Selective inhibition of cytokine-activated extracellular signal-regulated kinase by cyclic amp via Epac1-dependent induction of suppressor of cytokine signalling-3

Abstract

Here we demonstrate that elevation of cyclic AMP (cAMP) levels in human umbilical vein endothelial cells (HUVECs) specifically attenuates ERK1,2 activation in response to either leptin or a soluble interleukin IL-6 receptor-α/IL-6 (sIL-6R-α/IL-6) trans-signalling complex but not protein kinase C activator phorbol 12-myristate 13-acetate. The inhibitory effects of cAMP on sIL-6Ra/IL-6-stimulated phosphorylation of ERK1,2 and STAT3 were abolished by either short interfering (si) RNA-mediated knockdown or genetic ablation of suppressor of cytokine signalling-3 (SOCS-3). The inhibitory effect of cAMP could not be reversed by inhibition of cAMP-dependent protein kinase (PKA) but was blocked by depletion of the alternative intracellular cAMP sensor exchange protein activated by cAMP 1 (Epac1), which is also required to observe SOCS-3 accumulation in response to cAMP. Interestingly, the ability of cAMP elevation to inhibit IL-6 signalling was blocked by ERK inhibition. Consistent with this observation, cAMP elevation in HUVECs produced a transient yet robust activation of ERK, and subsequent phosphorylation of transcription factor C/EBPβ, both of which were resistant to PKA inhibition. However, siRNA depletion and immunoblotting experiments revealed that neither Epac1 nor Epac2 contributed to the PKA-independent activation of ERK1,2 observed following cAMP elevation. Together, these observations suggest that while SOCS-3 induction and subsequent inhibition of cytokine-mediated phosphorylation of ERK1,2 and STAT3 in response to cAMP require Epac1 and a transient PKA-independent activation of the ERK pathway, these two events are controlled by distinct mechanisms. In addition, it reveals a novel Epac-dependent mechanism by which cAMP can specifically inhibit ERK in response to cytokine receptor activation. Keywords: Cyclic AMP; exchange protein directly activated by cyclic AMP; interleukin-6; extracellular signalling-regulated kinase; suppressor of cytokine signalling Abbreviations: cAMP, cyclic AMP; HUVECs, human umbilical vein endothelial cells; sIL-6Ra, soluble IL-6 receptor a; ERK, extracellular signal-regulated kinase; STAT, signal transducer and activator of transcription; siRNA, short interfering RNA; SOCS, suppressor of cytokine signalling; Epac, exchange protein directly activated by cAMP; C/EBP, CCAAT/enhancer binding protein; JAK, Janus kinase; MEK, mitogen-activated protein/ERK kinase; SHP-2, SH2 domain-containing tyrosine phosphatase-2; PKA, cAMP-dependent protein kinase; GEF, guanine nucleotide exchange factor; PDE, phosphodiesterase; DNA-PK, DNA-dependent protein kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PMA, phorbol 12-myristate 13-acetate; CREB, cAMP response element binding protein; MEF, murine embryonic fibroblast