Ras-related GTPase RhoB Represses NF-
B Signaling*
Gerhard
Fritz
and
Bernd
Kaina
From the Division of Applied Toxicology, Institute of Toxicology,
University of Mainz, D-55131 Mainz, Germany
Received for publication, June 12, 2000, and in revised form, October 30, 2000
 |
ABSTRACT |
rhoB encoding a Ras-related GTPase is
immediate-early inducible by genotoxic treatments, indicating that it
is part of the cellular stress response. Here, we investigated the
influence of RhoB on signal pathways that are rapidly evoked by
genotoxic compounds. The data obtained show that wild-type RhoB neither affects activation of mitogen-activated protein kinases nor
AP-1-dependent gene expression. However, RhoB
inhibited both basal and genotoxic agent-stimulated activity of the
transcription factor nuclear factor
B (NF-
B). Thus, RhoB
attenuated alkylation-induced increase in the DNA binding activity of
NF-
B and abrogated NF-
B-driven gene expression. Furthermore, RhoB
inhibited decrease in the cellular amount of I
B
after
genotoxic stress as well as after tumor necrosis factor
and
12-O-tetradecanoylphorbol acetate treatment. This indicates that RhoB represses NF-
B activation by inhibiting
dissociation and subsequent degradation of I
B
. On the basis of
the data, we suggest that RhoB is a novel negative regulator of NF-
B signaling.
 |
INTRODUCTION |
Ras-related small GTPases (molecular mass, ~21 kDa) of the Rho
family (e.g. Rho, Rac, and Cdc42) are known to be involved in a large variety of cellular processes, such as the organization of
the microfilamental network (1-5), cell cycle progression (6, 7),
cellular transformation (8-10), and apoptosis (11-13). Furthermore,
Rac and Cdc42 have been shown to interfere with genotoxic stress-induced signaling by regulating the activation of N-terminal c-Jun kinases
(JNKs)1/stress-activated
protein kinases (SAPKs) and p38 mitogen-activated protein (MAP) kinase
(14-16). This regulatory function of Rac and Cdc42 is independent of
their influence on cytoskeleton or cell cycle (17). After exposure of
cells to genotoxic stress, Rac and Cdc42 trigger, via activation of MAP
kinases, the activity of transcription factors such as c-Jun, c-Fos,
Elk-1, and ATF-2 (18). Thereby, the pattern of gene expression
in cells exposed to DNA damage is controled. Another transcription
factor that is activated by different types of cellular stress is
nuclear factor
B (NF-
B; Refs. 19, 20). A prerequisite for
activation of NF-
B by tumor necrosis factor
(TNF
) is
IKK-dependent phosphorylation of the inhibitory
molecule I
B
(19). On its phosphorylation on Ser32,
NF-
B is released for nuclear translocation, and free I
B
is proteasomally degraded (19). In the case of treatment of cells with
ionizing radiation and UV light, activation of NF-
B occurs independent of phosphorylation of I
B
on Ser32 (21,
22). Moreover, UV-induced activation of NF-kB is even independent of
IKK (22). Thus, overall, activation of NF-
B by genotoxic stress is
different from activation triggered by TNF
. Previously, it was
reportet that Rho family GTPases interfere with the TNF
-induced
activation of NF-
B (23). Also, different members of nucleotide
exchange factors for Rho proteins are able to stimulate NF-
B (24),
indicating that multiple, Rho-regulated pathways are involved in
NF-
B regulation.
Recently, we have shown that the gene encoding the small GTPase RhoB
belongs to the group of immediate-early inducible genes (25). It is
activated by genotoxic stress (25, 26) as well as by growth factors
(25, 27). The regulation of rhoB induction appears to be
different from that of c-jun and c-fos (25-27).
The physiological function of RhoB is largely unknown. One interesting feature of RhoB is that it is essentially required for Ras-mediated transformation (8). Therefore, and because RhoB is subject to
modification by farnesylation (28), RhoB is discussed as a further
physiologically relevant target of farnesyltransferase inhibitors (29).
Originally, this type of drug was described as a promising
tumortherapeutic acting on Ras (30, 31). A variety of evidence strongly
indicates that the function of RhoB is distinguished from that of other
Rho species such as RhoA and RhoC. Thus, RhoB is distinguished from
RhoA/RhoC in its C-terminal modification by farnesylation (28),
intracellular localization (32) and concentration (25), association
with regulatory proteins (33), cell cycle-specific expression (34),
endosomal targeting of PRK1 (protein kinase C-related kinase 1)
(35), and inducibility by growth factors and genotoxic stress (25-27).
The latter finding is of particular interest because the rapidly
increased expression of rhoB in cells exposed to a genotoxic
agent indicates that RhoB plays a role in the cellular response to
induced DNA damage. This view gains support from the recent finding
that RhoB forces cells to alkylation-induced apoptotic cell death (36).
Because of its GTPase function, it is reasonble to assume that RhoB can
influence signal transmission in a very fast manner. Having this in
mind, we wondered whether RhoB interferes with early steps of cellular stress response, in particular with stress-induced gene expression participating in the regulation of apoptosis. To address this question,
we analyzed the effect of RhoB on the activation of MAP kinases and
NF-
B signaling by genotoxic agents. Here, we show that RhoB
represses NF-
B signaling without influencing MAP kinase-regulated pathways.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Rat rhoB cDNA was provided by T. Hunter. Hemagglutinin (HA) antibody was purchased from Roche Molecular
Biochemicals, and IKK
antibody was from Pharmingen (Hamburg,
Germany). Other antibodies used in the present study were from Santa
Cruz Biotechnology Inc. (Santa Cruz, CA). Methyl methanesulfonate (MMS)
was purchased from Sigma. Mafosfamide was from ASTA Medica (Frankfurt,
Germany). The NF-
B-specific reporter gene construct
(3xNF-
B-luciferase) was kindly provided by U. R. Rapp. The
human collagenase promoter-chloramphenicol acetyltrasferase (Coll-CAT)
construct containing the AP-1-regulated fragment (
73/+63) of human
interstitial collagenase gene (37) was obtained from H. J. Rahmsdorf, and the HA-JNK1 expression construct originates from M. Karin. Expression vectors encoding Rho GTPases RhoA, Cdc42, and Rac
were a gift from A. Hall.
Cell Culture and Transfection Experiments--
NIH 3T3 cells
were routinely grown in Dulbecco's modified Eagle's medium containing
10% fetal calf serum. Isolation of RhoB-overexpressing cells has been
described previously (36). For determination of
NF-
B-dependent gene expression, an NF-
B-specific
reporter gene construct was used (3xNF-
B-luciferase; Ref. 38).
24 h after transfection of NIH 3T3 cells together or without an
expression vector encoding the corresponding Rho GTPases, cells
were exposed to a given genotoxic compound. After an incubation period
of 24 h, cells were harvested, and luciferase activity was
analyzed using a luciferase assay (Promega). To analyze the effect of
Rho GTPases on the activity of the AP-1-regulated collagenase promoter, a human Coll-CAT construct (
73/+63; Ref. 37) was used for
transfection experiments. 24 h after transfection, cells were
harvested, and the amount of CAT protein was determined by the use of
an enzyme-linked immunosorbent assay-based method (Roche Molecular Biochemicals).
Western Blot Analysis--
20-30 µg of protein from nuclear
or total cell extracts (1000 × g supernatant) were
separated on 10% SDS-polyacrylamide gels. Proteins were wet-blotted to
nitrocellulose filters and were detected using the corresponding
primary antibodies and subsequently a peroxidase-coupled secondary
antibody. Incubation of filters with antibodies was performed for
2 h at room temperature in 5% dry milk in phosphate-buffered
saline and 0.1% Tween 20. For visualization of proteins, an ECL
detection system was used (PerkinElmer Life Sciences).
Gel Retardation Analysis--
32P labeling of
oligonucleotides was performed using T4 kinase. Total cell extracts
were prepared as described (39). Binding reactions were performed by
incubation of 10 µg of protein from NIH 3T3 extracts with ~5 fmol
of 32P-labeled oligonucleotide for 30 min at room
temperature (binding buffer, 10 mM Hepes, pH 7.9, 60 mM KCl, 5 mM MgCl2, 1 mM EDTA, 1 mM dithiothreitol, 1 µg/ml bovine
serum albumin, 10% glycerol, 0.5 µg poly(dI-dC)). Subsequently,
reaction mixtures were separated on 4% polyacrylamide gels at room
temperature. After electrophoresis, gels were dried and subjected to
autoradiography. The NF-
B specific oligonucleotide was purchased
from Promega (5'-AGTTGAGGGGACTTTCCCAGGC-3'). The sequence
of AP-1-specific oligonucleotide was derived from the mouse collagenase
promoter (5'-AGTGGTGACTCATCACT-3').
Kinase Assays--
JNK, p38, and extracellular signal-regulated
kinase (ERK) kinase activities were measured by the immune complex
assay system after immunoprecipitation of the corresponding kinases
from NIH 3T3 cells using monoclonal antibodies (Santa Cruz). As
substrate for the JNK reaction, glutathione
S-transferase-Jun (kindly provided by P. Angel), for
p38 kinase, glutathione S-transferase-ATF-2 (Santa Cruz),
and for the ERK reaction, myelin basic protein (Sigma) were used.
Reactions were performed at 30 °C for 30 min in a total volume of 40 µl of reaction buffer (JNK/p38 kinase reaction buffer: 25 mM Hepes, pH 7.6, 20 mM MgCl2, 20 mM
-glycerolphosphate, 0.1 mM sodium
orthovanadate, 2 mM dithiothreitol, 25 µM
ATP, 1 µCi of [
-32P]ATP; ERK reaction buffer: 20 mM Hepes, pH 7.1, 10 mM MgCl2, 1 mM sodium orthovanadate, 25 µM ATP, 1 µCi
of [
-32P-ATP]). Alternatively, ERK activity was
analyzed by a Western blot-based method as described previously (40).
Determination of IKK activity was performed by the use of an immune
complex assay system on immunoprecipitation of Ikk
with a specific
antibody (Pharmingen; reaction buffer: 20 mM Hepes, pH 7.4, 20 mM MgCl2, 10 mM
MnCl2, 1 mM EDTA, 1 mM
dithiothreitol, 0.1 mM sodium orthovanadate). As a
substrate for IKK, recombinant glutathione
S-transferase-I
B
was used. Reaction products were
separated by SDS-polyacrylamide gel electrophoresis and visualized by
autoradiography of the dried gels. For quantitation, autoradiograms
were densitometrically analyzed.
 |
RESULTS |
To analyze whether RhoB interferes with the early cellular
response to genotoxic stress, we made use of NIH 3T3 cells stably and
transiently overexpressing wild-type RhoB protein. For stable transfection, a pCDNA3neo expression vector containing the cDNA sequence for His-tagged wild-type RhoB was used. Cell clones
overexpressing RhoB were identified by Western blot analysis using a
His-specific antibody (36). Activation of MAP kinases represents one
type of early cellular response to genotoxic stress that affects gene expression (18). Comparing parental and RhoB-overexpressing cells with
regard to the activation of MAP kinases, we observed a similar level of
activation of JNK1, p38 MAP kinase, and ERK2 on treatment with MMS and
UV light (Fig. 1A). In line
with this, transient overexpression of RhoB (V14RhoB) also
failed to stimulate JNK1 activity (Fig. 1B). On the other
hand, as one would expect from data previously reported (15, 16), Cdc42
(V12Cdc42) was able to activate JNK1 (Fig. 1B).
The UV- and MMS-stimulated increase in the DNA binding activity of AP-1
was similar in RhoB-overexpressing and control cells (Fig.
1C). In accordance, the activity of an AP-1-regulated human
collagenase promoter fragment (
73/+63; Ref. 37) was not affected by
transient overexpression of RhoB, whereas overexpression of Rac and
RhoA caused its activation (Fig. 1D). A stimulatory effect
of Rac and RhoA on AP-1 transcription was reported recently for T cells
(41). Overall, the findings strongly indicate that RhoB is unable to
influence activation of MAP kinases and AP-1-regulated gene
expression.

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Fig. 1.
RhoB does not influence genotoxic
stress-induced signaling to MAP kinases and AP-1-regulated gene
expression. A, logarithmically growing cells were
treated with UV light (40 J/m2) or MMS (1 mM).
After an incubation period of 15 min for UV light and 30 min for MMS,
respectively, cells were harvested for determination of JNK1, p38 MAP
kinase, and ERK2 activity as described under "Experimental
Procedures." Control, NIH 3T3neo cells; RhoB,
RhoB-overexpressing cells. B, COS cells were transiently
transfected with the HA-JNK1 expression construct without
(Control) or together with an expression vector harboring
the cDNA sequence for constitutively activated RhoB
(V14RhoB), RhoA (V14RhoA), or Cdc42
(V12Cdc42). 24 h after transfection, HA-JNK1 was
immunoprecipitated by use of an HA-specific antibody (Roche Molecular
Biochemicals), and JNK1 activity was analyzed as described under
"Experimental Procedures." As a positive control,
HA-JNK1-transfected cells were treated with UV light (40 J/m2). C, Cells were treated as described under
A. After an incubation period of 4 h, AP-1 binding
activity was determined by gel retardation analysis as described under
"Experimental Procedures." D, NIH 3T3 wild-type cells
were transiently transfected with a human collagenase promoter CAT
construct (Coll-CAT; 73/+63) without (Control)
or together with expression vectors containing cDNA sequences
encoding wild-type RhoB (RhoB), Rac (Rac), or
constitutively activated RhoA (RhoA). 24 h after
transfection, cells were harvested, and the amount of CAT protein was
determined using an enzyme-linked immunosorbent assay-based
assay.
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Another important element involved in the regulation of gene expression
induced by cellular stresses is the transcription factor NF-
B (19).
To analyze whether RhoB affects NF-
B activity, a
32P-labeled oligonucleotide containing an NF-
B-specific
consensus sequence was incubated with extracts from wild-type and
RhoB-overexpressing cells. After gel electrophoresis, two
32P-labeled complexes, the specificity of which was shown
by competition experiments, were detected in wild-type cells (Fig.
2A). These complexes are known
to consist of p50/p65 heterodimers (complex 1) and p50/p50 homodimers
(complex 2), respectively (21, 42). In contrast to wild-type cells,
extracts from RhoB-overexpressing cells predominantly showed expression
of complex 2 (i.e. p50/p50; Fig. 2A), whereas
complex 1 (i.e. p50/p65) was only very poorly detectable
(Fig. 2A). Preincubation of the binding reaction with p50-specific antibody resulted in a strong supershift in control cells
(Fig. 2B). Appearance of the p50-induced supershift is
accompanied mainly by a decrease in the intensity of complex 1 (i.e. p50/p65) Under conditions of RhoB overexpression, this
supershift was largely reduced (>80%; Fig. 2B). Similar
results were obtained by the use of an anti-p65 antibody (data not
shown). When using the anti-c-Jun antibody as a control, a
supershifted, 32P-labeled complex was not observed (Fig.
2B). As determined by Western blot analysis, p50 and p65
protein levels are not changed in RhoB-overexpressing cells compared
with control cells (Fig. 2C). Therefore, quantitative
differences in p50 and p65 protein expression cannot be responsible for
the differences in DNA binding activity of NF-
B in RhoB
transfectants versus control cells.

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Fig. 2.
RhoB overexpression changes basal DNA binding
activity of NF- B. A, NF- B
binding activity of extracts from logarithmically growing NIH 3T3neo
(Control) and RhoB-overexpressing cells (RhoB)
was analyzed by gel retardation experiments. To this end, 2 µg of
protein from the corresponding extracts was incubated with a
32P-labeled NF- B-specific oligonucleotide for 30 min at
room temperature. Afterward, reaction products were separated on a 5%
nondenaturing polyacrylamide gel. For competition experiments, 2.5- and
25-fold molar excesses of unlabeled oligonucleotide were added to the
reaction mixture 5 min before addition of 32P-labeled
oligonucleotide. Shown is the autoradiography of the dried gel.
1 , p50/p65 heterodimer; 2 , p50/p50
homodimer. Control, NIH 3T3neo cells; RhoB,
RhoB-overexpressing cells. B, Gel retardation analysis was
performed as described under A. The reaction was
preincubated in the presence of either a p50-specific antibody (1 µg)
or a c-Jun-AP-1 antibody (1 µg) for 2 h at 4 °C. Binding of
an antibody to proteins involved in DNA-protein complex formation
becomes visible by formation of a supershift. Arrow
indicates the position of the p50-induced supershift. Shown is the
autoradiogram. C, 50 µg of protein from total cell
extracts was analyzed for p50 and p65 protein expression by Western
blot analysis. As a loading control, the filter was rehybridized with
an ERK2-specific antibody.
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The question arising next was whether RhoB is able to interfere with
drug-induced activation of NF-
B. As analyzed 2 h after exposure
of control cells to the alkylating mutagen MMS, DNA binding activity of
NF-
B was clearly enhanced. In contrast, MMS failed to stimulate
NF-
B binding activity in RhoB-overexpressing cells (Fig.
3A). A mechanism behind the
RhoB-triggered inhibition of NF-
B activation on alkylation could be
blockage of nuclear translocation of NF-
B. To test this hypothesis,
we analyzed the appearance of p50 protein in the nuclear fraction after
MMS treatment. As shown in Fig. 3B, an ~11-fold increase
in the amount of p50 protein in nuclear extracts of control cells was
detected within 8 h after MMS treatment. RhoB-overexpressing
cells, however, did not show elevation of p50 protein level (Fig.
3B). Basically the same results were obtained when
translocation of p65 was measured (data not shown). Thus, on the basis
of data obtained from both gel retardation experiments and Western blot
analysis, we conclude that RhoB impairs the activation of NF-
B in
cells exposed to alkylating agents.

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Fig. 3.
RhoB inhibits alkylating agent-induced
stimulation of the DNA binding activity of
NF- B and impairs nuclear translocation of
p50. A, 2 h after treatment with the alkylating
agent MMS (2 mM), cell extract was prepared for gel
retardation analysis using NF- B-specific 32P-labeled
oligonucleotide. Shown is the autoradiogram. Control, NIH
3T3neo cells; RhoB, RhoB-overexpressing cells. B,
control and RhoB-overexpressing cells were treated with MMS (2 mM, 1 h). Up to 8 h after exposure, the amount of
p50 protein in the nuclear fraction was analyzed by Western blot
analysis using a p50-specific antibody. For internal control, the
filter was rehybridized with Ref-1- and ERK2-specific antibodies. The
amount of p50 protein was related to that of ERK2, giving the relative
level of p50. The relative amount of p50 in the corresponding
nontreated control was set to 1.0.
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Recently, it was shown that the DNA binding activity of NF-
B and
nuclear translocation are not necessarily related to
NF-
B-dependent gene expression (43). Therefore, we
investigated whether RhoB exerts an effect on NF-
B-driven gene
expression. To this end, transfection experiments using an
NF-
B-specific minimal reporter gene construct (3xNF-
B-luciferase)
were performed. These studies revealed that both basal and MMS-induced
activation of gene expression triggered by NF-
B are largely blocked
in RhoB-overexpressing cells (Fig.
4A). We point out that the
same is true when the NF-
B reporter construct was transiently
coexpressed with wild-type RhoB (Fig. 4B). Whereas control
cells showed a ~3-5-fold increase in luciferase activity on MMS
exposure, stable as well as transient overexpression of wild-type RhoB
largely blocked this response (Fig. 4C). Neither the
coexpression of wild-type Rac nor that of RhoA blocked NF-
B
signaling (Fig. 4D). Overall, the data indicate that RhoB
acts as an inhibitory component of both basal and MMS-induced NF-
B-regulated gene expression.

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Fig. 4.
RhoB blocks basal and MMS-stimulated
NF- B-dependent gene
expression. A, NIH 3T3 and RhoB-overexpressing cell
variants (NIH 3T3-RhoB12 and NIH 3T3-RhoB14) were
transfected with 5 µg of NF- B specific reporter gene construct
(3xNF- B-luciferase). 24 h after transfection, cells were
treated with MMS (0.75 mM, 3 h). After a further
incubation period of 24 h, cells were harvested, and luciferase
activity was determined as described under "Experimental
Procedures." Con, not treated; MMS, treated
with the mutagen. B, NIH 3T3 wild-type cells were
transfected with 5 µg of the NF- B-specific reporter gene construct
together with (or without) 5 µg of expression vector encoding
wild-type RhoB. 24 h after transfection, cells were exposed to MMS
and harvested a further 24 h for determination of luciferase
activity. Data shown in A and B are mean
values ± S.D. from at least two independent experiments, each
performed in duplicate (n 4). C, NIH
3T3neo (3T3neo) and RhoB-overexpressing (RhoB12
and RhoB14) cells were transfected with the NF- B-specific
reporter construct. Furthermore, NIH 3T3 wild-type cells were
transiently transfected (TTF) with the NF- B specific
reporter without (Con) or together with expression vector
encoding wild-type RhoB (RhoB-WT). 24 h after
transfection, cells were treated with MMS as described under
A. Shown is the MMS-induced x-fold increase in
luciferase activity. Relative luciferase activity in the corresponding
untreated controls was set to 1.0. D, NIH 3T3 wild-type
cells were transfected with the NF- B-specific reporter without
(Control) or together with expression vectors encoding
wild-type RhoB (RhoB-WT), Rac (Rac-WT), or RhoA
(RhoA-WT). 24 h after transfection, cells were
harvested for determination of luciferase activity, which was set to
1.0 in the control. Data shown in C and D are
mean values ± S.D. from at least two independent experiments,
each performed in duplicate (n 4). RLU,
relative light units.
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A prerequisite for NF-
B activation, at least on treatment with
TNF
, is IKK-dependent phosphorylation of the inhibitory
molecule I
B
(19). On phosphorylation on Ser32,
I
B
dissociates from NF-
B and subsequently becomes degraded (19). Genotoxic stress-induced activation of NF-
B appears to be
regulated differently from that of TNF
(21, 22). This is illustrated
by the finding that stimulation of NF-
B by UV light results in
IKK-independent degradation of I
B
(21, 22). A possible mode of
action of RhoB would be that it inhibits MMS-induced IKK activation.
However, similarly, as reported for UV light (22), the alkylating agent
MMS failed to activate IKK (Fig. 5).
Thus, the inhibitory effect of RhoB on MMS-stimulated NF-
B activity cannot be explained by inhibition of IKK. As observed 8 h after MMS exposure, the I
B
protein level was reduced by ~40% in
control cells but remained unchanged in RhoB-overexpressing cells (Fig. 6). Obviously, RhoB attenuates the
MMS-induced decrease in I
B
.

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Fig. 5.
MMS does not stimulate IKK activity.
Logarithmically growing HeLa cells were treated with 2 mM
MMS. 1-4 h after exposure, cells were harvested for determination of
IKK activity as described under "Experimental Procedures." For
positive control, cells were treated with TNF , and IKK activity was
analyzed 10 min later. Shown is the autoradiography. Con,
untreated cells.
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Fig. 6.
RhoB inhibits MMS-induced degradation of
I B . Up to 8 h after exposure to MMS (1 mM), total cell extract was
prepared and analyzed by Western blot analysis with regard to the level
of I B protein. As control for the amounts of protein loaded,
filters were rehybridized with an ERK2-specific antibody.
Autoradiograms were densitometrically analyzed, and the relative amount
of I B protein in the untreated control was set to 1.0. Control, NIH 3T3neo cells; RhoB,
RhoB-overexpressing cells. Con, untreated cells.
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To address the question of whether the inhibitory effect of RhoB on
NF-
B is specific for alkylating agents, we investigated the effect
of RhoB on other NF-
B-activating compounds. As shown in Fig.
7A, RhoB attenuates both UV-
and 12-O-tetradecanoylphorbol acetate (TPA)-induced increase
in the DNA binding activity of NF-
B. Also, UV- and TPA-driven
reduction in I
B
was abolished by RhoB (Fig. 7, B and
C), yet RhoB did not impair activation of ERK2 by TPA, (Fig.
7D) as analyzed in identical extracts. This finding further
supports the view that RhoB specifically interferes with NF-
B
signaling without affecting MAP kinase-regulated pathways. An
inhibitory effect of RhoB on NF-
B-driven gene expression was observed not only for MMS but also for other genotoxic compounds, such
as mafosfamide, which is a cyclophosphamid analogue, and UV light (Fig.
8). This was shown both by the use of
stably transfected cells (Fig. 8A) and by transient
overexpression of wild-type RhoB (Fig. 8B). Compared with
MMS and mafosfamide, the repressive effect of RhoB on UV-induced
NF-
B activation was only partial (Fig. 8). This is in line with data
obtained from gel retardation experiments shown before (see Fig. 7).
Finally, we analyzed whether RhoB interferes with activation of NF-
B
by TNF
. As shown in Fig. 9, the
TNF
-induced increase in the DNA binding activity of NF-
B (Fig.
9A) as well as the TNF
-driven degradation of I
B
(Fig. 9B) were partially blocked by RhoB. Obviously,
compared with genotoxic agents and TPA, RhoB impairs TNF
-mediated
signaling to NF-
B to a lesser extent. Table
I summarizes the inhibitory function of
RhoB on the effectiveness of different types of agents to evoke
I
B
degradation. It turns out that RhoB exerts an inhibitory
effect on I
B
degradation stimulated by various types of
NF-
B-activating agents, including genotoxic and nongenotoxic
compounds. Thus, RhoB appears to interfere with the regulation of
different signal pathways converging in the activation of
NF-
B.

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Fig. 7.
RhoB impairs signaling to
NF- B stimulated by TPA or UV light.
A, NIH 3T3 (Control) and RhoB-overexpressing
cells were treated with UV light (60 J/m2) or TPA (2 × 10 7 M). 6 h (UV) and
2 h (TPA) after exposure, cells were harvested, and DNA binding
activity of NF- B was determined by gel retardation analysis. Shown
is the autoradiogram. B and C, 2 h after
irradiation (60 J/m2) and 15 min after treatment with TPA
(2 × 10 7 M), respectively,
cells were harvested, and the amount of I B protein was determined
by Western blot analysis. D, Identical extracts as used
under C were analyzed for ERK2 activation by Western blot as
described under "Experimental Procedures." Arrows
indicate the position of the inactive, nonphosphorylated
(2 ) and the active, phosphorylated (1 ) ERK2 species. Con, untreated cells.
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Fig. 8.
RhoB attenuates genotoxic stress-stimulated
gene expression regulated by NF- B.
A, NIH 3T3 (Control) and RhoB-overexpressing
(RhoB) cells were transfected with 5 µg of the
NF- B-specific reporter gene construct (3xNF- B-luciferase).
24 h after transfection, cells were treated with MMS (0.75 mM, 3 h), UV light (60 J/m2), and
mafosfamide (Maf; 60 µM, 3 h),
respectively. After a further incubation period of 24 h, cells
were harvested, and luciferase activity was determined as described
under "Experimental Procedures." Data shown are mean values ± S.D. from at least two independent experiments, each performed in
duplicate (n 4). Con, untreated cells.
B, NIH 3T3 wild-type cells were transfected with 5 µg of
the NF- B-specific reporter gene construct (3xNF- B-luciferase)
without (Control) or together with 5 µg of expression
vector encoding wild-type RhoB (RhoB-WT). 24 h later,
cells were exposed to genotoxic agents as described under A.
Data shown are mean values ± S.D. from at least two independent
experiments, each performed in duplicate (n 4).
Con, untreated cells.
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Fig. 9.
Activation of NF- B
by TNF is attenuated by RhoB.
A, 2 h after addition of TNF (10 ng/ml), NIH 3T3
(Control) and RhoB-overexpressing (RhoB) cells
were harvested, and extracts were analyzed for DNA binding activity of
NF- B as described under "Experimental Procedures."
Con, untreated cells. B, 15 min after TNF
treatment (10 ng/ml), the amount of I B protein was determined by
Western blot analysis. The relative amount of I B in the
corresponding untreated control was set to 1.0.
|
|
View this table:
[in this window]
[in a new window]
|
Table I
Decrease in I B protein level induced by different types of
agents is inhibited by RhoB
Control and RhoB-overexpressing cells were treated with different types
of genotoxic and nongenotoxic agents as indicated. Afterward, the
amount of I B protein was determined by Western blot analysis. The
relative amount of I B (I B level/ERK2 level) in the
corresponding untreated control was set to 1.0.
|
|
 |
DISCUSSION |
The gene coding for the small GTPase RhoB is
immediate-early-inducible by DNA-damaging agents such as UV irradiation
and alkylating compounds (25, 26). On the basis of this and having in
mind its GTPase function, which enables a very rapid modulation of signal mechanisms, we hypothesized that RhoB is important for the
regulation of early cellular responses to genotoxic stress. This
hypothesis was supported by our recent observation that overexpression of RhoB affects cellular sensitivity to induced DNA damage (36). In
particular, overexpression of RhoB renders cells hypersensitive to
alkylation-induced apoptotic cell death (36). Here, we analyzed the
influence of RhoB on signal mechanisms that are quickly activated by
DNA-damaging treatments, focusing on MAP kinase- and NF-
B-related mechanisms.
Analyzing the influence of RhoB on the stimulation of MAP kinases by UV
light and MMS, we found that RhoB-overexpressing cells were not
distinguished from the control. Genotoxic stress-induced activation of
ERK2, JNK1, and p38 MAP kinase were not affected by RhoB. In line with
this, the UV- and MMS-stimulated increase in the DNA binding activity
of AP-1 was not changed by RhoB. Furthermore, transient overexpression
of RhoB failed to stimulate JNK1 activity and did not increase the
expression of the AP-1-regulated human collagenase promoter. Obviously,
RhoB does not interfere with the genotoxic stress-induced activation of
MAP kinases and AP-1-regulated gene expression. In contrast, as
revealed by gel retardation experiments, RhoB overexpression causes
considerable changes in the basal DNA binding activity of the
transcription factor NF-
B. Thus, the NF-
B-specific DNA binding
complex p50/p65 was only very poorly detectable in the transfectants.
Furthermore, compared with control cells, an anti-p50-antibody-induced
supershift was largely reduced under conditions of RhoB overexpression.
Because the level of p50 and p65 protein was similar in wild-type and
variant cells, we suppose that RhoB overexpression gives rise to
posttranslational changes of p50, which in turn affect its interaction
with other members of the NF-
B family, in particular Rel A (p65).
Most interestingly, RhoB overexpression inhibited stimulation of the
DNA binding activity of NF-
B after exposure of cells to the
alkylating compound MMS. In line with the current view of NF-
B
activation, RhoB-mediated abrogation of NF-
B binding activity was
found to be accompanied by inhibition of nuclear translocation of
NF-
B in RhoB-overexpressing cells treated with MMS. As a consequence
of the inhibition of the ability of NF-
B to enter the nucleus and to
bind to DNA, basal and MMS-induced expression of NF-
B-regulated
genes is blocked by RhoB. This was shown in reporter gene transfection
experiments, which is important to note, because, as reported recently,
DNA binding activity of NF-
B is not necessarily related to its
capacity to stimulate promoter activity (43). The same inhibitory
effect on NF-
B-regulated gene expression as observed in
RhoB-overexpressing cell lines was found in cells transiently
coexpressing wild-type RhoB. This shows that stable overexpression of
wild-type RhoB is not associated with unphysiological side effects.
Inhibition of NF-
B-driven gene expression appears to be very
specific for RhoB because it was not observed with with Rac or RhoA. In
line with another report, NF-
B activity was even stimulated by RhoA
(23). Recently, we showed that overexpression of RhoB results in
hypersensitivity to alkylation-induced apoptotic cell death (36).
Having in mind that NF-
B is believed to act as an antiapoptotic
factor (44-46), the observed inhibitory effect of RhoB on
alkylation-triggered NF-
B-regulated gene expression provides an
attractive basis to explain the alkylation-hypersensitive phenotype of
RhoB-overexpressing cells.
With respect to the molecular mechanism underlying RhoB-mediated
inhibition of NF-
B, it appears unlikely that RhoB acts as a specific
inhibitor of IKK. This assumption is based on the fact that RhoB
impairs NF-
B activation by MMS and UV light, both of which exert
their stimulatory effect on NF-
B independent of IKK (Ref. 22; data
shown here). Furthermore, TNF
-induced activation of NF-
B, which
is regulated via IKK, is only partially inhibited by RhoB. A more
general event than stimulation of IKK going along with NF-
B
activation is proteasomal degradation of I
B
on its release from
complexation with NF-
B. Exposure to MMS causes a decrease in the
I
B
protein level in control cells but not in RhoB-overexpressing
cells. It is striking that the MMS-induced decrease in I
B
level
is quite weak compared with other types of stimuli activating NF-
B,
yet, discussing this point, one has to consider that activation of
NF-
B is basically also possible without I
B
degradation (47).
Notably, RhoB also prevented a decrease in I
B
protein level after
treatment of cells with other types of NF-
B-activating stimuli such
as TPA, ionizing radiation, UV light and, to a lesser extent, TNF
.
An explanation for the reduction in genotoxic stress-induced
degradation of I
B
under conditions of RhoB overexpression could
be that RhoB prevents proteasomal degradation of I
B
. This,
however, appears to be unlikely, because the amount of I
B
protein
in RhoB-overexpressing cells is very similar to that of control cells.
Interestingly, RhoB abolished TPA-induced degradation of I
B
but
did not affect the TPA-induced activation of ERK2. Thus, as already
observed for genotoxic stress-induced signaling, RhoB also specifically impairs TPA-stimulated signaling to NF-
B without influencing TPA-triggered activation of MAP kinase. Overall, on the basis of the
data available, we suggest that RhoB functions as a negative regulator
of NF-
B, counteracting the agent-triggered release of I
B
from
complexation with NF-
B. Detailed analysis of the underlying
molecular mechanisms will be the subject of further investigation.
In previous studies, the Rho family GTPases RhoA, Rac, and Cdc42 were
shown to activate NF-
B by stimulation of phosphorylation of I
B
(23). As shown here, RhoB differs from these Rho GTPases by exerting an
opposite, inhibitory effect on NF-
B. RhoB may thus be considered a
negative regulator of stress-induced gene expression. In this context,
it should be noted that a repressive effect of RhoB on gene expression
has indeed been reported previously. Thus, RhoB causes transcriptional
repression of its own gene (26), inhibits transforming growth factor
-stimulated gene expression (48), and impairs the activity of the
basal transcription factor DB1 (49). From these data we
hypothesize that the transient induction of RhoB observed after
DNA-damaging treatments (25) is a homeostatic mechanism, which is
important for subsequent down-modulation of genotoxic stress-stimulated
gene expression back to the basal level. This may be achieved by
inhibition of the activity of one or various transcription factors that
become activated by genotoxic treatment. One of these factors is
NF-
B. Therefore, we suggest that transient induction of RhoB in
cells exposed to a genotoxic agent counteracts coactivated gene
expression triggered by NF-
B. The hypothesis of a homeostatic role
of RhoB in gene expression is supported by the finding that stimulation of gene expression by transforming growth factor
is accompanied by
increase in RhoB protein level (because of RhoB stabilization), which
in turn provokes abrogation of the transforming growth factor
response (48).
 |
ACKNOWLEDGEMENTS |
We thank T. Hunter for the rhoB
cDNA and H. J. Rahmsdorf for the human Coll-CAT reporter gene
construct. Furthermore, we thank A. Hall for providing the Rho
expression vectors, M. Karin for the HA-JNK1 construct, and U. R. Rapp for the NF-
B reporter gene construct.
 |
FOOTNOTES |
*
This work was supported by the Deutsche
Forschungsgemeinschaft Grant Fr 1241/1-3.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: Division of Applied
Toxicology, Institute of Toxicology, Obere Zahlbacher Strasse 67, D-55131 Mainz, Germany. Tel.: 06131-39-33627; Fax: 06131-39-33421; E-mail: fritz@mail.uni-mainz.de.
Published, JBC Papers in Press, November 2, 2000, DOI 10.1074/jbc.M005058200
 |
ABBREVIATIONS |
The abbreviations used are:
JNK, N-terminal
c-Jun kinase;
AP-1, activator protein 1;
I
B, inhibitory protein
B;
IKK, I
B kinase;
MAP, mitogen-activated protein;
NF-
B, nuclear factor
B;
TNF, tumor necrosis factor;
HA, hemagglutinin;
MMS, methyl methanesulfonate;
Coll-CAT, collagenase
promoter-chloramphenicol acetyltrasferase;
ERK, extracellular
signal-regulated kinase;
TPA, 12-O-tetradecanoylphorbol
acetate.
 |
REFERENCES |
1.
|
Aktories, K.,
Rösener, S.,
Blaschke, U.,
and Chhatwal, G. S.
(1988)
Eur. J. Biochem.
172,
445-450[Abstract]
|
2.
|
Hall, A.
(1998)
Science
279,
509-514[Abstract/Free Full Text]
|
3.
|
Aktories, K.
(1997)
J. Clin. Invest.
99,
827-829[Free Full Text]
|
4.
|
Ridley, A. J.,
and Hall, A.
(1992)
Cell
70,
389-399[Medline]
[Order article via Infotrieve]
|
5.
|
Ridley, A. J.,
Paterson, H. F.,
Johnston, C. L.,
Diekmann, D.,
and Hall, A.
(1992)
Cell
70,
401-410[Medline]
[Order article via Infotrieve]
|
6.
|
Olson, M. F.,
Ashworth, A.,
and Hall, A.
(1995)
Science
269,
1270-1272[Medline]
[Order article via Infotrieve]
|
7.
|
Molnar, A.,
Theodoras, A. M.,
Zon, L. I.,
and Kyriakis, J. M.
(1997)
J. Biol. Chem.
272,
13229-13235[Abstract/Free Full Text]
|
8.
|
Prendergast, G. C.,
Khosravi-Far, R.,
Solski, P. A.,
Kurzawa, H.,
Lebowitz, P. F.,
and Der, C. J.
(1995)
Oncogene
10,
2289-2296[Medline]
[Order article via Infotrieve]
|
9.
|
Qiu, R.-G.,
Chen, J.,
Kirn, D.,
McCormick, F.,
and Symons, M.
(1995)
Nature
374,
457-459[CrossRef][Medline]
[Order article via Infotrieve]
|
10.
|
Westwick, J. K.,
Lambert, Q. T.,
Clark, G. J.,
Symons, M.,
Van-Aelst, L.,
Pestell, R. G.,
and Der, C. J.
(1997)
Mol. Cell. Biol.
17,
1324-1335[Abstract]
|
11.
|
Esteve, P.,
Del-Peso, L.,
and Lacal, J. C.
(1995)
Oncogene
11,
2657-2665[Medline]
[Order article via Infotrieve]
|
12.
|
Jimenez, B.,
Arends, M.,
Esteve, P.,
Perona, R.,
Aanchez, R.,
Ramon y Cajal, S.,
Wyllie, A.,
and Lacal, J. C.
(1995)
Oncogene
10,
811-816[Medline]
[Order article via Infotrieve]
|
13.
|
Esteve, P.,
Embade, N.,
Perona, R.,
Jiminez, B.,
del Peso, L.,
Leon, J.,
Arends, M.,
Miki, T.,
and Lacal, J. C.
(1998)
Oncogene
17,
1855-1869[CrossRef][Medline]
[Order article via Infotrieve]
|
14.
|
Zhang, S.,
Han, J.,
Sells, M. A.,
Chernoff, J.,
Knaus, U. G.,
Ulevitch, R. J.,
and Bokoch, G. M.
(1995)
J. Biol. Chem.
270,
23934-23936[Abstract/Free Full Text]
|
15.
|
Coso, A. A.,
Chiariello, M., Yu, J.-C.,
Teramoto, H.,
Crespo, P.,
Xu, N.,
Miki, T.,
and Gutkind, J. S.
(1995)
Cell
81,
1137-1146[Medline]
[Order article via Infotrieve]
|
16.
|
Minden, A.,
Lin, A.,
Claret, F.-X.,
Abo, A.,
and Karin, M.
(1995)
Cell
81,
1147-1157[Medline]
[Order article via Infotrieve]
|
17.
|
Lamarche, N.,
Tapon, N.,
Stowers, L.,
Burbelo, P. D.,
Aspenstrom, P.,
Bridges, T.,
Chant, J.,
and Hall, A.
(1996)
Cell
87,
519-529[Medline]
[Order article via Infotrieve]
|
18.
|
Canman, C. E.,
and Kastan, M. B.
(1996)
Nature
384,
213-214[CrossRef][Medline]
[Order article via Infotrieve]
|
19.
|
Baldwin, A. S., Jr.
(1996)
Annu. Rev. Immunol.
14,
649-681[CrossRef][Medline]
[Order article via Infotrieve]
|
20.
|
Das, K. C.,
and White, C. W.
(1997)
J. Biol. Chem.
272,
14914-14920[Abstract/Free Full Text]
|
21.
|
Bender, K.,
Göttlicher, M.,
Whiteside, S.,
Rahmsdorf, H. J.,
and Herrlich, P.
(1998)
EMBO J.
17,
5170-5181[Abstract/Free Full Text]
|
22.
|
Li, N.,
and Karin, M.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
13012-7[Abstract/Free Full Text]
|
23.
|
Perona, R.,
Montaner, S.,
Saniger, L.,
Sanchez-Perez, I.,
Bravo, R.,
and Lacal, J. C.
(1997)
Genes Dev.
11,
463-475[Abstract]
|
24.
|
Montaner, S.,
Perona, R.,
Saniger, L.,
and Lacal, J. C.
(1998)
J. Biol. Chem.
273,
12799-12785
|
25.
|
Fritz, G.,
Kaina, B.,
and Aktories, K.
(1995)
J. Biol. Chem.
270,
25172-25177[Abstract/Free Full Text]
|
26.
|
Fritz, G.,
and Kaina, B.
(1997)
J. Biol. Chem.
272,
30637-30644[Abstract/Free Full Text]
|
27.
|
Jähner, D.,
and Hunter, T.
(1991)
Mol. Cell. Biol.
11,
3682-3690[Medline]
[Order article via Infotrieve]
|
28.
|
Adamson, P.,
Marshall, C. J.,
Hall, A.,
and Tilbrook, P. A.
(1992)
J. Biol. Chem.
267,
20033-20038[Abstract/Free Full Text]
|
29.
|
Lebowitz, P. F.,
and Prendergast, G. C.
(1998)
Oncogene
17,
1439-1445[CrossRef][Medline]
[Order article via Infotrieve]
|
30.
|
Gibbs, J. B.,
Oliff, A.,
and Kohl, N. E.
(1994)
Cell
77,
175-178[Medline]
[Order article via Infotrieve]
|
31.
|
Gibbs, J. B.,
Kohl, N. E.,
Koblan, K. S.,
Omer, C. A.,
Sepp-Lorenzino, L.,
Rosen, N.,
Anthony, N. Y.,
Conner, M. W.,
deSolms, S. J.,
Williams, T. M.,
Graham, S. L.,
Hartman, G. D.,
and Oliff, A.
(1996)
Breast Cancer Res. Treat.
38,
75-83[Medline]
[Order article via Infotrieve]
|
32.
|
Adamson, P.,
Paterson, H. F.,
and Hall, A.
(1992)
J. Cell Biol.
119,
617-627[Abstract]
|
33.
|
Zalcman, G.,
Closson, V.,
Camonis, J.,
Honore, N.,
Rousseau-Merck, M. F.,
Tavitian, A.,
and Olofsson, B.
(1996)
J. Biol. Chem.
271,
30366-30374[Abstract/Free Full Text]
|
34.
|
Zalcman, G.,
Closson, V.,
Linares-Cruz, G.,
Lerebours, F.,
Honore, N.,
Tavitian, A.,
and Olofsson, B.
(1995)
Oncogene
10,
1935-1945[Medline]
[Order article via Infotrieve]
|
35.
|
Mellor, H.,
Flynn, P.,
Nobes, C. D.,
Hall, A.,
and Parker, P. J.
(1998)
J. Biol. Chem.
273,
4811-4814[Abstract/Free Full Text]
|
36.
|
Fritz, G.,
and Kaina, B.
(2000)
Biochem. Biophys. Res. Commun.
268,
784-789[CrossRef][Medline]
[Order article via Infotrieve]
|
37.
|
Angel, P.,
Baumann, I.,
Stein, B.,
Delius, H.,
Rahmsdorf, H. J.,
and Herrlich, P.
(1987)
Mol. Cell. Biol.
7,
2256-2266[Medline]
[Order article via Infotrieve]
|
38.
|
Baumann, B.,
Kistler, B.,
Kirillov, A.,
Bergman, Y.,
and Wirth, T.
(1998)
J. Biol. Chem.
273,
11448-11455[Abstract/Free Full Text]
|
39.
|
van Dam, H.,
Duyndam, M.,
Rottier, R.,
Bosch, A.,
de Vries-Smits, L.,
Herrlich, P.,
Zantema, A.,
Angel, P.,
and van-der-Eb, A. J.
(1993)
EMBO J.
12,
479-483[Abstract]
|
40.
|
Fritz, G.,
and Kaina, B.
(1999)
Mol. Cell. Biol.
19,
1768-1774[Abstract/Free Full Text]
|
41.
|
Chang, J. H.,
Pratt, J. C.,
Sawasdikosol, S.,
Kapeller, R.,
and Burakoff, S. J.
(1998)
Mol. Cell. Biol.
18,
4986-4993[Abstract/Free Full Text]
|
42.
|
Van Antwerp, D. J.,
Martin, S. J.,
Kafri, T.,
Green, D. R.,
and Verma, I. M.
(1996)
Science
274,
787-789[Abstract/Free Full Text]
|
43.
|
Bergmann, M.,
Hart, L.,
Lindsay, M.,
Barnes, P. J.,
and Newton, R.
(1998)
J. Biol. Chem.
273,
6607-6610[Abstract/Free Full Text]
|
44.
|
Beg, A. A.,
and Baltimore, D.
(1996)
Science
274,
782-784[Abstract/Free Full Text]
|
45.
|
Wang, C. Y.,
Mayo, M. W.,
and Baldwin, A. S., Jr.
(1996)
Science
274,
784-786[Abstract/Free Full Text]
|
46.
|
Wang, C.-Y.,
Cusack, J. C. J.,
Liu, R.,
and Baldwin, A. S. J.
(1999)
Nat. Med.
5,
412-417[CrossRef][Medline]
[Order article via Infotrieve]
|
47.
|
Imbert, V.,
Rupec, R. A.,
Livolsi, A.,
Pahl, H. L.,
Traenckner, E. B.,
Mueller-Dieckmann, C.,
Farahifar, D.,
Rossi, B.,
Auberger, P.,
Baeuerle, P. A.,
and Peyron, J. F.
(1996)
Cell
86,
787-798[Medline]
[Order article via Infotrieve]
|
48.
|
Engel, M. E.,
Datta, P. K.,
and Moses, H. L.
(1998)
J. Biol. Chem.
273,
9921-9926[Abstract/Free Full Text]
|
49.
|
Lebowitz, P. F.,
and Prendergast, G. C.
(1998)
Cell Adhes. Commun.
6,
277-288[Medline]
[Order article via Infotrieve]
|
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