From the 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
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ABSTRACT |
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Studies on the mechanisms of inducible and
constitutive activity of NF-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-
B
activity. Our analysis shows that these cells bear a specific defect
that interferes with NF-
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-
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-
B. Interestingly, both the potent
physiological inducer of NF-
B TNF
as well as endoplasmic
reticulum overload can induce NF-
B via a PDTC sensitive pathway. In
all cases, DNA-binding NF-
B complexes are comprised predominantly of
p50-RelA heterodimers, and NF-
B activation results in the induction
of transiently transfected or resident reporter genes. In summary,
these results suggest that the pathways for many NF-
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-
B induction. These
routes utilized by tumor necrosis factor
and endoplasmic reticulum
overload are still intact in this cell line.
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INTRODUCTION |
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The NF-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-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-
B family members are
localized in the cytoplasm due to the interaction with inhibitory
proteins, the I
Bs (8). These I
B proteins first of all inhibit
nuclear translocation of NF-
B proteins by masking the nuclear
localization signal and also directly block their DNA binding. The
I
B proteins again represent a protein family consisting of at least
six family members described so far. I
B
, I
B
, I
B
, and
Bcl3 represent independent genes, whereas I
B
and I
B
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-
B dimeric complexes.
IB
, I
B
, and I
B
are involved in regulation of the
inducible activity of the NF-
B complexes. A variety of external or internal stimuli lead to the initiation of a signal transduction cascade, which culminates in the activation of I
B kinase(s). Two
such I
B kinases (IKK
and
IKK
)1 have been cloned
recently, but the details of the activation of these kinase are not
known yet (11-15). I
B proteins become phosphorylated at specific
serine residues localized in an amino-terminal signal response domain
(16-19). These serine phosphorylated I
B molecules are subsequently
targeted by poly-ubiquitination for degradation via the proteasome
pathway (20-24). The degradation of I
B proteins leads to the
release of NF-
B complexes, which can migrate to the nucleus, bind to
DNA, and regulate transcription. Target genes regulated by NF-
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-B
in an inhibited form just like most other cell types, but later stages
of B cell development contain constitutive nuclear NF-
B (1-4). This
activation of NF-
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-
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
I
B
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 I
B
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 I
B inhibition (29, 30). A specific role
for I
B
has also been suggested recently (25, 31, 32). In the
process of B cell differentiation hypophosphorylated I
B
, which
can still interact with NF-
B but does not mask the nuclear
localization signal and fails to inhibit DNA binding, could shield
NF-
B from inhibition by I
B
(31, 32).
The constitutive activation of NF-B in B cells may have some
components in common with the inducible branch of NF-
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-
B
(33). In this cell line,
B-dependent transcription of
reporter genes is completely absent; the endogenous Ig
gene shows
normal expression, however. This is most likely due to the activity of
the 3'
-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-
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-B and I
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 I
B molecules
and treatment of cytoplasmic extracts with the detergent deoxycholate
released DNA-binding NF-
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
B-dependent
transcriptional activity. In addition, whereas the parental S107 cells
showed a striking defect in B cell-specific demethylation of a
transfected methylated Ig
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-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-
B induction.
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EXPERIMENTAL PROCEDURES |
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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 -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
-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 3x
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- enhancer consensus NF-
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
I
B
antibodies, which were a kind gift from Dr. Nancy Rice.
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RESULTS |
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S107 Cells Show a Specific Defect in NF-B Induction--
The
S107 cell line, in contrast to virtually all other known plasmacytoma
cell lines, lacks constitutive nuclear NF-
B proteins and is also
defective in induction on NF-
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-
B activation. Therefore, we analyzed a variety
of different stimuli for their ability to activate NF-
B and, as a
control, the AP-1 transcription factors. Untreated S107 cells lack
DNA-binding NF-
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-
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-
B activity, all treatments
resulted in the induction of NF-
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-
B pathway.
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Phosphatase Inhibitors Can Induce NF-B in S107 Cells--
A
mutant pre-B cell line that also failed to activate NF-
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-
B induction in S107 cells and as a control in Jurkat cells. Indeed, the inhibitors were able to
induce NF-
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-
B induction, 5 nM okadaic acid resulted in induced
NF-
B DNA binding, suggesting that phosphatase 2A is involved in the
activation (data not shown). Activation of NF-
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).
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TNF Is a Potent Inducer of NF-
B in S017 Cells--
The
analysis of the NF-
B induction by TNF
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-
B induction in these pathways. Interestingly, when
the catalytic subunits of the cytokine-inducible I
B kinase (IKK
and IKK
) were cloned (11-15), a direct physical interaction between
NIK and IKK
or IKK
-IKK
heterodimers was demonstrated.
Therefore, in this pathway the essential upstream components required
for NF-
B activation are known. We therefore asked whether TNF
could activate NF-
B in the S107 cells. Interestingly, in contrast to
the other physiological inducers of NF-
B tested before, TNF
was
readily capable of inducing NF-
B even in S107 cells (Fig.
3A). Because TNF
induction
of NF-
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-
B, whereas PDTC by itself had virtually no effect (Fig.
3B).
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TNF 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-
B (36, 37). We had furthermore
suggested that RelB is a target gene of NF-
B. We therefore asked
whether prolonged treatment of S107 cells with TNF
, which led to the
induction of NF-
B, would result in the expression of the endogenous
relB gene. Indeed, when extracts from Jurkat and S107 cells
induced with TNF
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 I
B
, another
known target gene of NF-
B, is degraded early after induction with
TNF
and then efficiently resynthesized. These results demonstrate
that NF-
B induced by TNF
in S107 cells is capable of activating
endogenous target genes.
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NF-B Induction in Response to ER Overload and MEKK1
Overexpression Is Intact--
An important intracellular stress
response pathway leading to NF-
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-
B. We overexpressed influenza virus hemagglutinin in S107 cells
and assayed NF-
B function by cotransfection of a
B-dependent reporter. This resulted in a potent
activation of
B-dependent transcription (Fig.
8A). Addition of PDTC could
block this activation efficiently. We therefore conclude that at least
two pathways for NF-
B activation, one induced by TNF
and the
other by ER overload, are still functionally intact in the mutant S107
cell line, and both pathways are PDTC-sensitive.
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DISCUSSION |
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The signal transduction pathways leading to activation of NF-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-
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-
B-inducing stimuli.
Furthermore, the observation that the defect in S107 cells affects both
the constitutive as well as the inducible activity of NF-
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-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-
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-B induction by interleukin-1 and TNF
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 IKK
or the IKK
-IKK
heterodimer, which
the catalytic subunits of the I
B kinase complex, and this
interaction is involved in activating the I
B kinase (12, 15).
Whereas in the mutant 70Z/3 cells this signaling pathway was defective,
we could demonstrate that TNF
efficiently induced NF-
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-
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 TNF
-induced NF-
B pathway could be that either
the TRAF-NIK-IKK
-IKK
interaction per se or one of the
kinases (NIK/IKK
-IKK
) are in fact sensitive to PDTC. The
observation that NF-
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 I
B
kinase is in fact a large multisubunit complex and the cloned
IKK
-IKK
only represent the catalytic subunits (11, 42, 47).
Our finding that ER overload can also activate NF-B in S107 cells
indicates that in addition to the direct activation by TNF
, 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-IKK
cascade. Earlier experiments had
demonstrated that in contrast to TNF
stimulation, ER overload
induction of NF-
B was sensitive to intracellular calcium chelators
(48, 49). An increase in intracellular Ca2+ concentration
is, however, not sufficient to induce NF-
B in S107 cells, because
ionomycin treatment did not result in NF-
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-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-
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-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-
B. Whereas Sun and colleagues reported that calyculin A induction
of NF-
B was not sensitive to PDTC treatment (50), Schmidt and
colleagues found that okadaic induction of NF-
B involves a PDTC
sensitive step (39). Our results suggest that the same stimuli can
induce NF-
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 I
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 I
B kinase complex (11). These data
demonstrate that the I
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 I
B kinase complex is hyperactive in S107 cells.
However, the finding that ER overload and TNF
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-
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 IB kinase. However, consistent with earlier observations, MEKK1, which can also directly phosphorylate and activate a biochemically purified I
B
kinase (42), is also capable of activating NF-
B in the mutant S107 cell
line. Although MEKK1 is also activated by TNF
, experiments with
dominant negative versions of MEKK1 have shown that this pathway is not
crucial for NF-
B activation by TNF
(43, 51). Nevertheless it has
been reported previously that transfection of a constitutively active
form of MEKK1 does result in the activation of NF-
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-B induction pathway. In addition, the fact that we can
make these cells NF-
B-positive by stably transfecting a RelB
expression vector makes these cells a valuable tool for identifying
specific NF-
B target genes in B cells.
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ACKNOWLEDGEMENTS |
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We thank M. Karin for the MEKK1 expression
plasmids, N. Rice for the IB
-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.
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FOOTNOTES |
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* 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, IB kinase;
LPS, lipopolysaccharide; PMA, phorbol 12-myristate 13-acetate; ER,
endoplasmic reticulum; TNF
, tumor necrosis factor
; EMSA,
electrophoretic mobility shift assay; PDTC, pyrrolidine
dithiocarbamate; ALLN, N-
acetyl-leucyl-leucyl-norleucinal.
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REFERENCES |
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