ICAM-1 expression is highly NF-jB-dependent in A549 cells No role for ERK and p38 MAPK Neil S. Holden 1 , Matthew C. Catley 2 , Lisa M. Cambridge 2 , Peter J. Barnes 2 and Robert Newton 1 1 Department of Biological Sciences, University of Warwick, Coventry, UK; 2 Thoracic Medicine, National Heart & Lung Institute, Imperial College Faculty of Medicine, London, UK The transcription factor nuclear factor jB(NF-jB) is an activator of multiple cytokines, chemokines and adhesion molecules, which are important in inflammatory diseases such as asthma, and is consequently considered as an attractive therapeutic target. In the present study, a con- stitutively active dominant version of IjBa,IjBaDN, was introduced into A549 pulmonary cells by adenovirus- mediated delivery. The dominant IjB, but not a null viral vector, prevented the induction of NF-jB-dependent transcription by both tumor necrosis factor a (TNFa)and interleukin-1b (IL-1b). Similarly, both TNFa and IL-1b strongly induced mRNA and protein expression of inter- cellular adhesion molecule (ICAM)-1 and in each case this was prevented by adenovirus expressing the dominant IjB, but not by the null virus, thereby establishing ICAM-1 as an NF-jB-dependent gene. Numerous studies have sug- gested key roles for the p38 and extracellular regulated kinase (ERK) mitogen-activated protein kinase (MAPK) cascades in the activation and transactivation of NF-jB. We show here that SB203580, a selective inhibitor of the p38 MAPK, and PD098059 and UO126, both selective inhibitors of the ERK MAPK cascade, have no effect on TNFa or IL-1b-induced translocation and DNA binding of NF-jB. Furthermore, these inhibitors showed no pharmacologically relevant effect on NF-jB-dependent transcription nor was there any effect on expression of ICAM-1. Taken together these data highlight the potential use of inhibition of the NF-jB signalling pathway in pulmonary inflammatory diseases and suggest that inhi- bitors of the p38 and ERK MAPK pathways may be of lesser effect. Keywords:NF-jB; transactivation; ICAM-1; ERK; p38. The transcription factor nuclear factor-jB(NF-jB) is a central regulator of the immune system and promotes the transcription of over 150 genes [1]. As many of these genes are inflammatory, and include cytokines, chemokines, adhesion molecules as well as other enzymes, such as inducible nitric oxide synthase and cyclooxengenase-2, NF-jB may provide an attractive target for therapeutic intervention in inflammatory diseases [2]. NF-jBexistsas a homo or heterodimer made up from subunits from the rel family of proteins, which in vertebrates, comprises of p65 (RelA), p50/p105, p52/p100, c-Rel and RelB [3]. Within resting cells, NF-jB is retained in the cytoplasm complexed to an inhibitor protein from the IjB family [3]. Stimulation with pro-inflammatory cytokines, such as tumour necrosis factor a (TNFa) or interleukin 1b (IL-1b), activates the IjB kinase (IKK) complex, which then phosphorylates the IjB [3]. This leads to ubiquitination of the IjB, targeting it for rapid degradation by the 26S proteosome [3]. Degradation of IjB reveals a nuclear localization signal (NLS) allowing NF-jB to interact with the nuclear import protein karyo- pherin a2 [4]. This allows NF-jB to translocate into the nucleus where it can bind to jB sequences in the promoters of NF-jB-dependent genes to up-regulate transcription [3]. There are still many aspects of the NF-jB activation pathway that have yet to be elucidated. For example, the mechanisms that enhance the transactivation of NF-jB once it is bound to its consensus sequence remain poorly defined and numerous studies have suggested the involve- ment of the p38 mitogen activated protein kinase (MAPK) and the extracellular signal-regulated kinases (ERK) in the activation or potentiation of NF-jB-dependent transcrip- tion [5]. Thus, the selective p38 inhibitor, SB203580, prevents NF-jB/p65-dependent, transactivation without affecting NF-jB DNA binding [6,7]. Possible downstream effects of the p38 MAPK include; phosphorylation of histone H3 to enhance recruitment of NF-jB [8], phos- phorylation of the TATA binding protein (TBP) to increase transactivation of the DNA bound NF-jB [9], or phos- phorylation of serine 276 of p65 or cAMP response element binding protein (CREB) binding protein (CBP), events Correspondence to R. Newton, Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK. Fax: + 44 247 652 3701, Tel.: + 44 247 657 4187, E-mail: RNewton@bio.warwick.ac.uk Abbreviations: CBP, CREB binding protein; ERK, extracellular signal-regulated kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ICAM-1, intercellular adhesion molecule 1; IKK, IjB kinase; IL-1b, interleukin 1b; MAPK, MAPK/ERK kinase; MEK, mitogen activated protein kinase; MOI, multiplicity of infection; NF-jB, nuclear factor jB; TBP, TATA binding protein; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide; TNFa, tumour necrosis factor a. Note: A web site is available at: http://www.bio.warwick.ac.uk/ molphys.asp (Received 12 September 2003, revised 4 December 2003, accepted 7 January 2004) Eur. J. Biochem. 271, 785–791 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.03982.x which have both been shown to increase the ability of p65 to interact with CBP/p300 [10,11]. Similarly, inhibition of the ERK pathway, with the MEK1 inhibitor, PD098059, abrogated IKK activity and dominant negative constructs of ERK prevented NF-jB DNA binding activity [12,13]. In addition, the ERK pathway has been suggested to both enhance DNA binding activity as well as to phosphorylate downstream cofactors including CBP/p300 and positive cofactor 1 to increase the transactivational potential of NF-jB [10,14]. The pulmonary epithelium is the primary site of contact with airborne allergens, irritants, pathogens and other proinflammatory agents that trigger exacerbation in airway diseases [15]. This epithelium is biosynthetically active and acts as a source of multiple inflammatory cytokines, chemokines, prostanoids and other mediators as well as adhesion molecules such as intercellular adhesion molecule 1 (ICAM-1) [15]. In this context, ICAM-1 is particularly important in inflammatory diseases such as asthma, where it is not only responsible for the recruitment of inflammatory cells from the blood to the airways, but also acts as a receptor for many viruses that may exacerbate asthma [16]. Numerous studies suggest a role for NF-jBinICAM-1 expression, but an unequivocal demonstration in pulmon- ary epithelial cells is currently lacking. As NF-jBis considered to be a candidate for therapeutic intervention in airway inflammation, we have investigated the role of NF-jB in the induction of ICAM-1 expression by pro- inflammatory cytokines. In addition, we have used NF-jB- dependent transcriptional reporters and ICAM-1 expression to address the role of p38 and ERK pathways in the activation of NF-jB dependent transcription in pulmonary epithelial cells. Materials and methods Cell culture, cytokines and drugs A549 cells were grown to confluency in six-well plates as described previously [17]. Cells were cultured overnight in serum free media before changing to fresh serum free media containing TNFa or IL-1b (both from R & D systems, Abingdon, UK). SB203580, PD098059, and U0126 (all from Calbiochem, Nottingham UK) were dissolved in dimethylsulfoxide. Final concentrations of dimethylsulfox- ide added to cells were < 0.1% and this had no effect on any of the responses (data not shown). NF-jB luciferase reporter cell lines The NF-jB-dependent reporter cells, A549 6jBtkluc, were grown as described previously [17]. These contain a stably integrated plasmid with three tandem repeats of the sequence 5¢-AGC TTA CAA GGG ACT TTC CGC TGG GGA CTT TCC AGG GA-3¢, which has two copies of the decameric NF-jB binding site (bold text) upstream of a minimal thymidine kinase promoter driving a luciferase gene. Confluent cells in 24-well plates were changed to serum-free medium and treated as indicated before harvest- ingat6hin1· reporter lysis buffer (Promega, South- ampton, UK). Luminescence was measured using the luciferase assay system (Promega). Adenovirus infection A549 cells were either infected with empty Ad5 expression vector or a vector expressing an IjBa proteinwithan N-terminal deletion (Ad5IjBaDN) [18]. This deletion renders IjBa immune to signal-induced proteolysis and generates a constitutive inhibitor of NF-jB. As previously described, cells were infected at a multiplicity of infection (MOI) of 10, a level which we have shown previously to infect > 95% of cells, and then cultured for 24 h prior to treatment with drugs or cytokines [19]. Cell viability assay Cell viability was assessed by using the 3-(4,5-dimethylthi- azol-2-yl)-2,5-diphenyl-tetrazolium bromide [thiazolyl blue tetrazolium bromide (MTT)] assay (Sigma, Poole, UK) according to the manufacturer’s instructions. Semi-quantitative reverse transcription PCR RNA isolation, reverse transcription, primers, PCR conditions, and cycling parameters for glyceraldehyde- 3-phosphate dehydrogenase (GAPDH) were as described previously [17]. Primer pairs for ICAM-1 (Accession No: BC015969) (5¢fi3¢) were (forward) CCG TGT ACT GGA CTC CAG AA, (reverse) AGG TGT AGC TGC ATG GCA TA. Cycling parameters for ICAM-1 were: 94 °C, 30 s; 58 °C, 30 s; 72 °C, 30 s. The number of amplification cycles used was that necessary to achieve exponential amplification where product formation was proportional to starting cDNA and was established empirically [17]. Following amplification, PCR products (10 lL) were run on 2.0% agarose gels stained with ethidium bromide. After densitometric analysis using TOTALLAB software, version 1 (Nonlinear Dynamics), data were expressed as the ratio of ICAM-1/GAPDH. Electrophoretic mobility shift assay (EMSA) Nuclear proteins were isolated 1 h after stimulation and the consensus NF-jB(5¢-AGT TGA GGG GAC TTT CCC AGG-3¢) probe (Promega) radioactively labelled as des- cribed previously [20]. Specificity of binding was determined by the prior addition of 100-fold excess unlabeled consensus oligonucleotide. Reactions were separated on 7% native acrylamide gels before vacuum drying and autoradiography. Western blot analysis Cells were harvested in 100 lLof10 m M Tris/HCl (pH 7.5), 0.15 M NaCl, 1.5 m M MgCl 2 , 0.65% (v/v) NP-40, 0.5 m M phenylmethylsulfonyl fluoride, 0.5 m M dithiothreitol. Sam- ples were run on 10% SDS polyacrylamide gels and transferred to Hybond-ECL nitro-cellulose paper (Amer- sham Pharmacia, Bucks, UK) using standard techniques. Membranes were probed with mouse monoclonal anti- human GAPDH Ig (#4699–9555) (Biogenesis Ltd, Poole, UK) or mouse polyclonal anti-ICAM-1 (sc-8439) Ig (Santa Cruz Biotechnology, CA, USA) at dilutions of 1 : 20 000 and 1 : 1000, respectively. Alternatively, membranes were probed for phospho-hsp27 (#2401), or ERK 1/2 (#9100) 786 N. S. Holden et al. (Eur. J. Biochem. 271) Ó FEBS 2004 (both phospho and pan ERK 1/2) (New England BioLabs, Hertfordshire, UK) at a dilution of 1 : 1000 and incubated overnight at 4 °C. In all cases, proteins were visualized using ECL (Amersham Pharmacia Biotech, Bucks, UK) accord- ing to the manufacturer’s instructions. Results The establishment of ICAM-1 as an NF-jB-dependent gene Western blot analysis revealed extremely low levels of ICAM-1 protein in untreated cells, but following treatment with either TNFa or IL-1b, ICAM-1 protein was rapidly induced (Fig. 1). This was first apparent by 2 h poststim- ulation and levels of ICAM-1 protein continued to rise over the 18 h of the experiment. To examine the role of NF-jB in the induction of ICAM-1, cells were infected with an adenoviral vector that over-expresses IjBaDN, a dominant inhibitor of NF-jB [18]. We have described previously the effectiveness of this construct in repressing both NF-jB DNA binding and transcriptional activity in A549 cells [19]. Both TNFa and IL-1b produced a robust increase in reporter activity, measured 6 h poststimulation as des- cribed previously [21]. In each case, this activity was reduced to basal levels by the IjBaDN expressing virus (Fig. 2). The null, or empty, viral vector showed no effect on induction of reporter activity (Fig. 2). Likewise, there was no effect of this virus on basal activation of the reporter (data not shown) [19]. Having established that IjBDN over-expression is effect- ive at preventing induction of NF-jB transcriptional activity, this virus was used to test the role of NF-jBin the induction of ICAM-1 expression. As in previous experiments, unstimulated cells expressed very little ICAM-1 protein and this was also true of ICAM-1 mRNA (Fig. 3). Upon stimulation with either TNFa or IL-1b, ICAM-1 mRNA and protein was dramatically increased. Prior infection with the IjBaDN expressing adenovirus, but not the null virus, totally prevented the expression of ICAM-1 in response to stimulation by both TNFa,and IL-1b. These data therefore establish that the induction of ICAM-1 by both TNFa and IL-1b is highly NF-jB- dependent in A549 cells. Effect of SB203580, PD098059 and U0126 on NF-jB DNA binding and NF-jB-dependent transcription To examine the role of the p38 and the ERK MAPK pathways, we examined the effects of the p38 MAPK inhibitor, SB203580, the MEK1 inhibitor, PD098059 and the MEK 1/2 inhibitor, U0126, on TNFa-andIL-1b- induced NF-jB translocation and DNA binding. As induction of NF-jB DNA binding was shown previously to be maximal at 1 h poststimulation [21], the cells were stimulated for 1 h with TNFa or IL-1b prior to analysis by EMSA. Following each treatment, two NF-jB DNA binding complexes were observed and these were both removed by competition with the addition of 100-fold excess of cold oligonucleotide (Fig. 4A). Densitometric analysis of these complexes indicated a 10-fold increase in NF-jB DNA binding when compared to unstimulated cells (Fig. 4A, lower panels). In each case the prior addition of SB203580, PD098059, or U0126 revealed no effect on the induction of NF-jB DNA binding. To investigate the potential role these MAPK cas- cades on NF-jB-dependent transcription, 6jBtk cells Fig. 1. Time course of ICAM-1 expression in TNFa and IL-1b stimu- lated cells. Cells were either not stimulated (NS) or stimulated with TNFa (10 ngÆmL )1 )orIL-1b (1 ngÆmL )1 ) and harvested at 0, 2, 6 and 18 h post stimulation, prior to Western blot analysis of ICAM-1. A representative blot is shown and optical densities (n ¼ 3) are plotted as arbitrary units as means ± SEM. Fig. 2. Validation of an adenovirus expressing a constitutively active IjB. A549 6jBtk cells were either infected or not at a MOI of 10 with either Ad5IjBDN or null virus as indicated. After changing to serum free media, cells were either unstimulated or treated with TNFa (10 ngÆmL )1 )orIL-1b (1 ngÆmL )1 ) as indicated. Cells were harvested after 6 h for luciferase activity determination. Data (n ¼ 4) are expressed as percentage of stimulated cells and are plotted as means ± SEM. Ó FEBS 2004 The role of NF-jB and MAPK in ICAM-1 expression (Eur. J. Biochem. 271) 787 were stimulated with either TNFa or IL-1b in the presence of absence of various concentrations of SB203580, PD098059 or UO126. At concentrations from 0.01 and 1 l M these inhibitors showed little or no effect on luciferase levels (Fig. 4B). However decreased luciferase activity was observedwith10 l M SB203850 and this appeared to reach a plateau by 100 l M . With PD098059 and UO126 a variable effect was observed at 10 l M and by 100 l M amarked repression was apparent (Fig. 4B). However, in each case, these concentrations were 10 to 100-fold greater than the reported K i values and so probably result from nonspecific effects of these kinase inhibitors ([22] and refs therein). To assess the effect of these compounds on cell viability, MTT assays were performed on cells preincubated with a range of concentrations of the inhibitors. With the exception of U0126, which showed a 30% loss of cell viability at 100 l M , MTT analysis revealed little or no effect of PD098059, SB203580 or U0126 on cell viability at any of the concentrations tested (0.01–100 l M ; data not shown). Effect of MEK and p38 MAPK inhibitors on ICAM-1 expression in A549 cells As previous reports implicating MAPK pathways in NF- jB-dependent transcription have suggested the involvement of mechanisms that impact on the promoter architecture, and this may not always be faithfully reproduced in a reporter system, we examined the effect of SB203580 and PD098059 on ICAM-1 expression as an endogenous indicator of NF-jB transcriptional activity. As in previous experiments, stimulation of A549 cells with TNFa or IL-1b caused a considerable up regulation of ICAM-1 expression at 6 h poststimulation compared to unstimulated cells (Fig. 5). Pre-incubation of cells with a range of concentra- tions of PD098059 or SB203580 showed little or no effect on ICAM-1 expression in either TNFa or IL-1b stimulated cells suggesting that neither the p38 MAPK nor the MEK1- ERK pathways are involved in the expression of ICAM-1 in this system (Fig. 5). Validation of the SB203580, PD098059 and U0126 in A549 cells To validate the inhibitory action of SB203580, the phos- phorylation of heat shock protein 27 (hsp27), a downstream target of p38 MAPK was analysed [22]. Both TNFa and IL-1b markedly induced hsp27 phosphorylation and this was prevented by preincubation with SB203580 (Fig. 6). Analysis of GAPDH expression confirmed equality of loading. Following densitometric analysis, EC 50 values of 0.21 and 0.23 l M were calculated for inhibition of TNFa- and IL-1b-stimulated hsp27 phosphorylation, respectively. These values are consistent with the published IC 50 for SB203580 (0.6 l M ) indicating the inhibition of p38 at pharmacologically relevant concentrations [23]. Similarly, the MEK inhibitors, PD098059 and U0126, were validated by analysis of phosphorylation of ERKs 1 and 2, which lie downstream of MEK1 [24]. Both TNFa and IL-1b upregulated phosphorylation of p42/44 ERKs and preincubation with PD098059 abrogated this response. Following densitometry, EC 50 values of 0.26 and 0.58 l M were calculated for TNFa-andIL-1b-stimulated cells, respectively. These are consistent with published values for MEK 1 inhibition (5 l M ) [25]. As a loading and expression control, the expression of total p42/44 was also examined. Likewise, U0126 completely prevented the phosphorylation of p42/44 with EC 50 values of 0.23 and 0.1 l M for TNFa and IL-1b stimulated cells, respectively. This is again consistent with the published IC 50 value for MEK1/2 inhibition (0.065 l M ) suggesting that both PD098059 and U0126 are functionally active in A549 cells [26]. Discussion The adhesion molecule ICAM-1 has been shown by numerous studies to be an important factor in many allergic diseases such as asthma, where it not only plays a critical role in airway inflammation and the development of hyper- responsiveness [27], but also acts as a receptor for infection by rhinoviral and respiratory syncytial viruses, which both increase exacerbations in asthma [28,29]. The regulation of ICAM-1 production is therefore a potential target for the development of new therapeutics in asthma. Various studies have implicated NF-jB in the transcriptional regulation of ICAM-1 in a variety of cells [30–33], however, these studies often rely on reporter systems, which may not faithfully mimic the architecture of the endogenous promoter. In the current study, we have over-expressed a dominant form of Fig. 3. Effect of an adenovirus expressing a constitutively active IjBon ICAM-1 expression. Cells were either infected or not at a MOI of 10 with either Ad5IjBDN or null virus as indicated. After changing to serum free media, cells were either not stimulated (NS) or treated with TNFa (10 ngÆmL )1 )orIL-1b (1 ngÆmL )1 ) as indicated. Cells were either harvested after 6 h for semiquantitative RT-PCR analysis or after 24 h for Western blot analysis of both ICAM-1 and GAPDH expression. Representative blots are shown, and data (n ¼ 6) were normalized to GAPDH expression and are plotted as means ± SEM. 788 N. S. Holden et al. (Eur. J. Biochem. 271) Ó FEBS 2004 IjBa to unequivocally demonstrate that the induction of ICAM-1 expression is NF-jB-dependent in TNFa-and IL-1b-stimulated pulmonary epithelial A549 cells. As noted above, both the p38 and ERK MAPK cascades are variously implicated in the activation of NF- jB. However, as many of these studies rely on transfection analysis of reporter plasmids, which are prone to artefacts, or on the effects of single high dose inhibitors, which may lead to pharmacologically unrelated events, to define functional roles for MAPK, they should be treated with a degree off caution ([22] and references therein). Thus, in contrast to the observation that dominant negative ERKs completely prevented NF-jB DNA binding [12,13], the present analysis showed no effect of either the MEK 1 inhibitor PD098059 or the MEK 1/2 inhibitor U0126 on NF-jB translocation and DNA binding. As a similar result was observed with the p38 MAPK inhibitor, SB203580, these data indicate that neither the ERK nor p38 MAPK pathways play a significant role in the activation of NF-jB DNA binding in A549 cells. However, numerous studies Fig. 4. Effect of PD098059, SB203580 and U0126 on NF-jB DNA binding, and NF-jB- dependent transcription. (A) Cells were pre- incubated with PD098059, SB203580 or U0126 (all at 10 l M ) and then stimulated with either TNFa (10 ngÆmL )1 )orIL-1b (1 ngÆmL )1 ).After1h,nuclearextractswere prepared and analysed by EMSA. Represen- tative blots are shown (n ¼ 4). XS indicates the presence of a 100-fold excess of cold NF-jB probe. Specific complexes, defined by competition (XS), are indicated, and data, expressed as a percentage of stimulated cells are plotted as means ± SEM. (B) 6jBtk A549 cells were preincubated with various concen- trations of PD098059, SB203580 or U0126 (0.01–100 l M ) before stimulation with either TNFa (10 ngÆmL )1 )orIL-1b (1 ngÆmL )1 ). Cells were harvested after 6 h for luciferase activity determination. Data (n ¼ 5) was expressed as percentage of stimulated cells and are plotted as means ± SEM. Fig. 5. Effect of p38 MAPK and MEK inhi- bitors on ICAM-1 expression. Cells were pre- incubated with various concentrations of PD098059, SB203580 or U0126 (0.01–30 l M ) before stimulation with either TNFa (10 ngÆmL )1 )orIL-1b (1 ngÆmL )1 ). Cells were harvested after 6 h for Western blot analysis of ICAM-1 expression. Representative blots are shown and data (n ¼ 2), expressed as percentage of stimulated cells, are plotted as means ± SEM. Ó FEBS 2004 The role of NF-jB and MAPK in ICAM-1 expression (Eur. J. Biochem. 271) 789 have implicated roles for p38 and MEK 1/2 cascades in the transactivation of NF-jB downstream of DNA binding [5–7], through modulation of various components of the basal transcriptional machinery such as TBP [9], or even NF-jB/p65 itself [10,11]. In addition, p38-dependent phosphorylation may lead to modification of histone proteins causing changes in the chromatin structure of NF-jB-dependent genes and allowing increased access of the basal transcription machinery to the DNA [8]. More recently, the p38 downstream kinase, mitogen- and stress- activated protein kinase-1 (MSK-1) was shown to phos- phorylate p65 at Ser276 and this was shown to be essential for association with CBP/p300 and thus up-regulation of NF-jB transactivation [10]. However, in the present study, inhibitors of the p38 and ERK MAPK pathways were all shown to be functionally active at the correct pharmaco- logical concentrations, yet had no effect on the activation of NF-jB-dependent transcription as determined by luci- ferase reporter assay. However, as transcriptional reporters may not mimic the true physiological architecture exhib- ited by an endogenous promoter within cells, ICAM-1 was also used as an endogenous NF-jB-dependent reporter. However, this analysis confirmed the reporter data indica- ting that neither the p38 nor the ERK MAPK pathways play a major role in NF-jB-dependent transcription in these cells. Finally, it should be noted that the use of the p38 MAPK inhibitor, SB203580, only implicates the a and b isofroms of p38, as SB203580 has little or no effect on p38 c and d and it is possible that this could explain discrepancies between over-expression and inhibitor based studies [34]. In conclusion, the data presented here firmly demon- strates that the adhesion molecule ICAM-1 is a highly NF-jB-dependent gene in A549 pulmonary epithelial cells and is therefore a useful endogenous reporter of NF-jB- dependent transcription. Furthermore, despite the extensive evidence documenting roles for the p38 and ERK MAPK pathways in NF-jB-dependent transcription, we found that highly selective inhibitors of these pathways had no effect on activation of NF-jB DNA binding, NF-jB-dependent transcription, or on the endogenous NF-jB-dependent gene ICAM-1. Taken together, these data highlight the potential utility of inhibiting the NF-jB signalling pathway in pulmonary inflammatory diseases and suggest that inhibi- tors of the p38 and ERK MAPK pathways may be of lesser effect in this cell type. Acknowledgements N. S. H. and M. C. C. are MRC collaborative students supported by Novartis Pharmaceuticals and Aventis Pharmaceuticals, respectively. L. M. C. was funded by a grant from Novartis Pharmaceuticals. References 1. Pahl, H.L. (1999) Activators and target genes of Rel/NF-kappaB transcription factors. Oncogene 18, 6853–6866. 2. Barnes, P.J. & Karin, M. (1997) Nuclear factor-kappaB: a pivotal transcription factor in chronic inflammatory diseases. N. Engl. J. Med. 336, 1066–1071. 3. Karin, M. & Ben-Neriah, Y. (2000) Phosphorylation meets ubi- quitination: the control of NF-[kappa]B activity. Annu. Rev. Immunol. 18, 621–663. 4. Cunningham, M.D., Cleaveland, J. & Nadler, S.G. 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