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Reactive Oxygen Species and p38MAPK Have a Role in the Smad2 Linker Region Phosphorylation Induced by TGF-β

BACKGROUND: Transforming growth factor-β (TGF-β) in addition to the C-terminal region can phosphorylate receptor-regulated Smads (R-Smads) in their linker region. The aim of the present study was to evaluate the role of signaling mediators such as NAD(P)H oxidases (reactive oxygen species [ROS] gene...

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Autores principales: Mehr, Reyhaneh Niayesh, Kheirollah, Alireza, Seif, Faezeh, Dayati, Parisa, Babaahmadi-Rezaei, Hossein
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Iranian Journal of Medical Sciences 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6055211/
https://www.ncbi.nlm.nih.gov/pubmed/30046209
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author Mehr, Reyhaneh Niayesh
Kheirollah, Alireza
Seif, Faezeh
Dayati, Parisa
Babaahmadi-Rezaei, Hossein
author_facet Mehr, Reyhaneh Niayesh
Kheirollah, Alireza
Seif, Faezeh
Dayati, Parisa
Babaahmadi-Rezaei, Hossein
author_sort Mehr, Reyhaneh Niayesh
collection PubMed
description BACKGROUND: Transforming growth factor-β (TGF-β) in addition to the C-terminal region can phosphorylate receptor-regulated Smads (R-Smads) in their linker region. The aim of the present study was to evaluate the role of signaling mediators such as NAD(P)H oxidases (reactive oxygen species [ROS] generators), ROS, and ROS-sensitive p38 mitogen-activated protein kinase (p38MAPK) in this signaling pathway in cultured human vascular smooth muscle cells (VSMCs). METHODS: The present in vitro study was performed on human VSMCs. Proteins were detected by western blotting utilizing an anti-phospho-Smad2 (Ser245/250/255) rabbit polyclonal antibody and a horseradish peroxidase-labeled secondary antibody. Glyceraldehyde-3-phosphate dehydrogenase was used as a loading control. The phospho-Smad2 linker region (pSmad2L) was detected in all the experimental groups: a control group (untreated group), a group treated with TGF-β (2 ng/mL), and a group treated with TGF-β plus different inhibitors. The data were normalized and presented as mean±SEM. The statistical analyses were performed using SPSS, version 16.0, and the nonparametric Kruskal-Wallis test. A P value smaller than 0.05 was considered statistically significant. RESULTS: The VSMCs treated with TGF-β (2 ng/mL) showed a time-dependent increase in the pSmad2L level. The highest level was observed at 15 minutes (P=0.03). The inhibitors of NAD(P)H oxidases (diphenyleneiodonium and apocynin) (P=0.04), ROS scavenger (N-acetylcysteine) (P=0.04), and p38MAPK inhibitor (SB-202190) (P=0.04) were able to reduce the increased level of the pSmad2L by TGF-β. CONCLUSION: Our results suggested that NAD(P)H oxidases played an important role in the Smad2L phosphorylation in the human VSMCs. Furthermore, our results confirmed that ROS and p38MAPK were involved in this signaling pathway. Thus, TGF-β via a ROS-dependent mechanism can transmit its signals to the pSmad2L.
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spelling pubmed-60552112018-07-25 Reactive Oxygen Species and p38MAPK Have a Role in the Smad2 Linker Region Phosphorylation Induced by TGF-β Mehr, Reyhaneh Niayesh Kheirollah, Alireza Seif, Faezeh Dayati, Parisa Babaahmadi-Rezaei, Hossein Iran J Med Sci Original Article BACKGROUND: Transforming growth factor-β (TGF-β) in addition to the C-terminal region can phosphorylate receptor-regulated Smads (R-Smads) in their linker region. The aim of the present study was to evaluate the role of signaling mediators such as NAD(P)H oxidases (reactive oxygen species [ROS] generators), ROS, and ROS-sensitive p38 mitogen-activated protein kinase (p38MAPK) in this signaling pathway in cultured human vascular smooth muscle cells (VSMCs). METHODS: The present in vitro study was performed on human VSMCs. Proteins were detected by western blotting utilizing an anti-phospho-Smad2 (Ser245/250/255) rabbit polyclonal antibody and a horseradish peroxidase-labeled secondary antibody. Glyceraldehyde-3-phosphate dehydrogenase was used as a loading control. The phospho-Smad2 linker region (pSmad2L) was detected in all the experimental groups: a control group (untreated group), a group treated with TGF-β (2 ng/mL), and a group treated with TGF-β plus different inhibitors. The data were normalized and presented as mean±SEM. The statistical analyses were performed using SPSS, version 16.0, and the nonparametric Kruskal-Wallis test. A P value smaller than 0.05 was considered statistically significant. RESULTS: The VSMCs treated with TGF-β (2 ng/mL) showed a time-dependent increase in the pSmad2L level. The highest level was observed at 15 minutes (P=0.03). The inhibitors of NAD(P)H oxidases (diphenyleneiodonium and apocynin) (P=0.04), ROS scavenger (N-acetylcysteine) (P=0.04), and p38MAPK inhibitor (SB-202190) (P=0.04) were able to reduce the increased level of the pSmad2L by TGF-β. CONCLUSION: Our results suggested that NAD(P)H oxidases played an important role in the Smad2L phosphorylation in the human VSMCs. Furthermore, our results confirmed that ROS and p38MAPK were involved in this signaling pathway. Thus, TGF-β via a ROS-dependent mechanism can transmit its signals to the pSmad2L. Iranian Journal of Medical Sciences 2018-07 /pmc/articles/PMC6055211/ /pubmed/30046209 Text en Copyright: © Iranian Journal of Medical Sciences http://creativecommons.org/licenses/by-nc-sa/3.0 This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-Share Alike 3.0 Unported, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Original Article
Mehr, Reyhaneh Niayesh
Kheirollah, Alireza
Seif, Faezeh
Dayati, Parisa
Babaahmadi-Rezaei, Hossein
Reactive Oxygen Species and p38MAPK Have a Role in the Smad2 Linker Region Phosphorylation Induced by TGF-β
title Reactive Oxygen Species and p38MAPK Have a Role in the Smad2 Linker Region Phosphorylation Induced by TGF-β
title_full Reactive Oxygen Species and p38MAPK Have a Role in the Smad2 Linker Region Phosphorylation Induced by TGF-β
title_fullStr Reactive Oxygen Species and p38MAPK Have a Role in the Smad2 Linker Region Phosphorylation Induced by TGF-β
title_full_unstemmed Reactive Oxygen Species and p38MAPK Have a Role in the Smad2 Linker Region Phosphorylation Induced by TGF-β
title_short Reactive Oxygen Species and p38MAPK Have a Role in the Smad2 Linker Region Phosphorylation Induced by TGF-β
title_sort reactive oxygen species and p38mapk have a role in the smad2 linker region phosphorylation induced by tgf-β
topic Original Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6055211/
https://www.ncbi.nlm.nih.gov/pubmed/30046209
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