Calcium-Dependent Regulation of NEMO Nuclear Export in Response to Genotoxic Stimuli

MOLECULAR AND CELLULAR BIOLOGY, Jan. 2007, p. 497–509 Vol. 27, No. 20270-7306/07/$08.00�0 doi:10.1128/MCB.01772-06Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Calcium-Dependent Regulation of NEMO Nuclear Export inResponse to Genotoxic Stimuli�

Craig M. Berchtold,1 Zhao-Hui Wu,1 Tony T. Huang,2 and Shigeki Miyamoto1*Department of Pharmacology, 301 SMI, 1300 University Avenue, University of Wisconsin, Madison, Wisconsin 53706,1 and

Dana-Farber Cancer Institute, Harvard Medical School, 44 Binney Street, M642, Boston, Massachusetts 021152

Received 19 September 2006/Accepted 20 October 2006

The mechanisms involved in activation of the transcription factor NF-�B by genotoxic agents are not wellunderstood. Previously, we provided evidence that a regulatory subunit of the I�B kinase (IKK) complex,NF-�B essential modulator (NEMO)/IKK�, is a component of a nuclear signal that is generated after DNAdamage to mediate NF-�B activation. Here, we found that etoposide (VP16) and camptothecin inducedincreases in intracellular free calcium levels at 60 min after stimulation of CEM T leukemic cells. Inhibitionof calcium increases by calcium chelators, BAPTA-AM and EGTA-AM, abrogated NF-�B activation by theseagents in several cell types examined. Conversely, thapsigargin and ionomycin attenuated the BAPTA-AMeffects and promoted NF-�B activation by the genotoxic stimuli. Analyses of nuclear NEMO levels in VP16-treated cells suggested that calcium was required for nuclear export of NEMO. Inhibition of the nuclearexporter CRM1 by leptomycin B did not interfere with NEMO nuclear export. Similarly, deficiency of aplausible calcium-dependent nuclear export receptor, calreticulin, failed to prevent NF-�B activation by VP16.However, temperature inactivation of the Ran guanine nucleotide exchange factor RCC1 in the tsBN2 cell lineharboring a temperature-sensitive mutant of RCC1 blocked NF-�B activation induced by genotoxic stimuli.Overexpression of Ran in this cell model showed that DNA damage stimuli induced formation of a complexbetween Ran and NEMO, suggesting that RCC1 regulated NF-�B activation through the modulation ofRanGTP. Indeed, evidence for VP16-inducible interaction between Ran-GTP and NEMO could be obtained bymeans of glutathione S-transferase (GST) pull-down assays using GST fused to the Ran binding domain ofRanBP2, which specifically interacts with the GTP-bound form of Ran. BAPTA-AM did not alter theseinteractions, suggesting that calcium is a necessary step beyond the formation of a Ran-GTP-NEMO complexin the nucleus. These results suggest that calcium has a unique role in genotoxic stress-induced NF-�Bsignaling by regulating nuclear export of NEMO subsequent to the formation of a nuclear export complexcomposed of Ran-GTP, NEMO, and presumably, an undefined nuclear export receptor.

The Rel/NF-�B family of transcription factors is regulatedby a wide variety of extracellular stimuli, including growthfactors, cytokines, and gentotoxic anticancer agents (2, 12, 34,46). The prototypical p50:p65(RelA) heterodimer is expressedin most mammalian cell types examined but kept inactivethrough the association of its inhibitor protein, such as I�B�.The inactive NF-�B is primarily localized in the cytoplasm, andits activation involves its release from I�B� and translocationto the nucleus. The release of NF-�B from I�B� can be ac-complished by different mechanisms, including the well-char-acterized “canonical” pathway (17, 28, 55). In this pathway,different signaling events lead to the activation of the cytoplas-mic I�B kinase (IKK) complex, which mediates site-specificphosphorylation of I�B�. This phosphorylation induces thedegradation of I�B� by the ubiquitin-proteasome system andthe release of NF-�B for nuclear translocation. In the nucleus,NF-�B binds to cognate decameric �B elements and regulatesthe transcription of target genes involved in a wide spectrum ofprocesses, including immune and inflammatory responses, pro-liferation, and apoptosis (2, 12, 81). NF-�B can also influence

the development of the cancer phenotype by regulating cellsurvival, angiogenesis, and metastasis (1, 47).

NF-�B activation is also elicited by genotoxic anticanceragents that cause double-strand breaks in DNA, such as topo-isomerase I and II inhibitors and ionizing radiation (IR) (19,37, 41). NF-�B can induce antiapoptotic genes resulting in achemo and radio resistance phenotype in cancer (3, 5, 20, 46).Thus, the understanding of the mechanisms important in theregulation of NF-�B activation may provide novel treatmentmodalities in preventing chemo and radio resistance in certaincancer settings (48). Like the canonical pathway, activation ofNF-�B by many genotoxic agents also involves the activation ofIKK and the release of NF-�B via IKK activation (40, 52).However, the genotoxic stress-induced pathway appears to in-volve a distinct series of upstream signaling steps. DNA-dam-aging agents induce the activation of the nuclear protein kinaseataxia telangiectasia mutated (ATM), and this appears to becritical for NF-�B activation by double-stranded break induc-ers (41, 42, 51, 69). Moreover, the nuclear localization of aregulatory subunit of the IKK complex, NF-�B essential mod-ulator (NEMO)/IKK�, also appears to be a critical event (41).This step of NEMO regulation appears independent of ATMand regulated by posttranslational modification by SUMO-1(small ubiquitin-like modifier 1). A recent study also suggeststhat the p53 inducible death domain protein and the receptor-interacting protein 1 participate in regulation of NEMO

* Corresponding author. Mailing address: Department of Pharma-cology, 301 SMI, 1300 University Avenue, University of Wisconsin,Madison, WI 53706. Phone: (608) 262-9281. Fax. (608) 262-1257. E-mail:[email protected].

� Published ahead of print on 30 October 2006.

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sumoylation (44). Receptor-interacting protein 1 was also pre-viously implicated in regulation of NF-�B activation via ATM(42). Nuclear NEMO associates with ATM and is further mod-ified by site-specific phosphorylation and ubiquitination to pro-mote export of ATM to the cytoplasm (91). In the cytoplasm,ATM appears to regulate IKK activity in a manner dependenton ELKS, an IKK regulatory subunit previously implicated incytokine signaling (23). Finally, the p90rsk kinase may alsoparticipate in this pathway prior to IKK activation (65).

Multiple stimuli can elicit increases in calcium levels thatmediate diverse molecular processes such as transcription, sig-nal transduction, and apoptosis (6, 9, 11). Calcium can alsoplay an important second messenger role for NF-�B activation.The stimulation of calcium-mediated signal transduction path-ways regulates NF-�B activation in B cells (21), T cells (22),and neuronal cells (58). In B cells, antigen binding to the B-cellreceptor induces the phospholipase C �2-mediated increase incalcium essential for the protein kinase C � (PKC �) activityrequired for NF-�B activation (90). Calcium-dependent PKC� phosphorylation of the scaffolding protein CARMA1 thenstimulates the IKK activity necessary for NF-�B translocationto the nucleus (79). Interestingly, IR also stimulates increasesin intracellular calcium (85) and causes an early (5 min) and atransient increase in the inducible isoform of nitric oxide (NO)synthetase, a calcium/calmodulin-dependent enzyme in Chi-nese hamster ovary cells (50). This calcium-dependent gener-ation of NO is important for the persistent cell stress signalinginitiated by acute IR exposure (60).

In the present study, we examined the role of intracellularcalcium in facilitating NF-�B activation by topoisomerase Iand II inhibitors, camptothecin (CPT), and etoposide (VP16),respectively. We found that intracellular calcium chelatorsBAPTA-AM and EGTA-AM inhibited NF-�B activation in-duced by these genotoxic agents. The effect of BAPTA-AMwas partly reversed by the addition of agents that increasedintracellular calcium levels, such as thapsigargin and ionomy-cin. Time course analyses indicated that the critical time periodwhere calcium imparted its NF-�B regulatory activity occurredbetween 60 and 90 min following CPT or VP16 treatment—thetime frame associated with the transition of NEMO into andout of the nucleus. We describe below our molecular analysesthat collectively suggest that calcium is critical for NEMOnuclear export via an RCC1-dependent, but CRM1- and cal-reticulin-independent, mechanism to mediate NF-�B activa-tion in response to CPT and VP16. Thus, our study provides aninitial insight into the role of calcium in regulating the NF-�Bactivation pathway induced by genotoxic stimuli.

MATERIALS AND METHODS

Chemicals and antibodies. N-Ethylmaleimide (NEM) was acquired fromSigma (St. Louis, MO), while INDO-1, MG132, and cycloheximide were fromCalbiochem (La Jolla, CA). The antibodies to ATM and CHKII were purchasedfrom Genetech (San Francisco, CA). The phospho-antibodies, ATM (pS1981)and I�B�, were from R& D Systems, Inc., (Minneapolis, MN) and UpstateBiotechnology (Charlottesville, VA), respectively, while the antibody to I�B�was from Santa Cruz (Santa Cruz, CA). The �-tubulin antibody was from On-cogene (San Diego, CA). The generation and culturing of the 1.3E2 murinepre-B cells has been described previously (41).

Cell culture and treatments. The American Type Culture Collection (ATCC,Manassas, VA) supplied the HEK293 and CEM cells. 1.3E2, Myc-NEMO-WT,SUMO-NEMO, and Ub-S85A-NEMO cells were generated previously (41, 91).BHK21 and tsBN2 cells were gifts from James Dahlberg (University of Wiscon-

sin—Madison). The HEK293, BHK21, and tsBN2 cells were cultured in Dul-becco’s modified Eagle’s medium with 10% fetal bovine serum (Sigma, St. Louis,MO) and penicillin/streptomycin (100 �g/ml). CEM cells were cultured in RPMImedia with 10% fetal bovine serum and penicillin/streptomycin (100 �g/ml).Unless specified, supplements were from Invitrogen (Carlsbad, CA). TheHEK293 cells were seeded at 6.5 � 105 cells/six-well dish approximately 18 hbefore treatment. The CEM cells were also seeded at 5 � 105 cells/ml approx-imately 18 h prior to treatment. The pH studies used Earle’s salt solution (pH7.2) supplemented with 10% bovine serum, NaHCO3, and L-glutamine. Thedifferent pHs were generated by using variations of NaHCO3 and HEPES (10mM) buffering systems incubated at 37°C in ambient air or 10% CO2. A pH of6.8 was acquired using HEPES and 10% CO2, a pH of 7.2 was acquired withHEPES/NaHCO3 and 10% CO2, a pH of 7.4 was acquired with NaHCO3 and10% CO2, and a pH of 8.2 was acquired with NaHCO3 and ambient air incu-bations. The pH of the media was measured with a pH meter at the terminationof the 2-h experiment and is the average of results from two determinations.Transient transfections of plasmid DNA in the HEK293 cells and tsBN2 cellswere done according to the method described by Huang et al. (40).

EMSA. The electrophoretic mobility shift assay (EMSA) analysis was doneaccording to the method of Huang et al. (40). The same sample was divided intotwo separate samples for analysis with the NF-�B (double stranded, 5�-TCAACA GAG GGG ACT TTC CGA GAG GCC-3�) and Oct-1 (double stranded,5�-TGT CGA ATG CAA ATC ACT AGA A-3�) radiolabeled probes. Separa-tion of the reaction was preformed on a 4% nondenaturing polyacrylamide gel,which was then dried and analyzed with a PhosphorImager (Amersham Bio-sciences, Piscataway, NJ).

Western blot analysis. Western blot analysis was done according to themethod of Huang et al. (40). The imaging analysis of Western blots was done byImage J software downloaded from the NIH imaging website.

Calcium analysis. The calcium analysis used cells (5 � 105/ml) preloaded withINDO-1-AM (2 �M) at 25°C for 30 min in buffer A (25 mM HEPES, 5.4 mMKCl, 0.8 mM MgCl2, 1.8 mM CaCl2, 121 mM NaCl, 5.5 mM glucose, 6 mMNaHCO3, pH 7.3, and 0.5% fetal bovine serum). Cells were isolated and resus-pended in buffer A. The cells were placed at room temperature for 20 min toallow de-esterfication of INDO-1-AM. The calcium analysis was carried out at37°C with flow cytometry (FacsVantage with Diva options; Becton Dickinson,Franklin Lakes, NJ). Propidium iodide (1 �g/ml) was added to the sample to gatelive and dead cells. A 325- to 360-nm wavelength excites the ratiometric INDO-1fluorophore into emitting light at 405 nm when bound to calcium and at 520 nmwhen free of calcium (87). To estimate cytoplasmic calcium concentrations, aprocedure according to Eastman was incorporated (24). Analysis using digitoninwith INDO-1 loading suggested that the calcium ionophore was predominantlyin the soluble fraction of the cells. A manganese-stimulated influx with ionomy-cin demonstrated that the majority of INDO-1-AM was processed to INDO-1(45). Collectively, the control (dimethyl sulfoxide [DMSO] and nontreatment)calcium estimations of 12 different experiments at 3 different time points (30, 60,and 180 min) had a mean of 102 nM 18 nM (standard deviation [SD]) and amedium of 106 nM. These values of calcium ranged from a minimum of 77 nMand a maximum at 129 nM. This is within the range of calcium estimated in thecontrols of nonstimulated cells in the literature (62, 67).

NEMO sumoylation analysis. Cells resuspended at 2 � 106 cells/ml in freshmedia 3 h prior to treatment were treated for 1 h with the indicated genotoxicagents. The isolated cells were pelleted (30 � 106 to 60 � 106/sample), washedonce with 1� phosphate-buffered saline, then quickly frozen on dry ice, andstored at �70°C until use. Samples were thawed on ice before resuspension inimmunoprecipitation (IP) buffer with 1% sodium dodecyl sulfate containing thefollowing protease inhibitors: 8 �M iodoacetamide, 10 �M N-ethylmaleimide,0.5 mM phenylmethylsulfonyl fluoride, 2 �M leupeptin, 9.5 TIU/ml aprotinin, 10mM sodium fluoride, 300 �M sodium orthovanadate. The samples were imme-diately boiled for 20 min to denature proteins, pelleted at 10,000 rpm for 10 minat room temperature, and diluted 1:10 in immunoprecipitation buffer containingprotease inhibitors for a total of 5 ml of reaction mixture. The NEMO antibody(BD Pharmingen, San Diego, CA) was added at 1.5 �g/ml, and samples weretumbled for 1 h at 4°C before the addition of protein G-Sepharose and furtherincubation overnight. The samples were then pelleted and washed four times inIP buffer. The samples were boiled in 2� sample buffer, run on a polyacrylamidegel electrophoresis gel, and transferred to a polyvinylidene difluoride filter (Mil-lipore, Bedford, MA). SUMO-1/GMP-1 (Zymed Laboratories, San FranciscoCA) or NEMO antibodies were used for analysis.

Immunofluorescence analysis. The staining of the 1.3E2 and Myc-NEMO-WTcells for nuclear localization NEMO was done according to the method of Wuet al. (91). The staining of the BHK21 and tsBN2 cells for p65 was performedsimilar to that previously described for mouse embryonic fibroblasts (39).

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Immuno-pull-down analysis. The Ran-GTP binding domain of AB1(RanBP2) (7) was subcloned into a pGST vector to express the glutathioneS-transferase (GST)-RanBP fusion protein in Escherichia coli. The carboxy ter-minus of AB1 not containing the Ran-GTP binding domain was also subclonedinto a pGST vector as a negative control. This negative control showed a markeddecrease in binding compared to the Ran-GTP binding domain (data notshown). The isolation and GST pull-down assay were done in a manner similarto previous studies (75). Briefly, cells treated for 90 min with VP16 were placedon ice, washed with phosphate-buffered saline, and lysed with buffer B (20 mMHEPES, pH 7.3, 120 mM potassium acetate, 5 mM MgCl2, 0.1% Tween 20, and100 mM of NEM) for 20 min. Dithiothreitol (200 mM) was then added, and thelysate was spun down at 13,000 rpm for 20 min at 4°C. The supernatant wasapplied at 4°C to a NAP-5 salt-exchange column equilibrated in buffer A (20 mMHEPES, pH 7.3, 120 mM NaCl, 10 mM magnesium acetate, 1 mM dithiothreitol,with phenylmethylsulfonyl fluoride and aprotinin). Equal concentrations of theeluate were then tumbled with GST-RanBP (7 �g/ml) for 1 h at 4°C after whichglutathione beads were applied for 1 h. The beads were pelleted, washed fourtimes in buffer B, and processed for Western blot analysis (see above).

Coimmunoprecipitation assay. The coimmunoprecipitation studies with tran-siently expressed hemagglutinin (HA)-tagged Ran-WT and Ran-G19V plasmidsin tsBN2 cells used a method described by Wu et al. (91).

Statistical analysis. The statistical analysis was conducted with Sigma Plot 8.0and Sigma Stat 3.0 software (SYSTAT Software, Inc., Richmond, CA).

RESULTS

BAPTA-AM blocks NF-�B activation in response to VP16and CPT. To test whether intracellular calcium is critical forNF-�B activation by genotoxic stimuli, HEK293 human em-bryonic kidney cells were exposed to the calcium chelatorBAPTA-AM 30 min prior to the addition of CPT or VP16 foran additional 2 h. BAPTA-AM effectively blocked NF-�B ac-tivation (Fig. 1A, lanes 3, 4, 7, and 8) (P � 0.001) withoutinterfering with its activation by tumor necrosis factor alpha(TNF-�) (Fig. 1A, lanes 11 and 12) (not significant [NS]).Western blot analyses indicated that both S32 and S36 phos-phorylation induced by IKK and I�B� degradation were inhib-ited by BAPTA-AM treatment (data not shown). BAPTA-AMalso blocked NF-�B activation by CPT, VP16, and IR (data notshown), but not by TNF-�, in CEM human T leukemic cells(Fig. 1B) (P � 0.001). BAPTA-AM similarly abrogated NF-�Bactivation by CPT, VP16, and IR, but not by lipopolysaccha-ride, in murine 70Z/3 pre-B cells (data not shown). Moreover,the use of a different intracellular calcium chelator in the samechemical family of BAPTA-AM, EGTA-AM (87), also inhib-ited the NF-�B activation with VP16 (Fig. 1C) (P � 0.01). Incontrast, the removal of extracellular calcium with cell-imper-meant EGTA had only a minor effect on NF-�B activation bygenotoxin treatment in HEK293 cells (Fig. 1D) (P � 0.05).These results suggested that calcium might have a selective andconserved role in NF-�B activation by the genotoxic stressagents in different cell systems.

VP16 and CPT induce increases in intracellular calciumlevels. Since BAPTA-AM prevented NF-�B activation byDNA-damaging agents, we next examined whether VP16 andCPT caused increases in intracellular free calcium levels. Gen-erally, calcium measurements are performed in a minimalbuffer formulated to maintain the viability of cells in an ambi-ent environment. However, we found that such buffer condi-tions used for intracellular calcium measurements were notpermissive for optimal NF-�B activation in response to geno-toxic agents. Cells treated with VP16 at room temperature(25°C) displayed reduced NF-�B activation compared tothose treated at 37°C (Fig. 2A). Furthermore, the medium pHs

of 6.8 and 8.3 increased and decreased, respectively, VP16-dependent activation of NF-�B compared to a normal pH of7.2 (Fig. 2B). Graded increases in the NaCl concentrationsfrom normal (110 mM) to hypertonic concentrations (170mM) in the culture buffer progressively abolished VP16-in-duced NF-�B activation without interfering with Oct-1 activity(Fig. 2C) (P � 0.001). Surprisingly, an NaCl concentration aslow as 130 mM NaCl could attenuate NF-�B activation inCEM T leukemic cells (data not shown). These preliminarystudies suggested that NF-�B activation by these genotoxicagents could be highly susceptible to cell culture conditions.Thus, we used a buffer condition with an NaCl concentration(121 mM) in agreement with Bootman and Berridge (13), anincubation temperature of 37°C, and a pH of 7.3 for thecalcium measurements below to ensure that conditions wereconducive for NF-�B activation by genotoxic agents.

To measure intracellular calcium increases, CEM cells wereloaded with INDO-1-AM, and the calcium analysis was carriedout at 37°C with flow cytometry. A 325- to 360-nm wavelengthexcites the ratiometric INDO-1 fluorophore into emitting lightat 405 nm when bound to calcium and at 520 nm when free ofcalcium (87). To estimate cytoplasmic calcium concentrations,

FIG. 1. BAPTA-AM inhibits NF-�B activation by VP16 and CPT.(A) HEK293 cells were exposed to BAPTA-AM (�M) 30 min prior toaddition of VP16 (10 �M, 2 h), CPT (10 �M, 2 h) or TNF-� (10 ng/ml,15 min). Total cell extracts were prepared and analyzed by EMSAusing an NF-�B probe (upper panels) or a Oct-1 probe (lower panels).The statistical analysis used analysis of variance for multiple compar-isons and Tukey’s test for multiple paired analysis. The gels representresults from one of three individual experiments. (B) A similar exper-iment as in panel A was performed using the CEM T leukemic cellline. (C) A similar experiment as in panel A was performed using theCEM T leukemic cell line with EGTA-AM treatment. (D) HEK293cells were exposed to EGTA (3 mM) 30 min prior to the addition ofVP16 and analyzed as in panel A.

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a procedure according to Eastman (24) was incorporated. Un-der these conditions, VP16 and CPT treatments demonstratedsignificant increases in calcium levels compared to the DMSOcontrol at 60 min (P � 0.05) and 180 min (P � 0.05), but notat 30 min (Fig. 2D; Table 1). Thus, steady-state increases inintracellular calcium levels could be observed in these leuke-mic cells between 60 and 180 min after exposure to theseDNA-damaging agents. TNF-� stimulation for 15 min did notincrease intracellular calcium levels, consistent with the lack offree calcium for NF-�B activation in this pathway (Fig. 2E;Table 2). BAPTA-AM alone or TNF-� and BAPTA-AM co-

FIG. 2. CPT and VP16 cause significant increases in steady-state cal-cium levels. (A) HEK293 cells were incubated either at 25°C or 37°C andexposed to VP16 (10 �M, 2 h) and analyzed by EMSA for NF-�B acti-vation. �, present; �, absent. (B) HEK293 cells were incubated in bufferswith different pHs as described in Materials and Methods and exposed toVP16 (10 �M) or TNF-� (10 ng/ml) for 2 h for EMSA of NF-�B activa-tion. (C) HEK293 cells were incubated in different salt concentrations andexposed to VP16 (10 �M, 2 h) for EMSA of NF-�B activation. The resultswere analyzed according to Fig. 1A. (D) CEM cells were preloaded withINDO-1-AM and exposed to the DMSO vehicle control, VP16 (10 �M),or CPT (10 �M) for the indicated times. At the end of each time point,intracellular calcium levels were estimated according to the method de-scribed in Materials and Methods. The data from Table 1 are depicted asthe percent increase over DMSO-vehicle calcium values at 30, 60, and 180min with VP16 (10 �M) and/or CPT (10 �M) treatment. The bar graphanalysis depicts the estimated steady-state calcium concentrations relativeto the DMSO-vehicle values times 100. Each bar is the average SD ofresults from three independent experiments at each time depicted on thegraph. (E) CEM cells were preloaded with INDO-1-AM and exposed toTNF-� (10 ng/ml) and other treatments for 15 min. The intracellularcalcium levels were estimated as described in Materials and Methods. Thedata from Table 2 are depicted as the percent increase or decrease overNT calcium values at 15 min with TNF-� (10 ng/ml) and BAPTA-AM (20�M) treatment. The bar graph analysis depicts the estimated steady-statecalcium concentrations relative to the NT values times 100. Each bar is theaverage SD of results from three independent experiments.

TABLE 1. Topoisomerase inhibitors increase calcium levels inCEM cells over timea

Stimulusb Time(min)

Estimated[Ca2�] (nM)c

ANOVA [Ca2�](P value)d

Multiple t test(P value)e

None 30 NS NDf

DMSO 125 4VP16 130 3

None 60 �0.001 �0.001DMSO 82 4VP16 96 6CPT 96 4 �0.05

None 180 �0.001 �0.05NT 97 14DMSO 105 9 NSVP16 132 9 �0.05CPT 158 9 �0.001

a Cells loaded with INDO-1-AM were exposed to the topoisomerase inhibitorsand analyzed by flow cytometry. The intracellular calcium levels were estimatedas described in Materials and Methods.

b DMSO is the vehicle control for VP16 (10 �M) and CPT (10 �M). TheDMSO concentration for treated and vehicle-control determinants was 0.01%.NT indicates the no treatment control.

c The estimated calcium concentration is the average of results from threeindividual experiments SD. Each experimental estimate of the calcium con-centration was derived from the average of 10,000 events or cells.

d The mean calcium concentrations (n 3) of the different treatment groupswere great enough to be statistically significant (P � 0.05) from multiple-com-parison analysis of variance (ANOVA).

e The individual calcium concentrations were statistically significant from theDMSO or NT values, as determined by Tukey’s multiple paired analysis.

f ND, not determined.

TABLE 2. TNF-� treatment does not alter calcium levels inCEM cellsa

Stimulusb Estimated [Ca2�](nM)c

ANOVA [Ca2�](P value)d

Multiple t test(P value)e

NT 55 4 �0.005TNF-� 58 2 NSTNF-� � BAPTA 34 1 �0.001BAPTA 33 1 �0.001

a Cells loaded with INDO-1-AM were exposed to TNF-� and analyzed by flowcytometry. The intracellular calcium levels were estimated as described inMaterials and Methods.

b NT indicates the no treatment control. TNF-� (10 ng/ml) was added for 15min. BAPTA-AM (20 �M) was added 10 min prior to the start of the experiment.

c The estimated calcium concentration is the average of results from threeindividual experiments SD. Each experimental estimate of the calcium con-centration was derived from the average of 10,000 events or cells.

d The mean calcium concentrations (n 3) of the different treatment groupswere great enough to be statistically significant (P � 0.01) from multiple-com-parison analysis of variance (ANOVA).

e The individual calcium concentrations were statistically significant from theNT values as determined by Tukey’s multiple paired analysis.



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treatment reduced calcium below the nontreated (NT) con-trols.

The increase in intracellular calcium is necessary for opti-mal NF-�B activation. To determine whether the rise in freecalcium levels around 60 min after exposure to VP16 is criticalfor NF-�B activation, we next compared the kinetics of NF-�Bactivation with that of ATM activation (4) in CEM andHEK293 cells. ATM activation, as measured by the level ofphospho-S1981-ATM antibody reactivity (4), reached maximallevels within 15 min of VP16 exposure. In contrast, NF-�Bactivation as measured by the EMSA in both cell types couldonly be detectable around 60 min following VP16 exposure butrequired about 120 min to reach maximal activation (Fig. 3A)(others not shown). Thus, the rise in calcium observed in Fig.2D appears to occur subsequent to ATM activation and priorto peak NF-�B activation.

If the calcium rise between 60 and 120 min is critical forNF-�B activation by VP16 treatment, it should be possible toprevent NF-�B activation by adding BAPTA-AM at a pointsubsequent to ATM activation. To test this idea, we addedBAPTA-AM at different times after VP16 addition, as de-picted in Fig. 3B, to evaluate the time point at which thiscalcium chelator causes NF-�B inhibition. Addition ofBAPTA-AM significantly reduced NF-�B activation comparedto DMSO controls at 30 min (37% 7.0%, P � 0.001) and 60min (47% 13%, P � 0.01) after VP16 addition (Fig. 3C).However, BAPTA-AM addition at 90 min after VP16 exposurefailed to inhibit NF-�B activation. The inhibition of NF-�Bactivation with BAPTA-AM addition at 30 min after VP16treatment did not inhibit ATM activation or the phosphoryla-tion of a substrate of ATM, CHK2 (Fig. 3D) (NS). Thus,BAPTA-AM inhibited NF-�B activation up to 60 min afterVP16 exposure subsequent to ATM activation. This timeframe correlated with the calcium increase observed at 60 min.

Calcium mobilizers can inhibit the effect of BAPTA-AM. Ifincreases in intracellular calcium are indeed critical for NF-�Bactivation by CPT and VP16, it may be possible to reverse theeffects of BAPATA-AM by increasing the levels of intracellu-lar calcium by calcium mobilizers, such as thapsigargin or iono-mycin. Thapsigargin releases calcium by inhibiting the calciumATPase pump responsible for maintaining the calcium storesin the endoplasmic reticulum (30, 84). The calcium ionophoreionomycin releases calcium from the extracellular mediathrough the plasma membrane and intracellular stores (30, 70).When HEK293 cells were cotreated with BAPTA-AM andthapsigargin or ionomycin, the BAPTA-AM-dependent inhi-bition of NF-�B activation was partially reversed by bothagents (Fig. 4A). Similarly, ionomycin also reversed the inhib-itory effect of BAPTA-AM in CEM cells with CPT treatment(data not shown). Moreover, when HEK293 cells were treatedwith a suboptimal dose of VP16 (3 �M), thapsigargin was ableto increase NF-�B activation in a dose-dependent manner(Fig. 4B). The thapsigargin-dependent enhancement of NF-�Bactivation was also observed at the higher concentration ofVP16 of 10 �M. Thapsigargin or ionomycin did not affectNF-�B activation by TNF-� (Fig. 4C) nor did treatment alonewith these calcium mobilizers activate NF-�B in this cell sys-tem (Fig. 4B and C). These observations further supported therole of intracellular calcium in promoting NF-�B activation bygenotoxic agents.

Calcium is required for nuclear export of NEMO. TheSUMO-1 modification of NEMO is one of the early molecularevents critical for the genotoxic stress-induced NF-�B activa-tion, which occurs around 60 min after VP16 exposure (41, 56).Since this time frame partly overlapped with the rise in intra-cellular calcium and inhibition of NF-�B activation byBAPTA-AM (Fig. 2 and 3), we next analyzed whetherBAPTA-AM treatment inhibited the SUMO-1 modification ofNEMO. Total cell extracts prepared from CEM cells exposedto VP16 with or without BAPTA-AM treatment were immu-

FIG. 3. BAPTA-AM exerts NF-�B inhibitory effects up to 60 minafter VP16 exposure. (A) HEK293 cells were exposed to VP16 (10�M) and processed at the indicated time points in duplicate. Theproteins were analyzed by EMSA for NF-�B activation and by Westernblot analysis with antibodies to pS1981-ATM, ATM, and �-tubulin.(B) Diagram depicting the treatment protocol used to applyBAPTA-AM after VP16 exposure. (C) HEK293 cells were exposed toVP16 (10 �M) and BAPTA-AM (30 �M) as depicted in panel C andanalyzed by EMSA for NF-�B activation. The results were analyzedaccording to the method described for Fig. 1A. (D) HEK293 cells wereexposed to BAPTA-AM (30 �M) 30 min after the addition of VP16(10 �M) and processed for Western blot analyses with antibodies topS1981-ATM, ATM, ChK2, and I�B�. The results were quantified byImage J software and analyzed according to the method described forFig. 1A.



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noprecipitated with an anti-NEMO antibody and analyzed byWestern blotting using an anti-SUMO-1 antibody. Induciblesumoylation of NEMO was detectable in the VP16 sample(Fig. 5A, lane 2). This sumoylation was not abrogated byBAPTA-AM treatment (lane 3 and 4). General sumoylatedprotein levels in total cell extracts appeared somewhat in-creased in the VP16- plus BAPTA-AM-treated samples (lane3 and lower panel). Consistent with the lack of inhibition ofsumoylation of NEMO by BAPTA-AM treatment, VP16-in-ducible NF-�B activation mediated by the SUMO-NEMO fu-sion protein expressed in the 1.3E2 NEMO-deficient pre-B cellline (41) was also inhibited by the calcium chelator (Fig. 5B)(P � 0.001). Moreover, NF-�B activation via a Ub-S85A-NEMO fusion protein that could bypass the necessity of ATM-dependent phosphorylation and ubiquitination of NEMO inresponse to VP16 exposure (91) was also sensitive to inhibitionby BAPTA-AM (Fig. 5C) (P � 0.001). These observations

suggested that calcium might be necessary for an event down-stream of NEMO ubiquitination.

A known event immediately downstream of NEMO ubiq-uitination is its nuclear export. This is previously observed as adecline in the percentage of cells displaying nuclear staining ofNEMO, which was transiently induced by VP16 exposure (91).To determine whether BAPTA-AM prevents the export ofNEMO, the subcellular localization of NEMO was monitoredin 1.3E2 cells stably reconstituted with Myc-NEMO protein asdescribed previously (91). VP16-inducible increases in NEMOnuclear staining could be observed at up to 2 h when the cellswere immobilized on a glass coverslip (Fig. 6A and B). Whencells were immobilized in this way, NF-�B activation, as mea-sured by p65 nuclear localization, peaked around 3 h afterVP16 treatment, which was about 1 h later than when the samecells were grown in suspension cultures (Fig. 3A and B) (91).The reason for this delay of NF-�B activation of immobilizedcells is unknown. Consistent with the lack of defect of sumoy-lation with BAPTA-AM treatment (Fig. 5A), nuclear accumu-lation of NEMO in response to VP16 exposure was also un-perturbed by BAPTA-AM (Fig. 6A, 2 h). In contrast, at the 3-htime point, BAPTA-AM-treated cells showed more NEMOnuclear staining than the VP16-treated cells (Fig. 6A, 3 h) (P �0.001). Thus, these results suggested that BAPTA-AM causedeither direct or indirect interference on NEMO nuclear exportinduced by genotoxic stress conditions without preventing itsearlier nuclear import process.

Nuclear export of NEMO is independent of CRM1 or cal-reticulin. Previous studies suggested a critical role for NEMOnuclear export in NF-�B activation by several genotoxic agents(91). However, the mechanism of this nuclear export remainsuncharacterized. While we previously showed that activation ofIKK by CPT and VP16 was not blocked by leptomycin B(LMB), an inhibitor of the nuclear export receptor CRM1(41), we did not report the effect of LMB on nuclear export ofNEMO. Thus, we tested whether LMB would modulateNEMO export in response to VP16 exposure. Consistent withthe lack of IKK inhibition by LMB treatment (41), we failed toobserve a marked inhibition of the decline in the percentage ofcells displaying nuclear staining of NEMO compared to VP16alone in a 3-h time course study (Fig. 6B). Because the 3-h timepoint showed a potential difference between the two condi-tions, we further focused on this time using BAPTA-AM as acontrol (Fig. 6C). There was no significant difference betweenthe percentage of cells displaying nuclear staining of NEMObetween the VP16 and VP16 plus LMB conditions. However,BAPTA-AM-treated cells showed a significantly higherNEMO staining than cells under these conditions (P � 0.01).In these experiments, LMB treatment caused nuclear accumu-lation of I�B� (Fig. 6D) and prevented its degradation afterVP16 exposure (Fig. 6E, lane 8) as described previously (41).In contrast, I�B�, which does not accumulate in the nucleuswith LMB treatment (39, 83), became more susceptible toVP16-inducible degradation (Fig. 6E, compare lanes 6 and 8),possibly due to the lack of competition by I�B� in the cyto-plasm for signal-induced degradation. These I�B protein anal-yses demonstrated that LMB was effectively inhibiting LMBactivity under the experimental conditions employed. Thus,these results collectively indicated that NEMO nuclear export

FIG. 4. Thapsigargin and ionomycin reverse the NF-�B inhibitoryeffects of BAPTA-AM. (A) HEK293 cells treated with VP16 (lane 2)for 2 h were cotreated with BAPTA-AM (30 �M) (lane 3) at 30 min inthe absence (�) or presence (�) of increasing amounts of thapsigargin(Tg) at 0.1 �M (lane 4), 0.5 �M (lane 5), or 1.0 �M (lane 6) orionomycin (Iono) at 0.1 �M (lane 7), 0.5 �M (lane 8), and 1.0 �M(lane 9). DMSO-treated cells (lane 1) were used as controls. The cellswere processed for NF-�B activation and the Oct-1 loading control.(B) HEK293 cells were exposed to VP16 (3 or 10 �M) in the absenceor presence of increasing amounts of thapsigargin (Tg, �M) for EMSAof NF-�B activation and an Oct-1 loading control. Cells exposed onlyto thapsigargin were also analyzed in parallel. (C) HEK293 cells wereexposed to TNF-� (3 or 10 ng/ml) in the absence or presence ofionomycin (Iono, 1.0 �M) or thapsigargin (Tg, 0.1 �M) for EMSA ofNF-�B activation and an Oct-1 loading control. Cells exposed only toionomycin or thapsigargin were also analyzed in parallel.



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induced by genotoxic stress was likely mediated by a CRM1-independent export mechanism.

Since our results found a potential role for calcium in pro-moting NEMO nuclear export, we next analyzed the role ofcalreticulin, the only nuclear export receptor that is reported todepend on free calcium for its activity (35, 36). We reduced theexpression of calreticulin by small interfering RNA (siRNA) inHEK293 cells. Even though we observed substantial silencingof calreticulin expression, as measured by Western blot anal-ysis (Fig. 6G), NF-�B activation by VP16 was not inhibited(Fig. 6F). Since a small amount of calreticulin that was presentin these knockdown cells could fully support NF-�B activation,we next examined NF-�B activation in fibroblasts derived fromcalreticulin knockout mice (59). However, we did not observea decrease in NF-�B activation in these cells (data not shown).Thus, like CRM1 above, calreticulin also appeared to be non-essential for NF-�B activation by genotoxic stress conditions.

Calcium is required for a nuclear export event downstreamof the assembly of the export complex containing Ran-GTPand NEMO. Many known nuclear export processes are regu-lated by an energy gradient in the form of Ran-GTP with itsgreatly higher level in the nucleus than in the cytoplasm (74).The nuclear Ran guanine nucleotide exchange factor RCC1maintains the high Ran-GTP levels in the nucleus (78). To gaininsight into the role of Ran-GTP in promoting NF-�B activa-tion in response to a genotoxic stress condition, we next exam-

ined the tsBN2 cell line that is derived from a baby hamsterkidney cell line (BHK21) and harbors a temperature-sensitivemutant of RCC1 protein (18). When these cells were grownunder the nonpermissive temperature of 39.5°C, RCC1 wasdegraded over the course of 3 h (Fig. 7A, lanes 8 and 11) (63).When these cells were then treated with CPT (at 33°C), NF-�Bactivation was inhibited (compare lanes 5 and 11). Returningthe cells to the permissive temperature of 33°C for 6 h restoredthe RCC1 expression before CPT treatment restored NF-�Bactivation (lane 12). Even though p65 can be a nucleocytoplas-mic shuttling protein (32, 38, 57, 82), incubation of tsBN2 cellsin the nonpermissive condition for 3 h did not alter the cyto-plasmic p65 localization (Fig. 7B). This demonstrated that thelack of NF-�B activation under the nonpermissive conditionwas not due to mislocalization of inactive p65 protein to thenucleus.

RCC1 regulates nuclear export by loading GTP to Ran toassemble an export complex composed of Ran-GTP, a specificexport receptor and the cargo protein (29). To examine ifDNA damage conditions could induce the formation of a sim-ilar nuclear transport complex containing NEMO, tsBN2 cellswere first transiently transfected with HA-tagged wild-typeRan (HA-Ran-WT) and HA-Ran-G19V, a mutated Ran(G19V) with diminished GTPase activity, and then stimulatedwith VP16 at nonpermissive temperature, and coimmunopre-cipitation experiments were performed with extracts derived

FIG. 5. BAPTA-AM does not inhibit NEMO sumoylation induced by VP16 and inhibits NF-�B activation mediated by SUMO-NEMO andUb-S85A-NEMO fusion proteins. (A) CEM cells were treated with VP16 (10 �M) for 1 h with (�) or without (�) BAPTA-AM (30 �M, 30 min)treatment or pretreatment (20 �M) and processed as described in Materials and Methods for NEMO immunoprecipitation and Western blotanalysis with anti-SUMO-1 (�-SUMO-1) antibody and anti-NEMO for a control. Total cell extracts were also analyzed by anti-SUMO-1 antibodyas an additional control. (B) 1.3E2 NEMO-deficient murine pre-B cells stably reconstituted with Myc-NEMO or SUMO-NEMO proteins weretreated with VP16 (10 �M) for 2 h with or without BAPTA-AM (10 �M, 30 min) treatment or pretreatment (20 �M) for EMSA of NF-�Bactivation and an Oct-1 control. The results were analyzed according to the method described for Fig. 1A. (C) 1.3E2 cells stably reconstituted withUb-S85A-NEMO protein were treated with VP16 (10 �M) for 2 h with or without BAPTA-AM (10 �M, 30�) treatment or pretreatment for EMSAof NF-�B activation and an Oct-1 control. The results were analyzed according to the method described for Fig. 1A.



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FIG. 6. NEMO nuclear export induced by VP16 treatment is blocked by BAPTA-AM. (A) 1.3E2 cells stably expressing Myc-NEMO wereplated on a glass coverslip overnight and then exposed to VP16 (10 �M) for 2 or 3 h, with coexposure to BAPTA-AM (30 �M) given 30 min afterVP16 exposure. Cells were fixed and stained with anti-Myc (�-Myc) antibody (9E10) as the primary antibody and fluorescein isothiocyanate-labeledsecondary anti-mouse antibody to examine nuclear localization of NEMO. 4�,6�-Diamidino-2-phenylindole (DAPI) staining was used for nuclearstaining. The bar graph represents the average SD of results from three independent experiments each at 2 h and 3 h for the percentage of cellsdisplaying nuclear NEMO staining. Each experiment consisted of counting at least 300 individual cells from multiple areas on the slide. Analysisof variance determined that there was a statistically significant difference among the means of the different treatment groups (P � 0.01) at 2 h.The Tukey’s multiple paired analysis determined that there was a statistically significant difference between the means of the DMSO-vehicle andVP16 (*, P � 0.05) but not VP16 and VP16 plus BAPTA (NS) at 2 h. Analysis of variance determined that there was a statistically significant



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from these treated cells. Exogenous Ran was transfected toincrease the cellular levels of Ran-GTP. Ran-G19V was alsoemployed, since the export function of microinjected Ran-G19V was previously shown to be insensitive to the decrease of

RCC1 activities in the tsBN2 cells (54) and thus provides ameans to further increase Ran-GTP levels at the nonpermis-sive condition. A small but reproducible amount of endoge-nous NEMO was coimmunoprecipitated using anti-HA anti-

difference among the means of the different treatment groups (P � 0.01) at 3 h. The Tukey’s multiple paired analysis determined that there was astatistically significant difference between the means of the DMSO-vehicle and VP16 (*, P � 0.05) and VP16 and VP16 plus BAPTA (**, P 0.01) at3 h. (B) Similar analysis as in panel A was done at 1, 2, and 3 h after exposure to VP16 with or without LMB (20 ng/ml). (C) Similar analysis as in panelA was done at 3 h after exposure to VP16 with or without LMB (10 ng/ml) or with or without BAPTA (10 �M). Both agents were given 30 min afterVP16 exposure. Analysis of variance determined that there was a statistically significant difference among the means of the different treatment groups(P � 0.01). The Tukey’s multiple paired analysis determined that there was a statistically significant difference between the means of the VP16 plusBAPTA and VP16 (*, P � 0.01) and also VP16 plus LMB (**, P � 0.01). However, VP16 and LMB were NS. (D) 1.3E2 cells stably expressing Myc-NEMOwere left untreated or exposed to LMB, fixed, and stained with anti-I�B�, anti-Myc, and DAPI to examine the nuclear accumulation of I�B� with LMBtreatment. (E) 1.3E2 cells stably expressing Myc-NEMO were left untreated or treated with LMB (20 ng/ml) for 30 min and then exposed to VP16 (10 �M, 2 h)or lipopolysaccharide (10 �g/ml, 30 min). Total cell extracts were then analyzed by EMSA for NF-�B activation and Western blotting with anti-I�B� andanti-I�B�. (F) HEK293 cells were transiently transfected with siRNA specific to calreticulin (CRT) or control nonspecific siRNA 24 h prior to exposure to VP16(10 �M, 2 h). Total cell extracts were then analyzed for NF-�B and Oct-1 activities by EMSA. (G) Serial dilutions of total cell extracts prepared from panel Fwere analyzed for the levels of calreticulin by Western blotting using anticalreticulin antibody. An antitubulin blot was also performed as a loading control.

FIG. 7. Calcium is required for a nuclear export event downstream of the assembly of the export complex containing Ran-GTP and NEMO. (A) BHK21and tsBN2 cells were incubated at permissive (33°C) or nonpermissive (39.5°C) temperature for 3 h to cause RCC1 degradation and then exposed to CPT (10�M, 2 h) at 33°C. Cells in lanes 3, 6, 9, and 12 were first incubated at nonpermissive temperature for 3 h and then returned to the permissive temperature foradditional 6 h prior to CPT treatment as above. Total cell extracts were prepared for NF-�B EMSA and Western blotting with anti-RCC1 (�-RCC1) andanti-Ran antibodies. (B) Indicated cells grown at the nonpermissive temperature for 3 h or left in the permissive condition were fixed and stained with anti-p65antibody. (C) tsBN2 cells were transfected with HA-Ran-WT or HA-Ran-G19V, and these transfected cells were incubated at the nonpermissive temperaturefor 4 h. Cell extracts were then isolated, and IP assays were performed using anti-HA antibody as described in Materials and Methods. (D) Myc-NEMO wastransiently transfected into HEK293 cells, and the cells were left untreated or treated with VP16 for 90 min. BAPTA-AM (30 �M) was also applied 30 min afterthe addition of VP16. The cell extracts were prepared and the GST pull-down assays were performed using GST-RanBP as described in Materials and Methods.Lanes 1 to 3 show Western blot analysis of indicated proteins after GST pull-down, while lanes 4 to 6 show Western blot analysis of 5% of the input controls.(F) HEK293 cells were treated with BAPTA-AM (20 �M, 120 min). The GST-RanBP pull-down assays were performed as for panel D. (E) HEK293 cells weretreated with VP16 (10 �M) for 90 min with BAPTA-AM (30 �M, 30 min). The GST-RanBP pull-down assays were done as described above.



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body following VP16 treatment (Fig. 7C). These resultssuggested that VP16 treatment induced the formation of acomplex between Ran and NEMO even in the absence ofRCC1 activity. Although endogenous NEMO could be immu-noprecipitated with HA-Ran proteins, it was unclear whetherNEMO associated with Ran-GTP, a form of Ran that is nec-essary for nuclear export. A Ran-binding domain (RanBD)from the Ran binding protein 1 or 2 can be employed to assessRan-GTP levels because the RanBD specifically binds to theGTP-bound form of Ran (7, 75). Moreover, buffers containinghigh levels (100 mM) of NEM can be used to maximally pre-serve cellular Ran-GTP levels in a GST pull-down assay, sincethe cytosolic Ran GTPase-activating protein and nuclearRCC1 activities, both of which are introduced by cell lysis, areblocked by high NEM levels (75). Using this GST pull-downassay, we found evidence that NEMO associated with Ran-GTP in a VP16-inducible manner (Fig. 7D). Significantly, thisinducible interaction was not blocked by BAPTA-AM treat-ment (lane 3). Additionally, the GST pull-down assay demon-strated that BAPTA-AM with or without VP16 treatment didnot alter the cellular levels of Ran-GTP (Fig. 7E and F). Theseresults suggested that the inhibitory effect of BAPTA-AM onNEMO nuclear export is unlikely due to RCC1 inhibition butrather due to inhibition of a nuclear export step downstream ofthe formation of Ran-GTP with a putative NEMO nuclearexporter and NEMO following genotoxic stress induction.

DISCUSSION

Studies of the signaling pathways induced by immune andinflammatory cytokines have provided fundamental insightsinto the mechanisms of activation of the cytoplasmically local-ized inactive NF-�B complexes. The consensus has emergedfrom these studies where the “canonical” NF-�B activationpathway is induced by activation of cytoplasmic IKK complexand degradation of NF-�B-associated inhibitor proteins to re-lease active NF-�B into the nucleus (17). NEMO plays anessential role in this canonical pathway. Similarly, a picture isalso emerging in which certain genotoxic stress inducers me-diate NF-�B signaling by triggering IKK activation (31, 42, 52,61, 65, 69). NEMO also plays an essential role in activation ofIKK and NF-�B by DNA-damaging agents (41, 44, 91). How-ever, unlike the immune and inflammatory modulators, whereNEMO remains in the cytoplasm and may be modified byunconventional K63-linked polyubiquitination (17), a smallamount of free NEMO appears to leave the cytoplasm eitherconstitutively (44) or in response to DNA damage stimuli toenter the nucleus in association with SUMO-1 modification(41, 91). In the nucleus, NEMO then undergoes a series ofmodifications to be ultimately exported to the cytoplasm tosomehow stimulate IKK activation (91). Thus, one of the im-portant questions to understand the NF-�B signal transductionpathway induced by nuclear DNA damage is: how is the trans-fer of the DNA damage signal from the nucleus to the cyto-plasm regulated? In the present study, our results collectivelysuggest that nuclear export of NEMO, a component of thenuclear DNA damage signal, in response to genotoxic stressrelies on a calcium-dependent process.

Previous studies have demonstrated that exposure of cells toIR can increase free calcium levels (85). In this case, the

release of intracellular calcium stores from the endoplasmicreticulum (ER) appeared necessary to induce an influx ofextracellular calcium, which resulted in a transient increase infree calcium levels within minutes. This relatively rapid andtransient increase in calcium levels was necessary for the in-crease in activity of the inducible form of calcium/calmodulin-dependent NO synthetase (50). However, calcium-stimulatedchanges in gene regulation generally occur over a period ofhours (10). To our knowledge, even though recent studiessuggested that calcium could be mobilized following cell stim-ulation with CPT (76) and VP16 (15), whether these agentscould cause activation of a calcium-dependent transcriptionfactor was unknown. Our calcium measurements using CEMcells demonstrated that the increases in intracellular calciumfollowing CPT and VP16 exposure can occur with relativelyslow kinetics, taking 60 min to reach significant levels (Fig.2D; Table 1). While we found evidence that rapid calciummobilization could occur transiently within 5 min after VP16treatment of HEK293 cells (C. M. Berchtold, unpublishedobservations), our time course analyses using BAPTA-AMclearly demonstrated the critical role for a calcium-dependentprocess occurring between 60 and 90 min post-genotoxic stressinduction to mediate NF-�B activation (Fig. 3). This timecourse of calcium requirement for the genotoxic stress-depen-dent NF-�B activation also contrasts with previous roles thatwere implicated in calcium-dependent NF-�B regulation. Pre-vious studies showed that the calcium-dependent activation ofNF-�B could be very rapid. For example, the activation ofNF-�B with ionomycin and a phorbol ester occurs within min-utes (15 min) of exposure in B cells through a calcium-mediated protein kinase C pathway (21, 79). Studies in neuro-nal cells also demonstrated a rapid calcium-induced NF-�Bactivation mediated by calmodulin-dependent kinase II withina similar time period (58). When we examined the role of PKCisoforms and calmodulin-dependent kinase II by means ofchemical inhibitors, we failed to find evidence for their involve-ment in NF-�B activation by genotoxic stimuli (C. M. Berch-told, unpublished observations) (16). Our data are more con-sistent with a calcium-dependent process occurring between 60and 90 min post-genotoxic stress induction (Fig. 3).

Is DNA damage per se required for calcium increases fol-lowing exposure to the genotoxic agents? CPT and VP16 in-teract with the reaction intermediates between DNA topo-isomerase I and II, respectively, leading to the generation of“cleavable complexes” in the nucleus (53). These lesions cangenerate DNA single- and double-strand breaks, thereby jus-tifying their designation as “DNA damaging agents.” However,these agents can also induce other cell stresses in the form ofoxidative stress (49), ceramide (77), and the ER stress proteinresponse (72, 73). These different stress conditions can stimu-late increases in intracellular calcium without the need forDNA damage (27). Moreover, the deregulation of calcium inisolated organelles such as mitochondria and ER can alsooccur with direct VP16 exposure (15). In the case of IR, oxi-dative stress induced by the ionization of water and macromol-ecules in cells, such as lipids and proteins (14), could be criticalfor calcium mobilization from the ER (85). While the magni-tude of EGTA effect was modest in our studies (Fig. 1C), wecannot exclude the possibility that extracellular calcium couldalso be a critical source for intracellular calcium increases



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following CPT and VP16 exposure under certain conditions.For example, genotoxic agents could elicit a decrease in theER calcium concentration, which could then signal for theinflux of extracellular calcium (66). Finally, studies showed thatcells containing a defective ATM were aberrant in the mobi-lization of calcium stimulated by serum (26). These studiessuggested the possibility that an ATM-dependent mechanismcould also contribute to the calcium increases induced by VP16and CPT treatment. When we reduced the expression levels ofATM by means of specific siRNAs, we found a modest inhib-itory effect on calcium mobilization in CEM cells (data notshown). We were unable to study the effect of chemical inhib-itors against ATM (e.g., wortmannin) due to perturbation ofthe calcium measurement. Thus, we cannot currently excludethe potential involvement of ATM in regulating calcium re-lease, and further in-depth analyses are required to determinethe source and the mechanism of intracellular calcium in-creases induced by CPT and VP16. These studies will help sortout which exact cell stresses beyond DNA damage per se arenecessary for activation of NF-�B by DNA-damaging agents.

Our molecular studies suggested that the step at which cal-cium imparts regulation on the NF-�B activation pathway in-duced by CPT and VP16 is likely at the NEMO export step.Ubiquitin appears to play a critical role in promoting NEMOnuclear export (91). A previous study suggested that mono-ubiquitination of the tumor suppressor p53 regulates its nu-clear export (1). A ubiquitin-p53 fusion strategy was employedto demonstrate the role of monoubiquitination and inhibitionby LMB, thus suggesting a role of CRM1 in mediating nuclearexport of monoubiquitinated p53. Unlike the case with p53,our analyses indicated that nuclear export of NEMO inducedby VP16 was insensitive to inhibition by LMB (Fig. 6B and C).This also contrasted with a previous study indicating that LMBcan cause nuclear accumulation of NEMO in unstressed HeLacells (89). To our knowledge, the only reported calcium-de-pendent nuclear export receptor is calreticulin (35, 36). How-ever, our results indicated that calreticulin is not essential forNF-�B activation by VP16.

Our studies suggest that NEMO is complexed to Ran-GTPfollowing a DNA damage stimulus and that BAPTA-AM doesnot appear to modulate these interactions (Fig. 7). Similarly,our molecular and biochemical analyses indicated that theupstream regulatory events invoked by genotoxic stress condi-tions, such as activation of ATM and NEMO sumoylation, arenot mediated by calcium. Because Ran-GTP is normally cou-pled to an export receptor which, in turn, plays a prominentrole in binding to the transporting cargo (29), we can inferfrom the VP16-inducible interaction between NEMO andRan-GTP that a similar trimeric complex is probably assem-bled with a putative nuclear export receptor for NEMO, whichis required for NEMO export and NF-�B activation. However,there is another regulatory step(s) described for the nuclearexport of a protein subsequent to the formation of a Ran-GTP/nuclear export receptor/cargo trimeric complex but prior to therelease of the cargo on the cytoplasmic surface of the nuclearpore complex. Nucleoporin proteins containing phenylalanine/glycine (FG) binding motifs line the inner surface of the nu-clear pore (86). Interactions between different FG-containingnucleoporins and Ran-GTP-mediated cargos appear to differ-entially facilitate multiple export pathways through the nuclear

pore (80). Interestingly, these FG-containing nucleoporins arein a native unfolded state which inherently makes the aqueousbinding surface of these proteins highly susceptible to changesin temperature, pH, salts, and calcium (68, 88). Moreover,more global conformation changes of the nuclear pore com-plex can also occur with different calcium-metabolizing com-pounds (25). Therefore, a calcium-dependent step for nuclearexport of NEMO may include any one of these known orunknown additional steps. Additionally, since BAPTA-AM didnot perturb TNF-�-dependent NF-�B activation, we suggestthat the calcium chelator is not a general inhibitor of nuclearexport. BAPTA-AM did not apparently block the CRM1-de-pendent nuclear export of I�B�/NF-�B complexes required forTNF�-dependent NF-�B activation (38). Further studies arerequired to investigate this interesting mechanism.

While it was not the focus of the current study, unexpectedly,the results shown in Fig. 2A to C revealed that a specificextracellular milieu could impart dramatic effects on NF-�Bactivation in response to genotoxic stress agents. These obser-vations suggest the possibility that NF-�B activation by certainDNA-damaging agents could be highly susceptible to subtlechanges in the cellular microenvironment. Coupled with thenotion that NF-�B can modulate survival and drug resistanceof tumor cells (3, 5, 20, 46), the results suggest a novel possi-bility that specific variations in tumor microenvironmentscould selectively attenuate or augment the NF-�B response toDNA-damaging anticancer agents. Changes in pH, intracellu-lar and extracellular calcium, osmolarity, and temperature ap-pear to modulate NF-�B activation by DNA-damaging agents.Interestingly, some of these changes can be observed withneoplastic transformation, within tumor microenvironments invarious cancer types, and during radiation therapy and chemo-therapy (8, 33, 43, 64, 71, 92). Therefore, understanding themechanisms responsible for enhancement (e.g., low pH, highertemperature) and the decrease (e.g., high osmolarity) ofNF-�B activation may provide novel strategies to preventNF-�B activation and to increase the efficacy of anticancertherapy.

ACKNOWLEDGMENTS

We thank James Dahlberg for tsBN2 and BHK21 cell lines anddiscussions regarding nuclear export receptors. We also thank M.Michalak for providing the calreticulin knockout and matched mouseembryonic fibroblast lines. We also thank Kathy Schell of the UWFlow Cytometry Laboratory for help with flow analyses and the mem-bers of the Miyamoto Laboratory for their helpful discussions.

This work was supported by R01-CA77474 and R01-CA81065 fromNIH and a Shaw Scientist Award from the Greater Milwaukee Foun-dation to S.M. Z.-H.W. is also funded by a special Fellowship from theLeukemia and Lymphoma Society.

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Calcium-Dependent Regulation of NEMO Nuclear Export in Response to Genotoxic Stimuli

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