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

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.

The Role of Cyclic AMP Signaling in Cardiac Fibrosis

“…However, it should be noticed that these effects might also result form off-target effects due to the possible interaction of these activators with Epac2 and PDEs [114]. Recently, novel small molecule activators have been identified that displays greater selectivity towards Epac1 [117,118]. Their impact on cardiac remodeling and fibrosis still need to be tested.…”

EPAC regulates von Willebrand factor secretion from endothelial cells in a PI3K/eNOS-dependent manner during inflammation

“…It has been proved that treatment of HUVECs with I942, which represents an effective tool to probe the function of cellular EPAC1, leads to alterations in the expression of a wide variety of genes associated with vascular function. In particular, I942 suppresses the expression of the proinflammatory adhesion molecules in HUVECs 75 . Upon an inflammatory trigger, ECs are activated, prompting the massive release of WPBs that contain vWF and P-selectin 76 .…”

“…EPAC1 is the major isoform in ECs, and Rap activation by EPAC1, but not EPAC2, contributes to the effects of cAMP-elevating hormones on endothelial barrier functions [42][43][44] . Growing evidence has revealed that the cAMP-EPAC signaling axis plays a regulatory role in suppressing inflammation 45,46 . The first identified non-cAMP EPAC1 specific agonist (ESA), I942, was shown to suppress IL-6 signaling and inflammatory gene expression in ECs in response to inflammatory stimuli 46 , suggesting EPAC1 playing an endothelial functionstabilizing role during inflammation 43,47 .…”

“…Growing evidence has revealed that the cAMP-EPAC signaling axis plays a regulatory role in suppressing inflammation 45,46 . The first identified non-cAMP EPAC1 specific agonist (ESA), I942, was shown to suppress IL-6 signaling and inflammatory gene expression in ECs in response to inflammatory stimuli 46 , suggesting EPAC1 playing an endothelial functionstabilizing role during inflammation 43,47 . In ECs, it has been documented that cAMP provokes the secretion of vWF via the cAMP-PKA pathway 21,35 .…”

The Epac1 Protein: Pharmacological Modulators, Cardiac Signalosome and Pathophysiology

“…In a functional fluorescence-based Rap1 GEF assay, I942 compound (Table 1) showed partial agonist activity towards Epac1 but not Epac2 or PKA, with an EC50 = 50 µM and a maximal potency of less than 10% of that of cAMP [30]. I942 was reported to promote Epac1-Rap activation in HEK293T cells stably expressing Epac1, and to induce SOCS3 expression and suppress Epac1-dependant IL6-stimulated JAK/STAT3 signalling in cultured vascular endothelial cells [31]. It may be noted that I942 shares a common chemical substructure (N-formyl-methylbenzenesulfonamide) with tolbutamide, whose Epac1-activating effect is discussed above.…”