Ras-related GTPase RhoB Represses NF-κB Signaling

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 κB (NF-κB). Thus, RhoB attenuated alkylation-induced increase in the DNA binding activity of NF-κB and abrogated NF-κB-driven gene expression. Furthermore, RhoB inhibited decrease in the cellular amount of IκBα after genotoxic stress as well as after tumor necrosis factor α and 12-O-tetradecanoylphorbol acetate treatment. This indicates that RhoB represses NF-κB activation by inhibiting dissociation and subsequent degradation of IκBα. On the basis of the data, we suggest that RhoB is a novel negative regulator of NF-κB signaling.

Ras-related small GTPases (molecular mass, ϳ21 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)(2)(3)(4)(5), cell cycle progression (6,7), cellular transformation (8 -10), and apoptosis (11)(12)(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 phospho-rylation of the inhibitory molecule IB␣ (19). On its phosphorylation on Ser 32 , NF-B is released for nuclear translocation, and free IB␣ 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 IB␣ on Ser 32 (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)(26)(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)(26)(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 alkylationinduced 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 Biotechnol-ogy 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 (Ϫ73/ϩ63) 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 (Ϫ73/ϩ63; 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 g of protein from nuclear or total cell extracts (1000 ϫ 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-32 P labeling of oligonucleotides was performed using T4 kinase. Total cell extracts were prepared as described (39). Binding reactions were performed by incubation of 10 g of protein from NIH 3T3 extracts with ϳ5 fmol of 32 P-labeled oligonucleotide for 30 min at room temperature (binding buffer, 10 mM Hepes, pH 7.9, 60 mM KCl, 5 mM MgCl 2 , 1 mM EDTA, 1 mM dithiothreitol, 1 g/ml bovine serum albumin, 10% glycerol, 0.5 g 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 (5Ј-AGTTGAGGGGACTTTCCCAGGC-3Ј). The sequence of AP-1-specific oligonucleotide was derived from the mouse collagenase promoter (5Ј-AGTGGTGACTCATCACT-3Ј).
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 l of reaction buffer (JNK/p38 kinase reaction buffer: 25 mM Hepes, pH 7.6, 20 mM MgCl 2 , 20 mM ␤-glycerolphosphate, 0.1 mM sodium orthovanadate, 2 mM dithiothreitol, 25 M ATP, 1 Ci of [␥-32 P]ATP; ERK reaction buffer: 20 mM Hepes, pH 7.1, 10 mM MgCl 2 , 1 mM sodium orthovanadate, 25 M ATP, 1 Ci of [␥-32 P-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 MgCl 2 , 10 mM MnCl 2 , 1 mM EDTA, 1 mM dithiothreitol, 0.1 mM sodium orthovanadate). As a substrate for IKK, recombinant glutathione S-transferase-IB␣ 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 con-taining 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 (Ϫ73/ϩ63; 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.
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 32 P-labeled oligonucleotide containing an NF-Bspecific consensus sequence was incubated with extracts from wild-type and RhoB-overexpressing cells. After gel electrophoresis, two 32 P-labeled complexes, the specificity of which was shown by competition experiments, were detected in wildtype 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 (Ͼ80%; 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, 32 P-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.
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 RhoBoverexpressing cells (Fig. 3A). A mechanism behind the RhoBtriggered 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 ϳ11fold 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.
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 . 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; Ϫ73/ϩ63) 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.

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 g of protein from the corresponding extracts was incubated with a 32 P-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 32 P-labeled oligonucleotide. Shown is the autoradiography of the dried gel. 1 3, p50/p65 heterodimer; 2 3, 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 g) or a c-Jun-AP-1 antibody (1 g) 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 g 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. (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 ϳ3-5-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.
A prerequisite for NF-B activation, at least on treatment with TNF␣, is IKK-dependent phosphorylation of the inhibitory molecule IB␣ (19). On phosphorylation on Ser 32 , IB␣ 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

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 g 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 g of the NF-B-specific reporter gene construct together with (or without) 5 g 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 Ϯ S.D. from at least two independent experiments, each performed in duplicate (n Ն 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 Ϯ S.D. from at least two independent experiments, each performed in duplicate (n Ն 4). RLU, relative light units.

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 32 P-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. illustrated by the finding that stimulation of NF-B by UV light results in IKK-independent degradation of IB␣ (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 MMSstimulated NF-B activity cannot be explained by inhibition of IKK. As observed 8 h after MMS exposure, the IB␣ protein level was reduced by ϳ40% in control cells but remained unchanged in RhoB-overexpressing cells (Fig. 6). Obviously, RhoB attenuates the MMS-induced decrease in IB␣.
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 IB␣ 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 IB␣ (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  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 IB␣ protein in the untreated control was set to 1.0. Control, NIH 3T3neo cells; RhoB, RhoB-overexpressing cells. Con, untreated cells.

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/m 2 ) or TPA (2 ϫ 10 Ϫ7 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/m 2 ) and 15 min after treatment with TPA (2 ϫ 10 Ϫ7 M), respectively, cells were harvested, and the amount of IB␣ 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 3) and the active, phosphorylated (1 3) ERK2 species. Con, untreated cells. lated 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.

DISCUSSION
The gene coding for the small GTPase RhoB is immediateearly-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-1regulated gene expression. In contrast, as revealed by gel retardation experiments, RhoB overexpression causes consider- 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 IB␣ protein was determined by Western blot analysis. The relative amount of IB␣ in the corresponding untreated control was set to 1.0. able 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 RhoBmediated 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 IB␣ on its release from complexation with NF-B. Exposure to MMS causes a decrease in the IB␣ protein level in control cells but not in RhoB-overexpressing cells. It is striking that the MMS-induced decrease in IB␣ 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 IB␣ degradation (47). Notably, RhoB also prevented a decrease in IB␣ 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 IB␣ under conditions of RhoB overexpression could be that RhoB prevents proteasomal degradation of IB␣. This, however, appears to be unlikely, because the amount of IB␣ protein in RhoB-overexpressing cells is very similar to that of control cells. Interestingly, RhoB abolished TPA-induced degradation of IB␣ but did not affect the TPA-induced activation of ERK2. Thus, as already observed for genotoxic stress-induced signal-ing, 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 IB␣ 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 IB␣ (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).