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The Role of Protein Arginine N-Methyltransferase 1 (PRMT1) in Inflammatory Gene Expression of Airway Smooth Muscle Cells in Asthma
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A7264 - The Role of Protein Arginine N-Methyltransferase 1 (PRMT1) in Inflammatory Gene Expression of Airway Smooth Muscle Cells in Asthma
Author Block: K. A. Kaczmarek1, R. L. Clifford1, J. Patel1, D. Shaw1, J. Dowden2, A. J. Knox1; 1Division of Respiratory Medicine, School of Medicine, University of Nottingham, Nottingham, United Kingdom, 2School of Chemistry, University of Nottingham, Nottingham, United Kingdom.
Introduction and objectives
Asthma affects at least 300 million people globally. Approximately 25% of asthma patients do not respond to currently available therapies; therefore we need to develop novel treatments. Airway smooth muscle (ASM) cells have a crucial role in asthma, contributing to airway remodelling, inflammation and airflow obstruction. Abnormalities in histone arginine methylation (HRme, the addition of a methyl group to arginine residues on the N-terminal tails of histones) have a role in inflammation in asthma, by increasing the secretion of inflammatory mediators from ASM cells. Protein arginine N-methyltransferases (PRMTs) are the enzymes which catalyse HRme and inhibiting them represents a strategy to reduce inflammation in asthmatic airways. PRMT1 is the most predominant PRMT in eukaryotic cells and carries out 85% of total protein arginine methylation. The aim of this study was to investigate the role of PRMT1-catalysed HRme in inflammatory gene expression.
Methods
Studies were performed in cultured human ASM cells from non-asthmatic donors at passage 6, under basal conditions and following stimulation with TNF-α (1ng/ml). The association of PRMT1 with the eotaxin, IL-6, IP-10 and CXCL8 promoters, as well as H4R3me2a (HRme mark catalysed by PRMT1) levels at these promoters, were investigated by chromatin immunoprecipitation (ChIP), followed by qPCR. The effect of inhibiting PRMT1 on the secretion of eotaxin, IL-6, IP-10 and CXCL8 from ASM cells, was investigated by ELISA. The effect of inhibiting PRMT1 on the levels of H4R3me2a at the eotaxin, IL-6, IP-10 and CXCL8 promoters was investigated by ChIP, followed by qPCR.
Results
The association of PRMT1 with eotaxin, IL-6, IP-10 and CXCL8 promoters, as well as H4R3me2a at these promoters, were observed under basal conditions. Stimulation with TNF- α moderately increased the association of PRMT1, while having a major impact on the increase in the levels of H4R3me2a, at these promoters. Inhibition of PRMT1 with a pharmacological inhibitor TCE5003 significantly reduced TNF-α induced secretion of eotaxin, IL-6, IP-10 and CXCL8 from ASM cells, and decreased the TNF-α induced levels of H4R3me2a at the CXCL8 promoter.
Conclusions
PRMT1 may have a role in regulating the inflammation in asthma, by catalysing a HRme mark involved in chemokine production from ASM cells. Therefore inhibiting PRMT1 could potentially alleviate the inflammation in asthmatic airways. This makes PRMT1 a promising target for future investigations in a search for novel treatments against asthma.
Author Block: K. A. Kaczmarek1, R. L. Clifford1, J. Patel1, D. Shaw1, J. Dowden2, A. J. Knox1; 1Division of Respiratory Medicine, School of Medicine, University of Nottingham, Nottingham, United Kingdom, 2School of Chemistry, University of Nottingham, Nottingham, United Kingdom.
Introduction and objectives
Asthma affects at least 300 million people globally. Approximately 25% of asthma patients do not respond to currently available therapies; therefore we need to develop novel treatments. Airway smooth muscle (ASM) cells have a crucial role in asthma, contributing to airway remodelling, inflammation and airflow obstruction. Abnormalities in histone arginine methylation (HRme, the addition of a methyl group to arginine residues on the N-terminal tails of histones) have a role in inflammation in asthma, by increasing the secretion of inflammatory mediators from ASM cells. Protein arginine N-methyltransferases (PRMTs) are the enzymes which catalyse HRme and inhibiting them represents a strategy to reduce inflammation in asthmatic airways. PRMT1 is the most predominant PRMT in eukaryotic cells and carries out 85% of total protein arginine methylation. The aim of this study was to investigate the role of PRMT1-catalysed HRme in inflammatory gene expression.
Methods
Studies were performed in cultured human ASM cells from non-asthmatic donors at passage 6, under basal conditions and following stimulation with TNF-α (1ng/ml). The association of PRMT1 with the eotaxin, IL-6, IP-10 and CXCL8 promoters, as well as H4R3me2a (HRme mark catalysed by PRMT1) levels at these promoters, were investigated by chromatin immunoprecipitation (ChIP), followed by qPCR. The effect of inhibiting PRMT1 on the secretion of eotaxin, IL-6, IP-10 and CXCL8 from ASM cells, was investigated by ELISA. The effect of inhibiting PRMT1 on the levels of H4R3me2a at the eotaxin, IL-6, IP-10 and CXCL8 promoters was investigated by ChIP, followed by qPCR.
Results
The association of PRMT1 with eotaxin, IL-6, IP-10 and CXCL8 promoters, as well as H4R3me2a at these promoters, were observed under basal conditions. Stimulation with TNF- α moderately increased the association of PRMT1, while having a major impact on the increase in the levels of H4R3me2a, at these promoters. Inhibition of PRMT1 with a pharmacological inhibitor TCE5003 significantly reduced TNF-α induced secretion of eotaxin, IL-6, IP-10 and CXCL8 from ASM cells, and decreased the TNF-α induced levels of H4R3me2a at the CXCL8 promoter.
Conclusions
PRMT1 may have a role in regulating the inflammation in asthma, by catalysing a HRme mark involved in chemokine production from ASM cells. Therefore inhibiting PRMT1 could potentially alleviate the inflammation in asthmatic airways. This makes PRMT1 a promising target for future investigations in a search for novel treatments against asthma.
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