Iron Causes Interactions of TAK1, p21ras, and Phosphatidylinositol 3-Kinase in Caveolae to Activate IκB Kinase in Hepatic Macrophages

Abstract

We recently discovered a novel signaling phenomenon involving a rapid and transient rise in intracellular low molecular weight iron complex(es) in activation of IκB kinase (IKK) in hepatic macrophages. We also showed direct treatment with ferrous iron substitutes for this event to activate IKK. The present study used this model to identify upstream kinases responsible for IKK activation. IKK activation induced by iron is abrogated by overexpression of a dominant negative mutant (DN) for transforming growth factor β-activated kinase-1 (TAK1), NF-κB-inducing kinase, or phosphatidylinositol 3-kinase (PI3K) and by treatment with the mitogen-activated protein kinase (MAPK) kinase-1 (MEK1) inhibitor. Iron increases AKT phosphorylation that is prevented by DNTAK1 or DNp21ras. Iron causes ERK1/2 phosphorylation that is attenuated by DN-PI3K, prevented by DNp21ras, but unaffected by DNTAK1. Iron-induced TAK1 activity is not affected by the PI3K or MEK1 inhibitor, suggesting TAK1 is upstream of PI3K and MEK1. Iron increases interactions of TAK1 and PI3K with p21ras as demonstrated by co-immunoprecipitation and co-localization of these proteins with caveolin-1 as shown by immunofluorescent microscopy. Finally, filipin III, a caveolae inhibitor, abrogates iron-induced TAK1 and IKK activation. In conclusion, MEK1, TAK1, NF-κ-inducing kinase, and PI3K are required for iron-induced IKK activation in hepatic macrophages and TAK1, PI3K, and p21ras physically interact in caveolae to initiate signal transduction. We recently discovered a novel signaling phenomenon involving a rapid and transient rise in intracellular low molecular weight iron complex(es) in activation of IκB kinase (IKK) in hepatic macrophages. We also showed direct treatment with ferrous iron substitutes for this event to activate IKK. The present study used this model to identify upstream kinases responsible for IKK activation. IKK activation induced by iron is abrogated by overexpression of a dominant negative mutant (DN) for transforming growth factor β-activated kinase-1 (TAK1), NF-κB-inducing kinase, or phosphatidylinositol 3-kinase (PI3K) and by treatment with the mitogen-activated protein kinase (MAPK) kinase-1 (MEK1) inhibitor. Iron increases AKT phosphorylation that is prevented by DNTAK1 or DNp21ras. Iron causes ERK1/2 phosphorylation that is attenuated by DN-PI3K, prevented by DNp21ras, but unaffected by DNTAK1. Iron-induced TAK1 activity is not affected by the PI3K or MEK1 inhibitor, suggesting TAK1 is upstream of PI3K and MEK1. Iron increases interactions of TAK1 and PI3K with p21ras as demonstrated by co-immunoprecipitation and co-localization of these proteins with caveolin-1 as shown by immunofluorescent microscopy. Finally, filipin III, a caveolae inhibitor, abrogates iron-induced TAK1 and IKK activation. In conclusion, MEK1, TAK1, NF-κ-inducing kinase, and PI3K are required for iron-induced IKK activation in hepatic macrophages and TAK1, PI3K, and p21ras physically interact in caveolae to initiate signal transduction. Iron accumulation often accompanies chronic inflammatory diseases such as arthrosclerosis (1Yuan X.M. Free Radic. Res. 1999; 30: 221-231Crossref PubMed Scopus (59) Google Scholar), pulmonary fibrosis (2Kennedy J.I. Chandler D.B. Jackson R.M. Fulmer J.D. Chest. 1986; 89: 123S-125SAbstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar), Parkinson disease (3Graham J.M. Paley M.N. Grunewald R.A. Hoggard N. Griffiths P.D. Brain. 2000; 123 (Pt. 12): 2423-2431Crossref PubMed Scopus (207) Google Scholar), and chronic liver disease (4Tsukamoto H. Lin M. Ohata M. Giulivi C. French S.W. Brittenham G. Am. J. Physiol. 1999; 277: G1240-G1250PubMed Google Scholar, 5Bonkovsky H.L. Troy N. McNeal K. Banner B.F. Sharma A. Obando J. Mehta S. Koff R.S. Liu Q. Hsieh C.C. J. Hepatol. 2002; 37: 848-854Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 6Uraz S. Aygun C. Sonsuz A. Ozbay G. Dig. Dis. Sci. 2005; 50: 964-969Crossref PubMed Scopus (19) Google Scholar). Mechanisms by which inflammation leads to increased iron storage have been enlightened by the recent revelation of induction of hepcidin by inflammatory cytokines (7Lee P. Peng H. Gelbart T. Wang L. Beutler E. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 1906-1910Crossref PubMed Scopus (452) Google Scholar) and the ability of this protein to bind to ferroportin for its internalization and degradation and consequently to reduce export of cellular iron (8Nemeth E. Tuttle M.S. Powelson J. Vaughn M.B. Donovan A. Ward D.M. Ganz T. Kaplan J. Science. 2004; 306: 2090-2093Crossref PubMed Scopus (3691) Google Scholar). Conversely, iron loading causes chronic tissue damage and inflammation. The most prevalent hypothesis for this causal link has been that the iron-catalyzed Fenton pathway generates a hydroxyl radical and causes oxidative tissue injury. However, emerging evidence indicates that the effects of iron may be more complex and intricate than simple oxidant injury and involve molecular mechanisms by which expression of proinflammatory cytokines is induced by enhanced iron-dependent signaling. To this end, we have demonstrated a novel signaling event of a transient rise in intracellular low molecular weightiron complex(es) in 2 min after the treatment of cultured hepatic macrophages (HM) 2The abbreviations used are: HM, hepatic macrophage; TAK1, transforming growth factor β-activated kinase-1; NIK, NF-κB-inducing kinase; IKK, IκB kinase; TNF, tumor necrosis factor; MAPK, mitogen-activating protein kinase; PI3K, phosphatidylinositol 3-kinase; AKT, protein kinase B; ERK, extracellular signal-regulated kinase; LPS, lipopolysaccharide; DN, dominant negative; m.o.i., multiplicity of infection; JNK, c-Jun N-terminal kinase; GST, glutathione S-transferase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase. with lipopolysaccharide (LPS) or tumor necrosis factor-α (TNFα) (9Xiong S. She H. Takeuchi H. Han B. Engelhardt J.F. Barton C.H. Zandi E. Giulivi C. Tsukamoto H. J. Biol. Chem. 2003; 278: 17646-17654Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). This unique event precedes and is required for activation of IκB kinase (IKK) and NF-κB (9Xiong S. She H. Takeuchi H. Han B. Engelhardt J.F. Barton C.H. Zandi E. Giulivi C. Tsukamoto H. J. Biol. Chem. 2003; 278: 17646-17654Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). Furthermore, an increase in intracellular non-heme iron store caused by a mutation in natural resistance-associated macrophage protein-1 results in coordinated accentuation of the low molecular weight-iron complex(es) response, IKK and NF-κB activation, and TNFα release in LPS-stimulated RAW264.7 cells (9Xiong S. She H. Takeuchi H. Han B. Engelhardt J.F. Barton C.H. Zandi E. Giulivi C. Tsukamoto H. J. Biol. Chem. 2003; 278: 17646-17654Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). Interestingly, the direct treatment with iron causes within 2 min an immediate and maximal quenching of RAW264.7 cells loaded with an iron-sensitive fluorescent probe, suggesting an immediate uptake of iron by the cells (10Atkinson P.G. Barton C.H. Immunology. 1999; 96: 656-662Crossref PubMed Scopus (71) Google Scholar). Addition of ferrous sulfate (5–50 μm) to cultured HM activates IKK but not JNK (11She H. Xiong S. Lin M. Zandi E. Giulivi C. Tsukamoto H. Am. J. Physiol. 2002; 283: G719-G726Crossref PubMed Scopus (109) Google Scholar). Thus, the low molecular weight-iron complex(es) response induced by a ligand-receptor interaction is substituted for by exogenous ferrous iron in transducing signals to activate IKK in primary cultures of HM. IKK plays a central role in a classical activation pathway of NF-κB through phosphorylation of IκB for subsequent polyubiquitination and proteasome-mediated degradation (see Ref. 12Bonizzi G. Karin M. Trends Immunol. 2004; 25: 280-288Abstract Full Text Full Text PDF PubMed Scopus (2100) Google Scholar for review). Three IKK subunits have been identified, IKKα and IKKβ as catalytic subunits and IKKγ as a regulatory unit. The most common form of IKK is composed of a heterodimer of IKKα and IKKβ bound to a dimer of IKKγ. IKK activation occurs when IKKβ is phosphorylated by mechanisms that appear to involve upstream kinases recruited by IKKγ. This phosphorylation triggers intra- and intermolecular trans-phosphorylation of IKK subunits. Much research has been done to identify these upstream kinases. NF-κB-inducing kinase (NIK) and mitogen-activated protein/extracellular signal-regulated kinase kinase 1 are shown to directly interact with and activate IKK (13Lin X. Cunningham Jr., E.T. Mu Y. Geleziunas R. Greene W.C. Immunity. 1999; 10: 271-280Abstract Full Text Full Text PDF PubMed Google Scholar, 14Mercurio F. Zhu H. Murray B.W. Shevchenko A. Bennett B.L. Li J. Young D.B. Barbosa M. 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Pagliari L.J. Pope R.M. J. Exp. Med. 2001; 194: 113-126Crossref PubMed Scopus (191) Google Scholar). On the other hand, conflicting results are available for the role of PI3-kinase pathway in regulation of NF-κB-dependent inflammatory gene expression. Inhibitors for PI3-kinase are shown to enhance (LPS)-induced expression of inducible nitric-oxide synthase (iNOS) in macrophages (20Park Y.C. Lee C.H. Kang H.S. Chung H.T. Kim H.D. Biochem. Biophys. Res. Commun. 1997; 240: 692-696Crossref PubMed Scopus (98) Google Scholar) and activation of PI3K conversely inhibits iNOS expression in glial cells (21Pahan K. Raymond J.R. Singh I. J. Biol. Chem. 1999; 274: 7528-7536Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). This negative regulation may be because of interference of LPS-induced MAPK pathways such as extracellular signal-regulated kinase-1/2 (ERK1/2), p38 MAPK, and JNK with PI3-kinase pathway (22Guha M. Mackman N. J. Biol. Chem. 2002; 277: 32124-32132Abstract Full Text Full Text PDF PubMed Scopus (646) Google Scholar). In contrast, opposing results are also available. PI3-kinase is shown to phosphorylate and increase transactivation activity of p65 (23Thomas K.W. Monick M.M. Staber J.M. Yarovinsky T. Carter A.B. Hunninghake G.W. J. Biol. Chem. 2002; 277: 492-501Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). Overexpression of a constitutively active AKT enhances NF-κB-dependent gene expression via IKK activation in fibroblasts (24Madrid L.V. Mayo M.W. Reuther J.Y. Baldwin Jr., A.S. J. Biol. Chem. 2001; 276: 18934-18940Abstract Full Text Full Text PDF PubMed Scopus (700) Google Scholar), and a dominant negative mutant of the p85 regulatory subunit of PI3-kinase blocks TNF-induced NF-κB-dependent gene expression (25Reddy S.A. Huang J.H. Liao W.S. J. Immunol. 2000; 164: 1355-1363Crossref PubMed Scopus (169) Google Scholar). The present study has investigated upstream kinases that are required for iron-induced IKK activation in HM. The use of dominant negative mutants (DN) and pharmacological inhibitors for potential upstream effector molecules demonstrated that multiple kinases, including MEK1, NIK, TAK1, and PI3K, are required for ferrous iron to activate IKK in HM. Further, TAK1 PI3K and p21ras are shown to physically interact in caveolae to initiate iron-induced signaling. Materials—RAW264.7 cells employed in this study were obtained from the American Tissue Culture Collection (Rockville, MD). Fetal bovine serum, ferrous sulfate, LPS (Escherichia coli 055:B5), filipin III, and phosphatase inhibitor mixture were purchased from Sigma. Adenoviral vectors Ad.DNTAK1, Ad.DNNIK, Ad.DNPI3K expressing DN mutants of TAK1 (K63W), NIK, and PI3K (Δp85) were obtained from Dr. David A. Brenner (Columbia University Medical Center, New York), and Ad.DNp21ras was given by Dr. L. F. Parada (University of Taxes Southwestern Medical Center). Antibodies against p-AKT and p-p44/p42 ERK1/2 were obtained from Cell Signaling Technology Inc. (Beverly, MA). Antibodies against p85α, TAK1, AKT, p21ras, and IKKα/β were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). A TNFα enzyme-linked immunosorbent assay kit was purchased from R&D Systems (Minneapolis, MN). [γ-32P]ATP was purchased from PerkinElmer Life Sciences. LY294002, U0126, and SB203580 were obtained from was obtained from Science. were obtained from kit was obtained from Inc. Iron and were from Dr. Brittenham of and Cell were from by the Cell of the for and as (4Tsukamoto H. Lin M. Ohata M. Giulivi C. French S.W. Brittenham G. Am. J. Physiol. 1999; 277: G1240-G1250PubMed Google Scholar, S. She H. Takeuchi H. Han B. Engelhardt J.F. Barton C.H. Zandi E. Giulivi C. Tsukamoto H. J. Biol. Chem. 2003; 278: 17646-17654Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, H. Xiong S. Lin M. Zandi E. Giulivi C. Tsukamoto H. Am. J. Physiol. 2002; 283: G719-G726Crossref PubMed Scopus (109) Google Scholar). the liver was in by with and and liver cells were by was by the to a of the cells were with LPS or or ferrous sulfate μm) in in the or of inhibitors as and to of cell for IKK and TAK1 and RAW264.7 cells were cultured in with and IKK and TAK1 assay the activity of IKK or TAK1, HM cultured in a were with ferrous sulfate μm) or LPS for min in the and of the iron or with and with a 1 and and The were in and with an expressing a dominant negative mutant of TAK1, NIK, PI3K, or p21ras, HM were with to effects iron-induced IKK and TAK1 IKK activity was as (9Xiong S. She H. Takeuchi H. Han B. Engelhardt J.F. Barton C.H. Zandi E. Giulivi C. Tsukamoto H. J. Biol. Chem. 2003; 278: 17646-17654Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). IKK was with IKKα/β and protein The assay was for 1 in a 2 2 of glutathione and [γ-32P]ATP The was by of and was by by a was by the with a TAK1 the was for the use of and as a the treatment with min iron treatment was to the HM the of and were as (9Xiong S. She H. Takeuchi H. Han B. Engelhardt J.F. Barton C.H. Zandi E. Giulivi C. Tsukamoto H. J. Biol. Chem. 2003; 278: 17646-17654Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, H. Xiong S. Lin M. Zandi E. Giulivi C. Tsukamoto H. Am. J. Physiol. 2002; 283: G719-G726Crossref PubMed Scopus (109) Google Scholar). were a to a with and by co-immunoprecipitation for TAK1, p21ras, and p85α, cell was with against p21ras p85 the complex was a and to a and the was with and The release of TNFα by HM was by a available TNFα enzyme-linked immunosorbent assay kit the HM were with and in for min after and the cells were by with in for treatment with for the was by of of against p21ras or in with The cells were with for min The cells were with the with or for 2 The were with for min after The cells were a with a and from Santa Cruz were the and were The HM were by a with of and and the for of from or Iron Iron-induced IKK iron-induced IKK activation in cultured HM. shown in the treatment with iron sulfate μm) increases IKK activity min This is abrogated by of the cells with or a or iron and Iron-induced IKK and TNFα by TAK1, NIK, PI3K, and TAK1, NIK, or PI3K is in iron-induced IKK activation, we used an expressing a DN mutant of kinase and a fluorescent as a of DN of iron-induced IKK activation Furthermore, use of the PI3K inhibitor results in of IKK activation Addition of U0126, the MEK1 inhibitor, also IKK activation the p38 MAPK inhibitor, not but IKK activity results the of TAK1, NIK, PI3K, and MEK1 in iron-induced IKK activation. The of IKK activity with the p38 MAPK inhibitor may be because of an of this MAPK via Han J. J. Res. 2003; Google Scholar, S. Cell. 18: PubMed Scopus Google Scholar). To these effects IKK activation are a we iron-induced release of TNFα by HM with or or pharmacological of the upstream kinases. Iron the release of TNFα by and this is not by a DN of TAK1, or PI3K, or by the use of the PI3K and inhibitors and TNFα Iron of and the MEK1 and PI3K in iron-induced signaling. Addition of ferrous sulfate AKT phosphorylation as as the treatment with LPS, PI3K activation by iron 2 and Iron blocks this AKT phosphorylation as prevented with but not with SB203580 has AKT phosphorylation We also ERK1/2 phosphorylation as a of MEK1. Iron ERK1/2 and this is by the iron blocks phosphorylation iron-induced phosphorylation SB203580 enhances results that MEK1 is not in iron-induced PI3K activity in HM. also that iron-induced phosphorylation is enhanced by PI3K but by p38 The the of IKK activity by SB203580 and may be caused by MEK1 by p38 MAPK as TAK1 and p21ras in Iron-induced PI3K in has been shown to be of TAK1 in signaling J. K. A. J. K. 1999; PubMed Scopus Google Scholar). However, the PI3K and TAK1 in IKK activation is not Thus, we PI3K activation is TAK1 activity in model of iron-induced IKK activation. To this end, we iron-induced AKT phosphorylation or with of DNTAK1. AKT phosphorylation is induced by iron in the cells with the fluorescent protein of DNTAK1 this as as expression This that TAK1 is in iron-induced PI3K We also the of AKT phosphorylation and IKK activation because this was shown to be by J. K. A. J. K. 1999; PubMed Scopus Google Scholar, R. A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) and to physically interact with PI3K T. B. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). with a expressing also blocks AKT phosphorylation and and IKK activation in HM that TAK1 and p21ras are in iron-induced PI3K the we shown in the of iron-induced phosphorylation is by the of DNTAK1 has iron-induced but this signaling results that iron-induced MAPK signaling via p21ras, but not TAK1, and is by we iron-induced TAK1 activity is affected by the PI3K or inhibitor. shown in iron TAK1 activity as by phosphorylation of but this induction is not by or U0126, suggesting TAK1 activity is upstream of PI3K and MEK1. We also iron-induced and IKK activation and and AKT phosphorylation are of we to the HM This treatment iron-induced TAK1 and IKK and and AKT phosphorylation TAK1 and p21ras with PI3K in of the a interaction p21ras and PI3K T. B. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar), of the of TAK1 and p21ras in PI3K activation that TAK1 may also interact with PI3K with p21ras in HM. This was by Cell from HM was to with an and the complex was and with an against TAK1 or the regulatory subunit of shown in iron treatment increases TAK1 with p21ras and Conversely, was with an against regulatory subunit and the complex was with an or Iron increases interactions of TAK1 and p21ras to the regulatory subunit Iron treatment not the of p21ras or A and results TAK1 and p21ras physically interact with PI3K in HM. To co-localization of p21ras and in HM, the cells were with the for p21ras and PI3K and fluorescent microscopy. shown in and p21ras to in min after of this is induced and co-localization are co-localization and with this co-localization phenomenon not We for TAK1, p21ras, and a for and microscopy. This co-localization of proteins iron treatment results interactions TAK1, PI3K, and p21ras and co-localization of these in Iron-induced IKK and TAK1 we of caveolae which and interactions of TAK1, PI3K, and p21ras, iron-induced TAK1 and IKK activation. this we used filipin III, which and inhibits caveolae P. E. J. J. Cell Biol. PubMed Scopus Google Scholar). this treatment abrogates iron-induced IKK and TAK1 activity A and suggesting that caveolae the of the signaling complex of p21ras, and PI3K for iron-induced IKK activation. iron-induced AKT phosphorylation is also by filipin but phosphorylation is signal and the of as intracellular have been (see Ref. J. 1997; PubMed Scopus Google Scholar for review). of the most classical of the of or is signaling induced by growth of growth such as growth growth and to kinase activity increases and via proteins molecules such as with a This results in activation of p21ras and the The kinase also the in the p85 regulatory subunit of PI3K and activates the PI3K via Thus, p21ras as the for the and pathways T. B. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). is this that or were to to results iron-induced signaling activation of and pathways with p21ras as a Further, study the evidence for the interactions by TAK1 with p21ras and PI3K A and This is to be because TAK1 plays a role in iron-induced AKT phosphorylation as for LPS-induced protein kinase activation J. L. J. Biol. 2000; PubMed Google Scholar). However, TAK1 with p21ras to have in the pathway because TAK1 has role in iron-induced phosphorylation as also shown in LPS-stimulated cells J. L. J. 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Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). of to a and iron of to S. M. M. 2004; PubMed Scopus Google Scholar). signaling induced by of ferrous sulfate is immediate as by co-localization of the effector proteins and activation of IKK (11She H. Xiong S. Lin M. Zandi E. Giulivi C. Tsukamoto H. Am. J. Physiol. 2002; 283: G719-G726Crossref PubMed Scopus (109) Google Scholar), be constitutively to in this of the cells with a p21ras mutant the role of results also the role of as iron-induced TAK1 and IKK is to be is a direct effector or with to initiate iron-induced signaling. results also the of caveolae as a of iron-induced signal involving the the for with TAK1 and subunit of PI3K and filipin III, an inhibitor for caveolae abrogates iron-induced TAK1 and IKK activation are that in cellular signal and regulation of to a (see Ref. I. Cell. Mol. Biol. 2004; Google Scholar for review). This is to many signaling including kinase growth factor growth factor protein kinases and and protein kinases A and C. Thus, of co-localization of in caveolae may not be However, the that the types of nitric-oxide synthase and are also in caveolae a that this an for iron-catalyzed signaling involving p21ras, and In we that iron activates IKK in hepatic macrophages through mechanisms involving of p21ras, PI3K, TAK1, NIK, and MEK1. Iron interactions of p21ras with PI3K and in caveolae to initiate of these and p21ras to as a for iron-induced activation of MAPK and PI3K