The role of histone arginine methylation in cytokine/chemokine gene expression in airway smooth muscle cells in asthma

Background: Asthma is a common chronic condition, which has a negative impact on life quality of those suffering and is also a burden to the national healthcare. Airway smooth muscle (ASM) cells have a crucial role in asthma, contributing to airway remodelling, airflow obstruction, and inflammation of the airways. Epigenetic changes, particularly histone lysine acetylation/methylation, have been shown to alter ASM functions. Histone arginine modifications have been less well studied. Hypothesis: We tested the hypothesis that histone arginine methylation is important in regulating ASM cytokine/chemokine production and that PRMTs, the enzymes which catalyse histone arginine methylation, are a therapeutic target in asthma. Aims: To characterise the expression of PRMTs in human ASM cells, to establish whether inhibiting PRMTs could reduce the cytokine/chemokine expression and their secretion from human ASM cells, and to establish the therapeutic potential of PRMT inhibitors in asthma. Methods: Studies were performed in cultured human ASM cells at passage six and 4hydroxytamoxifen (OHT)-inducible PRMT1FL/− ER-Cre mouse embryonic fibroblasts (MEFs). The activity of PRMT1 at the cytokine/chemokine promoters, as well as the impact of inhibiting PRMT1 on the inflammation in asthmatic airways, were investigated. TNF-α stimulation was used to simulate the conditions in asthmatic airways. Key results: The main findings of our study were that human ASM cells expressed mRNA and protein of all four PRMTs that methylate histones in vivo: PRMT1, PRMT4/CARM1, PRMT5 and PRMT6, but there was no significant difference in expression between ASM cells isolated from asthma patients and healthy subjects. We found that PRMT1 likely has a role in regulating the TNF-α-induced cytokine/chemokine production by ASM cells. Evidence supporting this role for PRMT1 came from studies showing that TNF-α-induced the PRMT1catalysed histone arginine methylation mark H4R3me2a, that a pharmacological inhibitor of PRMT1 inhibited cytokine/chemokine production by ASM, and that the molecular knockout by CRISPR showed comparable results, at least for IL-6, eotaxin and CXCL8, but not for IP-10. Further evidence for a role of PRMT1 was provided by our studies in PRMT1FL/− ER-Cre MEFs, as the loss of PRMT1 led to a reduction in TNF-α-induced secretion of a mouse chemokine KC. We also performed experiments studying an intermediate protein CNOT7, but overall the results regarding its involvement in TNF-α-induced PRMT1 mediated cytokine/chemokine production were inconclusive. Experiments with pharmacological inhibitors suggested that CARM1, but not PRMT5 or PRMT6, also had a regulatory role in cytokine/chemokine production in human ASM. Conclusions: Collectively, our results show that human ASM cells express several PRMTs and that PRMT1, and possibly also CARM1, should be investigated as potential targets for development of novel asthma treatments