Characterization of Calcineurin in Human Neutrophils
1999; Elsevier BV; Volume: 274; Issue: 1 Linguagem: Inglês
10.1074/jbc.274.1.93
ISSN1083-351X
AutoresModesto Carballo, Gracia Márquez, Manuel Conde, José Martı́n-Nieto, Javier Monteseirı́n, J. Condé, Elı́zabeth Pintado, Francisco Sobrino,
Tópico(s)Antimicrobial Peptides and Activities
ResumoWe describe here a specific calcineurin activity in neutrophil lysates, which is dependent on Ca2+, inhibited by trifluoroperazine, and insensitive to okadaic acid. Immunoblotting experiments using a specific antiserum recognized both the A and B chains of calcineurin. Neutrophils treated with cyclosporin A or FK 506 showed a dose-dependent inhibition of calcineurin activity. The effect of oxidant compounds on calcineurin activity was also investigated. Neutrophils treated with hydrogen peroxide (H2O2), where catalase was inhibited with aminotriazole, exhibited a specific inhibition of calcineurin activity. However, the addition of reducing agents to neutrophil extracts partially reversed the inhibition caused by H2O2. A similar inhibitory effect of H2O2 on calcineurin activity was observed to occur in isolated lymphocytes. This is the first demonstration that redox agents modulate calcineurin activity in a cellular system. In addition, electrophoretic mobility shift assays revealed that lipopolysaccharide-induced activation of NF-κB in human neutrophils is inhibited by cell pretreatment with H2O2 in a dose-dependent manner. These data indicate that calcineurin activity regulates the functional activity of lipopolysaccharide-induced NF-κB/Rel proteins in human neutrophils. These data indicate a role of peroxides in the modulation of calcineurin activity and that the H2O2-dependent NF-κB inactivation in neutrophils occurs in concert with inhibition of calcineurin. We describe here a specific calcineurin activity in neutrophil lysates, which is dependent on Ca2+, inhibited by trifluoroperazine, and insensitive to okadaic acid. Immunoblotting experiments using a specific antiserum recognized both the A and B chains of calcineurin. Neutrophils treated with cyclosporin A or FK 506 showed a dose-dependent inhibition of calcineurin activity. The effect of oxidant compounds on calcineurin activity was also investigated. Neutrophils treated with hydrogen peroxide (H2O2), where catalase was inhibited with aminotriazole, exhibited a specific inhibition of calcineurin activity. However, the addition of reducing agents to neutrophil extracts partially reversed the inhibition caused by H2O2. A similar inhibitory effect of H2O2 on calcineurin activity was observed to occur in isolated lymphocytes. This is the first demonstration that redox agents modulate calcineurin activity in a cellular system. In addition, electrophoretic mobility shift assays revealed that lipopolysaccharide-induced activation of NF-κB in human neutrophils is inhibited by cell pretreatment with H2O2 in a dose-dependent manner. These data indicate that calcineurin activity regulates the functional activity of lipopolysaccharide-induced NF-κB/Rel proteins in human neutrophils. These data indicate a role of peroxides in the modulation of calcineurin activity and that the H2O2-dependent NF-κB inactivation in neutrophils occurs in concert with inhibition of calcineurin. calcineurin nuclear factor of activated T cells nuclear factor κB cyclosporin A calcineurin A subunit calcineurin B subunit reactive oxygen intermediates hydrogen peroxide sodium oxodiperoxo(1,10-phenanthroline)vanadate(V) Hanks' balanced salt solution bovine serum albumin dithiothreitol phenylmethylsulfonyl fluoride pyrrolidine dithiocarbamate 3-amino-1,2,4-triazole polyacrylamide gel electrophoresis diisopropyl fluorophosphate 2′,7′-dichlorodihydrofluorescein diacetate electrophoretic mobility shift assay lipopolysaccharide. Calcineurin (CN)1 has recently been established as a key enzyme in the signal transduction cascade leading to T cell activation (1Liu J. Farmer J.D.J. Lane W.S. Friedman J. Weissman I. Schreiber S.L. Cell. 1991; 66: 807-815Abstract Full Text PDF PubMed Scopus (3634) Google Scholar, 2Fruman D.A. Klee C.B. Bierer B.E. Burakoff S.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3686-3690Crossref PubMed Scopus (757) Google Scholar, 3O'Keefe S.J. Tamura J. Kincaid R.L. Tocci M.J. O'Neill E. Nature. 1992; 357: 692-694Crossref PubMed Scopus (788) Google Scholar, 4Clipstone N.A. Crabtree G.R. Nature. 1992; 357: 695-697Crossref PubMed Scopus (1477) Google Scholar), and an important regulator of transcription factors such as NF-AT, NF-κB, and AP-1, which are involved in the expression of a number of important T cell early genes,i.e. interleukin-2, tumor necrosis factor-α, and interleukin-2R (5Sen R. Baltimore D. Cell. 1986; 46: 705-716Abstract Full Text PDF PubMed Scopus (1946) Google Scholar, 6Ullman K.S. Northrop J.P. Verweij C.L. Crabtree G.R. Annu. Rev. Immunol. 1990; 8: 421-452Crossref PubMed Scopus (491) Google Scholar, 7Karin M. Smeal T. Trends Biochem. Sci. 1992; 17: 418-422Abstract Full Text PDF PubMed Scopus (328) Google Scholar, 8Rao A. Immunol. Today. 1994; 15: 274-281Abstract Full Text PDF PubMed Scopus (490) Google Scholar). CN, also known as phosphatase 2B, is a calcium/calmodulin-dependent serine/threonine phosphatase (9Klee C.B. Krinks M.H. Biochemistry. 1978; 17: 120-126Crossref PubMed Scopus (313) Google Scholar, 10Klee C.B. Crouch T.H. Krinks M.H. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 6270-6273Crossref PubMed Scopus (611) Google Scholar, 11Stewart A.A. Ingebritsen T.S. Manalan A. Klee C.B. Cohen P. FEBS Lett. 1982; 137: 80-84Crossref PubMed Scopus (339) Google Scholar) and is composed of the following two subunits: a 59-kDa catalytic subunit (CNA), which contains a calmodulin-binding domain and an autoinhibitory region, and a 19-kDa intrinsic calcium-binding regulatory subunit (CNB) (12Aitkin A. Klee C.B. Cohen P. Eur. J. Biochem. 1984; 139: 663-671Crossref PubMed Scopus (115) Google Scholar, 13Perrino B.A. Ng L.Y. Soderling T.R. J. Biol. Chem. 1995; 270: 340-346Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 14Sikkink R. Haddy A. MacKelvie S. Mertz P. Litwiller R. Rusnak F. Biochemistry. 1995; 34: 8348-8356Crossref PubMed Scopus (40) Google Scholar). Human CN possesses a Fe-Zn active center. The assignment of stoichiometric amounts of Zn2+and Fe3+ in the CNA center is based on atomic absorption experiments (15King M.M. Huang C.Y. J. Biol. Chem. 1984; 259: 8847-8856Abstract Full Text PDF PubMed Google Scholar). The same assignments were made for the di-metal site in the structure of some CNA (16Griffith J.P. Kim J.L. Kim E.E. Sintchak M.D. Thomson J.A. Fitzgibbon M.J. Fleming M.A. Caron P.R. Hsiao K. Navia M.A. Cell. 1995; 82: 507-522Abstract Full Text PDF PubMed Scopus (774) Google Scholar).The central role of CN in T cell signaling was appreciated by its identification as the target of the immunosuppressive drugs cyclosporin A (CsA) and FK 506 (1Liu J. Farmer J.D.J. Lane W.S. Friedman J. Weissman I. Schreiber S.L. Cell. 1991; 66: 807-815Abstract Full Text PDF PubMed Scopus (3634) Google Scholar, 2Fruman D.A. Klee C.B. Bierer B.E. Burakoff S.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3686-3690Crossref PubMed Scopus (757) Google Scholar, 3O'Keefe S.J. Tamura J. Kincaid R.L. Tocci M.J. O'Neill E. Nature. 1992; 357: 692-694Crossref PubMed Scopus (788) Google Scholar, 4Clipstone N.A. Crabtree G.R. Nature. 1992; 357: 695-697Crossref PubMed Scopus (1477) Google Scholar). The phosphatase activity of CN is inhibited by either drug when complexed to intracellular binding proteins (immunophilins), i.e. CsA to cyclophilin and FK 506 to the FK 506-binding protein 12 (FKBP12), respectively. Neither drug nor immunophilin alone bind to or affect the activity of CN (1Liu J. Farmer J.D.J. Lane W.S. Friedman J. Weissman I. Schreiber S.L. Cell. 1991; 66: 807-815Abstract Full Text PDF PubMed Scopus (3634) Google Scholar). This phosphatase is expressed ubiquitously in eukaryotic cells. In mammals, CN is most abundant in the brain (17Stemmer P. Klee C.B. Curr. Opin. Neurobiol. 1991; 1: 53-64Crossref PubMed Scopus (59) Google Scholar) but has also been detected in T cells (1Liu J. Farmer J.D.J. Lane W.S. Friedman J. Weissman I. Schreiber S.L. Cell. 1991; 66: 807-815Abstract Full Text PDF PubMed Scopus (3634) Google Scholar, 2Fruman D.A. Klee C.B. Bierer B.E. Burakoff S.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3686-3690Crossref PubMed Scopus (757) Google Scholar, 3O'Keefe S.J. Tamura J. Kincaid R.L. Tocci M.J. O'Neill E. Nature. 1992; 357: 692-694Crossref PubMed Scopus (788) Google Scholar, 4Clipstone N.A. Crabtree G.R. Nature. 1992; 357: 695-697Crossref PubMed Scopus (1477) Google Scholar). On the other hand, it is known that NF-AT-mediated transactivation depends on the CN activity (18Liu J. Albers M.W. Wandless T.J. Luan S. Alberg D.G. Belshaw P.J. Cohen P. MacKintosh C. Klee C.B. Schereiber S.L Biochemistry. 1992; 31: 3896-3901Crossref PubMed Scopus (502) Google Scholar, 19Loh C. Shaw K.T.-Y. Carew J. Viola J.P.B. Luo C. Perrino B.A. Rao A. J. Biol. Chem. 1996; 271: 10884-10891Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar). Other findings suggest that NF-κB activity is also under CN control (20Schmidt A. Hennighausen L. Siebenlist U. J. Virol. 1990; 64: 4037-4041Crossref PubMed Google Scholar, 21Frantz B. Nordby E.C. Bren G. Steffan N. Paya C.V. Kincaid R.L. Tocci M.J. O'Keefe S.J. O'Neill E.A. EMBO J. 1994; 13: 861-870Crossref PubMed Scopus (304) Google Scholar, 22Gualberto A. Marquez G. Carballo M. Youngblood G.L. Hunt III, S.W. Baldwin A.S. Sobrino F. J. Biol. Chem. 1998; 273: 7088-7093Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). In neutrophils, only indirect evidence has been presented on the occurrence of the phosphatase CN. The treatment of these cells with inhibitors of CN (e.g. CsA and FK 506) inhibited the neutrophils chemokinesis on vitronectin matrix (23Hendey B. Klee C.B. Maxfield F.R. Science. 1992; 258: 296-299Crossref PubMed Scopus (173) Google Scholar, 24Hendey B. Maxfield F.R. Blood Cells. 1993; 19: 143-161PubMed Google Scholar). Furthermore, intracellular calcium and CN regulate neutrophil motility on vitronectin through a receptor identified by antibodies against the integrins αv and β3 (25Lawson M.A. Maxfield F.R. Nature. 1995; 377: 75-79Crossref PubMed Scopus (479) Google Scholar, 26Hendey B. Lawson M. Marcantonio E.E. Maxfield F.R. Blood. 1996; 87: 2038-2048Crossref PubMed Google Scholar). The first purpose of the present work was to assess the presence of CN in neutrophils using as a substrate a specific peptide corresponding to the phosphorylation site of the RII subunit of cyclic AMP-dependent protein kinase. Additionally, reactive oxygen intermediates (ROI) have been implicated in mediating signal transduction by a variety of stimuli in lymphoid cells, and transcription factors seem to be responsible for the inducible expression of a number of genes in response to oxidative stress (27Schreck R. Baeuerle P.A. Trends Cell Biol. 1991; 1: 39-42Abstract Full Text PDF PubMed Scopus (445) Google Scholar, 28Gomez del Arco P. Martinez-Martinez S. Calvo V. Armesilla A.L. Redondo J.M. J. Biol. Chem. 1996; 271: 26335-26340Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). In this context, the addition of H2O2 to the culture medium has been shown to activate NF-κB (29Schreck R. Rieber P. Baeuerle P.A. EMBO J. 1991; 10: 2247-2258Crossref PubMed Scopus (3430) Google Scholar). Hydroxyl radicals produced from H2O2 cannot function as diffusible intracellular messengers, since they can react with the nearest molecule in a nonspecific fashion. A more suitable ROI messenger would be the less reactive H2O2. However, arguments against ROI involvement in NF-κB activation have been published (30Brennan P. O'Neil L. Biochim. Biophys. Acta. 1995; 1260: 1670-1675Google Scholar, 31Suzuki Y. Mizuno M. Parker L. Biochem. Biophys. Res. Commun. 1995; 40: 537-541Crossref Scopus (43) Google Scholar, 32Suzuki Y.J. Mizuno M. Packer L. J. Immunol. 1994; 153: 5008-5015PubMed Google Scholar), and despite the fact that phorbol 12-myristate 13-acetate-dependent NF-κB stimulation is cancelled by antioxidants, it has been recently shown that phorbol 12-myristate 13-acetate does not increase intracellular ROI (33Courtois G. Whiteside S.T. Sibley C.H. Israel A. Mol. Cel. Biol. 1997; 17: 1441-1449Crossref PubMed Google Scholar). Peroxide-mediated stimulation of NF-κB appears to be cell line-specific, sinceN-acetylcysteine, an antioxidant, elicited up-regulation of NF-κB binding activity in monocyte-derived macrophages (34Nottet H.S.L.M. Moelans I.I.M.D. de Vos N.M. de Graaf L. Visser M.R. Verhoef J. J. Leukocyte Biol. 1997; 61: 33-39Crossref PubMed Scopus (10) Google Scholar). Moreover, NF-κB is not the only nuclear factor whose activity is altered by H2O2. In a fashion opposite to that observed for NF-κB, NF-AT has been shown to be actively suppressed by H2O2 in Jurkat T cells (35Beiqing L. Chen M. Whisler R.L. J. Immunol. 1996; 157: 160-169PubMed Google Scholar). Recently, the development of stabilized peroxovanadium compounds has provided the opportunity to more fully characterize the action of oxidants within the cell (36Posner B.I. Faure R. Burgess J.W. Bevan A.P. Lachance D. Zhang-Sun G. Fantus I.G. Ng J.B. Hall D.A. Lum B.S. J. Biol. Chem. 1994; 269: 4596-4604Abstract Full Text PDF PubMed Google Scholar). Treatment of lymphocytes with sodium oxodiperoxo(1,10-phenanthroline)vanadate (V), pV(phen), results in a large increase in intracellular oxidation, which correlates with a strong induction of cellular tyrosine phosphorylation and activation of kinases. The same authors (37Krejsa C.M. Nadler S.G. Esselstyn J.M. Kavanagh T.J. Ledbetter J.A. Schieven G.L. J. Biol. Chem. 1997; 272: 11541-11549Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar) have documented that antioxidant treatment does not prevent the activation of NF-κB by pV(phen). Therefore, we have also used pV(phen) as another tool to study the effect of oxidative stress on calcineurin activity. In summary, previous data suggest that, first, CN modulates the DNA binding activity of essential transcription factors (e.g.NF-AT and NF-κB), and second, ROI regulates positively or negatively those transcription factors. However, a link between both signals, that is CN and ROI, is lacking. In this paper we address this question and the implications of the ROI as universal messenger to activate NF-κB. Neutrophils were isolated from fresh heparinized blood of healthy human donors by dextran sedimentation, followed by Ficoll-Paque gradient centrifugation and hypotonic lysis of residual erythrocytes as indicated (38Monteseirin J. Camacho M.J. Montaño R. Llamas E. Conde M. Carballo M. Guardia P. Conde J. Sobrino F. J. Exp. Med. 1996; 183: 2571-2579Crossref PubMed Scopus (37) Google Scholar). Neutrophils were washed twice in Hanks' balanced salt solution (HBSS), suspended at a density of 1 × 107 cells/ml in HBSS supplemented with 0.1 mg/ml BSA, and maintained at 37 °C in an atmosphere of 5% CO2and 95% O2 for 1–2 h. Peripheral blood lymphocytes were obtained from heparinized venous blood of normal volunteers by Ficoll-Paque centrifugation. Dextran T-500 was obtained from Pharmacia Biotech (Barcelona, Spain). Ficoll-Paque, HBSS, and RPMI 1640 were obtained from BioWhittaker (Verviers, Belgium). CsA and FK 506 were kindly provided by Dr. S. F. Borel (Sandoz Ltd., Basel, Switzerland) and Fujisawa GmbH (München, Germany), respectively. Chemicals were of analytical grade from Merck (Darmstadt, Germany). Dithiothreitol (DTT) and phenylmethylsulfonyl fluoride (PMSF) were obtained from Boehringer Mannheim (Barcelona, Spain). Bovine serum albumin (BSA), okadaic acid, trifluoroperazine, pyrrolidine dithiocarbamate (PDTC), hydrogen peroxide (30% v/v), 3-amino-1,2,4-triazole (aminotriazole, AMT), soybean trypsin inhibitor, leupeptin, aprotinin, Nonidet P-40, and goat anti-rabbit IgG conjugated to horseradish peroxidase were purchased from Sigma (Madrid, Spain). Rabbit anti-bovine calcineurin IgG was kindly provided by C. B. Klee. The synthetic peptide used as a substrate for calcineurin was purchased from Peninsula Laboratories (Bellmont, CA). pV(phen) was synthesized as described previously (37Krejsa C.M. Nadler S.G. Esselstyn J.M. Kavanagh T.J. Ledbetter J.A. Schieven G.L. J. Biol. Chem. 1997; 272: 11541-11549Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). [γ-32P]ATP was obtained from NEN Life Science Products. 2-Mercaptoethanol, SDS, acrylamide,N,N′-methylene-bisacrylamide, Coomassie Brilliant Blue R-250, and blotting nitrocellulose membranes were purchased from Bio-Rad. Luminol (5-amino-2,3-dihydro-1,4-phthalazinedione) and diisopropyl fluorophosphate (DFP) were purchased from Serva (Madrid, Spain), and 4-iodophenol was from Aldrich (Madrid, Spain). Molecular weight standards (Rainbow markers) was obtained from Amersham Corp. (London, UK); Sephadex G-25 was from Pharmacia (Barcelona, Spain); and double-stranded oligonucleotide probe (5′-AGTTGAG GGGACTTTCC CAGGC-3′) containing NF-κB sites was from Boehringer Mannheim Gmbh (Mannheim, Germany). 2′,7′-Dichlorodihydro-fluorescein diacetate (H2DCFDA) was purchased from Molecular Probes (Leiden, The Netherlands). Untreated neutrophils (1 × 108 cells/ml) were lysed for 30 min on ice in 500 μl of buffer A (50 mm Tris, pH 8.0, 0.5% Triton X-100, 150 mm NaCl, 50 μg/ml PMSF, 50 μg/ml soybean trypsin inhibitor, 10 μg/ml leupeptin, and 10 μg/ml aprotinin) and disrupted by sonication. The homogenate was centrifuged at 12,000 × g for 10 min at 4 °C. The supernatant fluid (crude extract) was separated from low molecular weight material by passage through a 0.5 × 10-cm Sephadex G-25 column equilibrated with 50 mm Tris-Cl, pH 7.5, 0.1 mm EDTA, 0.1% β-mercaptoethanol, and a mixture of protease inhibitors as above (39Stemmer P.M. Wang X. Krinks M.H. Klee C.B. FEBS Lett. 1995; 374: 237-240Crossref PubMed Scopus (37) Google Scholar). Fractions containing most of the A 280 nmmaterial eluting with the void volume of the column were pooled and used to measure CN activity. Immunosuppressive agents were dissolved in dimethyl sulfoxide (Me2SO) at a concentration 1000-fold higher than that used for cell treatments. Neutrophils (7 × 106 cells/ml) were suspended in 1 ml of HBSS supplemented with 0.1 mg/ml BSA in microcentrifuge tubes, 1 μl of Me2SO or CsA or FK 506 was added, and the cells were incubated at 37 °C for 2 h. For experiments with H2O2 and other stimuli, neutrophils were incubated at 37 °C. The incubation times and concentration of agents are indicated in the figure legends. After incubation, the cells were washed once with 1 ml of HBSS on ice and lysed in 60 μl of buffer B (50 mm Tris, pH 7.5, 0.1 mm EGTA, 1 mm EDTA, 0.5 mm dithiothreitol, 50 μg/ml PMSF, 50 μg/ml soybean trypsin inhibitor, 10 μg/ml leupeptin, and 10 μg/ml aprotinin) and disrupted by sonication. Cell debris was removed by centrifugation at 4 °C for 10 min at 12,000 ×g, and supernatant was used as the source of CN. CN phosphatase activity was measured using an assay adapted from Hubbard and Klee (40Hubbard M.J. Klee C.B. Wheal H. Chad J. Molecular Neurobiology. Oxford University Press, Oxford1991: 135-137Google Scholar), basically as described (22Gualberto A. Marquez G. Carballo M. Youngblood G.L. Hunt III, S.W. Baldwin A.S. Sobrino F. J. Biol. Chem. 1998; 273: 7088-7093Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Neutrophils (7 × 106 cells/ml) were incubated for 2 h at 37 °C in the presence or absence of drugs as indicated in the text. Reaction mixtures containing 2 μm32P-labeled phosphopeptide, 500 nm okadaic acid (added to inhibit PP-1A and PP-2A type phosphatase activities), and 20 μl of cell lysate (about 80 μg of protein) were incubated in a total volume of 60 μl of assay buffer C (20 mm Tris, pH 8.0, 100 mm NaCl, 6 mm MgCl2, 0.5 mm dithiothreitol, and 0.1 mm CaCl2 or 5 mm EGTA, as indicated in the figures) for 15 min at 30 °C. After this time, reactions were terminated by the addition of 0.5 ml of 100 mm potassium phosphate buffer, pH 7.0, containing 5% trichloroacetic acid. The reaction mixture was passed through a 500-μl column of activated Dowex cation-exchange resin, and free inorganic phosphate was quantitated in the eluate by scintillation counting. It was verified that at 15 min of incubation time the assay was linear. Assays were performed in triplicate, and the counts/min measured in blank assay lacking cell lysate were subtracted. Data are expressed as the number of picomoles of 32PO4released in 15 min per mg of protein. The synthetic peptide (Asp-Leu-Asp-Val-Pro-Ile-Pro-Gly-Arg-Phe-Asp-Arg-Arg-Val-Ser-Val-Ala-Ala-Glu), corresponding to a segment of the RII subunit of cAMP-dependent kinase (41Blumenthal D.R. Takio K. Hansen R.S. Krebs E.G. J. Biol. Chem. 1986; 261: 8140-8145Abstract Full Text PDF PubMed Google Scholar), was phosphorylated on the unique serine residue by the catalytic subunit of cAMP-dependent protein kinase using [γ-32P]ATP, essentially as described (40Hubbard M.J. Klee C.B. Wheal H. Chad J. Molecular Neurobiology. Oxford University Press, Oxford1991: 135-137Google Scholar), and used as phosphatase substrate. The specific activity of fresh preparations of32P-labeled phosphopeptide was about 500 μCi/μmol peptide. Cells (7 × 106cells/ml) were lysed for 30 min on ice in 100 μl of buffer A (see above). Lysates were clarified by centrifugation at 4 °C for 2 min at 12,000 × g. Protein concentrations in the lysates were determined by the Bradford method (42Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217544) Google Scholar), using BSA as a standard. For the Western blot analysis of CN subunits A and B, neutrophil lysates were subjected to 12.5% SDS-PAGE followed by electroblotting onto nitrocellulose using the Bio-Rad Mini-blotting apparatus. Filters were blocked for 1 h in TBS (150 mm NaCl, 50 mm Tris-HCl, pH 7.5) containing 3% BSA. Furthermore, they were rinsed twice with TBS containing 0.1% Tween 20 (TBST), and they were incubated overnight with rabbit anti-bovine calcineurin IgG diluted 1:1000 in TBST. After three washes in TBST, the filters were incubated for 90 min with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:5000, final dilution). Filters were then washed twice with TBS and then twice with TBST for 10 min each time. The bound secondary antibody was detected by enhanced chemiluminescence (43Carballo M. Vilaplana R. Márquez G. Conde M. Bedoya F.J. González-Vı́lchez F. Sobrino F. Biochem. J. 1997; 328: 559-564Crossref PubMed Scopus (21) Google Scholar). Briefly, the membranes were incubated for 1 min in 10 ml of fresh luminescent reagent solution, composed of 10 mm Tris-HCl, pH 8.5, 2.25 mm luminol, 0.015% (v/v) H2O2, and 0.45 mm 4-iodophenol, the latter acting as an enhancer of the chemiluminescence reaction (44Hodgson M. Jones P. J. Biolum. Chemilumin. 1989; 3: 21-25Crossref PubMed Scopus (35) Google Scholar). These concentrations of luminol, H2O2, and 4-iodophenol were determined to be optimal for maximum light production. 2M. Carballo and F. Sobrino, unpublished data. Luminol and 4-iodophenol were freshly prepared in 10 ml of 10 mmTris-HCl, pH 8.5. Luminol was previously dissolved in 50 μl of 1m NaOH. The use of Me2SO as solvent should be avoided since in alkaline/Me2SO conditions, luminol autoxidizes with emission of intense luminescence (45White E.E. Zafirion O.C. Kagi H.M. Hill J.H.M. J. Am. Chem. Soc. 1964; 86: 940-941Crossref Scopus (196) Google Scholar). After 1 min of incubation, the membranes were placed on paper filter, covered with Saran Wrap, and exposed to x-ray films (X-Omat, Eastman Kodak Co.) in the dark for 1–5 min. Human neutrophils whose viability exceeded 98% after 3 h in culture, as determined by trypan blue exclusion, were resuspended at a density of 5 × 106 cells/ml in RPMI 1640 medium supplemented with 10% fetal calf serum. The cells were then incubated at 37 °C under a 5% CO2 atmosphere for 3 h, with occasional shaking in the absence or presence of 1 μg/ml LPS, 1 μg/ml CsA, or H2O2 at the concentrations indicated in each experiment. Then nuclear extracts were prepared basically as described by McDonald et al. (46McDonald P.P. Bald A. Cassatella M.A. Blood. 1997; 89: 3421-3433Crossref PubMed Google Scholar). With this purpose, cell suspensions were transferred into pre-cooled tubes containing an equivalent volume of ice-cold RPMI 1640 supplemented with DFP (2 mm, final concentration). After centrifugation at 4,000 × g for 1 min at 4 °C, the cells were resuspended in 100 μl of ice-cold relaxation buffer, consisting of 10 mm HEPES, pH 7.3, 30 mm KCl, 3 mm NaCl, 3.5 mmMgCl2, 1.25 mm EGTA, and 0.5 mmDTT, supplemented with an antiprotease mixture composed of 2 mm DFP, 1 mm PMSF, 10 mmiodoacetamide, 1 mm benzamidine, and 10 μg/ml each of aprotinin, leupeptin, and captopril. Cells were disrupted by short sonication (1 s), and the lysates were spun at 3,000 ×g for 10 min at 4 °C to pellet unbroken cells and intact nuclei. The pellets were resuspended in 100 μl of ice-cold relaxation buffer, again subjected to short (1 s) sonication, and respun as above to pellet intact nuclei. These were resuspended in 200 μl of relaxation buffer, and after new centrifugation, the nuclear pellet was resuspended in 25 μl of ice-cold relaxation buffer, additionally containing 10% (v/v) glycerol and 380 mm NaCl. Following a 20-min incubation on ice with occasional mixing, the samples were spun at 13,000 × g for 15 min at 4 °C, and the resulting supernatants were stored at −70 °C. EMSA was performed by using as a probe the double-stranded 22-base pair NF-κB consensus oligonucleotide indicated above, which was labeled with DIG using the labeling kit from Boehringer Mannheim. The nuclear extracts (5 μg of protein) were assayed for κB binding activity using the DIG gel shift assay kit (Boehringer Mannheim, GmbH). The reactions were performed in 15 μl of binding buffer (20 mm Tris-HCl, pH 7.5, 50 mm KCl, 1 mm EDTA, 1 mm DTT, 0.1% (v/v) Nonidet P-40, 6% glycerol) and allowed to proceed for 20 min at room temperature. For competition assays, binding reactions were performed in the presence of the unlabeled oligonucleotide (100-fold molar excess) for 20 min at room temperature. Supershift assays including anti-p50 and anti-p65 were carried out as described previously (46McDonald P.P. Bald A. Cassatella M.A. Blood. 1997; 89: 3421-3433Crossref PubMed Google Scholar). The samples were finally electrophoresed on 5% polyacrylamide native gels at 4 °C in 0.25× TBE. CN is a well characterized phosphatase that plays an important role in T cell activation pathways (1Liu J. Farmer J.D.J. Lane W.S. Friedman J. Weissman I. Schreiber S.L. Cell. 1991; 66: 807-815Abstract Full Text PDF PubMed Scopus (3634) Google Scholar, 2Fruman D.A. Klee C.B. Bierer B.E. Burakoff S.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3686-3690Crossref PubMed Scopus (757) Google Scholar, 3O'Keefe S.J. Tamura J. Kincaid R.L. Tocci M.J. O'Neill E. Nature. 1992; 357: 692-694Crossref PubMed Scopus (788) Google Scholar, 4Clipstone N.A. Crabtree G.R. Nature. 1992; 357: 695-697Crossref PubMed Scopus (1477) Google Scholar). In this work we have characterized the presence of CN activity and immunoreactive CN protein in human neutrophils. In crude neutrophil lysates, specific CN activity was observed, and a linear appearance of product during the assay was obtained in the range of 20–75 μg of lysate proteins (Fig.1 A). The time course of CN activity is shown in Fig. 1 B. Clearly, 32P release increased linearly along assay time until 15 min, and then a slow activity was found. Fig. 1 (inset) illustrates an immunoblotting analysis of CN expression in lymphocytes (lane 1) and different amounts of human neutrophil lysates (lanes 2–4). We used an antiserum that recognizes both the A (59 kDa) and B (19 kDa) chains of CN, confirming that both subunits are expressed in human neutrophils. As shown, neutrophil CNB migrates as a 16-kDa band in SDS-polyacrylamide gels, and it is detected along with a predominant CNA band migrating at 59 kDa. A third band, detected at 57 kDa in lymphocytes extracts, is probably a proteolytic fragment of CNA generated during preparation of the cell lysates. In some preparations of neutrophil lysates, a similar band of 55–57 kDa was also found (data not shown). Next experiments were addressed to analyze the regulation of CN activity in crude neutrophil lysates. Fig.2 illustrates that the dephosphorylation of the CN-specific substrate peptide by crude neutrophil lysates was Ca2+-dependent, as well as insensitive to okadaic acid, a potent and specific inhibitor of phosphatases 1A and 2A (reviewed in Ref. 47Cohen P. Holmes C.F.B. Tsukitani Y. Trends Biol. Sci. 1990; 15: 98-102Abstract Full Text PDF PubMed Scopus (1266) Google Scholar). When 500 nm okadaic acid was included in the assays, nearly all of the remaining phosphatase activity was Ca2+-dependent and could be eliminated by substituting 5 mm EGTA for Ca2+(Fig. 2). In contrast, the okadaic acid-sensitive component was resistant to EGTA, which is consistent with the reported Ca2+ independence of phosphatases 1A and 2A (17Stemmer P. Klee C.B. Curr. Opin. Neurobiol. 1991; 1: 53-64Crossref PubMed Scopus (59) Google Scholar). CN activity was abrogated in nominally calcium-free medium and in the presence of a known inhibitor of calmodulin, trifluoroperazine (48Stewart A.A. Ingebritsen T.S. Cohen P. Eur. J. Biochem. 1983; 132: 289-295Crossref PubMed Scopus (145) Google Scholar). However, trifluoroperazine did not inhibit calcium-independent, okadaic acid-sensitive phosphatases from neutrophil lysates (Fig. 2). Taken together, these data indicate that a specific Ca2+/calmodulin-dependent phosphatase activity is present in the neutrophil lysates.Figure 2Effect of calcium, okadaic acid, and trifluoroperazine on calcineurin activity from crude neutrophil lysates. Reaction mixtures contained 20 μl of crude neutrophil lysate (80 μg of protein), 2 μm32P-labeled phosphopeptide, and 40 μl of assay buffer. Other additions were
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