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

Kaczmarek, Klaudia (2019) The role of histone arginine methylation in cytokine/chemokine gene expression in airway smooth muscle cells in asthma. PhD thesis, University of Nottingham.

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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.

Item Type: Thesis (University of Nottingham only) (PhD)
Supervisors: Knox, Alan
Dowden, James
Clifford, Rachel
Keywords: Asthma, Airway smooth muscle cells, Epigenetic regulation, Histone modifications
Subjects: W Medicine and related subjects (NLM Classification) > WF Respiratory system
Faculties/Schools: UK Campuses > Faculty of Medicine and Health Sciences > School of Medicine
Item ID: 56695
Depositing User: Kaczmarek, Klaudia
Date Deposited: 26 Apr 2022 08:35
Last Modified: 26 Apr 2022 08:36

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