Iron Activates NFB in Kupffer Cells

Iron activates NF-B in Kupffer cells

HONGYUN SHE,1 SHIGANG XIONG,1 MIN LIN,1 EBRAHIM ZANDI,2

CECILIA GIULIVI,3 AND HIDEKAZU TSUKAMOTO11Departments of Pathology and 2Molecular Microbiology and Immunology, Keck Schoolof Medicine of the University of Southern California, Los Angeles, California 90033-9141;and 3Department of Chemistry, University of Minnesota, Duluth, Minnesota 55812Received 19 March 2002; accepted in final form 12 May 2002

She, Hongyun, Shigang Xiong, Min Lin, EbrahimZandi, Cecilia Giulivi, and Hidekazu Tsukamoto. Ironactivates NF-B in Kupffer cells. Am J Physiol GastrointestLiver Physiol 283: G719G726, 2002; 10.1152/ajpgi.00108.2002. Iron exacerbates various types of liver injury inwhich nuclear factor (NF)-B-driven genes are implicated.This study tested a hypothesis that iron directly elicits thesignaling required for activation of NF-B and stimulation oftumor necrosis factor (TNF)- gene expression in Kupffercells. Addition of Fe2 but not Fe3 (550 M) to culturedrat Kupffer cells increased TNF- release and TNF- pro-moter activity in a NF-B-dependent manner. Cu but notCu2 stimulated TNF- protein release and promoter activ-ity but with less potency. Fe2 caused a disappearance of thecytosolic inhibitor B, a concomitant increase in nuclearp65 protein, and increased DNA binding of p50/p50 andp65/p50 without affecting activator protein-1 binding. Addi-tion of Fe2 to the cells resulted in an increase in electronparamagnetic resonance-detectable OH peaking at 15 min,preceding activation of NF-B but coinciding with activationof inhibitor B kinase (IKK) but not c-Jun NH2-terminalkinase. In conclusion, Fe2 serves as a direct agonist toactivate IKK, NF-B, and TNF- promoter activity and toinduce the release of TNF- protein by cultured Kupffer cellsin a redox status-dependent manner. We propose that thisfinding offers a molecular basis for iron-mediated accentua-tion of TNF--dependent liver injury.

tumor necrosis factor-; free radical; promoter; inhibitor Bkinase; electron paramagnetic resonance; nuclear factor-B

IRON POTENTIATES VARIOUS FORMS of liver injury (4, 19, 28,41), and chelation of iron or decreasing iron contentconversely ameliorates the injury (9, 22, 30, 32). Themost accepted explanation for irons effects is an iron-catalyzed Fenton pathway resulting in the generationof OH and consequent oxidative tissue injury. In par-ticular, if the generation of reactive oxygen species(ROS) is already enhanced by underlying disease pro-cesses, a slight increase in hepatic iron content maysuffice for robust production of OH and accentuation ofoxidative damage, as exemplified in experimental alco-holic liver injury (41). This accentuation of liver injuryis accompanied by enhanced nuclear factor (NF)-Bactivation and expression of proinflammatory media-

tors (43). The latter events may merely reflect a con-sequence of enhanced hepatocellular necrosis or mayalso be considered as causal processes. In fact, at non-toxic concentrations, iron is known to promote macro-phage functions, including antimicrobial effects (18)and tumor necrosis factor (TNF)-mediated cytotoxicity(46). More specifically, recent evidence suggests therole of iron in promoting cytokine expression (7, 14)and NF-B activation (42) by hepatic macrophages.

Even though a catalytically active pool of iron isestimated to be extremely small in normal tissues, thepathological conditions may cause a transient releaseof iron from the intracellular compartments into themicroenvironment. For instance, oxidative stress isknown to release iron from ferritin through eitherreduction of Fe3 by O2

or oxidative destruction offerritin proteins (6, 39). Alternatively, NO may causemobilization of intracellular iron (11, 13, 21) by target-ing iron-sulfur groups contained in several key en-zymes (12, 17). Thus it is conceivable that in liverdiseases in which mild iron accumulation, oxidativestress, and TNF- induction commonly coexist, thetransient release of catalytically active iron may serveto facilitate oxidative signaling for proinflammatoryNF-B activation.

The present study tested whether direct addition ofionic iron to cultured Kupffer cells leads to activation ofNF-B and induction of TNF- expression. Our resultsdemonstrate that Fe2 but not Fe3 at concentrationsas low as 5 M stimulates TNF- release. It alsoinduces TNF- promoter activity in an NF-B-depen-dent manner, and this effect is associated with time-dependent activation of inhibitor B (IB) kinase (IKK)and NF-B without affecting activator protein (AP)-1binding. Collectively, these results support a notionthat iron can serve as a direct agonist to induce intra-cellular signaling for NF-B activation in Kupffer cellsin a redox status-dependent manner.

MATERIALS AND METHODS

Kupffer cell isolation and culture. Kupffer cells were iso-lated from normal Wistar rats by in situ sequential digestionof the liver with pronase and collagenase and arabinogalac-

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Am J Physiol Gastrointest Liver Physiol 283: G719G726, 2002;10.1152/ajpgi.00108.2002.

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tan gradient ultracentrifugation as previously described (22,42). The adherence purification method was performed toraise the purity of Kupffer cells cultured onto a 100-mm dishto 96% as determined by phagocytosis of 1-m latex beads.The viability was tested by the trypan blue exclusion test andalways exceeded 97%. The cells were incubated with DMEMcontaining 5% fetal calf serum for 2 days, following theadherence method for in vitro experiments. For iron or cop-per treatment, the cells were washed twice with PBS, incu-bated in serum-free DMEM, and exposed to ferrous sulfate,ferric ammonium sulfate, cuprous chloride, or cupric sulfate(150 M) for 4 h to assess their effects on the release ofTNF- and TNF- promoter activity. For activation of IKKand NF-B, as well as electron paramagnetic resonance(EPR) detection of radicals, the cells were incubated forshorter periods (from5 min to 4 h) as specified below and inthe figure legends. As a positive control, the cells weretreated with lipopolysaccharide (LPS; Escherichia coli 055:B5, 500 ng/ml, Sigma, St. Louis, MO).

Nuclear protein extraction and EMSA. To examine theeffects of Fe2 on DNA binding by NF-B and AP-1, nuclearproteins were extracted from cultured Kupffer cells by usingthe method of Schreiber et al. (35). The extracts (5 g) wereincubated in a reaction mixture [20 mM HEPES, pH 7.6, 100mM KCl, 0.2 mM EDTA, 2 mM dithiothreitol (DTT), 20%glycerol, and 200 g/ml poly(dI-dC)] on ice with the double-strand B consensus sequence (3), the B site from TNF-promoter (8), or the AP-1 binding site (2) labeled with 32P.After a 20-min incubation, the reaction mixture was resolvedon a 6% nondenaturing polyacrylamide gel and the gel wasdried for subsequent autoradiography. Densitometric analy-sis of the intensity of shifted bands was performed by usingthe Kodak Electrophoresis Documentation and Analysis Sys-tem and imaging analysis software (Eastman Kodak, Roch-ester, NY). For the supershift assays, antibodies against p50and p65 (Santa Cruz Biotechnology, Santa Cruz, CA) wereadded to the reaction mixture for an additional 30 min.

IB and p65 immunoblot analysis. Cytoplasmic and nu-clear extracts of iron-stimulated, cultured Kupffer cells wereexamined for IB and p65 levels by immunoblot analysis,respectively. Cytoplasmic or nuclear proteins (10 g) weremixed with 2 sample buffer (100 mM Tris HCl, pH 6.8, 4%SDS, 20% glycerol, and 10% -mercaptoethanol) and sepa-rated by 10% PAGE under reducing conditions. The proteinswere transferred to nitrocellulose filters (Bio-Rad, Hercules,CA) and treated overnight at 4C with 5% BLOTTO [5%nonfat milk with (in mM) 50 Tris HCl, pH 7.5, 50 NaCl, 1EDTA, and 1 DTT]. The filters were then incubated withrabbit polyclonal anti-human p65 (Biomol, Plymouth Meet-ing, PA) or anti-human IB (Santa Cruz Biotechnology) at1:1,000 dilution in TBST (10 mM Tris HCl, pH 8.0, 150 mMNaCl, and 0.05% Tween 20) with 1% BSA at room tempera-ture for 2 h, followed by three washes with TBS and 0.2%Tween 20. The filters were then incubated with horseradishperoxidase-conjugated goat anti-rabbit IgG (Sigma) at1:2,000 dilution at room temperature for 2 h. The immobi-lized p65 and IB antibody complexes were detected bychemiluminescence by using an enhanced chemilumines-cence kit (Amersham, Arlington Heights, IL).

EPR spectra of iron-treated Kupffer cells. To determinetime-dependent changes in the generation of free radicals byiron-treated Kupffer cells, the cells (107 cells/ml) were sus-pended in PBS containing 510 mM glucose with or withoutferrous sulfate (50 M). At different time points (0, 5, 10, 20,and 30 min), aliquots of the samples were withdrawn fromthe reaction mixtures, mixed with 50 mM -(4-pyridyl-1-oxide)-N-t-butylnitrone (POBN) and 0.1% (vol/vol) DMSO,

and immediately transferred to bottom-sealed Pasteur pi-pettes. The EPR spectra were recorded at room temperaturein a Bruker ECS 106 spectrometer operating at 9.8 GHz.Instrument conditions were as follows: modulation fre-quency, 100 kHz; time constant, 1.3s; sweep scan, 18 G/min;modulation amplitude, 0.9 G; and microwave power, 20 mW.The spectra were compared with simulated ones obtained byusing the published hyperfine splitting constants and thesimulation program from Oklahoma Research Center.

IKK and JNK assays. To assay the activity of IKK, Kupffercells cultured in 100-mm dishes were treated with ferroussulfate for 045 min or LPS (500 ng/ml) for 15 min, washedwith PBS once, and lysed with a lysis buffer (in mM: 20Tris HCl, pH 7.5, 20 NaF, 20 -glycerophosphate, 0.5Na3VO4, 2.5 metabisulfite, 5 benzamidine, 1 EDTA, 0.5EGTA, and 300 NaCl, with 10% glycerol and protease inhib-itors and 1.5% Triton X-100). The lysates were immediatelyfrozen in liquid nitrogen and stored at 80C until assay.IKK activity was determined as previously described (29).Briefly, IKK was immunoprecipitated by IKK antibodiesand protein G-Sepharose. The assay was performed at 30Cfor 1 h in buffer containing 20 mM Tris HCl, pH 7.5, 20 mMMgCl2, 2 mM DTT, 20 M ATP, 2 g/30 l glutathione-S-transferase (GST)-IB, and [-32P]ATP (0.5 Ci). The reac-tion was stopped by addition of Laemmli buffer and wasresolved by 10% SDS-PAGE followed by a transfer onto anitrocellulose membrane. Phosphate incorporated into GST-IB was visualized by exposing the membrane to a Phos-phorImager. The c-Jun NH2-terminal kinase (JNK) assaywas performed similarly, except that antibodies againstJNK-1 (Santa Cruz Biotechnology) and protein G-Sepharosewere used to immunoprecipitate JNK-1 and that GST-c-Jun(Santa Cruz Biotechnology) was used as a substrate. For bothIKK and JNK, total protein levels were assessed by immu-noblot analysis of the cell lysates.

Transfection and TNF- promoter analysis. To assess theeffects of ionic iron and copper on TNF- promoter activity,cultured Kupffer cells were transiently transfected with aTNF- promoter-luciferase construct using Targefect F-2(Targeting System, San Diego, CA). The construct was cre-ated by ligating a 1.4-kb mouse TNF- promoter (a KpnI andHindIII fragment) (15) into the pGL3-Basic plasmid (Pro-mega, Madison, WI). For determination of transfection effi-ciency, Renilla phRL-TK vector was used. For transfection,3-day-cultured Kupffer cells in six-well plates were treatedwith 2 g of the reporter gene, 0.02 g Renilla phRL-TK, and2 l of F-2 reagent in 1 ml serum-free RPMI for 2 h. Then 1ml of RPMI with 10% FCS was added to achieve the final FCSconcentration of 5% for overnight incubation. On the nextday, the medium was changed to new DMEM with 10% FCSand the cells were incubated for 24 h. During the last 14 h ofthe incubation, the medium was changed to serum-free RPMIwith or without ferrous sulfate, ferric ammonium sulfate,cuprous chloride, or cupric sulfate (10 or 50 M), and the celllysate was collected for luciferase assay by using the Dual-Luciferase Reporter assay system (Promega). Four experi-ments were performed independently, and all results werenormalized for transfection efficiency as determined by Re-nilla luciferase activity. To determine the dependence ofirons effects on NF-B, the cells were also cotransfected withthe IB super repressor plasmid, which expresses IBwith S32A/S36A mutations (16), or the empty vector. Theseplasmids were kindly provided by Dr. Richard Rippe (Uni-versity of North Carolina at Chapel Hill).

TNF- RT-PCR. For RT-PCR analysis for TNF-, 3 g oftotal RNA was reverse transcribed into cDNA by a Moloneymurine leukemia virus reverse transcriptase and oligo(dT)15

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AJP-Gastrointest Liver Physiol VOL 283 SEPTEMBER 2002 www.ajpgi.org

at 37C for 60 min. Synthesized cDNA was amplified bydenaturation at 94C for 4 min, followed by multiple (25 for-actin and 43 for TNF-) cycles of denaturation (95C, 30 s),annealing (58C, 30 s), and extension (72C, 60 s). Primersused for TNF- were sense, 5-ATGAGCACAGAAAGCAT-GATG and antisense, 5-TACAGGCTTGTCACTCGAATT,and for -actin they were sense, 5-CACGGCATTGTAAC-CAACTG and antisense, 5-AGGGCAACATAGCACAGCTT.

TNF- immunoassay. The effects of iron and copper on therelease of TNF- by cultured Kupffer cells were examined byanalyzing the TNF- protein in the media with a commer-cially available mouse TNF- immunoassay kit (R&D Sys-tems, Minneapolis, MN).

Statistical analysis. The numerical data were expressed asmeans SD, and comparison between treated and controlgroups was performed by Students t-test.

RESULTS

Fe2 but not Fe3 stimulates release of TNF-. Wefirst tested whether iron stimulates the release ofTNF- by cultured Kupffer cells. As shown in Fig. 1,the addition of Fe2 but not Fe3 increased TNF-release by twofold at 5 M and eightfold at 10 and 50M during the 4-h treatment period. Interestingly,Cu but not Cu2 also stimulated TNF- release at 10and 50 M, but its effect seemed less potent comparedwith Fe2. Thus these results demonstrate direct stim-ulation of Kupffer cell TNF- release by iron and cop-per in a redox status-dependent manner. It should alsobe noted that no toxicity was observed in Kupffer cellsexposed to 150 M of iron or copper as assessed bylactate dehydrogenase release or Sytox green nucleicacid staining (Molecular Probes, Eugene, OR).

Iron stimulates TNF- promoter activity. We thentested whether Fe2 stimulates the TNF- promoter in

cultured Kupffer cells. The promoter activity was in-deed increased23 fold with 10 and 50 M Fe2 (Fig.2A). Cu (50 M) also slightly increased TNF- pro-moter activity, but Cu2 and Fe3 did not (Fig. 2A).Cotransfection of a super repressor IB vector com-pletely abrogated the stimulation with 50 M Fe2,whereas cotransfection with a LacZ vector did not (Fig.

Fig. 1. Fe2 but not Fe3 stimulates tumor necrosis factor (TNF)-release. Cultured Kupffer cells in serum-free medium were treatedwith increasing concentrations of ferrous sulfate, ferric ammoniumsulfate, cuprous chloride, and cupric sulfate for 4 h, followed bydetermination of TNF- protein in the medium by ELISA. Notesignificantly increased release of TNF- protein with Fe2 but notFe3 at the concentrations as low as 5 M, reaching the maximal8-fold stimulation at 10 M. Cu also stimulates the release, but thedose response is shifted to the right, indicating less potency. Data areobtained from 36 different experiments and expressed as %con-trol (no metal addition). TNF- released under the control conditionwas 5.55 2.88 pg/ml (mean SD, n 6). *P 0.05 and **P 0.01vs. control.

Fig. 2. Fe2 increases TNF- promoter activity and mRNA level. A:cultured Kupffer cells were transfected with a TNF- promoter-luciferase construct followed by the treatment with Fe2, Fe3, Cu,or Cu2 for 14 h. The promoter activity was normalized by transfec-tion efficiency as determined by Renilla luciferase activity. Note thatFe2 induces the promoter activity by 2-fold at 10 and 50 M. Cuslightly induces the promoter, but the oxidized metals (Fe3 andCu2) do not. B: cells were cotransfected with the promoter-lucif-erase construct with a LacZ vector or dominant-negative inhibitorB (IB), followed by addition of Fe2 (50 M). Iron treatmentstimulated the TNF- promoter activity, and this effect was com-pletely blocked by cotransfection with a super repressor IB (DN-IB). Lipopolysaccharide (LPS)-stimulated promoter activity isshown as a positive control. *P 0.01 vs. control. C: iron treatmentincreases TNF-mRNA levels in Kupffer cells. The effects of Fe2 onTNF- mRNA levels in cultured Kupffer cells were examined byRT-PCR. Note increased mRNA levels with Fe2. The last laneshows a robust induction by LPS as a positive control.

G721IRON AND NF-B


2B). Stimulation of the promoter activity by 50 MFe2 was about half of the maximal response achievedby LPS (500 ng/ml) in a serum-free condition (Fig. 2B).These results establish that Fe2 activates TNF- pro-moter in a NF-B-dependent manner.

Fe2 increases TNF- mRNA levels. We then exam-ined whether TNF- promoter activity induced bytreatment with Fe2 is associated with increasedmRNA levels for this cytokine. As shown in RT-PCRdata in Fig. 2C, the iron treatment increased TNF-message. Densitometric analysis and standardizationwith -actin data showed 2.3- and 2.0-fold increases inTNF- message by 10 and 50 M Fe2, respectively.

Fe2 activates NF-B in cultured Kupffer cells. Next,we examined whether Fe2 increases the binding ofnuclear proteins to the B site in cultured rat Kupffercells. At 10 and 50 M, there was increased DNAbinding regardless of whether we used the consensussequence (Fig. 3) or the B site from the TNF- pro-moter (data not shown). Figure 3A shows the represen-tative EMSA results obtained with 50 M Fe2. In-creased binding was noted from 30 min following theiron addition and lasted for 24 h. Densitometric anal-

ysis of three sets of EMSA results demonstrated 3.4 1.0-fold and 2.1 0.8-fold increases (n 3, P 0.05) inp65/p50 and p50/p50 binding at 30 min after the treat-ment with Fe2, respectively. At 2 h, the intensities ofboth bands were only moderately increased by 67% forp65/p50 and 86% for p50/p50. AP-1 binding was ana-lyzed by using the same nuclear extracts, but nochanges were noted (Fig. 3A). Similar results wereobserved with 10 M Fe2 (data not shown). The su-pershift assay was performed to identify the proteinsencompassing the two sizes of the DNA-protein com-plexes detected by NF-B EMSA. This assay revealedthat they were a p50/p50 homodimer and a p65/p50heterodimer (Fig. 3B). To confirm that iron-inducedenhancement in NF-B DNA binding was due to acti-vation of the transcription factor, we performed West-ern blot analysis for cytosolic IB and nuclear p65. Asshown in the representative blots in Fig. 4, the cytoso-lic level of IB was transiently reduced at 30 min1 hwhile the nuclear p65 level increased from 30 min to24 h after the iron addition. Loading of cytosolic ornuclear proteins was equal, as shown by the staining ofthe proteins on the filters (Fig. 4). These results wereconfirmed in three independent experiments. These

Fig. 4. Iron treatment causes cytosolic IB degradation and nu-clear translocation of p65. Cytosolic and nuclear proteins preparedfrom Kupffer cells treated with iron sulfate (50 M) for 04 h wereanalyzed by immunoblotting for IB (A) and p65 (B) levels, respec-tively. These data, which are representative of 3 independent exper-iments, demonstrate that Fe2 induces a transient disappearance ofIB in cytosol and an increase of p65 in nuclear extracts at 30 minafter addition, the time point that correlates with the increasedNF-B binding shown in Fig. 3. Equal loading of proteins is sup-ported by the staining of fractionated proteins on the filters, asshown below the Western blot data.

Fig. 3. Treatment with Fe2 increases the binding of nuclear factor(NF)-B in cultured rat Kupffer cells. A: typical response of increasedbinding of both p65/p50 heterodimer and p50/p50 homodimer incultured Kupffer cells exposed to Fe2 (50 M) for 30 min2 h.However, activator protein (AP)-1 binding is not affected by thetreatment. B: supershift assays of the nuclear extracts from iron-treated Kupffer cells reveal the components of the NF-B bindingcomplexes to be a p65/p50 heterodimer and a p50/p50 homodimer.

G722 IRON AND NF-B


results support an interpretation that the iron treat-ment caused IB degradation, NF-B activation, andnuclear translocation of the RelA protein, resulting inincreased DNA binding by NF-B, all commencing at30 min. In addition, the lack of the AP-1 responsesuggests that the effect of Fe2 on NF-B is ratherselective.

Direct addition of Fe2 to nuclear proteins does notincrease RelA binding. Even though our Western blotresults strongly supported that activation of NF-Bwas most likely responsible for iron-induced enhance-ment in DNA binding of this transcription factor, itwas still possible that iron directly increased the asso-ciation of the nuclear NF-B to the B site in thenucleus. To test this possibility, Fe2 was added to thenuclear extracts prepared from the resting culturedKupffer cells at 0.1, 1, 10, and 50 M and the effectswere analyzed by EMSA. The results demonstratedthat the binding of p50/p50 but not of p65/p50 wasapparently increased by the treatment (Fig. 5), anddensitometric analysis of three sets of data showed25 7, 46 11, 97 18, and 121 21% increases inp50/p50 binding at 0.1, 1, 10, and 50 M, respectively,and confirmed no increase in p65/p50 binding. Thesedata suggested that this direct effect of iron on thenuclear extracts could not explain the increased bind-ing of p65/p50 observed in the iron-treated cells.

Iron activates IKK. To investigate the mechanisms ofiron-mediated activation of NF-B, we examined theeffect of Fe2 on IKK activity in cultured Kupffer cellsat different time points. As shown in Fig. 6, IKKactivity, as assessed by phosphorylation of GST-IB,was increased at 15 min, whereas the total IKK levelwas unchanged. As a positive control, LPS-stimulatedIKK activity is shown. The timing of IKK activationpreceded the disappearance of cytosolic IB at 30 minafter addition of iron (Fig. 4). In contrast, iron did notinduce JNK activity (Fig. 6), and this result corrobo-rated unchanged AP-1 binding by iron (Fig. 3A). An-other stress-activated mitogen-activated protein ki-nase (MAPK), p38, was also assessed. The level ofphosphorylated p38 was also unaffected by the irontreatment, suggesting that Fe2 did not activate thisMAPK (H. She, unpublished observations). The resultson IKK and JNK were confirmed in at least threeindependent experiments. Thus these results demon-strate for the first time that Fe2 activates IKK andsupport a notion that Fe2 serves as an agonist tostimulate signal transduction, which is rather selectivefor activation of NF-B.

Iron increases EPR-detectable radicals before NF-Bactivation. NF-B is a redox-sensitive transcriptionfactor, and ROS are implicated in its activation (1, 34,36, 38). Thus we postulated that Fe2 stimulates ROSproduction in Kupffer cells preceding activation of NF-B. In fact, Fe2 can react with oxygen in aqueoussolution to produce Fe3 and O2

, and this ROS may beresponsible for the observed effect. Fe2 may also cat-alyze the formation of OH from H2O2, which is gener-ated from basal NADPH oxidase activity of culturedKupffer cells. To address these possibilities, the cellswere treated with Fe2 for 030 min, ROS wastrapped with POBN, and EPR spectra were analyzed.Kupffer cells without iron treatment exhibited an EPRspectrum constituted by an equal mixture of three spinadducts: methyl, hydroxyl, and O2

(Fig. 7B). Additionof 50 M Fe2 to these cells resulted in an enhance-ment of the hydroxyl and methyl-POBN adduct signals(Fig. 7A). The formation of these adducts must haverelied on the production of hydroxyl radical from aniron-catalyzed Fenton reaction. The methyl adduct waslikely produced at the attack of the OH on the methylmoiety of DMSO and the subsequent trap of thismethyl radical by POBN. Both signals increased withincubation time (Fig. 7C) up to a maximum at 1520min, regaining the initial values after 30 min. Theseincreases in the steady-state concentration of theseradicals indicate that the transient increases probablyoccurred as part of a response mechanism or signaltransduction pathway on stimulus of exogenous iron.In particular, the fact that the peak of the radicalgeneration at 1520 min coincided with IKK activationand preceded activation of NF-B at 30 min suggests

Fig. 5. Direct addition of Fe2 to the nuclear proteins does notincrease RelA binding. Addition of ferrous sulfate (0.150 M) tothe nuclear extracts prepared from the resting Kupffer cells does notincrease the binding of p65/p50 but enhances p50/p50 binding.

Fig. 6. Fe2 activates IB kinase (IKK) but not c-Jun NH2-terminalkinase (JNK) before NF-B activation. IKK and JNK activity assayswere performed on the Kupffer cell lysate samples collected atdifferent time points after FeSO4 treatment. Note that IKK activityas assessed by phosphorylation of glutathione-S-transferase (GST)-IB (P-IB) is increased at 15 min after addition of FeSO4. Noactivation of JNK is seen after the iron treatment, as assessed byphosphorylation of GST-c-Jun (P-c-Jun). LPS-induced activation ofIKK (15 min) and JNK (30 min) is shown as positive controls in thelast lanes. Relatively equal levels of IKK, JNK p54, and JNK p46are shown by immunoblots.

G723IRON AND NF-B


the signaling role of the former in the latter events. Infact, this notion was developed in previous studies (37)that demonstrated activation of NF-B by OH-gener-ating systems and a reversal of this effect by OHscavengers or metal chelators in Jurkat cells.

DISCUSSION

Biological and mechanistic implications. The resultspresented by the current study demonstrate a directstimulatory effect of Fe2 on signal transduction forNF-B activation in cultured Kupffer cells. The effectis seen at least at the level of IKK activation andextended to the most downstream level of TNF- pro-tein expression. These results suggest a possibility thatiron may serve as an independent agonist for activa-tion of NF-B and induction of NF-B-responsive genesin Kupffer cells in vivo. In fact, iron supplementationaggravates liver injury induced by alcohol (41) or hep-atitis viral infection (4) in experimental animals. In aclinical setting, the increased hepatic iron content fre-

quently accompanies many different types of liver dis-ease, such as alcoholic liver disease (28), viral hepatitis(10), and nonalcoholic steatohepatitis (5, 25), and ironreduction modalities often ameliorate such liver dam-age (10). Acute iron loading to the isolated perfused ratliver results in early increases in Kupffer cell-depen-dent respiratory activity (40), and iron directly en-hances interleukin-1 secretion by macrophages stimu-lated by interferon- and LPS (7). We have previouslydemonstrated that the treatment of cultured Kupffercells with an iron chelator effectively suppressed acti-vation of NF-B (22). Therefore, the evidence pre-sented by the current study offers the pivotal molecu-lar basis for the link between iron and NF-Bactivation suggested by the earlier studies. Indeed, inpathological livers, iron that is compartmentalized intoprotein-bound forms may be released transiently intothe microenvironment due to oxidative (6, 39) or nitro-sative (11, 13, 21) stress. This catalytically active poolof iron may directly activate NF-B in Kupffer cells invivo.

It is also known that iron overload inhibits functionsof macrophages, including expression of proinflamma-tory cytokines (24, 26, 45). These effects are likely dueto cytotoxicity of the cells exposed to either high orchronic iron loading. Indeed, acute iron overload viaphagocytosis of erythrocytes is shown to cause celltoxicity in cultured Kupffer cells (20). In our study,Kupffer cells exposed to Fe2 iron at different concen-trations up to 50 M did not show signs of cytotoxicity,and under such conditions, the direct agonistic effecton NF-B was evident.

Our results also demonstrate that the peak of OHgeneration coincides with activation of IKK in iron-treated Kupffer cells, suggesting that either this mostpotent radical or downstream molecules may be thepotential effectors for IKK activation. It is presumedthat this radical is generated by Fe2 via a Fentonpathway catalyzing one electron reduction of H2O2.The role of metal-catalyzed generation of OH in NF-Bactivation has previously been proposed (22, 37, 42),and our present data further support the notion. How-ever, it remains to be determined whether and howOH indeed activates IKK. It may exert direct effects onIKK, such as oxidation of cysteine residues within theactivation loop of IKK and - and a tighter conforma-tion of the complex for phosphorylation of IB viadisulfide bond formation (33). It may also mediate IKKactivation via its effects on upstream kinases. Forinstance, thioredoxin can be oxidized by OH, and thismay cause a release of apoptosis signal-regulating ki-nase 1 (ASK1), which is usually bound to thioredoxinas an inactive form (31). Released ASK1 can then beoligomerized for activation of p38, which may in turnlead to activation of NF-B (23). However, since OH isextremely reactive, it is difficult to conceive that suchselective oxidation of target molecules can be achievedwith this radical without additional regulatory mech-anisms. OH may also target other unknown inhibitorsof IKK. Alternatively, OH may induce intracellularlipid peroxidation, and lipid peroxides or their end

Fig. 7. Iron increases electron paramagnetic resonance (EPR)-de-tectable radicals before NF-B activation. Cultured Kupffer cellswere treated with iron sulfate (50 M) for 030 min, reactiveoxygen species were trapped with -(4-pyridyl-1-oxide)-N-t-butylni-trone (POBN), and EPR spectra were analyzed. The cells withoutiron treatment exhibited an EPR spectrum constituted by an equalmixture of 3 spin adducts: methyl, hydroxyl, and superoxide anion(B). Addition of iron to the cells increased the hydroxyl and methyl-POBN adduct signals (A). The methyl adduct was likely produced onthe attack of hydroxyl radical on the methyl moiety of DMSO and thesubsequent trap of this methyl radical by POBN. Both signals in-creased with incubation time (C) up to a maximum at 15 min,regaining the initial values after 30 min.

G724 IRON AND NF-B


products, such as aldehydes, may regulate signalingfor IKK activation. Indeed, 4-hydroxynonenal, onesuch aldehydic product, has been shown to activateJNK (27, 44) and p38 MAPKs (44). However, in ourstudy, Fe2 activated IKK independently of JNK (Fig.6) or p38 (unpublished data) MAPK activities. Obvi-ously, future studies are needed to better delineate themolecular steps connecting iron and IKK activation.

This work was supported by National Institutes of Health grantsR37-AA-06603, P50-AA-11999 (USC-UCLA Research Center for Al-coholic Liver and Pancreatic Diseases), P30-DK-48522 (USC Re-search Center for Liver Diseases), R24-AA-12885 (Non-ParenchymalLiver Cell Core), and the Medical Research Service of the Depart-ment of Veterans Affairs. S. Xiong was supported by a CooleysAnemia Foundation Postdoctoral Award.

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Iron Activates NFB in Kupffer Cells