Nerve Growth Factor-dependent Survival of CESS B Cell Line Is Mediated by Increased Expression and Decreased Degradation of MAPK Phosphatase 1
2004; Elsevier BV; Volume: 279; Issue: 14 Linguagem: Inglês
10.1074/jbc.m305356200
ISSN1083-351X
AutoresPaolo Rosini, Giovanna De Chiara, С. Бонини, Maria Lucibello, Maria Elena Marcocci, Enrico Garaci, F Cozzolino, Maria Gabriella Torcia,
Tópico(s)Cell death mechanisms and regulation
ResumoThe sIgG+ lymphoblastoid B cell line CESS spontaneously produces a high amount of nerve growth factor (NGF) and expresses both high affinity (p140Trk-A) and low affinity (p75NTR) NGF receptors. Autocrine production of NGF maintains the survival of CESS cells through the continuous deactivation of p38 MAPK, an enzyme able to induce Bcl-2 phosphorylation and subsequent cytochrome c release and caspase activation. In this paper, we show that NGF induces transcriptional activation and synthesis of MAPK phosphatase 1 (MKP-1), a dual specificity phosphatase that dephosphorylates p38 MAPK, thus preventing Bcl-2 phosphorylation. Furthermore, NGF increases MKP-1 protein stability by preventing its degradation through the proteasome pathway. Following NGF stimulation, MKP-1 protein mainly localizes on mitochondria, suggesting an interaction with p38 MAPK in this compartment. Incubation of CESS cells with MKP-1-specific antisense oligonucleotides induces cell death, which was not prevented by exogenous NGF. By contrast, overexpression of native MKP-1, but not of its catalytically impaired form, inhibits apoptosis induced by NGF neutralization in CESS cells. Thus, the molecular mechanisms underlying the survival function of NGF in CESS B cell line predominantly consist in maintaining elevated levels of MKP-1 protein, which controls p38 MAPK activation. The sIgG+ lymphoblastoid B cell line CESS spontaneously produces a high amount of nerve growth factor (NGF) and expresses both high affinity (p140Trk-A) and low affinity (p75NTR) NGF receptors. Autocrine production of NGF maintains the survival of CESS cells through the continuous deactivation of p38 MAPK, an enzyme able to induce Bcl-2 phosphorylation and subsequent cytochrome c release and caspase activation. In this paper, we show that NGF induces transcriptional activation and synthesis of MAPK phosphatase 1 (MKP-1), a dual specificity phosphatase that dephosphorylates p38 MAPK, thus preventing Bcl-2 phosphorylation. Furthermore, NGF increases MKP-1 protein stability by preventing its degradation through the proteasome pathway. Following NGF stimulation, MKP-1 protein mainly localizes on mitochondria, suggesting an interaction with p38 MAPK in this compartment. Incubation of CESS cells with MKP-1-specific antisense oligonucleotides induces cell death, which was not prevented by exogenous NGF. By contrast, overexpression of native MKP-1, but not of its catalytically impaired form, inhibits apoptosis induced by NGF neutralization in CESS cells. Thus, the molecular mechanisms underlying the survival function of NGF in CESS B cell line predominantly consist in maintaining elevated levels of MKP-1 protein, which controls p38 MAPK activation. The lymphoblastoid CESS B cell line displays a CD19+, CD20-, CD44+, CD38+, CD77-, and IgGK+ surface phenotype (1Rosini P. De Chiara G. Lucibello M. Garaci E. Cozzolino F. Torcia M. Biochem. Biophys. Res. Commun. 2000; 278: 753-759Google Scholar), which suggests its origin from an antigen-selected, somatically hypermutated, and proliferating B lymphocyte, a stage ontogenetically close to that of memory B cells (2Ridderstad A. Tarlinton D.M. J. Immunol. 1998; 160: 4688-4695Google Scholar). Similar to memory B lymphocytes, CESS cells express both high affinity (p140Trk-A) and low affinity (p75NTR) NGF 1The abbreviations used are: NGF, nerve growth factor; MAPK, mitogen-activated protein kinase; MKP, mitogen-activated protein kinase phosphatase; JNK, c-Jun N-terminal kinase; ERK, extracellular signal-regulated kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; EGFP, enhanced green fluorescent protein; PE, phycoerythrin; PARP, poly(ADP-ribose) polymerase; NTR, neurotrophin receptor; hrNGF, human recombinant NGF; CMV, cytomegalovirus; dsPTPs, dual specificity phosphatases; TBS-T, Tris-buffered saline containing 0.05% Tween 20; PBS, phosphate-buffered saline; SAPK, stress-activated protein kinase. receptors, spontaneously produce high amounts of NGF, and utilize it for their own survival (1Rosini P. De Chiara G. Lucibello M. Garaci E. Cozzolino F. Torcia M. Biochem. Biophys. Res. Commun. 2000; 278: 753-759Google Scholar, 3Torcia M. Bracci-Laudiero L. Lucibello M. Nencioni L. Labardi D. Rubartelli A. Cozzolino F. Aloe L. Garaci E. Cell. 1996; 85: 345-356Google Scholar). In memory B lymphocytes and in the CESS B cell line, neutralization of endogenous NGF induces activation of p38 MAPK, its mitochondrial translocation, and phosphorylation of Bcl-2 protein (4Torcia M. De Chiara G. Nencioni L. Ammendola S. Labardi D. Lucibello M. Rosini P. Marlier L.N. Bonini P. Dello S.P. Palamara A.T. Zambrano N. Russo T. Garaci E. Cozzolino F. J. Biol. Chem. 2001; 276: 39027-39036Google Scholar). Besides p38 MAPK, several enzymes such as JNK, protein kinase Cα, Cdc2 kinase, CDK6, have been considered responsible for Bcl-2 phosphorylation (5Blagosklonny M.V. Giannakakou P. el Deiry W.S. Kingston D.G. Higgs P.I. Neckers L. Fojo T. Cancer Res. 1997; 57: 130-135Google Scholar, 6Chen C.Y. Faller D.V. J. Biol. Chem. 1996; 271: 2376-2379Google Scholar, 7Fan M. Goodwin M. Vu T. Brantley-Finley C. Gaarde W.A. Chambers T.C. J. Biol. Chem. 2000; 275: 29980-29985Google Scholar, 8Furukawa Y. Iwase S. Kikuchi J. Terui Y. Nakamura M. Yamada H. Kano Y. Matsuda M. J. Biol. Chem. 2000; 275: 21661-21667Google Scholar, 9Maundrell K. Antonsson B. Magnenat E. Camps M. Muda M. Chabert C. Gillieron C. Boschert U. Vial-Knecht E. Martinou J.C. Arkinstall S. J. Biol. Chem. 1997; 272: 25238-25242Google Scholar, 10Pathan N. Aime-Sempe C. Kitada S. Haldar S. Reed J.C. Neoplasia. 2001; 3: 70-79Google Scholar, 11Srivastava R.K. Mi Q.S. Hardwick J.M. Longo D.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3775-3780Google Scholar, 12Thomas A. Giesler T. White E. Oncogene. 2000; 19: 5259-5269Google Scholar, 13Ojala P.M. Yamamoto K. Castanos-Velez E. Biberfeld P. Korsmeyer S.J. Makela T.P. Nat. Cell Biol. 2000; 2: 819-825Google Scholar, 14Yamamoto K. Ichijo H. Korsmeyer S.J. Mol. Cell. Biol. 1999; 19: 8469-8478Google Scholar) during UV irradiation, exposure to microtubule-targeting drugs, or serum/growth factor deprivation. Bcl-2 phosphorylation, which occurs in serine and threonine residues located in a loop between α1 and α2 helices (11Srivastava R.K. Mi Q.S. Hardwick J.M. Longo D.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3775-3780Google Scholar), has been reported both as increasing or decreasing the anti-apoptotic potential of the protein (15Blagosklonny M.V. Leukemia. 2001; 15: 869-874Google Scholar). However, in memory B lymphocytes and in CESS cells, p38 MAPK-induced Bcl-2 phosphorylation, which occurs in Ser87 and Thr56 residues of the loop, (4Torcia M. De Chiara G. Nencioni L. Ammendola S. Labardi D. Lucibello M. Rosini P. Marlier L.N. Bonini P. Dello S.P. Palamara A.T. Zambrano N. Russo T. Garaci E. Cozzolino F. J. Biol. Chem. 2001; 276: 39027-39036Google Scholar), 2G. De Chiara, M. E. Marcocci, P. Rosini, M. Torica, and F. Cozzolino, manuscript in preparation. causes cytochrome c release from mitochondria, caspase activation, and apoptotic death. In contrast, the addition of exogenous NGF induces dephosphorylation of p38 MAPK, which prevents Bcl-2 phosphorylation and cell apoptosis. Continuous deactivation of p38 MAPK and maintenance of the functional and structural properties of Bcl-2 protein appear to be important mechanisms underlying the survival-promoting activity of endogenous NGF. MAPKs including ERK, JNK, and p38 MAPK are activated by upstream kinases such as MEKs 1 and 2 and mitogen kinase kinases 3, 4, and 6 (16Whitmarsh A.J. Davis R.J. Science's STKE. 1999; Google Scholar). However, once activated, MAPKs are rapidly inactivated by selected families of protein phosphatases. In particular, dual specificity protein phosphatases play crucial roles in the dephosphorylation/inactivation of MAPKs (17Hunter T. Cell. 1995; 80: 225-236Google Scholar, 18Sun H. Tonks N.K. Trends Biochem. Sci. 1994; 19: 480-485Google Scholar). MAPK phosphatase 1 (MKP-1), also termed CL100 or DUSP1, is a prototypic member of the family of inducible dual specificity phosphatases, dsPTPs (19Alessi D.R. Smythe C. Keyse S.M. Oncogene. 1993; 8: 2015-2020Google Scholar, 20Keyse S.M. Biochim. Biophys. Acta. 1995; 1265: 152-160Google Scholar). It selectively dephosphorylates tyrosine and threonine residues on MAPKs and inactivates them. Although all three MAPKs are potential targets of MKP-1 (21Franklin C.C. Kraft A.S. J. Biol. Chem. 1997; 272: 16917-16923Google Scholar, 22Liu Y. Gorospe M. Yang C. Holbrook N.J. J. Biol. Chem. 1995; 270: 8377-8380Google Scholar, 23Sun H. Charles C.H. Lau L.F. Tonks N.K. Cell. 1993; 75: 487-493Google Scholar), it has been reported that JNK and p38 MAPK are preferentially inactivated by this enzyme (21Franklin C.C. Kraft A.S. J. Biol. Chem. 1997; 272: 16917-16923Google Scholar). Other members of the family of inducible dsPTPs, such as MKP-3 and MKP-4, show higher selectivity to ERK, rather than JNK/SAPK or p38 MAPK, suggesting the presence of an expanding family of structurally homologous dsPTPs possessing distinct MAPK specificity and subcellular localization as well as diverse patterns of tissue expression (20Keyse S.M. Biochim. Biophys. Acta. 1995; 1265: 152-160Google Scholar, 24Muda M. Boschert U. Dickinson R. Martinou J.C. Martinou I. Camps M. Schlegel W. Arkinstall S. J. Biol. Chem. 1996; 271: 4319-4326Google Scholar). Serum and growth factor stimulation, tissue regeneration, oxidative stress and heat shock response, nitrogen starvation, and mitogen stimulation (20Keyse S.M. Biochim. Biophys. Acta. 1995; 1265: 152-160Google Scholar, 23Sun H. Charles C.H. Lau L.F. Tonks N.K. Cell. 1993; 75: 487-493Google Scholar, 25Keyse S.M. Emslie E.A. Nature. 1992; 359: 644-647Google Scholar, 26Rohan P.J. Davis P. Moskaluk C.A. Kearns M. Krutzsch H. Siebenlist U. Kelly K. Science. 1993; 259: 1763-1766Google Scholar) all induce transcription of dsPTPs. In particular, MKP-1 and MKP-2, which are not expressed in quiescent cells, are rapidly induced following serum addition, the proteins being detectable as early as 30 (MKP-1) or 60 min (MKP-2) after stimulation (27Brondello J.M. Brunet A. Pouyssegur J. McKenzie F.R. J. Biol. Chem. 1997; 272: 1368-1376Google Scholar). In this paper, we show that in CESS cells NGF is able to increase gene expression and synthesis of MKP-1, one of the most active p38 MAPK-dephosphorylating enzymes, and inhibits its degradation through the proteasome pathway, thus increasing the concentration of active MKP-1. Furthermore, we show that following NGF stimulation, MKP-1 localizes also on mitochondria, suggesting an interaction with p38 MAPK in this compartment. Incubation of CESS cells with MKP-1-specific antisense oligonucleotides induces cell death not prevented by exogenous NGF. By contrast, overexpression of native MKP-1, but not of its catalytically impaired form, inhibits apoptosis induced by NGF neutralization in CESS cells. Reagents—Human recombinant β-nerve growth factor, SB203580-p38 MAPK inhibitor, SB202474, K252a-Trk-A inhibitor, and proteasome inhibitor lactacystin were purchased by Calbiochem. Neutralizing rat anti-human NGF monoclonal antibodies (clone αD11) were kindly donated by Dr. A. Cattaneo (International School for Advanced Studies, Trieste, Italy) and always were used at 10 μg/ml. Rabbit anti-MKP-1 antibody and rabbit anti-ubiquitin were from Sigma. Rabbit anti-poly-(ADP-ribose) polymerase (PARP, H-250) and goat anti-actin antibody and immunoprecipitated rabbit anti-MKP-1 (V-15) antibodies were purchased from Santa Cruz Biotechnology. Rabbit anti-p38 MAPK, antiphospho-p38 MAPK, anti-phospho JNK, anti-JNK, anti-p44/42 MAPK, and anti-phospho-p44/42 MAPK were from New England Biolabs. Horseradish peroxidase-conjugated secondary antibodies were from Chemicon. Rabbit horseradish peroxidase-conjugated anti-EGFP antibody was from Clontech. ECL Plus Western blotting detection system was purchased from Amersham Biosciences. MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) and actinomycin D were from Sigma. All of the other materials were purchased from either Sigma or Merck-Eurolab. Cell Culture—CESS cell line was obtained from American Type Culture Collection and grown in RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mm l-glutamine, 50 IU/ml penicillin, and 50 μg/ml streptomycin. Reverse Transcription-PCR—Total RNA was isolated by using RNAFast reagent (Molecular Systems). Approximately 1 μg of RNA was reverse-transcribed in a total volume of 20 μl of specific buffer containing 50 ng of random hexamers, 1 mm dNTPs, 20 units of RNaseOUT (Invitrogen), and 20 units of avian myeloblastosis virus reverse transcriptase (Finnzymes) for 1 h at 37 °C. 2 μl of reverse transcription reaction was amplified by Dynazyme II DNA polymerase (Finnzymes) in the appropriate reaction buffer supplemented with 2 mm MgCl2 and 1 mm dNTPs using the following primers: 5′-GCG GGA AAT CGT GCG TGA CAT T-3′ and 5′-GAT GGA GTT GAA GGT AGT TTC GTG-3′ for β-actin; and 5′-CCG GAG CTG TGC AGC AAA-3′ and 5′-CTC CAC AGG GAT GCT CTT-3′ for MKP-1, yielding a PCR products of 234 and 282 bp, respectively. PCR products were separated on 2% agarose gels with 100-bp Ladder marker (Invitrogen) and visualized with ethidium bromide staining. Plasmid Construction and Cell Transfection—The human full-length MAPK phosphatase-1 coding sequence was amplified by PCR with primers DUSP1-FW (5′-GCT AGC AGA TCT ATG GTC ATG GAA GTG GGC ACC-3′) and DUSP1-RV (5′-GCT AGC AAG GAT CCG CAG CTG GGA GAG GTC GTA ATG GG-3′) using total cDNA of CESS cell line as template. The PCR fragment was subcloned in pCR2.1 vector (Invitrogen), yielding the p14-DUSP1 plasmid. pMEC10R plasmid coding for MKP-1/EGFP fusion protein under the CMV promoter was constructed by ligating in pEGFP-C1 (Clontech) in-frame to the N terminus of EGFP, the 1.1-kb NheI fragment from p14DUSP1 containing the MKP-1 sequence. Transiently transfected cells were obtained using the LipofectAMINE reagent (Invitrogen) by following manufacturer's instructions. Catalytically impaired form (Cys258 to Ser) of MKP-1 was obtained by using the QuikChange site-directed mutagenesis kit (Stratagene) with primers 5′-AGG GTG TTT GTC CAC AGC CAG GCA GGC ATT TCC-3′ and 5′-GGA AAT GCC TGC CTG GCT GTG GAC AAA CAC CCT-3′ and the pMEC10R plasmid as cDNA template. All of the products were sequenced by BigDye Terminator kit (Applied Biosystem). Immunoprecipitation Analysis—For immunoprecipitation studies, 107 cells cultured in the presence of 10 μm lactacystin with or without 10 nm hrNGF were lysed in 10 mm Hepes, 142.5 mm KCl, 5 mm MgCl2, 1mm EGTA, 50 μg/ml leupeptin, 30 μg/ml aprotinin, 1 mm phenylmethylsulfonyl fluoride, 0.25% Triton X-100, and 200 μg of cell lysate (as assessed by Bradford assay) were precleared with protein A-Sepharose and immunoprecipitated with 2 μg/ml rabbit anti-ubiquitin or rabbit anti-MKP-1 or control IgG followed by protein A-Sepharose. The immunoprecipitates were washed twice with PBS, boiled in Laemmli sample buffer, run on 10% SDS-PAGE, blotted onto nitrocellulose filter, and stained with anti-MKP-1 antibody or anti-ubiquitin antibody. Reaction was detected with ECL. Purification of Nuclear, Mitochondrial, and Cytosolic Fraction—108 cells were suspended in 5 mm Tris, pH 7.4, with 5 mm KCl, 1.5 mm MgCl2, and 0.1 mm EGTA, pH 8.0, containing 1 mm dithiothreitol, 0.2 mm phenylmethylsulfonyl fluoride, 5 μg/ml leupeptin, 5 μg/ml aprotinin, and 0.7 μg/ml pepstatin for 30 min on ice, Dounce-homogenized, and centrifuged at 750 × g at 4 °C to obtain the nuclear fraction. The supernatant was further centrifuged at 10,000 × g for 30 min at 4 °C to obtain the mitochondrial fraction (pellet) and the cytosolic fraction (supernatant). For Western blot analysis, the nuclear and mitochondrial fractions were directly lysed in sample buffer, whereas the cytosolic fraction was vacuum-concentrated and subsequently suspended in sample buffer. The purity of each fraction was assessed by staining aliquots with Abs to HSP60 for mitochondria, to actin for cytosol, and to PARP for nuclei. Immunoblot Analysis—2 × 106 cells were lysed on ice in 20 mm Tris-HCl, 150 mm NaCl, 1 mm EDTA, 50 μg/ml leupeptin, 30 μg/ml aprotinin, 1 mm phenylmethylsulfonyl fluoride, phosphatase inhibitor mixture (Sigma), and 0.25% Triton X-100, pH 7.4. Lysates were centrifuged at 15,000 × g for 10 min and stored at -80 °C for further analyses, and protein concentration was determined by Bradford assay. Equivalent amounts of proteins were diluted in Laemmli sample buffer, heated at 90 °C for 3 min, loaded on 8% (for PARP analysis) or 12% (for MKP-1 and p38 MAPK analysis) SDS-polyacrylamide gels, and transferred onto nitrocellulose membranes (Amersham Biosciences). Membranes were blocked with 5% nonfat dry milk in Tris-buffered saline containing 0.05% Tween 20 (TBS-T) and incubated with 1 μg/ml of primary antibodies diluted in TBS-T containing 5% nonfat dry milk for 1 h at room temperature. After washing with TBS-T, membranes were incubated with the appropriate horseradish peroxidase-conjugated secondary antibody diluted 1:1000 in TBS-T containing 5% nonfat dry milk for 1 h at room temperature, rinsed with TBS-T, and developed in ECL reagent. When necessary, membranes were stripped by heating at 56 °C in 62.5 mm Tris-HCl, pH 6.7, with 100 mm 2-mercaptoethanol and 2% SDS. Antisense Assays—CESS cells were plated in 6-well plates in serum-free medium at the concentration of 106cells/well. Phosphorothioate oligonucleotides (MWG Biotech) were used for all of the antisense transfection experiments. Appropriate amounts of the oligonucleotides were diluted in 200 μl of serum-free medium. 5 μl of LipofectAMINE were added to each tube and incubated at room temperature for 30 min to allow LipofectAMINE reagent-DNA complex formation. The transfection mixtures were added to the wells and incubated for 5 h in serum-free medium. 10% fetal bovine serum then was added, and incubation was prolonged for different times (24–72 h). The cultures were washed in PBS and lysed as described above. The phosphorothioate oligonucleotides used were as follows: p38 MAPK antisense, 5′-gtC TTG TTC AGC TCC tgc-3′; p38 MAPK sense, 5′-gcA GGA GCT GAA CAA gac-3′; p38 MAPK scrambled, 5′-tgC TTA GTT CTC GTC cgc-3′ (28Kiemer A.K. Weber N.C. Furst R. Bildner N. Kulhanek-Heinze S. Vollmar A.M. Circ. Res. 2002; 90: 874-881Google Scholar); MKP-1 antisense, 5′-ccC ACT TCC ATG ACC Atg g-3′; MKP-1 sense, 5′-ccA TGG TCA TGG AAG Tgg g-3′; and MKP-1 scrambled, 5′-gcA GGA CGT GCT AGA ggg-3′. Survival Assay—Approximately 5 × 103 cells/well were seeded in triplicate onto 96-well plates in RPMI 1640 medium and incubated for 24 h with 10 μm phosphorothioate anti-MKP-1 or control oligonucleotides. NGF then was added at the final concentration of 10 nm, and cells were incubated for an additional 48 h. Detection of MTT reduction was performed as described previously (29Shearman M.S. Ragan C.I. Iversen L.L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1470-1474Google Scholar). MTT was added to a final concentration of 0.5 mg/ml for 4 h. 100 μl of lysis buffer (20% SDS, 50% N,N-dimethylformamide, pH 7.4) and 100 μl of isopropyl alcohol were added to each well, and colorimetric reaction was measured by spectrophotometric analysis by using a 570-nm filter. Immunofluorescence Analysis—For immunofluorescence analysis, CESS cells were transiently transfected with pEMC10R or vector alone for 24 h, washed in PBS, and plated on poly-l-lysine-sensitized glass slides (Lab-Tek Chamber Slide, Nalge Nunc International) in serum-free medium. Cells were treated or not with 10 nm NGF for 30 min at 37 °C, labeled with 25 nm Mitotracker (Molecular Probes, Inc., Eugene, OR), washed twice with PBS, and fixed with 4% paraformaldehyde. After washing with PBS, nuclei were stained with Hoechst (Molecular Probes) and slides were analyzed by a confocal laser-scanning microscope. Relevance of p38 MAPK Activation in Cell Apoptosis Induced by NGF Withdrawal—The sIgG+ CESS B cell line originates from an antigen-selected somatically hypermutated, proliferating B lymphocyte, a stage ontogenetically close to that of memory B cells. Similar to memory B lymphocytes, CESS cells spontaneously produce NGF, express high and low affinity NGF receptors, and utilize NGF as an autocrine survival factor. Inhibition of endogenous NGF activity through neutralizing antibodies to NGF or use of the specific Trk-A inhibitor K252a (30Knusel B. Hefti F. J. Neurochem. 1992; 59: 1987-1996Google Scholar) induces apoptotic cell death via a p38 MAPK-mediated Bcl-2 phosphorylation, an event that induces cytochrome c release and caspase activation (1Rosini P. De Chiara G. Lucibello M. Garaci E. Cozzolino F. Torcia M. Biochem. Biophys. Res. Commun. 2000; 278: 753-759Google Scholar, 4Torcia M. De Chiara G. Nencioni L. Ammendola S. Labardi D. Lucibello M. Rosini P. Marlier L.N. Bonini P. Dello S.P. Palamara A.T. Zambrano N. Russo T. Garaci E. Cozzolino F. J. Biol. Chem. 2001; 276: 39027-39036Google Scholar). Fig. 1, panel A, shows that SB203580, a specific inhibitor of p38 MAPK (31Lee J.C. Kassis S. Kumar S. Badger A. Adams J.L. Pharmacol. Ther. 1999; 82: 389-397Google Scholar), is able to inhibit the apoptotic effect of NGF neutralization on CESS cells measured as caspase-3-like-mediated cleavage of PARP, whereas the inactive molecule SB202474 did not. The involvement of p38 MAPK in apoptosis induced by NGF neutralization was revealed also by using antisense oligonucleotides specific to p38 MAPK (28Kiemer A.K. Weber N.C. Furst R. Bildner N. Kulhanek-Heinze S. Vollmar A.M. Circ. Res. 2002; 90: 874-881Google Scholar). CESS cells were transfected for 48 h with 10 or 30 μm anti-p38 MAPK-specific oligonucleotides or with the same concentrations of sense or scrambled oligonucleotides as control. Fig. 1, panel B, shows that antisense oligonucleotides induced a decrease of p38 MAPK synthesis with a maximum effect at a concentration of 30 μm (≈60% decrease). Next, CESS cells were again transfected with 30 μm antisense oligonucleotides and incubated with 100 nm K252a as NGF-neutralizing agent for 6 h. The cells were then lysed and blotted with anti-PARP antibodies as a measure of caspase activation. Fig. 1, panel C, shows that antisense oligonucleotides to p38 MAPK completely abolished PARP cleavage induced by K252a incubation, whereas sense or scrambled oligonucleotides did not. The above data confirm that p38 MAPK activation is necessary in cell apoptosis induced by NGF neutralization and support the contention that endogenous NGF exerts its survival factor function through a pathway involving p38 MAPK deactivation. NGF Induces p38 MAPK Dephosphorylation and MKP-1 Expression—Stimulation of B lymphocytes with NGF induced tyrosine phosphorylation and activation of the two isoforms, p42 and p44, of Erk (32Franklin R.A. Brodie C. Melamed I. Terada N. Lucas J.J. Gelfand E.W. J. Immunol. 1995; 154: 4965-4972Google Scholar) as well as p38 MAPK dephosphorylation (4Torcia M. De Chiara G. Nencioni L. Ammendola S. Labardi D. Lucibello M. Rosini P. Marlier L.N. Bonini P. Dello S.P. Palamara A.T. Zambrano N. Russo T. Garaci E. Cozzolino F. J. Biol. Chem. 2001; 276: 39027-39036Google Scholar). To study the effect of NGF on phosphorylation status of MAPK, we cultured CESS cells in serum-free conditions for 4 h and stimulated them with 10 nm hrNGF for different times (from 10 min to 1 h). The phosphorylation status of ERK, p38 MAPK, and JNK was then studied by immunoblot analysis with antibodies specific for the phosphorylated enzymes. Fig. 2 shows that, under these experimental conditions, the activated forms of p38 MAPK and ERK were detected in unstimulated cultures. Activated JNK was never detected (data not shown). NGF stimulation had opposite effects on p38 and ERK, because it promptly induced p38 MAPK dephosphorylation, whereas ERK was instead strongly activated. 1 h after NGF stimulation, p38 MAPK continued to be dephosphorylated, whereas ERK phosphorylation kept returning to the base-line level. These results suggest the action of specific MAPK phosphatases with different activity toward ERK and p38 MAPK and are consistent with data generated using memory B lymphocytes (4Torcia M. De Chiara G. Nencioni L. Ammendola S. Labardi D. Lucibello M. Rosini P. Marlier L.N. Bonini P. Dello S.P. Palamara A.T. Zambrano N. Russo T. Garaci E. Cozzolino F. J. Biol. Chem. 2001; 276: 39027-39036Google Scholar). Among MAPK phosphatase family members known to deactivate p38 MAPK (33Keyse S.M. Curr. Opin. Cell Biol. 2000; 12: 186-192Google Scholar), MKP-1 and MKP-3 were reported to be induced by NGF in embryonic sympathetic neurons and in PC12 cells (34Peinado-Ramon P. Wallen A. Hallbook F. Brain Res. Mol. Brain Res. 1998; 56: 256-267Google Scholar). Whereas MKP-3 interacts preferentially with ERK MAPK (35Camps M. Nichols A. Gillieron C. Antonsson B. Muda M. Chabert C. Boschert U. Arkinstall S. Science. 1998; 280: 1262-1265Google Scholar), it has been reported that JNK and p38 MAPKs were preferentially inactivated by MKP-1 with the following order of affinity, p38 MAPK > JNK > ERK (21Franklin C.C. Kraft A.S. J. Biol. Chem. 1997; 272: 16917-16923Google Scholar). Furthermore, MKP-1 is encoded by an immediate early gene and is rapidly induced by many of the stimuli that activate MAPKs (20Keyse S.M. Biochim. Biophys. Acta. 1995; 1265: 152-160Google Scholar). For these reasons, we focused our attention on MKP-1. To investigate whether NGF is able to activate MKP-1 gene expression, CESS cells were starved from serum for 2 h and cultured in the presence or absence of 10 nm hrNGF for different times. mRNA was extracted, and MKP-1 gene expression was analyzed by PCR with specific oligonucleotides. Fig. 3 panel A shows that MKP-1 gene was rapidly induced in CESS cells after 30 min of incubation with exogenous NGF, reached a maximum expression after 1 h, and returned to the prestimulation levels after 6 h. Pretreatment of cells with 3 μg/ml actinomycin D completely blocked the induction of MKP-1 mRNA in the presence of NGF (Fig. 3, panel B), thus indicating a transcriptional regulation, rather than mRNA stabilization mechanism. We next examined the time-dependent effect of NGF on MKP-1 protein levels. CESS cells were cultured in serum-free conditions in the presence or absence of 10 nm hrNGF for different times, lysed, and analyzed by Western blot analysis with specific antibodies. Fig. 3, panel C, shows that MKP-1 protein was constitutively expressed by CESS cells but NGF was able to increase expression up to 1.5-fold as early as 30 min after stimulation, reaching a peak (>3-fold increase) after 2 h. The increased levels of protein were stable up to 6 h after NGF stimulation. These data suggest that, in basal conditions, the production of endogenous NGF contributes to continuously sustain MKP-1 gene expression and protein synthesis. NGF Decreases Degradation of MKP-1 through Proteasome Pathway—The early detection and the sustained levels of NGF-induced MKP-1 protein suggests that, in addition to the transcriptional regulation of MKP-1 gene expression, NGF can stabilize MKP-1 protein by inhibiting its degradation. Recently, it has been demonstrated that MKP-1 is degraded via the proteasome pathway (36Brondello J.M. Pouyssegur J. McKenzie F.R. Science. 1999; 286: 2514-2517Google Scholar). To investigate the effect of NGF on MKP-1 degradation, we cultured CESS cells in the presence of the proteasome inhibitor lactacystin with or without 10 nm hrNGF for different times (1–6 h). Cells were lysed and immunoprecipitated with rabbit anti-ubiquitin antibodies or with rabbit anti-MKP-1 or rabbit IgG as control. Anti-ubiquitin-immunoprecipitated proteins were blotted and stained with anti-MKP-1 antibodies. Anti-MKP-1-immunoprecipitated proteins were blotted and stained with anti-ubiquitin antibodies. Western blot analysis was also performed with the same anti-MKP-1 antibodies to detect the level of intact MKP-1. Fig. 4 shows that ubiquitinated MKP-1 molecules were increased in untreated cultures in comparison with NGF-stimulated cultures. These results indicate that, with its combined action on mRNA as well as protein levels, endogenous NGF contributes to maintain adequate amounts of active MKP-1 in CESS cells. Modulation of MKP-1 Protein Levels Changes the Phosphorylation Status of p38 MAPK—Because exogenous NGF increases MKP-1 synthesis and stability, we wanted to investigate whether NGF-neutralizing agents are able to modulate MKP-1 expression and whether the latter modulation is temporally related to the phosphorylation status of p38 MAPK. CESS cells were cultured in the presence of neutralizing antibodies to NGF for 12 h or with 100 nm K252 for 4 h, lysed, and blotted with anti-MKP-1 antibodies. Fig. 5, panel A, shows that incubation of cells with the above NGF-neutralizing agents inhibited the constitutive expression of MKP-1. In these experimental conditions, phosphorylation of p38 MAPK increased, whereas ERK phosphorylation decreased. Transfecting CESS cells with MKP-1-specific antisense oligonucleotides (37Duff J.L. Monia B.P. Berk B.C. J. Biol. Chem. 1995; 270: 7161-7166Google Scholar) yielded similar results. Fig. 5, panel B, shows that although p38 MAPK activation was increased under MKP-1 antisense oligonucleotides treatment, ERK activation was slightly down-modulated, suggesting that basal ERK activation escapes MKP-1 regulation and that another phosphatase (PP-2a) is possibly responsible for its dephosphorylation (38Jacob A. Molkentin J.D. Smolenski A. Lohmann S.M. Begum N. Am. J. Physiol. 2002; 283: C704-C713Google Scholar). However, these results clearly show that MKP-1 is involved in p38 MAPK deactivation in CESS cells and suggest its relevant role in NGF-dependent cell survival. MKP-1
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