioglycollate-elicited peritoneal macrophages were obtained from Swiss Webster, EP2 wild type, and EP2 null mice (34Kennedy C.R. Zhang Y. Brandon S. Guan Y. Coffee K. Funk C.D. Magnuson M.A. Oates J.A. Breyer M.D. Breyer R.M. Nat. Med. 1999; 5: 217-220Crossref PubMed Scopus (56) Google Scholar) by the method of Edelson and Cohn (35Edelson P.J. Cohn Z.A. Bloom B.R. David J.R. In vitro Methods in Cell Mediated Tumor Immunity. Academic Press, Inc, New York1976: 333-340Google Scholar) as described previously (36Falcone D.J. Ferenc M.J. J. Cell. Physiol. 1988; 135: 387-396Crossref PubMed Scopus (33) Google Scholar). Mice were injected intraperitoneally (3 ml/mouse) with 3% Brewer thioglycollate medium containing 0.3 mm thioglycollate (Difco). Four days later cells were harvested by lavage with cold DPBS. Peritoneal cells were recovered by centrifugation and resuspended in Dulbecco's modification of Eagle's medium (DMEM) supplemented with 10% Cellect Gold fetal bovine serum (FBS), penicillin (100 units/ml), streptomycin (100 μg/ml), and 4 mm glutamine (Invitrogen) and plated into tissue culture flasks or multiwell plates. Cells were allowed to adhere for 4 h and then were washed free of nonadherent cells. Experiments to determine the effect of matrix on macrophage proteinase expression were carried out in DMEM supplemented with 0.1% low endotoxin bovine serum albumin. Murine Macrophage Cell Line—RAW264.7 macrophages (37Raschke W.C. Baird S. Ralph P. Nakoinz I. Cell. 1978; 15: 261-267Abstract Full Text PDF PubMed Scopus (639) Google Scholar) were obtained from American Type Culture Collection and maintained as adherent cultures in DMEM-10% FBS. Preparation of Extracellular Matrix-coated Dishes—SMC-derived matrices were prepared as previously described (38Falcone D.J. McCaffrey T.A. Haimovitz-Friedman A. Vergilio J.-A. Nicholson A.C. J. Biol. Chem. 1993; 268: 11951-11958Abstract Full Text PDF PubMed Google Scholar) with the following modifications. Rat aortic smooth muscle cells (SMC; VEC Technologies, Inc.) were plated into 6-, 12-, or 24-well plates in DMEM supplemented with supplied growth medium. 3–4 days after reaching confluence, the cell layer was removed by sequential exposure to 0.1% Triton X-100 in DPBS (2 min at room temperature) and 0.2 mm NH4OH in DPBS (2 min at room temperature). The remaining insoluble matrices were washed 3× with DPBS and stored at 4 °C. RT-PCR—RNA was prepared using RNeasy Mini kits from Qiagen. RNA (2 μg) was reversed-transcribed using Moloney murine leukemia virus reverse transcriptase (Roche Applied Science) and oligo d(T)16 primer. The resulting cDNA was then used for amplification. The PCR reaction volume was 25 μl and contained 5 μl of cDNA, 50 mm KCl, 10 mm Tris-HCl (pH 8.3), 2 mm MgCl2, 0.4 mm dNTPs, 400 nm forward primer, 400 nm reverse primer, and 2.5 units of Taq polymerase (Applied Biosystems) in a thermocycler under the following conditions. For EP1, denature for 30 s at 95 °C, anneal for 30 s at 60 °C, and extend for 45 s at 70 °C; repeat for 35 cycles with a final extension for 10 min at 70 °C. For EP2 and EP4, denature for 30 s at 95 °C, anneal for 30 s at 62 °C, and extend for 45 s at 70 °C; repeat for 35 cycles with a final extension for 10 min at 70 °C. For EP3, denature for 30 s at 95 °C, anneal for 30 s at 60 °C, and extend for 45 s at 70 °C; repeat for 40 cycles with a final extension for 10 min at 70 °C. For MMP-9, denature for 20 s at 95 °C, anneal for 20 s at 60 °C, and extend for 45 s at 72 °C; repeat for 35 cycles with a final extension for 10 min at 72 °C. For COX-2, denature for 20 s at 94 °C, anneal for 20 s at 65 °C, and extend for 30 s at 72 °C; repeat for 35 cycles with a final extension for 10 min at 72 °C. Primers for murine EP1 were: sense, 5′-TTAACCTGAGCCTAGCGGAT-3′ (nucleotides 311–331), and antisense, 5′-CGCTGAGCGTATTGCACACTA-3′ (nucleotides 955–976); primers for murine EP2 were sense, 5′-GTGGCCCTGGCTCCCGAAA GTC-3′, (nucleotides 446–468) and antisense, 5′-GGCAAGGAGCATATGGCGAAGGTG-3′ (nucleotides 957–981); primers for murine EP3 were sense, 5′-CCGGGCACGTGGTGCTTCAT-3′ (nucleotides 538–557), and antisense, 5′-TAGCAGCAGATAAACCCAGG-3′ (nucleotides 956–975); primers for murine EP4 were sense, 5′-TTCCGCTCGTGGTGCGAGTGTTC-3′ (nucleo-tides 1074–1097), and antisense, 5′-GAGGTGGTG TCTGCTTGGGTCAG-3′ (nucleotides 1539–1562). Primers for murine MMP-9 were sense, 5′-CGTCGTGATCCCCACTTACT-3′ (nucleotides 651–671), and antisense, 5′-AACACACAGGGT TTGCCTTC-3′ (nucleotides 855–875). Primers for murine COX-2 were sense, 5′-GGTCTGGTGCCT GGTCTGATGATG-3′ (nucleotides 935–958), and antisense, 5′-GTCCTTTCAAGGAGAATGGTGC-3′ (nucleotides 1637–1658). The primers for β-actin were forward, 5′-GGTCACCCACACTGTGCCCAT-3′, and reverse, 5′-GGATGCCACAGGACTCCATGC-3′. PCR products were electrophoresed in a 1% agarose gel with 0.5 μg/ml ethidium bromide and photographed under UV light. The identity of each PCR product was confirmed by DNA sequencing. Preparation of Cell Lysates—Macrophages were lysed in Tris buffer, pH 7.5, containing 20 mm Tris-HCl, 137 mm NaCl, 2 mm EDTA, 1% Triton X-100, 25 mm β-glycerophosphate, 1 mm sodium vanadate, 2 mm sodium pyrophosphate, 1 mm phenylmethylsulfonyl fluoride, and 10 μg/ml aprotinin. Lysates were centrifuged (14,000 × g) for 20 min at 4 °C. The supernatants were recovered, normalized for protein, mixed with SDS sample buffer with β-mercaptoethanol, and boiled for 5 min. Equal amounts of cell lysates were applied to gels based on protein content. Determination of Metalloproteinase Activity—The presence of metalloproteinase activity in cellular-conditioned media was determined utilizing enzyme zymography as previously described (39Khan K.M.F. Falcone D.J. J. Biol. Chem. 1997; 272: 8270-8275Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Conditioned media were mixed with SDS sample buffer (without mercaptoethanol) and incubated for 30 min at 37 °C. Samples and molecular weight markers were electrophoresed in a 10% polyacrylamide gel containing 0.1% gelatin. The gel was then washed (2×) in 2.5% Triton X-100 to remove SDS. The gel was incubated at 37 °C for 48 h in 200 mm NaCl containing 40 mm Tris-HCl and 10 mm CaCl2 (pH 7.5) and stained with Coomassie Blue. The presence of gelatinolytic activity was identified as clear bands on a uniform blue background after destaining. Western Blot Identification of MMP-9—Macrophage-conditioned media were electrophoresed in 4–15% polyacrylamide gels, and proteins were transferred to a polyvinylidene difluoride membrane. After transfer, the membrane was placed in blocking buffer for 1 h, washed in PBS (1×), and incubated for 2 h in blocking buffer containing rabbit anti-mouse MMP-9 IgG (Chemicon). The membrane was washed (2×) in 25 mm Tris buffer (pH 8) containing 137 mm NaCl, 2.7 mm KCl, and 0.5% Tween (TTBS) and incubated for 1 h in blocking buffer containing 0.3 μg/ml goat anti-rabbit IgG conjugated to HRP (Transduction Laboratories). The membrane was washed (3×) in TTBS, and bound HRP was visualized utilizing enhanced chemiluminescence. Western Blot Identification of Phosphorylated MAPKerk1/2 and p38— Cell lysates were electrophoresed in gradient gels, and proteins were transferred to a polyvinylidene difluoride membrane, which was then blocked. Following 1 wash in TTBS, the membrane was incubated for 1 h in blocking buffer containing 75 ng/ml rabbit anti-phosphospecific p44/p42 MAPK IgG or rabbit anti-phosphospecific p38 MAPK IgG (Cell Signaling Technology). The membranes were washed (2×; TTBS) and incubated for 1 h in blocking buffer containing 0.3 μg/ml goat anti-rabbit IgG conjugated to HRP. After visualization of bound HRP, membranes were stripped in 62.5 mm Tris buffer (pH 6.7) containing 100 mm β-mercaptoethanol and 2% SDS for 30 min at 50 °C, washed, and probed for total MAPKerk1/2 or p38 (Cell Signaling Technology). Western Blot Identification of COX-2—Cell lysates were electrophoresed in gradient gels, and proteins were transferred to a polyvinylidene difluoride membrane. After transfer, the membrane was blocked, washed in PBS (1×), and incubated for 1 h in blocking buffer containing 0.5 μg/ml rabbit IgG raised against a peptide containing amino acids 584–598 of murine COX-2 (Cayman Chemical). The membrane was washed (2×; TTBS) and incubated for 1 h in blocking buffer containing 0.3 μg/ml goat anti-rabbit IgG conjugated to HRP. COX-2 siRNA Transfection—RAW264.7 macrophages were transfected with COX-2 siRNA (Santa Cruz) or mock-transfected according to the manufacturer's protocol, which was modified as follows. Macrophages (T-75 flask) were washed 3× with DPBS to remove serum, and medium was replaced with antibiotic-free DMEM. The cells were mechanically harvested, recovered by centrifugation, and resuspended in antibiotic-free DMEM supplemented with 10% FBS. The cells (2 × 106/well) were separated into aliquots in a 6-well plate and incubated overnight. The next morning, 9.1 μl of 10 μm COX-2 siRNA was added to 152 μl of transfection medium (Santa Cruz) and incubated for 5 min at room temperature. In another tube 9.1 μl of the transfection reagent (Santa Cruz) was added to the transfection medium and incubated for 5 min. The two tubes were combined and incubated for 20 min to form the siRNA transfection reagent complex. Immediately before transfection, macrophage medium was removed and replaced with 1.5 ml of antibiotic-free DMEM supplemented with 10% FBS. The plate was placed on a rocker plate in the laminar flow hood, and the transfection reagent complex was added dropwise. Cells were incubated for 30 h at 37 °C. EP4 siRNA Transfection—RAW264.7 macrophages were transfected with EP4 or control siRNA (Dharmacon RNA Technologies) according to the manufacturer's protocol, which was modified as follows. Macrophages (0.5 × 106/well) were separated into aliquots into a 6-well plate and incubated overnight in serum and antibiotic-free DMEM. The next morning 200 μl of 1 μm siRNA was added to 200 μl of diluted (1:50) DharmaFECT 4 transfection reagent, incubated for 20 min, and added to 1.6 ml of DMEM containing antibiotics and 10% FBS. Immediately before transfection, macrophage medium was removed and replaced with 2 ml of the transfection medium. Cells were incubated 24 h at 37 °C. ECM Stimulates Macrophage Expression of EP2 and EP4 Prostanoid Receptors—The diverse physiological and pathophysiological effects of PGE2 are mediated by engagement of the EP family of G-protein-coupled prostanoid receptors (40Breyer R.M. Bagdassarian C.K. Myers S.A. Breyer M.D. Annu. Rev. Pharmacol. Toxicol. 2001; 41: 661-690Crossref PubMed Scopus (853) Google Scholar, 41Hata A.N. Breyer R.M. Pharmacol. Ther. 2004; 103: 147-166Crossref PubMed Scopus (676) Google Scholar). The EP family is comprised of four subtypes (EP1–4), which exhibit varying affinities for PGE2 and trigger distinct signaling pathways. Differential expression of PGE2 receptors has been reported for macrophages stimulated with LPS (42Hubbard N.E. Lee S. Lim D. Erickson K.L. Prostaglandins Leukot. Essent. Fatty Acids. 2001; 65: 287-294Abstract Full Text PDF PubMed Scopus (33) Google Scholar, 43Ikegami R. Sugimoto Y. Segi E. Katsuyama M. Karahashi H. Amano F. Maruyama T. Yamane H. Tsuchiya S. Ichikawa A. J. Immunol. 2001; 166: 4689-4696Crossref PubMed Scopus (106) Google Scholar). However, the influence of ECM on macrophage PGE2 receptor expression is unknown. Therefore, we cultured thioglycollate-elicited peritoneal macrophages on an ECM deposited in situ by rat vascular smooth muscle cells and determined its effect on EP1–4 receptor mRNA utilizing RT-PCR (Fig. 1). Elicited macrophages cultured on tissue culture plastic expressed all PGE2 receptors (EP1–4). When plated on vascular ECM, macrophage EP2 and EP4 expression were markedly induced, whereas EP1 and EP3 expression was unchanged. Similar results were observed utilizing the murine macrophage cell line RAW264.7 (data not shown). Macrophage engagement of ECM triggers protein kinase C-dependent activation of MAPKerk1/2, which leads to the stimulation of COX-2 expression and MMP-9 synthesis (32Khan K.M. Howe L.R. Falcone D.J. J. Biol. Chem. 2004; 279: 22039-22046Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar), and activation with LPS triggers the p38-dependent increase in COX-2 expression and MMP-9 synthesis (44Chen B.C. Chen Y.H. Lin W.W. Immunology. 1999; 97: 124-129Crossref PubMed Scopus (220) Google Scholar). Thus, signaling through the MAPK cascade regulates COX-2 and MMP-9 expression by macrophages. Therefore, we determined whether the observed ECM-dependent increase in EP2 and EP4 expression (Fig. 1) was also dependent on the MAPK cascade. As observed in Fig. 2A, levels of phosphorylated MAPKerk1/2 and p38 were increased when cells were plated on SMC-ECM (Fig. 2A). Preincubation of cells with an inhibitor of protein kinase C (calphostin C) or MEK-1 (U0126) blocked ECM-induced activation of MAPKerk1/2. In contrast, ECM-induced levels of phosphorylated p38 were slightly increased in cells preincubated with calphostin C and decreased in cells incubated with U0126. Equal protein loading was confirmed by probing for total MAPKerk1/2 and p38. The causal relationship between MAPKerk1/2 activation and ECM-induced MMP-9 expression is shown in Fig. 2B. As previously reported, the expression of MMP-9 by macrophages cultured on SMC-ECM was markedly elevated as compared with that in cells cultured on plastic (32Khan K.M. Howe L.R. Falcone D.J. J. Biol. Chem. 2004; 279: 22039-22046Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Virtually all the MMP-9 secreted by murine macrophages was in the pro form (105 kDa), which is slightly larger than that expressed by human cells (92 kDa). High molecular weight multimers of MMP-9 are also visible in some preparations. Preincubation of macrophages with U0126 completely blocked ECM-induced MMP-9 expression; in contrast, proteinase expression was partially attenuated by exposure to a p38 inhibitor (SB202190; Calbiochem). We next determined the role of MAPKerk1/2 and p38 on ECM-induced EP2 and EP4 expression. RNA isolated from elicited macrophages plated on ECM in the presence of a MEK-1 or p38 inhibitor was probed for expression of EP2 and EP4 utilizing RT-PCR. ECM-induced expression of the PGE2 receptors was blocked by preincubation with the MEK-1 inhibitor, whereas inhibition of p38 had no effect (Fig. 2C). Taken together with earlier studies (32Khan K.M. Howe L.R. Falcone D.J. J. Biol. Chem. 2004; 279: 22039-22046Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar), these data demonstrate that ECM-induced activation of MAPKerk1/2 after macrophage engagement" @default.
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• W1991453413 date "2006-02-01" @default.
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• W1991453413 title "Targeting Prostaglandin E2 Receptors as an Alternative Strategy to Block Cyclooxygenase-2-dependent Extracellular Matrix-induced Matrix Metalloproteinase-9 Expression by Macrophages" @default.
• W1991453413 cites W1715047706 @default.
• W1991453413 cites W1745661527 @default.
• W1991453413 cites W1955246945 @default.
• W1991453413 cites W1964797510 @default.
• W1991453413 cites W1967678975 @default.
• W1991453413 cites W1968896068 @default.
• W1991453413 cites W1969158410 @default.
• W1991453413 cites W1972809962 @default.
• W1991453413 cites W1975248941 @default.
• W1991453413 cites W1975716287 @default.
• W1991453413 cites W1976372129 @default.
• W1991453413 cites W1979861588 @default.
• W1991453413 cites W1982865160 @default.
• W1991453413 cites W1985621460 @default.
• W1991453413 cites W1990099367 @default.
• W1991453413 cites W2000260906 @default.
• W1991453413 cites W2001364102 @default.
• W1991453413 cites W2001751920 @default.
• W1991453413 cites W2009682874 @default.
• W1991453413 cites W2017437032 @default.
• W1991453413 cites W2018716226 @default.
• W1991453413 cites W2020563114 @default.
• W1991453413 cites W2024233889 @default.
• W1991453413 cites W2024847459 @default.
• W1991453413 cites W2029941300 @default.
• W1991453413 cites W2030571587 @default.
• W1991453413 cites W2037405899 @default.
• W1991453413 cites W2038084627 @default.
• W1991453413 cites W2045648679 @default.
• W1991453413 cites W2053230224 @default.
• W1991453413 cites W2053425043 @default.
• W1991453413 cites W2054512672 @default.
• W1991453413 cites W2056726045 @default.
• W1991453413 cites W2058034672 @default.
• W1991453413 cites W2060351304 @default.
• W1991453413 cites W2061177902 @default.
• W1991453413 cites W2061857216 @default.
• W1991453413 cites W2062605111 @default.
• W1991453413 cites W2062861788 @default.
• W1991453413 cites W2063136699 @default.
• W1991453413 cites W2071041511 @default.
• W1991453413 cites W2076787271 @default.
• W1991453413 cites W2083169533 @default.
• W1991453413 cites W2083519527 @default.
• W1991453413 cites W2084120431 @default.
• W1991453413 cites W2084615605 @default.
• W1991453413 cites W2085775078 @default.
• W1991453413 cites W2094941678 @default.
• W1991453413 cites W2099955838 @default.
• W1991453413 cites W2106319388 @default.
• W1991453413 cites W2113892902 @default.
• W1991453413 cites W2117169037 @default.
• W1991453413 cites W2125376440 @default.
• W1991453413 cites W2129746956 @default.
• W1991453413 cites W2131116297 @default.
• W1991453413 cites W2138877935 @default.
• W1991453413 cites W2142651227 @default.
• W1991453413 cites W2146476085 @default.
• W1991453413 cites W2149876480 @default.
• W1991453413 cites W2153458404 @default.
• W1991453413 cites W2153853877 @default.
• W1991453413 cites W2164475521 @default.
• W1991453413 cites W2164807534 @default.
• W1991453413 cites W2172253009 @default.
• W1991453413 cites W4247652559 @default.
• W1991453413 cites W4252146505 @default.
• W1991453413 doi "https://doi.org/10.1074/jbc.m506846200" @default.
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