The novel exchange protein activated by cyclic AMP 1 (EPAC1) agonist, I942, regulates inflammatory gene expression in human umbilical vascular endothelial cells (HUVECs)

Jolanta Wiejak,Boy Van Basten,Urszula Luchowska-Stańska,Graham Hamilton,Stephen J Yarwood

doi:10.1016/j.bbamcr.2018.11.004

Abstract

Exchange protein activated by cyclic AMP (EPAC1) suppresses multiple inflammatory actions in vascular endothelial cells (VECs), partly due to its ability to induce expression of the suppressor of cytokine signalling 3 (SOCS3) gene, the protein product of which inhibits interleukin 6 (IL6) signalling through the JAK/STAT3 pathway. Here, for the first time, we use the non-cyclic nucleotide EPAC1 agonist, I942, to determine its actions on cellular EPAC1 activity and cyclic AMP-regulated gene expression in VECs. We demonstrate that I942 promotes EPAC1 and Rap1 activation in HEK293T cells and induces SOCS3 expression and suppresses IL6-stimulated JAK/STAT3 signalling in HUVECs. SOCS3 induction by I942 in HUVECs was blocked by the EPAC1 antagonist, ESI-09, and EPAC1 siRNA, but not by the broad-spectrum protein kinase A (PKA) inhibitor, H89, indicating that I942 regulates SOCS3 gene expression through EPAC1. RNA sequencing was carried out to further identify I942-regulated genes in HUVECs. This identified 425 I942-regulated genes that were also regulated by the EPAC1-selective cyclic AMP analogue, 007, and the cyclic AMP-elevating agents, forskolin and rolipram (F/R). The majority of genes identified were suppressed by I942, 007 and F/R treatment and many were involved in the control of key vascular functions, including the gene for the cell adhesion molecule, VCAM1. I942 and 007 also inhibited IL6-induced expression of VCAM1 at the protein level and blocked VCAM1-dependent monocyte adhesion to HUVECs. Overall, I942 represents the first non-cyclic nucleotide EPAC1 agonist in cells with the ability to suppress IL6 signalling and inflammatory gene expression in VECs.

Highlights

Atherosclerosis is a serious cardiovascular disease (CVD), which arises from chronic localised inflammation at coronary and carotid arterial branch points [1], and remains the principal cause of death in the developed world despite changes in lifestyle and the widespread use of anti-hypertensive and lipid-lowering drugs
We have previously shown that the EPAC1/Rap1 pathway mediates the induction of the suppressor of cytokine signalling 3 (SOCS3) gene in human umbilical vascular endothelial cells (HUVECs), independently of protein kinase A (PKA)
Results suggest that EPAC1 activation by I942 has the potential to suppress the pro-inflammatory gene expression through the inhibition of JAK/STAT3 signalling in HUVECs

Summary

Introduction

Atherosclerosis is a serious cardiovascular disease (CVD), which arises from chronic localised inflammation at coronary and carotid arterial branch points [1], and remains the principal cause of death in the developed world (http://www.who.int/cardiovascular_diseases/ en/) despite changes in lifestyle and the widespread use of anti-hypertensive and lipid-lowering drugs. IL6 localises to atherosclerotic plaques [5] and promotes chronic, low-level vascular inflammation, leading to neointimal thickening [6], vascular dysfunction [7], hypertension [8] and increased risk of myocardial infarction [2]. Initial engagement of monocytes with the endothelium involves rolling adhesion mediated by E-selectin (SELE) [11,12], followed by firm adhesion mediated by interactions between integrins expressed on leukocytes and adhesion molecules (ICAM1, VCAM1) expressed on endothelial cells [12]. Cadherin molecules at adherens junctions mediate trans-endothelial migration of

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