Genome-Wide Mapping Defines a Role for C/EBPβ and c-Jun in Non-Canonical Cyclic AMP Signalling

• Published in: Cells (Basel, Switzerland) Switzerland), 2019-10, Vol.8 (10), p.1253
• Main Authors: Wiejak, Jolanta, van Basten, Boy, Hamilton, Graham, Yarwood, Stephen J
• Format: Article
• Language: English
• Subjects: AKT protein; Antibodies; Binding sites; c-jun; c-Jun protein; c/ebpβ transcriptome; cell adhesion molecules; Cell cycle; Chemokines; chromatin; Cyclic AMP; Cytokines; Endothelial cells; epac1; Extracellular signal-regulated kinase; Forskolin; Gene expression; Gene mapping; Genomes; Inflammation; Intercellular adhesion molecule 1; Interleukin 6; Kinases; MAP kinase; Metabolism; Monocyte chemoattractant protein 1; NMR; Nuclear magnetic resonance; Phosphorylation; Physiology; Platelet aggregation; Proteins; Rolipram; SOCS-3 protein; Transcription factors; Umbilical vein; vascular endothelial cells; Vascular endothelial growth factor
• ISSN: 2073-4409; 2073-4409
• DOI: 10.3390/cells8101253

The novel exchange protein activated by cyclic AMP (EPAC1) activator, I942, induces expression of the suppressor of cytokine signalling 3 (SOCS3) gene, thereby inhibiting interleukin 6 (IL6) inflammatory processes in human umbilical vein endothelial cells (HUVECs). Here we use RNA-SEQ and ChIP-SEQ to determine global gene responses to I942, in comparison with cyclic AMP production promoted by forskolin and rolipram (F/R). We found that I942 promoted significant changes in the RNA expression of 1413 genes, largely associated with microtubule stability and cell cycle progression, whereas F/R regulated 197 genes linked to endothelial cell function, including chemokine production and platelet aggregation. A further 108 genes were regulated by both treatments, including endothelial regulatory genes involved in purinergic signalling and cell junction organization. ChIP-SEQ demonstrated that F/R induced genome-wide recruitment of C/EBPβ and c-Jun transcription factors, whereas I942 promoted recruitment of c-Jun to genes associated with IL6 signalling, with little effect on C/EBPβ activation. Despite this, certain key inflammatory genes, including IL6, VEGF, CCL2/MCP1, VCAM1, SELE and ICAM1 were regulated by I942 without significant c-Jun recruitment, suggesting an additional, indirect mode of action for I942. In this regard, SOCS3 induction by I942 was found to require c-Jun and was associated with suppression of IL6-promoted ERK MAP kinase and AKT activity and induction of ICAM1. Pharmacological inhibition of ERK and AKT also potentiated ICAM1 induction by I942. We therefore propose that c-Jun activation by I942 regulates endothelial gene expression in HUVECs through direct mechanisms, involving recruitment of c-Jun or, as for ICAM1, through indirect regulation of tertiary regulators, including SOCS3.