TGF-β1 Increases Resistance of NIH/3T3 Fibroblasts Toward Apoptosis Through Activation of Smad2/3 and Erk1/2 Pathways
Purpose: Excessive fibrosis has been suggested to result from persistence of fibroblasts in injured tissue due to impaired apoptosis, but signaling pathways are not fully defined. Methods: Suppression of apoptotic cell death following transforming growth factor-β1 (TGF-β1) exposure was studied using...
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Advocate Aurora Health
2016-11-01
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Series: | Journal of Patient-Centered Research and Reviews |
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Online Access: | http://digitalrepository.aurorahealthcare.org/cgi/viewcontent.cgi?article=1407&context=jpcrr |
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author | Ulugbek Negmadjanov Alisher Holmuhamedov Larisa Emelyanova Hao Xu Farhan Rizvi Gracious R. Ross A. Jamil Tajik Yang Shi Ekhson Holmuhamedov Arshad Jahangir |
author_facet | Ulugbek Negmadjanov Alisher Holmuhamedov Larisa Emelyanova Hao Xu Farhan Rizvi Gracious R. Ross A. Jamil Tajik Yang Shi Ekhson Holmuhamedov Arshad Jahangir |
author_sort | Ulugbek Negmadjanov |
collection | DOAJ |
description | Purpose: Excessive fibrosis has been suggested to result from persistence of fibroblasts in injured tissue due to impaired apoptosis, but signaling pathways are not fully defined. Methods: Suppression of apoptotic cell death following transforming growth factor-β1 (TGF-β1) exposure was studied using the culture of NIH/3T3 mouse embryonic fibroblasts. Caspase-3 activity, propidium iodide staining and annexin V binding induced by Fas-ligand (FasL) in NIH/3T3 fibroblasts in the absence and presence of TGF-β1 was determined, and relative contribution of signaling through Smad2/3 and noncanonical Erk1/2 and Akt pathways was dissected by assessing phosphorylation status of these kinases and caspase activity in the absence and presence of specific inhibitors (SB431542, PD0325901 and LY294002), respectively. Results: TGF-β1 treatment suppressed FasL-mediated fibroblasts apoptosis with a greater than threefold reduction of caspase-3 activity (from 894 ± 186 to 195 ± 56 nmol AFC/min/106 cells at 250 ng/mL of FasL) and reductions in cleaved caspase-8 and caspase-3 by 3.2-fold and 4.3-fold, respectively. The reduction in caspase activation was accompanied by a decrease in annexin V-positive cells by ~80%. TGF-β1 treatment phosphorylated Smad2/3, Erk1/2 and Akt, which were reduced by their selective inhibitors. Inhibition of Smad2/3 and Erk1/2 alone partially reduced the protective effect of TGF-β1 on caspase-3 activation, whereas inhibition of the Akt pathway had no significant effect. Concomitant inhibition of Smad2/3 and Erk1/2 completely reversed the protection by TGF-β1. Conclusions: TGF-β1-mediated suppression of apoptosis in fibroblasts involves both Smad2/3 and Erk1/2 pathways, but not the Akt pathway. A combined approach inhibiting Smad2/3 and Erk1/2 pathways can completely reverse the protective effect of TGF-β1 on apoptosis. These findings are proof of concept to help define strategies to reduce progression of fibrosis and resultant morbidities associated with conditions causing excessive fibrosis, including but not limited to keloid formation, transplant fibrosis and aging-associated fibrosis of the heart. |
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id | doaj.art-5f1e660c634a49f1a98becd7c11ea4d4 |
institution | Directory Open Access Journal |
issn | 2330-0698 |
language | English |
last_indexed | 2024-04-10T18:15:52Z |
publishDate | 2016-11-01 |
publisher | Advocate Aurora Health |
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series | Journal of Patient-Centered Research and Reviews |
spelling | doaj.art-5f1e660c634a49f1a98becd7c11ea4d42023-02-02T09:02:32ZengAdvocate Aurora HealthJournal of Patient-Centered Research and Reviews2330-06982016-11-013418719810.17294/2330-0698.1407TGF-β1 Increases Resistance of NIH/3T3 Fibroblasts Toward Apoptosis Through Activation of Smad2/3 and Erk1/2 PathwaysUlugbek Negmadjanov0Alisher Holmuhamedov1Larisa Emelyanova2Hao Xu3Farhan Rizvi4Gracious R. Ross5A. Jamil Tajik6Yang Shi7Ekhson Holmuhamedov8Arshad Jahangir9Aurora Research Institute, Milwaukee, WIAurora Research Institute, Milwaukee, WIAurora Research Institute, Milwaukee, WIAurora Research Institute, Milwaukee, WIAurora Research Institute, Milwaukee, WIAurora Research Institute, Milwaukee, WIAurora Health Care, Milwaukee, WIAurora Research Institute, Milwaukee, WIAurora Research Institute, Milwaukee, WIAurora Research Institute, Aurora Health Care, Milwaukee, WIPurpose: Excessive fibrosis has been suggested to result from persistence of fibroblasts in injured tissue due to impaired apoptosis, but signaling pathways are not fully defined. Methods: Suppression of apoptotic cell death following transforming growth factor-β1 (TGF-β1) exposure was studied using the culture of NIH/3T3 mouse embryonic fibroblasts. Caspase-3 activity, propidium iodide staining and annexin V binding induced by Fas-ligand (FasL) in NIH/3T3 fibroblasts in the absence and presence of TGF-β1 was determined, and relative contribution of signaling through Smad2/3 and noncanonical Erk1/2 and Akt pathways was dissected by assessing phosphorylation status of these kinases and caspase activity in the absence and presence of specific inhibitors (SB431542, PD0325901 and LY294002), respectively. Results: TGF-β1 treatment suppressed FasL-mediated fibroblasts apoptosis with a greater than threefold reduction of caspase-3 activity (from 894 ± 186 to 195 ± 56 nmol AFC/min/106 cells at 250 ng/mL of FasL) and reductions in cleaved caspase-8 and caspase-3 by 3.2-fold and 4.3-fold, respectively. The reduction in caspase activation was accompanied by a decrease in annexin V-positive cells by ~80%. TGF-β1 treatment phosphorylated Smad2/3, Erk1/2 and Akt, which were reduced by their selective inhibitors. Inhibition of Smad2/3 and Erk1/2 alone partially reduced the protective effect of TGF-β1 on caspase-3 activation, whereas inhibition of the Akt pathway had no significant effect. Concomitant inhibition of Smad2/3 and Erk1/2 completely reversed the protection by TGF-β1. Conclusions: TGF-β1-mediated suppression of apoptosis in fibroblasts involves both Smad2/3 and Erk1/2 pathways, but not the Akt pathway. A combined approach inhibiting Smad2/3 and Erk1/2 pathways can completely reverse the protective effect of TGF-β1 on apoptosis. These findings are proof of concept to help define strategies to reduce progression of fibrosis and resultant morbidities associated with conditions causing excessive fibrosis, including but not limited to keloid formation, transplant fibrosis and aging-associated fibrosis of the heart.http://digitalrepository.aurorahealthcare.org/cgi/viewcontent.cgi?article=1407&context=jpcrrfibroblastsTGF-β1stress kinasesSmad2/3apoptosiscell deathNIH/3T3caspaseErk1/2Akt |
spellingShingle | Ulugbek Negmadjanov Alisher Holmuhamedov Larisa Emelyanova Hao Xu Farhan Rizvi Gracious R. Ross A. Jamil Tajik Yang Shi Ekhson Holmuhamedov Arshad Jahangir TGF-β1 Increases Resistance of NIH/3T3 Fibroblasts Toward Apoptosis Through Activation of Smad2/3 and Erk1/2 Pathways Journal of Patient-Centered Research and Reviews fibroblasts TGF-β1 stress kinases Smad2/3 apoptosis cell death NIH/3T3 caspase Erk1/2 Akt |
title | TGF-β1 Increases Resistance of NIH/3T3 Fibroblasts Toward Apoptosis Through Activation of Smad2/3 and Erk1/2 Pathways |
title_full | TGF-β1 Increases Resistance of NIH/3T3 Fibroblasts Toward Apoptosis Through Activation of Smad2/3 and Erk1/2 Pathways |
title_fullStr | TGF-β1 Increases Resistance of NIH/3T3 Fibroblasts Toward Apoptosis Through Activation of Smad2/3 and Erk1/2 Pathways |
title_full_unstemmed | TGF-β1 Increases Resistance of NIH/3T3 Fibroblasts Toward Apoptosis Through Activation of Smad2/3 and Erk1/2 Pathways |
title_short | TGF-β1 Increases Resistance of NIH/3T3 Fibroblasts Toward Apoptosis Through Activation of Smad2/3 and Erk1/2 Pathways |
title_sort | tgf β1 increases resistance of nih 3t3 fibroblasts toward apoptosis through activation of smad2 3 and erk1 2 pathways |
topic | fibroblasts TGF-β1 stress kinases Smad2/3 apoptosis cell death NIH/3T3 caspase Erk1/2 Akt |
url | http://digitalrepository.aurorahealthcare.org/cgi/viewcontent.cgi?article=1407&context=jpcrr |
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