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• W2023891037 abstract "Macrophages exposed to receptor-recognized forms of α2-macroglobulin (α2M*) demonstrate increased DNA synthesis and cell division. In the current study, we have probed the role of cytosolic phospholipase A2 (cPLA2) activity in the cellular response to α2M*. Ligation of the α2M* signaling receptor by α2M*, or its receptor binding fragment, increased cPLA2 activity 2–3-fold in a concentration and time-dependent manner. This activation required a pertussis toxin-insensitive G protein. Cellular binding of α2M* also induced transient translocation of cPLA2 activity to nuclei and membrane fractions. Inhibition of protein kinase C activity or chelation of Ca2+ inhibited α2M*-induced increased cPLA2 activity. Binding of α2M* to macrophages, moreover, increased phosphorylation of MEK 1/2, ERK 1/2, p38 MAPK, and JNK. Incubation of macrophages with inhibitors of MEK 1/2 or p38 MAPK before stimulation with α2M* profoundly decreased phosphorylation of MAPKs, blocking cPLA2 activation. α2M*-induced increase in [3H]thymidine uptake and cell proliferation was completely abolished if activation of cPLA2 was prevented. The response of macrophages to α2M* requires transcription factors nuclear factor κB, and cAMP-responsive element-binding protein as well as expression of the proto-oncogenes c-fos and c-myc. These studies indicate that the activation of cPLA2 plays a crucial role in α2M*-induced mitogenesis and cell proliferation. Macrophages exposed to receptor-recognized forms of α2-macroglobulin (α2M*) demonstrate increased DNA synthesis and cell division. In the current study, we have probed the role of cytosolic phospholipase A2 (cPLA2) activity in the cellular response to α2M*. Ligation of the α2M* signaling receptor by α2M*, or its receptor binding fragment, increased cPLA2 activity 2–3-fold in a concentration and time-dependent manner. This activation required a pertussis toxin-insensitive G protein. Cellular binding of α2M* also induced transient translocation of cPLA2 activity to nuclei and membrane fractions. Inhibition of protein kinase C activity or chelation of Ca2+ inhibited α2M*-induced increased cPLA2 activity. Binding of α2M* to macrophages, moreover, increased phosphorylation of MEK 1/2, ERK 1/2, p38 MAPK, and JNK. Incubation of macrophages with inhibitors of MEK 1/2 or p38 MAPK before stimulation with α2M* profoundly decreased phosphorylation of MAPKs, blocking cPLA2 activation. α2M*-induced increase in [3H]thymidine uptake and cell proliferation was completely abolished if activation of cPLA2 was prevented. The response of macrophages to α2M* requires transcription factors nuclear factor κB, and cAMP-responsive element-binding protein as well as expression of the proto-oncogenes c-fos and c-myc. These studies indicate that the activation of cPLA2 plays a crucial role in α2M*-induced mitogenesis and cell proliferation. receptor-recognized forms of α2-macroglobulin α2-macroglobulin signaling receptor for receptor-recognized forms of α2-macroglobulin lipoprotein receptor-related protein cytosolic phospholipase A2 inositol 1,4,5-trisphosphate diacylglycerol protein kinase C receptor-associated protein phospholipase mitogen-activated protein kinase phosphatidylinositol 3-kinase epidermal growth factor platelet-derived growth factor Hanks' balanced salt solution containing HEPES and NaHCO3 bovine serum albumin the receptor binding fragment of α2-macroglobulin bromoenol lactone phosphatidylinositol-dependent phospholipase inositol 1,4,5-trisphosphate receptor guanyl-5′-yl thiodiphosphate guanosine 5′-3-O-(thio) triphosphate) lysophosphatidylcholine nuclear factor κB cAMP-responsive element-binding protein mitogen-activated protein kinase/extracellular signal-regulated kinase kinase extracellular signal-regulated kinase c-Jun N-terminal protein platelet-derived growth factor phorbol 12-myristate 13-acetate The plasma proteinase inhibitor α2-macroglobulin (α2M*)1undergoes a major conformational change when it binds proteinases (1Wu S. Pizzo S.V. Colman R.W. Hirsh J. Narder V.J. Clowes A.W. George J.N. Hemostasis and Thromboses: Basic Principles and Clinical Practice. 4th Ed. Lippincott Williams & Wilkins, Philadelphia2001: 367-386Google Scholar,2Krieger M. Herz J. Annu. Rev. Biochem. 1994; 63: 601-638Crossref PubMed Scopus (1058) Google Scholar). Each α2M subunit also contains an internal thiol ester, which can be directly attacked by small nucleophiles resulting in a similar conformational change (1Wu S. Pizzo S.V. Colman R.W. Hirsh J. Narder V.J. Clowes A.W. George J.N. Hemostasis and Thromboses: Basic Principles and Clinical Practice. 4th Ed. Lippincott Williams & Wilkins, Philadelphia2001: 367-386Google Scholar). In either event, receptor recognition sites are exposed in each of the subunits (1Wu S. Pizzo S.V. Colman R.W. Hirsh J. Narder V.J. Clowes A.W. George J.N. Hemostasis and Thromboses: Basic Principles and Clinical Practice. 4th Ed. Lippincott Williams & Wilkins, Philadelphia2001: 367-386Google Scholar). These receptor-recognized forms of α2M, termed α2M*, bind to the low density lipoprotein receptor-related protein (LRP) present on a variety of cells including macrophages (1Wu S. Pizzo S.V. Colman R.W. Hirsh J. Narder V.J. Clowes A.W. George J.N. Hemostasis and Thromboses: Basic Principles and Clinical Practice. 4th Ed. Lippincott Williams & Wilkins, Philadelphia2001: 367-386Google Scholar, 2Krieger M. Herz J. Annu. Rev. Biochem. 1994; 63: 601-638Crossref PubMed Scopus (1058) Google Scholar, 3Strickland D.K. Ashcom J.D. Williams S. Burgess W.H. Migliorini M. Argraves W.S. J. Biol. Chem. 1990; 265: 17401-17404Abstract Full Text PDF PubMed Google Scholar). In 1993, we demonstrated that the binding of α2M* to macrophages activated signaling cascades characterized by an inositol 1,4,5-trisphosphate (IP3)-dependent increase in [Ca2+]i (4Misra U.K. Chu C.T. Rubenstein D.S. Gawdi G. Pizzo S.V. Biochem. J. 1993; 290: 885-891Crossref PubMed Scopus (68) Google Scholar). Subsequent studies demonstrated that the binding to macrophages activates a pertussis toxin-insensitive phospholipase C, which hydrolyzes membrane phosphoinositides generating both IP3 and diacylglycerol (5Misra U.K. Chu C.T. Gawdi G. Pizzo S.V. J. Biol. Chem. 1994; 269: 12541-12547Abstract Full Text PDF PubMed Google Scholar, 6Misra U.K. Chu C.T. Gawdi G. Pizzo S.V. J. Biol. Chem. 1994; 269: 18303-18306Abstract Full Text PDF PubMed Google Scholar). These studies also demonstrated that α2M*-mediated signal transduction was not blocked by addition of receptor-associated protein (RAP). Because RAP blocks the binding of all known ligands to LRP, this observation suggested the presence of a second α2M* receptor on macrophages, which was termed the α2M* signaling receptor (α2MSR) (1Wu S. Pizzo S.V. Colman R.W. Hirsh J. Narder V.J. Clowes A.W. George J.N. Hemostasis and Thromboses: Basic Principles and Clinical Practice. 4th Ed. Lippincott Williams & Wilkins, Philadelphia2001: 367-386Google Scholar, 5Misra U.K. Chu C.T. Gawdi G. Pizzo S.V. J. Biol. Chem. 1994; 269: 12541-12547Abstract Full Text PDF PubMed Google Scholar, 6Misra U.K. Chu C.T. Gawdi G. Pizzo S.V. J. Biol. Chem. 1994; 269: 18303-18306Abstract Full Text PDF PubMed Google Scholar). In support of the identification of a distinct α2M* receptor were several other observations. Two classes of binding sites were identified on macrophages, one of high affinity and low capacity (Kd ∼ 50 pm and ∼1600 sites/cell) and the other LRP, which demonstrated lower affinity and higher binding capacity (Kd ∼2–5 nm and ∼70,000 sites/cell) (7Howard G.C. Misra U.K. DeCamp D.L. Pizzo S.V. J. Clin. Invest. 1996; 97: 1193-1203Crossref PubMed Scopus (39) Google Scholar, 8Howard G.C. Yamaguchi Y. Misra U.K. Gawdi G. Nelsen A. DeCamp D.L. Pizzo S.V. J. Biol. Chem. 1996; 271: 14105-14111Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 9Misra U.K. Gonzalez-Gronow M. Gawdi G. Pizzo S.V. J. Biol. Chem. 1997; 272: 497-502Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 10Misra U.K. Gawdi G. Gonzalez-Gronow M. Pizzo S.V. J. Biol. Chem. 1999; 274: 25785-25791Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Binding of other ligands to LRP, moreover, initiated signaling cascades that activated a pertussis toxin-sensitive G protein and were blocked by addition of RAP (4Misra U.K. Chu C.T. Rubenstein D.S. Gawdi G. Pizzo S.V. Biochem. J. 1993; 290: 885-891Crossref PubMed Scopus (68) Google Scholar, 5Misra U.K. Chu C.T. Gawdi G. Pizzo S.V. J. Biol. Chem. 1994; 269: 12541-12547Abstract Full Text PDF PubMed Google Scholar, 6Misra U.K. Chu C.T. Gawdi G. Pizzo S.V. J. Biol. Chem. 1994; 269: 18303-18306Abstract Full Text PDF PubMed Google Scholar, 7Howard G.C. Misra U.K. DeCamp D.L. Pizzo S.V. J. Clin. Invest. 1996; 97: 1193-1203Crossref PubMed Scopus (39) Google Scholar, 8Howard G.C. Yamaguchi Y. Misra U.K. Gawdi G. Nelsen A. DeCamp D.L. Pizzo S.V. J. Biol. Chem. 1996; 271: 14105-14111Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 9Misra U.K. Gonzalez-Gronow M. Gawdi G. Pizzo S.V. J. Biol. Chem. 1997; 272: 497-502Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 10Misra U.K. Gawdi G. Gonzalez-Gronow M. Pizzo S.V. J. Biol. Chem. 1999; 274: 25785-25791Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 11Chu C.T. Howard G.C. Misra U.K. Pizzo S.V. Ann. N. Y. Acad. Sci. 1994; 737: 291-307Crossref PubMed Scopus (47) Google Scholar, 12Misra U.K. Gawdi G. Pizzo S.V. Biochem. J. 1995; 309: 151-158Crossref PubMed Scopus (38) Google Scholar). More recent observations, however, suggest that α2M*-mediated signal transduction requires the presence of LRP on cells (13Bacskai B.J. Xia M.Q. Strickland D.K. Rebeck G.W. Hyman B.T. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 11551-11556Crossref PubMed Scopus (171) Google Scholar). Thus, Backsai et al. (13Bacskai B.J. Xia M.Q. Strickland D.K. Rebeck G.W. Hyman B.T. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 11551-11556Crossref PubMed Scopus (171) Google Scholar) have shown that α2M* binding to LRP on neuronal cells mediates signaling via N-methyl-d-aspartate receptors. These authors suggest that an adapter protein causes LRP to associate with this receptor, allowing α2M* to activate signal transduction. Furthermore, Herz and colleagues (14Trommsdorff M. Borg J.P. Margolis B. Herz J. J. Biol. Chem. 1998; 273: 33556-33560Abstract Full Text Full Text PDF PubMed Scopus (489) Google Scholar, 15Gotthardt M. Trommsdorff M. Nevitt M.F. Shelton J. Richardson J.A. Stockinger W. Nimpf J. Herz J. J. Biol. Chem. 2000; 275: 25616-25624Abstract Full Text Full Text PDF PubMed Scopus (395) Google Scholar) have identified a large number of adapter proteins that can associate with LRP, and Barnes et al. (16Barnes H. Larsen B. Tyers M. van Der Geer P. J. Biol. Chem. 2001; 276: 19119-19125Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar) have demonstrated that Tyr-phosphorylated LRP associates with the adapter protein SHC in SRC-transformed cells. Based on our observations with respect to activation of the p21ras-dependent MAPK and PI 3-kinase signaling cascades and subsequent cell proliferation, we have proposed that α2M* functions like a growth factor (9Misra U.K. Gonzalez-Gronow M. Gawdi G. Pizzo S.V. J. Biol. Chem. 1997; 272: 497-502Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar,17Misra U.K. Pizzo S.V. Biochim. Biophys. Acta. 1998; 1401: 121-128Crossref PubMed Scopus (26) Google Scholar, 18Misra U.K. Pizzo S.V. Cell. Signal. 1998; 10: 441-445Crossref PubMed Scopus (26) Google Scholar, 19Misra U.K. Pizzo S.V. J. Biol. Chem. 1998; 273: 13399-13402Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 20Asplin I.R. Misra U.K. Gawdi G. Gonzalez-Gronow M. Pizzo S.V. Arch. Biochem. Biophys. 2000; 383: 135-141Crossref PubMed Scopus (27) Google Scholar). Known growth factors activate cytosolic phospholipase A2 (cPLA2) and the products of cPLA2 hydrolysis are involved in the growth-promoting effects of these factors (21Exton J.H. J. Biol. Chem. 1990; 265: 1-4Abstract Full Text PDF PubMed Google Scholar, 22Dennis E.A. J. Biol. Chem. 1994; 269: 13057-13060Abstract Full Text PDF PubMed Google Scholar, 23Clark J.D. Schievella A.R. Nalefski E.A. Lin L.L. J. Lipid Mediat. Cell Signal. 1995; 12: 83-117Crossref PubMed Scopus (425) Google Scholar, 24Leslie C.C. J. Biol. Chem. 1997; 272: 16709-16712Abstract Full Text Full Text PDF PubMed Scopus (740) Google Scholar, 25Gijon M.A. Leslie C.C. J. Leukocyte Biol. 1999; 65: 330-336Crossref PubMed Scopus (250) Google Scholar, 26Kramer R.M. Sharp J.D. FEBS Lett. 1997; 410: 49-53Crossref PubMed Scopus (235) Google Scholar). We, therefore, have studied the effect on cPLA2 activation of ligating α2M* receptors on peritoneal macrophages. Specifically, we studied activation of PKC, MEK 1/2, ERK 1/2, p38 MAPK, JNK, and cPLA2; translocation to nuclei and membranes; modulation of cell division by inhibitors of MAPKs and cPLA2; activation of transcription factors NFκB and CREB; and expression of c-fos and c-myc proto-oncogenes. The sources of thioglycollate, cell culture materials, [3H]thymidine, BAPTA/AM, genistein, staurosporine, chelerythrine, manumycin A, SB 203580, PD98059, U0126, AACOCF3, wortmannin, and EGF have previously been described (6Misra U.K. Chu C.T. Gawdi G. Pizzo S.V. J. Biol. Chem. 1994; 269: 18303-18306Abstract Full Text PDF PubMed Google Scholar, 10Misra U.K. Gawdi G. Gonzalez-Gronow M. Pizzo S.V. J. Biol. Chem. 1999; 274: 25785-25791Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). 1-(6-C17β-3Methoxyestra-1,3,5(10)-trien-17-yl) aminohexyl)-1H-pyrrole-2,5-dione (U73122) was purchased from Biomol (Plymouth Meeting, PA). Bromoenol lactone (BEL) and pertussis toxin were procured from Sigma. Endotoxin-free α2M*, binding site mutants of α2M*, and RAP were prepared as described previously (9Misra U.K. Gonzalez-Gronow M. Gawdi G. Pizzo S.V. J. Biol. Chem. 1997; 272: 497-502Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 10Misra U.K. Gawdi G. Gonzalez-Gronow M. Pizzo S.V. J. Biol. Chem. 1999; 274: 25785-25791Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Antibodies against phosphorylated MEK 1/2, ERK 1/2, p38 MAPK, and JNK were purchased from New England Biolabs (Mississauga, Ontario, Canada). Antibodies against c-Fos, c-Myc, CREB, and NFκB proteins were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). [3H]Methylcholine with specific activity of 60–90 Ci/mol was purchased from ARC (St. Louis, MO). Silica gel G plates were from Analytical Technology (Newark, DE). Lipid standards were purchased from Avanti Polar Lipids (Alabaster, AL). Xestospongin C was purchased from Calbiochem (San Diego, CA), GDPβS and GTPγS were obtained fromRoche Molecular Biochemicals, and AlF3 was from Sigma. Culture media were from Invitrogen. All other chemicals and solvents used were of the highest available grade. Thioglycollate-elicited peritoneal macrophages were obtained from pathogen-free 6-week-old C57BL/6 mice (Charles River Laboratories, Raleigh, NC) in Hanks' balanced salt solution containing 10 mm HEPES, pH 7.4, and 3.5 mmNaHCO3 (HHBSS). The cells were washed with HHBSS and suspended in RPMI 1640 medium containing 2 mm glutamine, 12.5 units/ml penicillin, 6.25 μg/ml streptomycin, and 5% fetal bovine serum and plated at a cell density of 3.5–4 × 106 cells/4.5 cm2. The monolayers were incubated for 2 h at 37 °C in a humidified CO2(5%) incubator. The monolayers were washed with HHBSS three times to remove nonadherent cells, and monolayers were incubated overnight at 37 °C in RPMI 1640 medium containing the additions listed above except that 0.2% fatty acid-free BSA replaced the serum. Macrophage monolayers adhered for 2 h in RPMI 1640 medium were radiolabeled with [3H]methylcholine (2 μCi/ml) for 16–18 h at 37 °C in a humidified CO2 (5%) incubator. The monolayers were washed three times with cold HHBSS, a volume of the buffer (300 μl) added, and monolayers (3 × 106cells/well) incubated for 5 min at 37 °C prior to stimulation with 100 pm α2M, α2M*, the 17-kDa receptor-binding fragment of α2M* (RBF), or its mutant K1370A for various periods of time. Studies were also performed with EGF (10 ng/ml) as a control. The reaction was stopped by aspirating the medium and adding a volume of chilled methanol. The cells were scraped into screw cap glass tubes and lipids extracted according to Bligh and Dyer (27Bligh E.G. Dyer W.J. Can. J. Biochem. 1959; 37: 911-917Crossref PubMed Scopus (42694) Google Scholar). The organic layer was evaporated to dryness under N2 at 37 °C, dissolved in a volume of CHCl3:CH3OH (2:1, v/v), and processed for lipids fractionation. The choline-labeled lipids were fractionated on heat activated silica gel G plates in solvent system CHCl3:CH3OH:acetic acid:H2O (65:43:1:3, v/v/v/v) (28Misra U.K. Shackelford R.E. Florine-Casteel K. Thai S.F. Alford P.B. Pizzo S.V. Adams D.O. J. Leukocyte Biol. 1996; 60: 784-792Crossref PubMed Scopus (31) Google Scholar). Each sample was co-chromatographed with 5 μg of authentic lysoPC. The plates were dried, exposed to I2 vapors, and gel areas corresponding to the standard lysoPC scraped into scintillation vials and their radioactivity counted to detect [3H]lysoPC derived from cPLA2-catalyzed cleavage of [3H]methylcholine. In experiments, where the effects of RAP (1 μm/10 min) or BEL, an inhibitor of Ca2+-independent PLA2 (29Hazen S.L. Zupan L.A. Weiss R.H. Getman D.P. Gross R.W. J. Biol. Chem. 1991; 266: 7227-7232Abstract Full Text PDF PubMed Google Scholar), were examined, [3H]methylcholine-labeled macrophages (3 × 106 cells/well) were incubated with these agents for the specified time before adding α2M*. Other details were as described above. Macrophages (3 × 106 cells) were labeled with [3H]methylcholine as described above. The labeled monolayers were permeabilized with saponin as described previously and pretreated with GTPγS (20 μm) for 5 min at 37 °C prior to stimulation with α2M* (5Misra U.K. Chu C.T. Gawdi G. Pizzo S.V. J. Biol. Chem. 1994; 269: 12541-12547Abstract Full Text PDF PubMed Google Scholar). This concentration was chosen after studies to determine the maximal effect of GTPγS on cPLA2 activation by α2M* (data not shown). Experimental details for lipid isolation, fractionation, and counting were as described above. To further examine the involvement of heterotrimeric G proteins in α2M*-induced activation of cPLA2 activity, we employed GDPβS and AlF3. GDPβS, a nonhydrolyzable analog of GDP, competitively inhibits G protein activation by GTP and GTP analogs (30Gomperts B.D. Annu. Rev. Physiol. 1990; 52: 591-606Crossref PubMed Google Scholar). AlF3, on the other hand, mimics the terminal phosphate of GTP, such that the structures of the Gα-GDP-AlFn complexes resemble that of the GTP-bound form of the protein (31Gilman A.G. Annu. Rev. Biochem. 1987; 56: 615-649Crossref PubMed Scopus (4711) Google Scholar). [3H]Methylcholine-labeled macrophages were incubated overnight as above in RPMI 1640 medium, then washed twice with HHBSS. A volume of permeabilization buffer containing saponin (20 μg/ml) was added and the cells incubated for 10 min at 25 °C (32Jones L.G. Goldstein D. Brown J.H. Circ. Res. 1988; 62: 299-305Crossref PubMed Scopus (44) Google Scholar). The cells were washed three times with HHBSS and a volume of RPMI 1640 medium added. The cells were preincubated for temperature equilibration, GDPβS (1 mm) added, and the cells incubated at 37 °C for 10 min, followed by the addition of GTPγS (20 μm). The cells were incubated for 10 min and α2M* (100 pm) added, and incubation continued for 20 min as above. The reaction was terminated by aspirating the medium and adding a volume of methanol. Other details of lipid extraction, thin layer chromatography fractionation, and determining the radioactivity in the [3H]lysoPC fraction were as described above. In experiments where the effect of AlF3 was studied on activation of macrophage cPLA2 by α2M*, AlF3 was added (20 μm) to [3H]methylcholine-labeled and washed macrophages in RPMI 1640 medium. The cells were incubated for 10 min at 37 °C, followed by the addition of α2M* (100 pm). The cells were incubated as above for 20 min. The reaction was terminated by aspirating the medium and adding a volume of methanol. Other details of lipid extraction, thin layer chromatography fractionation, and determination of radioactivity in the [3H]lysoPC fraction were as described above. Macrophages (3 × 106 cells) were obtained and labeled with [3H]methylcholine as described above. Pertussis toxin (1 μg/ml) was added to the monolayers during the last 12 h of incubation (5Misra U.K. Chu C.T. Gawdi G. Pizzo S.V. J. Biol. Chem. 1994; 269: 12541-12547Abstract Full Text PDF PubMed Google Scholar). Pertussis toxin-treated macrophages were washed with cold HHBSS, a volume of the buffer added, and the cells preincubated for 5 min at 37 °C prior to stimulation with α2M*. Other details of lipid isolation, lipid fractionation, and counting were as described above. To understand the role of G protein-coupled PI-PLC activity on cPLA2 activation in α2M*-stimulated macrophages, we employed U73122, which is an relatively specific inhibitor of G protein-mediated PI-PLC activation and PI-PLC-linked events (33Yule D.I. Williams J.A. J. Biol. Chem. 1992; 267: 13830-13835Abstract Full Text PDF PubMed Google Scholar). To [3H]methylcholine-labeled and washed macrophages in RPMI 1640 medium, U73122 (2 μm) was added, cells incubated for 10 min as above and then stimulated with α2M* (100 pm/20 min). The reaction was terminated by aspirating the medium and adding a volume of methanol. Other details for quantifying radioactivity in the [3H]lysoPC fraction were as described above. We also examined the role of IP3 generated upon hydrolysis of phosphatidylinositol 4,5-bisphosphate by G protein-coupled PI-PLC in cPLA2 activation, by inhibiting its binding to IP3 receptors (IP3R) and hence release of endoplasmic reticulum sequestered Ca2+, with xestospongin C, an antagonist of IP3R (34Gafni J. Munsch J.A. Lam T.H. Catlin M.C. Costa L.G. Molinski T.F. Pessah I.N. Neuron. 1997; 19: 723-733Abstract Full Text Full Text PDF PubMed Scopus (513) Google Scholar). To [3H]methylcholine-labeled macrophages in RPMI 1640 medium was added xestospongin C (5 μm), and the cells were incubated for 10 min as above prior to addition of α2M* (100 pm/20 min). The reaction was terminated by aspirating the medium and adding a volume of methanol added. Other details of determining radioactivity in [3H]lysoPC fraction were as described above. Murine peritoneal macrophages harvested as above were allowed to adhere for 2 h in RPMI 1640 medium containing 0.2% fatty acid-free BSA, penicillin, streptomycin, and glutamine at 37 °C in a humidified CO2 (5%) incubator. The monolayers were washed twice with HHBSS and a volume of above RPMI medium added, followed by the addition of [3H]thymidine (2 μCi/ml) (9Misra U.K. Gonzalez-Gronow M. Gawdi G. Pizzo S.V. J. Biol. Chem. 1997; 272: 497-502Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 17Misra U.K. Pizzo S.V. Biochim. Biophys. Acta. 1998; 1401: 121-128Crossref PubMed Scopus (26) Google Scholar). To the respective wells α2M* (100 pm) or PDGF (10 ng/ml) were added. In experiments where the effect of AACOCF3 (20 μm/15 min), PD98059 (50 μm/90 min), or SB 203580 (15 μm/15 min) were studied, these were added to their respective wells and cells incubated for the specified time before adding α2M* or PDGF. The cells were incubated overnight in a humidified CO2 (5%) incubator. The incubations were terminated by aspirating the medium and washing macrophages twice first with 5% trichloroacetic acid (15 min/4 °C), and then three times with HHBSS. The monolayers were lysed with 1 n NaOH and an aliquot used for liquid scintillation counting and protein estimation (35Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (215653) Google Scholar). Because increased DNA synthesis is generally associated with an increase in total cellularity, the number of macrophages before and after overnight exposure to varying concentrations of α2M* was determined. Peritoneal macrophages were harvested and allowed to adhere in six-well plates in RPMI 1640 medium containing 5% fetal bovine serum for 2 h as described above. The adhered cells were carefully scraped, centrifuged at 1200 rpm for 5 min, and suspended in 15 ml of RPMI 1640 medium containing 0.2% fatty acid-free BSA, and 0.5-ml aliquots (2 × 105 cells) were pipetted into 15-ml siliconized polypropylene tubes. To the respective tubes, a specified concentration of α2M* was added, the contents mixed gently, and the tubes incubated overnight as above. After overnight incubation, 10 μl of trypan blue solution was added to each tube, the tubes gently shaken during incubation for 2 min, and a 10-μl aliquot employed for counting the number of cells in a hemocytometer. In experiments where the modulation in cell numbers of α2M*-exposed macrophages (2 × 105cells/tube) was studied, SB203580, a specific inhibitor of p38 MAPK (15 μm/30 min) (36Cuenda A. Cohen P. Buee-Scherrer V. Goedert M. EMBO J. 1997; 16: 295-305Crossref PubMed Scopus (315) Google Scholar); PD98059, a specific inhibitor of MEK 1/2 (50 μm/90 min) (37Alessi D.R. Cuenda A. Cohen P. Dudley D.T. Saltiel A.R. J. Biol. Chem. 1995; 270: 27489-27494Abstract Full Text Full Text PDF PubMed Scopus (3255) Google Scholar); U0126, a specific inhibitor of MEK 1/2 (1 μm/10 min) (38Favata M.F. Horiuchi K.Y. Manos E.J. Daulerio A.J. Stradley D.A. Feeser W.S. Van Dyk D.E. Pitts W.J. Earl R.A. Hobbs F. Copeland R.A. Magolda R.L. Scherle P.A. Trzaskos J.M. J. Biol. Chem. 1998; 273: 18623-18632Abstract Full Text Full Text PDF PubMed Scopus (2747) Google Scholar); AACOCF3 (20 μm/15 min) (39Street I.P. Lin H.-K. Laliberte F. Ghomashchi F. Wang Z. Perrier H. Tremblay N.M. Huang Z. Weech P.K. Gelb M.H. Biochemistry. 1993; 32: 5933-5940Crossref Scopus (419) Google Scholar); and wortmannin, a specific inhibitor of PI 3-kinase (30 nm/30 min) (40Vlahos C.J. Matter W.F. Hui K.Y. Brown R.F. J. Biol. Chem. 1994; 269: 5241-5248Abstract Full Text PDF PubMed Google Scholar) were added to the respective tubes, and tubes incubated for the specified time before adding α2M* (100 pm). The tubes were incubated and cell numbers counted as described above. Freshly harvested peritoneal macrophages in RPMI 1640 medium containing penicillin, streptomycin, glutamine, and 0.2% fatty acid-free BSA were allowed to adhere in six-well plates (3 × 106 cells/well) for 2 h as above. The monolayers were washed twice with HHBSS, a volume of above RPMI 1640 medium added, and plates incubated overnight as above. The monolayers were washed twice, a volume of RPMI medium containing 0.2% fatty acid-free BSA added, and the cells pretreated with specific inhibitors/modulators of MAPKs for the specified time period before exposing to α2M* (100 pm/20 min) or buffer. The incubations were terminated by aspirating the medium. The lysis of cells, their electrophoresis, and Western immunoblotting were performed according to the manufacturer's instruction. In each case, an equal amount of protein was employed for electrophoresis. The detection of phosphorylated MAPKs by enhanced chemifluorescence and quantification of their distribution was performed by phosphorimaging (Storm®). Freshly harvested peritoneal macrophages in RPMI 1640 medium containing penicillin, streptomycin, glutamine, and 0.2% BSA were allowed to adhere in six-well plates (3 × 106 cells/well) for 2 h as above. The monolayers were washed twice with HHBSSS, a volume of above RPMI 1640 medium added, and plates incubated overnight as above. The monolayers were washed, a volume of above RPMI medium added, and the cells incubated with specific inhibitors/modulators of MAPKs or a Ca2+ chelator, for the specified time period before exposing to α2M* (100 pm/20 min) or buffer. The incubations were terminated by aspirating the medium. The lysis of cells, their electrophoresis, and Western immunoblotting were performed according to the manufacturer's instruction. In each case, an equal amount of protein was employed for electrophoresis. The detection of immunoblots was performed by enhanced chemifluorescence, and quantitation of their distribution was done by phosphorimaging (Storm®). Two-h adhered cells (3 × 106 cells/well) in above RPMI 1640 medium were radiolabeled with [3H]methylcholine (2 μCi/ml) for 16–18 h at 37 °C in a humidified CO2 (5%) incubator. The radiolabeled monolayers were washed three times with cold HHBSS, a volume of the buffer added, and monolayers incubated for 5 min at 37 °C prior to stimulation with α2M* (100 pm) for various periods of time. The reaction was stopped by aspirating the medium, and a volume of “buffer A” containing 20 mm Tris-HCl, pH 7.4, 10 mm KCl, 2 mm MgCl2, 1 mm phenylmethylsulfonyl fluoride, 20 μg/ml leupeptin, 0.3 mm CaCl2, 1 mm NaF, and 1 mm sodium orthovanadate was added. Cells were allowed to swell for 10 min on ice, followed by the addition of three volumes of “buffer B” containing 50 mm Tris-HCl, pH 7.4, 25 mm KCl, 5 mm MgCl2, 0.25 m sucrose, 1 mm phenylmethylsulfonyl fluoride, 20 μg/ml leupeptin, 0.2 mm EGTA, 0.2 mm CaCl2, 1 mm NaF, 1 mm sodium orthovanadate, and 100 mm benzamidine. The cells were scraped into glass tubes containing sodium orthovanadate and 100 mm benzamidine and homogenized in a Potter-Elvehjem homogenizer by 15 up-and-down strokes at 4 °C. The homogenates were centrifuged for 10 min at 800 ×g, 40 °C. Supernatant was carefully removed, nuclear pellets washed with cold buffer B twice by centrifuging, and the pellets suspended in a volume of buffer B. The supernatant was layered on a 30–70% sucrose gradient and c" @default.
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• W2023891037 title "Regulation of Cytosolic Phospholipase A2 Activity in Macrophages Stimulated with Receptor-recognized Forms of α2-Macroglobulin" @default.
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