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Endothelin-1 Induces Increased Fibronectin Expression in Human Bronchial Epithelial Cells
JOBNAME: BBRC 220#3 PAGE: 1 SESS: 16 OUTPUT: Thu May 2 09:28:46 1996 /xypage/worksmart/tsp000/69829b/97
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
220, 896–899 (1996)
0502
Endothelin-1 Induces Increased Fibronectin Expression in Human Bronchial Epithelial Cells Maurizio Marini,* Sandro Carpi,*,† Alberto Bellini,† Francesco Patalano,‡ and Sabrina Mattoli*,1 *Institute of Experimental Medicine, Mail Boxes 6, Via Alessandria 4, I-20144 Milano, Italy; †Department of Pathology and Experimental Biology, University of Milano, I-20133 Milano, Italy; and ‡Ciba-Geigy Ltd., CH-4001 Basel, Switzerland Received February 23, 1996 Endothelin-1 may be involved in the pathogenesis of asthma by causing bronchial smooth muscle constriction and airway remodelling. Bronchial epithelial cells represent an important source of endothelin-1 in this disease, and increased release of epithelial cell-derived endothelin-1 may contribute to the genesis of subepithelial fibrosis by promoting fibroblast proliferation and collagen production. In this study, we demonstrate that endothelin-1 upregulates fibronectin gene expression and fibronectin release in bronchial epithelial cells via an ETA receptor. Fibronectin is an important component of the extracellular matrix which is deposited in excess in the subepithelial area of asthmatic bronchial mucosa, and it represents a potent chemotactic factor for fibroblasts. Thus, endothelin-1 may induce subepithelial fibrosis both directly and by the autocrine mechanism reported here. © 1996 Academic Press, Inc.
Many studies have demonstrated increased expression of endothelin (ET)-1 in the lung of patients with asthma (1–6), and some of them (2,5,6) have provided evidence that ET-1 may play a role in the pathogenesis of this disease. The potential deleterious effects of ET-1 in the airways are: I. the constriction of bronchial smooth muscle, both directly (7) and through the release of other mediators (8,9); II. enhancement of bronchial smooth muscle reactivity by promoting the proliferation of smooth muscle cells (10); III. airway remodelling by a mitogenic effect on fibroblasts and induction of collagen synthesis (11). Since epithelial cells represent an important source of ET-1 in asthma (3–5), epithelial cellderived ET-1 may also be involved in the genesis of the subepithelial fibrosis which contributes to airway narrowing in asthmatic patients (12). Subepithelial fibrosis is characterized by increased number of fibroblasts (13) and by deposition of type I, III and V collagen and fibronectin (14). As mentioned above, it has already been demonstrated that ET-1 can directly mediate two of these events, namely fibroblast proliferation and collagen deposition (11), but its effect on fibronectin production is unknown. The purpose of this study was to examine whether ET-1 can increase fibronectin mRNA expression and fibronectin release in human bronchial epithelial cells and which ET-1 receptor is involved in this potential autocrine mechanism. MATERIALS AND METHODS Cell cultures. Pulmonary tissue was part of that resected at the time of surgical operation from consenting patients with lung cancer. Soon after resection, macroscopically normal bronchial tissue fragments were taken as far away as possible from malignancy. The epithelium was stripped from the basement membrane under a dissecting microscope, and epithelial cells were isolated as described elsewhere (15). Cells were plated in 35-mm six-well culture plates and grown in LHC-8 medium (Biofluids Inc., Rockville, MD) supplemented with antibiotics. Cells were passaged every 5–7 days and those from passages 7–10 were used for each experiment. To induce growth arrest, the cell monolayers were rinsed and incubated for 24 hours before testing in DME/F12 medium devoid of serum and growth factors and supplemented with antibiotics. Then,
1
To whom correspondence should be addressed. FAX: (+39) 7 4458376. 896
0006-291X/96 $18.00 Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
the cell layers were rinsed again and reincubated for 48 hours in the same medium with different concentrations of recombinant human ET-1 (Peptide International, Louisville, KY) and in presence or absence of the selective antagonist of the ETA receptor BQ 123 (Peptide International) (17). In some experiments, the cells were reincubated for 48 hours with the selective agonist of the ETB receptor sarafotoxin 6c (Sigma Chemicals Co., St. Louis, MO) (18) alone. All the experiments were carried out in presence of 100 mM phosphoramidon (Sigma Chemicals Co.) to block endogenous ET-1 production (16). Northern blot analysis. Total RNA was extracted from cell layers using the guanidine-thiocianate/phenol method (19). Northern blot analysis was performed as previously described (20), using a 32P-labelled oligonucleotide synthesized on the basis of the published base pair sequence of the coding region for human fibronectin (21). To check the amounts of loaded RNA, the blots were rehybridized to a b-actin probe (15). Autoradiography was then performed (15,20) and the autoradiograms were scanned by laser densitometry. Evaluation of fibronectin release. The amounts of fibronectin released to the culture media were measured by an enzyme-linked immunosorbent assay, as previously published (22). The sensitivity of this assay was 8 ng/mL. Results were normalized for the number of viable cells in culture and expressed as mean values ± SEM. Statistical analysis. Differences across data groups were evaluated by the analysis of variance with a post-hoc analysis; p < 0.05 was considered significant.
RESULTS AND DISCUSSION Bronchial epithelial cells constitutively expressed fibronectin mRNA (Fig. 1). Stimulation of the cells with exogenous ET-1 for 48 hours induced a concentration-dependent increase in the level of fibronectin transcripts, which was maximal with 1 nM of ET-1 (Fig. 1). Higher concentrations did not cause additional increase (data not shown). The secretion of fibronectin was also augmented by ET-1 exposure from (Mean ± SEM) 31 ±
FIG. 1. Northern blot analysis showing the constitutive and ET-1 - induced expression of fibronectin mRNA in human bronchial epithelial cells after 48 hours of incubation. Endogenous ET-1 release was blocked by phosphoramidon, 100 mM (16). A: Autoradiography of fibronectin mRNA bands from a blot representative of 3 experiments. B: Densitometric analysis of that autoradiography. C: Autoradiography of b-actin mRNA bands from the same rehybridized blot showing equal loading of the lanes. 897
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FIG. 2. Constitutive and ET-1 - induced release of fibronectin in human bronchial epithelial cell cultures after 48 hours of incubation. Endogenous ET-1 release was blocked by phosphoramidon, 100 mM (16). The antagonist of the ETA receptor BQ 123 abolished the activity of exogenous ET-1 and the agonist of the ETB receptor sarafotoxin 6c did not affect fibronectin release. Results are expressed as Mean ± SEM (n 4 8). The horizontal line indicates the detection limit of the assay. v p < 0.05, vv p < 0.025 versus constitutive release.
5 ng/mL/106 cells up to a maximum of 126 ± 10 ng/mL/106 cells, and the treatment effect was significant (Fig. 2). The upregulation of fibronectin production was mediated by an ETA receptor, because it was reversed by excess amounts of the ETA receptor antagonist BQ 123 and could not be reproduced by the ETB receptor agonist sarafotoxin 6c (Fig. 2). Fibronectin is a potent chemotactic factor for fibroblasts (23), whose release is increased in the airways of asthmatic patients (22). It may direct the migration of fibroblasts toward the subepithelial areas of the bronchial mucosa thereby contributing to the genesis and persistence of subepithelial fibrosis in asthma. The increased production of fibronectin by bronchial epithelial cells can therefore represent one additional mechanism by which ET-1 might promote airway narrowing and remodelling in this disease. ACKNOWLEDGMENTS We thank Drs. S. Ancona and B. De Lellis, University of Milano, for providing the surgical material. This study was supported by the Italian National Research Council and the Italian Foundation of Experimental Medicine.
REFERENCES 1. Nomura, A., Uchida, Y., Kameyama, M., Saotome, M., Oki, K., and Hasegawa, S., (1989) Lancet 334, 746–747. 2. Mattoli, S., Soloperto, M., Marini, M., and Pasoli, A., (1991) J. Allergy Clin. Immunol. 88, 376–384. 3. Springall, D. R., Howarth, P. H., Counihan, H., Djukanovic, R., Holgate, S. T., and Polack, J. M., (1991) Lancet 337, 697–701. 4. Vittori, E., Marini, M., Fasoli, A., De Franchis, R., and Mattoli, S., (1992) Am. Rev. Respir. Dis. 146, 1320–1325. 5. Ackerman, V., Carpi, S., Bellini, A., Vassalli, G., Marini, M., and Mattoli, S., (1995) J. Allergy Clin. Immunol. 96, 618–627. 6. Redington, A. E., Springall, D. R., Ghatei, M. A., Lan, L. K. C., Bloom, S. R., Holgate, S. T., Polack, J. M., and Howarth, P. H., (1995) Am. J. Respir. Crit. Care Med. 151, 1034–1039. 7. McKay, K. O., Black, J. L., and Armour, C. L., (1991) Br. J. Pharmacol. 102, 422–428. 8. Wu, T., Rieves, R. D., Larivée, P., Logun, C., Lawrence, M. G., and Shelhamer, J. H., (1993) Am. J. Respir. Cell. Mol. Biol. 8, 282–290. 9. Egger, D., Geuenich, S., Denzlinger, C., Schmitt, E., Mailhammer, R., Ehrenreich, H., Dormer, P., and Hultner, L., (1995) J. Immunol. 154, 1830–1837. 10. Noveral, J. P., Rosemberg, R. A., Anbar, N. A., and Grunstein, M. M., (1992) Am. J. Physiol. 263, L317–L324. 11. Peacock, A. J., Dawes, K. E., Shock, A., Gray, A. J., Reeves, J. T. T., and Laurent, G. J., (1992) Am. J. Respir. Cell. Mol. Biol. 7, 492–499. 12. Matsuba, K., Wright, J. L., Wiggs, B. R., Paré, P. D., and Hogg, J. C., (1989) Eur. Respir. J. 2, 834–839. 898
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13. Brewster, C. E., Howarth, P. H., Djukanovich, J., Wilson, J., Holgate, S. T., and Roche, W. R., (1990) Am. J. Respir. Cell. Mol. Biol. 3, 507–511. 14. Roche, W. R., Beasley, R., Williams, J. H., and Holgate, S. T., (1991) Lancet 335, 520–524. 15. Soloperto, M., Mattoso, V. L., Fasoli, A., and Mattoli, S., (1991) Am. J. Physiol. 260, L530–L538. 16. Xu, D., Emoto, N., Giaid, A., Slaughter, C., Kaw, S., deWitt, D., and Yanagisawa, M., (1994) Cell 78, 473–485. 17. Ihara, M., Noguchi, K., Saeki, T., Fukuroda, T., Tsuchida, S., Kimura, S., Fukami, T., Ishikawa, K., Nishikibe, M., and Yano, M., (1991) Life Sci. 50, 225–247. 18. Williams, A. L., Jr., Jones, L., Pettibone, D. J., Lis, E. V., and Clineschmidt, V., (1991) Biochem. Biophys. Res. Commun. 175, 556–561. 19. Chomczynski, P., and Sacchi, N., (1987) Anal. Biochem. 162, 156–159. 20. Mattoli, S., Colotta, F., Fincato, G., Mezzetti, M., Mantovani, A., Patalano, F., and Fasoli, A., (1991) J. Cell. Physiol. 149, 260–268. 21. Kornblihtt, A. R., Vibe-Pedersen, K., and Baralle, F. E., (1983) Proc. Natl. Acad. Sci. USA 80, 3218–3222. 22. Mattoli, S., Mattoso, V. L., Soloperto, M., Allegra, L., and Fasoli, A., (1991) J. Allergy Clin. Immunol. 87, 794–802. 23. Bitterman, P. B., Rennard, S. I., Adelberg, S., and Crystal, R. G., (1983) J. Cell. Biol. 97, 1925–1932.
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
220, 896–899 (1996)
0502
Endothelin-1 Induces Increased Fibronectin Expression in Human Bronchial Epithelial Cells Maurizio Marini,* Sandro Carpi,*,† Alberto Bellini,† Francesco Patalano,‡ and Sabrina Mattoli*,1 *Institute of Experimental Medicine, Mail Boxes 6, Via Alessandria 4, I-20144 Milano, Italy; †Department of Pathology and Experimental Biology, University of Milano, I-20133 Milano, Italy; and ‡Ciba-Geigy Ltd., CH-4001 Basel, Switzerland Received February 23, 1996 Endothelin-1 may be involved in the pathogenesis of asthma by causing bronchial smooth muscle constriction and airway remodelling. Bronchial epithelial cells represent an important source of endothelin-1 in this disease, and increased release of epithelial cell-derived endothelin-1 may contribute to the genesis of subepithelial fibrosis by promoting fibroblast proliferation and collagen production. In this study, we demonstrate that endothelin-1 upregulates fibronectin gene expression and fibronectin release in bronchial epithelial cells via an ETA receptor. Fibronectin is an important component of the extracellular matrix which is deposited in excess in the subepithelial area of asthmatic bronchial mucosa, and it represents a potent chemotactic factor for fibroblasts. Thus, endothelin-1 may induce subepithelial fibrosis both directly and by the autocrine mechanism reported here. © 1996 Academic Press, Inc.
Many studies have demonstrated increased expression of endothelin (ET)-1 in the lung of patients with asthma (1–6), and some of them (2,5,6) have provided evidence that ET-1 may play a role in the pathogenesis of this disease. The potential deleterious effects of ET-1 in the airways are: I. the constriction of bronchial smooth muscle, both directly (7) and through the release of other mediators (8,9); II. enhancement of bronchial smooth muscle reactivity by promoting the proliferation of smooth muscle cells (10); III. airway remodelling by a mitogenic effect on fibroblasts and induction of collagen synthesis (11). Since epithelial cells represent an important source of ET-1 in asthma (3–5), epithelial cellderived ET-1 may also be involved in the genesis of the subepithelial fibrosis which contributes to airway narrowing in asthmatic patients (12). Subepithelial fibrosis is characterized by increased number of fibroblasts (13) and by deposition of type I, III and V collagen and fibronectin (14). As mentioned above, it has already been demonstrated that ET-1 can directly mediate two of these events, namely fibroblast proliferation and collagen deposition (11), but its effect on fibronectin production is unknown. The purpose of this study was to examine whether ET-1 can increase fibronectin mRNA expression and fibronectin release in human bronchial epithelial cells and which ET-1 receptor is involved in this potential autocrine mechanism. MATERIALS AND METHODS Cell cultures. Pulmonary tissue was part of that resected at the time of surgical operation from consenting patients with lung cancer. Soon after resection, macroscopically normal bronchial tissue fragments were taken as far away as possible from malignancy. The epithelium was stripped from the basement membrane under a dissecting microscope, and epithelial cells were isolated as described elsewhere (15). Cells were plated in 35-mm six-well culture plates and grown in LHC-8 medium (Biofluids Inc., Rockville, MD) supplemented with antibiotics. Cells were passaged every 5–7 days and those from passages 7–10 were used for each experiment. To induce growth arrest, the cell monolayers were rinsed and incubated for 24 hours before testing in DME/F12 medium devoid of serum and growth factors and supplemented with antibiotics. Then,
1
To whom correspondence should be addressed. FAX: (+39) 7 4458376. 896
0006-291X/96 $18.00 Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
JOBNAME: BBRC 220#3 PAGE: 2 SESS: 15 OUTPUT: Thu May 2 09:28:46 1996 /xypage/worksmart/tsp000/69829b/97
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
the cell layers were rinsed again and reincubated for 48 hours in the same medium with different concentrations of recombinant human ET-1 (Peptide International, Louisville, KY) and in presence or absence of the selective antagonist of the ETA receptor BQ 123 (Peptide International) (17). In some experiments, the cells were reincubated for 48 hours with the selective agonist of the ETB receptor sarafotoxin 6c (Sigma Chemicals Co., St. Louis, MO) (18) alone. All the experiments were carried out in presence of 100 mM phosphoramidon (Sigma Chemicals Co.) to block endogenous ET-1 production (16). Northern blot analysis. Total RNA was extracted from cell layers using the guanidine-thiocianate/phenol method (19). Northern blot analysis was performed as previously described (20), using a 32P-labelled oligonucleotide synthesized on the basis of the published base pair sequence of the coding region for human fibronectin (21). To check the amounts of loaded RNA, the blots were rehybridized to a b-actin probe (15). Autoradiography was then performed (15,20) and the autoradiograms were scanned by laser densitometry. Evaluation of fibronectin release. The amounts of fibronectin released to the culture media were measured by an enzyme-linked immunosorbent assay, as previously published (22). The sensitivity of this assay was 8 ng/mL. Results were normalized for the number of viable cells in culture and expressed as mean values ± SEM. Statistical analysis. Differences across data groups were evaluated by the analysis of variance with a post-hoc analysis; p < 0.05 was considered significant.
RESULTS AND DISCUSSION Bronchial epithelial cells constitutively expressed fibronectin mRNA (Fig. 1). Stimulation of the cells with exogenous ET-1 for 48 hours induced a concentration-dependent increase in the level of fibronectin transcripts, which was maximal with 1 nM of ET-1 (Fig. 1). Higher concentrations did not cause additional increase (data not shown). The secretion of fibronectin was also augmented by ET-1 exposure from (Mean ± SEM) 31 ±
FIG. 1. Northern blot analysis showing the constitutive and ET-1 - induced expression of fibronectin mRNA in human bronchial epithelial cells after 48 hours of incubation. Endogenous ET-1 release was blocked by phosphoramidon, 100 mM (16). A: Autoradiography of fibronectin mRNA bands from a blot representative of 3 experiments. B: Densitometric analysis of that autoradiography. C: Autoradiography of b-actin mRNA bands from the same rehybridized blot showing equal loading of the lanes. 897
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 2. Constitutive and ET-1 - induced release of fibronectin in human bronchial epithelial cell cultures after 48 hours of incubation. Endogenous ET-1 release was blocked by phosphoramidon, 100 mM (16). The antagonist of the ETA receptor BQ 123 abolished the activity of exogenous ET-1 and the agonist of the ETB receptor sarafotoxin 6c did not affect fibronectin release. Results are expressed as Mean ± SEM (n 4 8). The horizontal line indicates the detection limit of the assay. v p < 0.05, vv p < 0.025 versus constitutive release.
5 ng/mL/106 cells up to a maximum of 126 ± 10 ng/mL/106 cells, and the treatment effect was significant (Fig. 2). The upregulation of fibronectin production was mediated by an ETA receptor, because it was reversed by excess amounts of the ETA receptor antagonist BQ 123 and could not be reproduced by the ETB receptor agonist sarafotoxin 6c (Fig. 2). Fibronectin is a potent chemotactic factor for fibroblasts (23), whose release is increased in the airways of asthmatic patients (22). It may direct the migration of fibroblasts toward the subepithelial areas of the bronchial mucosa thereby contributing to the genesis and persistence of subepithelial fibrosis in asthma. The increased production of fibronectin by bronchial epithelial cells can therefore represent one additional mechanism by which ET-1 might promote airway narrowing and remodelling in this disease. ACKNOWLEDGMENTS We thank Drs. S. Ancona and B. De Lellis, University of Milano, for providing the surgical material. This study was supported by the Italian National Research Council and the Italian Foundation of Experimental Medicine.
REFERENCES 1. Nomura, A., Uchida, Y., Kameyama, M., Saotome, M., Oki, K., and Hasegawa, S., (1989) Lancet 334, 746–747. 2. Mattoli, S., Soloperto, M., Marini, M., and Pasoli, A., (1991) J. Allergy Clin. Immunol. 88, 376–384. 3. Springall, D. R., Howarth, P. H., Counihan, H., Djukanovic, R., Holgate, S. T., and Polack, J. M., (1991) Lancet 337, 697–701. 4. Vittori, E., Marini, M., Fasoli, A., De Franchis, R., and Mattoli, S., (1992) Am. Rev. Respir. Dis. 146, 1320–1325. 5. Ackerman, V., Carpi, S., Bellini, A., Vassalli, G., Marini, M., and Mattoli, S., (1995) J. Allergy Clin. Immunol. 96, 618–627. 6. Redington, A. E., Springall, D. R., Ghatei, M. A., Lan, L. K. C., Bloom, S. R., Holgate, S. T., Polack, J. M., and Howarth, P. H., (1995) Am. J. Respir. Crit. Care Med. 151, 1034–1039. 7. McKay, K. O., Black, J. L., and Armour, C. L., (1991) Br. J. Pharmacol. 102, 422–428. 8. Wu, T., Rieves, R. D., Larivée, P., Logun, C., Lawrence, M. G., and Shelhamer, J. H., (1993) Am. J. Respir. Cell. Mol. Biol. 8, 282–290. 9. Egger, D., Geuenich, S., Denzlinger, C., Schmitt, E., Mailhammer, R., Ehrenreich, H., Dormer, P., and Hultner, L., (1995) J. Immunol. 154, 1830–1837. 10. Noveral, J. P., Rosemberg, R. A., Anbar, N. A., and Grunstein, M. M., (1992) Am. J. Physiol. 263, L317–L324. 11. Peacock, A. J., Dawes, K. E., Shock, A., Gray, A. J., Reeves, J. T. T., and Laurent, G. J., (1992) Am. J. Respir. Cell. Mol. Biol. 7, 492–499. 12. Matsuba, K., Wright, J. L., Wiggs, B. R., Paré, P. D., and Hogg, J. C., (1989) Eur. Respir. J. 2, 834–839. 898
JOBNAME: BBRC 220#3 PAGE: 4 SESS: 15 OUTPUT: Thu May 2 09:28:46 1996 /xypage/worksmart/tsp000/69829b/97
Vol. 220, No. 3, 1996
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13. Brewster, C. E., Howarth, P. H., Djukanovich, J., Wilson, J., Holgate, S. T., and Roche, W. R., (1990) Am. J. Respir. Cell. Mol. Biol. 3, 507–511. 14. Roche, W. R., Beasley, R., Williams, J. H., and Holgate, S. T., (1991) Lancet 335, 520–524. 15. Soloperto, M., Mattoso, V. L., Fasoli, A., and Mattoli, S., (1991) Am. J. Physiol. 260, L530–L538. 16. Xu, D., Emoto, N., Giaid, A., Slaughter, C., Kaw, S., deWitt, D., and Yanagisawa, M., (1994) Cell 78, 473–485. 17. Ihara, M., Noguchi, K., Saeki, T., Fukuroda, T., Tsuchida, S., Kimura, S., Fukami, T., Ishikawa, K., Nishikibe, M., and Yano, M., (1991) Life Sci. 50, 225–247. 18. Williams, A. L., Jr., Jones, L., Pettibone, D. J., Lis, E. V., and Clineschmidt, V., (1991) Biochem. Biophys. Res. Commun. 175, 556–561. 19. Chomczynski, P., and Sacchi, N., (1987) Anal. Biochem. 162, 156–159. 20. Mattoli, S., Colotta, F., Fincato, G., Mezzetti, M., Mantovani, A., Patalano, F., and Fasoli, A., (1991) J. Cell. Physiol. 149, 260–268. 21. Kornblihtt, A. R., Vibe-Pedersen, K., and Baralle, F. E., (1983) Proc. Natl. Acad. Sci. USA 80, 3218–3222. 22. Mattoli, S., Mattoso, V. L., Soloperto, M., Allegra, L., and Fasoli, A., (1991) J. Allergy Clin. Immunol. 87, 794–802. 23. Bitterman, P. B., Rennard, S. I., Adelberg, S., and Crystal, R. G., (1983) J. Cell. Biol. 97, 1925–1932.
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