Abstract

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.

Highlights

  • Production of cyclic AMP in response to Gs-protein coupled receptor (GsPCR) activation leads to the regulation of a striking number of physiological processes, including control of metabolism, neuronal activity and immune cell processes through alterations in gene expression patterns in target cells [1]

  • Induction of suppressor of cytokine signalling 3 (SOCS3) by EPAC1 supports a paradigm for CREB-independent gene induction in VECs, which we have shown to involve cooperativity between c-Jun and CCAAT enhancer binding protein (C/EBP) transcription factors, which bind to a key AP1

  • We previously showed that when activated by cyclic AMP, the EPAC1/Rap1 pathway mediates the induction of the SOCS3 gene in human umbilical vein endothelial cells (HUVECs), independently of protein kinase A (PKA)

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Summary

Introduction

Production of cyclic AMP in response to Gs-protein coupled receptor (GsPCR) activation leads to the regulation of a striking number of physiological processes, including control of metabolism, neuronal activity and immune cell processes through alterations in gene expression patterns in target cells [1]. Elevations in intracellular cyclic AMP in response to activation of adenosine and prostaglandin GsPCRs in human umbilical vein endothelial cells (HUVECs) leads to inhibition of interleukin 6 (IL6) receptor activation of ERK and STAT3 [4]. This inhibition occurs independently of the classical route for cyclic

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