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• W2049310294 abstract "Macrophage migration inhibitory factor (MIF) is an important pro-inflammatory mediator with the unique ability to counter-regulate the inhibitory effects of glucocorticoids on immune cell activation. MIF is released from cells in response to glucocorticoids, certain pro-inflammatory stimuli, and mitogens and acts to regulate glucocorticoid action on the ensuing inflammatory response. To gain insight into the molecular mechanism of MIF action, we have examined the role of MIF in the proliferation and intracellular signaling events of the well characterized, NIH/3T3 fibroblast cell line. Both endogenously secreted and exogenously added MIFs stimulate the proliferation of NIH/3T3 cells, and this response is associated with the activation of the p44/p42 extracellular signal-regulated (ERK) mitogen-activated protein kinases (MAP). The MIF-induced activation of these kinases was sustained for a period of at least 24 h and was dependent upon protein kinase A activity. We further show that MIF regulates cytosolic phospholipase A2 activity via a protein kinase A and ERK dependent pathway and that the glucocorticoid suppression of cytokine-induced cytoplasmic phospholipase A2 activity and arachidonic acid release can be reversed by the addition of recombinant MIF. These studies indicate that the sustained activation of p44/p42 MAP kinase and subsequent arachidonate release by cytoplasmic phospholipase A2 are important features of the immunoregulatory and intracellular signaling events initiated by MIF and provide the first insight into the mechanisms that underlie the pro-proliferative and inflammatory properties of this mediator. Macrophage migration inhibitory factor (MIF) is an important pro-inflammatory mediator with the unique ability to counter-regulate the inhibitory effects of glucocorticoids on immune cell activation. MIF is released from cells in response to glucocorticoids, certain pro-inflammatory stimuli, and mitogens and acts to regulate glucocorticoid action on the ensuing inflammatory response. To gain insight into the molecular mechanism of MIF action, we have examined the role of MIF in the proliferation and intracellular signaling events of the well characterized, NIH/3T3 fibroblast cell line. Both endogenously secreted and exogenously added MIFs stimulate the proliferation of NIH/3T3 cells, and this response is associated with the activation of the p44/p42 extracellular signal-regulated (ERK) mitogen-activated protein kinases (MAP). The MIF-induced activation of these kinases was sustained for a period of at least 24 h and was dependent upon protein kinase A activity. We further show that MIF regulates cytosolic phospholipase A2 activity via a protein kinase A and ERK dependent pathway and that the glucocorticoid suppression of cytokine-induced cytoplasmic phospholipase A2 activity and arachidonic acid release can be reversed by the addition of recombinant MIF. These studies indicate that the sustained activation of p44/p42 MAP kinase and subsequent arachidonate release by cytoplasmic phospholipase A2 are important features of the immunoregulatory and intracellular signaling events initiated by MIF and provide the first insight into the mechanisms that underlie the pro-proliferative and inflammatory properties of this mediator. The protein known as macrophage migration inhibitory factor (MIF) 1The abbreviations used are: MIF, macrophage migration inhibitory factor; rMIF, recombinant MIF; ERK, extracellular-signal-regulated kinase; cPLA2, cytoplasmic phospholipase A2; MAP, mitogen-activated protein kinase; PKA, protein kinase A; TNF, tumor necrosis factor; DMEM, Dulbecco's modified Eagle's medium; mAb. monoclonal antibody, ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; MEK, MAP kinase. has emerged to play a central role in the control of the host inflammatory and immune response. In 1993, the structure of a peptide released by the anterior pituitary gland in response to stress was found to be that of MIF (1Bernhagen J. Calandra T. Mitchell R.A. Martin S.B. Tracey K.J. Voelter W. Manogue K.R. Cerami A. Bucala R. Nature. 1993; 365: 756-759Crossref PubMed Scopus (931) Google Scholar). Subsequent studies of MIF expression in vivo have established a critical role for MIF in the host response to endotoxic shock (1Bernhagen J. Calandra T. Mitchell R.A. Martin S.B. Tracey K.J. Voelter W. Manogue K.R. Cerami A. Bucala R. Nature. 1993; 365: 756-759Crossref PubMed Scopus (931) Google Scholar), the delayed type-hypersensitivity reaction (2Bernhagen J. Bacher M. Calandra T. Metz C.N. Doty S.B. Donnelly T. Bucala R. J. Exp. Med. 1996; 183: 277-282Crossref PubMed Scopus (250) Google Scholar), and the inflammatory pathologies responsible for arthritis (3Mikulowska A. Metz C.N. Bucala R. Holmdahl R. J. Immunol. 1997; 158: 5514-5517PubMed Google Scholar, 4Leech M. Metz C. Santos L. Peng T. Holdsworth S.R. Bucala R. Morand E.F. Arthritis Rheum. 1998; 41: 910-917Crossref PubMed Scopus (149) Google Scholar), glomerulonephritis (5Lan H.Y. Bacher M. Yang N. Mu W. Nikolic-Paterson D.J. Metz C. Meinhardt A. Bucala R. Atkins R.C. J. Exp. Med. 1997; 185: 1455-1465Crossref PubMed Scopus (257) Google Scholar), and the adult respiratory distress syndrome (6Donnelly S.C. Haslett C. Reid P.T. Grant I.S. Wallace W.A.H. Metz C.N. Bruce L.J. Bucala R. Nat. Med. 1997; 3: 320-323Crossref PubMed Scopus (394) Google Scholar). Monocytes and macrophages, which had originally been considered to be the target of MIF action, were identified to release MIF in response to various pro-inflammatory stimuli and to be a significant source of MIF release in vivo (7Calandra T. Bernhagen J. Mitchell R.A. Bucala R. J. Exp. Med. 1994; 179: 1895-1902Crossref PubMed Scopus (890) Google Scholar). MIF also is a required stimulus for T-cell activation and antibody production by B cells (8Bacher M. Metz C.N. Calandra T. Mayer K. Chesney J. Lohoff M. Gemsa D. Donnelly T. Bucala R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7849-7854Crossref PubMed Scopus (616) Google Scholar). In more recent studies, immune cells have been identified to secrete MIF in response to physiological increases in glucocorticoid levels, and once released, MIF can “override” the immunosuppressive effects of steroids on cytokine production and cellular activation (8Bacher M. Metz C.N. Calandra T. Mayer K. Chesney J. Lohoff M. Gemsa D. Donnelly T. Bucala R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7849-7854Crossref PubMed Scopus (616) Google Scholar, 9Calandra T. Bernhagen J. Metz C.N. Spiegel L.A. Bacher M. Donnelly T. Cerami A. Bucala R. Nature. 1995; 377: 68-71Crossref PubMed Scopus (1050) Google Scholar). MIF normally circulates at levels that have glucocorticoid regulatory properties in vitro, leading to the concept that the base-line state of immune cell responsiveness is mutually regulated by an active MIF/glucocorticoid dyad (9Calandra T. Bernhagen J. Metz C.N. Spiegel L.A. Bacher M. Donnelly T. Cerami A. Bucala R. Nature. 1995; 377: 68-71Crossref PubMed Scopus (1050) Google Scholar, 10Calandra T. Bucala R. Crit. Rev. Immunol. 1997; 17: 77-88Crossref PubMed Google Scholar). Cytokine mediators frequently function as mitogenic growth factors (11Lang R.A. Burgess A.W. Immunol. Today. 1990; 11: 244-249Abstract Full Text PDF PubMed Scopus (7) Google Scholar). The apparent requirement for MIF in T lymphocyte and endothelial cell proliferation (8Bacher M. Metz C.N. Calandra T. Mayer K. Chesney J. Lohoff M. Gemsa D. Donnelly T. Bucala R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7849-7854Crossref PubMed Scopus (616) Google Scholar, 12Chesney J. Metz C. Bacher M. Peng T. Meinhardt A. Bucala R. Mol. Med. 1999; 5: 179-189Crossref Google Scholar) as well as data implicating MIF mRNA expression as a delayed-early response gene (13Lanahan A. Williams J.B. Sanders L.K. Nathans D. Mol. Cell. Biol. 1992; 12: 3919-3929Crossref PubMed Scopus (299) Google Scholar) prompted us to examine more closely the role of MIF in the signaling pathways associated with cell proliferation. The cell surface receptor for MIF has not yet been identified and we reasoned that information concerning MIF-mediated signaling could provide insight into the intracellular pathways underlying the action of MIF. We describe herein the role of MIF in the proliferation and cell signaling events of the well characterized fibroblast cell line, NIH/3T3. We show that the sustained activation of p44/p42 MAP kinase and the release of arachidonic acid by cytoplasmic phospholipase A2 (cPLA2) are important features of MIF stimulation, and we present data on potential mechanisms for the pro-proliferative and glucocorticoid regulatory properties of this mediator. NIH/3T3 fibroblasts (1 × 105 cells/ml) were cultured until semi-confluent in 96-well plates containing DMEM and 10% heat-inactivated fetal bovine serum. The cells then were synchronized by overnight culture in 0.5% serum-containing media (DMEM). The medium was replaced with 0.5% serum-containing DMEM supplemented with purified, mouse recombinant MIF (rMIF) (14Bendrat K. Al-Abed Y. Callaway D.J. Peng T. Calandra T. Metz C.N. Bucala R. Biochemistry. 1997; 36: 15356-15362Crossref PubMed Scopus (143) Google Scholar) (<200 pg endotoxin/mg of protein) or 10% serum-containing DMEM together with a neutralizing anti-murine MIF mAb (14.15.5, IgG1 subclass) (9Calandra T. Bernhagen J. Metz C.N. Spiegel L.A. Bacher M. Donnelly T. Cerami A. Bucala R. Nature. 1995; 377: 68-71Crossref PubMed Scopus (1050) Google Scholar) or an isotype control mAb. The 14.15.5 mAb has been shown previously to neutralize both endogenously released (native) MIF and rMIF in a variety of in vitro and in vivo studies (3Mikulowska A. Metz C.N. Bucala R. Holmdahl R. J. Immunol. 1997; 158: 5514-5517PubMed Google Scholar, 4Leech M. Metz C. Santos L. Peng T. Holdsworth S.R. Bucala R. Morand E.F. Arthritis Rheum. 1998; 41: 910-917Crossref PubMed Scopus (149) Google Scholar, 5Lan H.Y. Bacher M. Yang N. Mu W. Nikolic-Paterson D.J. Metz C. Meinhardt A. Bucala R. Atkins R.C. J. Exp. Med. 1997; 185: 1455-1465Crossref PubMed Scopus (257) Google Scholar, 6Donnelly S.C. Haslett C. Reid P.T. Grant I.S. Wallace W.A.H. Metz C.N. Bruce L.J. Bucala R. Nat. Med. 1997; 3: 320-323Crossref PubMed Scopus (394) Google Scholar, 7Calandra T. Bernhagen J. Mitchell R.A. Bucala R. J. Exp. Med. 1994; 179: 1895-1902Crossref PubMed Scopus (890) Google Scholar, 8Bacher M. Metz C.N. Calandra T. Mayer K. Chesney J. Lohoff M. Gemsa D. Donnelly T. Bucala R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7849-7854Crossref PubMed Scopus (616) Google Scholar, 9Calandra T. Bernhagen J. Metz C.N. Spiegel L.A. Bacher M. Donnelly T. Cerami A. Bucala R. Nature. 1995; 377: 68-71Crossref PubMed Scopus (1050) Google Scholar). In separate experiments, this anti-MIF mAb also was determined not to recognize bovine MIF that is present in trace quantities in fetal bovine serum. [3H]Thymidine (5 μCi/ml, NEN Life Science Products) was added to each well, and the cells were allowed to proliferate for 16 h. The cells then were harvested, and the incorporation of [3H]thymidine into DNA was quantified by liquid scintillation counting (Packard Instrument Co.). The antisense MIF studies followed methods developed previously (15Waeber G. Calandra T. Roduit R. Haefliger J.-A. Bonny C. Thompson N. Thorens B. Temler E. Meinhardt A. Bacher M. Metz C.N. Nicod P. Bucala R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4782-4787Crossref PubMed Scopus (188) Google Scholar). In brief, the pREP9 episomal plasmid (Invitrogen, Carlsbad, CA) was used to clone an MIF polymerase chain reaction product spanning the open reading frame of murine MIF. Transfection was performed following a standard procedure (Life Technologies, Inc.), 2lifetech.com/cgi-online/techonline. and transfectants were selected for and maintained with 600 μg/ml Geneticin (Life Technologies, Inc.). Cell supernatants were assayed directly for MIF content by a sandwich ELISA employing a rabbit polyclonal antiserum and a mouse monoclonal IgG1 raised to purified, mouse rMIF (9Calandra T. Bernhagen J. Metz C.N. Spiegel L.A. Bacher M. Donnelly T. Cerami A. Bucala R. Nature. 1995; 377: 68-71Crossref PubMed Scopus (1050) Google Scholar). Briefly, 96-well microtiter plates (Dynatech, Type II) were coated with the anti-mouse MIF mAb (5 μg/ml in PBS), washed, and blocked with Superblock (Pierce) containing 2% goat serum (Sigma). Sixty-μl aliquots of each sample were added to wells for 30 min at 25 °C, and the incubation was then continued overnight at 4 °C. The wells were washed and incubated with rabbit polyclonal anti-MIF serum (1:250) (9Calandra T. Bernhagen J. Metz C.N. Spiegel L.A. Bacher M. Donnelly T. Cerami A. Bucala R. Nature. 1995; 377: 68-71Crossref PubMed Scopus (1050) Google Scholar) for 2 h at 25 °C. This was followed by the addition of a goat anti-rabbit IgG conjugated to alkaline phosphatase (Roche Molecular Biochemicals), and the antibody complexes were quantified after the addition of the alkaline phosphatase substrate,p-nitrophenyl phosphate. The MIF concentrations were calculated by extrapolation from a sigmoidal quadratic standard curve using mouse rMIF (range, 0–500 ng/ml; sensitivity, 150 pg/ml). Thioglycollate-elicited peritoneal macrophages were obtained from C3H/HeN and C3H/HeJ mice that were injected 3–4 days previously with 2 ml of sterile 3% thioglycollate broth. Cells were harvested under strict aseptic conditions by lavage of peritoneal cavities with 5 ml of an ice-chilled 11.6% sucrose solution. After centrifugation and washing with sterile PBS, cells were resuspended in RPMI, 10% fetal bovine serum, enumerated, and plated at a density of 2 × 106 cells/well. 2 h post-plating, cells were thoroughly washed and treated accordingly. Macrophage and NIH/3T3 whole cell extracts were prepared from 1 × 106 adherent cells. Cells first were washed in cold PBS and then 250 μl of ice-cold radioimmune precipitation buffer (containing 1 mm NaVO4, 2 mm NaF, and a protease inhibitor mixture (Roche Molecular Biochemicals)) were added. The cells were disrupted by repeated aspiration through a 21-gauge needle. After incubation on ice for 10 min and microcentrifugation at 3000 rpm for 15 min (4 °C), the supernatants were removed, the protein concentration was determined, and the lysates were stored at −80 °C. Equal amounts of cellular proteins were fractionated on 10% SDS-polyacrylamide electrophoresis gels (Bio-Rad) and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA). Immunoblotting with antibodies directed against either 85-kDa cPLA2 (Santa Cruz Biotechnology, Santa Cruz, CA), phospho-p44/p42, total p44/42, or Elk-1 was performed according to the manufacturer's instructions (New England Biolabs, Beverly, MA). The secondary antibodies were an anti-rabbit IgG conjugated to horseradish peroxidase (or anti-mouse-HRP for cPLA2 mAb), and detection was by chemiluminescence (Amersham Pharmacia Biotech). Densitometric analyses of Western blots was performed using the NIH Image software package. The films were scanned, and relative intensities were quantified by comparison to known quantities of electrophoresed standards. In selected experiments, 5 and 25 μm forskolin (16Galli C. Meucci O. Scorziello A. Werge T.M. Calissano P. Schettini G. J. Neurosci. 1995; 15: 1172-1179Crossref PubMed Google Scholar) (Calbiochem) was added to the cells at the same time as the neutralizing MIF monoclonal antibody. Whole cell extracts were prepared from 2 × 106 cells as described above. The p44/p42 MAP kinase assay was performed according to the manufacturer's directions (New England Biolabs). Briefly, equal amounts of lysate (200 μg in ∼200 μl) were incubated with 15 μl of an immobilized anti-phospho-p44/p42 MAP kinase mAb, and the samples were allowed to rotate overnight at 4 °C. The pellet was collected by centrifugation and washed with 500 μl of radioimmune precipitation buffer followed by 3 washes with 1× kinase buffer. The pellet then was resuspended in 50 μl of 1× kinase buffer supplemented with 200 μm ATP and 2 μg of Elk-1 fusion protein (New England Biolabs). After incubation at 37 °C for 30 min, the reaction was terminated by adding 25 μl of 3× Laemmli sample buffer. Thirty μl of each sample was electrophoresed on a 10% SDS-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane. The blot was then probed for phospho-Elk-1 protein by utilizing an anti-phospho-Elk-1 antibody. 2 × 106 cells were washed with ice-cold PBS and collected into homogenization buffer consisting of 50 mm HEPES, pH 7.4, 0.25 m sucrose, 1 mm EDTA, 1 mmEGTA, 5 mm CaCl2, and protease inhibitors. The cells then were disrupted by sonication and microcentrifuged at 3000 rpm for 5 min (4 °C). For each sample, 25 μg of total protein was added to the assay buffer (0.1 m Tris/HCl, pH 7.4, containing 1 mm EDTA, 1 mm EGTA, 5 mm CaCl2, 5 mm dithiothreitol, and protease inhibitors) followed by 2 nmol of [arachidonoyl-14C]phosphatidylcholine. The reaction was performed at 37 °C for 60 min in a total volume of 200 μl. The reactions were terminated by adding 400 μl of chloroform/methanol (2:1) and 2 μl of glacial acetic acid. The lower phase was chromatographed on TLC plates (Whatman) with chloroform/methanol/water (65:25:4, by vol) as the solvent system. The spot corresponding to free fatty acid was quantified by densitometric analysis with a phosphoimager (Packard Instruments) and comparison to [14C]arachidonic acid standards. The NIH/3T3 or the TNF-sensitive L929 mouse fibroblast cell lines (17Jayadev S. Hayter H.L. Andrieu N. Gamard C.J. Liu B. Balu R. Hayakawa M. Ito F. Hannun Y.A. J. Biol. Chem. 1997; 272: 17196-17203Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 18Hayakawa M. Ishida N. Takeuchi K. Shibamoto S. Hori T. Oku N. Ito F. Tsujimoto M. J. Biol. Chem. 1993; 268: 11290-11295Abstract Full Text PDF PubMed Google Scholar) were plated in 24-well plates in 10% fetal calf serum DMEM and allowed to grow to confluence. Medium then was changed to 0.5% serum-supplemented DMEM containing 1 μm[14C]arachidonic acid (NEN Life Science Products). After overnight culture, the cells were washed extensively with PBS, and then the appropriate samples were added in duplicate. At the indicated times, the supernatants were removed and cleared by centrifugation at 1000 × g for 5 min, and the radioactivity was measured by scintillation counting. For inhibitor studies employing H-89 (19Chijiwa T. Mishima A. Hagiwara M. Sano M. Hayashi K. Inoue T. Naito K. Toshioka T. Hidaka H. J. Biol. Chem. 1990; 265: 5267-5272Abstract Full Text PDF PubMed Google Scholar), AACOCF3 (20Street 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: 5935-5940Crossref PubMed Scopus (419) Google Scholar), SB203580 (21Waters S.B. Holt K.H. Ross S.E. Syu L.J. Guan K.L. Saltiel A.R. Koretzky G.A. Pessin J.E. J. Biol. Chem. 1995; 270: 20883-20886Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar), and PD98059 (22Lee J.C. Laydon J.T. McDonnell P.C. Gallagher T.F. Kumar S. Green D. McNulty D. Blumenthal M.J. Heys J.R. Landvatter S.W. Strickler J.E. McLaughlin M.M. Siemens I.R. Fisher S.M. George L.P. White J.R. Adams J.L. Young P.R. Nature. 1994; 372: 739-746Crossref PubMed Scopus (3138) Google Scholar) (Calbiochem), the compounds were pre-incubated with cells for 15 min before adding rMIF. Control wells contained either Me2SO or ethanol (depending on the solvent for each inhibitor). For antibody neutralization, anti-MIF mAb or an isotypic control mAb (100 μg/ml unless otherwise specified) were pre-incubated with mouse rMIF in medium for 15 min at room temperature and centrifuged at 14,000 rpm for 5 min before adding the solutions to cells. Calcium mobilization was measured by fluorescence spectroscopy of the Ca2+-sensitive dye, Fura 2-AM (Calbiochem). Briefly, 1 × 107 cells/ml were incubated for 45 min (at 370) in a 5 μm solution of Fura 2-AM in Hanks' balanced salt solution, pH 7.4, 1% fetal calf serum, and 2 mm Ca2+. Cells then were washed, resuspended in Hanks' balanced salt solution, 2 mm Ca2+ and treated with or without rMIF while monitoring fluorescence excitation at 380 nm. For the overriding of glucocorticoid suppression of arachidonic acid release, cell culture conditions followed methods described previously (9Calandra T. Bernhagen J. Metz C.N. Spiegel L.A. Bacher M. Donnelly T. Cerami A. Bucala R. Nature. 1995; 377: 68-71Crossref PubMed Scopus (1050) Google Scholar). The appropriate wells were preincubated for 1 h with 10−6m dexamethasone (Sigma) before the addition of the indicated amounts of mouse recombinant TNFα (R & D Systems, Minneapolis, MN) and mouse rMIF. We examined the role of MIF in cell proliferation by first investigating the effect of rMIF in the well characterized NIH/3T3 cell system. As shown in Fig.1 A, purified, mouse rMIF was found to stimulate the proliferation of quiescent NIH/3T3 fibroblasts in a dose-dependent manner. At a concentration of 50 ng/ml, exogenously added MIF stimulated thymidine incorporation by >50% when compared with unstimulated cells. MIF protein exists pre-formed in several cell types, and prior studies have indicated that endogenously released MIF can act to stimulate cellular responses in an autocrine fashion (8Bacher M. Metz C.N. Calandra T. Mayer K. Chesney J. Lohoff M. Gemsa D. Donnelly T. Bucala R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7849-7854Crossref PubMed Scopus (616) Google Scholar, 9Calandra T. Bernhagen J. Metz C.N. Spiegel L.A. Bacher M. Donnelly T. Cerami A. Bucala R. Nature. 1995; 377: 68-71Crossref PubMed Scopus (1050) Google Scholar, 15Waeber G. Calandra T. Roduit R. Haefliger J.-A. Bonny C. Thompson N. Thorens B. Temler E. Meinhardt A. Bacher M. Metz C.N. Nicod P. Bucala R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4782-4787Crossref PubMed Scopus (188) Google Scholar). Serum supplementation is known to induce the proliferation of quiescent cell populations (11Lang R.A. Burgess A.W. Immunol. Today. 1990; 11: 244-249Abstract Full Text PDF PubMed Scopus (7) Google Scholar), and we observed that the addition of 10% serum to NIH/3T3 cultures caused the release of immunoreactive MIF protein into culture supernatants in as little as 30 min (Fig. 1 B). The addition of a neutralizing monoclonal anti-MIF antibody inhibited by 40% the proliferative effect of serum addition to quiescent fibroblasts, indicating that the release of endogenous MIF contributes significantly to the mitogenic effect of serum stimulation (Fig. 1 C). Of note, cells treated with the neutralizing anti-MIF antibody appeared normal morphologically, and viability testing by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide showed no increase in cell death when compared with cells treated with an isotype control antibody (data not shown). Recent data indicates that MIF is expressed in higher concentrations in transformed cell lines when compared with primary cells (7Calandra T. Bernhagen J. Mitchell R.A. Bucala R. J. Exp. Med. 1994; 179: 1895-1902Crossref PubMed Scopus (890) Google Scholar, 23Takahashi N. Nishihira J. Sato Y. Kondo M. Ogawa H. Ohshima T. Une Y. Todo S. Mol. Med. 1998; 4: 707-714Crossref PubMed Google Scholar). We determined by ELISA that the amount of intracellular MIF present in resting NIH/3T3 cells was approximately 44 fg MIF/cell. Finally, in accordance with a recent report (23Takahashi N. Nishihira J. Sato Y. Kondo M. Ogawa H. Ohshima T. Une Y. Todo S. Mol. Med. 1998; 4: 707-714Crossref PubMed Google Scholar), the transfection of NIH/3T3 cells with an antisense MIF expression plasmid (15Waeber G. Calandra T. Roduit R. Haefliger J.-A. Bonny C. Thompson N. Thorens B. Temler E. Meinhardt A. Bacher M. Metz C.N. Nicod P. Bucala R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4782-4787Crossref PubMed Scopus (188) Google Scholar) was found to significantly decrease cell proliferation when compared with cells transfected with control vector (data not shown). One of the most widely studied protein phosphorylation cascades associated with cell proliferation is that of the Ras → Raf → MAP kinase/ERK kinase (MEK) → ERK pathway (24Denhardt D.T. Biochem. J. 1996; 318: 729-747Crossref PubMed Scopus (452) Google Scholar). To investigate ERK activation by MIF, quiescent NIH/3T3 cells were treated with rMIF, and the cell lysates were examined for ERK1/2 phosphorylation by Western blot analysis using phospho-specific anti-ERK antibodies. MIF was found to induce the phosphorylation of the p44/p42 ERK MAP kinases in both a time- and a dose-dependent fashion (Fig.2, A and B). ERK phosphorylation was detected as early as 30 min after MIF addition and, notably, was sustained for a period of at least 24 h (Fig. 2,A and D). Immunoneutralization of serum-stimulated cells with anti-MIF mAb resulted in a significant decrease in p44/p42 ERK MAP kinase phosphorylation, consistent with the decrease in proliferation with anti-MIF treatment (Fig. 2 C). We also examined if this sustained stimulation of p44/p42 ERK phosphorylation by rMIF produced an enhancement of p44/p42 ERK enzymatic activity. As shown in Fig. 2 D, cells treated for 24 h with increasing amounts of rMIF showed a dose-dependent increase in ERK enzymatic activity, as detected by the phosphorylation of the MAP kinase substrate, Elk-1, in an in vitro p44/p42 MAP kinase assay. This increase in enzymatic activity was associated with a corresponding increase in the phosphorylation of p44/p42 ERK present in the same cell lysates (Fig.2 D). To investigate if this MIF-mediated regulation of MAP kinase activity was similar in immune cells classically associated with MIF responsiveness, thioglycollate-elicited peritoneal macrophages from both endotoxin-sensitive (C3H/HeN) and endotoxin-resistant (C3H/HeJ) cells were stimulated with rMIF for 2 h. Fig. 2 E shows that rMIF induces ERK phosphorylation in macrophages from both mouse strains. The observation that an equivalent response occurs both in the endotoxin-sensitive and endotoxin-resistant macrophages indicates that there was no detectable contribution of trace lipopolysaccharide that may be present in bacterially expressed rMIF. A sustained, ligand-induced activation of MAP kinases has been described in one prior instance, the differentiation of neuronal PC12 cells by nerve growth factor (25Wu Y.Y. Bradshaw R.A. J. Biol. Chem. 1996; 271: 13023-13032Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 26Traverse S. Gomez N. Paterson H. Marshall C. Cohen P. Biochem. J. 1992; 288: 351-355Crossref PubMed Scopus (806) Google Scholar). It has been shown in this case that a prolonged response is dependent upon the activity of protein kinase A (PKA) (27Yao H. York R.D. Misra-Press A. Carr D.W. Stork P.J. J. Biol. Chem. 1998; 273: 8240-8247Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 28Vossler M.R. Yao H. York R.D. Pan M.G. Rim C.S. Stork P.J. Cell. 1997; 89: 73-82Abstract Full Text Full Text PDF PubMed Scopus (945) Google Scholar). To investigate a role for PKA in MIF-stimulated ERK activation, we tested the ability of MIF to induce p44/p42 MAP kinase activation in the presence of the specific PKA inhibitor, H-89 (19Chijiwa T. Mishima A. Hagiwara M. Sano M. Hayashi K. Inoue T. Naito K. Toshioka T. Hidaka H. J. Biol. Chem. 1990; 265: 5267-5272Abstract Full Text PDF PubMed Google Scholar). As shown in Fig.3 A, H-89-treated cells were resistant to MIF-stimulated ERK activation, and the base-line level of ERK2 phosphorylation decreased at 2 h. A similar level of MIF unresponsiveness was observed upon the addition to cultures of the cell-permeable PKA inhibitor,14–22 (29Eichholtz T. de Bont D.B. de Widt J. Liskamp R.M. Ploegh H.L. J. Biol. Chem. 1993; 268: 1982-1986Abstract Full Text PDF PubMed Google Scholar) (not shown). Conversely, we saw no effect on the MIF-induced phosphorylation of ERK in NIH/3T3 cells treated with either the protein kinase C or the phospholipase C inhibitors (R0–31-8220 and U-73122, respectively) (30Davis P.D. Elliott L.H. Harris W. Hill C.H. Hurst S.A. Keech E. Kumar M.K. Lawton G. Nixon J.S. Wilkinson S.E. J. Med. Chem. 1992; 35: 94-1001Crossref PubMed Scopus (41) Google Scholar, 31Thompson A.K. Mostafapour S.P. Denlinger L.C. Bleasdale J.E. Fisher S.K. J. Biol. Chem. 1991; 266: 23856-23862Abstract Full Text PDF PubMed Google Scholar) (data not shown). The addition of forskolin, an adenylate cyclase activator (16Galli C. Meucci O. Scorziello A. Werge T.M. Calissano P. Schettini G. J. Neurosci. 1995; 15: 1172-1179Crossref PubMed Google Scholar), also was found to reverse the inhibitory effects of anti-MIF on serum-induced ERK phosphorylation, further suggesting that MIF is acting by a PKA-dependent pathway (Fig. 3 B). The ERK pathway of activation results in the phosphorylation and activation of a number of cytosolic proteins. Among the best characterized substrates for the ERKs are P90 rsk, c-myc, and cPLA2(24Denhardt D.T. Biochem. J. 1996; 318: 729-747Crossref PubMed Scopus (452) Google Scholar). cPLA2 is a critical component of the pro-inflammatory cascade (18Hayakawa M. Ishida N. Takeuchi K. Shibamoto S. Hori T. Oku N. Ito F. Tsujimoto M. J. Biol. Chem. 1993; 268: 11290-11295Abstract Full Text PDF PubMed Google Scholar), and its product, arachidonic acid, is a precursor for the synthesis of prostaglandins and leukotrienes. Arachidonic acid also is known to activate the c-jun N-terminal kinase/stress-activated protein kinase pathway, which is also required for the translation of TNF mRNA (32Cui X.L. Douglas J.G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3771-3776Crossref PubMed Scopus (165) Google Scholar, 33Swantek J.L. Cobb M.H. Geppert T.D. Mol. Cell. Biol. 1997; 17: 6274-6282Crossref PubMed Google Scholar). The rMIF-stimulated increa" @default.
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