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Mechanosensitive Transcription Factors Involved in Endothelin B Receptor Expression

2001; Elsevier BV; Volume: 276; Issue: 40 Linguagem: Inglês

10.1074/jbc.m105158200

1083-351X

Marco Cattaruzza, Ina Eberhardt, Markus Hecker,

Nitric Oxide and Endothelin Effects

Growing evidence suggests an involvement of the endothelin B receptor (ETB-R) in blood pressure-dependent arterial remodeling. To study the molecular mechanisms leading to deformation-induced ETB-R expression, we have cultured rat aortic smooth muscle cells on flexible elastomers and, when grown to 70% confluence, periodically stretched them for 6 h (15% elongation, 0.5 Hz). The cells responded with an increase both in ETB-R mRNA (12-fold compared with control) and protein (4-fold). According to nuclear run-on analysis this increase in ETB-R expression occurred at the level of transcription. Among various kinase pathways, Rho kinase (ROCK) and p38 mitogen-activated protein kinase (p38 MAPK) mediated part of the deformation-induced increase in ETB-R expression, as judged by the inhibitory effect of Y27632 (1 μm, 38% inhibition) and SB202190 (10 μm, 44% inhibition), respectively. Gel shift assaying of the three transcription factors principally activated by these kinases revealed a transient deformation-induced activation of activator protein-1 (AP-1) and CCAAT/enhancer-binding protein (C/EBP), but not activating transcription factor, that was sensitive to both Y27632 and SB212190. The potential role of AP-1 and C/EBP in deformation-induced ETB-R expression was verified both by using decoy oligodeoxynucleotides mimicking the DNA-binding sites of these transcription factors and a nuclear run-on-based assay employing specific antibodies directed against AP-1 and C/EBP. Both techniques unequivocally demonstrated that activation of these transcription factors, namely that of C/EBPβ, contribute to the increase of ETB-R gene expression in response to cyclic stretch. Growing evidence suggests an involvement of the endothelin B receptor (ETB-R) in blood pressure-dependent arterial remodeling. To study the molecular mechanisms leading to deformation-induced ETB-R expression, we have cultured rat aortic smooth muscle cells on flexible elastomers and, when grown to 70% confluence, periodically stretched them for 6 h (15% elongation, 0.5 Hz). The cells responded with an increase both in ETB-R mRNA (12-fold compared with control) and protein (4-fold). According to nuclear run-on analysis this increase in ETB-R expression occurred at the level of transcription. Among various kinase pathways, Rho kinase (ROCK) and p38 mitogen-activated protein kinase (p38 MAPK) mediated part of the deformation-induced increase in ETB-R expression, as judged by the inhibitory effect of Y27632 (1 μm, 38% inhibition) and SB202190 (10 μm, 44% inhibition), respectively. Gel shift assaying of the three transcription factors principally activated by these kinases revealed a transient deformation-induced activation of activator protein-1 (AP-1) and CCAAT/enhancer-binding protein (C/EBP), but not activating transcription factor, that was sensitive to both Y27632 and SB212190. The potential role of AP-1 and C/EBP in deformation-induced ETB-R expression was verified both by using decoy oligodeoxynucleotides mimicking the DNA-binding sites of these transcription factors and a nuclear run-on-based assay employing specific antibodies directed against AP-1 and C/EBP. Both techniques unequivocally demonstrated that activation of these transcription factors, namely that of C/EBPβ, contribute to the increase of ETB-R gene expression in response to cyclic stretch. One of the most potent stimuli for the synthesis of endothelin-1 (ET-1) 1The abbreviations and trivial names used are: ET-1, endothelin-1; ETA-R, endothelin A receptor; ETB-R, endothelin B receptor; SMC, smooth muscle cells; ROBERTA, run on-basedregulation of transcriptionanalysis; AP-1, activator protein-1; ATF, activating transcription factor; C/EBP, CCAAT enhancer-binding protein; STAT, signal transducer and activator of transcription; MAPK, mitogen-activated protein kinase; SB212190, 4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)1H-imidazole; Y27632, (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide; RT-PCR, reverse transcription polymerase chain reaction; dODN, decoy oligodeoxynucleotide. is a high level of circumferential stretch to which preferentially vascular smooth muscle cells (SMC), but also endothelial, cells are exposed to by an increase in blood pressure (1Wang D.L. Wung B.S. Peng Y.C. Wang J.J. J. Cell. Physiol. 1995; 163: 400-406Crossref PubMed Scopus (70) Google Scholar). Besides its vasoconstrictor effect primarily mediated by the A type receptor (ETA-R), ET-1 has also been shown to be a potent co-mitogen for vascular SMC (2Janakidevi K. Fisher M.A. Del Vecchio P.J. Tiruppathi C. Figge J. Malik A.B. Am. J. Physiol. 1992; 263: 1295-1301Crossref PubMed Google Scholar). Significant expression of the B type receptor (ETB-R) in these cells is only detectable upon exposure to cyclic stretch (3Cattaruzza M. Dimigen C. Ehrenreich H. Hecker M. FASEB J. 2000; 14: 991-998Crossref PubMed Scopus (91) Google Scholar, 4Lauth M. Berger M.M. Cattaruzza M. Hecker M. Hypertension. 2000; 35: 648-654Crossref PubMed Scopus (39) Google Scholar). However, activation of this receptor seems to contribute to changes in medial SMC phenotype that are typical for the early phase of pressure-dependent remodeling of the vessel wall (3Cattaruzza M. Dimigen C. Ehrenreich H. Hecker M. FASEB J. 2000; 14: 991-998Crossref PubMed Scopus (91) Google Scholar,4Lauth M. Berger M.M. Cattaruzza M. Hecker M. Hypertension. 2000; 35: 648-654Crossref PubMed Scopus (39) Google Scholar). The endothelin system may thus act as a molecular switch between maintenance of vascular tone through ETA-R-mediated vasoconstriction and, in conditions of a prolonged supraphysiological increase in blood pressure, ETB-R-mediated adaptive remodeling. This process, when exaggerated, may play an important role,e.g. in the manifestation of arterial hypertension (5Hamet P. deBlois D. Dam T.V. Richard L. Teiger E. Tea B.S. Orlov S.N. Tremblay J. Can. J. Physiol. Pharmacol. 1996; 74: 850-861Crossref PubMed Scopus (53) Google Scholar), vein graft disease (6Weis M. von Scheidt W. Circulation. 1997; 96: 2069-2077Crossref PubMed Scopus (405) Google Scholar), or restenosis following angioplasty (7Pasterkamp G. de Kleijn D.P. Borst C. Cardiovasc. Res. 2000; 45: 843-852Crossref PubMed Scopus (165) Google Scholar) that often are resistant to therapeutic interventions. Preventing deformation-induced ETB-R expression in vascular SMC may thus constitute a feasible therapeutic option at the onset of these cardiovascular complications. To this end we have investigated the transcriptional control of ETB-R expression in vascular SMC exposed to cyclic stretch by using, e.g. a novel nuclear run-on-based technique that directly analyzes the functional significance of a given transcription factor in expression of the gene product of interest (runon-based regulation oftranscription analysis, ROBERTA). Antibodies against c-Jun (mouse monoclonal, 2 mg/ml), C/EBPβ (mouse monoclonal, 2 mg/ml), and the signal transducer and activator of transcription (STAT-1, mouse monoclonal, 2 mg/ml) were from Santa Cruz, Heidelberg, Germany. The antibody against rat fibronectin (mouse monoclonal, 1 mg/ml) was from BD Biosciences, Heidelberg, Germany. The antibody against β-actin (mouse monoclonal), secondary antibodies (goat anti-mouse, horseradish peroxidase-coupled), as well as laboratory chemicals were from Sigma, Deisenhofen, Germany. 4-(4-Fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)1H-imidazole (SB212190) was from Calbiochem, Bad Soden, Germany. (+)-(R)-trans-4-(1-Aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide (Y27632) was a kind gift of Yoshitomi Pharmaceutical Industries, Osaka, Japan. Isolation and culture of SMC derived from the rat aorta was done as described previously (3Cattaruzza M. Dimigen C. Ehrenreich H. Hecker M. FASEB J. 2000; 14: 991-998Crossref PubMed Scopus (91) Google Scholar). Isolation of RNA, reverse transcription, and subsequent analysis of relative amounts of cDNA by PCR were done as described previously (3Cattaruzza M. Dimigen C. Ehrenreich H. Hecker M. FASEB J. 2000; 14: 991-998Crossref PubMed Scopus (91) Google Scholar). The range of cycles to be in the exponential phase of the PCR reaction was newly established for each set of samples. Gel shift analyses were performed as described previously (8Cattaruzza M. Wachter R. Wagner A.H. Hecker M. Br. J. Pharmacol. 2000; 129: 1155-1162Crossref PubMed Scopus (19) Google Scholar). The custom made palindromic oligodeoxynucleotide (IBA, Goettingen, Germany) containing the consensus binding site for ATF had the following sequence: 5′-GACTTGACGTCAAGTC-3′. It was hybridized to the double-stranded form by standard procedures (9Ausubel, F. M. (ed) (1997) Current Protocols in Molecular Biology, John Wiley and Sons, New YorkGoogle Scholar). Incubation of SMC with the double-stranded dODN (10 μm final concentration) for 4 h was done as described previously (10Wagner A.H. Krzesz R. Schröder C. Gao D. Cattaruzza M. Hecker M. Mol. Pharmacol. 2000; 58: 1333-1340Crossref PubMed Scopus (27) Google Scholar). Exposure of the cells to two different dODN was done consecutively with an exchange of the medium in between. The sequence for the ATF dODN was exactly the same as that used for gel shift analysis except that the terminal 4 bases on either site of the molecule were linked by phosphorothioate esters for added stability. The RT-PCR-based nuclear run-on analysis was done as described previously (8Cattaruzza M. Wachter R. Wagner A.H. Hecker M. Br. J. Pharmacol. 2000; 129: 1155-1162Crossref PubMed Scopus (19) Google Scholar). Besides the de novo transcription of the ETB-R gene, transcription of glyceraldehyde-3-phosphate dehydrogenase as an internal standard was analyzed too. The ROBERTA is a modified nuclear run-on analysis. Prior to incubation with nucleotides (CTP, GTP, and UTP, 2 mmol/liter each) and the energy-regenerating system (creatine phosphate plus creatine kinase) for specific RNA synthesis, nuclei (150 μl of suspension) were incubated at 0 °C for 15 min with 0.5% Triton X-100, ATP (3 mmol/liter), and the antibody against the transcription factor of interest. Thereafter, the nuclear run-on procedure was started by the addition of the three ribonucleotides, creatine phosphate, and creatine kinase, exactly as described previously (8Cattaruzza M. Wachter R. Wagner A.H. Hecker M. Br. J. Pharmacol. 2000; 129: 1155-1162Crossref PubMed Scopus (19) Google Scholar). The amount as well as the type of antibody for effective inhibition of transcription had to be established for every transcription factor and antibody. In this study the final antibody concentrations were as follows: anti-c-Jun (20 μg/ml), anti-C/EBPβ (20 μg/ml), anti-STAT-1 (20 μg/ml), and anti-fibronectin (50 μg/ml). Western blot analysis was performed according to standard procedures (10Wagner A.H. Krzesz R. Schröder C. Gao D. Cattaruzza M. Hecker M. Mol. Pharmacol. 2000; 58: 1333-1340Crossref PubMed Scopus (27) Google Scholar) using PVDF membranes (PALL Gelman, Dreieich, Germany). For detection of ETB-R protein, a primary rabbit polyclonal antibody directed against a C-terminal peptide of the receptor (11Cramer H. Müller-Esterl W. Schröder C. Biochemistry. 1997; 36: 13325-13332Crossref PubMed Scopus (53) Google Scholar) was used together with a secondary horseradish peroxidase-conjugated anti-rabbit antibody and the Super Signal BlazeTM chemiluminescent reagent (Pierce, St. Augustin, Germany). Loading and transfer of equal amounts of protein in each lane was verified by re-probing the membrane (5-min incubation in 200 mmol/liter sodium hydroxide and consecutive neutralization by several washes with distilled water) with a monoclonal anti-β-actin antibody and visualization with a secondary horseradish peroxidase-conjugated anti-mouse antibody as described (9Ausubel, F. M. (ed) (1997) Current Protocols in Molecular Biology, John Wiley and Sons, New YorkGoogle Scholar). Unless indicated otherwise, results are expressed as means ± S.E. of n observations with cells obtained from different animals. One sample t test with two-sided p values or one-way analysis of variance followed by Dunnett post-test was used where appropriate to determine statistically significant differences between the means and and/or the means and control with p < 0.05 considered significant. ETB-R mRNA and protein abundance were both enhanced in the cultured SMC after 6-h exposure to a level of cyclic stretch similar to the in vivo situation (Ref. 12Stefanadis C. Dernellis J. Vlachopoulos C. Tsioufis C. Tsiamis E. Toutouzas K. Pitsavos C. Toutouzas P. Circulation. 1997; 96: 1853-1858Crossref PubMed Scopus (130) Google Scholar; Fig. 1). To evaluate whether the increase in ETB-R mRNA was due to transcriptional activation or enhanced mRNA stability, SMC were incubated with the transcriptional inhibitor actinomycin D (Fig.2A). In addition, a RT-PCR-based nuclear run-on assay was performed, demonstrating enhancedde novo synthesis of ETB-R mRNA in nuclei isolated from stretched SMC as compared with nuclei from SMC incubated under static conditions (Fig. 2B).Figure 2A, analysis of ETB-R mRNA stability. Cultured SMC were preincubated for 1 h with 1 μm actinomycin D (ActD) before exposure to cyclic stretch (15% elongation, 0.5 Hz, 6 h). ETB-R mRNA abundance was analyzed by RT-PCR 1 h after termination of the stretch protocol. B, nuclear run-on analysis of ETB-R gene expression. Nuclei isolated from SMC exposed to cyclic stretch (15% elongation, 0.5 Hz, 3 h) are compared with nuclei isolated from SMC incubated under static conditions. The negative control (0 min) is derived from the same amount of nuclei from the same batch of SMC lysed immediately. Both figures depict a representative result of a series of three identical experiments with different batches of SMC.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Signal transduction pathways thought to be activated upon mechanical stimulation in vascular cells were targeted by using specific protein kinase inhibitors. Neither PD98059 (1 μm, 1-h preincubation), a specific inhibitor of MEK1 located upstream of ERK/1/2, nor the protein kinase C inhibitor, Ro 31-8220 (1 μm, 1-h preincubation), affected deformation-induced ETB-R expression (n = 4, not shown). In contrast, the Rho kinase (ROCK) inhibitor Y27632 as well as SB 212190, an inhibitor of p38 MAPK, significantly attenuated ETB-R expression in response to cyclic stretch both at the mRNA and protein level (Fig. 3). Co-incubation of the SMC with both inhibitors, however, did not result in a more pronounced inhibition (Fig. 3A), indicating that both kinases are part of a common mechanotransduction pathway. According to TRANSFAC analysis (13Wingender E. Chen X. Hehl R. Karas H. Liebich I. Matys V. Meinhardt T. Prüβ M. Reuter I. Schacherer F. Nucleic Acids Res. 2000; 28: 316-319Crossref PubMed Scopus (1037) Google Scholar), the promoter of the human ETB-R gene (GenBankTM accession number AL139002) contains several consensus binding sites for the transcription factors ATF, AP-1, and C/EBP, the activation of which has been linked to the ROCK and/or p38 MAPK signaling pathways (14Kyriakis J.M. Gene Expr. 1999; 7: 217-231PubMed Google Scholar, 15Baldassare J.J. Bi Y. Bellone C.J. J. Immunol. 1999; 162: 5367-5373PubMed Google Scholar, 16Harris V.K. Kagan B.L. Ray R. Coticchia C.M. Liaudet-Coopman E.D. Wellstein A. Tate Riegel A. Oncogene. 2001; 20: 1730-1738Crossref PubMed Scopus (22) Google Scholar, 17Zhu T. Lobie P.E. J. Biol. Chem. 2000; 275: 2103-2114Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Therefore, nuclear translocation of these transcription factors in SMC exposed to cyclic stretch was monitored by gel shift analysis. Whereas the transcription factor ATF appeared to be inert to cyclic stretch (not shown), both AP-1 and C/EBP abundance in the nucleus were transiently enhanced (maximum 1 h, not shown). Moreover, translocation of AP-1 as well as C/EBP was sensitive to both Y27632 and SB212190 (Fig. 4). According to supershift analysis, primarily the β-isoform of C/EBP appeared to be stretch-sensitive. To confirm the involvement of AP-1 and C/EBPβ activation in stretch-induced ETB-R expression, two experimental approaches were chosen. First, SMC were preincubated for 4 h with dODN (10 μm), mimicking the consensus binding sites of the candidate transcription factors. Both dODN concentration and preincubation period were chosen according to previous experiments with the cultured SMC, demonstrating maximum neutralization of the transcription factors under these conditions (cf. Ref. 10Wagner A.H. Krzesz R. Schröder C. Gao D. Cattaruzza M. Hecker M. Mol. Pharmacol. 2000; 58: 1333-1340Crossref PubMed Scopus (27) Google Scholar). As shown in Fig. 5, dODN blockade of AP-1 and C/EBP, but not ATF (not shown), significantly attenuated stretch-induced expression of the ETB-R gene. Consecutive incubation of the SMC with both the AP-1 and C/EBP dODN, on the other hand, did not result in a further inhibition as compared with the effect of the C/EBP dODN alone (n = 4, not shown). As a second method to verify the involvement of AP-1 and C/EBP in stretch-induced expression of the ETB-R gene, ROBERTA was employed. The principle of this method, described in detail in the methods section, is outlined in Fig.6. Incubation of nuclei isolated from stretched SMC with a specific anti-C/EBPβ antibody attenuated ETB-R gene expression in response to cyclic stretch, whereas the c-Jun antibody (AP-1) did not reveal a significant effect (Fig. 7A). The inhibitory effect of the antibody directed against C/EBPβ appeared to be specific, for neither an antibody directed against a transcription factor not functional in ETB-R expression (STAT-1) nor an antibody directed against an unrelated protein (fibronectin) revealed an effect on stretch-induced de novo synthesis of ETB-R mRNA (Fig. 7B). As a matter of principle, however, the anti-STAT-1 antibody was clearly active, for it efficiently attenuated cytokine-induced de novo mRNA synthesis of the STAT-1-dependent gene CD40 (∼50% inhibition) in nuclei isolated from cultured SMC under static conditions (not shown).Figure 7Stretch-induced ETB-R expression in SMC as analyzed by ROBERTA. Nuclei from SMC exposed to cyclic stretch (15% elongation, 0.5 Hz, 3 h) were isolated and incubated with the appropriate antibodies (20–50 μg/ml) for 30 min. Thereafter, nucleotides and the ATP-regenerating system were added, and the nuclei were either lysed immediately (negative control; 0 min) or incubated for 30 min at 30 °C. A, analysis of the role of C/EBPβ. B, negative control experiments. Left panel, lack of effect of the anti-STAT-1α antibody on stretch-induced ETB-R mRNA expression compared with that of the anti-C/EBPβ antibody. Right panel, lack of effect of an anti-fibronectin (FN) antibody.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Vascular remodeling per se is an adaptive process enabling the vessel wall to accommodate an increased hemodynamic load. If exaggerated, however, this process becomes pathologic such as, e.g. in manifest hypertension, ultimately leading to fixation of peripheral resistance and eventually occlusion of the blood vessel. There is increasing evidence that the endothelin system is crucially involved in the onset of deformation-induced vascular remodeling (1Wang D.L. Wung B.S. Peng Y.C. Wang J.J. J. Cell. Physiol. 1995; 163: 400-406Crossref PubMed Scopus (70) Google Scholar, 3Cattaruzza M. Dimigen C. Ehrenreich H. Hecker M. FASEB J. 2000; 14: 991-998Crossref PubMed Scopus (91) Google Scholar), and that this effect is mediated primarily via the ETB-R rather than the ETA-R expressed on vascular SMC. Although there are several ETA-R-specific and mixed antagonists available, these substances for several reasons do not provide an attractive means for challenging ET-1-induced vascular remodeling. Most importantly, in conditions of enhanced blood pressure, ETA-R antagonism may have an adverse effect by weakening the ETA-R-mediated vasoconstrictive capacity of the SMC that is normally relieving the hemodynamic load of the vessel wall to some extent, thereby promoting deformation-induced ETB-R expression. Activation of the ETB-R, in turn, induces or supports the remodeling process (4Lauth M. Berger M.M. Cattaruzza M. Hecker M. Hypertension. 2000; 35: 648-654Crossref PubMed Scopus (39) Google Scholar, 18Wang X. Douglas S.A. Louden C. Vickery-Clark L.M. Feuerstein G.Z. Ohlstein E.H. Circ. Res. 1996; 78: 322-328Crossref PubMed Scopus (138) Google Scholar). Therefore, in the absence of a specific nontoxic ETB-R antagonist, understanding stretch-induced ETB-R expression at the molecular level might lead to the definition of a novel target for interfering with the onset of an exaggerated remodeling process. Using inhibitors against kinases potentially involved in mechanotransduction, ROCK and p38-MAPK were characterized to contribute to stretch-induced ETB-R expression, while protein kinase C or the classical MAP kinase pathway was not. This finding was unprecedented, for inhibition of protein kinase C, which often integrates multiple extracellular signals, was without effect, whereas ROCK located upstream of protein kinase C (19Iizuka K. Yoshii A. Samizo K. Tsukagoshi H. Ishizuka T. Dobashi K. Nakazawa T. Mori M. Br. J. Pharmacol. 1999; 128: 925-933Crossref PubMed Scopus (104) Google Scholar), and p38 MAPK, one of the best characterized downstream kinases of protein kinase C (20Ito T. Kozawa O. Tanabe K. Niwa M. Matsuno H. Sakai N. Ito H. Kato K. Uematsu T. Hypertension. 2000; 35: 673-678Crossref PubMed Scopus (23) Google Scholar), both constituted part of the signal transduction pathway mediating stretch-induced ETB-R expression. Moreover, the inhibitors of these two kinases were similarly effective but when combined did not even produce an additive effect. The most plausible explanation for these results is that both kinases are part of the same signal transduction pathway. For example, ROCK may directly or indirectly activate p38-MAPK, bypassing protein kinase C. Indeed, such a direct signal transduction pathway has already been documented in monocytes/macrophages (21Ashida N. Arai H. Yamasaki M. Kita T. J. Biol. Chem. 2001; 276: 16555-16560Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar) and therefore may represent a fast and effective way to transmit mechanical deformation signals to the nucleus of vascular SMC. If this hypothesis holds true, p38 MAPK, besides its well documented ability to phosphorylate c-Jun, hence activate AP-1, must be able to activate C/EBP, too, and this has in fact been documented in other cell types (15Baldassare J.J. Bi Y. Bellone C.J. J. Immunol. 1999; 162: 5367-5373PubMed Google Scholar, 22Wang X.Z. Ron D. Science. 1996; 272: 1347-1349Crossref PubMed Scopus (753) Google Scholar). The aforementioned characterization of ROCK and p38 MAPK and analysis of the ETB-R gene promoter suggested three candidate transcription factors for stretch-induced ETB-R expression,i.e. AP-1, ATF, and C/EBP (or NF-IL-6). According to gel shift analysis, both AP-1 and C/EBP indeed translocated to the nucleus of the cultured SMC upon exposure to cyclic stretch. Moreover, both kinase inhibitors attenuated the nuclear translocation of the two transcription factors to a similar degree, providing further evidence for the notion that ROCK acts through p38-MAPK and not via alternative pathways such as ERK1/2 and protein kinase C activation, which also mediate AP-1 translocation to the nucleus (19Iizuka K. Yoshii A. Samizo K. Tsukagoshi H. Ishizuka T. Dobashi K. Nakazawa T. Mori M. Br. J. Pharmacol. 1999; 128: 925-933Crossref PubMed Scopus (104) Google Scholar, 23Chihara K. Amano M. Nakamura N. Yano T. Shibata M. Tokui T. Ichikawa H. Ikebe R. Ikebe M. Kaibuchi K. J. Biol. Chem. 1997; 272: 25121-25217Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). Nuclear translocation of ATF, on the other hand, was not detectable in the stretched SMC, even though phosphorylation, hence activation of this transcription factor, has been reported in SMC exposed to cyclic stretch (24Hipper A. Isenberg G. Eur. J. Physiol. 2001; 441 (abstr.): R255Google Scholar). As a consequence, this finding per se did not exclude ATF from being involved in deformation-induced ETB-R expression, especially because proteins of the CREB/ATF family of transcription factors are not necessarily regulated by nuclear translocation. Therefore, the role of all three transcription factors in deformation-induced expression of the ETB-R in SMC was investigated further by using the dODN technique. Both the AP-1 and C/EBP family dODN significantly inhibited ETB-R expression in response to cyclic stretch, whereas the consensus dODN for the ATF/CREB family of transcription factors was without effect. Combined treatment of the SMC with the AP-1 and C/EBP dODN, however, did not produce a greater inhibitory effect. The most likely reason for this finding is that both dODNs must compete for a common uptake mechanism in the cultured SMC. 2M. Hecker, unpublished observation.Nonetheless, one can safely conclude from the dODN data that both AP-1 and C/EBP, but not ATF, are involved in stretch-induced up-regulation of ETB-R expression in vascular SMC. At least in the case of C/EBP, this conclusion was supported by ROBERTA, a direct method for monitoring transcription factor activity in the nucleus. This method, which was devised on the basis of a previously published nuclear run-on assay (9Ausubel, F. M. (ed) (1997) Current Protocols in Molecular Biology, John Wiley and Sons, New YorkGoogle Scholar), seems to be a valuable tool, complimenting other methods such as reporter gene analysis (25Schenborn E. Groskreutz D. Mol. Biotechnol. 1999; 13: 29-44Crossref PubMed Scopus (71) Google Scholar) or the dODN technique (26Morishita R. Higaki J. Tomita N. Ogihara T. Circ. Res. 1998; 82: 1023-1028Crossref PubMed Scopus (196) Google Scholar) for functional analysis of gene expression. ROBERTA is an antibody-based, hence specific, tool for demonstrating the involvement of a single transcription factor in the expression of the gene of interest. The advantage of this approach is that the genes and transcription factors remain in their intact functional environment, i.e. the nuclear chromatin. Perhaps the only factor limiting ROBERTA is the binding capacity (and specificity) of the antibody for the chosen transcription factor. This obviously was a problem with AP-1 that consists of different heterodimeric proteins. For factors belonging to the C/EBP family of transcription factors, on the other hand, gel supershift analysis and ROBERTA clearly suggested a role for C/EBPβ rather than another C/EBP family member in deformation-induced ETB-R expression. The specificity of this novel technique can be inferred from the fact that the inhibitory effect of the anti-C/EBPβ antibody was not mimicked by two different control antibodies directed against an unrelated transcription factor (STAT-1) or just another protein (fibronectin). Taken together, the aforementioned findings demonstrate that stretch-induced expression of the ETB-R in cultured vascular SMC is mediated at least in part by two different mechanosensitive transcription factors, AP-1 and C/EBPβ. It remains to be determined whether deformation-induced activation of these transcription factors fully accounts for the observed increase in ETB-R expression or whether other truly mechanosensitive transcription factors also play a role therein. At present, C/EBPβ appears to be the most feasible target for limiting deformation-induced ETB-R expression and thus an exaggerated remodeling of the vessel wall in response to an increased pressure load.