CXCL1-Triggered PAD4 Cytoplasmic Translocation Enhances Neutrophil Adhesion through Citrullination of PDIA1
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- Aoyama Jiro
- Department of Life Science and Bioethics, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University Department of Neurosurgery, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University
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- Osaka Mizuko
- Department of Life Science and Bioethics, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University Department of Nutrition and Metabolism in Cardiovascular Disease, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University
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- Deushi Michiyo
- Department of Life Science and Bioethics, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University
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- Hosoya Shoichi
- Research Core, Research Facility Cluster, Institute of Research, Tokyo Medical and Dental University
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- Ishigami Akihito
- Research Team for Functional Biogerontology, Tokyo Metropolitan Institute of Gerontology
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- Maehara Taketoshi
- Department of Neurosurgery, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University
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- Yoshida Masayuki
- Department of Life Science and Bioethics, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University
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Abstract
<p> Aims: Vascular inflammation is critical for the development and progression of atherosclerosis. Previously, we reported that neutrophils adhere to the vascular endothelium in low-density lipoprotein receptor null mice fed a high-fat diet through hypercitrullination of histone H3 by peptidylarginine deiminase 4 (PAD4) in neutrophils. However, the involvement of PAD4 and citrullination of proteins other than histone H3 in neutrophil adhesion is not well known. In this study, we investigated the function of PAD4 and identified citrullinated proteins during vascular inflammation.</p><p>Methods: We pefformed flow assay under physiological flow conditions using differentiated HL-60 (dHL-60) cells stimulated with CXCL1 and human umbilical vein endothelial cells (HUVECs). Furthermore, phalloidin stain for dHL-60 stimulated with CXCL1 to observe F-actin polymerization and immunohistochemistry for the activated β2-integrin was conducted. To identify a target of citrullination in the cytoplasm of dHL-60 cells, liquid chromatography-mass spectrometry (LC-MS/MS) for dHL-60 stimulated with CXCL1 was performed.</p><p> Results: Inhibition or knockdown of PAD4 significantly decreased adhesion of under physiological flow conditions. Thr-Asp-F-amidine trifluoroacetate salt (TDFA), a PAD4 inhibitor, inhibited cytoplasmic translocation of PAD4 by CXCL1. TDFA or knockdown of PAD4 significantly decreased expression of β2-integrin and F-actin polymerization activated by CXCL1. Moreover, LC-MS/MS identified protein disulfide isomerase A1 (PDIA1) as a target of citrullination in the cytoplasm of dHL-60 cells. Knockdown of PDIA1 significantly decreased adhesion of dHL-60 cells to HUVECs, expression of β2-integrin, and F-actin polymerization.</p><p>Conclusions: Cytoplasmic translocation of PAD4 by CXCL1 induces neutrophil adhesion to vascular endothelial cells and citrullination of PDIA1.</p>
Journal
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- Journal of Atherosclerosis and Thrombosis
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Journal of Atherosclerosis and Thrombosis 29 (9), 1307-1318, 2022-09-01
Japan Atherosclerosis Society