(Received for publication, June 5, 1995; and in revised form, July 31, 1995)
From the
The low molecular weight GTP-binding proteins RhoA, RhoB, and
RhoC are characterized as specific substrates for the
ADP-ribosyltransferase C3 from Clostridium botulinum and are
supposed to be involved in the organization of the microfilamental
network and transformation. rhoB is known to be
immediate-early inducible by growth factors and protein-tyrosine
kinases. Since increasing evidence indicates overlapping of growth
factor- and UV-induced signal pathways, we studied the effect of UV
light and other genotoxic agents on early rhoB transcription.
Within 30 min after UV irradiation of NIH3T3 cells, the amount of rhoB mRNA increased 3-4-fold. Elevated rhoB mRNA was accompanied by an increase in RhoB protein, as detected
by C3-mediated [P]ADP-ribosylation. The
transcription inhibitor actinomycin D prevented the UV-induced increase
in rhoB mRNA and proved rhoB mRNA to be unstable with
a half-life of
20 min. Transcriptional activation of rhoB by UV light was confirmed by run-on analysis. The increase in rhoB mRNA after UV irradiation was prevented by inhibitors of
protein kinase A (H9) and C (H7, Gö18). The
tyrosine kinase inhibitor genistein did not affect UV induction of rhoB. In addition to UV, N-methyl-N-nitrosourea and the cytostatic drug
cisplatin evoked rhoB response. Cycloheximide was likewise
effective in increasing the amount of rhoB mRNA, whereas
Bt
cAMP, 12-O-tetradecanoylphorbol-13-acetate, and
retinoic acid were without effect. Prior down-regulation of signaling
by 12-O-tetradecanoylphorbol-13-acetate and serum pretreatment
reduced UV-stimulated rhoB expression. The data indicate that rhoB represents a novel DNA damage-inducible function involved
in early steps of signal transduction upon genotoxic stress.
Signaling after mitogenic stimulation of cells has been
extensively
investigated(1, 2, 3, 4, 5, 6) .
It is characterized by rapid and transient transcriptional activation
of genes such as c-fos, fosB, fra-1, c-jun, junB, and NGFI-A, all of which encode DNA-binding
proteins(7, 8, 9, 10, 11, 12, 13) .
On the other hand, regulation of cellular responses after exposure to
DNA damaging agents is still not well understood. This is particularly
true for the early effects caused by DNA damage. Various mammalian gene
functions inducible by DNA damaging treatments have been described,
such as human collagenase and human plasminogen
activator(14, 15) , metallothionein (16, 17) , p53(18, 19) ,
EPIF(20) , thymidylate synthase(21) , the DNA-repair
protein O-methylguanine-DNA-methyltransferase(22, 23) ,
-polymerase(24, 25) , and the proto-oncogenes
c-fos and
c-jun(26, 27, 28, 29, 30, 31) .
Among these inducible functions, only the proto-oncogenes c-fos and c-jun are transcriptionally activated within minutes
after induction of DNA damage. Therefore, these two genes, whose
products act as trans-activators by forming the transcription factor
AP-1(26, 32, 33) , are referred to as
``immediate-early'' inducible.
Recently it has been shown
that rhoB is immediate-early inducible by growth factors and
v-Fps(34) . Interestingly, the gene product of rhoB belongs to the family of Ras homologous small
GTP-binding proteins. The Rho ()protein family consists of
at least three highly homologous members (RhoA, -B, and
-C(35, 36) ). RhoA and RhoC are known to be involved
in the regulation of the actin
cytoskeleton(37, 38, 39, 40, 41, 42, 43, 44) .
In addition, RhoA has been reported to interfere with cell adhesion (45) and transformation(46) . Apparently, Rho proteins
(RhoA, -B, and -C) have different intracellular locations (47) , indicating different physiological functions. Consistent
with this is the observation that the expression of rhoB, but
not of rhoA or rhoC is rapidly stimulated by growth
factors(34) . Transcriptional stimulation of rhoB by
mitogens appears to differ from other immediate-early genes, like
c-fos(34) , suggesting a novel pathway for
mitogen-induced cellular responses. Interestingly, RhoB has recently
been suggested to be involved in cell growth control and Ras-mediated
oncogenic transformation(48) .
So far, GTP-binding proteins have not been examined as regulators in early signaling after DNA damage, although they could rapidly control adaptive cellular reactions by GTP binding and GTP hydrolysis, thereby changing the activity of various downstream targets. Since the immediate-early genes c-fos and c-jun are not only inducible by mitogens but also by UV light, the question arose whether this would be true for rhoB too. Thus, the recently published data on rhoB regulation (34) prompted us to investigate whether rhoB is part of the early cellular response to DNA damage. Here we show that UV light and other DNA-damaging agents increase RhoB very rapidly by transcriptional activation of the gene, apparently utilizing a pathway not common for previously described early-responsive genes.
To address the question, whether rhoB is inducible
by genotoxic agents, NIH3T3 cells were exposed to UV light and rhoB mRNA levels were measured various times after irradiation. As
shown in Fig. 1A, a 3-4-fold increase in the
amount of rhoB mRNA was observed 30 min after UV treatment, as
detected by hybridization with a rhoB-specific probe. This
hybridization probe mainly consists of the 3`-noncoding region of rat rhoB cDNA and does not cross-hybridize to rhoA or rhoC mRNA. As expected, the amount of c-fos mRNA
which was taken as internal standard, was also enhanced upon UV
irradiation. Furthermore, rehybridization of the filter was performed
with a hybridization probe covering the whole coding region of rhoA. Because of the high homology (>85%) of the diverse
Rho species(36) , this hybridization probe cross-hybridizes
with all rho mRNAs (rhoA, -B, and
-C). Using this probe, no UV-induced change in total rho mRNA was observed, indicating that rhoB mRNA most likely
represents only a minor portion of total rho mRNA (rhoA, -B, and -C). In contrast to UV, TPA did not
increase rhoB mRNA (Fig. 1A). Kinetic analysis
of the UV response showed maximal amounts of rhoB mRNA 30 min
after irradiation (Fig. 1B). 2 h after exposure to UV, rhoB mRNA returned to basal level again. Increase in rhoB mRNA was already observed with low doses (10 J/m) of
UV (Fig. 1C), exerting only slight toxic effects (90%
cell survival). The level of rhoB mRNA was similarly increased
after UV treatment of serum-starved or confluent cells, indicating that
the UV response of rhoB did not depend on proliferation (not
shown). Additionally, we analyzed constitutive and UV-induced rhoB expression on protein level using the specific ADP-ribosylation of
Rho proteins by C. botulinum exoenzyme
C3(37, 38, 39, 40, 41) .
Separation of [
P]ADP-ribosylated cell extracts
by two-dimensional gel electrophoresis showed that RhoA and RhoC are
the major Rho proteins constitutively expressed in NIH3T3 cells (Fig. 2). In contrast, basal amounts of RhoB are very low. 1 h
after UV treatment, the amount of ADP-ribosylated RhoB protein
increased about 2-3-fold, as compared with either ADP-ribosylated
RhoA (RhoC) or RhoA and RhoC proteins. As related to RhoA, the level of
RhoC was not significantly (<1.5-fold) changed after UV treatment.
However, in spite of its inducibility, the amount of RhoB remained
significantly below RhoA and RhoC. Thus, the calculation of the
relative amount of RhoB protein deduced from ADP-ribosylation
experiments is in line with our data obtained from Northern blot
analysis. 4 h after UV irradiation, the level of
[
P]ADP-ribosylated RhoB protein decreased again
(not shown).
Figure 1:
UV light causes a time- and
dose-dependent increase in rhoB mRNA. A,
logarithmically growing NIH3T3 cells were UV-irradiated (30
J/m) or treated with TPA (2
10
M). After 30 min, total RNA was prepared and subjected
to Northern blot analysis as described under ``Experimental
Procedures.'' After hybridization with a rhoB specific
hybridization probe, filters were rehybridized with c-fos, GAPDH, and rhoA. With the latter probe all rho mRNA species are detectable (see ``Experimental
Procedures''). B, logarithmically growing NIH3T3 cells
were irradiated with 30 J/m
. Cells were harvested for
Northern blot analysis 30-240 min after treatment. Autoradiograms
were densitometrically analyzed and the amount of rhoB mRNA
was related to the amount of GAPDH mRNA, giving the relative rhoB mRNA level. The relative rhoB mRNA level of
control cells (not treated) was set to 1.0. C, cells were
irradiated with 10-60 J/m
and total RNA isolated 30
min after treatment. Relative rhoB mRNA was determined from
Northern blot analysis as described in B.
Figure 2:
Induction of RhoB protein after UV
treatment of NIH3T3 cells. Logarithmically growing NIH3T3 cells were
irradiated with 30 J/m and cells were harvested 1 h later.
50 µg of protein from total cell extracts was ADP-ribosylated by C3
and [
P]ADP-ribosylated proteins were separated
by two-dimensional gel electrophoresis. Autoradiograms of dried gels
are shown. Control, control extract from non-irradiated cells. Arrows indicate increase in the H
-gradient. Numbers 1, 2, and 3 (marked with arrows)
indicate the position of Rho species RhoA, RhoC, and RhoB,
respectively.
Next we studied the effect of the transcription
inhibitor actinomycin D on rhoB mRNA. As shown in Fig. 3A, actinomycin D prevented the UV-induced
increase in rhoB mRNA, indicating that the rhoB gene
was transcriptionally activated upon UV irradiation. This was confirmed
by run-on analysis showing transcriptional activation of rhoB within 15 min after UV irradiation (Fig. 3B). To
determine the stability of rhoB mRNA, logarithmically growing
NIH3T3 cells were treated for various periods of time with actinomycin
D. As shown in Fig. 3C, 90 min after actinomycin D
addition rhoB mRNA was not longer detectable. Densitometric
analysis of the autoradiogram indicated a half-life of rhoB mRNA of 20 min (Fig. 3D). In contrast to rhoB mRNA, total rho mRNA did not decrease in the
presence of actinomycin D (Fig. 3, C and D).
Figure 3:
RhoB is transcriptionally activated by UV
light. A, logarithmically growing NIH3T3 cells were
UV-irradiated (30 J/m) in the presence (UV+ActD) or absence (UV) of actinomycin D (ActD, 5 µg/ml) which was added 5 min before irradiation.
30 min after treatment with UV, total RNA was isolated for Northern
blot analysis. Con, untreated cells. B, 15 min after
UV irradiation (30 J/m
) of logarithmically growing NIH3T3
cells nuclei were prepared and run-on analysis performed as described
under ``Experimental Procedures.'' C,
logarithmically growing NIH3T3 cells were treated with 5 µg/ml
actinomycin D. After an incubation period of 30-240 min, total
RNA was isolated for Northern blot analysis. Con, untreated
cells. D, densitometric analysis of the autoradiograms shown
in C. Relative rhoB mRNA is shown as a function of
time after addition of actinomycin D. Relative amounts of rhoB and rho mRNA were determined as described in the legend
to Fig. 1.
To further analyze the regulation of rhoB expression, we investigated the kinetics of rhoB mRNA increase and its subsequent degradation after treatment with serum or cycloheximide, both of them are well known inducers of c-fos. Serum stimulation of quiescent NIH3T3 cells and cycloheximide treatment of exponentially growing NIH3T3 cells both resulted in a rapid increase in rhoB mRNA. Notably, the level of rhoB mRNA remained enhanced for a longer period of time than c-fos mRNA (Fig. 4, A and B). As already observed after UV treatment, total rho mRNA level did not change after serum stimulation or cycloheximide treatment. Furthermore, neither cycloheximide nor serum influenced the level of the C3-mediated ADP-ribosylation of Rho proteins (not shown).
Figure 4: Cycloheximide and serum increase the amount of rhoB mRNA. A, logarithmically growing NIH3T3 cells were treated with cycloheximide (5 µg/ml) and cells were harvested 1-4 h later for Northern blot analysis. The autoradiograms were densitometrically analyzed and the relative amounts of rhoB, rho, and c-fos mRNA were determined by referring to the level of GAPDH mRNA. Relative rhoB, rho, and c-fos mRNA in non-treated cells was set to 1.0. B, serum-starved (24 h, 0.5% FCS) subconfluent NIH3T3 cells were stimulated by addition of fetal calf serum (final concentration 20%). After 1-4 h, cells were harvested for Northern blot analysis. Quantitation of the autoradiograms was performed as described in A.
Since protein kinases interfere with the regulation of the UV stimulated expression of c-fos(9, 16, 26, 53) , we analyzed the involvement of protein kinases in the UV induction of rhoB. NIH3T3 cells were treated with different protein kinase inhibitors before UV irradiation and then the level of rhoB mRNA was assayed. As shown in Table 1, the UV-stimulated increase in the amount of rhoB mRNA was blocked after inhibition of protein kinase C by the protein kinase C-inhibitors H7 and Gö18. Likewise, UV stimulated expression of the c-fos gene was inhibited by H7 (not shown). Pretreatment of cells with the protein kinase A inhibitor H9 reduced both the basal and the UV stimulated level of rhoB mRNA (Table 1). These data indicate that protein kinases A and C are involved in the UV-stimulated expression of the rhoB gene. The tyrosine kinase inhibitor genistein did not inhibit UV induction of rhoB (Table 1). In contrast to UV, serum-stimulated expression of rhoB was partially inhibited by protein kinase C inhibitors H7 and Gö18 as well as by the tyrosine kinase inhibitor genistein, but not by H9 (Table 1). To further analyze components interfering with UV-stimulated rhoB expression, protein kinase C-dependent signaling cascade which was shown to be non-refractory upon repeated treatments was down-modulated by pretreatment with TPA or growth factors as described previously(54) . Pretreatment of NIH3T3 cells with TPA or serum caused a 50-70% reduction in a subsequent UV stimulation of rhoB, as compared to non-pretreated control cells (Table 2). These data indicate that, with respect to rhoB expression, UV-, TPA-, and growth factor-induced signaling share common pathways.
Interestingly, in addition to UV light, the
alkylating agent MNU also caused a dose-dependent increase in the
amount of rhoB mRNA (Fig. 5). Under identical
conditions c-fos expression was not stimulated (not shown). Table 3summarizes the effects of various treatments on the amount
of rhoB mRNA. Cisplatin, hydroxyurea, and dexamethasone also
elicited rhoB induction, whereas retinoic acid and
BtcAMP, given either by its own or in combination with TPA,
were without effect.
Figure 5: N-Methyl-N-nitrosourea causes a dose-dependent increase in the amount of rhoB mRNA. A, logarithmically growing NIH3T3 cells were treated with various concentrations of MNU. After an incubation period of 30 min cells were harvested and RNA isolated for Northern blot analysis. B, increase of rhoB mRNA level as a function of MNU concentration. Data are from quantitative evaluation of the autoradiograms shown in A.
RhoA and RhoC have been shown to interfere with
the regulation of the actin cytoskeleton, especially in the
organization of growth factor-induced focal adhesions and stress fiber
formation(39, 42, 45) . So far, the
involvement of RhoB in the organization of actin cytoskeleton has not
been demonstrated convincingly. Therefore, we were interested to see
whether the UV induced increase in RhoB was accompanied by change in
actin cytoskeleton. Cells were UV-irradiated and, thereafter, the actin
cytoskeleton was fixed and stained by FITC-phalloidin. In a second
approach to identify changes of actin cytoskeleton, e.g. depolymerization of F-actin, we used C. botulinum C2
toxin, that ADP-ribosylates specifically monomeric G-actin, but not
F-actin. Neither F-actin staining by FITC-phalloidin nor the specific
[P]ADP-ribosylation of G-actin were changed
after UV treatment (not shown). Furthermore, no change in F-actin was
detectable after transient transfection of rhoB sense and
antisense expression vectors followed by FITC staining (not shown).
These data indicate that no major alteration (polymerization or
depolymerization) of the actin cytoskeleton had occurred after changing
RhoB expression. Overall, these findings indicate that RhoB very likely
does not play a crucial role in the regulation of actin microfilaments.
In this study we have shown that rhoB, encoding a
Ras-related GTP-binding protein is a novel, immediate-early DNA-damage
inducible gene. Similar to c-fos, the rhoB gene can
be transcriptionally activated by UV light. Other mutagens such as MNU
and cisplatin, as well as serum factors and the protein synthesis
inhibitor cycloheximide, also evoked rhoB response. However,
cycloheximide which apparently interferes with signaling (55) did not induce rhoB in Rat2 cells(34) ,
indicating that cycloheximide-mediated rhoB induction is not a
general phenomenon. Obviously, there are cell-type specific differences
in signaling on which cycloheximide converges. The pathway of rhoB induction appears to overlay only partially that regulating
c-fos expression. For example, c-fos expression is
enhanced by TPA and BtcAMP
treatment(26, 30) , whereas rhoB expression
was not ( (34) and our data). Additionally, we found rhoB expression to be induced by the alkylating agent MNU in NIH3T3
cells, whereas under identical experimental conditions, c-fos expression was not enhanced. Furthermore, c-fos and rhoB mRNA showed different kinetics in that rhoB mRNA
decreased at a slower rate than c-fos mRNA upon stimulation of
NIH3T3 cells with cycloheximide and serum factors, respectively. These
data indicate that the genes encoding c-fos and rhoB differ in respect to the factors regulating their inducible
expression. On the other hand, UV-induced increase in both rhoB and c-fos mRNA was blocked by inhibitors of protein
kinase A or protein kinase C, indicating a general involvement of these
kinases in DNA damage-induced cellular responses. It appears that
protein kinase A and C are necessary, but not sufficient components of
the rhoB response. This hypothesis is based on the following
findings: (i) inhibition of both protein kinases A and C prevented rhoB induction, whereas stimulation of protein kinase A and C
by Bt
cAMP and TPA did not elicit an increase of rhoB expression; (ii) down-modulation of protein kinase C signaling by
TPA pretreatment reduced UV induction of rhoB as compared with
non-pretreated cells. Furthermore, there are differences in signal
transduction pathways after mitogen- and DNA damage-stimulated rhoB expression. This was concluded from the observation that
inhibition of protein kinase A by H9 only interfered with UV, but not
with serum-stimulated rhoB expression. Additionally,
serum-stimulated rhoB expression was sensitive to the tyrosine
kinase inhibitor genistein whereas UV-mediated rhoB induction
was not sensitive. Thus, a regulatory significance of tyrosine kinases
in UV response, as deduced from the analysis of the UV induction of
c-jun(56) , appears to be questionable for rhoB. On the other hand, serum pretreatment reduced the level
of a subsequent UV stimulation of rhoB, indicating overlapping
of mitogen- and UV-induced signaling of rhoB induction. It is
unlikely that the epidermal growth factor receptor is involved in the
regulation of rhoB expression by UV because it is activated
not earlier than 30-60 min after UV irradiation(57) .
Cloning of the rhoB gene to analyze its regulatory elements is
required in order to clarify its obviously complex regulation. Another
kinase recently shown to be activated by UV light is JNK1
kinase(58) . The substrate for this kinase has been identified
to be c-Jun(58) . Whether JNK1 kinase and c-Jun also interfere
with the UV-stimulated expression of rhoB remains to be
elucidated.
The identification of rhoB as an immediate-early gene indicates that RhoB activity is regulated not only by a GTPase cycle but also on the transcriptional level. The high homology between various Rho proteins (RhoA, RhoB, and RhoC) and their characterization as Ras homologous indicates that RhoB is also involved in signal transduction. Rho proteins are believed to participate in the regulation of the actin cytoskeleton(38, 39, 40, 41, 42, 43, 44) . This was suggested from the results of microinjection experiments with purified and recombinant RhoA and RhoC protein and from the application of Rho inactivating bacterial ADP-ribosyltransferases. However, because RhoB, but not RhoA and RhoC, has been localized on prelysosomal membranes (47) and only rhoB is induced by mitogens(34) , the physiological function of RhoB appears to be distinct from that of RhoA and RhoC. This hypothesis is supported by our finding that only rhoB but not the other rho genes behaved as inducible upon treatment with DNA-damaging agents. Furthermore, UV-stimulated rhoB mRNA and protein expression, as well as transient transfection of rhoB expression vectors were not accompanied by changes in actin cytoskeleton. Thus, a major role of RhoB in the formation of actin microfilaments determining cell morphology or adhesion appears to be unlikely. Beside its involvement in cytoskeleton organization, RhoA has additionally been shown to have oncogenic activity(46, 59) . In this context it is interesting that very recently RhoB has been suggested to play a role in cell growth regulation and to be necessary for transformation by oncogenic Ras(48) .
Summarizing, the GTP-binding protein RhoB which is immediate-early inducible upon genotoxic stress appears to be a candidate for a regulator that directly interferes with early steps of signaling after DNA-damaging treatments. The well known immediate-early inducible proto-oncogenes c-fos and c-jun encode transcription factors that act by trans-activating late responsive genes, some of which may exhibit a protective function(60, 61, 62, 63) . Another gene product which is involved in cell cycle control and accumulates after UV irradiation is p53(18, 19) . A UV-stimulated increase in p53 is not observed earlier than 3-5 h after UV irradiation(18) . Thus, this response appears to occur too late in order to mediate rapid cellular reactions, such as the block of replication that is maximal already 1-2 h after UV irradiation (63) . The immediate-early induction of RhoB indicates the existence of a new regulatory pathway which might enable cells to react very rapidly upon induction of DNA damage. It is, to our knowledge, the first evidence for a possible involvement of an inducible GTP-binding protein in the very fast acute response of mammalian cells to environmental stress.