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• W2050503669 abstract "NF-κB is a redox-sensitive transcription factor known to be activated by oxidative stress as well as chemical and biological reductants. Its DNA binding activity requires reduced cysteines present in the p65 subunit of the dimer. Thioredoxin (Trx) is an endogenous disulfide oxidoreductase known to modulate several redox-dependent functions in the cell. NF-κB was activated by addition of Escherichia coli thioredoxin in a redox-dependent manner in A549 cells. Such activation was accompanied by degradation of IκB in the cytosol. In addition, only the reduced form of thioredoxin activated NF-κB, whereas the oxidized form was without any effect. Overexpression of human thioredoxin also caused activation of NF-κB and degradation of IκB. On the contrary, dominant-negative redox-inactive mutant thioredoxin expression did not activate NF-κB, further confirming the redox-dependent activation of NF-κB. We also investigated the mechanism of activation of NF-κB by thioredoxin. We demonstrate that thioredoxin activates c-Jun NH2-terminal kinase (JNK)-signaling cascade, and dominant-negative expression of mitogen-activated protein kinase kinase kinase 1 (MEKK1), JNK kinase, or JNK inhibits NF-κB activation by thioredoxin. In contrast, wild-type MEKK1 or JNK kinase induced NF-κB activation alone or in combination with thioredoxin expression plasmid. These findings were also confirmed by NF-κB-dependent luciferase reporter gene transcription. NF-κB is a redox-sensitive transcription factor known to be activated by oxidative stress as well as chemical and biological reductants. Its DNA binding activity requires reduced cysteines present in the p65 subunit of the dimer. Thioredoxin (Trx) is an endogenous disulfide oxidoreductase known to modulate several redox-dependent functions in the cell. NF-κB was activated by addition of Escherichia coli thioredoxin in a redox-dependent manner in A549 cells. Such activation was accompanied by degradation of IκB in the cytosol. In addition, only the reduced form of thioredoxin activated NF-κB, whereas the oxidized form was without any effect. Overexpression of human thioredoxin also caused activation of NF-κB and degradation of IκB. On the contrary, dominant-negative redox-inactive mutant thioredoxin expression did not activate NF-κB, further confirming the redox-dependent activation of NF-κB. We also investigated the mechanism of activation of NF-κB by thioredoxin. We demonstrate that thioredoxin activates c-Jun NH2-terminal kinase (JNK)-signaling cascade, and dominant-negative expression of mitogen-activated protein kinase kinase kinase 1 (MEKK1), JNK kinase, or JNK inhibits NF-κB activation by thioredoxin. In contrast, wild-type MEKK1 or JNK kinase induced NF-κB activation alone or in combination with thioredoxin expression plasmid. These findings were also confirmed by NF-κB-dependent luciferase reporter gene transcription. nuclear transcription factor κB thioredoxin thioredoxin reductase mitogen-activated protein c-Jun NH2-terminal kinase JNK kinase MAP kinase kinase kinase dominant-negative electrophoretic mobility shift assay human pulmonary artery endothelial cells protein kinase C Nuclear transcription factor κB (NF-κB)1 is a multi-subunit factor that can rapidly activate the expression of genes involved in inflammatory, immune, and acute phase responses (1Gilmore T.D. Cell. 1990; 62: 841-843Abstract Full Text PDF PubMed Scopus (163) Google Scholar). Although multiple forms can exist, the principal active form appears to be a heterodimer consisting of 50- and 65-kDa subunits (2Baldwin A.S. Annu. Rev. Immunol. 1996; 14: 649-681Crossref PubMed Scopus (5592) Google Scholar). The heterodimer remains bound to an inhibitor protein IκB in the cytoplasm. In response to a variety of stimuli, IκB is phosphorylated and ubiquitinated, followed by degradation by the 26 S proteosome (3Chen Z.J. Hagler J. Palombella V. Melandri F. Scherer D. Ballard D. Maniatis T. Genes Dev. 1996; 9: 1586-1597Crossref Scopus (1172) Google Scholar). This process exposes the nuclear localization signal, allowing the heterodimeric complex to interact with the nuclear transport machinery and to translocate to the nucleus (2Baldwin A.S. Annu. Rev. Immunol. 1996; 14: 649-681Crossref PubMed Scopus (5592) Google Scholar). A characteristic of NF-κB is that many different agents can induce its DNA binding activity (2Baldwin A.S. Annu. Rev. Immunol. 1996; 14: 649-681Crossref PubMed Scopus (5592) Google Scholar). Oxidants (4Meyer M. Schreck R. Baeuerle P.A. EMBO J. 1991; 12: 2005-2015Crossref Scopus (1271) Google Scholar) as well as reductants (5Toledano M.B. Leonard W.J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4328-4332Crossref PubMed Scopus (579) Google Scholar, 6Staal F.J.T. Roederer M. Herzenberg L. Herzenberg L.A. Proc. Natl. Acad. Sci., U. S. A. 1990; 87: 9943-9947Crossref PubMed Scopus (898) Google Scholar, 7Das K.C. Lewis-Molock Y. White C.W. Am. J. Physiol. 1995; 269: L588-L602PubMed Google Scholar) are known to activate NF-κB. Although redox-dependent activation of NF-κB is widely recognized, little is known about how cellular redox status could modulate the signaling events that are associated with the activation of NF-κB. Thioredoxin (Trx) is a potent protein disulfide reductase that catalyzes protein reduction using reducing equivalents from NADPH in conjunction with thioredoxin reductase (TR). Remarkably low concentrations of thioredoxin are effective in reducing disulfides in insulin, fibrinogen, human chorionic gonadotropin, nitric-oxide synthase, ribonucleotide reductase, glucocorticoid receptors, and other proteins (8Holmgren A. Annu. Rev. Biochem. 1985; 54: 237-271Crossref PubMed Google Scholar, 9Holmgren A. J. Biol. Chem. 1979; 254: 9113-9119Abstract Full Text PDF PubMed Google Scholar, 10Karoly K. Yee B.C. Buchanan B.B. J. Biol. Chem. 1991; 266: 16135-16140Abstract Full Text PDF PubMed Google Scholar, 11Patel J.M. Zhang J. Block E.R. Am. J. Respir. Cell Mol. Biol. 1996; 15: 410-419Crossref PubMed Scopus (78) Google Scholar). The rate of reduction of insulin disulfide by thioredoxin was found to be 10,000 times higher than that by dithiothreitol (8Holmgren A. Annu. Rev. Biochem. 1985; 54: 237-271Crossref PubMed Google Scholar). Thus, reduced thioredoxin is an extremely potent protein disulfide reductase. Intracellularly, most of this ubiquitous low molecular mass (12 kDa) protein remains reduced (12Spector A. Yan G.Z. Huang C.R. McDermott M.J. Gascoyne P.R.C. Pigiet V. J. Biol. Chem. 1988; 263: 4984-4990Abstract Full Text PDF PubMed Google Scholar, 13Fernando M.R. Nanri H. Yoshitake S. Nagata-kuno K. Minakami S. Eur. J. Biochem. 1992; 209: 917-922Crossref PubMed Scopus (201) Google Scholar). Thioredoxin has two critical cysteine residues at the active site, which in the oxidized protein, form a disulfide bridge located in a protrusion from the three-dimensional structure of the protein (8Holmgren A. Annu. Rev. Biochem. 1985; 54: 237-271Crossref PubMed Google Scholar). The flavoprotein thioredoxin reductase catalyzes the NADPH-dependent reduction of this disulfide (8Holmgren A. Annu. Rev. Biochem. 1985; 54: 237-271Crossref PubMed Google Scholar). Small increases in thioredoxin can cause profound changes in sulfhydryl-disulfide redox status in protein (8Holmgren A. Annu. Rev. Biochem. 1985; 54: 237-271Crossref PubMed Google Scholar). Additionally, thioredoxin was shown to restore DNA binding activity of NF-κB in a cell-free system (14Hayashi T. Ueno Y. Okamoto T. J. Biol. Chem. 1993; 268: 11380-11388Abstract Full Text PDF PubMed Google Scholar). In the same report, it was shown that redox regulation of NF-κB activity appeared to be exerted after dissociation of IκB from the NF-κB complex. Moreover, thioredoxin was shown to form a complex with p50 subunit of NF-κB (15Qin J. Clore G.M. Kennedy W.M.P. Huth J.R. Gronenborn A.M. Structure (Lond.). 1995; 3: 289-297Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). In addition, other reports have shown that critical cysteine 62 in NF-κB is required to be reduced by thioredoxin for its activation (16Matthews J.R. Wakasugi N. Virelizier J.L. Yodoi J. Hay T.R. Nucleic Acids Res. 1992; 20: 3821-3830Crossref PubMed Scopus (729) Google Scholar). We reported earlier that reducing thiols can activate NF-κB in intact cells (7Das K.C. Lewis-Molock Y. White C.W. Am. J. Physiol. 1995; 269: L588-L602PubMed Google Scholar). We also showed that sulfhydryl oxidation or alkylation can inhibit tumor necrosis factor-α- or interleukin-1-induced NF-κB activation (17Das K.C. Molock-Lewis Y. White C.W. Mol. Cell. Biochem. 1995; 148: 45-57Crossref PubMed Scopus (118) Google Scholar). Mitogen-activated protein (MAP) kinases are serine/threonine kinases activated by dual phosphorylation on both a tyrosine and a threonine (18Minden A. Karin M. Biochim. Biophys. Acta. 1997; 1333: 85-104PubMed Google Scholar). These enzymes are important components of signaling pathways that transduce extracellular stimuli into intracellular responses. There are three major forms of MAP kinases; extracellular signal-regulating kinase, c-Jun-NH2-terminal Kinase (JNK) or stress-activated protein kinase), and p38 MAP kinase. Extracellular signal-regulating kinase pathway is activated by growth factors and phorbol esters (18Minden A. Karin M. Biochim. Biophys. Acta. 1997; 1333: 85-104PubMed Google Scholar). JNK/stress-activated protein kinase pathway is activated in response to cellular stresses such as heat shock, UV irradiation, or inflammatory cytokines (19Ip Y.T. Davis R.J Curr. Opin. Cell Biol. 1998; 10: 205-219Crossref PubMed Scopus (1386) Google Scholar). Inflammatory cytokines as well as environmental stresses such as osmotic shock activate p38 MAP kinase (20Davis R.J Biochem. Soc. Symp. 1998; 64: 1-12Google Scholar). The JNK-signaling cascade functions through the activation of an initiating kinase such as MAP kinase kinase kinase (MEKK1), which in turn phosphorylates the MAP kinase kinase (MKK4/SEK1), and MKK4 finally activates the JNK by phosphorylating the serine and threonine residues on it (reviewed in Ref. 19Ip Y.T. Davis R.J Curr. Opin. Cell Biol. 1998; 10: 205-219Crossref PubMed Scopus (1386) Google Scholar). Although the signal transduction cascade leading to the activation of JNK is relatively well defined, the steps leading to the phosphorylation of IκBα are poorly understood. Recent studies demonstrate that IκBα can be phosphorylated by MEKK1, an upstream kinase of the JNK pathway (21Lee F.S. Hagler J. Chen J. Maniatis T. Cell. 1997; 88: 213-222Abstract Full Text Full Text PDF PubMed Scopus (662) Google Scholar). Moreover, many of the stimuli that induce NF-κB activation, such as tumor necrosis factor-α, UV radiation, and lipopolysaccharide, also activate the JNK signaling cascade. Since phosphorylation of, IκBα is required for its degradation and Trx can activate NF-κB in intact cells, we hypothesized a potential role of JNK in Trx-mediated IκB degradation and activation of NF-κB. In this report, we demonstrate that thioredoxin activates NF-κB and causes degradation of IκB. Additionally, we have shown that MEKK1 is the initiating kinase of the JNK pathway that mediates the NF-κB activation by thioredoxin. Moreover, we also demonstrate that JNK subgroup of MAP kinases is activated by redox-active thioredoxin. Furthermore, we have also shown that thioredoxin can induce NF-κB-dependent reporter gene expression, and such transcription can be abrogated by inhibition of JNK-signaling intermediates using dominant-negative constructs. Escherichia coli thioredoxin was obtained from Promega Corp. (Madison, WI). Thioredoxin reductase and human thioredoxin were obtained from American Diagnostica, Inc. (Greenwich, CT). Anti-p50, anti-p65, pJNK, JNK, and anti-IκB antibodies were obtained from Santa Cruz Biotechnology (Sant Cruz, CA). All other materials were obtained in the highest available grade. A549 cells (adenocarcinoma cells) were obtained from ATCC. Lactacystin was obtained from Sigma. A549 cells were cultured in Kaigan's modified F-12K medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum and 100 units of penicillin/streptomycin. Confluent monolayers were treated with various concentrations of thioredoxin for different time periods as indicated in the figure legends. Cell viability was determined by the trypan blue exclusion method. Human pulmonary artery endothelial cells were obtained from Clonetics Corp. and propagated in endothelial growth medium (Clonetics, CA). MEKK1 (pcDNA3-MEKK1) and the kinase-dead dnMEKK1 were generous gifts of Dr. Tom Maniatis (Harvard University, Boston) and have been described (21Lee F.S. Hagler J. Chen J. Maniatis T. Cell. 1997; 88: 213-222Abstract Full Text Full Text PDF PubMed Scopus (662) Google Scholar). The dominant-negative JNKK expression plasmid (pSRα-dnJNKK) and dominant-negative JNK (pSRα-AFPJNK) expression plasmid were generous gifts of Dr. Gary L. Johnson (National Jewish Medical Center, Denver, CO) and have been described (22Wang T.H. Wnag H.S Ichijo H Giannakakou P. Foster J.S. Fojo T. Wimalasena J. J. Biol. Chem. 1998; 273: 4928-4936Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar). Transfection of various expression plasmids into A549 cells was carried out using Transfectam reagent (Promega) or Geneporter reagent (Gene Therapy Systems Inc. San Diego, CA) as per manufacturer's protocol. Site-directed mutagenesis of the redox-active Cys-32 and Cys-35 to serine was performed by a synthesized oligonucleotide (5′-TCATTTT G GAAGGCCCA G ACCACGTGGC-3′) and quick-change mutagenesis kit (Stratagene, La Jolla, CA) as per manufacturer's protocol. Briefly, double-stranded mutagenic oligonucleotide was synthesized (Genosys) and purified by polyacrylamide gel electrophoresis. Mutagenic oligonucleotide was added to the double-stranded plasmid pcDNA3-Trx. The plasmid was denatured, and the mutagenic-oligonucleotide was annealed by temperature cycling using Pfu Turbo DNA polymerase. After temperature cycling, the methylated nonmutated parental template DNA was digested with Dpn I. XL1-Blue supercompetent cells (Stratagene) were transformed with mutated plasmid. Base substitution in the mutagenic thioredoxin open reading frame was verified by sequencing (University of Texas Medical Branch, Galveston, TX). The mutagenic plasmid (pcDNA3-dnTrx) was amplified for transfection experiments. Nuclear extracts were prepared as described previously (23Das K.C. White CW. J. Biol. Chem. 1997; 272: 14914-14920Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar). Briefly, 107 cells were washed in 10 ml of phosphate-buffered saline and centrifuged (1,500 × g for 5 min). The pellet was resuspended in phosphate-buffered saline (1 ml), transferred into an Eppendorf tube, and centrifuged again (16,000 × g; 15 s). Phosphate-buffered saline was removed, and the cell pellet was resuspended in 400 μl of buffer A (10 mm HEPES, pH 7.8, 10 mm KCl, 0.1 mm EDTA, 2 mmdithiotheitol, 1 mm phenylmethylsulfonyl fluoride, leupeptin (0.5 mg/ml), antipain (0.3 mg/ml)) by gentle pipetting. The cells were allowed to swell on ice for 15 min, after which 25 μl of 10% Nonidet-P40 (Sigma) was added, and the tube was vortexed vigorously for 10 s. The homogenate was centrifuged for 30 s in a microcentrifuge. The nuclear pellet was resuspended in buffer C (20 mm HEPES, pH 7.8, 0.42 m NaCl, 5 mm EDTA, 5 mm dithiotheitol, 1 mmphenylmethylsulfonyl fluoride, 10% (v/v) glycerol), and the tube was rocked gently at 4 °C for 30 min on a shaking platform. The nuclear extract was centrifuged for 10 min in a microcentrifuge at 4 °C, and the supernatant was frozen at −70 °C in aliquots until the electrophoretic mobility shift assay (EMSA) was performed. Protein was quantified by Bradford protein assay (Bio-Rad; Ref. 24Bradford M. Anal. Biochem. 1976; 72: 248-251Crossref PubMed Scopus (217544) Google Scholar). For the EMSA, the NFκB specific oligonucleotide was obtained from Promega Corp.Oligonucleotide was end-labeled using T4 polynucleotide kinase (Promega) and [γ-32P]ATP (NEN) in 10× kinase buffer (0.5 m Tris-HCl, pH 7.5, 0.1 mMgCl2, 50 mm dithiotheitol, 1 mmspermidine, and 1 mm EDTA). For competition studies, 3.5 pmol of unlabeled oligonucleotide was used. Nuclear extract without labeled oligonucleotide was preincubated for 15 min at 4 °C followed by a 20-min incubation at room temperature after the addition of labeled oligonucleotide. The binding reaction contained 10 μg of sample protein, 5 μl of 5× incubation buffer (20% glycerol, 5 mm MgCl2, 5 mm EDTA, 5 mm dithiotheitol, 500 mm NaCl, 50 mm Tris-HCl, pH 7.5, 0.4 mg/ml calf thymus DNA). In some of the binding reactions poly(dI-dC) (Amersham Pharmacia Biotech) was added to a final concentration of 2 μg. The nuclear protein-32P-oligonucleotide complex was separated from free32P-labeled oligonucleotide by electrophoresis through a 6% native polyacrylamide gel in a running buffer of 0.25× TBE (5× TBE = 500 mm Tris, pH 8.0, 450 mm borate, 5 mm EDTA). For the supershift assay, some of the binding reactions contained 200 ng of anti-p50 or anti-p65 antibody (Santa Cruz) along with 2 μg of poly(dI-dC) (Amersham Pharmacia Biotech). Post-nuclear supernatant was treated as the cytosolic extract and quantified with Bradford assay (Bio-Rad, Ref. 24Bradford M. Anal. Biochem. 1976; 72: 248-251Crossref PubMed Scopus (217544) Google Scholar). Equal amounts of protein were resolved on a 10% or 12% SDS-polyacrylamide gel electrophoresis. After electorphoresis, protein was transferred to a nitrocellulose membrane (Hybond-ECL, Amersham Pharmacia Biotech) or polyvinylidene difluoride membrane (Bio-Rad), immunoblotted with anti-IκB (Santa Cruz), and visualized by the ECL system (Amersham Pharmacia Biotech) using anti-rabbit-HRP IgG (Santa Cruz). The activity of JNK was assayed by a nonradioactive assay kit as per the manufacturer's protocol (New England Biolabs, Beverly, MA). Briefly, stress-activated protein kinase/JNK was precipitated from the cell lysate by c-Jun fusion protein bound to glutathione-Sepharose beads. c-Jun contains a high affinity binding site for stress-activated protein kinase/JNK, NH2-terminal to the two phosphorylation sites, Ser-63 and Ser-73. After selectively pulling down JNK using c-Jun fusion protein beads, the beads were extensively washed, and the kinase reaction was carried out in the presence of cold ATP in a final volume of 25 μl. The reaction was stopped with 25 μl of 2× SDS sample buffer and loaded onto a 10% polyacrylamide gel. Protein was transferred to nitrocellulose by electroblotting, and c-Jun phosphorylation was selectively measured using phospho-c-Jun antibody. This antibody specifically measures JNK-induced phosphorylation of c-Jun at Ser-63, a site important for the c-Jun-dependent transcriptional activity (20Davis R.J Biochem. Soc. Symp. 1998; 64: 1-12Google Scholar). p-TAL-NF-κB-luciferase reporter vector was obtained from CLONTECH Inc., Palo Alto, CA. A549 cells were transiently transfected with various expression plasmids by Transfectam (Promega) as per manufacturer's protocol. Cells were lysed 48 h post-transfection using reporter lysis buffer (Promega). After lysis, the lysates were centrifuged for 2 min at 21,000 × g in a microcentrifuge, and the supernatant was kept frozen at −80 °C. Luciferase activity was assayed with an automatic microplate luminometer using a luciferase assay kit (Promega) as per manufacturer's protocol (Promega). Data was normalized to protein concentration, and transfection efficiency was normalized with β-gal expression. β-Galactosidase expression was determined by a luminescence assay (Tropix) as per manufacturer's protocol and measured in an automatic microplate luminometer. Data were presented as fold induction. A549 cells were incubated with various amounts of E. coli thioredoxin as shown in Fig.1 A. After incubation, nuclear extract was prepared, and EMSA was performed using a consensus oligonucleotide for NF-κB. As demonstrated in Fig. 1 A, the oxidized form of E. coli thioredoxin did not activate NF-κB. On the contrary, incubation of cells with a thioredoxin-reducing system (thioredoxin, TR, and NADPH) activated NF-κB in a time-dependent manner. Maximal activation of NF-κB occurred at about 2 h in A549 cells (Fig. 1 A). In the dose-response study, oxidized thioredoxin at a concentration of 1–5 μm did not activate NF-κB. On the other hand, a thioredoxin-reducing system activated NF-κB in a dose-dependent manner (Fig. 1 B). Thioredoxin reductase or NADPH individually or in combination did not activate NF-κB, suggesting the activation of NF-κB by reduced Trx. NF-κB complex is formed by p65/p50 heterodimer, cRel/p50 heterodimer, or p50 homodimer. Thus, to determine the type of NF-κB complex that is formed by thioredoxin, we performed gel-supershift assay using anti-p50 or anti-p65 antibodies. As demonstrated in Fig. 2, the use of anti-p50 supershifted the NF-κB band, confirming a p50 subunit in the complex. The NF-κB band was abolished by the use of p65 antibody, indicating that the DNA contact is exclusively a function of p65 subunit. Use of both antibodies lessened the intensity of the band, suggesting inhibition of DNA binding. A549 cells are derived from pulmonary epithelial cells and are highly dedifferentiated. Hence, to demonstrate the activation of NF-κB by thioredoxin in primary cell cultures, we used pulmonary artery endothelial cells (HPAEC). HPAEC were cultured as described under “Experimental Procedures” and were incubated with oxidized or reduced thioredoxin in a similar manner as that described for the A549 cells. As demonstrated in Fig. 3, oxidized thioredoxin did not activate NF-κB in HPAEC. On the contrary, the reduced thioredoxin system induced NF-κB activation in a time-dependent manner. Maximal activation occurred at 3 h. Therefore, thioredoxin activated NF-κB in primary cells as well as in transformed cell lines. Thioredoxin has been shown to activate NF-κB in a cell-free system (14Hayashi T. Ueno Y. Okamoto T. J. Biol. Chem. 1993; 268: 11380-11388Abstract Full Text PDF PubMed Google Scholar). Activation of NF-κB by thioredoxin was also shown to occur after the dissociation of IκB complex form NF-κB. Recent studies have also demonstrated the activation of NF-κB-dependent reporter in MCF-7 cells stably expressing thioredoxin (25Freemerman A.J. Gallegos A. Powis G. Cancer Res. 1999; 59: 4090-4094PubMed Google Scholar). However, the mechanism of such activation has not been elucidated. In this study, we have shown that externally addedE. coli thioredoxin could activate NF-κB in intact cells. Thus, to demonstrate whether NF-κB is activated as a consequence of degradation of IκB by thioredoxin redox, we assayed for IκB in the cytoplasmic extracts. As shown in the Fig.4 A, IκB was degraded in the cytoplasm at 2 h when the A549 cells were incubated with a thioredoxin-reducing system (lane 6). Additionally, thioredoxin treatment caused IκB degradation in HPAEC after 3 h (Fig. 4 B, lane 7). These time points correlate to the EMSA studies, demonstrating that degradation of IκB and NF-κB DNA binding occurring simultaneously. Human thioredoxin is similar to E. coli thioredoxin in its redox function (8Holmgren A. Annu. Rev. Biochem. 1985; 54: 237-271Crossref PubMed Google Scholar). However, the human thioredoxin contains two catalytic cysteines at positions 32 and 35 and three other structural cysteines (8Holmgren A. Annu. Rev. Biochem. 1985; 54: 237-271Crossref PubMed Google Scholar). In addition, human thioredoxin is unable to enter the cell (26Gasdaska JR. Kirkpatrick D.L. Montfort W. Kuperus M. Hill S.R. Berggren M. Powis G. Biochem. Pharmacol. 1996; 52: 1741-1747Crossref PubMed Scopus (67) Google Scholar) unlike the bacterial thioredoxin. Thus, to investigate whether human thioredoxin is similar to E. coli thioredoxin in causing the activation of NF-κB, we overexpressed human thioredoxin by transfecting an expression vector containing Trx open reading frame (pcDNA3-Trx) as described under “Experimental Procedures.” Overexpression of thioredoxin induced the activation of NF-κB, as demonstrated in Fig. 5 A(lane 2). IκB was also degraded in response to Trx overexpression (Fig. 5 B, third lane). To further verify the role of redox-active cysteines of thioredoxin in NF-κB activation, we mutated the Cys-32 and Cys-35 by site-directed mutagenesis as described under “Experimental Procedures.” The redox-inactive thioredoxin was produced in a dominant-negative manner when such mutagenic cDNA was cloned to an overexpression vector (dnTrx) and transfected to A549 cells. Transfection of A549 cells with dnTrx did not activate NF-κB (Fig. 5 A, thirdlane) or degrade IκB (Fig. 5 B, secondlane), confirming that the redox activity of Trx is required for the activation of NF-κB (Fig. 5 B) in intact cells. Thioredoxin has been shown to activate PKC (27Biguet C. Wakasugi N. Mishal Z. Holmgren A. Chouaib S. Tursz T. Wakasugi H. J. Biol. Chem. 1994; 269: 28865-28870Abstract Full Text PDF PubMed Google Scholar). Recent reports also indicate that PKC can mediate NF-κB activation in a variety of cell types (28Anrather J. Csizmadia V. Soares M.P. Winkler H. J. Biol. Chem. 1999; 274: 13594-13604Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). Therefore, we hypothesized that PKC may mediate the NF-κB activation by thioredoxin. We incubated cells with specific inhibitors of PKC, calphostin C (29Tamaoki T. Nomoto H. Takahashi I. Kato Y. Morimoto M,. Tomita F. Biochem. Biophys. Res. Commun. 1992; 135: 397-402Crossref Scopus (2224) Google Scholar) or GF109203X (30Toullec D. Pianettis P. Coste H. Bellevergue P. Grand-Perret T. Ajakanes M. Baudet V. Boissin P. Boursier E. Loriolle F. Duhamel L. Charon D. Kirilovsky J. J. Biol. Chem. 1991; 266: 15478-15771Abstract Full Text PDF Google Scholar), and then stimulated the cells with E. coli thioredoxin-reducing system. In this experiment, we specifically sought to determine the degradation of IκB by thioredoxin and the inhibition of thioredoxin-mediated IκB degradation by PKC inhibitors. As demonstrated in Fig. 6 (lane 2), thioredoxin-reducing system degraded IκB. However, specific PKC inhibitors calphostin C or GF109203X did not prevent the IκB degradation by thioredoxin, indicating that the PKC pathway is not involved in the thioredoxin-mediated degradation of IκB and, hence, the activation of NF-κB. Since JNK pathway is activated by cytokines or UV radiation, which also activates NF-κB, we hypothesized that the external addition of E. coli thioredoxin may activate JNK, and such activation may mediate NF-κB activation. The cytosolic extracts of E. coli thioredoxin-treated cells were subjected to phospho-JNK detection using phospho-specific antibodies from Santa Cruz. As demonstrated in the Fig. 7 A (lanes 2–4), oxidized E. coli thioredoxin did not activate the JNK; however, the thioredoxin-reducing system activated JNK after 2 h of incubation (lane 6), a time point similar to the activation of NF-κB (Fig. 1 A). Although we detected phospho-JNK in thioredoxin-treated cells, there is reason to believe that total JNK protein may increase due to thioredoxin treatment, and the phospho-specific antibody may loose its specificity at higher JNK levels. To investigate these possibilities, we detected total JNK level by using a polyclonal antibody to JNK (Santa Cruz). As demonstrated in Fig. 7 B, there was no change in the total JNK levels, confirming the specific JNK phosphorylation in response to thioredoxin treatment. To determine the specificity of JNK activation by thioredoxin, we also determined the activation of extracellular signal-regulating kinase or p38 MAP kinases by thioredoxin using phospho-specific antibodies. We did not detect phospho-extracellular signal-regulating kinase or phospho-p38 in the thioredoxin-treated cells (data not shown). These results suggest that thioredoxin specifically activates JNK-signaling cascade. Since reducedE. coli thioredoxin activated JNK phosphorylation, we sought to determine whether the overexpression of human thioredoxin could also activate JNK. Moreover, since human thioredoxin is not permeable into the cells, overexpression of thioredoxin in the cell is an appropriate method to study the effect of human thioredoxin on JNK activity. We have demonstrated that only the redox-active thioredoxin is able to activate NF-κB. Hence, we also sought to determine the effect of redox-inactive thioredoxin on JNK activity. A549 cells were transfected with pcDNA3-Trx or pcDNA3-dnTrx for 24 h followed by cell lysis, and JNK activity assay was performed as described under “Experimental Procedures.” Redox-active thioredoxin potently activated JNK, as determined by its ability to phosphorylate c-Jun (Fig. 7 C, lane 3). On the other hand, redox-inactive mutant thioredoxin was unable to activate JNK (Fig.7 C, lane 4). Thus, the data suggest a redox-control of JNK activation by thioredoxin. If JNK activation is responsible for NF-κB activation by thioredoxin, then blocking the JNK activation by dominant-negative expression of JNK or dominant-negative expression of JNK kinase should inhibit NF-κB activation by thioredoxin. Therefore, to delineate the role of the JNK-signaling pathway in NF-κB activation by thioredoxin, we cotransfected pcDNA3-Trx and dnJNKK (MKK4/SEK1) or dnJNK expression plasmids into A549 cells. As demonstrated in Fig. 8 A, both dnJNKK and dnJNK inhibited NF-κB activation by thioredoxin. MEKK1 is an initiating kinase that activates the JNK cascade through phosphorylation of MKK4 or the p38 MAP kinase cascade by phosphorylating MKK3/MKK6 (19Ip Y.T. Davis R.J Curr. Opin. Cell Biol. 1998; 10: 205-219Crossref PubMed Scopus (1386) Google Scholar). Additionally, MEKK1 can directly phosphorylate IκBα kinase, which can cause the phosphorylation of IκBα and the activation of" @default.
• W2050503669 created "2016-06-24" @default.
• W2050503669 creator A5065370839 @default.
• W2050503669 date "2001-02-01" @default.
• W2050503669 modified "2023-09-30" @default.
• W2050503669 title "c-Jun NH2-terminal Kinase-mediated Redox-dependent Degradation of IκB" @default.
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