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Activation of NFκB Is Essential but Not Sufficient to Stimulate Mitogenesis of Vascular Smooth Muscle Cells
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
235, 365–368 (1997)
RC976788
Activation of NFkB Is Essential but Not Sufficient to Stimulate Mitogenesis of Vascular Smooth Muscle Cells Ellen Bretschneider,* Michael Wittpoth,† Artur-Aron Weber,† Erika Glusa,* and Karsten Schro¨r†,1 *Zentrum fu¨r Vaskula¨re Biologie und Medizin Erfurt der Friedrich-Schiller-Universita¨t Jena; and †Institut fu¨r Pharmakologie, Heinrich-Heine-Universita¨t Du¨sseldorf, Germany
Received April 29, 1997
This study investigates the role of the transcription factor NFkB in thrombin- and thrombin receptor activating peptide (TRAP, SFLLRNPNDKYEPYF)-induced mitogenesis of cultured bovine coronary artery smooth muscle cells (SMC). Stimulation of resting cells by thrombin (10 nM) or TRAP (10-100 mM) resulted in a comparable time-dependent activation of NFkB as detected by Western blotting and electrophoretic mobility shift assay (EMSA) of nuclear extracts. The NFkB activation was antagonized by N-acetyl-L-cysteine (20 mM) and pentoxifylline (0.5 mM). Thrombin caused a 3-4-fold increase in [3H]thymidine incorporation within 24 h which was prevented by inhibitors of NFkB activation. In contrast, TRAP did not cause any mitogenic response. These results demonstrate that activation of NFkB is an essential but not a sufficient signal for SMC mitogenesis. q 1997 Academic Press
Members of the nuclear factor-kB family of transcription factors play an important role in transcriptional regulation of a variety of genes (1-3). Among these genes is also the proto-oncogene c-myc, a factor that is associated with cell proliferation (4-6). Recently, Bellas et al., (6) have demonstrated that inhibitors of NFkB, such as N-acetyl-L-cysteine or pentoxifylline, inhibit growth of cultured vascular smooth muscle cells (SMC). In addition, microinjection of either purified IkB-a or double-stranded oligonucleotides harboring NFkB elements selectively inhibited SMC proliferation (6). Similarly, antisense oligonucleotides to the p65 subunit of NFkB have been found to inhibit SMC proliferation and neointima formation in rat carotid arteries (7). 1 To whom correspondence should be addressed: Institut fu¨r Pharmakologie, Heinrich-Heine-Universita¨t, Moorenstrasse 5, 40225 Du¨sseldorf, Germany. Fax: /49/211/8114781. E-mail: schroer@ pharma.uni-duesseldorf.de.
From these data the conclusion was drawn that activation of NFkB is essential for proliferation of SMC. Activation of NFkB in SMC can be triggered by growth factors such as thrombin (8). Thrombin activates its receptor through the proteolytic cleavage of the N-terminal extracellular domain of the receptor. This cleavage exposes a new N-terminus, which activates the receptor as a ‘‘tethered‘‘ ligand (9,10). Synthetic peptides, corresponding to the sequence of the new N-terminus, mimic cellular effects of thrombin in several cell types, including platelets and endothelial cells (11). The present study was initiated to compare thrombin and TRAP with respect to activation of of NFkB and mitogenesis in coronary artery SMC. MATERIALS AND METHODS Materials. TRAP (SFLLRNPNDKYEPYF) was synthesized by BioGenes (Berlin, Germany), N-acetyl-L-cysteine was purchased from Sigma (Deisenhofen, Germany), PDGF-BB was from Boehringer Mannheim (Mannheim, Germany). The NFkB-consensus-sequence 5*-TCG ACC TCT CGG AAA GTC CCC TCT GA-3* and 5*AGC TTC AGA GGG GAC TTT CCG AGA GG-3* was synthesized by MWG-Biotech (Eberberg, Germany). All other chemicals were from Merck (Darmstadt, Germany) or Sigma (Deisenhofen, Germany), respectively. The following were gifts: Indomethacin (Luitpold Pharma, Mu¨nchen, Germany); purified a-thrombin (Dr. J. Stu¨rzebecher, Zentrum fu¨r Vaskula¨re Biologie und Medizin Erfurt der FSU Jena, Germany); pentoxifylline (HMR, Frankfurt, Germany). Cell culture. Coronary artery SMC were isolated from adult cattles and cultivated at 377C and 5% CO2 in a humidified atmosphere in 80% HAM’s F12-medium and 20% DMEM supplemented with 100 U/ml penicillin, 0.1 mg/ml streptomycin and 10% fetal calf serum (FCS) as previously described (12). Subconfluent cells at passages 46 were used for the experiments. [3H]Thymidine incorporation. SMC were seeded into 24-well plates (51104 cells/well) and allowed to grow to subconfluency. The cells were cultivated for 24 h in FCS-free medium in order to obtain growth arrest. All experiments were performed in the presence of indomethacin (3 mM) to avoid interactions with endogenously synthesized prostaglandins. The cells were pulse-labeled with [3H]thymidine (2 mCi/ml) 4 h prior to the end of the incubation period
365
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
(24 h). Media were removed and the cell monolayer was washed sequentially with cold PBS, HClO4 (0.3 M) and again with cold PBS. The cells were solubilized by the addition of 0.3 ml NaOH (0.1 M) for 20 minutes at 377C. Aliquots of 0.2 ml were added to 3 ml of scintillant. [3H]Thymidine incorporation was determined by liquid scintillation spectrometry. N-acetyl-L-cysteine was added 20 h, pentoxifylline 1 h prior to the stimulation of SMC. Electrophoretic mobility shift assay (EMSA). Nuclear extracts were prepared according to Dignam et al. (13) with minor modifications. Briefly, cells were scraped, resuspended in buffer A and allowed to swell for 15 min on ice. Then, cell nuclei were sedimented by centrifugation for 10 min at 10,000 1 g. The supernatants were discarded and the nuclei resuspended in buffer C. Following a brief sonification, the samples were centrifuged for 30 min at 10,000 1 g. The supernatants, containing nuclear proteins, were removed, shockfrozen in liquid nitrogen and stored at 0807C until use. The protein content was measured using the BioRad-assay according to Bradford (14). Ten mg of nuclear protein were used for each binding reaction in binding buffer and incubated in a final volume of 20 ml for 10 min in the presence of 1 mg poly-dI-dC (Boehringer Mannheim, Germany) to saturate non-specific binding sites. This was followed by a 20 min incubation period with [32P]-labeled double-stranded oligonucleotide containing the NFkB-consensus sequence. The resulting DNA protein complexes were analyzed using a 4% native polyacrylamide gel electrophoresis. Bands were visualized by autoradiography. Western blotting. Immunoblot analysis of NFkB was performed by separation of 20 mg nuclear protein on a 8% sodium dodecyl polyacrylamide gel. Separated proteins were transferred onto nitrocellulose membranes and blocked in Blotto A (TBS, 0.05% Tween-20, 5% dry-milk) for 30 min. NFkB-antigens were detected with polyclonal antibodies against the p65 component (Santa Cruz Biotechnology, Heidelberg, Germany) according to the manufacturer’s instructions. Bands were visualized by ECL (Amersham, Buckinghamshire, England). Statistics. The data are mean { SEM of n independent experiments. Statistical analysis was performed by the two-tailed t-test. P levels of £ 0.05 were considered significant.
RESULTS Effects of Thrombin and TRAP on NFkB Activation Both thrombin (10 nM) and TRAP (10 or 100 mM) caused a significant, time-dependent NFkB activation as dectected by Western blotting and EMSA (Fig. 1). Thrombin- or TRAP-induced NFkB activation was prevented by pretreatment with N-acetyl-L-cysteine (20 mM) or pentoxifylline (0.5 mM). Figure 2 demonstrates the inhibition of thrombin (10 nM)-induced NFkB activity by N-acetyl-L-cysteine. Inhibition of thrombinor TRAP-induced NFkB activity by N-acetyl-L-cysteine and pentoxifylline, respectively, was also seen in Western blotting of the p65 NFkB component in nuclear extracts (not shown). Effects of Thrombin and TRAP on SMC [3H]Thymidine Incorporation Stimulation of SMC by thrombin (10 nM) resulted in a 2.5-4-fold (mean threefold) increase in [3H]thymidine incorporation as compared to unstimulated controls. In contrast, TRAP, even at a concentration of 100 mM did not significantly increase [3H]thymidine incorporation in SMC (Fig. 3). In order to exclude a proteolytic degradation of TRAP by cell-derived peptidases during the incubation of the SMC with TRAP for 24 h, additional experiments were carried out in the presence of the aminopeptidase inhibitor amastatin (10 mM). Again, no increase in [3H]thymidine incorporation in TRAPstimulated cells was observed (Fig. 3). Furthermore,
FIG. 1. Time-dependent stimulation of NFkB by thrombin (10 nM) and TRAP (10 mM) as detected by EMSA (upper panel) or Western blot (lower panel). 366
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Vol. 235, No. 2, 1997
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 4. Inhibition of thrombin (Thr, 10 nM)-induced [3H]thymidine incorporation by pentoxifylline (PEX, 0.5 mM) and Nacetyl-L-cysteine (NAC, 20 mM). Data are mean { SEM of n Å 3-9 experiments * P õ 0.05 vs Con. FIG. 2. EMSA demonstrating the inhibition of thrombin (10 nM)-induced activation of NFkB by N-acetyl-L-cysteine (NAC, 20 mM) in SMC.
DISCUSSION full platelet aggregating potency of TRAP was preserved after the incubation of SMC with TRAP for 24 h (not shown). Effect of NFkB Inhibition on Thrombin-Induced Mitogenesis Pretreatment of SMC with N-acetyl-L-cysteine (20 mM) completely prevented thrombin-induced mitogenic responses (Fig. 4). Similar results were obtained when the activation of NFkB was inhibited by pentoxifylline (0.5 mM). The inhibitory effects of N-acetyl-Lcysteine and pentoxifylline on [3H]thymidine uptake were not due to cytotoxicity since trypan blue exclusion analysis demonstrated no decrease in cell viability (§ 95%) after treatment of the cells with these substances for 24 h or 48 h (not shown).
FIG. 3. Stimulation of [3H]thymidine incorporation by thrombin (Thr, 10 nM) and TRAP (100 mM) in the presence and absence of amastatin (Amast, 10 mM). Data are mean { SEM of n Å 4-9 experiments, * P õ 0.05 vs Con.
Nakajima et al. (8) have shown that both thrombin and TRAP activate NFkB and stimulate proliferation of human umbilical vein SMC. They have concluded that NFkB is essential for thrombin-induced mitogenesis. The present study confirms the activation of NFkB by thrombin and TRAP in SMC from coronary arteries. The question was whether NFkB activation by thrombin or TRAP is followed by a mitogenic response. The present experiments demonstrate that NFkB is an essential signal for thrombin-induced mitogenesis in these cells since two structurally different inhibitors of NFkB activation, N-acetyl-L-cysteine and pentoxifylline (6) antagonized thrombin-induced [3H]thymidine incorporation. However, although the time course and extent of NFkB activation by TRAP was comparable to that by thrombin, TRAP did not cause any significant mitogenic response. These data demonstrate that activation of NFkB is not a sufficient signal to stimulate SMC mitogenesis. Previous studies on the mitogenic potency of TRAP have provided conflicting results. Herbe´rt et al. (15) observed an equal mitogenic response to thrombin and TRAP in rabbit aortic SMC. Kanthou et al. (16) found that the efficacy of TRAP in human abdominal aortic SMC was maximally 30% of that of thrombin. These conflicting findings may be due, in part, to species differences. McNamara et al. reported that TRAP stimulated the proliferation of rat (10) but not of rabbit (17) arterial SMC, whereas thrombin was effective in both species. The authors suggested that although the rabbit thrombin receptor is catalytically activated, the resultant tethered ligand or ligand binding site may contain a sequence that is divergent from the human or rodent thrombin receptor. In the present study, TRAP, like thrombin, stimulated NFkB, indicating activation of the bovine thrombin receptor by human TRAP.
367
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692f$$$381
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Vol. 235, No. 2, 1997
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
In summary, the present data indicate that NFkB activation is an essential signal for thrombin-induced mitogenesis. TRAP also activated NFkB but did not induce mitogenesis. Therefore, activation of NFkB is essential for SMC proliferation but does not predict a mitogenic response. ACKNOWLEDGMENTS The authors are grateful to Marlies Laube for competent technical assistance and to Erika Lohmann for secretarial help. The study was supported by the Deutsche Forschungs gemeinschaft (Grants GL 178/3-1, Schr 194/10-1).
REFERENCES 1. Sen, R., and Baltimore, D. (1986) Cell 47, 921–928. 2. Nabel, G., and Baltimore, D. (1987) Nature 326, 711–713. 3. Visvanathan, K. V., and Goodbourn, S. (1989) EMBO J. 8, 1129– 1138. 4. La Rosa, F., Pierce, J., and Sonenshein. G. E. (1993) Mol. Cell. Biol. 14, 1039–1045. 5. Ji, L., Arcinas, M., and Boxer, L. (1994) Mol. Cell. Biol. 14, 7967– 7977.
6. Bellas, R. E., Lee, J. S., and Sonenshein, G. E. (1995) J. Clin. Invest. 696, 2521–2527. 7. Autieri, M. V., Yue, T.-L., Ferstein, G. Z., and Ohlstein, E. (1995) Biochem. Biophys. Res. Comm. 213, 827–836. 8. Nakajima, T., Kitajima, I., Shin, H., Takasaki, I., Shigeta, K., Abeyama, K., Yamashita, Y., Tokioka, T., Soejima, Y., and Maruyama, I. (1994) Biochem. Biophys. Res. Comm. 204, 950–955. 9. Vu, T.-K., Hung, D. T., Wheaton, V. I., Coughlin, S. R. (1991) Cell 64, 1057–1068. 10. McNamara, C. A., Sarembock, I. J., Gimple, L. W., Fenton, J. W. II, Coughlin, S. R., and Owens, G. K., (1993) J. Clin. Invest. 91, 94–98. 11. Grand, R. J. A., Turnell, A. S., and Grabham, P. W. (1996) Biochem. J. 313, 353–368. 12. Großer, T., Zucker, T.-P., Weber, A.-A., Schulte, K., Sachinidis, A., Vetter, H., and Schro¨r, K. (1997) Eur. J. Pharmacol. 319, 327–332. 13. Dignam, J. D., Lebovitz, R. M., and Roeder, R. G. (1983) Nucl. Acid. Res. 11, 1475–1489. 14. Bradford, M. (1976) Anal. Biochem. 72, 248–254. 15. Herbe´rt, J. M., Lamarche, I., and Dol, F. (1992) FEBS Lett. 301, 155–158. 16. Kanthou, C., Benzakour, O., Patel, G., Deadman, J., Kakkar, V. V., and Lupu, F. (1995) Thromb. Haemost. 74, 1340–1347. 17. McNamara, C. A., Sarembock, I. J., Gimple, L. W., Fenton, I. I., and Owens, G. K. (1995) Drug Develop. Res. 35, 7–12.
368
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BBRC 6788
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692f$$$381
06-04-97 08:11:52
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AP: BBRC
235, 365–368 (1997)
RC976788
Activation of NFkB Is Essential but Not Sufficient to Stimulate Mitogenesis of Vascular Smooth Muscle Cells Ellen Bretschneider,* Michael Wittpoth,† Artur-Aron Weber,† Erika Glusa,* and Karsten Schro¨r†,1 *Zentrum fu¨r Vaskula¨re Biologie und Medizin Erfurt der Friedrich-Schiller-Universita¨t Jena; and †Institut fu¨r Pharmakologie, Heinrich-Heine-Universita¨t Du¨sseldorf, Germany
Received April 29, 1997
This study investigates the role of the transcription factor NFkB in thrombin- and thrombin receptor activating peptide (TRAP, SFLLRNPNDKYEPYF)-induced mitogenesis of cultured bovine coronary artery smooth muscle cells (SMC). Stimulation of resting cells by thrombin (10 nM) or TRAP (10-100 mM) resulted in a comparable time-dependent activation of NFkB as detected by Western blotting and electrophoretic mobility shift assay (EMSA) of nuclear extracts. The NFkB activation was antagonized by N-acetyl-L-cysteine (20 mM) and pentoxifylline (0.5 mM). Thrombin caused a 3-4-fold increase in [3H]thymidine incorporation within 24 h which was prevented by inhibitors of NFkB activation. In contrast, TRAP did not cause any mitogenic response. These results demonstrate that activation of NFkB is an essential but not a sufficient signal for SMC mitogenesis. q 1997 Academic Press
Members of the nuclear factor-kB family of transcription factors play an important role in transcriptional regulation of a variety of genes (1-3). Among these genes is also the proto-oncogene c-myc, a factor that is associated with cell proliferation (4-6). Recently, Bellas et al., (6) have demonstrated that inhibitors of NFkB, such as N-acetyl-L-cysteine or pentoxifylline, inhibit growth of cultured vascular smooth muscle cells (SMC). In addition, microinjection of either purified IkB-a or double-stranded oligonucleotides harboring NFkB elements selectively inhibited SMC proliferation (6). Similarly, antisense oligonucleotides to the p65 subunit of NFkB have been found to inhibit SMC proliferation and neointima formation in rat carotid arteries (7). 1 To whom correspondence should be addressed: Institut fu¨r Pharmakologie, Heinrich-Heine-Universita¨t, Moorenstrasse 5, 40225 Du¨sseldorf, Germany. Fax: /49/211/8114781. E-mail: schroer@ pharma.uni-duesseldorf.de.
From these data the conclusion was drawn that activation of NFkB is essential for proliferation of SMC. Activation of NFkB in SMC can be triggered by growth factors such as thrombin (8). Thrombin activates its receptor through the proteolytic cleavage of the N-terminal extracellular domain of the receptor. This cleavage exposes a new N-terminus, which activates the receptor as a ‘‘tethered‘‘ ligand (9,10). Synthetic peptides, corresponding to the sequence of the new N-terminus, mimic cellular effects of thrombin in several cell types, including platelets and endothelial cells (11). The present study was initiated to compare thrombin and TRAP with respect to activation of of NFkB and mitogenesis in coronary artery SMC. MATERIALS AND METHODS Materials. TRAP (SFLLRNPNDKYEPYF) was synthesized by BioGenes (Berlin, Germany), N-acetyl-L-cysteine was purchased from Sigma (Deisenhofen, Germany), PDGF-BB was from Boehringer Mannheim (Mannheim, Germany). The NFkB-consensus-sequence 5*-TCG ACC TCT CGG AAA GTC CCC TCT GA-3* and 5*AGC TTC AGA GGG GAC TTT CCG AGA GG-3* was synthesized by MWG-Biotech (Eberberg, Germany). All other chemicals were from Merck (Darmstadt, Germany) or Sigma (Deisenhofen, Germany), respectively. The following were gifts: Indomethacin (Luitpold Pharma, Mu¨nchen, Germany); purified a-thrombin (Dr. J. Stu¨rzebecher, Zentrum fu¨r Vaskula¨re Biologie und Medizin Erfurt der FSU Jena, Germany); pentoxifylline (HMR, Frankfurt, Germany). Cell culture. Coronary artery SMC were isolated from adult cattles and cultivated at 377C and 5% CO2 in a humidified atmosphere in 80% HAM’s F12-medium and 20% DMEM supplemented with 100 U/ml penicillin, 0.1 mg/ml streptomycin and 10% fetal calf serum (FCS) as previously described (12). Subconfluent cells at passages 46 were used for the experiments. [3H]Thymidine incorporation. SMC were seeded into 24-well plates (51104 cells/well) and allowed to grow to subconfluency. The cells were cultivated for 24 h in FCS-free medium in order to obtain growth arrest. All experiments were performed in the presence of indomethacin (3 mM) to avoid interactions with endogenously synthesized prostaglandins. The cells were pulse-labeled with [3H]thymidine (2 mCi/ml) 4 h prior to the end of the incubation period
365
0006-291X/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
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Vol. 235, No. 2, 1997
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
(24 h). Media were removed and the cell monolayer was washed sequentially with cold PBS, HClO4 (0.3 M) and again with cold PBS. The cells were solubilized by the addition of 0.3 ml NaOH (0.1 M) for 20 minutes at 377C. Aliquots of 0.2 ml were added to 3 ml of scintillant. [3H]Thymidine incorporation was determined by liquid scintillation spectrometry. N-acetyl-L-cysteine was added 20 h, pentoxifylline 1 h prior to the stimulation of SMC. Electrophoretic mobility shift assay (EMSA). Nuclear extracts were prepared according to Dignam et al. (13) with minor modifications. Briefly, cells were scraped, resuspended in buffer A and allowed to swell for 15 min on ice. Then, cell nuclei were sedimented by centrifugation for 10 min at 10,000 1 g. The supernatants were discarded and the nuclei resuspended in buffer C. Following a brief sonification, the samples were centrifuged for 30 min at 10,000 1 g. The supernatants, containing nuclear proteins, were removed, shockfrozen in liquid nitrogen and stored at 0807C until use. The protein content was measured using the BioRad-assay according to Bradford (14). Ten mg of nuclear protein were used for each binding reaction in binding buffer and incubated in a final volume of 20 ml for 10 min in the presence of 1 mg poly-dI-dC (Boehringer Mannheim, Germany) to saturate non-specific binding sites. This was followed by a 20 min incubation period with [32P]-labeled double-stranded oligonucleotide containing the NFkB-consensus sequence. The resulting DNA protein complexes were analyzed using a 4% native polyacrylamide gel electrophoresis. Bands were visualized by autoradiography. Western blotting. Immunoblot analysis of NFkB was performed by separation of 20 mg nuclear protein on a 8% sodium dodecyl polyacrylamide gel. Separated proteins were transferred onto nitrocellulose membranes and blocked in Blotto A (TBS, 0.05% Tween-20, 5% dry-milk) for 30 min. NFkB-antigens were detected with polyclonal antibodies against the p65 component (Santa Cruz Biotechnology, Heidelberg, Germany) according to the manufacturer’s instructions. Bands were visualized by ECL (Amersham, Buckinghamshire, England). Statistics. The data are mean { SEM of n independent experiments. Statistical analysis was performed by the two-tailed t-test. P levels of £ 0.05 were considered significant.
RESULTS Effects of Thrombin and TRAP on NFkB Activation Both thrombin (10 nM) and TRAP (10 or 100 mM) caused a significant, time-dependent NFkB activation as dectected by Western blotting and EMSA (Fig. 1). Thrombin- or TRAP-induced NFkB activation was prevented by pretreatment with N-acetyl-L-cysteine (20 mM) or pentoxifylline (0.5 mM). Figure 2 demonstrates the inhibition of thrombin (10 nM)-induced NFkB activity by N-acetyl-L-cysteine. Inhibition of thrombinor TRAP-induced NFkB activity by N-acetyl-L-cysteine and pentoxifylline, respectively, was also seen in Western blotting of the p65 NFkB component in nuclear extracts (not shown). Effects of Thrombin and TRAP on SMC [3H]Thymidine Incorporation Stimulation of SMC by thrombin (10 nM) resulted in a 2.5-4-fold (mean threefold) increase in [3H]thymidine incorporation as compared to unstimulated controls. In contrast, TRAP, even at a concentration of 100 mM did not significantly increase [3H]thymidine incorporation in SMC (Fig. 3). In order to exclude a proteolytic degradation of TRAP by cell-derived peptidases during the incubation of the SMC with TRAP for 24 h, additional experiments were carried out in the presence of the aminopeptidase inhibitor amastatin (10 mM). Again, no increase in [3H]thymidine incorporation in TRAPstimulated cells was observed (Fig. 3). Furthermore,
FIG. 1. Time-dependent stimulation of NFkB by thrombin (10 nM) and TRAP (10 mM) as detected by EMSA (upper panel) or Western blot (lower panel). 366
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06-04-97 08:11:52
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Vol. 235, No. 2, 1997
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 4. Inhibition of thrombin (Thr, 10 nM)-induced [3H]thymidine incorporation by pentoxifylline (PEX, 0.5 mM) and Nacetyl-L-cysteine (NAC, 20 mM). Data are mean { SEM of n Å 3-9 experiments * P õ 0.05 vs Con. FIG. 2. EMSA demonstrating the inhibition of thrombin (10 nM)-induced activation of NFkB by N-acetyl-L-cysteine (NAC, 20 mM) in SMC.
DISCUSSION full platelet aggregating potency of TRAP was preserved after the incubation of SMC with TRAP for 24 h (not shown). Effect of NFkB Inhibition on Thrombin-Induced Mitogenesis Pretreatment of SMC with N-acetyl-L-cysteine (20 mM) completely prevented thrombin-induced mitogenic responses (Fig. 4). Similar results were obtained when the activation of NFkB was inhibited by pentoxifylline (0.5 mM). The inhibitory effects of N-acetyl-Lcysteine and pentoxifylline on [3H]thymidine uptake were not due to cytotoxicity since trypan blue exclusion analysis demonstrated no decrease in cell viability (§ 95%) after treatment of the cells with these substances for 24 h or 48 h (not shown).
FIG. 3. Stimulation of [3H]thymidine incorporation by thrombin (Thr, 10 nM) and TRAP (100 mM) in the presence and absence of amastatin (Amast, 10 mM). Data are mean { SEM of n Å 4-9 experiments, * P õ 0.05 vs Con.
Nakajima et al. (8) have shown that both thrombin and TRAP activate NFkB and stimulate proliferation of human umbilical vein SMC. They have concluded that NFkB is essential for thrombin-induced mitogenesis. The present study confirms the activation of NFkB by thrombin and TRAP in SMC from coronary arteries. The question was whether NFkB activation by thrombin or TRAP is followed by a mitogenic response. The present experiments demonstrate that NFkB is an essential signal for thrombin-induced mitogenesis in these cells since two structurally different inhibitors of NFkB activation, N-acetyl-L-cysteine and pentoxifylline (6) antagonized thrombin-induced [3H]thymidine incorporation. However, although the time course and extent of NFkB activation by TRAP was comparable to that by thrombin, TRAP did not cause any significant mitogenic response. These data demonstrate that activation of NFkB is not a sufficient signal to stimulate SMC mitogenesis. Previous studies on the mitogenic potency of TRAP have provided conflicting results. Herbe´rt et al. (15) observed an equal mitogenic response to thrombin and TRAP in rabbit aortic SMC. Kanthou et al. (16) found that the efficacy of TRAP in human abdominal aortic SMC was maximally 30% of that of thrombin. These conflicting findings may be due, in part, to species differences. McNamara et al. reported that TRAP stimulated the proliferation of rat (10) but not of rabbit (17) arterial SMC, whereas thrombin was effective in both species. The authors suggested that although the rabbit thrombin receptor is catalytically activated, the resultant tethered ligand or ligand binding site may contain a sequence that is divergent from the human or rodent thrombin receptor. In the present study, TRAP, like thrombin, stimulated NFkB, indicating activation of the bovine thrombin receptor by human TRAP.
367
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BBRC 6788
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692f$$$381
06-04-97 08:11:52
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In summary, the present data indicate that NFkB activation is an essential signal for thrombin-induced mitogenesis. TRAP also activated NFkB but did not induce mitogenesis. Therefore, activation of NFkB is essential for SMC proliferation but does not predict a mitogenic response. ACKNOWLEDGMENTS The authors are grateful to Marlies Laube for competent technical assistance and to Erika Lohmann for secretarial help. The study was supported by the Deutsche Forschungs gemeinschaft (Grants GL 178/3-1, Schr 194/10-1).
REFERENCES 1. Sen, R., and Baltimore, D. (1986) Cell 47, 921–928. 2. Nabel, G., and Baltimore, D. (1987) Nature 326, 711–713. 3. Visvanathan, K. V., and Goodbourn, S. (1989) EMBO J. 8, 1129– 1138. 4. La Rosa, F., Pierce, J., and Sonenshein. G. E. (1993) Mol. Cell. Biol. 14, 1039–1045. 5. Ji, L., Arcinas, M., and Boxer, L. (1994) Mol. Cell. Biol. 14, 7967– 7977.
6. Bellas, R. E., Lee, J. S., and Sonenshein, G. E. (1995) J. Clin. Invest. 696, 2521–2527. 7. Autieri, M. V., Yue, T.-L., Ferstein, G. Z., and Ohlstein, E. (1995) Biochem. Biophys. Res. Comm. 213, 827–836. 8. Nakajima, T., Kitajima, I., Shin, H., Takasaki, I., Shigeta, K., Abeyama, K., Yamashita, Y., Tokioka, T., Soejima, Y., and Maruyama, I. (1994) Biochem. Biophys. Res. Comm. 204, 950–955. 9. Vu, T.-K., Hung, D. T., Wheaton, V. I., Coughlin, S. R. (1991) Cell 64, 1057–1068. 10. McNamara, C. A., Sarembock, I. J., Gimple, L. W., Fenton, J. W. II, Coughlin, S. R., and Owens, G. K., (1993) J. Clin. Invest. 91, 94–98. 11. Grand, R. J. A., Turnell, A. S., and Grabham, P. W. (1996) Biochem. J. 313, 353–368. 12. Großer, T., Zucker, T.-P., Weber, A.-A., Schulte, K., Sachinidis, A., Vetter, H., and Schro¨r, K. (1997) Eur. J. Pharmacol. 319, 327–332. 13. Dignam, J. D., Lebovitz, R. M., and Roeder, R. G. (1983) Nucl. Acid. Res. 11, 1475–1489. 14. Bradford, M. (1976) Anal. Biochem. 72, 248–254. 15. Herbe´rt, J. M., Lamarche, I., and Dol, F. (1992) FEBS Lett. 301, 155–158. 16. Kanthou, C., Benzakour, O., Patel, G., Deadman, J., Kakkar, V. V., and Lupu, F. (1995) Thromb. Haemost. 74, 1340–1347. 17. McNamara, C. A., Sarembock, I. J., Gimple, L. W., Fenton, I. I., and Owens, G. K. (1995) Drug Develop. Res. 35, 7–12.
368
AID
BBRC 6788
/
692f$$$381
06-04-97 08:11:52
bbrcg
AP: BBRC