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The Essential Role of Peptidylarginine Deiminases 2 for Cytokines Secretion, Apoptosis, and Cell Adhesion in Macrophage

Objective: The study aims to investigate the functional roles of peptidylarginine deiminase 2 (PADI2) in macrophages. Methods: The clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein-9 nuclease (Cas9) system was used to knockout PADI2 in U937 cells. U937 cell...

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Autores principales: Yu, Hui-Chun, Tung, Chien-Hsueh, Huang, Kuang-Yung, Huang, Hsien-Bin, Lu, Ming-Chi
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7460808/
https://www.ncbi.nlm.nih.gov/pubmed/32785008
http://dx.doi.org/10.3390/ijms21165720
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author Yu, Hui-Chun
Tung, Chien-Hsueh
Huang, Kuang-Yung
Huang, Hsien-Bin
Lu, Ming-Chi
author_facet Yu, Hui-Chun
Tung, Chien-Hsueh
Huang, Kuang-Yung
Huang, Hsien-Bin
Lu, Ming-Chi
author_sort Yu, Hui-Chun
collection PubMed
description Objective: The study aims to investigate the functional roles of peptidylarginine deiminase 2 (PADI2) in macrophages. Methods: The clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein-9 nuclease (Cas9) system was used to knockout PADI2 in U937 cells. U937 cells were introduced to differentiate macrophages and were stimulated with lipopolysaccharides (LPS). The protein expression of PADI2, PADI4, and citrullinated proteins were analyzed by Western blotting. The mRNA and protein levels of interleukin 1 beta (IL-1β), IL-6, and tumor necrosis factor-alpha (TNF-α) were analyzed using RT-PCR and ELISA, respectively. Cell apoptosis was analyzed using flow cytometry. Cell adhesion assay was performed using a commercially available fibrinogen-coated plate. Results: PADI2 knockout could markedly suppress the PADI2 protein expression, but not the PADI4 protein expression. PADI2 knockout decreased the protein levels of citrullinated nuclear factor κB (NF-κB) p65, but not those of citrullinated histone 3, resulting in the decreased mRNA expression levels of IL-1β and TNF-α in the U937 cells and IL-1β and IL-6 in the differentiated macrophages and the macrophages stimulated with LPS. The cytokines levels of IL-1β, IL-6, and TNF-α were all dramatically decreased in the PADI2 knockout group compared with in the controls. PADI2 knockout prevented macrophages apoptosis via the decreased caspase-3, caspase-2, and caspase-9 activation. PADI2 knockout also impaired macrophages adhesion capacity through the decreased protein levels of focal adhesion kinase (FAK), phospho-FAK, paxillin, phospho-paxillin, and p21-activated kinase 1. Conclusion: This study showed that PADI2 could promote IL-1β, IL-6, and TNF-α production in macrophages, promote macrophage apoptosis through caspase-3, caspase-2, and caspase-9 activation and enhance cell adhesion via FAK, paxillin, and PAK1. Therefore, targeting PADI2 could be used as a novel strategy for controlling inflammation caused by macrophages.
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spelling pubmed-74608082020-09-03 The Essential Role of Peptidylarginine Deiminases 2 for Cytokines Secretion, Apoptosis, and Cell Adhesion in Macrophage Yu, Hui-Chun Tung, Chien-Hsueh Huang, Kuang-Yung Huang, Hsien-Bin Lu, Ming-Chi Int J Mol Sci Article Objective: The study aims to investigate the functional roles of peptidylarginine deiminase 2 (PADI2) in macrophages. Methods: The clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein-9 nuclease (Cas9) system was used to knockout PADI2 in U937 cells. U937 cells were introduced to differentiate macrophages and were stimulated with lipopolysaccharides (LPS). The protein expression of PADI2, PADI4, and citrullinated proteins were analyzed by Western blotting. The mRNA and protein levels of interleukin 1 beta (IL-1β), IL-6, and tumor necrosis factor-alpha (TNF-α) were analyzed using RT-PCR and ELISA, respectively. Cell apoptosis was analyzed using flow cytometry. Cell adhesion assay was performed using a commercially available fibrinogen-coated plate. Results: PADI2 knockout could markedly suppress the PADI2 protein expression, but not the PADI4 protein expression. PADI2 knockout decreased the protein levels of citrullinated nuclear factor κB (NF-κB) p65, but not those of citrullinated histone 3, resulting in the decreased mRNA expression levels of IL-1β and TNF-α in the U937 cells and IL-1β and IL-6 in the differentiated macrophages and the macrophages stimulated with LPS. The cytokines levels of IL-1β, IL-6, and TNF-α were all dramatically decreased in the PADI2 knockout group compared with in the controls. PADI2 knockout prevented macrophages apoptosis via the decreased caspase-3, caspase-2, and caspase-9 activation. PADI2 knockout also impaired macrophages adhesion capacity through the decreased protein levels of focal adhesion kinase (FAK), phospho-FAK, paxillin, phospho-paxillin, and p21-activated kinase 1. Conclusion: This study showed that PADI2 could promote IL-1β, IL-6, and TNF-α production in macrophages, promote macrophage apoptosis through caspase-3, caspase-2, and caspase-9 activation and enhance cell adhesion via FAK, paxillin, and PAK1. Therefore, targeting PADI2 could be used as a novel strategy for controlling inflammation caused by macrophages. MDPI 2020-08-10 /pmc/articles/PMC7460808/ /pubmed/32785008 http://dx.doi.org/10.3390/ijms21165720 Text en © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Yu, Hui-Chun
Tung, Chien-Hsueh
Huang, Kuang-Yung
Huang, Hsien-Bin
Lu, Ming-Chi
The Essential Role of Peptidylarginine Deiminases 2 for Cytokines Secretion, Apoptosis, and Cell Adhesion in Macrophage
title The Essential Role of Peptidylarginine Deiminases 2 for Cytokines Secretion, Apoptosis, and Cell Adhesion in Macrophage
title_full The Essential Role of Peptidylarginine Deiminases 2 for Cytokines Secretion, Apoptosis, and Cell Adhesion in Macrophage
title_fullStr The Essential Role of Peptidylarginine Deiminases 2 for Cytokines Secretion, Apoptosis, and Cell Adhesion in Macrophage
title_full_unstemmed The Essential Role of Peptidylarginine Deiminases 2 for Cytokines Secretion, Apoptosis, and Cell Adhesion in Macrophage
title_short The Essential Role of Peptidylarginine Deiminases 2 for Cytokines Secretion, Apoptosis, and Cell Adhesion in Macrophage
title_sort essential role of peptidylarginine deiminases 2 for cytokines secretion, apoptosis, and cell adhesion in macrophage
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7460808/
https://www.ncbi.nlm.nih.gov/pubmed/32785008
http://dx.doi.org/10.3390/ijms21165720
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