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• W1974653642 abstract "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 [32P]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 Bt2cAMP, 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. 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 [32P]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 Bt2cAMP, 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(1Berra E. Diaz Meco M.T. Dominguez I. Munico M. Sanz L. Lozano J. Chapkin R.S. Moscat J. 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Biol. 1987; 7: 2256-2266Crossref PubMed Scopus (585) Google Scholar, 15Miskin R. Ben-Ishai R. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 6236-6240Crossref PubMed Scopus (69) Google Scholar), metallothionein (16Angel P. Pöting A. Mallick U. Rahmsdorf H.J. Schorpp M. Herrlich P. Mol. Cell. Biol. 1986; 6: 1760-1766Crossref PubMed Scopus (172) Google Scholar, 17Fornace Jr., A.J. Schalch H. Alamo Jr., I. Mol. Cell. Biol. 1988; 8: 4716-4720Crossref PubMed Scopus (45) Google Scholar), p53(18Lu X. Lane D.P. Cell. 1993; 75: 765-778Abstract Full Text PDF PubMed Scopus (775) Google Scholar, 19Maltzman W. Czyzyk L. Mol. Cell. Biol. 1984; 4: 1689-1694Crossref PubMed Scopus (817) Google Scholar), EPIF(20Schorpp M. Mallick U. Rahmsdorf H.J. Herrlich P. Cell. 1984; 37: 861-868Abstract Full Text PDF PubMed Scopus (126) Google Scholar), thymidylate synthase(21Kashani-Sabet M. Wang W. Scanlon K.J. J. Biol. Chem. 1990; 265: 11285-11288Abstract Full Text PDF PubMed Google Scholar), the DNA-repair protein O6-methylguanine-DNA-methyltransferase(22Fritz G. Tano K. Mitra S. Kaina B. Mol. Cell. Biol. 1991; 11: 4660-4668Crossref PubMed Scopus (130) Google Scholar, 23Lefebvre P. Zak P. Laval F. DNA-Cell Biol. 1993; 12: 233-241Crossref PubMed Scopus (44) Google Scholar), β-polymerase(24Fornace A.J. Zmudzka B. Hollander C. Wilson S.H. Mol. Cell. Biol. 1989; 9: 851-853Crossref PubMed Scopus (148) Google Scholar, 25Kedar P.S. Widen S.G. Englander E.W. Fornace A.J. Wilson S.H. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3729-3733Crossref PubMed Scopus (44) Google Scholar), and the proto-oncogenes c-fos and c-jun(26Büscher M. Rahmsdorf H. Litfin M. Karin M. Herrlich P. Oncogene. 1988; 3: 301-311PubMed Google Scholar, 27Ghosh R. Amstad P. Cerutti O. Mol. Cell. Biol. 1993; 13: 6992-6999Crossref PubMed Scopus (39) Google Scholar, 28Greenberg M.E. Ziff E.B. Nature. 1984; 311: 433-437Crossref PubMed Scopus (2010) Google Scholar, 29Hollander C. Fornace A.J. Cancer Res. 1989; 49: 1687-1692PubMed Google Scholar, 30Krämer M. Stein B. Mai S. Kunz E. König H. Loferer H. Grunicke H.H. Ponta H. Herrlich P. Rahmsdorf H.F. Radiat. Environ. Biophys. 1990; 29: 303-313Crossref PubMed Scopus (41) Google Scholar, 31Sherman M. Datt L.R. Hallahan D.E. Weichselbaum R.R. Kufe D.W. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5663-5666Crossref PubMed Scopus (278) Google Scholar). 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(26Büscher M. Rahmsdorf H. Litfin M. Karin M. Herrlich P. Oncogene. 1988; 3: 301-311PubMed Google Scholar, 32Ryseck R.-P. Bravo R. Oncogene. 1991; 6: 533-542PubMed Google Scholar, 33Schütte J. Viallet J. Nau M. Segal S. Fedorko J. Minna J.D. Cell. 1989; 59: 987-997Abstract Full Text PDF PubMed Scopus (385) Google Scholar), are referred to as “immediate-early” inducible. Recently it has been shown that rhoB is immediate-early inducible by growth factors and v-Fps(34Jähner D. Hunter T. Mol. Cell. Biol. 1991; 11: 3682-3690Crossref PubMed Scopus (151) Google Scholar). Interestingly, the gene product of rhoB belongs to the family of Ras homologous small GTP-binding proteins. The Rho 1The abbreviations used are: RhoRas homologousBt2cAMPdibutyrylic cyclic AMPFCSfetal calf serumGAPDHglyceraldehyde-3-phosphate dehydrogenaseMNUN-methyl-N-nitrosoureaTPA12-O-tetradecanoylphorbol-13-acetatePBSphosphate-buffered salineFITCfluorescein isothiocyanate. protein family consists of at least three highly homologous members (RhoA, -B, and -C(35Chardin P. Boquet P. Madaule P. Popoff M.R. Rubin E.J. Gill D.M. EMBO J. 1989; 8: 1087-1092Crossref PubMed Scopus (422) Google Scholar, 36Yeramian P. Chardin P. Madaule P. Tavitian A. Nucleic Acids Res. 1987; 15: 1869Crossref PubMed Scopus (88) Google Scholar)). RhoA and RhoC are known to be involved in the regulation of the actin cytoskeleton(37Aktories K. Weller U. Chhatwal G.S. FEBS Lett. 1987; 212: 109-113Crossref PubMed Scopus (172) Google Scholar, 38Aktories K. Rösener S. Blaschke U. Chhatwal G.S. Eur. J. Biochem. 1988; 172: 445-450Crossref PubMed Scopus (122) Google Scholar, 39Chardin P. Madaule P. Tavitian A. Nucleic Acids Res. 1988; 16: 2727Crossref PubMed Scopus (93) Google Scholar, 40Kikuchi A. Yamamoto K. Fujita T. Takai Y. J. Biol. Chem. 1988; 263: 16303-16308Abstract Full Text PDF PubMed Google Scholar, 41Narumiya S. Sekine A. Fujiwara M. J. Biol. Chem. 1988; 263: 17255-17257Abstract Full Text PDF PubMed Google Scholar, 42Paterson H.F. Self A.J. Garrett M.S. Just I. Aktories K. Hall A. J. Cell Biol. 1990; 111: 1001-1007Crossref PubMed Scopus (571) Google Scholar, 43Rubin E.J. Gill M. Boquet P. Popoff M.P. Mol. Cell. Biol. 1988; 8: 418-426Crossref PubMed Scopus (235) Google Scholar, 44Wiegers W. Just I. Müller H.M. Hellwig A. Traub P. Aktories K. Eur. J. Cell Biol. 1991; 54: 237-245PubMed Google Scholar). In addition, RhoA has been reported to interfere with cell adhesion (45Ridley A.J. Hall A. Cell. 1992; 70: 389-399Abstract Full Text PDF PubMed Scopus (3832) Google Scholar) and transformation(46Avraham H. Weinberg R.A. Mol. Cell. Biol. 1989; 9: 2058-2066Crossref PubMed Scopus (95) Google Scholar). Apparently, Rho proteins (RhoA, -B, and -C) have different intracellular locations (47Adamson P. Paterson H.F. Hall A. J. Cell Biol. 1992; 119: 617-627Crossref PubMed Scopus (329) Google Scholar), 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(34Jähner D. Hunter T. Mol. Cell. Biol. 1991; 11: 3682-3690Crossref PubMed Scopus (151) Google Scholar). Transcriptional stimulation of rhoB by mitogens appears to differ from other immediate-early genes, like c-fos(34Jähner D. Hunter T. Mol. Cell. Biol. 1991; 11: 3682-3690Crossref PubMed Scopus (151) Google Scholar), 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(48Prendergast G.C. Khosravi-Far R. Solski P.A. Kurzawa H. Lebowitz P.F. Der C.J. Oncogene. 1995; 10: 2289-2296PubMed Google Scholar). Ras homologous dibutyrylic cyclic AMP fetal calf serum glyceraldehyde-3-phosphate dehydrogenase N-methyl-N-nitrosourea 12-O-tetradecanoylphorbol-13-acetate phosphate-buffered saline fluorescein isothiocyanate. 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 (34Jähner D. Hunter T. Mol. Cell. Biol. 1991; 11: 3682-3690Crossref PubMed Scopus (151) Google Scholar) 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. N-Methyl-N-nitrosourea (MNU) and cisplatin were purchased from Sigma. Protein kinase inhibitors H7 and H9 were obtained from RBI (Research Biochemicals Inc.). Protein kinase inhibitor Gö18 was kindly provided by Dr. Schächtele (Gödecke, Freiburg, 47). The tyrosine kinase inhibitor genistein was obtained from Sigma. Mouse NIH3T3 cells were routinely grown in Dulbecco's modified Eagle's medium containing 10% heat-inactivated FCS, 2 mML-glutamate, 100 units/ml penicillin, and 100 μg/ml streptomycin. For serum starvation, cells were washed twice with phosphate-buffered saline (PBS) and cultured in the presence of Dulbecco's modified Eagle's medium containing 0.5% FCS for 24 h. Before UV treatment (254 nm), medium was removed. All other treatments were performed by adding the drug directly to the medium. After treatment of exponentially growing NIH3T3 cells, medium was removed and cell layer washed twice with ice-cold PBS. Subsequently, cells were lysed onto the plates with guanidinium thiocyanate and total RNA was prepared as described(49Chomzynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Google Scholar). After separation on 1.2% agarose gels, RNA was transferred to Hybond N+ membranes overnight (transfer buffer: 50 mM NaOH). Prehybridization was performed in 0.5 M phosphate buffer (pH 7.0) containing 7% SDS and 1 mM EDTA for 2 h. Hybridization was done overnight in the same solution additionally containing 1% bovine serum albumin and 32P-labeled probe (106 cpm/ml). Filters were washed 2 × 30 min in a solution containing decreasing salt concentrations (2 × SSC (1 × SSC) + 0.5% SDS + 1 mM EDTA). All steps were performed at 65°C. Rat rhoB-cDNA was kindly provided by Dr. T. Hunter (San Diego, CA), the human rhoA-cDNA by Dr. A. Hall (London, United Kingdom). For rhoB-specific hybridization we used a 0.95-kilobase EcoRI-XhoI fragment from the 3′-region of rhoB-cDNA (containing rhoB-specific coding and noncoding sequences). Amounts of total rho mRNAs were determined by hybridization with the whole coding sequence of rhoA cDNA, which cross-hybridizes to all rho mRNA species because of their high homology(36Yeramian P. Chardin P. Madaule P. Tavitian A. Nucleic Acids Res. 1987; 15: 1869Crossref PubMed Scopus (88) Google Scholar). The c-fos and GAPDH cDNA hybridization probes were obtained from Dr. H. J. Rahmsdorf (Institute of Genetics, Research Center, Karlsruhe, Federal Republic of Germany). For quantitation of the data, densitometrical analysis was performed. Relative gene expression was calculated by referring rhoB (rho c-fos) mRNA to the amount of GAPDH mRNA and by relating to control cells included in each experiment. Run-on experiments were essentially performed as described(28Greenberg M.E. Ziff E.B. Nature. 1984; 311: 433-437Crossref PubMed Scopus (2010) Google Scholar). 5 μg of plasmid DNA containing rhoB-, c-fos-, c-jun-, and GAPDH-cDNA sequences were heat-denatured (10 min, 95°C) and blotted onto Hybond N+ filter using a slot-blot apparatus. Nuclei from UV-irradiated and non-irradiated NIH3T3 cells were incubated in a buffer containing 10 mM Tris-HCl (pH 8.0), 5 mM MgCl2, 300 mM KCl, 0.5 mM of each dATP, dCTP, and dGTP, and 100 μCi of [α-32P]dUTP for 30 min at 30°C. Reactions were stopped by DNase I treatment (20 μg/ml, 5 min, 30°C) followed by proteinase K digestion (30 min, 42°C). After phenol/chloroform extraction, 32P-labeled RNA was precipitated by trichloroacetic acid and filtered on BA85 filters (Millipore). After elution from the filters, 32P-labeled RNA was ethanol-precipitated. Hybridization of the blots with [32P]RNA was performed as described (see “Northern Blot Analysis”). A 1.6-kilobase EcoRI fragment from rat rhoB cDNA (27Ghosh R. Amstad P. Cerutti O. Mol. Cell. Biol. 1993; 13: 6992-6999Crossref PubMed Scopus (39) Google Scholar) was cloned both in sense and antisense orientation into the eukaryotic expression vector pSVT7 (gift of Dr. U. Günthert, Freidrich-Mieseler Institute, Basel) and pMAMneo (Clontech), respectively. Transfection of NIH3T3 cells was performed with 20 μg of DNA of rhoB expression plasmid using the calcium phosphate co-precipitation technique(50Kaina B. van Zeeland A.A. Backendorf C. Thielmann H.W. van de Putte P. Mol. Cell. Biol. 1987; 7: 2024-2030Crossref PubMed Scopus (26) Google Scholar). 16 h after transfection, cells were fixed for FITC staining as described below. NIH3T3 cells were disrupted by sonication in ice-cold buffer containing 10 mM Tris-HCl (pH 7.4), 1 mM EDTA, 1 mM MgCl2, 0.1 mM phenylmethylsulfonyl fluoride. After centrifugation (10 min, 600 × g, 4°C), supernatant was used for protein determination according to Bradford (51Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216440) Google Scholar). ADP-ribosylation of cell lysates was performed with either Clostridium botulinum C3 exoenzyme (Rho-specific [32P]ADP-ribosylation) or C. botulinum C2 exoenzyme ([32P]ADP-ribosylation of G-actin). 20-50 μg of protein from total extracts were incubated for 30 min at 37°C in buffer containing 20 mM Tris-HCl (pH 7.4), 1 mM EDTA, 1 mM MgCl2, 1 mM dithiothreitol, 10 mM thymidine, 0.2 μM NAD, 0.5 μCi of [32P]NAD, and 0.1 μg of C3 (0.5 μg of C2). Reaction products were then analyzed by one-dimensional SDS-gel electrophoresis according to Laemmli (52Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207233) Google Scholar) or by two-dimensional gel electrophoresis(53Harris E.L.V. Angal S. Harris E.L.V. Protein Purification Methods, A Practical Approach. IRL Press, Oxford, United Kingdom1989: 29Google Scholar). [32P]ADP-ribosylated proteins were detected after exposure of dried gels on Kodak X-Omat films. Cells were fixed on dishes with 4% formaldehyde, 0.2% Triton X-100 in 0.1 M phosphate-buffer (pH 7.4) for 1 h at room temperature. After washing with PBS, cells were incubated with FITC-labeled phalloidin (0.5 μM in PBS) for 1 h at room temperature. Subsequently cells were washed three times with PBS and actin filaments were detected by fluorescence microscopy. 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(36Yeramian P. Chardin P. Madaule P. Tavitian A. Nucleic Acids Res. 1987; 15: 1869Crossref PubMed Scopus (88) Google Scholar), 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/m2) 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(37Aktories K. Weller U. Chhatwal G.S. FEBS Lett. 1987; 212: 109-113Crossref PubMed Scopus (172) Google Scholar, 38Aktories K. Rösener S. Blaschke U. Chhatwal G.S. Eur. J. Biochem. 1988; 172: 445-450Crossref PubMed Scopus (122) Google Scholar, 39Chardin P. Madaule P. Tavitian A. Nucleic Acids Res. 1988; 16: 2727Crossref PubMed Scopus (93) Google Scholar, 40Kikuchi A. Yamamoto K. Fujita T. Takai Y. J. Biol. Chem. 1988; 263: 16303-16308Abstract Full Text PDF PubMed Google Scholar, 41Narumiya S. Sekine A. Fujiwara M. J. Biol. Chem. 1988; 263: 17255-17257Abstract Full Text PDF PubMed Google Scholar). Separation of [32P]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 [32P]ADP-ribosylated RhoB protein decreased again (not shown).Figure 2Induction of RhoB protein after UV treatment of NIH3T3 cells. Logarithmically growing NIH3T3 cells were irradiated with 30 J/m2 and cells were harvested 1 h later. 50 μg of protein from total cell extracts was ADP-ribosylated by C3 and [32P]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.View Large Image Figure ViewerDownload Hi-res image Download (PPT) 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). 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). Since protein kinases interfere with the regulation of the UV stimulated expression of c-fos(9Jähner D. Hunter T. Oncogene. 1991; 6: 1259-1268PubMed Google Scholar, 16Angel P. Pöting A. Mallick U. Rahmsdorf H.J. Schorpp M. Herrlich P. Mol. Cell. Biol. 1986; 6: 1760-1766Crossref PubMed Scopus (172) Google Scholar, 26Büscher M. Rahmsdorf H. Litfin M. Karin M. Herrlich P. Oncogene. 1988; 3: 301-311PubMed Google Scholar, 53Harris E.L.V. Angal S. Harris E.L.V. Protein Purification Methods, A Practical Approach. IRL Press, Oxford, United Kingdom1989: 29Google Scholar), 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(54Sachsenmaier C. Radler-Pohl A. Zinck R. Nordheim A. Herrlich P. Rahmsdorf H.J. Cell. 1994; 78: 963-972Abstract Full Text PDF PubMed Scopus (406) Google Scholar). 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.Tabled 1 Open table in a new tab Tabled 1View Large Image Figure ViewerDownload Hi-res image Download (PPT) Open table in a new tab 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 3 summarizes the effects of various treatments on the amount of rhoB mRNA. Cisplatin, hydroxyurea, and dexamethasone also elicited rhoB induction, whereas retinoic acid and Bt2cAMP, given either by its own or in combination with TPA, were without effect.Tabled 1View Large Image Figure ViewerDownload Hi-res image Download (PPT) Open table in a new tab 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(39Chardin P. Madaule P. Tavitian A. Nucleic Acids Res. 1988; 16: 2727Crossref PubMed Scopus (93) Google Scholar, 42Paterson H.F. Self A.J. Garrett M.S. Just I. Aktories K. Hall A. J. Cell Biol. 1990; 111: 1001-1007Crossref PubMed Scopus (571) Google Scholar, 45Ridley A.J. Hall A. Cell. 1992; 70: 389-399Abstract Full Text PDF PubMed Scopus (3832) Google Scholar). 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 [32P]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 (55Mahadevan L.C. Edwards D.R. Nature. 1991; 349: 747-749Crossref PubMed Scopus (155) Google Scholar) did not induce rhoB in Rat2 cells(34Jähner D. Hunter T. Mol. Cell. Biol. 1991; 11: 3682-3690Crossref PubMed Scopus (151) Google Scholar), 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 Bt2cAMP treatment(26Büscher M. Rahmsdorf H. Litfin M. Karin M. Herrlich P. Oncogene. 1988; 3: 301-311PubMed Google Scholar, 30Krämer M. Stein B. Mai S. Kunz E. König H. Loferer H. Grunicke H.H. Ponta H. Herrlich P. Rahmsdorf H.F. Radiat. Environ. Biophys. 1990; 29: 303-313Crossref PubMed Scopus (41) Google Scholar), whereas rhoB expression was not ((34Jähner D. Hunter T. Mol. Cell. Biol. 1991; 11: 3682-3690Crossref PubMed Scopus (151) Google Scholar) 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 Bt2cAMP 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(56Devary Y. Gottlieb R.A. Smeal T. Karin M. Cell. 1992; 71: 1081-1091Abstract Full Text PDF PubMed Scopus (795) Google Scholar), 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(57Miller C.C. Hale P. Pentland P.A. J. Biol. Chem. 1994; 269: 3529-3533Abstract Full Text PDF PubMed Google Scholar). 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(58Derijard B. Hibi M. Wu I.H. Barrett T. Su B. Deng T. Karin M. Davis R.J. Cell. 1994; 76: 1025-1037Abstract Full Text PDF PubMed Scopus (2955) Google Scholar). The substrate for this kinase has been identified to be c-Jun(58Derijard B. Hibi M. Wu I.H. Barrett T. Su B. Deng T. Karin M. Davis R.J. Cell. 1994; 76: 1025-1037Abstract Full Text PDF PubMed Scopus (2955) Google Scholar). 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(38Aktories K. Rösener S. Blaschke U. Chhatwal G.S. Eur. J. Biochem. 1988; 172: 445-450Crossref PubMed Scopus (122) Google Scholar, 39Chardin P. Madaule P. Tavitian A. Nucleic Acids Res. 1988; 16: 2727Crossref PubMed Scopus (93) Google Scholar, 40Kikuchi A. Yamamoto K. Fujita T. Takai Y. J. Biol. Chem. 1988; 263: 16303-16308Abstract Full Text PDF PubMed Google Scholar, 41Narumiya S. Sekine A. Fujiwara M. J. Biol. Chem. 1988; 263: 17255-17257Abstract Full Text PDF PubMed Google Scholar, 42Paterson H.F. Self A.J. Garrett M.S. Just I. Aktories K. Hall A. J. Cell Biol. 1990; 111: 1001-1007Crossref PubMed Scopus (571) Google Scholar, 43Rubin E.J. Gill M. Boquet P. Popoff M.P. Mol. Cell. Biol. 1988; 8: 418-426Crossref PubMed Scopus (235) Google Scholar, 44Wiegers W. Just I. Müller H.M. Hellwig A. Traub P. Aktories K. Eur. J. Cell Biol. 1991; 54: 237-245PubMed Google Scholar). 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 (47Adamson P. Paterson H.F. Hall A. J. Cell Biol. 1992; 119: 617-627Crossref PubMed Scopus (329) Google Scholar) and only rhoB is induced by mitogens(34Jähner D. Hunter T. Mol. Cell. Biol. 1991; 11: 3682-3690Crossref PubMed Scopus (151) Google Scholar), 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(46Avraham H. Weinberg R.A. Mol. Cell. Biol. 1989; 9: 2058-2066Crossref PubMed Scopus (95) Google Scholar, 59Perona R. Esteve P. Jimenez B. Ballestero R.P. Ramón y Cajal S. Lacal J.C. Oncogene. 1993; 8: 1285-1292PubMed Google Scholar). 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(48Prendergast G.C. Khosravi-Far R. Solski P.A. Kurzawa H. Lebowitz P.F. Der C.J. Oncogene. 1995; 10: 2289-2296PubMed Google Scholar). 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(60Kaina B. Schönthal A. Rahmsdorf H.J. Ponta H. Herrlich P. Lambert M.W. Laval J. DNA Repair Mechanisms and Their Biological Implications in Mammalian Cells. Plenum Publishing Corp., New York1990: 149-165Google Scholar, 61Scanlon K.J. Ishida H. Kashani-Sabet M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11123-11127Crossref PubMed Scopus (85) Google Scholar, 62Scanlon K.J. Jiao L. Funato T. Wnag W. Tone T. Rossi J.J. Kashani-Sabet M. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10591-10595Crossref PubMed Scopus (205) Google Scholar, 63Haas S. Kaina B. Carcinogenesis. 1995; 16: 985-991Crossref PubMed Scopus (72) Google Scholar). Another gene product which is involved in cell cycle control and accumulates after UV irradiation is p53(18Lu X. Lane D.P. Cell. 1993; 75: 765-778Abstract Full Text PDF PubMed Scopus (775) Google Scholar, 19Maltzman W. Czyzyk L. Mol. Cell. Biol. 1984; 4: 1689-1694Crossref PubMed Scopus (817) Google Scholar). A UV-stimulated increase in p53 is not observed earlier than 3-5 h after UV irradiation(18Lu X. Lane D.P. Cell. 1993; 75: 765-778Abstract Full Text PDF PubMed Scopus (775) Google Scholar). 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 (63Haas S. Kaina B. Carcinogenesis. 1995; 16: 985-991Crossref PubMed Scopus (72) Google Scholar). 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. We thank Dr. T. Hunter for providing the rat rhoB cDNA and Dr. A. Hall for rhoA cDNA. Furthermore, we thank Dr. Rahmsdorf for c-fos and GAPDH cDNA as well as Dr. Schächtele for the gift of the protein kinase C inhibitor Gö18." @default.
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