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• W2034603447 abstract "The transcription factor NF-κB is critical for the expression of multiple genes involved in inflammatory responses and apoptosis. However, the signal transduction pathways regulating NF-κB activation in human neutrophils in response to stimulation with tumor necrosis factor-α (TNFα) are undefined. Since recent studies implicated activation of NF-κB as well as protein kinase C-δ (PKCδ) in neutrophil apoptosis, we investigated involvement of PKCδ in the activation of NF-κB in TNFα-stimulated neutrophils. Specific inhibition of PKCδ by rottlerin prevented IκBα degradation and NF-κB activation in TNFα-stimulated neutrophils. This regulation of NF-κB activation by PKCδ was specific only for TNFα signaling, since lipopolysaccharide- or interleukin-1β-induced NF-κB activation and IκBα degradation were not inhibited by rottlerin. In addition, we show that in human neutrophils, but not monocytes, IκBα localizes in significant amounts in the nucleus of unstimulated cells, and the amount of IκBα in the nucleus, as well as in the cytoplasm, correlates with the NF-κB DNA binding. These results suggest that in human neutrophils, the presence of IκBα in the nucleus may function as a safeguard against initiation of NF-κB dependent transcription of pro-inflammatory and anti-apoptotic genes, and represents a distinct and novel mechanism of NF-κB regulation. The transcription factor NF-κB is critical for the expression of multiple genes involved in inflammatory responses and apoptosis. However, the signal transduction pathways regulating NF-κB activation in human neutrophils in response to stimulation with tumor necrosis factor-α (TNFα) are undefined. Since recent studies implicated activation of NF-κB as well as protein kinase C-δ (PKCδ) in neutrophil apoptosis, we investigated involvement of PKCδ in the activation of NF-κB in TNFα-stimulated neutrophils. Specific inhibition of PKCδ by rottlerin prevented IκBα degradation and NF-κB activation in TNFα-stimulated neutrophils. This regulation of NF-κB activation by PKCδ was specific only for TNFα signaling, since lipopolysaccharide- or interleukin-1β-induced NF-κB activation and IκBα degradation were not inhibited by rottlerin. In addition, we show that in human neutrophils, but not monocytes, IκBα localizes in significant amounts in the nucleus of unstimulated cells, and the amount of IκBα in the nucleus, as well as in the cytoplasm, correlates with the NF-κB DNA binding. These results suggest that in human neutrophils, the presence of IκBα in the nucleus may function as a safeguard against initiation of NF-κB dependent transcription of pro-inflammatory and anti-apoptotic genes, and represents a distinct and novel mechanism of NF-κB regulation. tumor necrosis factor α diacylglycerol electrophoretic mobility shift assay glucocorticoid receptor inhibitor κB IκB kinase interleukin lactate dehydrogenase lipopolysaccharide nuclear factor κB NF-κB inducing kinase PI-PLC, phosphatidylinositol-specific phospholipase C protein kinase C small ubiquitin-related modifier Neutrophils (polymorphonuclear leukocytes) are short-lived terminally differentiated blood cells that play a vital role in the inflammatory response; they are one of the first cells recruited to the site of injury or infection (1Weiss S.J. N. Engl. J. Med. 1989; 320: 365-376Crossref PubMed Scopus (3847) Google Scholar, 2Henson P.M. Tonnesen M.G. Worthen G.S. Parsons P.E. Maronne G. Lichtenstein L.M. Kondorelli M. Fauci A. Human Inflammatory Disease, Clinical Immunology. 1. B. C. Decker, Inc., Toronto1988: 69-76Google Scholar). In addition to their phagocytic and killing properties, neutrophils synthesize numerous proinflammatory cytokines and chemokines, including TNFα,1 interleukin (IL)-1α and IL-1β, IL-8, and macrophage inflammatory protein α, that may amplify the inflammatory process (3Tiku K. Tiku M.L. Skosey J.L. J. Immunol. 1986; 136: 3677-3685PubMed Google Scholar, 4Dubravec D.B. Spriggs D.R. Mannick J.A. Rodrick M.L. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6758-6761Crossref PubMed Scopus (237) Google Scholar, 5Strieter R.M. Kasahara K. Allen R.M. Standiford T.J. Rolfe M.W. Becker F.S. Chensue S.W. Kunkel S.L. Am. J. Pathol. 1992; 141: 397-407PubMed Google Scholar, 6Tan N.D. Davidson D. Pediatr. Res. 1995; 38: 11-16Crossref PubMed Scopus (38) Google Scholar, 7Zentay Z. Sharaf M. Qadir M. Drafta D. Davidson D. Pediatr. Res. 1999; 46: 406-410Crossref PubMed Scopus (38) Google Scholar, 8Cassatella M.A. Gasperini S. Russo M.P. Ann. N. Y. Acad. Sci. 1997; 832: 233-242Crossref PubMed Scopus (80) Google Scholar). Expression of many of these proinflammatory proteins is regulated at the level of gene transcription by transcription factor NF-κB (9Barnes P.J. Karin M. N. Engl. J. Med. 1997; 336: 1066-1071Crossref PubMed Scopus (4271) Google Scholar, 10Blackwell T.S. Christman J.W. Am. J. Respir. Cell Mol. Biol. 1997; 17: 3-9Crossref PubMed Scopus (908) Google Scholar). Since the knowledge that neutrophils are an important source of cytokines is relatively new, the molecular mechanisms regulating cytokine expression in these cells have only begun to be investigated (11McDonald P.P. Bald A. Cassatella M.A. Blood. 1997; 89: 3421-3433Crossref PubMed Google Scholar, 12Nick J.A. Avdi N.J. Young S.K. Lehman L.A. McDonald P.P. Frasch S.C. Billstrom M.A. Henson P.M. Johnson G.L. Worthen G.S. J. Clin. Invest. 1999; 103: 851-858Crossref PubMed Scopus (264) Google Scholar). We have previously shown that NF-κB activity in human neutrophils consists of p50/50 homodimers and p50/65 heterodimers, and that their activation in TNFα-stimulated neutrophils is inhibited by dexamethasone, an anti-inflammatory drug (13Vancurova I. Bellani I. Davidson D. Pediatr. Res. 2001; 49: 257-262Crossref PubMed Scopus (39) Google Scholar). Using pharmacological inhibitors Nick et al. (12Nick J.A. Avdi N.J. Young S.K. Lehman L.A. McDonald P.P. Frasch S.C. Billstrom M.A. Henson P.M. Johnson G.L. Worthen G.S. J. Clin. Invest. 1999; 103: 851-858Crossref PubMed Scopus (264) Google Scholar) demonstrated that lipopolysaccharide (LPS)-induced activation of NF-κB in neutrophils is mediated by p38α mitogen-activated protein kinase. However, the signaling pathways leading to NF-κB activation in response to neutrophil stimulation with TNFα are undefined. Interestingly, a recent study demonstrated that NF-κB also regulates both constitutive and TNFα-induced apoptosis in human neutrophils (14Ward C. Chilvers E.R. Lawson M.F. Pryde J.G. Fujihara S. Farrow S.N. Haslett C. Rossi A.G. J. Biol. Chem. 1999; 274: 4309-4318Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar). This neutrophil apoptosis has been recently shown to be mediated by a novel isoform of protein kinase C (PKC), PKCδ (15Pongracz J. Webb P. Wang K. Deacon E. Lunn O.J. Lord J.M. J. Biol. Chem. 1999; 274: 37329-37334Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 16Khwaja A. Tatton L. Blood. 1999; 94: 291-301Crossref PubMed Google Scholar). Therefore, we sought to investigate involvement of PKCδ in TNFα-induced activation of NF-κB in human neutrophils. PKCδ is selectively inhibited by rottlerin (15Pongracz J. Webb P. Wang K. Deacon E. Lunn O.J. Lord J.M. J. Biol. Chem. 1999; 274: 37329-37334Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 17Gschwendt M. Muller H.J. Kielbassa K. Zang R. Kittstein W. Rincke G. Marks F. Biochem. Biophys. Res. Commun. 1994; 199: 93-98Crossref PubMed Scopus (759) Google Scholar, 18Reyland M.E. Anderson S.M. Matassa A.A. Barzen K.A. Quissell D.O. J. Biol. Chem. 1999; 274: 19115-19123Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar, 19Frasch S.C. Henson P.M. Kailey J.M. Richter D.A. Janes M.S. Fadok V.A. Bratton D.L. J. Biol. Chem. 2000; 275: 23065-23073Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar), and as any other novel PKC, it is activated in a Ca2+-independent manner by diacylglycerol (DAG), which is produced by activated phospholipase C (PLC) (20Kent J.D. Sergeant S. Burns D.J. McPhail L.C. J. Immunol. 1996; 157: 4647-4651Google Scholar). In this study, we show that inhibition of phosphatidylinositol-specific phospholipase C (PI-PLC) and PKCδ blocks activation of NF-κB in TNFα-stimulated human neutrophils by inhibiting degradation of IκBα. The regulation of NF-κB activation by PKCδ is specific only for TNFα signaling, since LPS- or IL-1β-induced activation of NF-κB and degradation of IκBα are not inhibited by rottlerin. In addition, we show that in human neutrophils, but not monocytes, IκBα localizes in significant amounts in the nucleus of resting unstimulated cells. The NF-κB DNA binding in the neutrophil does not correlate with nuclear translocation of NF-κB subunits, as is the case in most mammalian cells (21Baeuerle P.A. Baltimore D. Science. 1988; 242: 540-546Crossref PubMed Scopus (1685) Google Scholar, 22Hay R.T. Vuillard L. Desterro J.M. Rodriguez M.S. Philos. Trans. R. Soc. Lond. B Biol. Sci. 1999; 354: 1601-1609Crossref PubMed Scopus (82) Google Scholar, 23Karin M. Ben-Neriah Y. Annu. Rev. Immunol. 2000; 18: 621-663Crossref PubMed Scopus (4073) Google Scholar), but rather with the amount of IκBα in the nucleus, as well as in the cytoplasm. Ficoll-Paque PLUS, dextran T-500, T4 polynucleotide kinase, poly(dI-dC), and Sephadex G25 spin columns were purchased from Amersham Pharmacia Biotech (Piscataway, NJ). Hank's balanced salt solution, RPMI 1640 medium, and endotoxin tested, heat-inactivated fetal calf serum (FCS) were obtained from Life Technologies (Grand Island, NY). Escherichia coli expressed purified recombinant human TNFα and IL-1β were purchased from R & D Systems (Minneapolis, MN). [32P]ATP was purchased from PerkinElmer Life Sciences (Boston, MA). Histone H1, U-73122, U-73343, D-609, Et-18-OCH3, rottlerin, and Ro-31-8425 were purchased from Calbiochem (La Jolla, CA). Protein A/G Plus-agarose, purified polyclonal antibodies to human p50 (sc-7178X), IκBα (sc-371), PKCδ (sc-937), and glucocorticoid receptor (GR, sc-1003), and mouse monoclonal anti-actin antibody (sc-8432) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Polyclonal antibody to p65 (SA-171) was obtained from Biomol (Plymouth Meeting, PA). Monoclonal mouse anti-SUMO-1 antibody was from Zymed Laboratories Inc.(San Francisco, CA), and polyclonal lactate dehydrogenase (LDH) antibody (20-LG22) was purchased from Fitzgerald Industries International (Concord, MA). Horseradish peroxidase-conjugated anti-rabbit, anti-mouse, and anti-goat IgG secondary antibodies were from Amersham Pharmacia Biotech (Arlington Heights, IL). All other reagents were molecular biology grade and were purchased from Sigma. All reagents and plasticware used throughout the experiments were pyrogen-free. Fresh blood was obtained from healthy adult human volunteers and collected in heparinized preservative-free tubes. Neutrophils and monocytes (95–98% purity) were separated under endotoxin-free conditions using Ficoll-Paque centrifugation (24Boyum A. Scand. J. Immunol. 1976; 5: 9-15Crossref PubMed Scopus (1332) Google Scholar), and the neutrophils were subsequently purified by dextran sedimentation and hypotonic lysis of residual erythrocytes as described previously (6Tan N.D. Davidson D. Pediatr. Res. 1995; 38: 11-16Crossref PubMed Scopus (38) Google Scholar). Purified cells were resuspended in RPMI 1640 supplemented with 5% low endotoxin fetal calf serum, at a final concentration of 5 × 106 cells/ml, and incubated at 37 °C in polypropylene tubes with gentle agitation. For the inhibition experiments, the inhibitors were dissolved in dimethyl sulfoxide, and the cells were pretreated 15 min with either the inhibitor or with Me2SO alone, before stimulation with TNFα. The incubations were terminated by placing cells on ice and rapid centrifugation (1 min, 5,000 × g, 4 °C). Nuclear and cytoplasmic extracts were prepared from 5 × 106 cells as described previously (13Vancurova I. Bellani I. Davidson D. Pediatr. Res. 2001; 49: 257-262Crossref PubMed Scopus (39) Google Scholar). Briefly, the pelleted cells were resuspended in 300 μl of hypotonic buffer (buffer A: 10 mm Hepes, pH 7.5, 10 mm KCl, 3 mmNaCl, 3 mm MgCl2, 1 mm EDTA, 1 mm EGTA, and 2 mm dithiothreitol) containing the following protease and phosphatase inhibitors: 2 mmphenylmethylsulfonyl fluoride, 100 μg/ml soybean trypsin inhibitor, 1 mm benzamidine, 2 mm levamisole, 1 mm Na3VO4, 10 mm NaF, 20 mm glycerophosphate, and protease inhibitor mixture from Sigma (P-8340), used at concentration 60 μl/5 × 106cells). After 15 min incubation on ice, 0.05 volumes of 10% Nonidet P-40 were added, the cells were vortexed (10 s) and immediately centrifuged at 500 × g for 10 min at 4 °C. The supernatants were collected, designated as cytoplasmic extracts, aliquoted, and stored at −80 °C. The nuclear pellets were washed in 200 μl of buffer A containing the protease inhibitors, and re-centrifuged. The pelleted nuclei were resuspended in 50 μl of ice-cold nuclear buffer (NE buffer: 20 mm Hepes, pH 7.5, 25% glycerol, 0.8 m KCl, 1 mm MgCl2, 1% Nonidet P-40, 0.5 mmEDTA, 2 mm dithiothreitol) containing the protease and phosphatase inhibitors as described above. Following a 20-min incubation on ice (with occasional mixing), the samples were centrifuged (14,000 × g, 15 min, 4 °C), and the resulting supernatants (nuclear extracts) were aliquoted and stored at −80 °C. Protein concentration was measured using the Pierce Coomassie Plus protein assay kit (Pierce, Rockford, IL). Contamination of nuclear and cytoplasmic fractions by cytoplasmic and nuclear proteins, respectively, was determined by Western analysis using LDH and SUMO-1 as specific markers. The oligonucleotide used as a probe for EMSA was a 42-base pair double-stranded construct (5′-TTGTTACAAGGGGACTTTCCGCTGGGGACTTTCCAGGGAGGC-3′) containing two tandemly repeated NF-κB-binding sites (underlined). Mutant oligonucleotide used for competition studies was 5′-TTGTTACAATCTCACTTTCCGCTTCTCACTTTCCAGGGAGGC-3′. End labeling was accomplished by treatment with T4 kinase in the presence of [γ-32P]ATP, and the labeled oligonucleotide was purified on a Sephadex G-25 column, as described elsewhere (25Carter T.H. Vancurova I. Sun I. Lou W. DeLeon S. Mol. Cell. Biol. 1990; 10: 6460-6471Crossref PubMed Scopus (246) Google Scholar). Nuclear extracts (containing 4–6 μg of protein in 5–7 μl) were incubated (20 min at room temperature) with 5–10 fmol of radiolabeled oligonucleotide (∼70,000 cpm) in 20 μl of binding buffer (20 mm Tris-Cl, pH 7.5, 150 mm KCl, 1 mm EDTA, 1 mm dithiothreitol, 0.1% Nonidet P-40, 6% glycerol) supplemented with 20 μg of acetylated bovine serum albumin and 2 μg of poly(dI-dC). For competition or supershift experiments, binding reactions were performed in the presence of 30m excess of unlabeled oligonucleotide or 1 μg of specific polyclonal antibody, respectively, and incubated 15 min at room temperature before adding 32P-labeled oligonucleotide. The resulting complexes were resolved on 5% nondenaturing polyacrylamide gels that had been pre-run at 100 V for 30 min in 0.5 × TBE buffer. Electrophoresis was conducted at 180 V for 2.5 h. After electrophoresis, gels were transferred to Whatman DE-81 paper, dried, and exposed to autoradiographic film (Kodak BioMax MS) with intensifier screen at −80 °C. PKCδ enzymatic activity was assayed in whole cell lysates immunoprecipitated by PKCδ specific polyclonal antibody as follows. Neutrophils (5 × 106) were lysed in 0.3 ml of lysis buffer (50 mm Tris-Cl, pH 8.0, 250 mm NaCl, 1.5 mm MgCl2, 1 mm EDTA, 1% Triton X-100, 10% glycerol, 2 mmdithiothreitol, 2 mm phenylmethylsulfonyl fluoride, 100 μg/ml soybean trypsin inhibitor; 1 mm benzamidine, 2 mm levamisole, 1 mmNa3VO4, 20 mm glycerophosphate, 10 mm NaF and protease inhibitor mixture from Sigma (P-8340), used at concentration 60 μl/5 × 106 cells). Soluble proteins were pre-cleared by a 1-h incubation (4 °C) with 10 μl of Protein A/G Plus-agarose. The precleared supernatants were incubated with 1 μg of anti-PKC-δ or control anti-GR antibody (2 h, 4 °C), and immunoprecipitated with 10 μl of Protein A/G Plus-agarose for an additional 1 h. The immune complexes were washed 5 times with lysis buffer and 1 time with kinase buffer (20 mm Hepes, pH 7.5, 10 mm MgCl2, 2 mmMnCl2, 20 μm ATP), and resuspended in 20 μl of kinase buffer. Five μl of 5 × reaction buffer (1 mg/ml histone H1, 20 μm 1,2-dioleoyl-sn-glycerol, and 0.25 mg/ml l-α-phosphatidyl-l-serine) and 5 μCi of [γ-32P]ATP were added, and the samples were incubated for 5 min at 30 °C. Reactions were stopped by the addition of 8 μl of 5 × sample buffer, the samples were boiled and resolved on a 12% SDS-polyacrylamide gel. The gels were stained with Coomassie, and the extent of histone H1 phosphorylation was determined by both autoradiography and scintillation counting of the excised Coomassie-stained histone polypeptide bands. In experiments examining the effect of rottlerin on PKCδ activity in vitro, rottlerin was added to the PKCδ immunoprecipitates in concentrations given in the text before the addition of 1 μm ATP. Denatured proteins were separated on 12% denaturing polyacrylamide gels and transferred to nitrocellulose membrane (Hybond C; Amersham Pharmacia Biotech). Membranes were blocked overnight with a 5% (w/v) nonfat dry milk solution containing 10 mm Tris-Cl, pH 7.5, 140 mm NaCl, 1.5 mm MgCl2, and 0.1% Tween 20 (TBSTM) before incubating with primary antibodies (1 h for IκBα, p65, p50, SUMO-1, and LDH antibodies, and overnight for actin antibody). Primary antibodies were diluted in TBSTM (1:250 for IκBα and actin, 1:700 for p65, 1:300 for p50, and 1:200 for LDH and SUMO-1). After washing, the membranes were incubated 1 h with horseradish peroxidase-labeled secondary antibody diluted 1:2000 in TBSTM, and the labeled proteins were detected using enhanced chemiluminescence (ECL) reagents as described by the manufacturer (Amersham Pharmacia Biotech). To confirm equivalent amounts of loaded proteins, or to re-probe the membrane with another antibody, the membranes were stripped with 100 mm 2-mercaptoethanol, 2% SDS, and 62.5 mmTris-Cl (pH 6.7) for 30 min at 50 °C, and incubated with the appropriate primary antibody diluted in TBSTM. The signal was developed using secondary IgG-horseradish peroxidase and ECL detection as described above. Data presented here represent a minimum of three experiments, and, where appropriate, data are expressed as mean ± S.E. Statistical significance was evaluated by using ANOVA. To investigate whether the TNFα-induced NF-κB activation involves PLC- and PKCδ-dependent pathways, we used inhibitors of phosphatidylcholine (PC)- and phosphatidylinositol (PI)-specific PLC, and PKCδ: D-609 (26Schutze S. Potthoff K. Machleidt T. Berkovic D. Wiegmann K. Kronke M. Cell. 1992; 71: 765-776Abstract Full Text PDF PubMed Scopus (971) Google Scholar), U-73122, and Et-18-OCH3 (27Smith R.J. Justen J.M. McNab A.R. Rosenbloom C.L. Steele A.N. Detmers P.A. Anderson D.C. Manning A.M. J. Pharmacol. Exp. Ther. 1996; 278: 320-329PubMed Google Scholar, 28Wang J.P. Hsu M.F. Kuo S.C. Eur. J. Pharmacol. 1997; 319: 131-136Crossref PubMed Scopus (14) Google Scholar, 29Powis G. Seewald M.J. Gratas C. Melder D. Riebow J. Modest E.J. Cancer Res. 1992; 52: 2840-2853Google Scholar), and rottlerin (15Pongracz J. Webb P. Wang K. Deacon E. Lunn O.J. Lord J.M. J. Biol. Chem. 1999; 274: 37329-37334Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 17Gschwendt M. Muller H.J. Kielbassa K. Zang R. Kittstein W. Rincke G. Marks F. Biochem. Biophys. Res. Commun. 1994; 199: 93-98Crossref PubMed Scopus (759) Google Scholar, 18Reyland M.E. Anderson S.M. Matassa A.A. Barzen K.A. Quissell D.O. J. Biol. Chem. 1999; 274: 19115-19123Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar, 19Frasch S.C. Henson P.M. Kailey J.M. Richter D.A. Janes M.S. Fadok V.A. Bratton D.L. J. Biol. Chem. 2000; 275: 23065-23073Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar), respectively. Neutrophils were preincubated 15 min with or without the corresponding inhibitor, stimulated 30 min with TNFα, and the NF-κB DNA binding activity was measured in nuclear extracts by EMSA. As seen in Fig. 1 A, neutrophil stimulation with TNFα induced activation of the p50/65 heterodimer, and to a lower extent also the p50/50 homodimer. The specificity and identity of these complexes was confirmed using competition and supershift assay as shown in panel B. Since the NF-κB form responsible for induction of inflammatory and apoptotic genes is the p50/65 heterodimer, whereas the cellular function of the p50/50 homodimer is not fully understood (30Siebenlist U. Franzoso G. Brown K. Annu. Rev. Cell Biol. 1994; 10: 405-455Crossref PubMed Scopus (2015) Google Scholar), we focused on DNA binding activity of the p50/65 NF-κB heterodimer. The p50/65 NF-κB DNA binding activity was inhibited by U-73122 (5 μm) and Et-18-OCH3 (50 μm), inhibitors of PI-PLC, and by PKCδ inhibitor rottlerin (50 μm). In contrast, inhibitor of PC-PLC, D-609 at 50 μmconcentration previously shown to be selectively effective to inhibit PC-PLC activity (26Schutze S. Potthoff K. Machleidt T. Berkovic D. Wiegmann K. Kronke M. Cell. 1992; 71: 765-776Abstract Full Text PDF PubMed Scopus (971) Google Scholar, 31Kozawa O. Suzuki A. Kaida T. Tokuda H. Uematsu T. J. Biol. Chem. 1997; 272: 25099-25104Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar) did not reduce NF-κB DNA binding (Fig.1 A). The inhibition of NF-κB DNA binding by U-73122 was dose dependent (Fig. 2 A). The complete inhibition of p50/65 NF-κB was achieved at 5 μm U-73122 concentration, and the IC50 was ∼2 μm. This IC50 value is consistent with the previously reported IC50 for PLC specific inhibition by U-73122 in the neutrophil (27Smith R.J. Justen J.M. McNab A.R. Rosenbloom C.L. Steele A.N. Detmers P.A. Anderson D.C. Manning A.M. J. Pharmacol. Exp. Ther. 1996; 278: 320-329PubMed Google Scholar, 28Wang J.P. Hsu M.F. Kuo S.C. Eur. J. Pharmacol. 1997; 319: 131-136Crossref PubMed Scopus (14) Google Scholar). The inactive structural analogue of U-73122, U-73343, in the range of 0.1–5 μmconcentrations, had no effect on NF-κB DNA binding (data not shown). Fig. 2 B shows a dose response of the PKCδ-specific inhibitor rottlerin on NF-κB DNA binding in TNFα-stimulated neutrophils. The complete inhibition of the p50/65 NF-κB heterodimer was achieved by rottlerin concentrations of 50 μm, and the IC50 was ∼10 μm. These values correlate well with the previously reported rottlerin IC50 for PKCδ inhibition 3–6 μm, whereas the IC50 values for other PKC isoforms were 40–100 μm (17Gschwendt M. Muller H.J. Kielbassa K. Zang R. Kittstein W. Rincke G. Marks F. Biochem. Biophys. Res. Commun. 1994; 199: 93-98Crossref PubMed Scopus (759) Google Scholar, 18Reyland M.E. Anderson S.M. Matassa A.A. Barzen K.A. Quissell D.O. J. Biol. Chem. 1999; 274: 19115-19123Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar, 19Frasch S.C. Henson P.M. Kailey J.M. Richter D.A. Janes M.S. Fadok V.A. Bratton D.L. J. Biol. Chem. 2000; 275: 23065-23073Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). In contrast, neutrophil pretreatment with Ro-31–8425 (100 nm), which inhibits the classical isoforms of PKC but not PKCδ (32Merritt J.E. Sullivan J.A. Tse J. Wilkinsin S.E. Nixon J.S. Cell. Signal. 1997; 9: 53-57Crossref PubMed Scopus (19) Google Scholar), had no inhibitory effect on TNFα-induced NF-κB DNA binding even at concentration 10 times higher than the reported IC50 (data not shown). Importantly, the inhibitory effect of rottlerin on NF-κB DNA binding was specific only for TNFα induction, since LPS, as well as IL-1β-induced NF-κB activation was not inhibited by 50 μm rottlerin (Fig.3, panel A). These results demonstrate that the rottlerin effect is specific only for the TNFα signaling pathway, and indicate that the NF-κB activation in response to neutrophil stimulation with TNFα is mediated by PI-PLC and PKCδ dependent pathways. Rottlerin was originally reported to inhibit PKCδ by competing for ATP binding (17Gschwendt M. Muller H.J. Kielbassa K. Zang R. Kittstein W. Rincke G. Marks F. Biochem. Biophys. Res. Commun. 1994; 199: 93-98Crossref PubMed Scopus (759) Google Scholar). To confirm that the same rottlerin concentrations inhibiting NF-κB activation in TNFα-stimulated neutrophils can also inhibit activity of PKCδ, the PKCδ was immunoprecipitated from whole cell lysates using PKCδ specific polyclonal antibody, and PKCδ kinase activity was measured using histone H1 as a substrate. For comparative purposes, immunoprecipitation using irrelevant glucocorticoid receptor (GR) antibody was performed as a control. As seen in Fig.4 A, while no histone phosphorylation was detected in lysates prepared from TNFα-stimulated neutrophils and immunoprecipitated with GR antibody (lane 1), immunoprecipitation with PKCδ antibody resulted in strong phosphorylation of histone H1 (lanes 2–5), demonstrating that the immunoprecipitation of PKCδ was specific. To determine whether rottlerin inhibits activity of PKCδ directly, or whether it inhibits events upstream of PKCδ, we performed two types of experiments. In the first set of experiments, PKCδ was immunoprecipitated from TNFα-stimulated neutrophils and incubated with rottlerin in vitro (Fig. 4 B). Rottlerin inhibited PKCδ activity in a dose-dependent manner, the IC50 being about 10 μm, which is consistent with the rottlerin inhibition of NF-κB activation demonstrated above (Fig. 2 B). In the second set of experiments, neutrophils were preincubated with varying concentrations of rottlerin in vivo, prior to stimulation with TNFα, and PKCδ was immunoprecipitated from corresponding cell lysates and assayed for histone phosphorylation (panel A). Since it is very likely that the extensive washing of the immunoprecipitates efficiently removes rottlerin from PKCδ, this experiment differentiates between the direct and indirect effect of rottlerin on PKCδ activity. If rottlerin targets protein(s) (for example, another protein kinase(s)) upstream of PKCδ, then neutrophil preincubation with rottlerin in vivo would result in a reduced activity of the immunoprecipitated PKCδ. However, as seen in Fig. 4 A, neutrophil preincubation with varying concentrations of rottlerin in vivo did not significantly inhibit histone phosphorylation by the immunoprecipitated PKCδ. These results demonstrate that the effect of rottlerin on PKCδ is direct, and further suggest that the activity of PKCδ is required for NF-κB activation in response to neutrophil stimulation with TNFα. To determine whether PKCδ activates NF-κB through regulating cellular pools of the IκBα inhibitor, neutrophils were stimulated with TNFα (15 min, 10 ng/ml) in the presence of varying concentrations of rottlerin, and cytoplasmic extracts were analyzed by Western blotting using IκBα specific polyclonal antibody (Fig. 5 A). Consistent with a previous report (11McDonald P.P. Bald A. Cassatella M.A. Blood. 1997; 89: 3421-3433Crossref PubMed Google Scholar), neutrophil stimulation with TNFα substantially reduced the cytosolic pool of IκBα (lane 2). Importantly, neutrophil pretreatment with rottlerin inhibited, in a dose-dependent manner, the TNFα-induced depletion of cytosolic IκBα (Fig. 5 A). The lower laneshows reprobing the membrane with control anti-actin antibody, demonstrating equal protein loading and transfer to nitrocellulose. To determine whether the increased cytoplasmic pools of IκBα by rottlerin resulted from new protein synthesis or increased protein stability, neutrophils were pretreated with cycloheximide (100 μg/ml, 10 min) prior to incubation with rottlerin (50 μm, 15 min) and stimulation with TNFα (10 ng/ml, 30 min). As seen in Fig. 5,panels B and C, no new protein synthesis was required for the rottlerin up-regulation of IκBα and inhibition of NF-κB DNA binding, respectively. These results indicate that PKCδ is involved in the activation of NF-κB in response to neutrophil stimulation with TNFα by activating pathway(s) leading to degradation of IκBα. To confirm that the PKCδ involvement in NF-κB activation is specific for TNFα signaling (Fig. 3), neutrophils were preincubated with rottlerin (50 μm, 15 min) and stimulated 30 min with TNFα, LPS, or IL-1β, and cytoplasmic extracts were analyzed for IκBα expression (Fig. 3 B). Consistent with NF-κB DNA binding (Fig. 3 A), the rottlerin effect was specific only for TNFα, since the LPS- and IL-1β-induced IκBα degradation were not inhibited (Fig. 3 B). In most mammalian cells, activation of NF-κB has been shown to be controlled at the level of nuclear translocation of NF-κB proteins through their tightly regulated association with IκBα anchored in the cytoplasm (21Baeuerle P.A. Baltimore D. Science. 1988; 242: 540-546Crossref PubMed Scopus (1685) Google Scholar, 22Hay R.T. Vuillard L. Desterro J.M. Rodriguez M.S. Philos. Trans. R. Soc. Lond. B Biol. Sci. 1999; 354: 1601-1609Crossref PubMed Scopus (82) Google Scholar, 23Karin M. Ben-Neriah Y. Annu. Rev. Immunol. 2000; 18: 621-663Crossref PubMed Scopus (4073) Google Scholar). Therefore, we sought to determine whether the inhibition of PKCδ-dependent activation of NF-κB in response to neutrophil stimulation with TNFα is mediated by cytoplasmic retention of p50 and p65 NF-κB subunits. Neutrophils were stimulated with TNFα with and without pretreatment with rottlerin, and the cytoplasmic and nuclear fractions were analyzed by Western blotting using p50- and p65-specific antibodies. Surprisingly, both the cytoplasmic and the nuclear levels of p50 and p65 NF-κB subunits were not significantly affected by neutrophil stimulation with TNFα or pretreatment with rottlerin (Fig.6). Moreover, both NF-κB proteins were present in significant amounts in the nucleus even under conditions when the NF-κB DNA binding is inhi" @default.
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