Hydrogen Peroxide Activates NF-κB through Tyrosine Phosphorylation of IκBα and Serine Phosphorylation of p65

Although it is well established that reactive oxygen intermediates mediate the NF-κB activation induced by most agents, how H2O2 activates this transcription factor is not well understood. We found that treatment of human myeloid KBM-5 cells with H2O2 activated NF-κB in a dose- and time-dependent manner much as tumor necrosis factor (TNF) did but unlike TNF, H2O2 had no effect on IκBα degradation. Unexpectedly, however, like TNF-induced activation, H2O2-induced NF-κB activation was blocked by the calpain inhibitor N-Ac-Leu-Leu-norleucinal, suggesting that a proteosomal pathway was involved. Although H2O2 activated IκBα kinase, it did not induce the serine phosphorylation of IκBα. Like TNF, H2O2 induced the serine phosphorylation of the p65 subunit of NF-κB, leading to its nuclear translocation. We found that H2O2 induced the tyrosine phosphorylation of IκBα, which is needed for NF-κB activation. We present several lines of evidence to suggest that the Syk protein-tyrosine kinase is involved in H2O2-induced NF-κB activation. First, H2O2 activated Syk in KBM-5 cells; second, H2O2 failed to activate NF-κB in cells that do not express Syk protein; third, overexpression of Syk increased H2O2-induced NF-κB activation; and fourth, reduction of Syk transcription using small interfering RNA inhibited H2O2-induced NF-κB activation. We also showed that Syk induced the tyrosine phosphorylation of IκBα, which caused the dissociation, phosphorylation, and nuclear translocation of p65. Thus, overall, our results demonstrate that H2O2 induces NF-κB activation, not through serine phosphorylation or degradation of IκBα, but through Syk-mediated tyrosine phosphorylation of IκBα Although it is well established that reactive oxygen intermediates mediate the NF-κB activation induced by most agents, how H2O2 activates this transcription factor is not well understood. We found that treatment of human myeloid KBM-5 cells with H2O2 activated NF-κB in a dose- and time-dependent manner much as tumor necrosis factor (TNF) did but unlike TNF, H2O2 had no effect on IκBα degradation. Unexpectedly, however, like TNF-induced activation, H2O2-induced NF-κB activation was blocked by the calpain inhibitor N-Ac-Leu-Leu-norleucinal, suggesting that a proteosomal pathway was involved. Although H2O2 activated IκBα kinase, it did not induce the serine phosphorylation of IκBα. Like TNF, H2O2 induced the serine phosphorylation of the p65 subunit of NF-κB, leading to its nuclear translocation. We found that H2O2 induced the tyrosine phosphorylation of IκBα, which is needed for NF-κB activation. We present several lines of evidence to suggest that the Syk protein-tyrosine kinase is involved in H2O2-induced NF-κB activation. First, H2O2 activated Syk in KBM-5 cells; second, H2O2 failed to activate NF-κB in cells that do not express Syk protein; third, overexpression of Syk increased H2O2-induced NF-κB activation; and fourth, reduction of Syk transcription using small interfering RNA inhibited H2O2-induced NF-κB activation. We also showed that Syk induced the tyrosine phosphorylation of IκBα, which caused the dissociation, phosphorylation, and nuclear translocation of p65. Thus, overall, our results demonstrate that H2O2 induces NF-κB activation, not through serine phosphorylation or degradation of IκBα, but through Syk-mediated tyrosine phosphorylation of IκBα Nuclear factor-κB (NF-κB) 1The abbreviations used are: NF-κB, nuclear factor kappa B; IκB, inhibitory subunit of NF-κB; IKK, IκBα kinase; TNF, tumor necrosis factor; Syk, spleen tyrosine kinase; p56lck, lymphocyte-specific protein-tyrosine kinase; EMSA, electrophoretic mobility shift assay; ALLN, N-acetyl-leucyl-leucyl-norleucinal; siRNA, small interfering RNA. is a transcription factor consisting of a group of five proteins, namely c-Rel, RelA (p65), Rel B, NF-κB1 (p50 and p105), and NF-κB2 (p52) (1Ghosh S. Karin M. Cell. 2002; 109: S81-S96Google Scholar). In the resting state, NF-κB is sequestered in the cytoplasm through its tight association with specific inhibitory proteins, called inhibitors of NF-κB (IκB), belonging to a gene family consisting of IκBα, IκBβ, IκBϵ, IκBγ, Bcl-3, p100, and p105 (1Ghosh S. Karin M. Cell. 2002; 109: S81-S96Google Scholar). On activation by agents such as TNF, IκBα is phosphorylated at serine residues 32 and 36, ubiquitinated at lysine residues 21 and 22, and degraded through the proteosomal pathway, thus exposing the nuclear localization signals on the p50-p65 heterodimer. Then p65 undergoes phosphorylation, leading to nuclear translocation and binding to a specific sequence in DNA, which in turn results in gene transcription. The phosphorylation of IκBα is catalyzed by IκBα kinase (IKK), which consists of IKK-α, IKK-β, and IKK-γ (also called NF-κB essential modulator (NEMO)) (1Ghosh S. Karin M. Cell. 2002; 109: S81-S96Google Scholar). Gene deletion studies have established that IKK-β is essential for NF-κB activation by TNF (2Li Z.W. Chu W. Hu Y. Delhase M. Deerinck T. Ellisman M. Johnson R. Karin M. J. Exp. Med. 1999; 189: 1839-1845Google Scholar, 3Li Q. Estepa G. Memet S. Israel A. Verma I.M. Genes Dev. 2000; 14: 1729-1733Crossref Google Scholar, 4Li Q. Antwerp D.V. Mercurio F. Lee K.F. Verma I.M. Science. 1999; 284: 321-325Google Scholar). IKK-α deletion, however, has no effect on NF-κB activation by most agents. Which kinase induces the phosphorylation of p65 is controversial, but protein kinase A, casein kinase II, IKK-α, and IKK-β have all been implicated (5Hayashi T. Sekine T. Okamoto T. J. Biol. Chem. 1993; 268: 26790-26795Google Scholar, 6Zhong H. SuYang H. Erdjument-Bromage H. Tempst P. Ghosh S. Cell. 1997; 89: 413-424Google Scholar, 7Zhong H. Voll R.E. Ghosh S. Mol. Cell. 1998; 1: 661-671Google Scholar, 8Wang D. Westerheide S.D. Hanson J.L. Baldwin Jr., A.S. J. Biol. Chem. 2000; 275: 32592-32597Google Scholar, 9Sakurai H. Chiba H. Miyoshi H. Sugita T. Toriumi W. J. Biol. Chem. 1999; 274: 30353-30356Google Scholar, 10Sizemore N. Lerner N. Dombrowski N. Sakurai H. Stark G.R. J. Biol. Chem. 2002; 277: 3863-3869Google Scholar). The phosphorylation of p65 at serine 529 has been shown to be required for the TNF-induced transcriptional activity of NF-κB (11Wang D. Baldwin Jr., A.S. J. Biol. Chem. 1998; 273: 29411-29416Google Scholar). NF-κB is activated by a wide variety of agents, including all 18 members of the TNF superfamily, interleukin-1, interleukin-17, interleukin-18, lipopolysaccharide, H2O2, ceramide, phorbol esters, growth factors, UV, X-rays, and γ-radiation (12Garg A. Aggarwal B.B. Leukemia. 2002; 16: 1053-1068Google Scholar). Whether all these agents activate NF-κB through the same pathway as described above is not clear. Certain agents activate NF-κB not through serine phosphorylation but through tyrosine phosphorylation of IκBα: nerve growth factor, erythropoietin, pervanadate, hypoxia, and silica (13Bui N.T. Livolsi A. Peyron J.F. Prehn J.H. J. Cell Biol. 2001; 152: 753-764Google Scholar, 14Digicaylioglu M. Lipton S.A. Nature. 2001; 412: 641-647Google Scholar, 15Imbert V. Rupec R.A. Livolsi A. Pahl H.L. Traenckner E.B. Mueller-Dieckmann C. Farahifar D. Rossi B. Auberger P. Baeuerle P.A. Peyron J.F. Cell. 1996; 86: 787-798Google Scholar, 16Singh S. Darnay B.G. Aggarwal B.B. J. Biol. Chem. 1996; 271: 31049-31054Google Scholar, 17Kang J.L. Pack I.S. Hong S.M. Lee H.S. Castranova V. Toxicol. Appl. Pharmacol. 2000; 169: 59-65Google Scholar, 18Koong A.C. Chen E.Y. Giaccia A.J. Cancer Res. 1994; 54: 1425-1430Google Scholar). The tyrosine phosphorylation of IκBα by most agents does not lead to IκBα degradation. Pervanadate-induced NF-κB activation, however, leads to tyrosine phosphorylation and degradation of IκBα (19Mukhopadhyay A. Manna S.K. Aggarwal B.B. J. Biol. Chem. 2000; 275: 8549-8555Google Scholar). Surprisingly, UV-C-induced NF-κB activation is mediated through the degradation of IκBα that involves phosphorylation of neither serine nor the tyrosine residue of IκBα (20Li N. Karin M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13012-13017Google Scholar). Pervanadate-induced tyrosine phosphorylation of IκBα blocks the TNF-induced serine phosphorylation of IκBα and NF-κB activation (16Singh S. Darnay B.G. Aggarwal B.B. J. Biol. Chem. 1996; 271: 31049-31054Google Scholar, 21Singh S. Aggarwal B.B. J. Biol. Chem. 1995; 270: 10631-10639Google Scholar), indicating potential stereochemical hindrance. NF-κB activation of most agents has been shown to require the generation of reactive oxygen intermediates, in studies that used either reactive oxygen intermediate quenchers, such as N-acetylcysteine, or antioxidant enzymes, such as glutathione peroxidase, superoxide dismutase, γ-glutamylcysteine synthetase, and thioredoxin (22Staal F.J. Roederer M. Herzenberg L.A. Herzenberg L.A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9943-9947Google Scholar, 23Kretz-Remy C. Mehlen P. Mirault M.E. Arrigo A.P. J. Cell Biol. 1996; 133: 1083-1093Google Scholar, 24Schreck R. Rieber P. Baeuerle P.A. EMBO J. 1991; 10: 2247-2258Google Scholar, 25Giri D.K. Aggarwal B.B. J. Biol. Chem. 1998; 273: 14008-14014Google Scholar, 26Manna S.K. Zhang H.J. Yan T. Oberley L.W. Aggarwal B.B. J. Biol. Chem. 1998; 273: 13245-13254Google Scholar, 27Manna S.K. Kuo M.T. Aggarwal B.B. Oncogene. 1999; 18: 4371-4382Google Scholar, 28Shrivastava A. Aggarwal B.B. Antioxid Redox Signal. 1999; 1: 181-191Google Scholar, 29Matthews J.R. Wakasugi N. Virelizier J.L. Yodoi J. Hay R.T. Nucleic Acids Res. 1992; 20: 3821-3830Google Scholar). Additionally, there are reports that H2O2 activates NF-κB (23Kretz-Remy C. Mehlen P. Mirault M.E. Arrigo A.P. J. Cell Biol. 1996; 133: 1083-1093Google Scholar, 24Schreck R. Rieber P. Baeuerle P.A. EMBO J. 1991; 10: 2247-2258Google Scholar, 30Meyer M. Schreck R. Baeuerle P.A. EMBO J. 1993; 12: 2005-2015Google Scholar). Although it has been shown that H2O2-induced NF-κB is blocked by N-acetylcysteine (24Schreck R. Rieber P. Baeuerle P.A. EMBO J. 1991; 10: 2247-2258Google Scholar), how H2O2 activates NF-κB is not fully understood (31Livolsi A. Busuttil V. Imbert V. Abraham R.T. Peyron J.F. Eur. J. Biochem. 2001; 268: 1508-1515Google Scholar, 32Schoonbroodt S. Ferreira V. Best-Belpomme M. Boelaert J.R. J. Immunol. 2000; 164: Scholar, H. T. S. H. 2002; Scholar). In the present the of H2O2-induced NF-κB activation. We found that H2O2-induced NF-κB activation degradation of IκBα. H2O2 activated Syk protein-tyrosine kinase, which in turn induced tyrosine phosphorylation of IκBα, leading to NF-κB activation. human to with a specific activity of was by and H2O2 was and was Cell and by and that the serine of p65 was as described M.T. M. A.J. Nature. 2000; Scholar), was by C. D. of was used for of of human Syk or human Syk and to which the and of The sequence used was are in are in and is Cell KBM-5 is myeloid with The lines and human cells human cells and cells the and Cell cells with the gene by of The of these cells has been A. Cell. 1992; Scholar). KBM-5 cells in with and cells in with and NF-κB activation, as described A. Aggarwal B.B. 2000; Scholar). nuclear cells with NF-κB of protein with of the human NF-κB binding for at and the was on was used to the of binding of NF-κB to the The of binding was also by with the nuclear cells with either or p65 of NF-κB for at the was by and as The and by a using the of protein in cytoplasm or nuclear S. Aggarwal B.B. J. Immunol. 2001; cells and by the to with and by The of the was using a and IκBα was by a described S.K. A. Aggarwal B.B. J. Immunol. 2000; Scholar). cytoplasm was with IKK-α, by treatment with protein a the with and in kinase of and of the was at for the was by with for the protein was on the was and the by the of IKK-α and IKK-β in of the protein was on to a and with either or Syk the activity of protein-tyrosine kinase Syk induced by H2O2, with and the in kinase using the as the cells with A.C. N. S. Aggarwal B.B. for with H2O2 for and in the and The kinase protein was using by protein the with the and in a kinase and of and at the with for to and the and by a using The tyrosine phosphorylation of IκBα by Syk was also by using in the kinase as described and using Nuclear of p65 NF-κB by effect of H2O2 on the nuclear translocation of p65 was by the as described A. C. M. 1992; Scholar). cells on a by using a with and with of in blocked with for and with p65 or IκBα at at the with at for and for with for with and a using and for on in and of in was with of was using protein for at with and in for and in and the of Syk protein-tyrosine kinase in the H2O2-induced NF-κB activation, cells either with small interfering or with of in was in of and and with of was to the cells and for was and for cells used to the of Syk protein by NF-κB activity by EMSA, and Syk activity by the kinase as described In this the effect of H2O2 on NF-κB activation, IκBα phosphorylation, IκBα p65 phosphorylation, and nuclear and the of protein-tyrosine kinase Syk in H2O2-induced NF-κB activation. NF-κB activation by TNF is well used TNF as a for most by and the treatment of cells with H2O2 for had no effect on was TNF of NF-κB in a and the effect of H2O2 on the activation of NF-κB, KBM-5 cells with of H2O2 for or TNF for Nuclear for of NF-κB activation by H2O2 and TNF induced NF-κB activation in a manner in KBM-5 cells activation with H2O2 at NF-κB was activated by agents in a time-dependent manner TNF induced NF-κB activation and activation for H2O2-induced NF-κB activation at at and at The TNF-induced NF-κB activation was with H2O2 it was Thus, H2O2 induced NF-κB activation did The and in the of activation suggest that the of NF-κB activation by H2O2 is that of NF-κB is a of proteins, of protein NF-κB that to a specific sequence in (1Ghosh S. Karin M. Cell. 2002; 109: S81-S96Google Scholar). that the by in cells was NF-κB, nuclear cells with to either the or the p65 subunit of the to a thus suggesting that the of and p65 nor had NF-κB caused of the and a of NF-κB did not NF-κB binding NF-κB TNF-induced NF-κB activation the degradation of IκBα S. Karin M. Cell. 2002; 109: S81-S96Google Scholar). Whether H2O2-induced NF-κB activation is also mediated through IκBα degradation was cells with TNF or H2O2 for the the and for IκBα on using TNF induced IκBα degradation and IκBα was at H2O2 did not induce IκBα degradation at TNF but induces phosphorylation of 32 and of IκBα S. Karin M. Cell. 2002; 109: S81-S96Google Scholar). H2O2 induces serine phosphorylation of IκBα. the phosphorylated IκBα, blocked degradation of IκBα using the inhibitor A.C. N. S. Aggarwal B.B. Scholar). using showed that TNF induced the phosphorylation of IκBα but H2O2 had no effect on the serine phosphorylation of IκBα. NF-κB by TNF and blocked not TNF-induced NF-κB activation, but also H2O2-induced NF-κB activation IκBα phosphorylation and degradation are in TNF-induced NF-κB activation. and of the effect of TNF and H2O2 on translocation and phosphorylation of p65 by TNF and H2O2 induced nuclear translocation of p65 in a time-dependent On TNF p65 nuclear translocation TNF treatment and In the of H2O2, p65 translocation was induced at and TNF and H2O2 induced the phosphorylation of p65 in a time-dependent but the of H2O2-induced phosphorylation of p65 was that for TNF showed that in p65 was in the TNF and H2O2 induced translocation of p65 the H2O2-induced phosphorylation and translocation of p65 to the with TNF-induced IκBα our results that H2O2-induced NF-κB activation is not mediated through the phosphorylation and degradation of IκBα, H2O2 activate has been shown that is required not for TNF-induced phosphorylation of IκBα but also for the phosphorylation of p65 H. Chiba H. Miyoshi H. Sugita T. Toriumi W. J. Biol. Chem. 1999; 274: 30353-30356Google Scholar, 10Sizemore N. Lerner N. Dombrowski N. Sakurai H. Stark G.R. J. Biol. Chem. 2002; 277: 3863-3869Google Scholar). in kinase using as the showed that TNF and H2O2 activated as as TNF but activation H2O2-induced activation however, that induced by TNF nor H2O2 had effect on the of either IKK-α or IKK-β results suggest that H2O2 activated but had no effect on the serine phosphorylation of IκBα. NF-κB in the of Syk in H2O2-induced NF-κB activation, used cells to A. Cell. 1992; and Syk protein S. S. T. Cell 1998; 10: Scholar). that cells Syk protein but cells or no Syk The was specific as it did not protein in cells TNF activated NF-κB in and cells and induced IκBα degradation in the lines The of TNF-induced NF-κB activation, however, was in cells in cells and the of activation was also In H2O2 activated NF-κB in cells but not in cells indicating essential of Syk protein in H2O2-induced activation. We also the of H2O2 to activate NF-κB in human and which be activated for Syk and cells with H2O2 for the nuclear and for NF-κB activation by for Syk activation. We found that H2O2 failed to activate Syk and this with the of activation of NF-κB in and Whether H2O2 activate NF-κB in cells was this cells for the with H2O2 and for NF-κB activation by and IκBα degradation by shown in H2O2 induced NF-κB activation in cells and this was with the degradation and of IκBα. results with a S. Ferreira V. Best-Belpomme M. Boelaert J.R. J. Immunol. 2000; 164: but that in human NF-κB in cells have been shown to and Syk protein-tyrosine A. Cell. 1992; Scholar). have shown that is required for and NF-κB activation S.K. Aggarwal B.B. J. Biol. Chem. 2000; 275: Scholar, S.K. Aggarwal B.B. J. Immunol. 2000; 164: Scholar). of cells had been that H2O2 activated NF-κB in cells but not in cells had been IκBα phosphorylation was in either of the lines In was not required for H2O2-induced NF-κB activation or for IκBα of Syk in but in and cells with H2O2 for the with and to using H2O2-induced tyrosine phosphorylation of Syk in but not in cells of IκBα in our and have shown that agents activate NF-κB through tyrosine phosphorylation it does not lead to the degradation of IκBα (13Bui N.T. Livolsi A. Peyron J.F. Prehn J.H. J. Cell Biol. 2001; 152: 753-764Google Scholar, 14Digicaylioglu M. Lipton S.A. Nature. 2001; 412: 641-647Google Scholar, 15Imbert V. Rupec R.A. Livolsi A. Pahl H.L. Traenckner E.B. Mueller-Dieckmann C. Farahifar D. Rossi B. Auberger P. Baeuerle P.A. Peyron J.F. Cell. 1996; 86: 787-798Google Scholar, 16Singh S. Darnay B.G. Aggarwal B.B. J. Biol. Chem. 1996; 271: 31049-31054Google Scholar, 17Kang J.L. Pack I.S. Hong S.M. Lee H.S. Castranova V. Toxicol. Appl. Pharmacol. 2000; 169: 59-65Google Scholar, 18Koong A.C. Chen E.Y. Giaccia A.J. Cancer Res. 1994; 54: 1425-1430Google Scholar). H2O2 did not induce IκBα H2O2 induces tyrosine phosphorylation of IκBα. cells with H2O2 tyrosine phosphorylation of IκBα is the of of cells with tyrosine phosphorylation of IκBα results suggest that H2O2 induced tyrosine phosphorylation of IκBα by of Syk tyrosine Syk of the of Syk-mediated phosphorylation of IκBα, the in kinase using as the We found that Syk IκBα, and this phosphorylation was by treatment the Syk-mediated tyrosine phosphorylation of IκBα, the in kinase using as the and The IκBα was by using and results suggest that Syk protein-tyrosine kinase IκBα on tyrosine of IκBα p65 serine phosphorylation leads to the degradation of IκBα, tyrosine phosphorylation does not (13Bui N.T. Livolsi A. Peyron J.F. Prehn J.H. J. Cell Biol. 2001; 152: 753-764Google Scholar, 14Digicaylioglu M. Lipton S.A. Nature. 2001; 412: 641-647Google Scholar, 15Imbert V. Rupec R.A. Livolsi A. Pahl H.L. Traenckner E.B. Mueller-Dieckmann C. Farahifar D. Rossi B. Auberger P. Baeuerle P.A. Peyron J.F. Cell. 1996; 86: 787-798Google Scholar, 16Singh S. Darnay B.G. Aggarwal B.B. J. Biol. Chem. 1996; 271: 31049-31054Google Scholar, 17Kang J.L. Pack I.S. Hong S.M. Lee H.S. Castranova V. Toxicol. Appl. Pharmacol. 2000; 169: 59-65Google Scholar, 18Koong A.C. Chen E.Y. Giaccia A.J. Cancer Res. 1994; 54: 1425-1430Google Scholar). Whether H2O2-induced tyrosine phosphorylation of IκBα was for the of p65 was cells with TNF or with H2O2 and for IκBα by TNF induced the degradation of IκBα in the and the translocation of p65 to the TNF also induced the of p65 IκBα In the p65 was the degradation of IκBα We to that H2O2 did not induce of p65 IκBα H2O2 induced a of IκBα and IκBα be in neither nor p65 showed that p65 and IκBα in the of cells The cells with TNF IκBα in the and p65 was to the in these Although H2O2 induced p65 translocation the IκBα in the results suggest that H2O2 induced translocation of p65 of Syk H2O2-induced NF-κB and of the of Syk protein-tyrosine kinase in H2O2-induced NF-κB activation, the cells with the Syk shown in these cells showed in of Syk protein H2O2-induced NF-κB activation and in the IκBα tyrosine phosphorylation the these results suggest that Syk protein-tyrosine kinase mediate H2O2-induced NF-κB through the tyrosine phosphorylation of IκBα. of Syk by H2O2-induced NF-κB and of also used to Syk T. 2002; 20: Scholar). and H2O2-induced NF-κB activation and IκBα tyrosine shown in the of Syk protein H2O2-induced NF-κB activation and tyrosine phosphorylation of IκBα as with the results suggest that Syk in NF-κB activation induced by using inhibitors and antioxidant enzymes, it has been well established that oxygen are involved in NF-κB activation by most agents. Additionally, that H2O2 activate NF-κB has been for a but the has We demonstrate in the present that H2O2 activates NF-κB the IκBα and its serine it induced tyrosine phosphorylation of activated IKK-α and Syk kinase, and this activation of Syk kinase to be for tyrosine phosphorylation of IκBα and for NF-κB activation. TNF and H2O2 activated NF-κB, but the of NF-κB activation was with TNF that with results in that the of NF-κB activation by TNF are that of Although reactive oxygen intermediates has been implicated in TNF-induced NF-κB activation R. B. W. Baeuerle P.A. J. Exp. Med. 1992; Scholar, H.J. B. J. Biol. Chem. 2001; Scholar), this not be for NF-κB activation. The in also be to the of TNF-induced activation H2O2 activity is We have in that the of NF-κB activation by H2O2 is that of TNF induced IκBα degradation but H2O2 did agents are also to activate NF-κB IκBα including pervanadate, hypoxia, erythropoietin, and nerve growth factor (13Bui N.T. Livolsi A. Peyron J.F. Prehn J.H. J. Cell Biol. 2001; 152: 753-764Google Scholar, 14Digicaylioglu M. Lipton S.A. Nature. 2001; 412: 641-647Google Scholar, 15Imbert V. Rupec R.A. Livolsi A. Pahl H.L. Traenckner E.B. Mueller-Dieckmann C. Farahifar D. Rossi B. Auberger P. Baeuerle P.A. Peyron J.F. Cell. 1996; 86: 787-798Google Scholar, 16Singh S. Darnay B.G. Aggarwal B.B. J. Biol. Chem. 1996; 271: 31049-31054Google Scholar, 17Kang J.L. Pack I.S. Hong S.M. Lee H.S. Castranova V. Toxicol. Appl. Pharmacol. 2000; 169: 59-65Google Scholar, 18Koong A.C. Chen E.Y. Giaccia A.J. Cancer Res. 1994; 54: 1425-1430Google Scholar). results with F. A. Biol. Med. 2001; and S.A. Biochem. 1998; but S. Ferreira V. Best-Belpomme M. Boelaert J.R. J. Immunol. 2000; 164: Scholar), showed that H2O2 induces IκBα degradation in Whether these are to is not clear. We used lines and myeloid cells and found results also suggest unlike TNF, H2O2 does not induce serine phosphorylation of IκBα. S. Ferreira V. Best-Belpomme M. Boelaert J.R. J. Immunol. 2000; 164: also showed that phosphorylation of serine 32 and of IκBα is not required for NF-κB activation by was the for TNF, H2O2-induced NF-κB activation was by ALLN, a proteosomal H2O2-induced NF-κB activation is not clear. The has been implicated in the degradation of IκBα and in the of p105 and is that H2O2-induced NF-κB activation through the of degradation of IκBα or of NF-κB The inhibitors and have also been shown to H2O2-induced NF-κB activation IκBα, or in cells 1998; Scholar). results suggest the of factor in H2O2-induced NF-κB activation. results also demonstrate that H2O2 did not induce serine phosphorylation of IκBα, it did activate IKK, phosphorylated p65 at serine residue and p65 to the it was that p65 H. Chiba H. Miyoshi H. Sugita T. Toriumi W. J. Biol. Chem. 1999; 274: 30353-30356Google Scholar, 10Sizemore N. Lerner N. Dombrowski N. Sakurai H. Stark G.R. J. Biol. Chem. 2002; 277: 3863-3869Google Scholar). it is that H2O2-induced is needed not for the phosphorylation of IκBα but for the phosphorylation of p65. results are in with a by H. T. S. H. 2002; Scholar), showed that H2O2 activate and this activation is essential for NF-κB activation. H2O2-induced activates NF-κB, however, was not by these results S. Ferreira V. Best-Belpomme M. Boelaert J.R. J. Immunol. 2000; 164: Scholar), showed of activation of by results that H2O2 induces tyrosine phosphorylation of IκBα. results are in with reports by Livolsi (31Livolsi A. Busuttil V. Imbert V. Abraham R.T. Peyron J.F. Eur. J. Biochem. 2001; 268: 1508-1515Google and S. Ferreira V. Best-Belpomme M. Boelaert J.R. J. Immunol. 2000; 164: Scholar). kinase induces the tyrosine phosphorylation of IκBα, however, is not clear. results evidence that Syk protein kinase a in this First, H2O2 activated Syk in KBM-5 cells; H2O2 failed to activate NF-κB in cells that do not express Syk protein; third, overexpression of Syk increased H2O2-induced NF-κB activation; and fourth, reduction of Syk transcription using inhibited H2O2-induced NF-κB activation. We also showed that Syk induced the tyrosine phosphorylation of IκBα, which caused the dissociation, phosphorylation, and nuclear translocation of p65. Lee R.A. J. Biol. Chem. 2002; 277: showed that H2O2-induced phosphorylation of IκBα through in H2O2 has been shown to activate Syk H. Biochem. Res. 1998; Scholar), a kinase to be in and and We found that cells that do not express Syk, such as of failed to activate NF-κB on treatment with V. J. Biol. Chem. 1998; 273: showed that in human cells or human TNF activate NF-κB but H2O2 The of H2O2 to activate NF-κB in these cells have been of a of of Syk kinase in these it has been shown that H2O2 activate Proc. Natl. Acad. Sci. U. S. A. 1995; Scholar). Although has been implicated in the NF-κB activation by and by S.K. Aggarwal B.B. J. Biol. Chem. 2000; 275: Scholar, S.K. Aggarwal B.B. J. Immunol. 2000; 164: Scholar), our results that H2O2 does not induce NF-κB activation through activation of as cells did not to H2O2 for NF-κB activation. We found that in all the of IκBα was degraded and the p65 was to the In in cells IκBα was phosphorylated but not to p65 in the a of the p65 was to the results thus suggest that H2O2-induced NF-κB activation through a that of In our that H2O2 induces a Syk kinase, which in turn induces tyrosine phosphorylation of IκBα. IκBα induces the p65 NF-κB leading to p65 phosphorylation and nuclear translocation.