The Mutant Plasmacytoma Cell Line S107 Allows the Identification of Distinct Pathways Leading to NF-kappa B Activation*

Bernd BaumannDagger , Barbara KistlerDagger , Andrei Kirillov§, Yehudit Bergman§, and Thomas WirthDagger

From the Dagger  Institut für Medizinische Strahlenkunde und Zellforschung, Universität Würzburg, Versbacher Strasse 5, 97078 Würzburg, Germany and the § Hubert Humphrey Center for Experimental Medicine and Cancer Research, Hebrew University Hadassah Medical School, Jerusalem 91120, Israel

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Studies on the mechanisms of inducible and constitutive activity of NF-kappa B transcription factors have been hampered by the lack of appropriate mutant cell lines. We have analyzed the defect in the murine S107 plasmacytoma cell line, which was previously found to lack both constitutive and inducible NF-kappa B activity. Our analysis shows that these cells bear a specific defect that interferes with NF-kappa B induction by many diverse stimuli, such as lipopolysaccharide, phorbol 12-myristate 13-acetate, UV light, x-rays, and H2O2. This does not however represent a general signal transduction defect, because AP-1 transcription factors are readily induced by the same stimuli. Phosphatase inhibitors such as okadaic acid as well as calyculin A can efficiently induce NF-kappa B in S107 cells via a pathway apparently insensitive to the radical scavenger pyrrolidine dithiocarbamate. Furthermore, MEKK1 a protein kinase supposedly induced by some of the above stimuli, is also capable of activating NF-kappa B. Interestingly, both the potent physiological inducer of NF-kappa B TNFalpha as well as endoplasmic reticulum overload can induce NF-kappa B via a PDTC sensitive pathway. In all cases, DNA-binding NF-kappa B complexes are comprised predominantly of p50-RelA heterodimers, and NF-kappa B activation results in the induction of transiently transfected or resident reporter genes. In summary, these results suggest that the pathways for many NF-kappa B-inducing stimuli converge at a specific junction, and this pivotal step is mutated in the S107 cell line. Yet there are alternative routes bypassing this critical step that also lead to NF-kappa B induction. These routes utilized by tumor necrosis factor alpha  and endoplasmic reticulum overload are still intact in this cell line.

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

The NF-kappa B/Rel transcription factors consist of five mammalian family members that bind to DNA as homo- and/or heterodimers (1-4). Two of the family members, NFKB1 and NFKB2, are synthesized as precursor proteins (p105 and p100, respectively), which are proteolytically processed to obtain the mature proteins p50 and p52. The other three members, RelA, RelB, and c-Rel, are produced directly without such a processing step. These latter family members contain efficient transactivation domains in their COOH-terminal domains (RelA and c-Rel) or both NH2- and COOH-terminal domains (RelB), and therefore they are largely responsible for the transactivation capacity of homo- and heterodimers containing these subunits.

NF-kappa B was originally identified as a transcription factor constitutively present in the nuclei of mature B and plasma cells, yet present in a latent but inducible form in pre-B cells as well as most other cell types (5-7). The mechanism leading to this inducibility was resolved to some extent. In most cell types, NF-kappa B family members are localized in the cytoplasm due to the interaction with inhibitory proteins, the Ikappa Bs (8). These Ikappa B proteins first of all inhibit nuclear translocation of NF-kappa B proteins by masking the nuclear localization signal and also directly block their DNA binding. The Ikappa B proteins again represent a protein family consisting of at least six family members described so far. Ikappa Balpha , Ikappa Bbeta , Ikappa Bepsilon , and Bcl3 represent independent genes, whereas Ikappa Bgamma and Ikappa Bdelta are derived from the COOH-terminal domains of the NFKB1 and NFKB2 precursor proteins, respectively (2, 4, 9, 10). The different inhibitor proteins show some selectivity toward different NF-kappa B dimeric complexes.

Ikappa Balpha , Ikappa Bbeta , and Ikappa Bepsilon are involved in regulation of the inducible activity of the NF-kappa B complexes. A variety of external or internal stimuli lead to the initiation of a signal transduction cascade, which culminates in the activation of Ikappa B kinase(s). Two such Ikappa B kinases (IKKalpha and IKKbeta )1 have been cloned recently, but the details of the activation of these kinase are not known yet (11-15). Ikappa B proteins become phosphorylated at specific serine residues localized in an amino-terminal signal response domain (16-19). These serine phosphorylated Ikappa B molecules are subsequently targeted by poly-ubiquitination for degradation via the proteasome pathway (20-24). The degradation of Ikappa B proteins leads to the release of NF-kappa B complexes, which can migrate to the nucleus, bind to DNA, and regulate transcription. Target genes regulated by NF-kappa B play important roles in immune, inflammatory or stress responses, cell adhesion, and protection against apoptosis (1, 3, 4).

Within the B cell lineage, pro-B cells and pre-B cells contain NF-kappa B in an inhibited form just like most other cell types, but later stages of B cell development contain constitutive nuclear NF-kappa B (1-4). This activation of NF-kappa B as a consequence of B cell development is also observed in a primary B cell differentiation system (25). The mechanisms leading to the constitutive activation of NF-kappa B complexes in mature B cells and plasma cells remain largely unresolved. Several observations have been made over the past years, which addressed this constitutive activity. It was demonstrated that the half-life of the Ikappa Balpha protein is significantly decreased in mature B and plasma cells as compared with pre-B cells, but at the same time there is an increased rate of de novo synthesis (26, 27). Furthermore, it could be shown that some heterodimer complexes are less efficiently targeted by Ikappa Balpha as compared with others (28), and in addition, at least the p50-RelB complexes in mature B cells appear to be modified in a way that makes them escape Ikappa B inhibition (29, 30). A specific role for Ikappa Bbeta has also been suggested recently (25, 31, 32). In the process of B cell differentiation hypophosphorylated Ikappa Bbeta , which can still interact with NF-kappa B but does not mask the nuclear localization signal and fails to inhibit DNA binding, could shield NF-kappa B from inhibition by Ikappa Balpha (31, 32).

The constitutive activation of NF-kappa B in B cells may have some components in common with the inducible branch of NF-kappa B activation. However, a detailed analysis of these two mechanisms was hampered by the lack of appropriate mutant cell lines, which would allow a thorough analysis. The murine plasmacytoma cell line, S107, was described several years ago as a cell line lacking constitutive nuclear NF-kappa B (33). In this cell line, kappa B-dependent transcription of reporter genes is completely absent; the endogenous Igkappa gene shows normal expression, however. This is most likely due to the activity of the 3' kappa -enhancer element (34). Early somatic cell fusion studies of S107 cells with pre-B cell lines had already suggested that most components of the machinery leading to constitutive nuclear NF-kappa B were still intact in this cell line, because the defect could be restored in the fusion hybrids (35).

Analysis of the expression levels of NF-kappa B and Ikappa B family members had revealed that most of the subunits of these families are in fact expressed in S107 cells (36). Interestingly, p50-RelA complexes were found in the cytoplasm of these cells associated with Ikappa B molecules and treatment of cytoplasmic extracts with the detergent deoxycholate released DNA-binding NF-kappa B complexes. However, these complexes could not be mobilized by LPS or PMA treatment of the intact cells, suggesting some defect in the inducible signal transduction pathway. Interestingly, S107 cells lack expression of the relB gene and upon stable transfection of these cells with a RelB expression vector but not a RelA expression vector resulted in the appearance of nuclear p50-RelB complexes and restored the kappa B-dependent transcriptional activity. In addition, whereas the parental S107 cells showed a striking defect in B cell-specific demethylation of a transfected methylated Igkappa locus, the RelB transfectants had also regained the demethylation capacity (36, 37).

Here we have analyzed the defect of the S107 cell line in much more detail. We provide evidence that S107 cells are specifically defective in responding to a subset of NF-kappa B-inducing stimuli. Because other inducers still work in these cells this suggests the existence of at least two distinct signal transduction pathways, which both lead to NF-kappa B induction.

    EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Cell Culture and Transfection Experiments-- All cells were grown in Dulbecco's modified Eagle's medium/Glutamax (Life Technologies, Inc.) supplemented with 10% fetal bovine serum, 50 µM beta -mercaptoethanol, and antibiotics. For transient transfections, 3 µg of the luciferase reporter and 1 µg of Rous sarcoma virus LacZ were used. Cells were transfected by electroporation using a Bio-Rad gene pulser at 960 microfarad and 220 V. For cotransfection experiments, 10 µg of the MEKK1 expression vector, the influenza hemagglutinin expression vector, or the parental empty expression vectors were included. The Rous sarcoma virus-driven eucaryotic expression vector for the hemagglutinin gene of the human influenza strain A/Mongolia/231/85 was kindly provided by Dr. S. Ludwig. 16 h after transfection cells were treated as indicated, and cells were harvested 6-8 h later. Luciferase enzyme activity was determined, and beta -galactosidase levels were used to normalize for differences in transfection efficiencies. For generation of stable transfectants of the S107 cell line, cells were electroporated with 20 µg of the 3xkappa B-reporter plasmid together with 1 µg of a pSV.puro vector. Stably transfected cell clones were selected in 2 µg/ml puromycin.

Preparation and Analysis of Protein Extracts-- Whole cell extracts were prepared by the freeze-thaw method described previously (29). Conditions for EMSA and EMSA antibody supershifts have also been described before. In all cases, a radiolabeled probe containing the Ig-kappa enhancer consensus NF-kappa B site was used. Quality of the extracts was verified by parallel EMSA experiments with an octamer probe detecting the Oct-1 (and Oct-2) transcription factors. For Western immunoblots, 50-100 µg of protein extract were separated on 12.5% SDS-polyacrylamide gels. After transfer to polyvinylidene difluoride membranes, proteins were detected with the indicated antibodies, and blots were developed using the ECL system (Amersham Pharmacia Biotech). Antibodies used for supershift and Western immunoblot assays were purchased from Santa Cruz, except for the Ikappa Bepsilon antibodies, which were a kind gift from Dr. Nancy Rice.

    RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References

S107 Cells Show a Specific Defect in NF-kappa B Induction-- The S107 cell line, in contrast to virtually all other known plasmacytoma cell lines, lacks constitutive nuclear NF-kappa B proteins and is also defective in induction on NF-kappa B after treatment with a variety of stimuli (33, 35, 37). We wanted to assess whether these cells harbor a pleiotropic signal transduction defect or are specifically defective in signals mediating NF-kappa B activation. Therefore, we analyzed a variety of different stimuli for their ability to activate NF-kappa B and, as a control, the AP-1 transcription factors. Untreated S107 cells lack DNA-binding NF-kappa B proteins, and stimulation with different agents such as LPS, hydrogen peroxide (H2O2), phorbol ester together with a calcium ionophore (PMA and ionomycin), x-ray, and UV irradiation all fail to induce NF-kappa B in S107 cells (Fig. 1A). In contrast, when these same stimulating conditions were analyzed with the pre-B cell line PD31, which also lacks constitutive NF-kappa B activity, all treatments resulted in the induction of NF-kappa B complexes. However, the same stimuli readily activated AP-1 transcription factors in both PD31 as well as S107 cells (Fig. 1A, lower panels). Therefore, the signaling defect in S107 cells appears to be specific for the NF-kappa B pathway.


View larger version (38K):
[in this window]
[in a new window]
 
Fig. 1.   S107 cells show a specific defect in NF-kappa B activation. A, whole cell extracts (5 µg) from PD31 pre-B cells (left panels) and S107 cells (right panels) treated as indicated on top were analyzed for NF-kappa B (upper panels) and AP-1 (lower panels) DNA binding activity in EMSA. Only the part of the autoradiograph containing the relevant complexes (marked on the left) are shown. Treatments were performed as follows: co, control; LPS, 2 h with 50 µg/ml; H2O2, 2 h with 250 µM; PMA and ionomycin (P/I), 2 h with 10 ng/ml each; x-ray, irradiated with 20 Gy, harvested after 2 h; UV, irradiated with 50 J/m2 (UVC) and harvested after 4 h. The position of a nonspecific band is indicated by an asterisk. B, prolonged stimulation of S107 cells does not lead to NF-kappa B activation. Whole cell extracts (5 µg) of PD31 and S107 cells treated for the indicated times with LPS were analyzed by EMSA with the kappa B probe as in A.

To exclude the possibility that the response to these various stimuli was just delayed in S107 cells, we analyzed several inducers over an extended time course. Importantly, even continuous treatment with NF-kappa B-inducing stimuli such as LPS, PMA, and H2O2 for up to 24 h did not result in a measurable induction in S107 cells, whereas PD31 cells showed a rapid and persistent NF-kappa B activation (Fig. 1B and data not shown).

Phosphatase Inhibitors Can Induce NF-kappa B in S107 Cells-- A mutant pre-B cell line that also failed to activate NF-kappa B in response to a variety of stimuli was described recently (38). The defect in these cells could be overcome by treating them with phosphatase inhibitors such as okadaic acid and calyculin A. Interestingly, this induction by phosphatase inhibitors was insensitive to treatment with the antioxidant PDTC. We therefore investigated whether these phosphatase inhibitors would lead to NF-kappa B induction in S107 cells and as a control in Jurkat cells. Indeed, the inhibitors were able to induce NF-kappa B DNA binding activity in both cell lines (Fig. 2A). Whereas at high concentrations okadaic acid inhibits both phosphatase 1 and phosphatase 2A, at low concentration okadaic acid specifically inhibits phosphatase 2A. When different concentrations of okadaic acid were tested for NF-kappa B induction, 5 nM okadaic acid resulted in induced NF-kappa B DNA binding, suggesting that phosphatase 2A is involved in the activation (data not shown). Activation of NF-kappa B by okadaic acid and calyculin A was rapid and could be detected after only 15 min of treatment (Fig. 2A and data not shown).


View larger version (28K):
[in this window]
[in a new window]
 
Fig. 2.   Induction of NF-kappa B by phosphatase inhibitors in S107 cells. A, S107 cells and as a control (Co) Jurkat T cells were treated with 200 nM okadaic acid (OA) or 25 nM calyculin A (CyA) for the indicated times. NF-kappa B binding was determined by EMSA using whole cell extracts (5 µg). In some experiments an additional upper nonspecific band marked by an asterisk occurs sporadically in Jurkat cells and S107 cells (see panel B and Fig. 3A). This binding activity does not react with antibodies recognizing all the known members of the NF-kappa B family. B, effect of the radical scavenger PDTC on NF-kappa B induction by okadaic acid and calyculin A in S107 and Jurkat cells. In the experiment shown in the left panel, S107 cells were preincubated with increasing amounts of PDTC (25, 50, 100, and 200 µM) for 60 min prior to induction by calyculin A for 3 h. For the right panel, S107 and Jurkat cells were preincubated with 100 µM PDTC for 60 min, and the cells were then treated with 200 nM okadaic acid for 6 h. Whole cell extracts (5 µg) were used, and kappa B-specific complexes revealed by EMSA are shown. The complex marked by the arrow is due to partial proteolysis. C, the effect of phosphatase inhibitors as well as PDTC was evaluated in PD31 cells as described for the other cell types in B. D, NF-kappa B induction by okadaic acid can be inhibited by proteasome inhibitors. The upper panels show the effect of 100 µM ALLN pretreatment (60 min) on okadaic acid (3 h)-induced NF-kappa B analyzed by EMSA. The lower panels show protein immunoblots for Ikappa Balpha with the same extracts used for EMSA. 50 µg of each extract were loaded on the SDS-polyacrylamide gel. *, nonspecific band.

From previous results it was not absolutely clear at what stage of this signal transduction cascade okadaic acid and calyculin A would interfere. Whereas it had been reported that the activation of NF-kappa B by okadaic acid involves the generation of reactive oxygen intermediates and could be blocked by addition of radical scavengers, such as PDTC (39), the results in the mutant pre-B cell line demonstrated that induction of NF-kappa B by phosphatase inhibitors can be resistant to PDTC treatment (38). We therefore analyzed the effect of PDTC on induction of NF-kappa B by okadaic acid and calyculin A in S107 and Jurkat cells. Interestingly, whereas NF-kappa B induction was completely resistant to the addition of the radical scavenger in S107 cells (Fig. 2B, left and right panels), NF-kappa B induction by okadaic acid in Jurkat cells could be completely blocked by pretreating the cells with PDTC (Fig. 2B, right panel). To analyze whether induction of NF-kappa B by phosphatase inhibitors would be sensitive to PDTC in other cells, we analyzed PD31 cells and found a virtual complete block by PDTC (Fig. 2C). These results demonstrate that the same inducing stimuli may activate NF-kappa B in different cell types via distinct pathways.

In the typical pathway of NF-kappa B activation, the phosphorylated Ikappa Balpha protein becomes poly-ubiquitinated and degraded by the proteasome. To test whether treatment of cells with okadaic acid does indeed result in the accumulation of phosphorylated Ikappa Balpha , which is targeted for subsequent degradation, we analyzed the effect of a well known proteasome inhibitor, ALLN. Addition of ALLN to okadaic acid-treated Jurkat or S107 cells resulted in a strong reduction of induced NF-kappa B complexes (Fig. 2D, right upper panel). When the status of Ikappa Balpha was analyzed by protein immunoblotting, ALLN treatment resulted in the accumulation of a slower migrating form of Ikappa Balpha , which represents the phosphorylated form of this protein (Fig. 2D, lower right panel). The identical results were obtained for Jurkat T cells (Fig. 2D, left panel). These results suggest that the downstream signaling events, starting from phosphorylation of Ikappa Balpha to proteolytic degradation of this protein by the proteasome pathway, are apparently intact in the mutant S107 cell line. This localizes the defect to a common signaling intermediate utilized by all the stimuli unable to induce NF-kappa B.

TNFalpha Is a Potent Inducer of NF-kappa B in S017 Cells-- The analysis of the NF-kappa B induction by TNFalpha and interleukin-1 clearly represents the best understood pathway to date. The receptors for these ligands recruit TRAF proteins (TRAF2 and TRAF6, respectively) upon activation, and these TRAF proteins directly associate with NIK, a mitogen-activated protein kinase kinase kinase shown to be critically involved in NF-kappa B induction in these pathways. Interestingly, when the catalytic subunits of the cytokine-inducible Ikappa B kinase (IKKalpha and IKKbeta ) were cloned (11-15), a direct physical interaction between NIK and IKKalpha or IKKalpha -IKKbeta heterodimers was demonstrated. Therefore, in this pathway the essential upstream components required for NF-kappa B activation are known. We therefore asked whether TNFalpha could activate NF-kappa B in the S107 cells. Interestingly, in contrast to the other physiological inducers of NF-kappa B tested before, TNFalpha was readily capable of inducing NF-kappa B even in S107 cells (Fig. 3A). Because TNFalpha induction of NF-kappa B in most cell lines is also sensitive to the radical scavenger PDTC, we analyzed whether the observed induction in S107 cells might be PDTC-sensitive. Indeed we found that pretreatment of S107 cells with this radical scavenger completely abolished induction of NF-kappa B, whereas PDTC by itself had virtually no effect (Fig. 3B).


View larger version (32K):
[in this window]
[in a new window]
 
Fig. 3.   Induction of NF-kappa B by TNFalpha in S107 cells. A, S107 and Jurkat cells were treated with 80 ng/ml TNFalpha for 60 min, and NF-kappa B binding was determined by EMSA using 5 µg of whole cell extracts. *, nonspecific bands. B, NF-kappa B induction by TNFalpha can be inhibited by PDTC. S107 cells were preincubated with PDTC (100 µM) prior to to TNFalpha treatment. kappa B-specific complexes revealed by EMSA were shown. C, no difference in the NF-kappa B dimer composition was induced by okadaic acid (OA) and TNFalpha . Antibody characterization of kappa B-specific complexes from S107 cells treated with TNFalpha and okadaic acid for 60 min. Cell extracts were preincubated with the indicated antibodies as shown. PI, preimmune serum; ss, supershifted complexes. The reason for the appearance of two supershifted complexes is unclear.

The observation that TNFalpha induces NF-kappa B via a PDTC-sensitive pathway, whereas okadaic acid and calyculin A induced NF-kappa B via a PDTC-insensitive pathway, posed the question of whether the induced NF-kappa B complexes observed after induction by these two stimuli were identical. To address this question, we performed antibody supershift experiments using antibodies recognizing the different members of the NF-kappa B/Rel-family. This analysis revealed that in both cases predominantly RelA-containing complexes were induced (Fig. 3C).

We then asked whether the observed induction of NF-kappa B by TNFalpha was a direct effect or whether the induction might be indirect, e.g. TNFalpha might induce some secreted molecules that activate NF-kappa B in an autocrine/paracrine fashion. We therefore analyzed the kinetic of NF-kappa B induction after TNFalpha treatment in S107 cells and Jurkat T cells. In both cell lines, strong NF-kappa B induction could be detected within 15 min of TNFalpha treatment (Fig. 4). This suggests that the activation of NF-kappa B is indeed a direct consequence of TNFalpha treatment in S107 cells and not the consequence of activation of an autocrine loop. We do note a difference between the two cell lines, however. Upon prolonged induction (more than 2 h) of Jurkat cells, the NF-kappa B activity is attenuated (see Fig. 4). In contrast, S107 cells show a persistent stimulation of NF-kappa B DNA binding.


View larger version (45K):
[in this window]
[in a new window]
 
Fig. 4.   Kinetic of NF-kappa B induction after TNFalpha treatment. S107 and Jurkat cells were treated with TNFalpha for the indicated time points. NF-kappa B binding in whole cell extracts (5 µg) was analyzed by EMSA. The slowly migrating complex marked by an arrow contains the RelB protein (data not shown).

To assess whether the TNFalpha -induced NF-kappa B complexes would be transcriptionally active, we analyzed the transactivation capacity on a kappa B-dependent reporter (3xkappa B.luc). When S107 cells were transiently transfected with this reporter, activity could be induced about 40-fold by treatment of the cells with TNFalpha (Fig. 5A). When this reporter was stably integrated into S107 cells it showed only weak basal activity. Upon induction of S107 cells with TNFalpha , however, activity of the kappa B-dependent reporter was again induced about 40-50-fold (Fig. 5B). Consistent with the results obtained in the EMSA experiments, treatment of the transfected S107 cells with PDTC completely abolished the induction of reporter gene activity by TNFalpha in both transient and stable transfections. Likewise, when cells were treated with a proteasome inhibitor ALLN, TNFalpha failed to induce reporter gene activity. Neither PDTC nor ALLN had any effect on the reporter in the absence of TNFalpha treatment (Fig. 5, A and B).


View larger version (8K):
[in this window]
[in a new window]
 
Fig. 5.   Effect of TNFalpha on NF-kappa B-dependent transactivation in S107 cells. A, S107 cells were transiently transfected with 3xkappa B-luc reporter gene (three copies of the Igkappa -kappa B-motif immediately upstream of the beta -globin TATA-box (36)). 16 h after transfection cells were treated with TNFalpha alone or after 60 min of PDTC pretreatment. Luciferase activity was measured 6 h after stimulation. B, S107 cells containing a stably integrated 3xkappa B-luc reporter gene were treated with PDTC or ALLN 60 min prior to stimulation with TNF-alpha . 6 h post-stimulation luciferase activity was determined. The fold induction over untreated cells is shown.

TNFalpha Treatment Activates the Endogenous relB Gene-- We had previously shown that the S107 cell line lacks expression of the relB gene, and we had suggested that this might be a consequence of the lack of induced NF-kappa B (36, 37). We had furthermore suggested that RelB is a target gene of NF-kappa B. We therefore asked whether prolonged treatment of S107 cells with TNFalpha , which led to the induction of NF-kappa B, would result in the expression of the endogenous relB gene. Indeed, when extracts from Jurkat and S107 cells induced with TNFalpha for 4 h and longer were analyzed for RelB expression by protein immunoblot, a significant induction was seen in both cell lines (Fig. 6A). From this analysis it is furthermore evident that Ikappa Balpha , another known target gene of NF-kappa B, is degraded early after induction with TNFalpha and then efficiently resynthesized. These results demonstrate that NF-kappa B induced by TNFalpha in S107 cells is capable of activating endogenous target genes.


View larger version (43K):
[in this window]
[in a new window]
 
Fig. 6.   Induction of the endogenous relB and ikappa balpha genes by TNFalpha . A, S107 and Jurkat cells were treated with TNF-alpha for the indicated time points, and a protein immunoblot with 50 µg of whole cell extract was performed with using RelB- and Ikappa Balpha -specific antibodies. *, nonspecific band; hu, human; m, murine. B, analysis of Ikappa Bbeta and Ikappa Bepsilon protein levels in stimulated S107 cells. The experiment was performed as in A.

Interestingly, in S107 cells Ikappa Balpha levels decreased early after TNFalpha induction but resumed to starting levels within 1 h, whereas induced NF-kappa B levels persisted for much longer times. We therefore analyzed whether degradation of other members of the Ikappa B protein family might be responsible for this continuous activation. Interestingly, whereas Ikappa Bepsilon levels also decreased early after TNFalpha stimulation and then resumed, the amounts of Ikappa Bbeta where persistently reduced upon prolonged stimulation (Fig. 6B).

We had previously shown that the S107 defect in kappa B-dependent transcription can be overcame by ectopic expression of RelB but not the RelA protein (36, 37). The induction of relB gene expression by TNFalpha prompted us to investigate whether RelB, once expressed in these cells, would give positive feedback and up-regulate its own synthesis. The result of such a positive feedback loop would be a conversion of the NF-kappa B-deficient S107 cells to a RelB-positive descendent, which should behave similarly to the stable RelB transfectants. Indeed, upon prolonged induction by TNFalpha a slowly migrating DNA-binding complex containing the RelB protein was detected by EMSA (Fig. 4, complex marked by arrow). A similarly migrating RelB complex was previously noted in some plasmacytoma cells (27). We therefore stimulated S107 cells bearing the stably transfected kappa B-dependent luciferase reporter for 16 h with TNFalpha and then asked whether RelB/NF-kappa B transcriptional activity could be measured after TNFalpha withdrawal. Continuous treatment of the S107 cells for up to 64 h had no effect on the growth characteristics of these cells and resulted in a stable up-regulation of reporter gene activity. However, whenever TNFalpha was removed, reporter gene activity dropped to almost base line within 48 h (Fig. 7A). Consistent with the lack of activity of the kappa B-dependent promoter, no NF-kappa B DNA binding activity could be scored in extracts of S107 48 h after TNFalpha withdrawal (Fig. 7B). This suggests that TNFalpha -induced NF-kappa B was not sufficient to sustain the synthesis of NF-kappa B proteins, i.e. the RelB protein, and therefore did not result in the continuous presence of nuclear NF-kappa B. Consistent with this interpretation we found a loss of RelB protein expression in Western immunoblots from cells after TNFalpha withdrawal (Fig. 7C).


View larger version (29K):
[in this window]
[in a new window]
 
Fig. 7.   TNFalpha treatment does not result in a persistent induction of NF-kappa B activity in S107 cells. S107 cells containing a stably integrated 3xkappa B-luc reporter gene were treated with TNFalpha or left untreated. After 16 h cells were washed twice with phosphate-buffered saline and were incubated for 48 h either in the presence or absence of TNFalpha . Cell extracts were prepared at the indicated time points and used for determination of Luciferase activity (A), analysis of kappa B-specific DNA binding by EMSA as before (B), and Western immunoblot (50 µg of whole cell extract) with RelB- and Ikappa Balpha -specific antibodies (C). *, nonspecific bands; Co, control.

NF-kappa B Induction in Response to ER Overload and MEKK1 Overexpression Is Intact-- An important intracellular stress response pathway leading to NF-kappa B activation was discovered a few years ago (40, 41). Overexpression of secreted proteins leading to overload of the endoplasmic reticulum results in a strong activation of NF-kappa B. We overexpressed influenza virus hemagglutinin in S107 cells and assayed NF-kappa B function by cotransfection of a kappa B-dependent reporter. This resulted in a potent activation of kappa B-dependent transcription (Fig. 8A). Addition of PDTC could block this activation efficiently. We therefore conclude that at least two pathways for NF-kappa B activation, one induced by TNFalpha and the other by ER overload, are still functionally intact in the mutant S107 cell line, and both pathways are PDTC-sensitive.


View larger version (10K):
[in this window]
[in a new window]
 
Fig. 8.   ER overload and MEKK1 can induce NF-kappa B activity in S107 cells. A, S107 cells were transiently cotransfected with 3xkappa B-luc together with empty expression vector or expression vector for the hemagglutinin gene (HA) of the human influenza virus. 16 h post-transfection cells were treated for 6 h with PDTC as indicated. Luciferase activities were determined and normalized on the basis of beta -galactosidase expression. The fold induction over the activity of untreated cells cotransfected with reporter plasmid and empty expression vector is shown. B, activation of NF-kappa B by MEKK1. S107 cells were transiently cotransfected with a kappa B-dependent reporter plasmid together with the empty expression vector or an expression vector for a constitutively active version of MEKK1 (51). 16 h after transfection, cells were treated for 6 h with PDTC before harvesting as indicated. Luciferase activities were determined and normalized on the basis of beta -galactosidase expression. The fold induction over the activity of untreated cells cotransfected with reporter plasmid and empty expression vector is shown.

MEKK1, like NIK, is a member of the mitogen-activated protein kinase kinase kinases. It had been previously suggested to be involved in the induction of the stress kinase pathway as well as NF-kappa B activation. Furthermore, MEKK1 was recently shown to directly phosphorylate subunits of a biochemically purified Ikappa Balpha kinase complex in vitro (42), and MEKK1 is present in the partially purified IKK complex (14). To determine whether MEKK1 could activate NF-kappa B in S107 cells, a constitutively active version of MEKK1 was cotransfected together with the kappa B-dependent reporter. Similar to results obtained in other cell lines (42, 43), MEKK1 efficiently activated this reporter, and this induction could not be blocked by treatment of the cells with PDTC (Fig. 8B). This result suggests that MEKK1 interacts with the NF-kappa B induction pathway downstream of the PDTC-sensitive step. Furthermore, the defect in the S107 cells is apparently localized upstream of the MEKK1 protein or similar integrators of the signaling pathways.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

The signal transduction pathways leading to activation of NF-kappa B are clearly quite complex. Here we have analyzed the defect in a mutant plasmacytoma cell line, and we could demonstrate that the specific mutation affects several signal transduction pathways leading to NF-kappa B activation, whereas others are still intact. Therefore the defect in these cells most likely affects a common signaling intermediate for a subset of different NF-kappa B-inducing stimuli. Furthermore, the observation that the defect in S107 cells affects both the constitutive as well as the inducible activity of NF-kappa B suggests that these two pathways are in fact interwoven or at least share a critical common component.

Too little is known about the critical signal transduction components of such diverse stimuli as PMA, LPS, H2O2, x-rays, or UV irradiation to pinpoint a specific target molecule that could explain the defect in S107 cells. However, it was shown in the past that NF-kappa B activation by all these stimuli can be blocked by the addition of the radical scavenger PDTC (44, 45). Therefore, the PDTC-sensitive step in the signaling relay was a prime candidate for the defect in these cells. Such a conclusion was drawn from the analysis of the mutant 70Z/3 pre-B cell line, which was analyzed by Courtois and colleagues recently (38). They did indeed find that all inducers that were able to activate NF-kappa B in these cells, namely phosphatase inhibitors, osmotic shock, and the human T-cell lymphotrophic virus Tax protein, were not blocked by the anti-oxidant (38).

We do not believe that the defect in the mutant 70Z/3 cell line and the S107 plasmacytoma cells is identical. The signal transduction pathways leading to NF-kappa B induction by interleukin-1 and TNFalpha are thought to converge at a membrane-proximal step. Both receptors interact directly or indirectly with members of the TRAF protein family (TRAF2 and TRAF6, respectively), which then physically interact with NIK (46). NIK itself can then contact either IKKalpha or the IKKalpha -IKKbeta heterodimer, which the catalytic subunits of the Ikappa B kinase complex, and this interaction is involved in activating the Ikappa B kinase (12, 15). Whereas in the mutant 70Z/3 cells this signaling pathway was defective, we could demonstrate that TNFalpha efficiently induced NF-kappa B in S107 cells. This would put the defect in S107 cells upstream of the defect in the variant 70Z/3 cells, because only a subset of PDTC-sensitive pathways are affected in this cell. As a cautionary note it should be stressed that it is at present unclear whether there is something like "the" PDTC sensitive step in NF-kappa B activation or whether PDTC might affect many different components of the cell and thereby interfere at different levels. Potential explanations for the PDTC sensitivity of the TNFalpha -induced NF-kappa B pathway could be that either the TRAF-NIK-IKKalpha -IKKbeta interaction per se or one of the kinases (NIK/IKKalpha -IKKbeta ) are in fact sensitive to PDTC. The observation that NF-kappa B induction by phophatase inhibitors and overexpression of the MEKK1 kinase are not affected by PDTC suggests that the IKK complex itself is not sensitive to the radical scavenger. In this respect it is of interest that the biochemically purified Ikappa B kinase is in fact a large multisubunit complex and the cloned IKKalpha -IKKbeta only represent the catalytic subunits (11, 42, 47).

Our finding that ER overload can also activate NF-kappa B in S107 cells indicates that in addition to the direct activation by TNFalpha , most likely via the described TRAF-NIK pathway, other pathways also work in S107 cells. It is unlikely, however, that ER overload also directly funnels into the TRAF-NIK-IKKalpha cascade. Earlier experiments had demonstrated that in contrast to TNFalpha stimulation, ER overload induction of NF-kappa B was sensitive to intracellular calcium chelators (48, 49). An increase in intracellular Ca2+ concentration is, however, not sufficient to induce NF-kappa B in S107 cells, because ionomycin treatment did not result in NF-kappa B activation (Fig. 1).

The localization of the defect in S107 cells upstream of the defect in the variant 70Z/3 cell line is of specific interest with respect to the fact that S107 cells show a dual defect in both inducible and constitutive NF-kappa B activity. As a consequence one might speculate that the signaling intermediate, important for such diverse stimuli as PMA, LPS, H2O2, UV light, and x-rays, is also involved in regulating constitutive NF-kappa B activity in B cells.

A further important result of our analysis is the demonstration that identical stimuli, such as okadaic acid and calyculin A, can induce NF-kappa B via distinct pathways in different cell types. Induction in Jurkat cells and PD31 cells was completely reverted by pretreatment of the cells with PDTC; no effect of the radical scavenger was detected in S107 cells. It should be noted that different results have been reported with respect to the PDTC sensitivity of okadaic acid induced NF-kappa B. Whereas Sun and colleagues reported that calyculin A induction of NF-kappa B was not sensitive to PDTC treatment (50), Schmidt and colleagues found that okadaic induction of NF-kappa B involves a PDTC sensitive step (39). Our results suggest that the same stimuli can induce NF-kappa B by different pathways, apparently depending on the cell type. In the case of the Jurkat and PD31 cells, the phosphatase inhibitors elicit an "upstream" signal relayed through the conventional Ikappa B kinase pathway, whereas in the S107 cells and mutant 70Z/3 cells, these inhibitors function more downstream. In fact it was recently shown that phosphatases sensitive to okadaic acid can inactivate the purified Ikappa B kinase complex (11). These data demonstrate that the Ikappa B kinase complex itself is regulated by phosphorylation events. The details of this regulation need to be elucidated, however. It could be envisaged that the phosphatase inactivating the Ikappa B kinase complex is hyperactive in S107 cells. However, the finding that ER overload and TNFalpha signaling still function completely normally in these cells and the observation that even continuous treatment with stimuli such as LPS or H2O2 did not induce NF-kappa B more likely point to an upstream signal transduction defect.

Clearly, NIK is one of the upstream activators and might in fact directly phosphorylate and activate the Ikappa B kinase. However, consistent with earlier observations, MEKK1, which can also directly phosphorylate and activate a biochemically purified Ikappa Balpha kinase (42), is also capable of activating NF-kappa B in the mutant S107 cell line. Although MEKK1 is also activated by TNFalpha , experiments with dominant negative versions of MEKK1 have shown that this pathway is not crucial for NF-kappa B activation by TNFalpha (43, 51). Nevertheless it has been reported previously that transfection of a constitutively active form of MEKK1 does result in the activation of NF-kappa B (42, 43, 52).

It will be important to characterize the S107 cell defect at the molecular level. The previous cell fusion experiments already suggested that the defect is recessive. It should be interesting to fuse the S107 cells with the mutant 70Z/3 cell line see whether the two defects can complement each other. Furthermore, these cells can be used in a complementation screening approach to identify this critical component of the NF-kappa B induction pathway. In addition, the fact that we can make these cells NF-kappa B-positive by stably transfecting a RelB expression vector makes these cells a valuable tool for identifying specific NF-kappa B target genes in B cells.

    ACKNOWLEDGEMENTS

We thank M. Karin for the MEKK1 expression plasmids, N. Rice for the Ikappa Bepsilon -specific antibodies, S. Ludwig for the hemagglutinin expression vector, R. Schreck and A. Denk for critical reading of the manuscript and helpful suggestions, R. Röder for help typing the manuscript, and S. Pfränger for preparation of the figures.

    FOOTNOTES

* This work was supported by Grant DFG Wi 789/2-1 from the Deutsche Forschungsgemeinschaft and Grant 97.031.1 from the Wilhelm Sander Stiftung (to T. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 49-931-201-5145; Fax: 49-931-201-5147; E-mail: t.wirth{at}rzbox.uni-wuerzburg.de.

1 The abbreviations used are: IKK, Ikappa B kinase; LPS, lipopolysaccharide; PMA, phorbol 12-myristate 13-acetate; ER, endoplasmic reticulum; TNFalpha , tumor necrosis factor alpha ; EMSA, electrophoretic mobility shift assay; PDTC, pyrrolidine dithiocarbamate; ALLN, N- acetyl-leucyl-leucyl-norleucinal.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

  1. Baeuerle, P. A., and Henkel, T. (1994) Annu. Rev. Immunol. 12, 141-179[CrossRef][Medline] [Order article via Infotrieve]
  2. Baldwin, A. S., Jr. (1996) Annu. Rev. Immunol. 14, 649-681[CrossRef][Medline] [Order article via Infotrieve]
  3. Siebenlist, U., Franzoso, G., and Brown, K. (1994) Annu. Rev. Cell. Biol. 10, 405-455[CrossRef]
  4. Schreck, R., Kistler, B., and Wirth, T. (1997) in The NF-Transcription Factors in Eukaryotes (Papavassiliou, A. G., ed), pp. 153-188, R. G. Landes Company, Austin, TX
  5. Sen, R., and Baltimore, D. (1986) Cell 46, 705-716[Medline] [Order article via Infotrieve]
  6. Sen, R., and Baltimore, D. (1986) Cell 47, 921-928[Medline] [Order article via Infotrieve]
  7. Grilli, M., Chiu, J. J.-S., and Lenardo, M. J. (1993) Int. Rev. Cytol. 143, 1-62[Medline] [Order article via Infotrieve]
  8. Baeuerle, P. A., and Baltimore, D. (1988) Science 242, 540-546[Medline] [Order article via Infotrieve]
  9. Whiteside, S. T., Epinat, J.-C., Rice, N. R., and Israel, A. (1997) EMBO J. 16, 1413-1426[Abstract/Free Full Text]
  10. Li, Z., and Nabel, G. J. (1997) Mol. Cell. Biol. 17, 6184-6190[Abstract]
  11. DiDonato, J. A., Hayakawa, M., Rothwarf, D. M., Zandi, E., and Karin, M. (1997) Nature 388, 548-554[CrossRef][Medline] [Order article via Infotrieve]
  12. Régnier, C. H., Song, H. Y., Gao, X., Goeddel, D. V., Cao, Z., and Rothe, M. (1997) Cell 90, 373-383[Medline] [Order article via Infotrieve]
  13. Zandi, E., Rothwarf, D. M., Delhase, M., Hayakawa, M., and Karin, M. (1997) Cell 91, 243-252[Medline] [Order article via Infotrieve]
  14. Mercurio, F., Zhu, H., Murray, B. W., Shevchenko, A., Bennett, B. L., Li, J. W., Young, D. B., Barbosa, M., Mann, M., Manning, A., and Rao, A. (1997) Science 278, 860-866[Abstract/Free Full Text]
  15. Woronicz, J. D., Gao, X., Cao, Z., Rothe, M., and Goeddel, D. V. (1997) Science 278, 866-869[Abstract/Free Full Text]
  16. Brown, K., Gerstberger, S., Carlson, L., Franzoso, G., and Siebenlist, U. (1995) Science 267, 1485-1488[Medline] [Order article via Infotrieve]
  17. Brockman, J., Scherer, D., McKinsey, T., Hall, S., Qi, X., Lee, W., and Ballard, D. (1995) Mol. Cell. Biol. 15, 2809-2918[Abstract]
  18. Traenckner, E. B.-M., Pahl, H. L., Henkel, T., Schmidt, K. N., Wilk, S., and Baeuerle, P. A. (1995) EMBO J. 14, 2876-2883[Abstract]
  19. Whiteside, S. T., Ernst, M. K., LeBail, O., Laurent-Winter, C., Rice, N., and Israel, A. (1995) Mol. Cell. Biol. 15, 5339-5345[Abstract]
  20. Miyamoto, S., Maki, M., Schmitt, M. J., Hatanaka, M., and Verma, I. M. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 12740-12744[Abstract/Free Full Text]
  21. Traenckner, E. B., Wilk, S., and Baeuerle, P. A. (1994) EMBO J. 13, 5433-5441[Abstract]
  22. Alkalay, I., Yaron, A., Hatzubai, A., Orian, A., Ciehanover, A., and Ben-Neriah, Y. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 10599-10603[Abstract]
  23. DiDonato, J. A., Mercurio, F., and Karin, M. (1995) Mol. Cell. Biol. 15, 1302-1311[Abstract]
  24. Lin, Y. C., Brown, K., and Siebenlist, U. (1995) Proc. Natl. Acad. Sci U. S. A. 92, 552-556[Abstract]
  25. Kistler, B., Rolink, A., Marienfeld, R., Neumann, M., and Wirth, T. (1998) J. Immunol. 160, 2308-2317[Abstract/Free Full Text]
  26. Miyamoto, S., Chiao, P. J., and Verma, I. M. (1994) Mol. Cell. Biol. 14, 3276-3282[Abstract]
  27. Liou, H. C., Sha, W. C., Scott, M. L., and Baltimore, D. (1994) Mol. Cell. Biol. 14, 5349-5359[Abstract]
  28. Dobrzanski, P., Ryseck, R.-P., and Bravo, R. (1994) EMBO J. 13, 4608-4616[Abstract]
  29. Lernbecher, T., Müller, U., and Wirth, T. (1993) Nature 365, 767-770[CrossRef][Medline] [Order article via Infotrieve]
  30. Lernbecher, T., Kistler, B., and Wirth, T. (1994) EMBO J. 13, 4060-4069[Abstract]
  31. Suyang, H., Phillips, R., Douglas, I., and Ghosh, S. (1996) Mol. Cell. Biol. 16, 5444-5449[Abstract]
  32. Phillips, R. J., and Ghosh, S. (1997) Mol. Cell. Biol. 17, 4390-4396[Abstract]
  33. Atchison, M. L., and Perry, R. P. (1987) Cell 48, 121-128[Medline] [Order article via Infotrieve]
  34. Meyer, K. B., and Neuberger, M. S. (1989) EMBO J. 8, 1959-1964[Abstract]
  35. Atchison, M. L., and Perry, R. P. (1988) EMBO J. 7, 4213-4220[Abstract]
  36. Kirillov, A., Kistler, B., Mostoslavsky, R., Cedar, H., Wirth, T., and Bergman, Y. (1996) Nat. Genet. 13, 435-441[Medline] [Order article via Infotrieve]
  37. Kistler, B., Baumann, B., Bergman, Y., and Wirth, T. (1997) Immunobiology 198, 24-34[Medline] [Order article via Infotrieve]
  38. Courtois, G., Whiteside, S. T., Sibley, C. H., and Israel, A. (1997) Mol. Cell. Biol. 17, 1441-1449[Abstract]
  39. Schmidt, K. N., Traenckner, E. B.-M., Meier, B., and Baeuerle, P. A. (1995) J. Biol. Chem. 270, 27136-27142[Abstract/Free Full Text]
  40. Pahl, H. L., and Baeuerle, P. A. (1995) J. Virol. 69, 1480-1484[Abstract]
  41. Pahl, H. L., and Baeuerle, P. A. (1995) EMBO J. 14, 2580-2588[Abstract]
  42. Lee, F. S., Hagler, J., Chen, Z. J., and Maniatis, T. (1997) Cell 88, 213-222[Medline] [Order article via Infotrieve]
  43. Song, H. Y., Regnier, C. H., Kirschning, C. J., Goeddel, D. V., and M., R. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 9792-9796[Abstract/Free Full Text]
  44. Schreck, R., Rieber, P., and Baeuerle, P. A. (1991) EMBO J. 10, 2247-2258[Abstract]
  45. Schreck, R., Meier, B., Maennel, D. N., Droege, W., and Baeuerle, P. A. (1992) J. Exp. Med. 175, 1181-1194[Abstract]
  46. Malinin, N. L., Boldin, M. P., Kovalenko, A. V., and Wallach, D. (1997) Nature 385, 540-544[CrossRef][Medline] [Order article via Infotrieve]
  47. Chen, Z. J., Parent, L., and Maniatis, T. (1996) Cell 84, 853-862[Medline] [Order article via Infotrieve]
  48. Pahl, H. L., and Baeuerle, P. A. (1996) FEBS Lett. 392, 129-136[CrossRef][Medline] [Order article via Infotrieve]
  49. Pahl, H. L., and Baeuerle, P. A. (1997) Trends Biochem. Sci. 22, 63-67[CrossRef][Medline] [Order article via Infotrieve]
  50. Sun, S.-C., Maggirwar, S. B., and Harhaj, E. (1995) J. Biol. Chem. 270, 18347-18351[Abstract/Free Full Text]
  51. Liu, Z.-G., Hsu, H., Goeddel, D. V., and Karin, M. (1996) Cell 87, 565-576[Medline] [Order article via Infotrieve]
  52. Meyer, C. F., Wang, X., Chang, C., Templeton, D., and Tan, T.-H. (1996) J. Biol. Chem. 271, 8971-8976[Abstract/Free Full Text]


Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.