The mode of action and role of cGMP-mediated gene transcription in macrophages
File(s)
Author(s)
Cave, Luke
Type
Thesis
Abstract
Background: Nitric oxide (NO) and carbon monoxide (CO) gaseous signalling molecules are important factors in the pathogenesis of inflammatory disease as they regulate leukocyte activation, platelet aggregation and vasodilation. Cyclic-3’-5’guanosine monophosphate (cGMP) is a second messenger for NO, CO and several other mediators and pharmaceutical agents. cGMP has many well-described effects mediated by post-translational modifications, such as inhibition of vascular smooth muscle contraction. Its analogue, cyclic-3’-5’-adenosine monophosphate (cAMP) is well-known to modulate gene expression via the transcription factor cAMP-response element binding protein (CREB). In contrast, the gene regulatory effects of cGMP were until now essentially unknown. Hypothesis: cGMP regulates gene expression in macrophages via a CREB-dependent pathway. Methods and Results: Experiments were carried out on human blood-derived macrophages (HMDMs) from normal donors. In these, cGMP cell permeable analogue, dibutyryl-cGMP (dbcGMP), induced well-known CREB-target genes, such as NR4A2 and FOS. This effect was replicated with the co-treatment of CO-releasing molecule 3 (CORM3) and phosphodiesterase5 inhibitor, sildenafil but not with the treatment of NO-donors S-nitroso-Nacetylpenicillamine (SNAP) and sodium nitroprusside. dbcGMP, SNAP and CORM3 with sildenafil induced activating phosphorylation of CREB. Additionally, dbcGMP treatment induced the direct binding of CREB to the NR4A2 enhancer, as measured by ChIP-qPCR. dbcGMP-induced NR4A2 expression was attenuated by siRNAmediated CREB knockdown. Next, CREB gene was deleted in human monocytic U937 cell line via CRISPR/Cas9 technology. NR4A2 was induced by dbcGMP stimulation in control, but not in CREB-deficient U937 cells. RNA-sequencing of HMDMs revealed 59 genes regulated by dbcGMP only, 184 genes regulated by dbcAMP only and 114 genes regulated by both. The effect of dbcAMP and dbcGMP on gene expression were closely related, with both gene sets positively correlating with each other (p<0.05), as measured by gene set enrichment analysis. In contrast, dbcGMP and dbcAMP gene sets negatively correlated with an in vivo human inflammatory gene set (p<0.05). RNA-Sequencing revealed important gene targets not previously known to be cGMP-regulated that may mediate downstream functions such as NR1D1, CCR2, TLR5 and HSPA1. Conclusion: This data defines cGMP-mediated gene regulation, an almost completely novel area, and characterises CREB as a predominant mediating transcription factor in human macrophages. This adds significant insights to both inflammation biology and gene regulation.
Version
Open Access
Date Issued
2021-07
Date Awarded
2021-11
Copyright Statement
Creative Commons Attribution NonCommercial Licence
License URL
Advisor
Boyle, Joseph
Sponsor
British Heart Foundation
Publisher Department
National Heart & Lung Institute
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)